**Part 2**

**From the Laboratory to the Clinic: HIV and the Immune System** 

196 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

Wilson, S.J., Webb, B.L., Ylinen, L.M., Verschoor, E., Heeney, J.L., and Towers, G.J. (2008).

*the National Academy of Sciences of the United States of America 105*, 3557-3562. Wu, X., Anderson, J.L., Campbell, E.M., Joseph, A.M., and Hope, T.J. (2006). Proteasome

Xu, H., Svarovskaia, E.S., Barr, R., Zhang, Y., Khan, M.A., Strebel, K., and Pathak, V.K.

Yap, M.W., Nisole, S., Lynch, C., and Stoye, J.P. (2004). Trim5alpha protein restricts both

Ylinen, L.M., Keckesova, Z., Wilson, S.J., Ranasinghe, S., and Towers, G.J. (2005). Differential

Yu, X., Yu, Y., Liu, B., Luo, K., Kong, W., Mao, P., and Yu, X.F. (2003). Induction of

Zhang, F., Perez-Caballero, D., Hatziioannou, T., and Bieniasz, P.D. (2008). No effect of

Zhang, H., Yang, B., Pomerantz, R.J., Zhang, C., Arunachalam, S.C., and Gao, L. (2003). The

Zhao, Y., Chen, Y., Schutkowski, M., Fischer, G., and Ke, H. (1997). Cyclophilin A

Zheng, Y.H., Irwin, D., Kurosu, T., Tokunaga, K., Sata, T., and Peterlin, B.M. (2004). Human

virus SIVmac by TRIM5alpha alleles. *Journal of virology 79*, 11580-11587. Yu, Q., Konig, R., Pillai, S., Chiles, K., Kearney, M., Palmer, S., Richman, D., Coffin, J.M., and

*America 103*, 7465-7470.

*the United States of America 101*, 10786-10791.

*Science* (New York, NY *302*, 1056-1060.

5652-5657.

435-442.

*reply* 236-238.

DNA. *Nature 424*, 94-98.

activity. *Structure 5*, 139-146.

1 replication. *Journal of virology 78*, 6073-6076.

Independent evolution of an antiviral TRIMCyp in rhesus macaques. *Proceedings of* 

inhibitors uncouple rhesus TRIM5alpha restriction of HIV-1 reverse transcription and infection. *Proceedings of the National Academy of Sciences of the United States of* 

(2004). A single amino acid substitution in human APOBEC3G antiretroviral enzyme confers resistance to HIV-1 virion infectivity factor-induced depletion. *Proceedings of the National Academy of Sciences of the United States of America 101*,

HIV-1 and murine leukemia virus. *Proceedings of the National Academy of Sciences of* 

restriction of human immunodeficiency virus type 2 and simian immunodeficiency

Landau, N.R. (2004). Single-strand specificity of APOBEC3G accounts for minusstrand deamination of the HIV genome. *Nature structural & molecular biology 11*,

APOBEC3G ubiquitination and degradation by an HIV-1 Vif-Cul5-SCF complex.

endogenous TRIM5alpha on HIV-1 production. *Nature medicine 14*, 235-236; *author* 

cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1

complexed with a fragment of HIV-1 gag protein: insights into HIV-1 infectious

APOBEC3F is another host factor that blocks human immunodeficiency virus type

**8** 

*USA* 

**HIV Without AIDS: The Immunological** 

Human Immunodeficiency Virus (HIV) infection causes Acquired Immune Deficiency Syndrome (AIDS), while its ape and monkey progenitor Simian Immunodeficiency Virus (SIV) does not cause AIDS in its nonhuman primate natural reservoir hosts. Astonishingly, AIDS is avoided despite findings in a number of these host primate species indicating they too harbor high levels of virus replication that kills off CD4 T cells. These primate species that are so called "natural hosts" of SIV, essentially have HIV without ever getting AIDS. The elucidation of the exact mechanisms allowing natural SIV hosts to avoid disease progression may prove decisive in the battle to understand HIV pathogenesis for the purpose of preventative or curative HIV and AIDS therapy. Comparative studies in natural and nonnatural hosts of lentiviral infections (i.e. HIV or SIV) have defined essential distinguishing features, opening up new avenues for possible therapeutic and preventative

In this chapter, we will describe recent and past breakthroughs that come from comparing lentiviral infections in AIDS-free natural hosts, to immunocompromised nonnatural hosts. In addition, we will discuss how the knowledge derived from the study of natural hosts may inform the design of novel therapies and vaccine strategies for HIV-infected humans.

The observation in the late '70s and early '80s of a previously unrecognized adult-onset immunodeficiency associated with Kaposi's sarcoma (a skin cancer now known to be caused by Herpes Virus 8) and *Pneumocystis carinii* (a yeast-like fungus now known as *Pneumocystis jiroveci*) pneumonia signaled the beginning of one of the most devastating tragedies of modern times: the AIDS epidemic. Early epidemiological hypotheses on the etiologic agent of this disease included sexually transmitted pathogens as well as toxic "street" drugs (Centers for Disease Control (CDC), 1982; Harris et al., 1983). During these early years, basic and clinical researchers alike began furiously searching for the causes of AIDS, which culminated in 1983 with the discovery of the Human Immunodeficiency

A series of studies in molecular virology and epidemiology conducted in subsequent years have delineated both the timing and geographical origin of the AIDS pandemic. The African city of Kinshasa (formerly known as Leopoldville), in the Democratic Republic of the Congo,

**1. Introduction** 

intervention.

**1.1 A brief history of HIV** 

Virus (Barre-Sinoussi et al., 1983).

Zachary Ende, Michelle Bonkosky and Mirko Paiardini

**Secrets of Natural Hosts** 

*Emory University, Atlanta,* 

## **HIV Without AIDS: The Immunological Secrets of Natural Hosts**

Zachary Ende, Michelle Bonkosky and Mirko Paiardini *Emory University, Atlanta, USA* 

## **1. Introduction**

Human Immunodeficiency Virus (HIV) infection causes Acquired Immune Deficiency Syndrome (AIDS), while its ape and monkey progenitor Simian Immunodeficiency Virus (SIV) does not cause AIDS in its nonhuman primate natural reservoir hosts. Astonishingly, AIDS is avoided despite findings in a number of these host primate species indicating they too harbor high levels of virus replication that kills off CD4 T cells. These primate species that are so called "natural hosts" of SIV, essentially have HIV without ever getting AIDS.

The elucidation of the exact mechanisms allowing natural SIV hosts to avoid disease progression may prove decisive in the battle to understand HIV pathogenesis for the purpose of preventative or curative HIV and AIDS therapy. Comparative studies in natural and nonnatural hosts of lentiviral infections (i.e. HIV or SIV) have defined essential distinguishing features, opening up new avenues for possible therapeutic and preventative intervention.

In this chapter, we will describe recent and past breakthroughs that come from comparing lentiviral infections in AIDS-free natural hosts, to immunocompromised nonnatural hosts. In addition, we will discuss how the knowledge derived from the study of natural hosts may inform the design of novel therapies and vaccine strategies for HIV-infected humans.

#### **1.1 A brief history of HIV**

The observation in the late '70s and early '80s of a previously unrecognized adult-onset immunodeficiency associated with Kaposi's sarcoma (a skin cancer now known to be caused by Herpes Virus 8) and *Pneumocystis carinii* (a yeast-like fungus now known as *Pneumocystis jiroveci*) pneumonia signaled the beginning of one of the most devastating tragedies of modern times: the AIDS epidemic. Early epidemiological hypotheses on the etiologic agent of this disease included sexually transmitted pathogens as well as toxic "street" drugs (Centers for Disease Control (CDC), 1982; Harris et al., 1983). During these early years, basic and clinical researchers alike began furiously searching for the causes of AIDS, which culminated in 1983 with the discovery of the Human Immunodeficiency Virus (Barre-Sinoussi et al., 1983).

A series of studies in molecular virology and epidemiology conducted in subsequent years have delineated both the timing and geographical origin of the AIDS pandemic. The African city of Kinshasa (formerly known as Leopoldville), in the Democratic Republic of the Congo,

HIV Without AIDS: The Immunological Secrets of Natural Hosts 201

SIVagm.ver SIVagm.gri SIVagm.sab SIVagm.tan

SIVcol SIVwrc SIVolc

SIVcpz.ptt SIVcpz.pts

SIVsyk SIVlhoest SIVsun SIVdeb SIVmon SIVmus

SIVdrl

SIVsmm SIVrcm

SIVmnd/SIVmnd2

**Genus Species/subspecies Virus** 

Vervet monkey (*C. pygerythrus*) Grivet monkey (*C. aethiops*) Green monkey (*C. sabaeus*) Tantalus monkey (*C. tantalus*)

Manted guereza (*C. guereza*) Western red colobus (*Piliocolobus* 

Olive colobus (*Procolobus verus)* 

Western chimpanzee (*P. troglodytes* 

Eastern chimpanzee (*P. troglodytes* 

Mustached monkey (*C. cephus*)

*Lophocebus* Black mangabey (*Lophocebus aterrimus*) SIVbkm

Drill (*M. leucophaeus*)

(*Miopithecus*) Angolan talapoin (*M. talapoin*) SIVtal

Table 1. Natural SIV hosts (reviewed on (VandeWoude & Apetrei, 2006))

Sooty mangabey (*C. atys*)

Red-capped mangabey (*C. torquatus*)

Most of the naturally occurring SIVs do not cause disease in their natural hosts (Paiardini, et al., 2009); however, they can be highly pathogenic when replicating in nonnatural hosts, such as rhesus macaques and humans. Wild chimpanzee studies of SIV prevalence and pathogenicity, made possible by testing stool samples, have recently demonstrated that SIV positive chimpanzees die at a faster rate than their uninfected counterparts –a 9.8-15.6-fold increased death hazard) (Keele et al., 2009). While searching for the age and origins of the chimpanzee SIV, a major breakthrough came when it was noticed that the 5' region of the chimpanzee SIV genome closely matches that found in red-capped mangabeys (*Cercocebus torquatus*), but the 3' end closely resembles SIVs found in greater spot-nosed (*Cercopithecus nictitans*), mustached (*Cercopithecus cephus*) and mona monkeys (*Cercopithecus mona*). Based on these findings, SIVcpz is thought to be a recombination of viruses ancestral to those found in red-capped mangabeys, mona, spot-nosed and mustached monkeys (Paul M Sharp, Shaw, & Beatrice H Hahn, 2005). The data suggest that chimpanzees have not evolved along with their own SIV for very long, and may represent a necessary evolutionary stage for the virus to enable cross-species transmission into humans. In a recent study, Worobey et al. established that SIV is at least 32,000 years old, based on Bioko Island geography and SIV relatedness of the various African nonhuman primates on the island. The authors conclude

Sykes' monkey (*C. mitis*) L'Hoest monkey (*C. lhoesti*) Sun-tailed monkey (*C. solatus*) De Brazza monkey (*C. neglectus*)

*badius)* 

*troglodytes*)

*schweinfirthii*)

Mona (*C. mona*)

Mandrills (*Mandrillus*) Mandrill (*M. sphinx*)

African green monkeys (*Chlorocebus*)

Black and white

Chimpanzee

colobus (*Colobus*)

(*Pan*)

Guenons (*Cercopithecus*)

Talapoins

White-eyelid mangabeys (*Cercocebus*)

is the place where the oldest known HIV-infected samples were discovered in a lymph node biopsy from 1960 and a blood-plasma sample from 1959 (Paul M Sharp & Beatrice H Hahn, 2008). In 1998, Zhu et al. estimated that HIV-1 originated in the 1940's or early 1950's, and also proposed that the split between HIV-1 and HIV-2 must have occurred considerably earlier (T. Zhu et al., 1998). Ten years later, Worobey et al. proposed the origin of HIV-1 to be anywhere from 1884-1933, a range corresponding to the rise of urban populations in the Leopoldville/Kinshasa area (Worobey et al., 2008). Collectively, these observations and models have refuted the controversial speculation that experimental polio vaccine formulations from the 1950's were responsible for the AIDS epidemic (Worobey et al., 2004). Subsequent studies indicated that HIV infection in humans arose from cross-species transmission of viruses that naturally infect African nonhuman primates (NHP), which are referred to as natural hosts for SIV (P M Sharp & B H Hahn, 2010). The next section will describe the different African nonhuman primates infected with SIV, focusing on those that infected giving rise to HIV-1 and HIV-2.

#### **1.2 Introduction to natural hosts**

At least 40 monkey species in Africa have been found to be naturally infected with speciesspecific strains of SIVs, and usually with a high prevalence. In the vast majority of cases the virus is designated by a three-letter abbreviation of the infected nonhuman primates (NHP) species name to differentiate between SIV strains (VandeWoude and Apetrei, 2006). For example, SIVcpz is the virus isolated from chimpanzees (*Pan troglodytes*), SIVsmm from sooty mangabeys (*Cercocebus atys*), SIVmnd from mandrills (*Mandrillus sphinx*), SIVagm from African green monkeys (AGM), and so on. The viruses that infect the different species of AGM are named SIVagm.ver (Vervet monkey), SIVagm.gri (Grivet monkey), SIVagm.sab (Green monkey), SIVagm.tan (Tantalus monkey). Table 1 lists the different African nonhuman primates infected with SIV.

These natural hosts for SIV represent an extremely large reservoir of lentiviruses potentially infecting other species. Phylogenetic analyses revealed that there have been cross-species transmissions of divergent viral strains since the beginning of the evolution of primate lentiviruses (Courgnaud et al., 2003; Hirsch, Dapolito, Goeken, & Campbell, 1995; P M Sharp & B H Hahn, 2010). For instance, HIV-2 emerged from the west African natural host sooty mangabey (*Cercocebus atys*) in at least eight cross-species events into the human population (Wertheim & Worobey, 2009), while the origins of HIV-1 have been more controversial. Four HIV-1 lineages originating in chimpanzees have been independently transmitted across species to infect humans, though one or two may have come via gorillas (P M Sharp & B H Hahn, 2010; Takehisa & Miura, 2010). Most HIV-1 isolates resemble viruses found in a chimpanzee subspecies (*Pan troglodytes troglodytes*) native to areas in and around Cameroon, Gabon and Equatorial Guinea, including the areas around Kinshasa (Gao et al., 1999; P M Sharp & B H Hahn, 2010). HIV-1 group M, responsible for a suggested 98% of the global epidemic, as well as rare groups N and O, are endemic in the aforementioned areas. Hunting chimpanzees for food, which is thought to be the method of cross-species transmission is also common in this central African region (Gao et al., 1999). In addition, the SIVs that have been isolated from Asian macaques, the most commonly used primate model of HIV infection, appear to have been transmitted from captive sooty mangabeys (reviewed in I. Pandrea, Sodora, Silvestri, & Apetrei, 2008).

is the place where the oldest known HIV-infected samples were discovered in a lymph node biopsy from 1960 and a blood-plasma sample from 1959 (Paul M Sharp & Beatrice H Hahn, 2008). In 1998, Zhu et al. estimated that HIV-1 originated in the 1940's or early 1950's, and also proposed that the split between HIV-1 and HIV-2 must have occurred considerably earlier (T. Zhu et al., 1998). Ten years later, Worobey et al. proposed the origin of HIV-1 to be anywhere from 1884-1933, a range corresponding to the rise of urban populations in the Leopoldville/Kinshasa area (Worobey et al., 2008). Collectively, these observations and models have refuted the controversial speculation that experimental polio vaccine formulations from the 1950's were responsible for the AIDS epidemic (Worobey et al., 2004). Subsequent studies indicated that HIV infection in humans arose from cross-species transmission of viruses that naturally infect African nonhuman primates (NHP), which are referred to as natural hosts for SIV (P M Sharp & B H Hahn, 2010). The next section will describe the different African nonhuman primates infected with SIV, focusing on those that

At least 40 monkey species in Africa have been found to be naturally infected with speciesspecific strains of SIVs, and usually with a high prevalence. In the vast majority of cases the virus is designated by a three-letter abbreviation of the infected nonhuman primates (NHP) species name to differentiate between SIV strains (VandeWoude and Apetrei, 2006). For example, SIVcpz is the virus isolated from chimpanzees (*Pan troglodytes*), SIVsmm from sooty mangabeys (*Cercocebus atys*), SIVmnd from mandrills (*Mandrillus sphinx*), SIVagm from African green monkeys (AGM), and so on. The viruses that infect the different species of AGM are named SIVagm.ver (Vervet monkey), SIVagm.gri (Grivet monkey), SIVagm.sab (Green monkey), SIVagm.tan (Tantalus monkey). Table 1 lists the different African

These natural hosts for SIV represent an extremely large reservoir of lentiviruses potentially infecting other species. Phylogenetic analyses revealed that there have been cross-species transmissions of divergent viral strains since the beginning of the evolution of primate lentiviruses (Courgnaud et al., 2003; Hirsch, Dapolito, Goeken, & Campbell, 1995; P M Sharp & B H Hahn, 2010). For instance, HIV-2 emerged from the west African natural host sooty mangabey (*Cercocebus atys*) in at least eight cross-species events into the human population (Wertheim & Worobey, 2009), while the origins of HIV-1 have been more controversial. Four HIV-1 lineages originating in chimpanzees have been independently transmitted across species to infect humans, though one or two may have come via gorillas (P M Sharp & B H Hahn, 2010; Takehisa & Miura, 2010). Most HIV-1 isolates resemble viruses found in a chimpanzee subspecies (*Pan troglodytes troglodytes*) native to areas in and around Cameroon, Gabon and Equatorial Guinea, including the areas around Kinshasa (Gao et al., 1999; P M Sharp & B H Hahn, 2010). HIV-1 group M, responsible for a suggested 98% of the global epidemic, as well as rare groups N and O, are endemic in the aforementioned areas. Hunting chimpanzees for food, which is thought to be the method of cross-species transmission is also common in this central African region (Gao et al., 1999). In addition, the SIVs that have been isolated from Asian macaques, the most commonly used primate model of HIV infection, appear to have been transmitted from captive sooty mangabeys (reviewed in I. Pandrea, Sodora,

infected giving rise to HIV-1 and HIV-2.

nonhuman primates infected with SIV.

Silvestri, & Apetrei, 2008).

**1.2 Introduction to natural hosts** 


Table 1. Natural SIV hosts (reviewed on (VandeWoude & Apetrei, 2006))

Most of the naturally occurring SIVs do not cause disease in their natural hosts (Paiardini, et al., 2009); however, they can be highly pathogenic when replicating in nonnatural hosts, such as rhesus macaques and humans. Wild chimpanzee studies of SIV prevalence and pathogenicity, made possible by testing stool samples, have recently demonstrated that SIV positive chimpanzees die at a faster rate than their uninfected counterparts –a 9.8-15.6-fold increased death hazard) (Keele et al., 2009). While searching for the age and origins of the chimpanzee SIV, a major breakthrough came when it was noticed that the 5' region of the chimpanzee SIV genome closely matches that found in red-capped mangabeys (*Cercocebus torquatus*), but the 3' end closely resembles SIVs found in greater spot-nosed (*Cercopithecus nictitans*), mustached (*Cercopithecus cephus*) and mona monkeys (*Cercopithecus mona*). Based on these findings, SIVcpz is thought to be a recombination of viruses ancestral to those found in red-capped mangabeys, mona, spot-nosed and mustached monkeys (Paul M Sharp, Shaw, & Beatrice H Hahn, 2005). The data suggest that chimpanzees have not evolved along with their own SIV for very long, and may represent a necessary evolutionary stage for the virus to enable cross-species transmission into humans. In a recent study, Worobey et al. established that SIV is at least 32,000 years old, based on Bioko Island geography and SIV relatedness of the various African nonhuman primates on the island. The authors conclude

HIV Without AIDS: The Immunological Secrets of Natural Hosts 203

Natural host species (sooty mangabeys −−−, African green monkeys −−−) and nonnatural host species (humans −−−, rhesus macaques −−−) have similar levels of viremia in the acute and chronic phase of infection. Originally published in *Blood* Online. Brenchley JM & Paiardini M. Immunodeficiency lentiviral infections in natural and nonnatural hosts. *Blood*.

The depletion of CD4 T cells is the immunological hallmark of progressive HIV infection. The loss of circulating CD4 T cell numbers at levels below 200 cells/ml of blood coincides with onset of opportunistic infections. As such, a better understanding of the dynamics of CD4 T cell depletion is essential when studying the pathogenesis of HIV infection. Depletion of CD4 T cells from the peripheral blood is generally quite slow, with HIV-infected humans losing approximately 40 CD4 T cells per l of blood per year during the chronic phase of infection (Moore, Keruly, Richman, Creagh-Kirk, &

The study of SIV-infected macaques has provided important information on the dynamics of CD4 T cell depletion, particularly in the early phase of infection and in anatomical locations difficult to study in HIV-infected humans. In particular, a series of influential studies have elucidated the early consequences of pathogenic HIV and SIV infections at the level of mucosal tissues, showing that the depletion of CD4 T cells is more rapid and severe at this site than in the peripheral blood or secondary lymphoid organs (figure 2) (Brenchley, Schacker, et al., 2004a; Douek, Mario Roederer, & Koup, 2009; Guadalupe et al., 2003; Haase, 2005; Mehandru et al., 2004; T Schneider et al., 1995; Veazey et al., 1998). Other observations underpin the reasons why the mucosal tissues undergo such stress: (i) the large majority of CD4 T cells located in the effector mucosal sites show a memory, activated, CCR5+

Fig. 1. Viral load in natural and nonnatural host species.

**2.2.1 Nonnatural hosts for HIV and SIV infections** 

**2.2 CD4 T cell homeostasis** 

Chaisson, 1992).

Prepublished April 19, 2011; DOI:10.1182/blood-2010-12-325936.

that humans may have had previous encounters with this virus over time, and that natural hosts that show low pathogenicity to SIV have arisen likely as "a consequence of long-term host-virus coevolution" (P M Sharp & B H Hahn, 2010; Worobey et al., 2010) .

As above mentioned, and in obvious contrast with HIV infection in humans, which almost invariably leads to AIDS if left untreated, SIV infection in natural African NHP hosts is typically non-progressive. The infected animals live an apparently normal lifespan, without experiencing any signs of illness whether in the wild or captivity (Paiardini ann rev med). The fact that HIV causes a deadly disease in humans while its simian counterparts are virtually non-pathogenic in their natural hosts remains one of the fundamental mysteries of modern medicine, and it is widely recognized that the elucidation of the exact mechanisms allowing natural SIV hosts to avoid disease progression may prove critical in terms of HIV pathogenesis, therapy, and vaccines. Over the past few years, comparative studies in natural and nonnatural hosts of lentiviral infections have shed light on a number of critical distinguishing features.

## **2. Immunology and virology of HIV and SIV infections**

#### **2.1 Viral loads**

#### **2.1.1 Nonnatural hosts for HIV and SIV infections**

As previously noted, HIV and SIV infections in humans and Asian macaques were generated from cross-species transmissions of viruses that naturally infect nonhuman primates in Africa. These primate lentiviruses replicate very efficiently *in vivo*, with the vast majority of HIV-infected humans and SIV-infected Asian macaques showing approximately 108 virions per milliliter of plasma during the acute phase of infection (Picker, 2006) (figure 1). Tracking SIV-infected macaques has been and continues to be indispensible for our understanding of virus kinetics at all stages of infection and in key tissues (i.e. gut and lymph node). Information obtained early in the infection process when virus replication begins and the adaptive immune response is underway, is vital to our ability to rationally design effective treatment and preventative strategies.

As the infection advances into the chronic phase, viral load in plasma declines and stabilizes to its "set point" (figure 1). This stage is reached once the immune system develops antibodies in an attempt to fight the virus. The behavior of the virus at set point is characterized by three major factors: (i) viral load remains relatively stable for several years; (ii) individuals who have a higher set point level have faster progression to AIDS; and (iii) shortly before the development of clinical AIDS, viral load increases. Despite declining levels of viral replication from peak viremia to set point, other factors persist, such as generalized immune activation, that play important roles in damaging a progressively dysfunctional immune system.

#### **2.1.2 Natural hosts for SIV infections**

Worth noting is the point that both in the acute and chronic phases of infection, the levels of plasma viremia are similar in HIV-infected humans and SIV-infected natural hosts, such as sooty mangabeys and African green monkeys (figure 1) (Picker, 2006) . The implication of the data is clear and extremely important: the presence of a cytophatic virus that replicates at high levels is not sufficient, by itself, to induce AIDS. In other words, additional factors are required for disease progression in HIV-infected humans and SIV-infected rhesus macaques.

that humans may have had previous encounters with this virus over time, and that natural hosts that show low pathogenicity to SIV have arisen likely as "a consequence of long-term

As above mentioned, and in obvious contrast with HIV infection in humans, which almost invariably leads to AIDS if left untreated, SIV infection in natural African NHP hosts is typically non-progressive. The infected animals live an apparently normal lifespan, without experiencing any signs of illness whether in the wild or captivity (Paiardini ann rev med). The fact that HIV causes a deadly disease in humans while its simian counterparts are virtually non-pathogenic in their natural hosts remains one of the fundamental mysteries of modern medicine, and it is widely recognized that the elucidation of the exact mechanisms allowing natural SIV hosts to avoid disease progression may prove critical in terms of HIV pathogenesis, therapy, and vaccines. Over the past few years, comparative studies in natural and nonnatural hosts of lentiviral infections have shed light on a number of critical

As previously noted, HIV and SIV infections in humans and Asian macaques were generated from cross-species transmissions of viruses that naturally infect nonhuman primates in Africa. These primate lentiviruses replicate very efficiently *in vivo*, with the vast majority of HIV-infected humans and SIV-infected Asian macaques showing approximately 108 virions per milliliter of plasma during the acute phase of infection (Picker, 2006) (figure 1). Tracking SIV-infected macaques has been and continues to be indispensible for our understanding of virus kinetics at all stages of infection and in key tissues (i.e. gut and lymph node). Information obtained early in the infection process when virus replication begins and the adaptive immune response is underway, is vital to our ability to rationally

As the infection advances into the chronic phase, viral load in plasma declines and stabilizes to its "set point" (figure 1). This stage is reached once the immune system develops antibodies in an attempt to fight the virus. The behavior of the virus at set point is characterized by three major factors: (i) viral load remains relatively stable for several years; (ii) individuals who have a higher set point level have faster progression to AIDS; and (iii) shortly before the development of clinical AIDS, viral load increases. Despite declining levels of viral replication from peak viremia to set point, other factors persist, such as generalized immune activation, that play important roles in damaging a

Worth noting is the point that both in the acute and chronic phases of infection, the levels of plasma viremia are similar in HIV-infected humans and SIV-infected natural hosts, such as sooty mangabeys and African green monkeys (figure 1) (Picker, 2006) . The implication of the data is clear and extremely important: the presence of a cytophatic virus that replicates at high levels is not sufficient, by itself, to induce AIDS. In other words, additional factors are required for disease progression in HIV-infected humans and SIV-infected rhesus

host-virus coevolution" (P M Sharp & B H Hahn, 2010; Worobey et al., 2010) .

**2. Immunology and virology of HIV and SIV infections** 

**2.1.1 Nonnatural hosts for HIV and SIV infections** 

design effective treatment and preventative strategies.

progressively dysfunctional immune system.

**2.1.2 Natural hosts for SIV infections** 

macaques.

distinguishing features.

**2.1 Viral loads** 

Fig. 1. Viral load in natural and nonnatural host species.

Natural host species (sooty mangabeys −−−, African green monkeys −−−) and nonnatural host species (humans −−−, rhesus macaques −−−) have similar levels of viremia in the acute and chronic phase of infection. Originally published in *Blood* Online. Brenchley JM & Paiardini M. Immunodeficiency lentiviral infections in natural and nonnatural hosts. *Blood*. Prepublished April 19, 2011; DOI:10.1182/blood-2010-12-325936.

## **2.2 CD4 T cell homeostasis**

## **2.2.1 Nonnatural hosts for HIV and SIV infections**

The depletion of CD4 T cells is the immunological hallmark of progressive HIV infection. The loss of circulating CD4 T cell numbers at levels below 200 cells/ml of blood coincides with onset of opportunistic infections. As such, a better understanding of the dynamics of CD4 T cell depletion is essential when studying the pathogenesis of HIV infection. Depletion of CD4 T cells from the peripheral blood is generally quite slow, with HIV-infected humans losing approximately 40 CD4 T cells per l of blood per year during the chronic phase of infection (Moore, Keruly, Richman, Creagh-Kirk, & Chaisson, 1992).

The study of SIV-infected macaques has provided important information on the dynamics of CD4 T cell depletion, particularly in the early phase of infection and in anatomical locations difficult to study in HIV-infected humans. In particular, a series of influential studies have elucidated the early consequences of pathogenic HIV and SIV infections at the level of mucosal tissues, showing that the depletion of CD4 T cells is more rapid and severe at this site than in the peripheral blood or secondary lymphoid organs (figure 2) (Brenchley, Schacker, et al., 2004a; Douek, Mario Roederer, & Koup, 2009; Guadalupe et al., 2003; Haase, 2005; Mehandru et al., 2004; T Schneider et al., 1995; Veazey et al., 1998). Other observations underpin the reasons why the mucosal tissues undergo such stress: (i) the large majority of CD4 T cells located in the effector mucosal sites show a memory, activated, CCR5+

HIV Without AIDS: The Immunological Secrets of Natural Hosts 205

rhesus macaques, SIV-infected sooty mangabeys manifest a rapid and severe depletion of mucosal CD4 T cells (figure 2). In the first study, Gordon et al. showed that memory CD4 T cells are rapidly and severely depleted from the mucosal sites (but not from peripheral blood or lymph nodes) of SIV infected sooty mangabeys, with kinetics remarkably similar to those observed during pathogenic SIVmac infection of macaques (Gordon et al., 2007). In the second study, Pandrea et al. observed a similar level of mucosal CD4 T cell depletion in African green monkeys compared to rhesus macaques during the acute phase of SIV infection (I. V. Pandrea et al., 2007b). Notably, the early loss of mucosal CD4 T cells does not progress further after reaching a stable plateau in sooty mangabeys and is followed by a partial recovery of these cells in African green monkeys—trends that contrast with that described in pathogenic HIV and SIV infections in humans and rhesus macaques where mucosal CD4 T cell depletion becomes increasingly more severe as disease progresses to

Intriguingly, despite levels of CD4 T cells in the gut comparable to those described in HIVinfected humans who progress to AIDS, sooty mangabeys and African green monkeys maintain normal mucosal immune function, as indicated by the maintenance of an intact mucosal barrier, the complete absence of any increased susceptibility to infections, and the lack of microbial translocation (Brenchley, Price, Schacker, Asher, et al., 2006a; Estes et al., 2010; Gordon et al., 2007; I. V. Pandrea et al., 2007b). These findings raise an important question of how SIV-infected natural hosts maintain mucosal immunity and avoid progression to AIDS despite the loss of mucosal CD4 T cells. One might hypothesize that in sooty mangabeys, preservation of CD4 T cell homeostasis in the peripheral blood compensates for the loss of mucosal CD4 T cells, and is sufficient to maintain a functional immune system. This hypothesis, however, is not consistent with the observation that a fraction of naturally and experimentally infected sooty mangabeys experience a variable but significant (with animals showing <100 cells/ul blood) loss of CD4 T cell in blood and tissues, while still remaining healthy and AIDS-free (Milush et al., 2007; Mir, Gasper, Sundaravaradan, & Sodora, 2011; Sumpter et al., 2007; Taaffe et al., 2010). The evidence indicates that even a generalized depletion of CD4 T cells, per se, is not sufficient to induce progression to AIDS in natural hosts for SIV. This leaves unanswered the question of how SIV-infected sooty mangabeys can afford to lose CD4 T cells but maintain mucosal

To answer this question, several, non-mutually exclusive mechanisms have been suggested in the past few years. One possibility is that the lack of other pathogenic factors, in particular chronic immune activation, protects the CD4 T cell depleted mucosa of sooty mangabeys (Mirko Paiardini et al., 2009b). An alternative possibility is that the immune system of natural hosts evolved to be less dependent on CD4 T cells, with other cell types carrying on the CD4 T cell helper functions. In particular, two recently published studies of sooty mangabeys and African green monkeys showed the presence of a significant fraction of that despite lacking CD4 expression, indeed act as CD4 T cells; this allows the immune system to maintain "classical" helper functions that otherwise would be lost (Milush et al., 2011, Beaumier et al., 2009). A third possibility is that despite being quantitatively similar, the depletion of CD4 T cells is qualitatively different in pathogenic and nonpathogenic lentiviral infections. This last possibility implies that natural hosts for SIV are able to preserve certain critical CD4 T cell subsets, in the context of generalized CD4 T cell

AIDS.

immunity and avoid progression to AIDS.

depletion, sufficient for maintaining a functional immune system.

phenotype, (ii) the majority of newly transmitted HIV and SIV strains are CCR5-tropic, and (iii) primate lentiviruses preferentially infect activated CD4 T cells (Z. Zhang et al., 1999; Brenchley, Hill, et al., 2004b; Brenchley, Silvestri, & Douek, 2010; Veazey et al., 2000; Y . Zhang et al., 2000). As such, a large fraction of mucosal-resident CD4 T cells represent a highly susceptible target for virus replication, especially at a time when no antiviral adaptive immune response has yet been generated. Using the macaque SIV model, it was demonstrated that mucosal memory CD4+CCR5+ T cells are the earliest targets of the virus regardless of the route of infection (Veazey et al., 1998), and the majority (70-95%) of CD4 T cells in the jejunum, ileum, and colon are depleted in less than three weeks post infection (Li et al., 2005; Mattapallil et al., 2005). Due to the large surface area of the gastrointestinal (GI) tract, this severe loss of mucosal CD4 T cells during the acute phase of infection likely translates to depletion of most CD4 T cells within the body.

While there is a general consensus on the dramatic loss of mucosal CD4 T cells, the exact mechanisms accounting for this depletion are not completely clear, with evidence pointing in different directions. Direct virus-mediated killing of infected CD4 T cells is responsible for the earliest (within days of infection) loss of CD4 T cells (Mattapallil et al., 2005) and CD95-mediated activation induced cell death of uninfected bystander CD4 T cells (Li et al., 2005) accounts for the subsequent depletion (within weeks). Of note, recent studies comparing multiple GI sites have shown anatomic-specific differences in the extent of CD4 T cell loss in chronically SIV-infected rhesus macaques, with CD4 T cell depletion being more severe in the small intestine compared to the large intestine (L. D. Harris, Klatt, et al., 2010a). Due to the complexity of performing longitudinal mucosal collections and sampling multiple anatomic sites, similar comparative analysis has not, systematically, been performed in humans. Therefore it is debatable whether this phenomenon extends to HIVinfected individuals.

Based on these findings, a new model of AIDS pathogenesis has been proposed. That is to say, the early and complex dysfunction of the mucosal immune system induces a significant impairment of mucosal barrier integrity resulting in a series of pathogenic sequelae that become mostly apparent during chronic infection. The best characterized consequences of damage to the mucosal barrier are the translocation of microbial products from the intestinal lumen into systemic circulation, and the establishment of high levels of chronic immune activation. From this relatively new point of view, the depletion of CD4 T cells from mucosal tissues during acute HIV or SIV infection is a keydeterminant of disease progression [1, 49-52].

#### **2.2.2 Natural hosts for SIV infection**

One of the most peculiar features of natural hosts of SIV infection is their ability to preserve healthy levels of peripheral CD4 T cells, despite levels of plasma viremia similar or even higher than those described in HIV-infected individuals (Chakrabarti et al., 2000; Rey-Cuillé et al., 1998; Silvestri et al., 2003). For instance, approximately 90% of SIV-infected sooty mangabeys maintain CD4 T cell counts comparable to those observed in uninfected animals (Sumpter et al., 2007). This is a clear difference compared to the progressive depletion of circulating CD4 T cells that characterize pathogenic HIV and SIV infections in humans and rhesus macaques (figure 2).

Intriguingly, two recent studies aimed at investigating the kinetics of mucosal CD4 T cells during SIV infection of sooty mangabeys and African green monkeys (Gordon et al., 2007; I. V. Pandrea et al., 2007b) demonstrated that just like HIV-infected humans and SIV-infected

phenotype, (ii) the majority of newly transmitted HIV and SIV strains are CCR5-tropic, and (iii) primate lentiviruses preferentially infect activated CD4 T cells (Z. Zhang et al., 1999; Brenchley, Hill, et al., 2004b; Brenchley, Silvestri, & Douek, 2010; Veazey et al., 2000; Y . Zhang et al., 2000). As such, a large fraction of mucosal-resident CD4 T cells represent a highly susceptible target for virus replication, especially at a time when no antiviral adaptive immune response has yet been generated. Using the macaque SIV model, it was demonstrated that mucosal memory CD4+CCR5+ T cells are the earliest targets of the virus regardless of the route of infection (Veazey et al., 1998), and the majority (70-95%) of CD4 T cells in the jejunum, ileum, and colon are depleted in less than three weeks post infection (Li et al., 2005; Mattapallil et al., 2005). Due to the large surface area of the gastrointestinal (GI) tract, this severe loss of mucosal CD4 T cells during the acute phase of infection likely

While there is a general consensus on the dramatic loss of mucosal CD4 T cells, the exact mechanisms accounting for this depletion are not completely clear, with evidence pointing in different directions. Direct virus-mediated killing of infected CD4 T cells is responsible for the earliest (within days of infection) loss of CD4 T cells (Mattapallil et al., 2005) and CD95-mediated activation induced cell death of uninfected bystander CD4 T cells (Li et al., 2005) accounts for the subsequent depletion (within weeks). Of note, recent studies comparing multiple GI sites have shown anatomic-specific differences in the extent of CD4 T cell loss in chronically SIV-infected rhesus macaques, with CD4 T cell depletion being more severe in the small intestine compared to the large intestine (L. D. Harris, Klatt, et al., 2010a). Due to the complexity of performing longitudinal mucosal collections and sampling multiple anatomic sites, similar comparative analysis has not, systematically, been performed in humans. Therefore it is debatable whether this phenomenon extends to HIV-

Based on these findings, a new model of AIDS pathogenesis has been proposed. That is to say, the early and complex dysfunction of the mucosal immune system induces a significant impairment of mucosal barrier integrity resulting in a series of pathogenic sequelae that become mostly apparent during chronic infection. The best characterized consequences of damage to the mucosal barrier are the translocation of microbial products from the intestinal lumen into systemic circulation, and the establishment of high levels of chronic immune activation. From this relatively new point of view, the depletion of CD4 T cells from mucosal tissues during acute HIV or SIV infection is a keydeterminant of disease progression [1, 49-52].

One of the most peculiar features of natural hosts of SIV infection is their ability to preserve healthy levels of peripheral CD4 T cells, despite levels of plasma viremia similar or even higher than those described in HIV-infected individuals (Chakrabarti et al., 2000; Rey-Cuillé et al., 1998; Silvestri et al., 2003). For instance, approximately 90% of SIV-infected sooty mangabeys maintain CD4 T cell counts comparable to those observed in uninfected animals (Sumpter et al., 2007). This is a clear difference compared to the progressive depletion of circulating CD4 T cells that characterize pathogenic HIV and SIV infections in humans and

Intriguingly, two recent studies aimed at investigating the kinetics of mucosal CD4 T cells during SIV infection of sooty mangabeys and African green monkeys (Gordon et al., 2007; I. V. Pandrea et al., 2007b) demonstrated that just like HIV-infected humans and SIV-infected

translates to depletion of most CD4 T cells within the body.

infected individuals.

**2.2.2 Natural hosts for SIV infection** 

rhesus macaques (figure 2).

rhesus macaques, SIV-infected sooty mangabeys manifest a rapid and severe depletion of mucosal CD4 T cells (figure 2). In the first study, Gordon et al. showed that memory CD4 T cells are rapidly and severely depleted from the mucosal sites (but not from peripheral blood or lymph nodes) of SIV infected sooty mangabeys, with kinetics remarkably similar to those observed during pathogenic SIVmac infection of macaques (Gordon et al., 2007). In the second study, Pandrea et al. observed a similar level of mucosal CD4 T cell depletion in African green monkeys compared to rhesus macaques during the acute phase of SIV infection (I. V. Pandrea et al., 2007b). Notably, the early loss of mucosal CD4 T cells does not progress further after reaching a stable plateau in sooty mangabeys and is followed by a partial recovery of these cells in African green monkeys—trends that contrast with that described in pathogenic HIV and SIV infections in humans and rhesus macaques where mucosal CD4 T cell depletion becomes increasingly more severe as disease progresses to AIDS.

Intriguingly, despite levels of CD4 T cells in the gut comparable to those described in HIVinfected humans who progress to AIDS, sooty mangabeys and African green monkeys maintain normal mucosal immune function, as indicated by the maintenance of an intact mucosal barrier, the complete absence of any increased susceptibility to infections, and the lack of microbial translocation (Brenchley, Price, Schacker, Asher, et al., 2006a; Estes et al., 2010; Gordon et al., 2007; I. V. Pandrea et al., 2007b). These findings raise an important question of how SIV-infected natural hosts maintain mucosal immunity and avoid progression to AIDS despite the loss of mucosal CD4 T cells. One might hypothesize that in sooty mangabeys, preservation of CD4 T cell homeostasis in the peripheral blood compensates for the loss of mucosal CD4 T cells, and is sufficient to maintain a functional immune system. This hypothesis, however, is not consistent with the observation that a fraction of naturally and experimentally infected sooty mangabeys experience a variable but significant (with animals showing <100 cells/ul blood) loss of CD4 T cell in blood and tissues, while still remaining healthy and AIDS-free (Milush et al., 2007; Mir, Gasper, Sundaravaradan, & Sodora, 2011; Sumpter et al., 2007; Taaffe et al., 2010). The evidence indicates that even a generalized depletion of CD4 T cells, per se, is not sufficient to induce progression to AIDS in natural hosts for SIV. This leaves unanswered the question of how SIV-infected sooty mangabeys can afford to lose CD4 T cells but maintain mucosal immunity and avoid progression to AIDS.

To answer this question, several, non-mutually exclusive mechanisms have been suggested in the past few years. One possibility is that the lack of other pathogenic factors, in particular chronic immune activation, protects the CD4 T cell depleted mucosa of sooty mangabeys (Mirko Paiardini et al., 2009b). An alternative possibility is that the immune system of natural hosts evolved to be less dependent on CD4 T cells, with other cell types carrying on the CD4 T cell helper functions. In particular, two recently published studies of sooty mangabeys and African green monkeys showed the presence of a significant fraction of that despite lacking CD4 expression, indeed act as CD4 T cells; this allows the immune system to maintain "classical" helper functions that otherwise would be lost (Milush et al., 2011, Beaumier et al., 2009). A third possibility is that despite being quantitatively similar, the depletion of CD4 T cells is qualitatively different in pathogenic and nonpathogenic lentiviral infections. This last possibility implies that natural hosts for SIV are able to preserve certain critical CD4 T cell subsets, in the context of generalized CD4 T cell depletion, sufficient for maintaining a functional immune system.

HIV Without AIDS: The Immunological Secrets of Natural Hosts 207

CD45RA and CD62L expression are lost in parallel with CD4 T cells, regardless of the stage of disease progression and despite rises in total numbers of CD8 T cells (M Roederer et al., 1995). Low levels of CD69, an early marker of activation, and increased T regulatory cells have been associated with HIV-resistant individuals (Card et al., 2009), along with low levels of HLA-DR+CD38+ CD4 T cells and Ki-67+ CD4 and CD8 T cells (Koning et al., 2005). Upon stimulation, activation markers CD80, CD86 and CD70 are increased in HIV infected patients (Wolthers et al., 1996). Other soluble activation markers have also been found in serum and plasma to be increased in HIV infected patients including beta2-microglobulin (Grieco et al., 1984), IL-2 receptor (Sethi & Näher, 1986; Pizzolo et al., 1987), tumor necrosis factor (Reddy, Sorrell, Lange, & Grieco, 1988) tumor necrosis factor receptor II (Fahey et al.,

A recurrent trend in research focusing on immune activation is the consistent importance of CD38 as a marker of disease prognosis. CD38, otherwise known as cyclic ADP ribose hydrolase, is an ectoenzyme transmembrane glycoprotein that correlates with other cell activation markers and is associated with enhanced cell to cell adhesion, cytokine production and T-cell activation (Deeks et al., 2004). According to a Giorgi et al. study referenced over 330 times (ISI Web of KnowledgeSM), CD4 and CD8 T cell expression of CD38 is increased in clinically defined AIDS patients who survived less than 6 months versus those who survived greater than 18 months (J V Giorgi et al., 1999; Sandler et al., 2011). While the level of HIV RNA is a good predictor of disease progression early in infection, and CD4 T cell count is as good if not better later in infection, CD38 levels on CD8 T cells is a good early and late predictor (Janis V Giorgi et al., 2002). Activated CD8+CD38+CD45RO+ T cells predict CD4 T cell decline (Bofill et al., 1996), though CD8+HLA-DR+ cannot (J V Giorgi et al., 1993). An activation set-point measured by CD38 expression on CD4 and especially CD8 T cells arises early in infection and is relatively stable and able to predict subsequent CD4 T cell decline even without considering viral load (Deeks et al., 2004). Also, increased HLADR+CD38+ T cells in elite controllers with low plasma virus loads is associated with decreased CD4 counts (Hunt et al., 2008), in tune with

the idea that T cell activation promotes HIV disease progression (Fahey et al., 1998).

Soluble markers of immune activation, that are more easily measurable than cellular activation, have also been shown to have prognostic value and predict HIV disease progression with comparable efficiency to CD4 counts and viral load measurements (Liu et al., 1997). In particular, neopterin, produced by macrophages upon IFNg stimulation (Melmed, Taylor, Detels, Bozorgmehri, & Fahey, 1989), beta2-microglobulin for general lymphoid activation (Chitra, Bakthavatsalam, & Palvannan, 2011; Fahey et al., 1990), and soluble IL-2 receptor (Sethi & Näher, 1986) have all been shown to be elevated and predictive of disease progression to varying degrees (Fahey et al., 1998). Increased soluble CD14 levels, a marker of monocyte activation that also correlated with IL-6, C-reactive protein, serum amyloid A and D-dimer, independently predicts mortality in HIV patients

In summary, the HIV-associated immune activation (i) is characterized by high frequencies of numerous immune cell types expressing markers of activation, proliferation, and apoptosis; (ii) predicts the tempo of progression to AIDS independently from, and more accurately than viral load; (iii) strongly correlates with the efficacy of antiretroviral therapy (ART) in reconstituting the immune system of HIV-infected individuals. Although the benefits of being able to predict or modify the course of disease during acute HIV infection

1998) and others.

(Sandler et al., 2011).

Two of these mechanisms, i.e. the lack of immune activation and the preservation of the homeostasis of selective CD4 T cell subsets, are described in more details in the next sections.

Fig. 2. CD4 T cell homeostasis in natural and nonnatural hosts. In both pathogenic (humans −−−, rhesus macaques −−−) and nonpathogenic (sooty mangabeys −−− and African green monkeys −−−) HIV/SIV infection, CD4 T cells are rapidly lost in the mucosal associated lymphoid tissue (MALT, dotted lines). In contrast to pathogenic infection, CD4 T cells are generally preserved in the peripheral blood (PB, solid lines) of natural host species. Originally published in *Blood* Online. Brenchley JM & Paiardini M. Immunodeficiency lentiviral infections in natural and nonnatural hosts. *Blood*. Prepublished April 19, 2011; DOI:10.1182/blood-2010-12-325936.

### **3. Immune activation**

#### **3.1 Immune activation markers and their role as predictors of disease progression**

The establishment of a state of chronic, generalized immune activation is a characteristic feature of pathogenic HIV infection in humans and SIV infection in macaques (Douek D Ann REv Med 2009; Sodora DL AIDS 2009). A large number of scientific evidence clearly shows that HIV infection is associated with high frequencies of numerous immune cell types, including CD4 and CD8 T cells, B cells, NK cells, and monocytes, that express markers of activation, proliferation, and apoptosis (reviewed in Sodora et al., 2008).

The strong association between immune activation and AIDS pathogenesis is well documented. A large 2006 study that took place over 20 years probing 2,801 treatment naïve HIV-1 infected patients concluded that only a small percent of CD4 loss variability could be attributed to HIV-1 RNA plasma viral loads, suggesting other factors, mainly immune activation, were likely responsible for CD4 T cell decline (Rodríguez et al., 2006). CD4 T cell recovery during antiretroviral treatment is mitigated when there are higher frequencies of CD4 and CD8 CD38+HLA-DR+ T cells (Hunt et al., 2003). Naïve CD8 T cells defined by

Two of these mechanisms, i.e. the lack of immune activation and the preservation of the homeostasis of selective CD4 T cell subsets, are described in more details in the next sections.

Fig. 2. CD4 T cell homeostasis in natural and nonnatural hosts.

Prepublished April 19, 2011; DOI:10.1182/blood-2010-12-325936.

**3. Immune activation** 

In both pathogenic (humans −−−, rhesus macaques −−−) and nonpathogenic

M. Immunodeficiency lentiviral infections in natural and nonnatural hosts. *Blood*.

(sooty mangabeys −−− and African green monkeys −−−) HIV/SIV infection, CD4 T cells are rapidly lost in the mucosal associated lymphoid tissue (MALT, dotted lines). In contrast to pathogenic infection, CD4 T cells are generally preserved in the peripheral blood (PB, solid lines) of natural host species. Originally published in *Blood* Online. Brenchley JM & Paiardini

**3.1 Immune activation markers and their role as predictors of disease progression**  The establishment of a state of chronic, generalized immune activation is a characteristic feature of pathogenic HIV infection in humans and SIV infection in macaques (Douek D Ann REv Med 2009; Sodora DL AIDS 2009). A large number of scientific evidence clearly shows that HIV infection is associated with high frequencies of numerous immune cell types, including CD4 and CD8 T cells, B cells, NK cells, and monocytes, that express

markers of activation, proliferation, and apoptosis (reviewed in Sodora et al., 2008).

The strong association between immune activation and AIDS pathogenesis is well documented. A large 2006 study that took place over 20 years probing 2,801 treatment naïve HIV-1 infected patients concluded that only a small percent of CD4 loss variability could be attributed to HIV-1 RNA plasma viral loads, suggesting other factors, mainly immune activation, were likely responsible for CD4 T cell decline (Rodríguez et al., 2006). CD4 T cell recovery during antiretroviral treatment is mitigated when there are higher frequencies of CD4 and CD8 CD38+HLA-DR+ T cells (Hunt et al., 2003). Naïve CD8 T cells defined by CD45RA and CD62L expression are lost in parallel with CD4 T cells, regardless of the stage of disease progression and despite rises in total numbers of CD8 T cells (M Roederer et al., 1995). Low levels of CD69, an early marker of activation, and increased T regulatory cells have been associated with HIV-resistant individuals (Card et al., 2009), along with low levels of HLA-DR+CD38+ CD4 T cells and Ki-67+ CD4 and CD8 T cells (Koning et al., 2005). Upon stimulation, activation markers CD80, CD86 and CD70 are increased in HIV infected patients (Wolthers et al., 1996). Other soluble activation markers have also been found in serum and plasma to be increased in HIV infected patients including beta2-microglobulin (Grieco et al., 1984), IL-2 receptor (Sethi & Näher, 1986; Pizzolo et al., 1987), tumor necrosis factor (Reddy, Sorrell, Lange, & Grieco, 1988) tumor necrosis factor receptor II (Fahey et al., 1998) and others.

A recurrent trend in research focusing on immune activation is the consistent importance of CD38 as a marker of disease prognosis. CD38, otherwise known as cyclic ADP ribose hydrolase, is an ectoenzyme transmembrane glycoprotein that correlates with other cell activation markers and is associated with enhanced cell to cell adhesion, cytokine production and T-cell activation (Deeks et al., 2004). According to a Giorgi et al. study referenced over 330 times (ISI Web of KnowledgeSM), CD4 and CD8 T cell expression of CD38 is increased in clinically defined AIDS patients who survived less than 6 months versus those who survived greater than 18 months (J V Giorgi et al., 1999; Sandler et al., 2011). While the level of HIV RNA is a good predictor of disease progression early in infection, and CD4 T cell count is as good if not better later in infection, CD38 levels on CD8 T cells is a good early and late predictor (Janis V Giorgi et al., 2002). Activated CD8+CD38+CD45RO+ T cells predict CD4 T cell decline (Bofill et al., 1996), though CD8+HLA-DR+ cannot (J V Giorgi et al., 1993). An activation set-point measured by CD38 expression on CD4 and especially CD8 T cells arises early in infection and is relatively stable and able to predict subsequent CD4 T cell decline even without considering viral load (Deeks et al., 2004). Also, increased HLADR+CD38+ T cells in elite controllers with low plasma virus loads is associated with decreased CD4 counts (Hunt et al., 2008), in tune with the idea that T cell activation promotes HIV disease progression (Fahey et al., 1998).

Soluble markers of immune activation, that are more easily measurable than cellular activation, have also been shown to have prognostic value and predict HIV disease progression with comparable efficiency to CD4 counts and viral load measurements (Liu et al., 1997). In particular, neopterin, produced by macrophages upon IFNg stimulation (Melmed, Taylor, Detels, Bozorgmehri, & Fahey, 1989), beta2-microglobulin for general lymphoid activation (Chitra, Bakthavatsalam, & Palvannan, 2011; Fahey et al., 1990), and soluble IL-2 receptor (Sethi & Näher, 1986) have all been shown to be elevated and predictive of disease progression to varying degrees (Fahey et al., 1998). Increased soluble CD14 levels, a marker of monocyte activation that also correlated with IL-6, C-reactive protein, serum amyloid A and D-dimer, independently predicts mortality in HIV patients (Sandler et al., 2011).

In summary, the HIV-associated immune activation (i) is characterized by high frequencies of numerous immune cell types expressing markers of activation, proliferation, and apoptosis; (ii) predicts the tempo of progression to AIDS independently from, and more accurately than viral load; (iii) strongly correlates with the efficacy of antiretroviral therapy (ART) in reconstituting the immune system of HIV-infected individuals. Although the benefits of being able to predict or modify the course of disease during acute HIV infection

HIV Without AIDS: The Immunological Secrets of Natural Hosts 209

HIV infection, with individuals falling in the highest quartile of sCD14 levels having a 6 fold higher risk of death than those in the lowest quartile, even after adjusting for inflammatory markers, CD4 T cell count, and HIV RNA level (Sandler et al., 2011). Moreover, another study demonstrated that microbial translocation was detected by the presence of 16S ribosomal DNA in 95% of untreated HIV-infected patients observed (Jiang et al., 2009). Interestingly, plasma LPS levels were found to be higher with drug abuse, or co-

Due to the immediacy of these events, and the fact that translocating products are bioactive in vivo, the gut breakdown and associated microbial translocation cascade has been thought to stoke the fire of, or at least contribute to, the establishment of high levels of innate and adaptive immune activation (Brenchley, Price, & Douek, 2006b; Douek et al., 2009). Evidence supporting this model comes from the fact that plasma levels of LPS are significantly increased, and correlate with the level of systemic immune activation in chronically HIV infected individuals and SIV infected rhesus macaques. Even uninfected CD4 T cells in the gut dive to extremely low numbers after just weeks of infection, as bacterial products rise in the blood of HIV infected patients. In a more recent study, Nowroozalizadeh and collaborators found elevated levels of plasma LPS in both individuals infected with HIV type 1 and HIV type 2. Furthermore, they showed that the severity of microbial translocation correlates with CD4 T cell count and viral load independently of HIV type, as well as with defective innate and mitogen responsiveness (Nowroozalizadeh et

Due to its broad impacts on several cell types of the innate and adaptive immune response, HIV-associated immune activation may damage the immune system in many different ways. Depletion of CD4 T cells in the gut and peripheral blood in the acute phase and beyond leads to vacancies in the T cell receptor repertoire that threatens immune resources normally in reserve to fight new, latent or mutating infections (Simons et al., 2008). Certain CD4 T cell specificities are preferentially lost. For instance, CD4 T cells specific for *Mycobacterium tuberculosis* (MTB) are lost quickly compared to those for CMV, likely due to lower expression of CCR5 ligand MIP-1b on MTB specific CD4 T cells (Geldmacher et al., 2010). Cytokines and other soluble factors (as described in the section about activation markers) are at dangerously abnormal levels. Th17, an IL-17 producing CD4 T cell subset critical for mucosal immunity are preferentially depleted (Brenchley et al., 2008). B cell dysfunction is also pronounced as HIV impacts activation states, hypergammaglobulinemia, exhaustion, and impaired antibody production against vaccination and infections (reviewed

In spite of the large number of immune abnormalities that have been described, there are still unanswered questions about the exact mechanisms by which this virus causes

An important mechanism that appears to link loss of mucosal barrier integrity, microbial translocation and the establishment of immune activation is the preferential depletion of Th17 cells, a recently identified CD4 T cell subset that produce IL-17 and IL-22 and play a critical role in antimicrobial mucosal immunity. In particular, IL-17 and IL-22 (i) induce epithelial cells to express cytokines (i.e., IL-6 and GM-CSF), chemokines (i.e., IL-8, CXCL1, CXCL10, and CCL20) and metalloproteinases critical for the recruitment, activation and

progressive disease, possibly because so many constituents are impacted.

**4. Depletion of Th17 cells and loss of mucosal barrier integrity** 

infection with hepatitis-c virus (HCV) (Ancuta et al., 2008).

al., 2010).

in Shen & Tomaras, 2011).

would likely be substantial, the value of immune activation biomarkers has largely been detected during chronic HIV infection due to the obvious constraints of human studies.

#### **3.2 Causes and consequence of HIV-associated chronic immune activation**

The causes of the chronic immune activation and subsequent immunopathogenesis in HIV infected patients is unsettled. Whether or not immunopathogenesis is mainly caused by the virus or the immune response to the virus has been the object of a long scientific debate. While some have focused on the virus and its direct cytophathicity by claiming "it's the virus stupid" (Cohen, 1993), others counterclaimed, "it's the immune system, stupid" (STEP perspective, 1999; Smith, 2006). Further studies in humans, natural hosts of SIV, rhesus macaque models of progressive infection and even mice models of immune activation have helped to clarify that the cause of HIV pathogenesis is multifactorial, with both viral and host factors contributing to progression to AIDS. Moreover, many arms of the immune system aside from infected CD4 T cells are dysregulated.

A particularly salient example of how immune activation alone damages the immune system comes from a transgenic mouse model of chronic immune activation triggered by CD27-CD70 costimulation. The mice showed uncanny familiarity with HIV disease without a virus present, with constant costimulation and TCR antigen stimulation leading to thymic involution, T cell turnover, loss of naïve T cell populations, and progressive inability of T cells to respond ex vivo upon stimulation (Tesselaar et al., 2003).

Possible explanations for HIV-associated chronic immune activation is a long list: gut damage and microbial translocation (Brenchley, Price, & Douek, 2006b), loss of T helper 17 (Th17) cells (Brenchley et al., 2008), loss of regulatory T cells (Hunt et al., 2011, Card et al., 2009), expansion and exhaustion of HIV-specific T cells (Khaitan & Unutmaz, 2011) decreased lymphopoiesis and increased depletion of central memory CD4 T cells (TCM), both resulting in increases in homeostatic proliferation (Brenchley et al., 2010; Okoye et al., 2007; M Paiardini et al., 2009a; Picker et al., 2004; Sauce et al., 2011) and latent or newly acquired infections due to general immunodeficiency (Ford, Puronen, & Sereti, 2009).

In particular, special emphasis has been recently placed on the role played by the complex dysfunction of the mucosal immune system typical of pathogenic HIV and SIV infections in humans and rhesus macaques. The HIV-associated mucosal immune dysfunction is characterized by the loss of integrity of the mucosal barrier and the translocation of microbial products from the intestinal lumen into systemic circulation. Alexander and collaborators defined microbial translocation as "passage of both viable and nonviable microbes and microbial products, such as endotoxin across the intestinal barrier." They show that microbial translocation of microbes and microbial products occurred because of alterations in mucosal balance (Alexander et al., 1990).

Numerous evidences demonstrated the translocation of bacteria and bacterial products into the bloodstream in pathogenic HIV and SIV infections. Lipopolysacharides (LPS), which is excreted from gram-negative bacteria and act as an endotoxin, is one of the bacterial products that is translocated into the bloodstream and can therefore be used as an indicator of microbial translocation. Circulating LPS levels were increased in chronically HIV-infected individuals and SIV-infected rhesus macaques during the chronic phase of the disease, and LPS levels were associated with increased levels of soluble CD14, a marker of monocyte response to LPS (Brenchley, Price, Schacker, Asher, et al., 2006a). Of note, a recent casecontrol study demonstrated that soluble CD14 is an independent predictor of mortality in

would likely be substantial, the value of immune activation biomarkers has largely been detected during chronic HIV infection due to the obvious constraints of human studies.

The causes of the chronic immune activation and subsequent immunopathogenesis in HIV infected patients is unsettled. Whether or not immunopathogenesis is mainly caused by the virus or the immune response to the virus has been the object of a long scientific debate. While some have focused on the virus and its direct cytophathicity by claiming "it's the virus stupid" (Cohen, 1993), others counterclaimed, "it's the immune system, stupid" (STEP perspective, 1999; Smith, 2006). Further studies in humans, natural hosts of SIV, rhesus macaque models of progressive infection and even mice models of immune activation have helped to clarify that the cause of HIV pathogenesis is multifactorial, with both viral and host factors contributing to progression to AIDS. Moreover, many arms of the immune

A particularly salient example of how immune activation alone damages the immune system comes from a transgenic mouse model of chronic immune activation triggered by CD27-CD70 costimulation. The mice showed uncanny familiarity with HIV disease without a virus present, with constant costimulation and TCR antigen stimulation leading to thymic involution, T cell turnover, loss of naïve T cell populations, and progressive inability of T

Possible explanations for HIV-associated chronic immune activation is a long list: gut damage and microbial translocation (Brenchley, Price, & Douek, 2006b), loss of T helper 17 (Th17) cells (Brenchley et al., 2008), loss of regulatory T cells (Hunt et al., 2011, Card et al., 2009), expansion and exhaustion of HIV-specific T cells (Khaitan & Unutmaz, 2011) decreased lymphopoiesis and increased depletion of central memory CD4 T cells (TCM), both resulting in increases in homeostatic proliferation (Brenchley et al., 2010; Okoye et al., 2007; M Paiardini et al., 2009a; Picker et al., 2004; Sauce et al., 2011) and latent or newly acquired

In particular, special emphasis has been recently placed on the role played by the complex dysfunction of the mucosal immune system typical of pathogenic HIV and SIV infections in humans and rhesus macaques. The HIV-associated mucosal immune dysfunction is characterized by the loss of integrity of the mucosal barrier and the translocation of microbial products from the intestinal lumen into systemic circulation. Alexander and collaborators defined microbial translocation as "passage of both viable and nonviable microbes and microbial products, such as endotoxin across the intestinal barrier." They show that microbial translocation of microbes and microbial products occurred because of

Numerous evidences demonstrated the translocation of bacteria and bacterial products into the bloodstream in pathogenic HIV and SIV infections. Lipopolysacharides (LPS), which is excreted from gram-negative bacteria and act as an endotoxin, is one of the bacterial products that is translocated into the bloodstream and can therefore be used as an indicator of microbial translocation. Circulating LPS levels were increased in chronically HIV-infected individuals and SIV-infected rhesus macaques during the chronic phase of the disease, and LPS levels were associated with increased levels of soluble CD14, a marker of monocyte response to LPS (Brenchley, Price, Schacker, Asher, et al., 2006a). Of note, a recent casecontrol study demonstrated that soluble CD14 is an independent predictor of mortality in

**3.2 Causes and consequence of HIV-associated chronic immune activation** 

system aside from infected CD4 T cells are dysregulated.

alterations in mucosal balance (Alexander et al., 1990).

cells to respond ex vivo upon stimulation (Tesselaar et al., 2003).

infections due to general immunodeficiency (Ford, Puronen, & Sereti, 2009).

HIV infection, with individuals falling in the highest quartile of sCD14 levels having a 6 fold higher risk of death than those in the lowest quartile, even after adjusting for inflammatory markers, CD4 T cell count, and HIV RNA level (Sandler et al., 2011). Moreover, another study demonstrated that microbial translocation was detected by the presence of 16S ribosomal DNA in 95% of untreated HIV-infected patients observed (Jiang et al., 2009). Interestingly, plasma LPS levels were found to be higher with drug abuse, or coinfection with hepatitis-c virus (HCV) (Ancuta et al., 2008).

Due to the immediacy of these events, and the fact that translocating products are bioactive in vivo, the gut breakdown and associated microbial translocation cascade has been thought to stoke the fire of, or at least contribute to, the establishment of high levels of innate and adaptive immune activation (Brenchley, Price, & Douek, 2006b; Douek et al., 2009). Evidence supporting this model comes from the fact that plasma levels of LPS are significantly increased, and correlate with the level of systemic immune activation in chronically HIV infected individuals and SIV infected rhesus macaques. Even uninfected CD4 T cells in the gut dive to extremely low numbers after just weeks of infection, as bacterial products rise in the blood of HIV infected patients. In a more recent study, Nowroozalizadeh and collaborators found elevated levels of plasma LPS in both individuals infected with HIV type 1 and HIV type 2. Furthermore, they showed that the severity of microbial translocation correlates with CD4 T cell count and viral load independently of HIV type, as well as with defective innate and mitogen responsiveness (Nowroozalizadeh et al., 2010).

Due to its broad impacts on several cell types of the innate and adaptive immune response, HIV-associated immune activation may damage the immune system in many different ways. Depletion of CD4 T cells in the gut and peripheral blood in the acute phase and beyond leads to vacancies in the T cell receptor repertoire that threatens immune resources normally in reserve to fight new, latent or mutating infections (Simons et al., 2008). Certain CD4 T cell specificities are preferentially lost. For instance, CD4 T cells specific for *Mycobacterium tuberculosis* (MTB) are lost quickly compared to those for CMV, likely due to lower expression of CCR5 ligand MIP-1b on MTB specific CD4 T cells (Geldmacher et al., 2010). Cytokines and other soluble factors (as described in the section about activation markers) are at dangerously abnormal levels. Th17, an IL-17 producing CD4 T cell subset critical for mucosal immunity are preferentially depleted (Brenchley et al., 2008). B cell dysfunction is also pronounced as HIV impacts activation states, hypergammaglobulinemia, exhaustion, and impaired antibody production against vaccination and infections (reviewed in Shen & Tomaras, 2011).

In spite of the large number of immune abnormalities that have been described, there are still unanswered questions about the exact mechanisms by which this virus causes progressive disease, possibly because so many constituents are impacted.

## **4. Depletion of Th17 cells and loss of mucosal barrier integrity**

An important mechanism that appears to link loss of mucosal barrier integrity, microbial translocation and the establishment of immune activation is the preferential depletion of Th17 cells, a recently identified CD4 T cell subset that produce IL-17 and IL-22 and play a critical role in antimicrobial mucosal immunity. In particular, IL-17 and IL-22 (i) induce epithelial cells to express cytokines (i.e., IL-6 and GM-CSF), chemokines (i.e., IL-8, CXCL1, CXCL10, and CCL20) and metalloproteinases critical for the recruitment, activation and

HIV Without AIDS: The Immunological Secrets of Natural Hosts 211

which are normally attributed to chronic immune activation (Silvestri et al., 2003). Furthermore, naturally SIV-infected sooty mangabeys preserve the ability to properly regulate

cell cycle progression when compared to SIV-infected macaques (Paiardini M JV 2006).

Fig. 3. Immune activation in natural and nonnatural hosts.

April 19, 2011; DOI:10.1182/blood-2010-12-325936.

In contrast to pathogenic HIV/SIV infection (humans −−− , rhesus macaques −−−),

African green monkeys −−−) is associated with the resolution of immune activation during chronic infection. Originally published in *Blood* Online. Brenchley JM & Paiardini M. Immunodeficiency lentiviral infections in natural and nonnatural hosts. *Blood*. Prepublished

Interestingly, the consistently low levels of chronic immune activation in natural hosts does not result from intrinsically attenuated innate immune responses, but rather from active immuno-regulatory mechanisms that allow these animals to tune-down the immune response during the transition from the acute to the chronic phase of infection (figure 3). The initial studies of natural SIV infections were performed during chronic infection and were not able to inform early events. Indeed, more recent studies designed to characterize the acute phase of SIV infection consistently show that, as described for progressive infection, nonprogressive SIV infection is also associated with an early increase in T cell proliferation and activation (Gordon et al., 2007; Kornfeld et al., 2005; I. V. Pandrea et al., 2007b; Silvestri et al., 2005). This phenotype is very common among several natural hosts, even those less characterized than sooty mangabeys and African green monkeys. For instance, transient levels of immune activation have been described in Mandrills, in which CD4 and CD8 HLA-DR+ cells at first increase but then return to normal levels by day 60 post-infection (Onanga et al., 2006), as well as in Caribbean African green monkeys, which show a rapid increase in CD8 HLA-DR+ T cells and then a rapid return to baseline 2-3 weeks post-infection, while having no changes in CD4 HLA-DR+ T cell frequencies (Kornfeld et al., 2005; I. Pandrea et al., 2006). Furthermore, the rapid resolution of acute immune activation has also been shown at a genetic level in sooty

nonpathogenic SIV infection in natural hosts (sooty mangabeys −−− and

migration of neutrophils to areas of bacterial infection; (ii) promote the production of antimicrobial molecules, such as defensins; and (iii) regulate the integrity of the epithelial barrier by stimulating the proliferation and survival of GI enterocyte and the transcription of tight junction proteins (Aujla et al., 2008; Dandekar, George, & Bäumler, 2010; Guglani & Khader, 2010; Liang et al., 2006; Milner, Sandler, & Douek, 2010; Ouyang & Valdez, 2008; Romagnani, 2008; Zheng et al., 2008). Consistent with their important role in antimicrobial immunity, Th17 cells confer protection against several extracellular pathogens, such as *Candida albicans*, *Klebsiella pneumoniae*, *Citrobacter rodentium*, *Mycobacteria tuberculosis*, *Staphylococcus aureus*, *Bacteroides fragilis*, *Escherichia coli* (Huang, Na, Fidel, & Schwarzenberger, 2004; Khader et al., 2007; Ouyang & Valdez, 2008). Given the role of Th17 cells in mucosal immunity, and the observed mucosal immune dysfunction associated with HIV infection, we and others investigated the homeostasis of Th17 during pathogenic lentiviral infection, showing that Th17 cells are preferentially depleted in the gastrointestinal tracts of HIV-infected humans and SIV-infected macaques (Brenchley et al., 2008; Cecchinato et al., 2008; d'Ettorre, Mirko Paiardini, Ceccarelli, Silvestri, & Vullo, 2011; Favre et al., 2009; Gordon et al., 2010; Raffatellu et al., 2008). Moreover, Raffatellu and colleagues showed that in healthy SIV-negative rhesus macaques, the gene expression profile induced by S. typhimurium in ileal loops is dominated by Th17 responses, including the expression of IL-17 and IL-22; and severe depletion of mucosal Th17 cells in SIV-infected rhesus macaques resulted in an impaired mucosal barrier function and increased S. *typhimurium* dissemination (Raffatellu et al., 2008). Furthermore, loss of mucosal Th17 cells has been associated with increased systemic immune activation and disease progression in both HIVinfected humans and SIV-infected rhesus macaques (Cecchinato et al., 2008; Gordon et al., 2010; Hartigan-O'connor, Hirao, McCune, & Dandekar, 2011). Consistent with the model linking depletion of Th17 cells with compromised antimicrobial immunity, it has been shown that patients with dominant negative stat3 gene mutations, common in hyperimmunoglobulin E syndrome or the more biblical Job's syndrome, in which CD4 T cells are unable to differentiate into Th17 cells, are exquisitely susceptible to bacterial infections (Milner et al., 2008).

Collectively, these studies demonstrate that pathogenic HIV and SIV infections are associated with a preferential and sustained depletion of mucosal Th17 cells, the severity of which correlates with the structural and immunological maintenance of the mucosal barrier, the levels of immune activation, and progression to AIDS. These observations further elucidate the immunodeficiency of HIV disease and provide a mechanistic basis for the mucosal barrier breakdown that characterizes HIV infection.

#### **5. Immunology of natural hosts for SIV**

#### **5.1 Absence of chronic immune activation**

A very large body of evidence clearly demonstrated that, in sharp contrast with all the known models of pathogenic HIV infection, nonpathogenic SIV infection of natural hosts is characterized by the absence of high levels of chronic immune activation, assessed as the fraction of cells expressing markers of activation and proliferation, in the context of continuous virus replication (figure 3) (Mirko Paiardini et al., 2009b; Silvestri et al., 2003; Silvestri, Mirko Paiardini, I. Pandrea, Lederman, & Sodora, 2007). Consistent with their lower levels of immune activation, infected sooty mangabeys show no increase in lymphocyte apoptosis, lymph node structural damage, thymic involution, or loss of naïve T cell populations—all of

migration of neutrophils to areas of bacterial infection; (ii) promote the production of antimicrobial molecules, such as defensins; and (iii) regulate the integrity of the epithelial barrier by stimulating the proliferation and survival of GI enterocyte and the transcription of tight junction proteins (Aujla et al., 2008; Dandekar, George, & Bäumler, 2010; Guglani & Khader, 2010; Liang et al., 2006; Milner, Sandler, & Douek, 2010; Ouyang & Valdez, 2008; Romagnani, 2008; Zheng et al., 2008). Consistent with their important role in antimicrobial immunity, Th17 cells confer protection against several extracellular pathogens, such as *Candida albicans*, *Klebsiella pneumoniae*, *Citrobacter rodentium*, *Mycobacteria tuberculosis*, *Staphylococcus aureus*, *Bacteroides fragilis*, *Escherichia coli* (Huang, Na, Fidel, & Schwarzenberger, 2004; Khader et al., 2007; Ouyang & Valdez, 2008). Given the role of Th17 cells in mucosal immunity, and the observed mucosal immune dysfunction associated with HIV infection, we and others investigated the homeostasis of Th17 during pathogenic lentiviral infection, showing that Th17 cells are preferentially depleted in the gastrointestinal tracts of HIV-infected humans and SIV-infected macaques (Brenchley et al., 2008; Cecchinato et al., 2008; d'Ettorre, Mirko Paiardini, Ceccarelli, Silvestri, & Vullo, 2011; Favre et al., 2009; Gordon et al., 2010; Raffatellu et al., 2008). Moreover, Raffatellu and colleagues showed that in healthy SIV-negative rhesus macaques, the gene expression profile induced by S. typhimurium in ileal loops is dominated by Th17 responses, including the expression of IL-17 and IL-22; and severe depletion of mucosal Th17 cells in SIV-infected rhesus macaques resulted in an impaired mucosal barrier function and increased S. *typhimurium* dissemination (Raffatellu et al., 2008). Furthermore, loss of mucosal Th17 cells has been associated with increased systemic immune activation and disease progression in both HIVinfected humans and SIV-infected rhesus macaques (Cecchinato et al., 2008; Gordon et al., 2010; Hartigan-O'connor, Hirao, McCune, & Dandekar, 2011). Consistent with the model linking depletion of Th17 cells with compromised antimicrobial immunity, it has been shown that patients with dominant negative stat3 gene mutations, common in hyperimmunoglobulin E syndrome or the more biblical Job's syndrome, in which CD4 T cells are unable to differentiate into Th17 cells, are exquisitely susceptible to bacterial

Collectively, these studies demonstrate that pathogenic HIV and SIV infections are associated with a preferential and sustained depletion of mucosal Th17 cells, the severity of which correlates with the structural and immunological maintenance of the mucosal barrier, the levels of immune activation, and progression to AIDS. These observations further elucidate the immunodeficiency of HIV disease and provide a mechanistic basis for the

A very large body of evidence clearly demonstrated that, in sharp contrast with all the known models of pathogenic HIV infection, nonpathogenic SIV infection of natural hosts is characterized by the absence of high levels of chronic immune activation, assessed as the fraction of cells expressing markers of activation and proliferation, in the context of continuous virus replication (figure 3) (Mirko Paiardini et al., 2009b; Silvestri et al., 2003; Silvestri, Mirko Paiardini, I. Pandrea, Lederman, & Sodora, 2007). Consistent with their lower levels of immune activation, infected sooty mangabeys show no increase in lymphocyte apoptosis, lymph node structural damage, thymic involution, or loss of naïve T cell populations—all of

infections (Milner et al., 2008).

mucosal barrier breakdown that characterizes HIV infection.

**5. Immunology of natural hosts for SIV 5.1 Absence of chronic immune activation** 

which are normally attributed to chronic immune activation (Silvestri et al., 2003). Furthermore, naturally SIV-infected sooty mangabeys preserve the ability to properly regulate cell cycle progression when compared to SIV-infected macaques (Paiardini M JV 2006).

Fig. 3. Immune activation in natural and nonnatural hosts. In contrast to pathogenic HIV/SIV infection (humans −−− , rhesus macaques −−−), nonpathogenic SIV infection in natural hosts (sooty mangabeys −−− and African green monkeys −−−) is associated with the resolution of immune activation during chronic infection. Originally published in *Blood* Online. Brenchley JM & Paiardini M. Immunodeficiency lentiviral infections in natural and nonnatural hosts. *Blood*. Prepublished April 19, 2011; DOI:10.1182/blood-2010-12-325936.

Interestingly, the consistently low levels of chronic immune activation in natural hosts does not result from intrinsically attenuated innate immune responses, but rather from active immuno-regulatory mechanisms that allow these animals to tune-down the immune response during the transition from the acute to the chronic phase of infection (figure 3). The initial studies of natural SIV infections were performed during chronic infection and were not able to inform early events. Indeed, more recent studies designed to characterize the acute phase of SIV infection consistently show that, as described for progressive infection, nonprogressive SIV infection is also associated with an early increase in T cell proliferation and activation (Gordon et al., 2007; Kornfeld et al., 2005; I. V. Pandrea et al., 2007b; Silvestri et al., 2005). This phenotype is very common among several natural hosts, even those less characterized than sooty mangabeys and African green monkeys. For instance, transient levels of immune activation have been described in Mandrills, in which CD4 and CD8 HLA-DR+ cells at first increase but then return to normal levels by day 60 post-infection (Onanga et al., 2006), as well as in Caribbean African green monkeys, which show a rapid increase in CD8 HLA-DR+ T cells and then a rapid return to baseline 2-3 weeks post-infection, while having no changes in CD4 HLA-DR+ T cell frequencies (Kornfeld et al., 2005; I. Pandrea et al., 2006). Furthermore, the rapid resolution of acute immune activation has also been shown at a genetic level in sooty

HIV Without AIDS: The Immunological Secrets of Natural Hosts 213

is still unclear, several non-mutually exclusive mechanisms have been proposed, including the increased susceptibility to HIV/SIV infection of Th17 cells and its CD4+CCR6+ and CD4+CD161+ T cell precursors (Gosselin et al., 2010; Kader et al., 2009; Monteiro et al., 2011; Prendergast et al., 2010) and the defective generation of Th17 cells in nonnatural versus natural hosts. Very recent and unpublished observations suggest that loss of CD4+IL-21+ T cells and CD103+ dendritic cells, with reduced availability of IL-21 or retinoic acid, respectively, may significantly contribute to Th17 cell depletion in SIV-infected rhesus macaques (Cervasi B et al, CROI 2011; Klatt N et al, Keystone 2011). Consistent with their important role in Th17 cell homeostasis, CD4+IL-21+ T cells and CD103+ dendritic cells are preserved in SIV-infected SM (Cervasi B et al, CROI 2011; Klatt N et al, Keystone 2011). Collectively, these data indicate that by preserving the balance of IL-17 and IL-22 producing Th17 cells, natural hosts for SIV maintain mucosal barrier integrity and avoid the establishment of aberrant immune activation (figure 4). As such, the data suggest that differential regulation of Th17 cell homeostasis may be central in determining the

pathogenic or nonpathogenic outcome of HIV and SIV infections in primates.

Fig. 4. Th17 cell homeostasis and mucosal immunity in natural and nonnatural hosts. Mucosal Th17 cells are preferentially depleted in nonnatural hosts (humans and RM) but preserved at healthy frequencies in natural hosts (sooty mangabeys and African green monkeys) for lentiviral infections. Th17 cells regulate antimicrobial immunity, i.e. recruiting neutrophils, maintaining tight junction integrity and stimulating antimicrobial molecule production. As such, the preservation of Th17 cells is one of the key factors limiting microbial translocation and chronic immune activation, thus contributing to the ability of

natural hosts to remain AIDS-free. Adapted from (Mirko Paiardini, 2010).

mangabeys and African green monkeys from microarray data of early infection revealing that interferon stimulated genes are upregulated early in both natural and nonnatural hosts. Only natural hosts reduce their expression in blood and lymph nodes to near pre-infection levels in the acute to chronic phase transition (4-6 weeks), while macaques fail to resolve their early interferon stimulated gene response (Bosinger et al., 2009; Jacquelin et al., 2009; Lederer et al., 2009). Finally, immunohistochemical and immunofluorescent analyses recently demonstrated a robust IFN- response in the lymph nodes of sooty mangabeys, African green monkeys, and rhesus macaques in the acute phase of SIV infection, which is later resolved only in mangabeys and African green monkeys (L. D. Harris, Tabb, et al., 2010b).

The finding that naturally SIV-infected sooty mangabeys do not experience elevated levels of chronic immune activation in the context of high levels of viral replication further confirms the association between chronic immune activation and disease progression, and highlights the clinical importance of defining the mechanisms accounting for the establishment of high levels of chronic activation, or lack thereof, in pathogenic and nonpathogenic lentiviral infections.

#### **5.2 Preservation of Th17 cells and mucosal integrity**

Homeostasis of mucosal Th17 cells is a feature that distinguishes pathogenic HIV/SIV infections of humans and rhesus macaques, where these cells are preferentially depleted, from nonprogressive SIV infection of sooty mangabeys and African green monkeys, wherein Th17 cells are preserved at healthy frequencies (Brenchley et al., 2008; Cecchinato et al., 2008; Favre et al., 2009; Hartigan-O'connor et al., 2011; Mirko Paiardini, 2010; Raffatellu et al., 2008).

As previously described, studies in natural hosts demonstrated that a significant depletion of mucosal CD4 T cells alone is not sufficient to cause AIDS (Gordon et al., 2007; I. V. Pandrea et al., 2007b), suggesting that preservation of a specific CD4 T cell subset may allow the maintenance of mucosal integrity in the context of generalized CD4 T cell depletion. An increasing number of experimental evidence suggests that Th17 cells represent this specific subset. Indeed, Th17 cells are depleted in all the known models of pathogenic HIV/SIV infection, and preserved in all the known models of nonprogressive HIV/SIV infection including natural hosts for SIV, human long-term non-progressors and rhesus macaque elite controllers (Brenchley et al., 2008; Cecchinato et al., 2008; Favre et al., 2009; Mirko Paiardini, 2010). Specifically, we showed that whereas human Th17 cells are preferentially diminished compared to IFNg secreting Th1 cells in the gastrointestinal tracts of HIV-infected people, sooty mangabey Th17 cells are maintained in blood and the gastrointestinal tract (Brenchley et al., 2008). Likewise, while pigtailed macaques lose most IL-17 producing CD4 T cells by day 10 post-infection, African green monkeys show no decline (Favre et al., 2009). Intriguingly, in nonprogressive infections of sooty mangabeys and African green monkeys preservation of healthy frequencies of Th17 cells is associated with maintenance of mucosal immunity, absence of microbial translocation and low levels of chronic immune activation (figure 4) (Brenchley, Price, Schacker, Asher, et al., 2006a). Finally, Th17 cells were measured in human long-term non-progressors (n=14) and were found to be at levels equivalent to uninfected controls and those successfully (i.e., viral loads <50 copies/mL) treated with antiretroviral therapy in the colon and peripheral blood (Ciccone et al., 2011).

To understand how natural hosts preserve Th17 cells and mucosal immunity might be central to the development of therapeutic interventions aimed at improving mucosal immunity in HIV-infected individuals. While the exact cause accounting for this phenotype

mangabeys and African green monkeys from microarray data of early infection revealing that interferon stimulated genes are upregulated early in both natural and nonnatural hosts. Only natural hosts reduce their expression in blood and lymph nodes to near pre-infection levels in the acute to chronic phase transition (4-6 weeks), while macaques fail to resolve their early interferon stimulated gene response (Bosinger et al., 2009; Jacquelin et al., 2009; Lederer et al., 2009). Finally, immunohistochemical and immunofluorescent analyses recently demonstrated a robust IFN- response in the lymph nodes of sooty mangabeys, African green monkeys, and rhesus macaques in the acute phase of SIV infection, which is later resolved only in mangabeys

The finding that naturally SIV-infected sooty mangabeys do not experience elevated levels of chronic immune activation in the context of high levels of viral replication further confirms the association between chronic immune activation and disease progression, and highlights the clinical importance of defining the mechanisms accounting for the establishment of high levels of chronic activation, or lack thereof, in pathogenic and

Homeostasis of mucosal Th17 cells is a feature that distinguishes pathogenic HIV/SIV infections of humans and rhesus macaques, where these cells are preferentially depleted, from nonprogressive SIV infection of sooty mangabeys and African green monkeys, wherein Th17 cells are preserved at healthy frequencies (Brenchley et al., 2008; Cecchinato et al., 2008; Favre et al., 2009; Hartigan-O'connor et al., 2011; Mirko Paiardini, 2010; Raffatellu et al., 2008). As previously described, studies in natural hosts demonstrated that a significant depletion of mucosal CD4 T cells alone is not sufficient to cause AIDS (Gordon et al., 2007; I. V. Pandrea et al., 2007b), suggesting that preservation of a specific CD4 T cell subset may allow the maintenance of mucosal integrity in the context of generalized CD4 T cell depletion. An increasing number of experimental evidence suggests that Th17 cells represent this specific subset. Indeed, Th17 cells are depleted in all the known models of pathogenic HIV/SIV infection, and preserved in all the known models of nonprogressive HIV/SIV infection including natural hosts for SIV, human long-term non-progressors and rhesus macaque elite controllers (Brenchley et al., 2008; Cecchinato et al., 2008; Favre et al., 2009; Mirko Paiardini, 2010). Specifically, we showed that whereas human Th17 cells are preferentially diminished compared to IFNg secreting Th1 cells in the gastrointestinal tracts of HIV-infected people, sooty mangabey Th17 cells are maintained in blood and the gastrointestinal tract (Brenchley et al., 2008). Likewise, while pigtailed macaques lose most IL-17 producing CD4 T cells by day 10 post-infection, African green monkeys show no decline (Favre et al., 2009). Intriguingly, in nonprogressive infections of sooty mangabeys and African green monkeys preservation of healthy frequencies of Th17 cells is associated with maintenance of mucosal immunity, absence of microbial translocation and low levels of chronic immune activation (figure 4) (Brenchley, Price, Schacker, Asher, et al., 2006a). Finally, Th17 cells were measured in human long-term non-progressors (n=14) and were found to be at levels equivalent to uninfected controls and those successfully (i.e., viral loads <50 copies/mL) treated with

antiretroviral therapy in the colon and peripheral blood (Ciccone et al., 2011).

To understand how natural hosts preserve Th17 cells and mucosal immunity might be central to the development of therapeutic interventions aimed at improving mucosal immunity in HIV-infected individuals. While the exact cause accounting for this phenotype

and African green monkeys (L. D. Harris, Tabb, et al., 2010b).

**5.2 Preservation of Th17 cells and mucosal integrity** 

nonpathogenic lentiviral infections.

is still unclear, several non-mutually exclusive mechanisms have been proposed, including the increased susceptibility to HIV/SIV infection of Th17 cells and its CD4+CCR6+ and CD4+CD161+ T cell precursors (Gosselin et al., 2010; Kader et al., 2009; Monteiro et al., 2011; Prendergast et al., 2010) and the defective generation of Th17 cells in nonnatural versus natural hosts. Very recent and unpublished observations suggest that loss of CD4+IL-21+ T cells and CD103+ dendritic cells, with reduced availability of IL-21 or retinoic acid, respectively, may significantly contribute to Th17 cell depletion in SIV-infected rhesus macaques (Cervasi B et al, CROI 2011; Klatt N et al, Keystone 2011). Consistent with their important role in Th17 cell homeostasis, CD4+IL-21+ T cells and CD103+ dendritic cells are preserved in SIV-infected SM (Cervasi B et al, CROI 2011; Klatt N et al, Keystone 2011). Collectively, these data indicate that by preserving the balance of IL-17 and IL-22 producing Th17 cells, natural hosts for SIV maintain mucosal barrier integrity and avoid the establishment of aberrant immune activation (figure 4). As such, the data suggest that differential regulation of Th17 cell homeostasis may be central in determining the pathogenic or nonpathogenic outcome of HIV and SIV infections in primates.

Fig. 4. Th17 cell homeostasis and mucosal immunity in natural and nonnatural hosts. Mucosal Th17 cells are preferentially depleted in nonnatural hosts (humans and RM) but preserved at healthy frequencies in natural hosts (sooty mangabeys and African green monkeys) for lentiviral infections. Th17 cells regulate antimicrobial immunity, i.e. recruiting neutrophils, maintaining tight junction integrity and stimulating antimicrobial molecule production. As such, the preservation of Th17 cells is one of the key factors limiting microbial translocation and chronic immune activation, thus contributing to the ability of natural hosts to remain AIDS-free. Adapted from (Mirko Paiardini, 2010).

HIV Without AIDS: The Immunological Secrets of Natural Hosts 215

Another feature distinguishing natural and nonnatural hosts for lentiviral infections is the expression of CCR5, the main co-receptor used by HIV and SIV in vivo, to enter CD4 T cells. A comparative, cross sectional analysis of CCR5 expression in blood, lymph nodes and rectal biopsies obtained from several natural (sooty mangabeys, African green monkeys, and others) and nonnatural (human, rhesus macaques, and others) primate host species demonstrated that natural hosts for SIV infection consistently show a paucity of CD4 T cells expressing CCR5 (I. Pandrea et al., 2007a). This lower fraction of CD4 T cells expressing CCR5 was confirmed in both infected and uninfected animals, and in all sampled tissues, including those representing the major sites of viral replication (mucosa and lymph node) and CD4 T cell depletion (mucosa) during pathogenic HIV/SIV infection. Moreover, a five year longitudinal study of SIV-infected and uninfected sooty mangabeys showed stable median fractions (between 2-4%) of CD4 T cells expressing CCR5, independent of SIV (Taaffe et al., 2010). While this observation is very consistent and clear, its interpretation has been difficult, since naturally SIVinfected sooty mangabeys show levels of virus replication comparable to those of pathogenic infections. In an ongoing effort to better understand the pathophysiologic role of this decreased fraction of CCR5+ CD4 T cells in sooty mangabeys, we recently compared the levels and kinetics of CCR5 expression in sooty mangabey and rhesus macaque CD4 T cells, as well as the phenotype in their naïve, central memory, and effector memory subsets, following in vitro and in vivo activation. By doing this, we found CD4 T cells from sooty mangabeys failed to up-regulate CCR5 as do rhesus macaques in spite of activation and proliferation found to be equal in both species upon stimulation in vitro. Intriguingly, this phenomenon was more evident in CD4 T cells with a central-memory phenotype (TCM), and associated with a markedly reduced susceptibility of these cells to SIV infection. Since recent findings indicated the depletion of CD4 TCM cells as a critical step in the loss of CD4 T cell homeostasis and disease progression in SIV-infected rhesus macaques (Okoye et al., 2007; Picker et al., 2004), our recent data suggests that partial protection of CD4 TCM cells from SIV infection is one mechanism contributing to maintenance of a healthy immune system and avoidance of

progression to AIDS in SIV-infected sooty mangabeys (Paiardini et al., 2011).

**approaches for HIV-infected humans** 

clinical management of HIV-infected humans.

**6. How natural hosts may inform the design of novel vaccine and therapeutic** 

The pathogenesis of HIV infection results from a complex interaction between virus and host. Studies aimed at characterizing the virus-host interactions in natural hosts have led to important findings for understanding HIV pathogenesis in humans and, even more important, have many implications for new therapies and vaccines, giving us the opportunity to stop disease progression by understanding what nature has already discovered over millennia (Sodora et al., 2009). Table 2, along with the section above, summarizes several therapeutic approaches that could attempt to mimic the critical features of nonpathogenic infection in sooty mangabeys, which could be beneficial if included in the

1. *Targeting chronic immune activation to slow disease progression*. Considering that chronic immune activation is a key player in HIV pathogenesis, being associated with CD4 T cell depletion and the overall functionality of the immune system, and it

**5.4 Lower expression of CCR5 on CD4 T cells** 

#### **5.3 Preservation of bone marrow based T cell renewal**

As stated earlier, the mechanisms leading to CD4 T cell loss in HIV infection are multifactorial and still not completely defined. In addition to direct viral infection and bystander cell death, evidence has exhibited that insufficient T cell reconstitution may play a key role. Within the bone marrow, a major site of hematopoiesis and T cell proliferation, a suppression of function common in HIV-infected humans is associated with AIDS related neutropenia, thrombocytopenia and lymphopenia (Bain, 1997; Isgrò et al., 2005; Moses, Nelson, & Bagby, 1998; Silvestri et al., 2003).

Our group recently aimed to address the hypothesis that the preservation of bone marrow based proliferation and regeneration of T cells could be an important factor in regulating CD4 T cell homeostasis in progressive and nonprogressive lentiviral infections. To test this hypothesis, we utilized carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling during in vitro stimulations, along with flow-cytometric intracellular measurements of the cell cycle marker Ki-67, to measure proliferation in sooty mangabeys and rhesus macaques; these assessments were performed also in the experimental setting of in vivo antibodymediated CD4 or CD8 lymphocyte depletion (M Paiardini et al., 2009a). We discovered that SIV positive rhesus macaques have diminished proliferative capacity in bone marrow CD4 and CD8 T cells, while SIV positive SM had no decline compared to uninfected monkeys. Intriguingly, the rare subset of SIV-infected SM with low CD4 T cell count showed significantly lower levels of bone marrow proliferation when compared to SM that preserve the homeostasis of the CD4 T cell compartment (M Paiardini et al., 2009a). In addition, we found a correlation between Ki-67+ CD4 T cells and CD4 T cell count in the bone marrow but not in the peripheral blood (figure 5)(M Paiardini et al., 2009a).

Fig. 5. Bone Marrow based CD4 T cell proliferation in sooty mangabeys. In SIV-infected SM, blood CD4 T cell count correlates directly with the percentage of proliferating CD4 T cells in the bone marrow (BM, left panel) and inversely with the percentage of proliferating CD4 T cells in the peripheral blood (PB, right panel). This research was originally published in *Blood*. Paiardini M, *Blood*. 2009; *113*(3), 612-621. © the American Society of Hematology.

These findings suggest that the bone marrow is a major site of T cell proliferation in nonhuman primates, and the ability of SIV-infected sooty mangabeys to preserve the bone marrow based CD4 T cell proliferation is important for maintaining the homeostasis of the CD4 T cell compartment and avoiding progression to AIDS.

#### **5.4 Lower expression of CCR5 on CD4 T cells**

214 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

As stated earlier, the mechanisms leading to CD4 T cell loss in HIV infection are multifactorial and still not completely defined. In addition to direct viral infection and bystander cell death, evidence has exhibited that insufficient T cell reconstitution may play a key role. Within the bone marrow, a major site of hematopoiesis and T cell proliferation, a suppression of function common in HIV-infected humans is associated with AIDS related neutropenia, thrombocytopenia and lymphopenia (Bain, 1997; Isgrò et al., 2005; Moses,

Our group recently aimed to address the hypothesis that the preservation of bone marrow based proliferation and regeneration of T cells could be an important factor in regulating CD4 T cell homeostasis in progressive and nonprogressive lentiviral infections. To test this hypothesis, we utilized carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling during in vitro stimulations, along with flow-cytometric intracellular measurements of the cell cycle marker Ki-67, to measure proliferation in sooty mangabeys and rhesus macaques; these assessments were performed also in the experimental setting of in vivo antibodymediated CD4 or CD8 lymphocyte depletion (M Paiardini et al., 2009a). We discovered that SIV positive rhesus macaques have diminished proliferative capacity in bone marrow CD4 and CD8 T cells, while SIV positive SM had no decline compared to uninfected monkeys. Intriguingly, the rare subset of SIV-infected SM with low CD4 T cell count showed significantly lower levels of bone marrow proliferation when compared to SM that preserve the homeostasis of the CD4 T cell compartment (M Paiardini et al., 2009a). In addition, we found a correlation between Ki-67+ CD4 T cells and CD4 T cell count in the bone marrow

**5.3 Preservation of bone marrow based T cell renewal** 

but not in the peripheral blood (figure 5)(M Paiardini et al., 2009a).

Fig. 5. Bone Marrow based CD4 T cell proliferation in sooty mangabeys.

© the American Society of Hematology.

CD4 T cell compartment and avoiding progression to AIDS.

In SIV-infected SM, blood CD4 T cell count correlates directly with the percentage of proliferating CD4 T cells in the bone marrow (BM, left panel) and inversely with the percentage of proliferating CD4 T cells in the peripheral blood (PB, right panel). This research was originally published in *Blood*. Paiardini M, *Blood*. 2009; *113*(3), 612-621.

These findings suggest that the bone marrow is a major site of T cell proliferation in nonhuman primates, and the ability of SIV-infected sooty mangabeys to preserve the bone marrow based CD4 T cell proliferation is important for maintaining the homeostasis of the

Nelson, & Bagby, 1998; Silvestri et al., 2003).

Another feature distinguishing natural and nonnatural hosts for lentiviral infections is the expression of CCR5, the main co-receptor used by HIV and SIV in vivo, to enter CD4 T cells. A comparative, cross sectional analysis of CCR5 expression in blood, lymph nodes and rectal biopsies obtained from several natural (sooty mangabeys, African green monkeys, and others) and nonnatural (human, rhesus macaques, and others) primate host species demonstrated that natural hosts for SIV infection consistently show a paucity of CD4 T cells expressing CCR5 (I. Pandrea et al., 2007a). This lower fraction of CD4 T cells expressing CCR5 was confirmed in both infected and uninfected animals, and in all sampled tissues, including those representing the major sites of viral replication (mucosa and lymph node) and CD4 T cell depletion (mucosa) during pathogenic HIV/SIV infection. Moreover, a five year longitudinal study of SIV-infected and uninfected sooty mangabeys showed stable median fractions (between 2-4%) of CD4 T cells expressing CCR5, independent of SIV (Taaffe et al., 2010). While this observation is very consistent and clear, its interpretation has been difficult, since naturally SIVinfected sooty mangabeys show levels of virus replication comparable to those of pathogenic infections. In an ongoing effort to better understand the pathophysiologic role of this decreased fraction of CCR5+ CD4 T cells in sooty mangabeys, we recently compared the levels and kinetics of CCR5 expression in sooty mangabey and rhesus macaque CD4 T cells, as well as the phenotype in their naïve, central memory, and effector memory subsets, following in vitro and in vivo activation. By doing this, we found CD4 T cells from sooty mangabeys failed to up-regulate CCR5 as do rhesus macaques in spite of activation and proliferation found to be equal in both species upon stimulation in vitro. Intriguingly, this phenomenon was more evident in CD4 T cells with a central-memory phenotype (TCM), and associated with a markedly reduced susceptibility of these cells to SIV infection. Since recent findings indicated the depletion of CD4 TCM cells as a critical step in the loss of CD4 T cell homeostasis and disease progression in SIV-infected rhesus macaques (Okoye et al., 2007; Picker et al., 2004), our recent data suggests that partial protection of CD4 TCM cells from SIV infection is one mechanism contributing to maintenance of a healthy immune system and avoidance of progression to AIDS in SIV-infected sooty mangabeys (Paiardini et al., 2011).

### **6. How natural hosts may inform the design of novel vaccine and therapeutic approaches for HIV-infected humans**

The pathogenesis of HIV infection results from a complex interaction between virus and host. Studies aimed at characterizing the virus-host interactions in natural hosts have led to important findings for understanding HIV pathogenesis in humans and, even more important, have many implications for new therapies and vaccines, giving us the opportunity to stop disease progression by understanding what nature has already discovered over millennia (Sodora et al., 2009). Table 2, along with the section above, summarizes several therapeutic approaches that could attempt to mimic the critical features of nonpathogenic infection in sooty mangabeys, which could be beneficial if included in the clinical management of HIV-infected humans.

1. *Targeting chronic immune activation to slow disease progression*. Considering that chronic immune activation is a key player in HIV pathogenesis, being associated with CD4 T cell depletion and the overall functionality of the immune system, and it

HIV Without AIDS: The Immunological Secrets of Natural Hosts 217

CD4+CCR5+ T cells Very low Normal CCR5 blockade Mucosal integrity Preserved Lost Sure up mucosal

Table 2. Critical features distinguishing pathogenic from non nonpathogenic SIV infection in nonnatural and natural hosts, respectively. The last column includes general targets for intervention derived from studying natural hosts. These approaches mimic critical features of nonprogressive lentiviral infection and could improve the clinical management of HIV-

We firmly believe that a comprehensive elucidation of how natural hosts for SIV have coevolved to avoid disease progression is critical for understanding the mechanisms of AIDS pathogenesis in HIV-infected humans. The elucidation of these mechanisms may translate

Alexander, J. W., Boyce, S. T., Babcock, G. F., Gianotti, L., Peck, M. D., Dunn, D. L., Pyles, T.,

Ancuta, P., Kamat, A., Kunstman, K. J., Kim, E., Autissier, P., Wurcel, A., Zaman, T., et al.

Aujla, S. J., Chan, Y. R., Zheng, M., Fei, M., Askew, D. J., Pociask, D. A., Reinhart, T. A., et al.

Bain, B. J. (1997). The haematological features of HIV infection *British journal of haematology*,

Barré-Sinoussi, F., Chermann, J. C., Rey, F., Nugeyre, M. T., Chamaret, S., Gruest, J.,

pneumonia *Nature medicine*, *14*(3), 275-281. doi:10.1038/nm1710

et al. (1990). The process of microbial translocation *Annals of surgery*, *212*(4), 496-

(2008). Microbial translocation is associated with increased monocyte activation and dementia in AIDS patients *PloS one*, *3*(6), e2516.

(2008). IL-22 mediates mucosal host defense against Gram-negative bacterial

Dauguet, C., et al. (1983). Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS) *Science (New York, N.Y.)*,

into major advances in prevention and therapy of HIV infection and AIDS.

activation No Yes Immune modulators of

**hosts** 

**Possible therapeutic intervention** 

activation

CD4 T cell renewal strategies; IL-7 and other homeostatic cytokines

Increase Th17 cell differentiation; IL-21 and other Th17-driving factors

boundaries

**infections Natural hosts Nonnatural** 

peripheral CD4 T cells No Yes

Mucosal Th17 cells Preserved Lost

**Feature of HIV or SIV** 

Chronic immune

Progressive loss of

Frequency of

infected humans.

**7. Final remarks** 

**8. References** 

510; discussion 511-2.

*99*(1), 1-8.

*220*(4599), 868-871.

doi:10.1371/journal.pone.0002516

is absent in nonprogressive SIV infection of natural hosts, there is a strong rationale for introducing immune suppressive molecules in the treatment of HIV-infected individuals. In this context, it is important to note that in HIV-infected humans chronic immune activation is not fully resolved even in the setting of successful antiretroviral therapy (ART), and that this residual immune activation is considered the major cause for the increased "non-AIDS" morbidity and mortality observed in individuals undergoing long-term ART (Grund, Neuhaus, Phillips, INSIGHT SMART Study Group, 2009). Since the exact mechanisms and signaling pathways responsible for chronic immune activation in HIV-infected humans are still unclear, approaches have mostly focused on using drugs with a generic immune suppressive ability, such as Cyclosporin, Rapamycin, and Hydroxychloroquine, already in use for individuals with autoimmune disorders or recipients of transplants. Hydroxychloroquine, an antimalarial drug also used to reduce inflammation in rheumatoid arthritis and lupus, has already been shown to reduce the expression of the immune activation markers CD38, Ki-67 and HLA-DR on CD8 T-cells and to decrease viral loads in HIV infected patients (Murray et al., 2010, Sperber et al., 1995).



Table 2. Critical features distinguishing pathogenic from non nonpathogenic SIV infection in nonnatural and natural hosts, respectively. The last column includes general targets for intervention derived from studying natural hosts. These approaches mimic critical features of nonprogressive lentiviral infection and could improve the clinical management of HIVinfected humans.

## **7. Final remarks**

216 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

2. *Preserving Th17 cell homeostasis by increasing their differentiation and survival*. IL-21, a multifunctional cytokine that initiates the induction of Th17 cells (Korn et al., 2007; Nurieva et al., 2007; Yang et al., 2008) could be used to test the hypothesis that increased levels of Th17 cells will sure up gut permeability, thus preventing continuous microbial translocation and immune activation. The rationale for using this cytokine comes from several findings, including the following: (i) plasma levels of IL-21 are significantly decreased in HIV infected patients (Iannello et al., 2008); (ii) CD8 T cells producing IL-21 are increased in elite controllers, (Williams et al. 2011); (iii) circulating CD4 T cells expressing IL-21 are severely lost in pathogenic SIV infection of rhesus macaques, with the extent of this depletion being associated with that of Th17 cells (Cervasi B, CROI 2011),; (iv) CD4 T cells producing IL-21 are preserved at healthy frequencies in SIV-infected sooty mangabeys (Cervasi B, CROI 2011) ; (v) finally, IL-21 is already in clinical trials for the use against renal cell

3. *Targeting of CCR5 expression*. Specifically targeting expression of CCR5 and other coreceptors for HIV may be critical in preventing AIDS. A unique bone marrow transplantation demonstrated the attainability of an HIV cure, despite the unusual and unrepeatable events that led to that cure: harsh chemotherapy, total body irradiation and an unlikely hematopoietic stem cell transplantation match of a homozygous CCR5Δ32 donor (Hütter, Nowak, Mossner, Ganepola, et al., 2009a). This case report of one patient has justifiably led to excitement about future therapies using CCR5Δ32 donors as well as other entry blocking strategies in HIV infection (Hütter, Thomas Schneider, & Thiel, 2009b). Other less strenuous methods to target CCR5 have been made possible by zinc finger nuclease-mediated gene disruption, maraviroc, small interfering RNA molecules, and a number of new molecular nanotechnologies (reviewed in Cannon & June, 2011). Data obtained in sooty mangabeys suggest that these treatments may be significantly enhanced upon

carcinoma and melanoma (Hashmi & Van Veldhuizen, 2010).

targeting of CCR5 expression on CD4 TCM cells specifically.

1995).

is absent in nonprogressive SIV infection of natural hosts, there is a strong rationale for introducing immune suppressive molecules in the treatment of HIV-infected individuals. In this context, it is important to note that in HIV-infected humans chronic immune activation is not fully resolved even in the setting of successful antiretroviral therapy (ART), and that this residual immune activation is considered the major cause for the increased "non-AIDS" morbidity and mortality observed in individuals undergoing long-term ART (Grund, Neuhaus, Phillips, INSIGHT SMART Study Group, 2009). Since the exact mechanisms and signaling pathways responsible for chronic immune activation in HIV-infected humans are still unclear, approaches have mostly focused on using drugs with a generic immune suppressive ability, such as Cyclosporin, Rapamycin, and Hydroxychloroquine, already in use for individuals with autoimmune disorders or recipients of transplants. Hydroxychloroquine, an antimalarial drug also used to reduce inflammation in rheumatoid arthritis and lupus, has already been shown to reduce the expression of the immune activation markers CD38, Ki-67 and HLA-DR on CD8 T-cells and to decrease viral loads in HIV infected patients (Murray et al., 2010, Sperber et al.,

> We firmly believe that a comprehensive elucidation of how natural hosts for SIV have coevolved to avoid disease progression is critical for understanding the mechanisms of AIDS pathogenesis in HIV-infected humans. The elucidation of these mechanisms may translate into major advances in prevention and therapy of HIV infection and AIDS.

## **8. References**


HIV Without AIDS: The Immunological Secrets of Natural Hosts 219

Chakrabarti, L. A., Lewin, S. R., Zhang, L., Gettie, A., Luckay, A., Martin, L. N., Skulsky, E.,

immunodeficiency virus infection *Journal of virology*, *74*(3), 1209-1223. Chitra, P., Bakthavatsalam, B., & Palvannan, T. (2011). Beta-2 microglobulin as an

*international journal of clinical chemistry*. doi:10.1016/j.cca.2011.01.037 Ciccone, E. J., Greenwald, J. H., Lee, P. I., Biancotto, A., Read, S. W., Yao, M. A., Hodge, J. N.,

doi:10.1128/JVI.02643-10

*Science (New York, N.Y.)*, *260*(5106), 292-293.

*retroviruses*. doi:10.1089/AID.2010.0342

doi:10.1097/COH.0b013e328335eda3

doi:10.1146/annurev.med.60.041807.123549

166-172. doi:10.1056/NEJM199001183220305

doi:10.1182/blood-2003-09-3333

doi:10.1371/journal.ppat.1001052

doi:10.1371/journal.ppat.1000295

et al. (2000). Normal T-cell turnover in sooty mangabeys harboring active simian

immunological marker to assess the progression of human immunodeficiency virus infected patients on highly active antiretroviral therapy *Clinica chimica acta;* 

et al. (2011). CD4+ T Cells, Including Th17 and Cycling Subsets, are Intact in the Gut Mucosa of HIV-1 Infected Long-Term Non- Progressors *Journal of virology*.

M. (2003). Identification of a new simian immunodeficiency virus lineage with a vpu gene present among different cercopithecus monkeys (C. mona, C. cephus, and

Associated Immune Activation: From Bench to Bedside *AIDS research and human* 

barrier *Current opinion in HIV and AIDS*, *5*(2), 173-178.

(2004). Immune activation set point during early HIV infection predicts subsequent CD4+ T-cell changes independent of viral load *Blood*, *104*(4), 942-947.

immunopathogenesis of AIDS *Annual review of medicine*, *60*, 471-484.

al. (2010). Damaged intestinal epithelial integrity linked to microbial translocation in pathogenic simian immunodeficiency virus infections *PLoS pathogens*, *6*(8).

(1990). The prognostic value of cellular and serologic markers in infection with human immunodeficiency virus type 1 *The New England journal of medicine*, *322*(3),

Prognostic significance of plasma markers of immune activation, HIV viral load

Critical loss of the balance between Th17 and T regulatory cell populations in pathogenic SIV infection *PLoS pathogens*, *5*(2), e1000295.

Cohen, J. (1993, April 16). AIDS research. Keystone's blunt message: 'it's the virus, stupid'

Courgnaud, V., Abela, B., Pourrut, X., Mpoudi-Ngole, E., Loul, S., Delaporte, E., & Peeters,

Dandekar, S., George, M. D., & Bäumler, A. J. (2010). Th17 cells, HIV and the gut mucosal

Deeks, S. G., Kitchen, C. M. R., Liu, L., Guo, H., Gascon, R., Narváez, A. B., Hunt, P., et al.

Douek, D. C., Roederer, Mario, & Koup, R. A. (2009). Emerging concepts in the

Estes, J. D., Harris, L. D., Klatt, N. R., Tabb, B., Pittaluga, S., Paiardini, M., Barclay, G. R., et

Fahey, J. L., Taylor, J. M., Detels, R., Hofmann, B., Melmed, R., Nishanian, P., & Giorgi, J. V.

Fahey, J. L., Taylor, J. M., Manna, B., Nishanian, P., Aziz, N., Giorgi, J. V., & Detels, R. (1998).

and CD4 T-cell measurements *AIDS (London, England)*, *12*(13), 1581-1590. Favre, D., Lederer, S., Kanwar, B., Ma, Z., Proll, S., Kasakow, Z., Mold, J., et al. (2009).

C. nictitans) from Cameroon *Journal of virology*, *77*(23), 12523-12534. d'Ettorre, G., Paiardini, Mirko, Ceccarelli, G., Silvestri, G., & Vullo, V. (2011). HIV-


Bofill, M., Mocroft, A., Lipman, M., Medina, E., Borthwick, N. J., Sabin, C. A., Timms, A., et

Bosinger, S. E., Li, Q., Gordon, S. N., Klatt, N. R., Duan, L., Xu, L., Francella, N., et al. (2009).

Brenchley, J. M., Schacker, T. W., Ruff, L. E., Price, D. A., Taylor, J. H., Beilman, G. J.,

Brenchley, J. M., Hill, B. J., Ambrozak, D. R., Price, D. A., Guenaga, F. J., Casazza, J. P.,

Brenchley, J. M., Price, D. A., Schacker, T. W., Asher, T. E., Silvestri, G., Rao, S., Kazzaz, Z., et

Brenchley, J. M., Price, D. A., & Douek, D. C. (2006b). HIV disease: fallout from a mucosal

Brenchley, J. M., Silvestri, G., & Douek, D. C. (2010). Nonprogressive and progressive

Cannon, P., & June, C. (2011). Chemokine receptor 5 knockout strategies *Current opinion in* 

Card, C. M., McLaren, P. J., Wachihi, C., Kimani, J., Plummer, F. A., & Fowke, K. R. (2009).

Cecchinato, V., Trindade, C. J., Laurence, A., Heraud, J. M., Brenchley, J. M., Ferrari, M. G.,

Centers for Disease Control (CDC). (1982). A cluster of Kaposi's sarcoma and Pneumocystis

catastrophe *Nature immunology*, *7*(3), 235-239. doi:10.1038/ni1316

*HIV and AIDS*, *6*(1), 74-79. doi:10.1097/COH.0b013e32834122d7

*infectious diseases*, *199*(9), 1318-1322. doi:10.1086/597801

*Mucosal immunology*, *1*(4), 279-288. doi:10.1038/mi.2008.14

Boston, Feb 27-Mar 2, 2011 Abstract # 243.

*medicine*, *200*(6), 749-759. doi:10.1084/jem.20040874

*England)*, *10*(8), 827-834.

doi:10.1172/JCI40115

1160-1168.

2008-05-159301

doi:10.1016/j.immuni.2010.06.004

al. (1996). Increased numbers of primed activated CD8+CD38+CD45RO+ T cells predict the decline of CD4+ T cells in HIV-1-infected patients *AIDS (London,* 

Global genomic analysis reveals rapid control of a robust innate response in SIVinfected sooty mangabeys *The Journal of clinical investigation*, *119*(12), 3556-3572.

Nguyen, P. L., et al. (2004a). CD4+ T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract *The Journal of experimental* 

Kuruppu, J., et al. (2004b). T-cell subsets that harbor human immunodeficiency virus (HIV) in vivo: implications for HIV pathogenesis *Journal of virology*, *78*(3),

al. (2006a). Microbial translocation is a cause of systemic immune activation in chronic HIV infection *Nature medicine*, *12*(12), 1365-1371. doi:10.1038/nm1511 Brenchley, J. M., Paiardini, M., Knox, K. S., Asher, A. I., Cervasi, B., Asher, T. E., Scheinberg,

P., et al. (2008). Differential Th17 CD4 T-cell depletion in pathogenic and nonpathogenic lentiviral infections *Blood*, *112*(7), 2826-2835. doi:10.1182/blood-

primate immunodeficiency lentivirus infections *Immunity*, *32*(6), 737-742.

Decreased immune activation in resistance to HIV-1 infection is associated with an elevated frequency of CD4(+)CD25(+)FOXP3(+) regulatory T cells *The Journal of* 

Zaffiri, L., et al. (2008). Altered balance between Th17 and Th1 cells at mucosal sites predicts AIDS progression in simian immunodeficiency virus-infected macaques

carinii pneumonia among homosexual male residents of Los Angeles and Orange Counties, California *MMWR. Morbidity and mortality weekly report*, *31*(23), 305-307. Cervasi B, Reyes-Aville E, Ende Z, Else J, Silvestri G, & Paiardini M. Differential Regulation

of IL-21-producing CD4+ T Cells in Natural and Non-natural Hosts for SIV [abstract]. In: 18th Conference on Retroviruses and Opportunistic Infections,


HIV Without AIDS: The Immunological Secrets of Natural Hosts 221

Guglani, L., & Khader, S. A. (2010). Th17 cytokines in mucosal immunity and inflammation

Haase, A. T. (2005). Perils at mucosal front lines for HIV and SIV and their hosts *Nature* 

Harris, C., Small, C. B., Klein, R. S., Friedland, G. H., Moll, B., Emeson, E. E., Spigland, I., et

Harris, L. D., Klatt, N. R., Vinton, C., Briant, J. A., Tabb, B., Ladell, K., Lifson, J., et al. (2010a).

Hartigan-O'connor, D. J., Hirao, L. A., McCune, J. M., & Dandekar, S. (2011). Th17 cells and

Hashmi, M. H., & Van Veldhuizen, P. J. (2010). Interleukin-21: updated review of Phase I

Huang, W., Na, L., Fidel, P. L., & Schwarzenberger, P. (2004). Requirement of interleukin-

Hunt, P. W., Martin, J. N., Sinclair, E., Bredt, B., Hagos, E., Lampiris, H., & Deeks, S. G.

Hunt, P. W., Brenchley, J., Sinclair, E., McCune, J. M., Roland, M., Page-Shafer, K., Hsue, P.,

Hütter, G., Nowak, D., Mossner, M., Ganepola, S., Müssig, A., Allers, K., Schneider, T., et al.

macaques *Blood*, *116*(20), 4148-4157. doi:10.1182/blood-2010-05-283549 Harris, L. D., Tabb, B., Sodora, D. L., Paiardini, M., Klatt, N. R., Douek, D. C., Silvestri, G., et

*reviews. Immunology*, *5*(10), 783-792. doi:10.1038/nri1705

1184. doi:10.1056/NEJM198305193082001

*84*(15), 7886-7891. doi:10.1128/JVI.02612-09

*diseases*, *190*(3), 624-631. doi:10.1086/422329

doi:10.1086/374786

doi:10.1371/journal.pone.0015924

doi:10.1056/NEJMoa0802905

*AIDS*, *6*(3), 221-227. doi:10.1097/COH.0b013e32834577b3

*therapy*, *10*(5), 807-817. doi:10.1517/14712598.2010.480971

11717.

following highly active antiretroviral therapy *Journal of virology*, *77*(21), 11708-

*Current opinion in HIV and AIDS*, *5*(2), 120-127. doi:10.1097/COH.0b013e328335c2f6

al. (1983). Immunodeficiency in female sexual partners of men with the acquired immunodeficiency syndrome *The New England journal of medicine*, *308*(20), 1181-

Mechanisms underlying γδ T-cell subset perturbations in SIV-infected Asian rhesus

al. (2010b). Downregulation of robust acute type I interferon responses distinguishes nonpathogenic simian immunodeficiency virus (SIV) infection of natural hosts from pathogenic SIV infection of rhesus macaques *Journal of virology*,

regulatory T cells in elite control over HIV and SIV *Current opinion in HIV and* 

and II clinical trials in metastatic renal cell carcinoma, metastatic melanoma and relapsed/refractory indolent non-Hodgkin's lymphoma *Expert opinion on biological* 

17A for systemic anti-Candida albicans host defense in mice *The Journal of infectious* 

(2003). T cell activation is associated with lower CD4+ T cell gains in human immunodeficiency virus-infected patients with sustained viral suppression during antiretroviral therapy *The Journal of infectious diseases*, *187*(10), 1534-1543.

et al. (2008). Relationship between T cell activation and CD4+ T cell count in HIVseropositive individuals with undetectable plasma HIV RNA levels in the absence of therapy *The Journal of infectious diseases*, *197*(1), 126-133. doi:10.1086/524143 Hunt, P. W., Landay, A. L., Sinclair, E., Martinson, J. A., Hatano, H., Emu, B., Norris, P. J., et

al. (2011). A low T regulatory cell response may contribute to both viral control and generalized immune activation in HIV controllers *PloS one*, *6*(1), e15924.

(2009a). Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation *The New England journal of medicine*, *360*(7), 692-698.


Ford, E. S., Puronen, C. E., & Sereti, I. (2009). Immunopathogenesis of asymptomatic chronic

Gao, F., Bailes, E., Robertson, D. L., Chen, Y., Rodenburg, C. M., Michael, S. F., Cummins, L.

Geldmacher, C., Ngwenyama, N., Schuetz, A., Petrovas, C., Reither, K., Heeregrave, E. J.,

Giorgi, J V, Liu, Z., Hultin, L. E., Cumberland, W. G., Hennessey, K., & Detels, R. (1993).

Giorgi, J V, Hultin, L. E., McKeating, J. A., Johnson, T. D., Owens, B., Jacobson, L. P., Shih,

Giorgi, Janis V, Lyles, R. H., Matud, J. L., Yamashita, T. E., Mellors, J. W., Hultin, L. E.,

Gordon, S. N., Klatt, N. R., Bosinger, S. E., Brenchley, J. M., Milush, J. M., Engram, J. C.,

Gordon, S. N., Cervasi, B., Odorizzi, P., Silverman, R., Aberra, F., Ginsberg, G., Estes, J. D., et

*(Baltimore, Md. : 1950)*, *185*(9), 5169-5179. doi:10.4049/jimmunol.1001801 Gosselin, A., Monteiro, P., Chomont, N., Diaz-Griffero, F., Said, E. A., Fonseca, S., Wacleche,

Grieco, M. H., Reddy, M. M., Kothari, H. B., Lange, M., Buimovici-Klein, E., & William, D.

Grund, B., Neuhaus, J., Phillips, A., INSIGHT SMART Study Group. (2009). Relative risk of

Guadalupe, M., Reay, E., Sankaran, S., Prindiville, T., Flamm, J., McNeil, A., & Dandekar, S.

*1950)*, *184*(3), 1604-1616. doi:10.4049/jimmunol.0903058

214. doi:10.1097/COH.0b013e328329c68c

904-912.

184.

*Nature*, *397*(6718), 436-441. doi:10.1038/17130

*diseases*, *179*(4), 859-870. doi:10.1086/314660

*deficiency syndromes*, *29*(4), 346-355.

doi:10.1016/S1473-3099(09)70302-0

*(Baltimore, Md. : 1950)*, *179*(5), 3026-3034.

*medicine*, *207*(13), 2869-2881. doi:10.1084/jem.20100090

HIV Infection: the calm before the storm *Current opinion in HIV and AIDS*, *4*(3), 206-

B., et al. (1999). Origin of HIV-1 in the chimpanzee Pan troglodytes troglodytes

Casazza, J. P., et al. (2010). Preferential infection and depletion of Mycobacterium tuberculosis-specific CD4 T cells after HIV-1 infection *The Journal of experimental* 

Elevated levels of CD38+ CD8+ T cells in HIV infection add to the prognostic value of low CD4+ T cell levels: results of 6 years of follow-up. The Los Angeles Center, Multicenter AIDS Cohort Study *Journal of acquired immune deficiency syndromes*, *6*(8),

R., et al. (1999). Shorter survival in advanced human immunodeficiency virus type 1 infection is more closely associated with T lymphocyte activation than with plasma virus burden or virus chemokine coreceptor usage *The Journal of infectious* 

Jamieson, B. D., et al. (2002). Predictive value of immunologic and virologic markers after long or short duration of HIV-1 infection *Journal of acquired immune* 

Dunham, R. M., et al. (2007). Severe depletion of mucosal CD4+ T cells in AIDS-free simian immunodeficiency virus-infected sooty mangabeys *Journal of immunology* 

al. (2010). Disruption of intestinal CD4+ T cell homeostasis is a key marker of systemic CD4+ T cell activation in HIV-infected individuals *Journal of immunology* 

V., et al. (2010). Peripheral blood CCR4+CCR6+ and CXCR3+CCR6+CD4+ T cells are highly permissive to HIV-1 infection *Journal of immunology (Baltimore, Md. :* 

(1984). Elevated beta 2-microglobulin and lysozyme levels in patients with acquired immune deficiency syndrome *Clinical immunology and immunopathology*, *32*(2), 174-

death in the SMART study *The Lancet infectious diseases*, *9*(12), 724-725.

(2003). Severe CD4+ T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy *Journal of virology*, *77*(21), 11708- 11717.


HIV Without AIDS: The Immunological Secrets of Natural Hosts 223

Lederer, S., Favre, D., Walters, K., Proll, S., Kanwar, B., Kasakow, Z., Baskin, C. R., et al.

Li, Q., Duan, L., Estes, J. D., Ma, Z., Rourke, T., Wang, Y., Reilly, C., et al. (2005). Peak SIV

Liang, S. C., Tan, X., Luxenberg, D. P., Karim, R., Dunussi-Joannopoulos, K., Collins, M., &

Liu, Z., Cumberland, W. G., Hultin, L. E., Prince, H. E., Detels, R., & Giorgi, J. V. (1997).

Mattapallil, J. J., Douek, D. C., Hill, B., Nishimura, Y., Martin, M., & Roederer, M. (2005).

Mehandru, S., Poles, M. A., Tenner-Racz, K., Horowitz, A., Hurley, A., Hogan, C., Boden, D.,

Melmed, R. N., Taylor, J. M., Detels, R., Bozorgmehri, M., & Fahey, J. L. (1989). Serum

Milner, J. D., Brenchley, J. M., Laurence, A., Freeman, A. F., Hill, B. J., Elias, K. M., Kanno, Y.,

Milner, J. D., Sandler, N. G., & Douek, D. C. (2010). Th17 cells, Job's syndrome and HIV:

Milush, J. M., Reeves, J. D., Gordon, S. N., Zhou, D., Muthukumar, A., Kosub, D. A., Chacko,

Milush, J. M., Mir, K. D., Sundaravaradan, V., Gordon, S. N., Engram, J., Cano, C. A.,

*Journal of clinical investigation*, *121*(3), 1102-1110. doi:10.1172/JCI44876 Mir, K. D., Gasper, M. A., Sundaravaradan, V., & Sodora, D. L. (2011). SIV infection in

SIV infection *Nature*, *434*(7037), 1093-1097. doi:10.1038/nature03501

*experimental medicine*, *200*(6), 761-770. doi:10.1084/jem.20041196

*5*(2), 179-183. doi:10.1097/COH.0b013e328335ed3e

*experimental medicine*, *203*(10), 2271-2279. doi:10.1084/jem.20061308

*pathogens*, *5*(2), e1000296. doi:10.1371/journal.ppat.1000296

cells *Nature*, *434*(7037), 1148-1152. doi:10.1038/nature03513

*Association*, *16*(2), 83-92.

*syndromes*, *2*(1), 70-76.

3056.

(2009). Transcriptional profiling in pathogenic and non-pathogenic SIV infections reveals significant distinctions in kinetics and tissue compartmentalization *PLoS* 

replication in resting memory CD4+ T cells depletes gut lamina propria CD4+ T

Fouser, L. A. (2006). Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides *The Journal of* 

Elevated CD38 antigen expression on CD8+ T cells is a stronger marker for the risk of chronic HIV disease progression to AIDS and death in the Multicenter AIDS Cohort Study than CD4+ cell count, soluble immune activation markers, or combinations of HLA-DR and CD38 expression *Journal of acquired immune deficiency syndromes and human retrovirology : official publication of the International Retrovirology* 

Massive infection and loss of memory CD4+ T cells in multiple tissues during acute

et al. (2004). Primary HIV-1 infection is associated with preferential depletion of CD4+ T lymphocytes from effector sites in the gastrointestinal tract *The Journal of* 

neopterin changes in HIV-infected subjects: indicator of significant pathology, CD4 T cell changes, and the development of AIDS *Journal of acquired immune deficiency* 

et al. (2008). Impaired T(H)17 cell differentiation in subjects with autosomal dominant hyper-IgE syndrome *Nature*, *452*(7188), 773-776. doi:10.1038/nature06764

opportunities for bacterial and fungal infections *Current opinion in HIV and AIDS*,

E., et al. (2007). Virally induced CD4+ T cell depletion is not sufficient to induce AIDS in a natural host *Journal of immunology (Baltimore, Md. : 1950)*, *179*(5), 3047-

Reeves, J. D., et al. (2011). Lack of clinical AIDS in SIV-infected sooty mangabeys with significant CD4+ T cell loss is associated with double-negative T cells *The* 

natural hosts: resolution of immune activation during the acute-to-chronic


Hütter, G., Schneider, Thomas, & Thiel, E. (2009b). Transplantation of selected or transgenic

Iannello, A., Tremblay, C., Routy, J., Boulassel, M., Toma, E., & Ahmad, A. (2008). Decreased

+T-Cell Counts. *Viral Immunology*, *21*(3), 385-388. doi:10.1089/vim.2008.0025 Isgrò, A., Aiuti, A., Leti, W., Gramiccioni, C., Esposito, A., Mezzaroma, I., & Aiuti, F. (2005).

Jacquelin, B., Mayau, V., Targat, B., Liovat, A., Kunkel, D., Petitjean, G., Dillies, M., et al.

Jiang, W., Lederman, M. M., Hunt, P., Sieg, S. F., Haley, K., Rodríguez, B., Landay, A., et al.

Keele, B. F., Jones, J. H., Terio, K. A., Estes, J. D., Rudicell, R. S., Wilson, M. L., Li, Y., et al.

infected with SIVcpz *Nature*, *460*(7254), 515-519. doi:10.1038/nature08200 Khader, S. A., Bell, G. K., Pearl, J. E., Fountain, J. J., Rangel-Moreno, J., Cilley, G. E., Shen, F.,

Khaitan, A., & Unutmaz, D. (2011). Revisiting immune exhaustion during HIV infection

Klatt, Nichole. Short Talk: Mechanisms Underlying Damage to the Mucosal Barrier and

Koning, F. A., Otto, S. A., Hazenberg, M. D., Dekker, L., Prins, M., Miedema, F., &

Korn, T., Bettelli, E., Gao, W., Awasthi, A., Jäger, A., Strom, T. B., Oukka, M., et al. (2007). IL-

Kornfeld, C., Ploquin, M. J., Pandrea, I., Faye, A., Onanga, R., Apetrei, C., Poaty-

*Journal of clinical investigation*, *115*(4), 1082-1091. doi:10.1172/JCI23006

*Current HIV/AIDS reports*, *8*(1), 4-11. doi:10.1007/s11904-010-0066-0

*Nature immunology*, *8*(4), 369-377. doi:10.1038/ni1449

*448*(7152), 484-487. doi:10.1038/nature05970

*Society*, *12*(1), 10. doi:10.1186/1758-2652-12-10

*4*(8), 486-490. doi:10.1016/j.autrev.2005.04.014

3544-3555. doi:10.1172/JCI40093

doi:10.1038/mi.2009.90

Whistler, British Columbia.

6117-6122.

blood stem cells - a future treatment for HIV/AIDS *Journal of the International AIDS* 

Levels of Circulating IL-21 in HIV-Infected AIDS Patients: Correlation with CD4

Immunodysregulation of HIV disease at bone marrow level *Autoimmunity reviews*,

(2009). Nonpathogenic SIV infection of African green monkeys induces a strong but rapidly controlled type I IFN response *The Journal of clinical investigation*, *119*(12),

(2009). Plasma levels of bacterial DNA correlate with immune activation and the magnitude of immune restoration in persons with antiretroviral-treated HIV infection *The Journal of infectious diseases*, *199*(8), 1177-1185. doi:10.1086/597476 Kader, M., Wang, X., Piatak, M., Lifson, J., Roederer, M., Veazey, R., & Mattapallil, J. J.

(2009). Alpha4(+)beta7(hi)CD4(+) memory T cells harbor most Th-17 cells and are preferentially infected during acute SIV infection *Mucosal immunology*, *2*(5), 439-449.

(2009). Increased mortality and AIDS-like immunopathology in wild chimpanzees

et al. (2007). IL-23 and IL-17 in the establishment of protective pulmonary CD4+ T cell responses after vaccination and during Mycobacterium tuberculosis challenge

Immune Activation after Pathogenic SIV Infection of Rhesus Macaques. Keystone Symposia, HIV Evolution, Genomics and Pathogenesis, March 20-25, 2011,

Schuitemaker, H. (2005). Low-level CD4+ T cell activation is associated with low susceptibility to HIV-1 infection *Journal of immunology (Baltimore, Md. : 1950)*, *175*(9),

21 initiates an alternative pathway to induce proinflammatory T(H)17 cells *Nature*,

Mavoungou, V., et al. (2005). Antiinflammatory profiles during primary SIV infection in African green monkeys are associated with protection against AIDS *The* 


HIV Without AIDS: The Immunological Secrets of Natural Hosts 225

Paiardini, Mirko, Pandrea, I., Apetrei, C., & Silvestri, G. (2009b). Lessons learned from the

Pandrea, I., Apetrei, C., Dufour, J., Dillon, N., Barbercheck, J., Metzger, M., Jacquelin, B., et

Pandrea, I. V., Gautam, R., Ribeiro, R. M., Brenchley, J. M., Butler, I. F., Pattison, M.,

Pandrea, I., Sodora, D. L., Silvestri, G., & Apetrei, C. (2008). Into the wild: simian

Picker, L. J. (2006). Immunopathogenesis of acute AIDS virus infection *Current opinion in* 

Picker, L. J., Hagen, S. I., Lum, R., Reed-Inderbitzin, E. F., Daly, L. M., Sylwester, A. W.,

*Journal of experimental medicine*, *200*(10), 1299-1314. doi:10.1084/jem.20041049 Pizzolo, G., Vinante, F., Sinicco, A., Chilosi, M., Agostini, C., Perini, A., Zuppini, B., et al.

Prendergast, A., Prado, J. G., Kang, Y., Chen, F., Riddell, L. A., Luzzi, G., Goulder, P., et al.

Raffatellu, M., Santos, R. L., Verhoeven, D. E., George, M. D., Wilson, R. P., Winter, S. E.,

Reddy, M. M., Sorrell, S. J., Lange, M., & Grieco, M. H. (1988). Tumor necrosis factor and

Rey-Cuillé, M. A., Berthier, J. L., Bomsel-Demontoy, M. C., Chaduc, Y., Montagnier, L.,

Rodríguez, B., Sethi, A. K., Cheruvu, V. K., Mackay, W., Bosch, R. J., Kitahata, M., Boswell, S.

*Association*, *296*(12), 1498-1506. doi:10.1001/jama.296.12.1498

doi:10.1146/annurev.med.60.041807.123753

*1950)*, *179*(5), 3035-3046.

*5*(4), 180-183.

419-428. doi:10.1016/j.it.2008.05.004

502. doi:10.1097/QAD.0b013e3283344895

*medicine*, *14*(4), 421-428. doi:10.1038/nm1743

*immune deficiency syndromes*, *1*(5), 436-440.

*virology*, *72*(5), 3872-3886.

*Blood*, *109*(3), 1069-1076. doi:10.1182/blood-2006-05-024364

*immunology*, *18*(4), 399-405. doi:10.1016/j.coi.2006.05.001

natural hosts of HIV-related viruses *Annual review of medicine*, *60*, 485-495.

al. (2006). Simian immunodeficiency virus SIVagm.sab infection of Caribbean African green monkeys: a new model for the study of SIV pathogenesis in natural hosts *Journal of virology*, *80*(10), 4858-4867. doi:10.1128/JVI.80.10.4858-4867.2006 Pandrea, I., Apetrei, C., Gordon, S., Barbercheck, J., Dufour, J., Bohm, R., Sumpter, B., et al.

(2007a). Paucity of CD4+CCR5+ T cells is a typical feature of natural SIV hosts

Rasmussen, T., et al. (2007b). Acute loss of intestinal CD4+ T cells is not predictive of simian immunodeficiency virus virulence *Journal of immunology (Baltimore, Md. :* 

immunodeficiency virus (SIV) infection in natural hosts *Trends in immunology*, *29*(9),

Walker, J. M., et al. (2004). Insufficient production and tissue delivery of CD4+ memory T cells in rapidly progressive simian immunodeficiency virus infection *The* 

(1987). Increased levels of soluble interleukin-2 receptor in the serum of patients with human immunodeficiency virus infection *Diagnostic and clinical immunology*,

(2010). HIV-1 infection is characterized by profound depletion of CD161+ Th17 cells and gradual decline in regulatory T cells *AIDS (London, England)*, *24*(4), 491-

Godinez, I., et al. (2008). Simian immunodeficiency virus-induced mucosal interleukin-17 deficiency promotes Salmonella dissemination from the gut *Nature* 

HIV P24 antigen levels in serum of HIV-infected populations *Journal of acquired* 

Hovanessian, A. G., & Chakrabarti, L. A. (1998). Simian immunodeficiency virus replicates to high levels in sooty mangabeys without inducing disease *Journal of* 

L., et al. (2006). Predictive value of plasma HIV RNA level on rate of CD4 T-cell decline in untreated HIV infection *JAMA : the journal of the American Medical* 

transition phase *Microbes and infection / Institut Pasteur*, *13*(1), 14-24. doi:10.1016/j.micinf.2010.09.011


Monteiro, P., Gosselin, A., Wacleche, V. S., El-Far, M., Said, E. A., Kared, H., Grandvaux, N.,

Moore, R. D., Keruly, J., Richman, D. D., Creagh-Kirk, T., & Chaisson, R. E. (1992). Natural

Murray, S. M., Down, C. M., Boulware, D. R., Stauffer, W. M., Cavert, W. P., Schacker, T. W.,

Nowroozalizadeh, S., Månsson, F., da Silva, Z., Repits, J., Dabo, B., Pereira, C., Biague, A., et

Nurieva, R., Yang, X. O., Martinez, G., Zhang, Y., Panopoulos, A. D., Ma, L., Schluns, K., et

Okoye, A., Meier-Schellersheim, M., Brenchley, J. M., Hagen, S. I., Walker, J. M.,

Onanga, R., Souquière, S., Makuwa, M., Mouinga-Ondéme, A., Simon, F., Apetrei, C., &

Ouyang, W., & Valdez, P. (2008). IL-22 in mucosal immunity *Mucosal immunology*, *1*(5), 335-

Paiardini, Mirko. (2010). Th17 cells in natural SIV hosts *Current opinion in HIV and AIDS*,

Paiardini, M, Cervasi, B., Engram, J. C., Gordon, S. N., Klatt, N. R., Muthukumar, A., Else, J.,

Paiardini M, Cervasi B, Reyes-Aviles E, Micci L, Ortiz AM, Chahroudi A, Vinton C, Gordon

T cells *Nature*, *448*(7152), 480-483. doi:10.1038/nature05969

doi:10.1016/j.micinf.2010.09.011

doi:10.1128/JVI.01466-10

doi:10.1084/jem.20070567

338. doi:10.1038/mi.2008.26

doi:10.1128/JVI.80.7.3301-3309.2006

doi:10.1182/blood-2008-06-159442

Medicine 17(7):830-6. doi: 10.1038/nm.2395

*5*(2), 166-172. doi:10.1097/COH.0b013e328335c161

doi:10.1086/651430

virus-1 on hematopoiesis *Blood*, *91*(5), 1479-1495.

transition phase *Microbes and infection / Institut Pasteur*, *13*(1), 14-24.

et al. (2011). Memory CCR6+CD4+ T Cells Are Preferential Targets for Productive HIV Type 1 Infection Regardless of Their Expression of Integrin {beta}7 *Journal of immunology (Baltimore, Md. : 1950)*, *186*(8), 4618-4630. doi:10.4049/jimmunol.1004151

history of advanced HIV disease in patients treated with zidovudine. The Zidovudine Epidemiology Study Group *AIDS (London, England)*, *6*(7), 671-677. Moses, A., Nelson, J., & Bagby, G. C. (1998). The influence of human immunodeficiency

Brenchley, J. M., et al. (2010). Reduction of immune activation with chloroquine therapy during chronic HIV infection *Journal of virology*, *84*(22), 12082-12086.

al. (2010). Microbial translocation correlates with the severity of both HIV-1 and HIV-2 infections *The Journal of infectious diseases*, *201*(8), 1150-1154.

al. (2007). Essential autocrine regulation by IL-21 in the generation of inflammatory

Rohankhedkar, M., Lum, R., et al. (2007). Progressive CD4+ central memory T cell decline results in CD4+ effector memory insufficiency and overt disease in chronic SIV infection *The Journal of experimental medicine*, *204*(9), 2171-2185.

Roques, P. (2006). Primary simian immunodeficiency virus SIVmnd-2 infection in mandrills (Mandrillus sphinx) *Journal of virology*, *80*(7), 3301-3309.

et al. (2009a). Bone marrow-based homeostatic proliferation of mature T cells in nonhuman primates: implications for AIDS pathogenesis. *Blood*, *113*(3), 612-621.

SN, Bosinger SE, Francella N, Hallberg PL, Cramer E, Schlub T, Chan ML, Riddick NE, Collman RG, Apetrei C, Pandrea I, Else J, Munch J, Kirchhoff F, Davenport MP, Brenchley JM, Silvestri G. (2011). Low levels of SIV infection in sooty mangabey central memory CD4(+) T cells are associated with limited CCR5 expression Nature


HIV Without AIDS: The Immunological Secrets of Natural Hosts 227

Smith, S. M. (2006). The pathogenesis of HIV infection: stupid may not be so dumb after all

Sodora, D. L., Allan, J. S., Apetrei, C., Brenchley, J. M., Douek, D. C., Else, J. G., Estes, J. D., et

Sodora, D. L., & Silvestri, G. (2008). Immune activation and AIDS pathogenesis *AIDS (London, England)*, *22*(4), 439-446. doi:10.1097/QAD.0b013e3282f2dbe7 Sumpter, B., Dunham, R., Gordon, S., Engram, J., Hennessy, M., Kinter, A., Paiardini, M., et

Taaffe, J., Chahroudi, A., Engram, J., Sumpter, B., Meeker, T., Ratcliffe, S., Paiardini, M., et

activation *Journal of virology*, *84*(11), 5476-5484. doi:10.1128/JVI.00039-10 Takehisa, J., & Miura, T. (2010). [The origin and evolution of HIV] *Nippon rinsho. Japanese* 

Tesselaar, K., Arens, R., van Schijndel, G. M. W., Baars, P. A., van der Valk, M. A., Borst, J.,

VandeWoude, S., & Apetrei, C. (2006). Going wild: lessons from naturally occurring T-

Veazey, R. S., DeMaria, M., Chalifoux, L. V., Shvetz, D. E., Pauley, D. R., Knight, H. L.,

Wertheim, J. O., & Worobey, M. (2009). Dating the age of the SIV lineages that gave rise to

Williams LD, Bansal A, Sabbaj S, Heath SL, Song W, Tang J, Zajac AJ, Goepfert PA. (2011).

*European journal of immunology*, *26*(8), 1700-1706. doi:10.1002/eji.1830260806 Worobey, M., Santiago, M. L., Keele, B. F., Ndjango, J. N., Joy, J. B., Labama, B. L., Dhed'A,

controllers. *Journal of virology*, 85(5), 2316-24. doi:10.1128/JVI.01476-10 Wolthers, K. C., Otto, S. A., Lens, S. M., Kolbach, D. N., van Lier, R. A., Miedema, F., &

al. (2009). Toward an AIDS vaccine: lessons from natural simian immunodeficiency virus infections of African nonhuman primate hosts *Nature medicine*, *15*(8), 861-865.

al. (2007). Correlates of preserved CD4(+) T cell homeostasis during natural, nonpathogenic simian immunodeficiency virus infection of sooty mangabeys: implications for AIDS pathogenesis *Journal of immunology (Baltimore, Md. : 1950)*,

al. (2010). A five-year longitudinal analysis of sooty mangabeys naturally infected with simian immunodeficiency virus reveals a slow but progressive decline in CD4+ T-cell count whose magnitude is not predicted by viral load or immune

van Oers, M. H. J., et al. (2003). Lethal T cell immunodeficiency induced by chronic costimulation via CD27-CD70 interactions *Nature immunology*, *4*(1), 49-54.

lymphotropic lentiviruses *Clinical microbiology reviews*, *19*(4), 728-762.

Rosenzweig, M., et al. (1998). Gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection *Science (New York, N.Y.)*, *280*(5362),

HIV-1 and HIV-2 *PLoS computational biology*, *5*(5), e1000377.

Interleukin-21-producing HIV-1-specific CD8 T cells are preferentially seen in elite

Meyaard, L. (1996). Increased expression of CD80, CD86 and CD70 on T cells from HIV-infected individuals upon activation in vitro: regulation by CD4+ T cells

B. D., et al. (2004). Origin of AIDS: contaminated polio vaccine theory refuted

*Retrovirology*, *3*, 60. doi:10.1186/1742-4690-3-60

*journal of clinical medicine*, *68*(3), 410-414.

doi:10.1038/nm.2013

*178*(3), 1680-1691.

doi:10.1038/ni869

427-431.

doi:10.1128/CMR.00009-06

doi:10.1371/journal.pcbi.1000377

*Nature*, *428*(6985), 820. doi:10.1038/428820a


It's the immune system, stupid. (1999). It's the immune system, stupid *STEP perspective*,

Roederer, M, Dubs, J. G., Anderson, M. T., Raju, P. A., Herzenberg, L. A., & Herzenberg, L.

*The Journal of clinical investigation*, *95*(5), 2061-2066. doi:10.1172/JCI117892 Romagnani, S. (2008). Human Th17 cells *Arthritis research & therapy*, *10*(2), 206.

Sandler, N. G., Wand, H., Roque, A., Law, M., Nason, M. C., Nixon, D. E., Pedersen, C., et al.

Schneider, T, Jahn, H. U., Schmidt, W., Riecken, E. O., Zeitz, M., & Ullrich, R. (1995). Loss of

Sethi, K. K., & Näher, H. (1986). Elevated titers of cell-free interleukin-2 receptor in serum

Sharp, Paul M, & Hahn, Beatrice H. (2008). AIDS: prehistory of HIV-1 *Nature*, *455*(7213), 605-

Sharp, P M, & Hahn, B H. (2010). The evolution of HIV-1 and the origin of AIDS.

Sharp, Paul M, Shaw, G. M., & Hahn, Beatrice H. (2005). Simian immunodeficiency virus

Shen, X., & Tomaras, G. D. (2011). Alterations of the B-cell response by HIV-1 replication *Current HIV/AIDS reports*, *8*(1), 23-30. doi:10.1007/s11904-010-0064-2 Silvestri, G., Sodora, D. L., Koup, R. A., Paiardini, M., O'Neil, S. P., McClure, H. M.,

Silvestri, G., Fedanov, A., Germon, S., Kozyr, N., Kaiser, W. J., Garber, D. A., McClure, H., et

Simons, B. C., Vancompernolle, S. E., Smith, R. M., Wei, J., Barnett, L., Lorey, S. L., Meyer-

variants *Journal of immunology (Baltimore, Md. : 1950)*, *181*(7), 5137-5146.

*clinical investigation*, *117*(11), 3148-3154. doi:10.1172/JCI33034

Diarrhea/Wasting Syndrome Study Group *Gut*, *37*(4), 524-529.

syndrome *Immunology letters*, *13*(4), 179-184.

606. doi:10.1038/455605a

2494. doi:10.1098/rstb.2010.0031

doi:10.1128/JVI.79.7.3891-3902.2005

viremia *Immunity*, *18*(3), 441-452.

A. (1995). CD8 naive T cell counts decrease progressively in HIV-infected adults

(2011). Plasma levels of soluble CD14 independently predict mortality in HIV infection *The Journal of infectious diseases*, *203*(6), 780-790. doi:10.1093/infdis/jiq118 Sauce, D., Larsen, M., Fastenackels, S., Pauchard, M., Ait-Mohand, H., Schneider, L., Guihot,

A., et al. (2011). HIV disease progression despite suppression of viral replication is associated with exhaustion of lymphopoiesis *Blood*. doi:10.1182/blood-2011-01-

CD4 T lymphocytes in patients infected with human immunodeficiency virus type 1 is more pronounced in the duodenal mucosa than in the peripheral blood. Berlin

and cerebrospinal fluid specimens of patients with acquired immunodeficiency

*Philosophical Transactions of the Royal Society B: Biological Sciences*, *365*(1552), 2487-

infection of chimpanzees *Journal of virology*, *79*(7), 3891-3902.

Staprans, S. I., et al. (2003). Nonpathogenic SIV infection of sooty mangabeys is characterized by limited bystander immunopathology despite chronic high-level

al. (2005). Divergent host responses during primary simian immunodeficiency virus SIVsm infection of natural sooty mangabey and nonnatural rhesus macaque hosts *Journal of virology*, *79*(7), 4043-4054. doi:10.1128/JVI.79.7.4043-4054.2005 Silvestri, G., Paiardini, Mirko, Pandrea, I., Lederman, M. M., & Sodora, D. L. (2007).

Understanding the benign nature of SIV infection in natural hosts *The Journal of* 

Olson, D., et al. (2008). Despite biased TRBV gene usage against a dominant HLA B57-restricted epitope, TCR diversity can provide recognition of circulating epitope

*99*(1), 5.

331306

doi:10.1186/ar2392


**9** 

**Immunotherapies and Vaccines** 

Human Immunodeficiency Virus (HIV) was first isolated in 1983 by Barre-Sinoussi and Gallo in parallel at two independent institutions (Barre-Sinoussi, Chermann et al. 1983; Gallo, Sarin et al. 1983). The following year, HIV was established as the causative agent of Acquired Immunodeficiency Syndrome (AIDS). Given such a monumental discovery, there were expectations that an effective vaccine or treatment was not far from being marketed. Unfortunately these expectations have yet to become reality and HIV has become a global epidemic. In 2010, the World Health Organization (WHO) reported that 33.3 million individuals worldwide were living with HIV/AIDS (World Health Organization 2009). The discovery of HIV as the causative agent of AIDS stimulated many areas of basic virological and immunological research. Researchers continue to stress the need for more integrated approaches for development of HIV antiviral treatments and vaccines. In this chapter, the viral and immune challenges, criteria for evaluating clinical studies, candidate therapies and

Several factors have contributed to the delay of HIV vaccines and therapeutics. These factors can be grouped into two main categories; 1) intrinsic viral characteristics and 2) viral and host interactions. The intrinsic viral properties of HIV, such as rapid replication and virus mutation, virus recombination and viral integration have been obstacles in drug and vaccine development (Aaron N. Endsley 2008; Montagnier 2010). One of the major problems in HIV vaccine development is the high sequence variability of viral isolates (Monteiro, Alcantara et al. 2009; Cuevas, Fernandez-Garcia et al. 2010). The classification of HIV into clades is covered in the HIV nomenclature proposal now found on the Los Alamos HIV Sequence database website (Robertson, Anderson et al. 2000). A major contributor to the high variability of the virus is the lack of proof reading activity present in the viral polymerase (reverse transcriptase) combined with rapid replication rate. Such a combination allows for emergence of viral isolates that can evade the immune response elicited to older viral sequences. The constant escape from immune surveillance results in a constant need for "catch up" by the immune response. Another reason for increase variability is the ability of the virus to genetically recombine (Brown, Peters et al. 2011). Due to the possibility of superinfection, viruses from different clades can be present in the same cell during replication and may result in recombinant viruses. For example, a virus classified A/E has an envelope derived from a clade A virus and Gag proteins derived from a clade E virus.

**1. Introduction** 

vaccines will be reviewed.

**1.1 Viral and immune challenges** 

Hermancia S. Eugene and Ted M. Ross

*University of Pittsburgh* 

*United States* 


(n.d.).

## **Immunotherapies and Vaccines**

Hermancia S. Eugene and Ted M. Ross *University of Pittsburgh United States* 

#### **1. Introduction**

228 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

Worobey, M., Gemmel, M., Teuwen, D. E., Haselkorn, T., Kunstman, K., Bunce, M.,

Yang, L., Anderson, D. E., Baecher-Allan, C., Hastings, W. D., Bettelli, E., Oukka, M.,

human T(H)17 cells *Nature*, *454*(7202), 350-352. doi:10.1038/nature07021 Zheng, Y., Valdez, P. A., Danilenko, D. M., Hu, Y., Sa, S. M., Gong, Q., Abbas, A. R., et al.

bacterial pathogens *Nature medicine*, *14*(3), 282-289. doi:10.1038/nm1720 Zhu, T., Korber, B. T., Nahmias, A. J., Hooper, E., Sharp, P. M., & Ho, D. D. (1998). An

Kinshasa by 1960 *Nature*, *455*(7213), 661-664. doi:10.1038/nature07390 Worobey, M., Telfer, P., Souquière, S., Hunter, M., Coleman, C. A., Metzger, M. J., Reed, P.,

*N.Y.)*, *329*(5998), 1487. doi:10.1126/science.1193550

*Nature*, *391*(6667), 594-597. doi:10.1038/35400

(n.d.).

Muyembe, J., et al. (2008). Direct evidence of extensive diversity of HIV-1 in

et al. (2010). Island biogeography reveals the deep history of SIV *Science (New York,* 

Kuchroo, V. K., et al. (2008). IL-21 and TGF-beta are required for differentiation of

(2008). Interleukin-22 mediates early host defense against attaching and effacing

African HIV-1 sequence from 1959 and implications for the origin of the epidemic

Human Immunodeficiency Virus (HIV) was first isolated in 1983 by Barre-Sinoussi and Gallo in parallel at two independent institutions (Barre-Sinoussi, Chermann et al. 1983; Gallo, Sarin et al. 1983). The following year, HIV was established as the causative agent of Acquired Immunodeficiency Syndrome (AIDS). Given such a monumental discovery, there were expectations that an effective vaccine or treatment was not far from being marketed. Unfortunately these expectations have yet to become reality and HIV has become a global epidemic. In 2010, the World Health Organization (WHO) reported that 33.3 million individuals worldwide were living with HIV/AIDS (World Health Organization 2009). The discovery of HIV as the causative agent of AIDS stimulated many areas of basic virological and immunological research. Researchers continue to stress the need for more integrated approaches for development of HIV antiviral treatments and vaccines. In this chapter, the viral and immune challenges, criteria for evaluating clinical studies, candidate therapies and vaccines will be reviewed.

#### **1.1 Viral and immune challenges**

Several factors have contributed to the delay of HIV vaccines and therapeutics. These factors can be grouped into two main categories; 1) intrinsic viral characteristics and 2) viral and host interactions. The intrinsic viral properties of HIV, such as rapid replication and virus mutation, virus recombination and viral integration have been obstacles in drug and vaccine development (Aaron N. Endsley 2008; Montagnier 2010). One of the major problems in HIV vaccine development is the high sequence variability of viral isolates (Monteiro, Alcantara et al. 2009; Cuevas, Fernandez-Garcia et al. 2010). The classification of HIV into clades is covered in the HIV nomenclature proposal now found on the Los Alamos HIV Sequence database website (Robertson, Anderson et al. 2000). A major contributor to the high variability of the virus is the lack of proof reading activity present in the viral polymerase (reverse transcriptase) combined with rapid replication rate. Such a combination allows for emergence of viral isolates that can evade the immune response elicited to older viral sequences. The constant escape from immune surveillance results in a constant need for "catch up" by the immune response. Another reason for increase variability is the ability of the virus to genetically recombine (Brown, Peters et al. 2011). Due to the possibility of superinfection, viruses from different clades can be present in the same cell during replication and may result in recombinant viruses. For example, a virus classified A/E has an envelope derived from a clade A virus and Gag proteins derived from a clade E virus.

Immunotherapies and Vaccines 231

2010; Sabado, O'Brien et al. 2010; Kolte, Gaardbo et al. 2011). Therefore, the HIV immunotherapeutic field does not only have to establish the correlate for preventing disease progression but has to overcome the immune dysfunction caused by the viral infection. The moderate success of the Thailand study and the failure of the STEP trial have brought into question both humoral and cellular immunity as the correlates of protection. New vaccine designs are now aimed at inducing both of humoral and cellular responses. The key to overcoming these obstacles faced by drug and vaccine development is continued research not only in terms of treatment, but also basic research of HIV and human immunology.

Since there is a lack of protective correlate(s), standardized evaluation of clinical responses is needed to develop preventative AIDS vaccines and improved immunotherapies (Pantaleo and Koup 2004). Usually, when a correlate of protection is lacking, a vaccine's ability to provide clinical benefit by reducing mortality and morbidity gives precedent for licensing. Due to the availability of an FDA approved therapy, HAART, any therapy that will be approved is compared to benefits given by HAART. HAART reduces viral loads and restores some level of CD4+T cells in individuals that benefit from therapy. In a clinical setting, the hallmarks or surrogate markers of efficacy are reduction in viral loads and increase in the number of CD4+ T cells (Peto 1996; Peters 2000). While CD4 +T cells increase during HAART therapy leads to better prognosis of disease. Recent Proleukin (rIL-2) clinical trials, SILCAAT and ESPRIT volunteers showed increase in CD4 +T cells but the time to disease progression was not increased. These results have brought into question the validity of increase CD4+T cells as a readout for better disease outcome (Peters and Samuel 2010). The composition of the CD4+ T cell population recovered after therapy was evaluated at the end of the study to determine if the increase CD4+ T cells population was any different from populations seen after HAART therapy. Other T cells have also come into light in the last few years, Th17 cells and cells that secret IL-21 may play a role in the non-disease progression seen in African green monkeys and Sooty Mangabeys (Ciccone, Greenwald et al. 2011; Hartigan-O'Connor, Hirao et al. 2011; Milush, Mir et al. 2011). These trends are also being reported in long- term non-progressors and elite controllers (Hartigan-O'Connor, Hirao et al. 2011; Salgado, Rallun et al. 2011; Salgado, RallÛn et al. 2011). Results in animal studies have shown the possible role of Th17 cells in the gut mucosa in reduced bacterial translocation and slower disease progression (Cecchinato, Trindade et al. 2008; Maloy and

There is a lack of consensus on the hallmarks or surrogate markers for preventative vaccine trials. The ideal standard for a preventative HIV vaccine would be sterilizing protection. Sterilizing protection is complete protection from HIV infection, no detectable HIV at any time and no transmission of HIV. In 2007 an NIH workshop on vaccine efficacy resulted in a reported by Follman et al. which stated three parameters for evaluating HIV vaccine efficacy(Follmann, Duerr et al. 2007). The three parameters for evaluating vaccine efficacy are 1) reduction in risk of acquiring HIV 2) the reduction in cumulative risk of progressing to AIDS from the time of infection to diagnosis and 3) reduction in the risk of transmission of HIV to others. Vaccine endpoints are based on years of clinical studies (Peto 1996; Follmann, Duerr et al. 2007; MacLachlan, Mayer et al. 2009). Preclinical (usually NHP) studies have established surrogate markers for vaccine efficacy. Thus far, the most relevant marker identified as a determinant of disease

**1.2 Evaluation of immunotherapies and vaccines in human trials** 

Kullberg 2008; Hofer and Speck 2009).

The other major viral property that works against effective of therapy and viral clearance is viral integration. HIV contains a viral integrase responsible for integration of the HIV provirus into the DNA of an infected cell (Delelis, Carayon et al. 2008). Provirus integration is an essential part of replication (Engelman, Englund et al. 1995). This integrated viral DNA results in both establishment of viral reservoirs in the host and disruption of the immune responses against the infecting virus (Finzi, Blankson et al. 1999; Miedema 2008; Carter, Onafuwa-Nuga et al. 2010; Virgin and Walker 2010). These viral reservoirs are a source of actively replicating viruses in individuals who have controlled infection and have undetectable levels of virus(Wong, Hezareh et al. 1997; Chun, Nickle et al. 2008; Lerner, Guadalupe et al. 2011). Authors Siliciano J.D and Siliciano,R F discuss HIV reservoirs and how they contribute to the lack of virus eradication and the need for continuous HAART therapy by HIV infected individuals to prevent virus rebound (Siliciano and Siliciano 2004).

The lack of a defined correlate(s) of protection for HIV is a major obstacle in vaccine and therapeutic development. Humoral immune responses were initially proposed as a correlate of protection. Studies in experimental animals have shown that passively administering anti-HIV antibodies results in protection from infection (Prince, Reesink et al. 1991; Putkonen, Thorstensson et al. 1991; Emini, Schleif et al. 1992). The first prophylaxis vaccine to enter phase III trials, AIDSVAX by VaxGen, induced antibodies to HIV vaccine components but vaccine was not efficacious (Ltd. 2003). The failure of the initial studies has not changed the viewpoint of everyone on the role of humoral responses as the correlate of protection. The antigens used in these studies, monomeric gp120 and monomeric gp160, are not the functional unit of the HIV envelope. The HIV envelope is trimeric on the surface of the virus particle. Studies using trimeric envelope immunogens have been used to improve the humoral responses (Nkolola, Peng et al. 2010; Sundling, Forsell et al. 2010; Sundling, O'Dell et al. 2010). Also, the recent vaccine trial in Thailand, has provided some data to support the possibility that humoral responses may be the HIV correlate of protection (Rerks-Ngarm, Pitisuttithum et al. 2009).

Many investigators are designing preventative vaccines for HIV that induces cellular response (Nanjundappa, Wang et al. 2011; Ranasinghe, Eyers et al. 2011; Sistigu, Bracci et al. 2011). Data from preclinical studies, as well as infected individuals, showed that an effective cellular response was able to control viral replication and resulted in reduce progression to diseases (Wilson, Keele et al. 2009; Streeck and Nixon 2010). Coming off the heels of failed humorally-driven trials, the certainty of developing a preventative vaccine is questioned and some in the field believed that preventing progression to disease to be a viable alternate focus. Vaccines aimed at eliciting cellular responses for preventing infection or disease progression have also not been successful. In 2007, the Merck HIV vaccine trial used adenovirus to deliver HIV genes *gag, pol* and *nef* .The trial was stopped after intermediate data analysis showed no supportive evidence to continue (Sekaly 2008). It appears that preexisting immune responses to the adenovirus may have increased susceptibility to infection. Other alternative theories of the immune correlates of HIV/AIDS protection need to be considered. The immune correlate(s) for preventing infection and prevention of disease symptoms (*i.e.* control of infection) may be different (Jose Esparza 1996). In the case of preventative vaccines, an effective initial response to the virus is needed. At the time of the initial assault, the immune system is not dysfunctional. In contrast, during HIV therapy the immune system is in a state of dysregulation due to the constant tug of war with the virus infection. HIV infection causes a dysregulation of the immune response (Kuhrt, Faith et al.

The other major viral property that works against effective of therapy and viral clearance is viral integration. HIV contains a viral integrase responsible for integration of the HIV provirus into the DNA of an infected cell (Delelis, Carayon et al. 2008). Provirus integration is an essential part of replication (Engelman, Englund et al. 1995). This integrated viral DNA results in both establishment of viral reservoirs in the host and disruption of the immune responses against the infecting virus (Finzi, Blankson et al. 1999; Miedema 2008; Carter, Onafuwa-Nuga et al. 2010; Virgin and Walker 2010). These viral reservoirs are a source of actively replicating viruses in individuals who have controlled infection and have undetectable levels of virus(Wong, Hezareh et al. 1997; Chun, Nickle et al. 2008; Lerner, Guadalupe et al. 2011). Authors Siliciano J.D and Siliciano,R F discuss HIV reservoirs and how they contribute to the lack of virus eradication and the need for continuous HAART therapy by HIV infected individuals to prevent virus rebound (Siliciano and Siliciano 2004). The lack of a defined correlate(s) of protection for HIV is a major obstacle in vaccine and therapeutic development. Humoral immune responses were initially proposed as a correlate of protection. Studies in experimental animals have shown that passively administering anti-HIV antibodies results in protection from infection (Prince, Reesink et al. 1991; Putkonen, Thorstensson et al. 1991; Emini, Schleif et al. 1992). The first prophylaxis vaccine to enter phase III trials, AIDSVAX by VaxGen, induced antibodies to HIV vaccine components but vaccine was not efficacious (Ltd. 2003). The failure of the initial studies has not changed the viewpoint of everyone on the role of humoral responses as the correlate of protection. The antigens used in these studies, monomeric gp120 and monomeric gp160, are not the functional unit of the HIV envelope. The HIV envelope is trimeric on the surface of the virus particle. Studies using trimeric envelope immunogens have been used to improve the humoral responses (Nkolola, Peng et al. 2010; Sundling, Forsell et al. 2010; Sundling, O'Dell et al. 2010). Also, the recent vaccine trial in Thailand, has provided some data to support the possibility that humoral responses may be the HIV correlate of protection

Many investigators are designing preventative vaccines for HIV that induces cellular response (Nanjundappa, Wang et al. 2011; Ranasinghe, Eyers et al. 2011; Sistigu, Bracci et al. 2011). Data from preclinical studies, as well as infected individuals, showed that an effective cellular response was able to control viral replication and resulted in reduce progression to diseases (Wilson, Keele et al. 2009; Streeck and Nixon 2010). Coming off the heels of failed humorally-driven trials, the certainty of developing a preventative vaccine is questioned and some in the field believed that preventing progression to disease to be a viable alternate focus. Vaccines aimed at eliciting cellular responses for preventing infection or disease progression have also not been successful. In 2007, the Merck HIV vaccine trial used adenovirus to deliver HIV genes *gag, pol* and *nef* .The trial was stopped after intermediate data analysis showed no supportive evidence to continue (Sekaly 2008). It appears that preexisting immune responses to the adenovirus may have increased susceptibility to infection. Other alternative theories of the immune correlates of HIV/AIDS protection need to be considered. The immune correlate(s) for preventing infection and prevention of disease symptoms (*i.e.* control of infection) may be different (Jose Esparza 1996). In the case of preventative vaccines, an effective initial response to the virus is needed. At the time of the initial assault, the immune system is not dysfunctional. In contrast, during HIV therapy the immune system is in a state of dysregulation due to the constant tug of war with the virus infection. HIV infection causes a dysregulation of the immune response (Kuhrt, Faith et al.

(Rerks-Ngarm, Pitisuttithum et al. 2009).

2010; Sabado, O'Brien et al. 2010; Kolte, Gaardbo et al. 2011). Therefore, the HIV immunotherapeutic field does not only have to establish the correlate for preventing disease progression but has to overcome the immune dysfunction caused by the viral infection. The moderate success of the Thailand study and the failure of the STEP trial have brought into question both humoral and cellular immunity as the correlates of protection. New vaccine designs are now aimed at inducing both of humoral and cellular responses. The key to overcoming these obstacles faced by drug and vaccine development is continued research not only in terms of treatment, but also basic research of HIV and human immunology.

#### **1.2 Evaluation of immunotherapies and vaccines in human trials**

Since there is a lack of protective correlate(s), standardized evaluation of clinical responses is needed to develop preventative AIDS vaccines and improved immunotherapies (Pantaleo and Koup 2004). Usually, when a correlate of protection is lacking, a vaccine's ability to provide clinical benefit by reducing mortality and morbidity gives precedent for licensing. Due to the availability of an FDA approved therapy, HAART, any therapy that will be approved is compared to benefits given by HAART. HAART reduces viral loads and restores some level of CD4+T cells in individuals that benefit from therapy. In a clinical setting, the hallmarks or surrogate markers of efficacy are reduction in viral loads and increase in the number of CD4+ T cells (Peto 1996; Peters 2000). While CD4 +T cells increase during HAART therapy leads to better prognosis of disease. Recent Proleukin (rIL-2) clinical trials, SILCAAT and ESPRIT volunteers showed increase in CD4 +T cells but the time to disease progression was not increased. These results have brought into question the validity of increase CD4+T cells as a readout for better disease outcome (Peters and Samuel 2010). The composition of the CD4+ T cell population recovered after therapy was evaluated at the end of the study to determine if the increase CD4+ T cells population was any different from populations seen after HAART therapy. Other T cells have also come into light in the last few years, Th17 cells and cells that secret IL-21 may play a role in the non-disease progression seen in African green monkeys and Sooty Mangabeys (Ciccone, Greenwald et al. 2011; Hartigan-O'Connor, Hirao et al. 2011; Milush, Mir et al. 2011). These trends are also being reported in long- term non-progressors and elite controllers (Hartigan-O'Connor, Hirao et al. 2011; Salgado, Rallun et al. 2011; Salgado, RallÛn et al. 2011). Results in animal studies have shown the possible role of Th17 cells in the gut mucosa in reduced bacterial translocation and slower disease progression (Cecchinato, Trindade et al. 2008; Maloy and Kullberg 2008; Hofer and Speck 2009).

There is a lack of consensus on the hallmarks or surrogate markers for preventative vaccine trials. The ideal standard for a preventative HIV vaccine would be sterilizing protection. Sterilizing protection is complete protection from HIV infection, no detectable HIV at any time and no transmission of HIV. In 2007 an NIH workshop on vaccine efficacy resulted in a reported by Follman et al. which stated three parameters for evaluating HIV vaccine efficacy(Follmann, Duerr et al. 2007). The three parameters for evaluating vaccine efficacy are 1) reduction in risk of acquiring HIV 2) the reduction in cumulative risk of progressing to AIDS from the time of infection to diagnosis and 3) reduction in the risk of transmission of HIV to others. Vaccine endpoints are based on years of clinical studies (Peto 1996; Follmann, Duerr et al. 2007; MacLachlan, Mayer et al. 2009). Preclinical (usually NHP) studies have established surrogate markers for vaccine efficacy. Thus far, the most relevant marker identified as a determinant of disease

Immunotherapies and Vaccines 233

(World Health Organization 2006) : 1) suitability of drug combination, 2) licensing of drugs by national regulatory department and recommended dose, 3) toxicity profile of the drug, 4) availability of laboratory monitoring, 5) potential of maintenance and adherence to treatment , 6) prevalence of co-existing infections (*e.g.* Tuberculosis), 7) child bearing age, 8) availability of local and international manufacturers, and 9) price and effectiveness

Stages of replication cycle where drugs act: 1) Receptor binding and membrane fusion, 2) RNA genome reverse transcribed into DNA, 3) Provirus integration, 4) Virion egress and maturation. Fig. 1. Diagram showing the licensed antiretroviral HIV drugs and the step in the replication

One of the complications that an individual can experience on HAART is immune reconstitution inflammatory syndrome (IRIS) (Letang, Miro et al. 2011). IRIS is seen in individuals recovering from immunodeficiency. Criteria for IRIS 1) Response to antiviral therapy by: viral loads >1 log10/ml decrease in RNA level 2) clinical deterioration of inflammatory of infectious condition upon antiviral treatment and 3) symptoms cannot be alleviated by: clinical course of treatment, medication side effects or toxicity, treatment failure or complete non adherence (Tappuni 2011). IRIS is also recorded in individuals with HIV co-infections such as tuberculosis (Lin, Lai et al. 2010). Additionally, individuals on HAART develop other diseases such as cardiac and metabolic complications that are affiliated with aging (Broder 2010). The side effects of HAART treatment and limited availability in HIV endemic areas are the drive for development of new immunotherapies

of drug.

cycle they act on.

for HIV.

outcome is the reduction of plasma HIV genome RNA levels following infection (Lavreys, Baeten et al. 2006). The viral set point is a consistent marker for determining disease progression; i.e. the higher the viral set point, the more likely a patient will progress to AIDS (Lavreys, Baeten et al. 2006; Kelley, Barbour et al. 2007) The levels of CD4+T cells in the blood of infected individuals can also act as a surrogate of disease progression (Chouquet, Autran et al. 2002). However, the correlation between CD4+ T cell levels in the blood and disease progression becomes more significant closer to the onset of AIDS. Modeling studies have concluded that a reduction in blood viral titers of 1-1.5 log10 compared to peak infection leads to a significantly positive impact on progression to disease (Davenport, Ribeiro et al. 2004). The other aspect to a preventative vaccine is reduction of viral load leading to reduce transmission. A vaccine that results in a reduction in the rate or probability of transmission would have a positive impact on the HIV global epidemic. In the absence of a sterilizing vaccine, to have a vaccine, that not only lowers viral set point, but also reduces transmission rate would be beneficial (Gurunathan, Habib et al. 2009).

## **2. Immunotherapies**

#### **2.1 Highly Active Antiviral Therapy (HAART)**

Two years after the discovery of the causative agent of AIDS, the first sign of possible treatment was reported in 1985 with the development of the first antiretroviral compound (Mitsuya, Weinhold et al. 1985). This compound was called Retrovir (zidovudine, AZT) and became the first drug in the family of nucleoside reverse transcriptase inhibitors (NRTI). AZT targets the reverse transcription process of HIV's replication cycle. Since AZT, several NRTI and other families of drugs targeting the replication cycle of HIV have been discovered. As of 2010, the FDA has licensed twenty-five antiretroviral drugs. These drugs can be grouped based on the mode of action and are placed into one of the following groups: nucleoside reverse transcriptase inhibitors (NRTI), nucleotide reverse transcriptase inhibitor (NtRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI), protease inhibitor (PI), co-receptor inhibitor (CRI), integrase inhibitor (INI), and fusion inhibitor (FI) (De Clercq 2010). Figure 1 highlights licensed drugs and the mode of action of each group of drugs.

In 1996, the first use of combination drug therapy was attempted using a protease inhibitor that was combined with an NRTI (Gulick, Mellors et al. 1997). Combinational therapy confirmed that there was a longer period of undetectable or reduced viral loads as well as recovery of CD4+T cells in the blood. Combination therapy or HAART is now the treatment of choice for HIV infected individuals and results in various clinical outcomes (Greenbaum, Wilson et al. 2008; Crabtree-Ramirez, Villasis-Keever et al. 2010). Individuals on HAART need to be monitored at all times to ensure the combinational therapy is effective and does not result in viral mutants. Individuals on HAART will need their treatment regimen adjusted upon development of viral resistance (Paci, Martini et al. 2011; Von Kleist, Menz et al. 2011). In addition to viral resistance, HAART is highly toxic to patients resulting in reduced patient compliance (John, Moore et al. 2001; Kronenberg, Riehle et al. 2001). Moreover, HAART treatment is expensive and therefore people living in developing countries have less access to drugs even though these are the locations where the epidemic is greatest. Based on surveys and clinical research the WHO has identified factors that need to be considered to determine HAART treatment in an adult

outcome is the reduction of plasma HIV genome RNA levels following infection (Lavreys, Baeten et al. 2006). The viral set point is a consistent marker for determining disease progression; i.e. the higher the viral set point, the more likely a patient will progress to AIDS (Lavreys, Baeten et al. 2006; Kelley, Barbour et al. 2007) The levels of CD4+T cells in the blood of infected individuals can also act as a surrogate of disease progression (Chouquet, Autran et al. 2002). However, the correlation between CD4+ T cell levels in the blood and disease progression becomes more significant closer to the onset of AIDS. Modeling studies have concluded that a reduction in blood viral titers of 1-1.5 log10 compared to peak infection leads to a significantly positive impact on progression to disease (Davenport, Ribeiro et al. 2004). The other aspect to a preventative vaccine is reduction of viral load leading to reduce transmission. A vaccine that results in a reduction in the rate or probability of transmission would have a positive impact on the HIV global epidemic. In the absence of a sterilizing vaccine, to have a vaccine, that not only lowers viral set point, but also reduces transmission rate would be beneficial

Two years after the discovery of the causative agent of AIDS, the first sign of possible treatment was reported in 1985 with the development of the first antiretroviral compound (Mitsuya, Weinhold et al. 1985). This compound was called Retrovir (zidovudine, AZT) and became the first drug in the family of nucleoside reverse transcriptase inhibitors (NRTI). AZT targets the reverse transcription process of HIV's replication cycle. Since AZT, several NRTI and other families of drugs targeting the replication cycle of HIV have been discovered. As of 2010, the FDA has licensed twenty-five antiretroviral drugs. These drugs can be grouped based on the mode of action and are placed into one of the following groups: nucleoside reverse transcriptase inhibitors (NRTI), nucleotide reverse transcriptase inhibitor (NtRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI), protease inhibitor (PI), co-receptor inhibitor (CRI), integrase inhibitor (INI), and fusion inhibitor (FI) (De Clercq 2010). Figure 1 highlights licensed drugs and the mode of action of each group of

In 1996, the first use of combination drug therapy was attempted using a protease inhibitor that was combined with an NRTI (Gulick, Mellors et al. 1997). Combinational therapy confirmed that there was a longer period of undetectable or reduced viral loads as well as recovery of CD4+T cells in the blood. Combination therapy or HAART is now the treatment of choice for HIV infected individuals and results in various clinical outcomes (Greenbaum, Wilson et al. 2008; Crabtree-Ramirez, Villasis-Keever et al. 2010). Individuals on HAART need to be monitored at all times to ensure the combinational therapy is effective and does not result in viral mutants. Individuals on HAART will need their treatment regimen adjusted upon development of viral resistance (Paci, Martini et al. 2011; Von Kleist, Menz et al. 2011). In addition to viral resistance, HAART is highly toxic to patients resulting in reduced patient compliance (John, Moore et al. 2001; Kronenberg, Riehle et al. 2001). Moreover, HAART treatment is expensive and therefore people living in developing countries have less access to drugs even though these are the locations where the epidemic is greatest. Based on surveys and clinical research the WHO has identified factors that need to be considered to determine HAART treatment in an adult

(Gurunathan, Habib et al. 2009).

**2.1 Highly Active Antiviral Therapy (HAART)** 

**2. Immunotherapies** 

drugs.

(World Health Organization 2006) : 1) suitability of drug combination, 2) licensing of drugs by national regulatory department and recommended dose, 3) toxicity profile of the drug, 4) availability of laboratory monitoring, 5) potential of maintenance and adherence to treatment , 6) prevalence of co-existing infections (*e.g.* Tuberculosis), 7) child bearing age, 8) availability of local and international manufacturers, and 9) price and effectiveness of drug.

Stages of replication cycle where drugs act: 1) Receptor binding and membrane fusion, 2) RNA genome reverse transcribed into DNA, 3) Provirus integration, 4) Virion egress and maturation.

Fig. 1. Diagram showing the licensed antiretroviral HIV drugs and the step in the replication cycle they act on.

One of the complications that an individual can experience on HAART is immune reconstitution inflammatory syndrome (IRIS) (Letang, Miro et al. 2011). IRIS is seen in individuals recovering from immunodeficiency. Criteria for IRIS 1) Response to antiviral therapy by: viral loads >1 log10/ml decrease in RNA level 2) clinical deterioration of inflammatory of infectious condition upon antiviral treatment and 3) symptoms cannot be alleviated by: clinical course of treatment, medication side effects or toxicity, treatment failure or complete non adherence (Tappuni 2011). IRIS is also recorded in individuals with HIV co-infections such as tuberculosis (Lin, Lai et al. 2010). Additionally, individuals on HAART develop other diseases such as cardiac and metabolic complications that are affiliated with aging (Broder 2010). The side effects of HAART treatment and limited availability in HIV endemic areas are the drive for development of new immunotherapies for HIV.

Immunotherapies and Vaccines 235

Table 1. Evidence of dysfunction of the immune system of HIV infected individuals

HIV Viral Protein Remune, rgp160 (VaxSyn), rgp120,

Components of Immune Systems rIL-2, rIL-7, primed dendritic cells

HIV viral proteins used for immunotherapy include the viral envelope protein, core protein (Gag), reverse transcriptase, tat, nef, polymerase, as well as the whole killed virus (Tsoukas, Raboud et al. 1998; Gorse, Simionescu et al. 2006; Ensoli, Bellino et al. 2010). Viral proteins have been delivered as recombinant proteins, DNA vaccines, on virus-like-particles or in viral vectors (Buonaguro, Tornesello et al. 2009; Rosenberg, Graham et al. 2010). CD8+ T cells with cytolytic activity appeared to control viral titers in both non-human primate

HIV regulatory protein inhibitors sCD4 , Indinavir, nevirapine

Current experimental therapies are grouped based on components and or modes of action, such as the HIV viral proteins targeted, the components of the immune system effected, fusion inhibitors, viral inhibitors, and HIV regulatory protein inhibitors, (Kilby 1999; Peters

(Chun, Carruth et al. 1997),(Douek,

(Finkel, Tudor-Williams et al. 1995)

(D'Orsogna, Krueger et al. 2007),(Fauci,

Brenchley et al. 2002)

Mavilio et al. 2005),

(M Roederer 1995)

(Michel, Balde et al. 2000)

p24VLP, ALVAC1452

(Baenziger, Heikenwalder et al. 2009)

**Dysfunction References** 

The frequency of CD+ T cells infected by HIV in vivo is too low to account for the

Most apoptotic CD4+T cells in peripheral blood and lymph nodes of patients with chronic HIV infection are infected HIV

Naïve CD8+T cells, memory B and NK cells as well as CD4+T cells decline in HIV

SIV-infected macaques exhibit a persistently activated immune system and rapidly progress to AIDS, while SIV-infected sooty mangabeys show normal T cell division rates and do not progress to AIDS.

HIV-2 infection is associated with lower levels of immune activation, which may explain the slower decline of CD4+Tcells

In mice, TLR7 stimulation unrelated to a virus infection induces immune activation and immunopathology similar to that in

2000; Pett 2009). Examples of these can be seen in Table 2.

**Type of Immunotherapy Examples** 

Fusion inhibitors T-20, T-1249

DNAzymes DzV3-9

Table 2. Some examples of Immunotherapies

compared with HIV-1 infection

(Fernandez, Lim et al. 2009)

CD4 T cell loss

infection

HIV infection

Viral inhibitors

## **2.2 Expanding HIV therapy from HAART**

During HIV infection, the immune system does provide a defense aimed at eradicating the infection. This mounted immune response is insufficient and allows the viral infection to impair the immune system and persist. The use of therapeutics in infected individuals is aimed at overcoming the immune systems impairment to allow for viral control leading to decrease progression to AIDS. Human studies of long term non-progressor and elite progressors have observed a slower progression to disease in these individuals (Rodes, Toro et al. 2004; Okulicz and Lambotte 2011). Long term non-progressors are characterized based on absence of disease, low viral loads, and stable or increasing CD4 T cells.(Paroli, Propato et al. 2001). There is great need for drugs and vaccine strategies that reduce viral loads in infected individuals. Development of an effective HIV therapy needs to incorporate the knowledge that the immune system of an infected individual is dysfunctional. Figure 2 below simplifies the current knowledge of immune dysfunction and pathogenesis of HIV infection and table1(Fernandez, Lim et al. 2009). Immune dysfunction has been identified in Lymphocytes (T and B cells), NK cells, macrophages and even cytokine secretion.

Fig. 2. Pathogenesis of immune dysfunction associated with HIV (Fernandez, Lim et al. 2009)

During HIV infection, the immune system does provide a defense aimed at eradicating the infection. This mounted immune response is insufficient and allows the viral infection to impair the immune system and persist. The use of therapeutics in infected individuals is aimed at overcoming the immune systems impairment to allow for viral control leading to decrease progression to AIDS. Human studies of long term non-progressor and elite progressors have observed a slower progression to disease in these individuals (Rodes, Toro et al. 2004; Okulicz and Lambotte 2011). Long term non-progressors are characterized based on absence of disease, low viral loads, and stable or increasing CD4 T cells.(Paroli, Propato et al. 2001). There is great need for drugs and vaccine strategies that reduce viral loads in infected individuals. Development of an effective HIV therapy needs to incorporate the knowledge that the immune system of an infected individual is dysfunctional. Figure 2 below simplifies the current knowledge of immune dysfunction and pathogenesis of HIV infection and table1(Fernandez, Lim et al. 2009). Immune dysfunction has been identified in

Lymphocytes (T and B cells), NK cells, macrophages and even cytokine secretion.

Fig. 2. Pathogenesis of immune dysfunction associated with HIV (Fernandez, Lim et al. 2009)

**2.2 Expanding HIV therapy from HAART** 


Table 1. Evidence of dysfunction of the immune system of HIV infected individuals (Fernandez, Lim et al. 2009)

Current experimental therapies are grouped based on components and or modes of action, such as the HIV viral proteins targeted, the components of the immune system effected, fusion inhibitors, viral inhibitors, and HIV regulatory protein inhibitors, (Kilby 1999; Peters 2000; Pett 2009). Examples of these can be seen in Table 2.


Table 2. Some examples of Immunotherapies

HIV viral proteins used for immunotherapy include the viral envelope protein, core protein (Gag), reverse transcriptase, tat, nef, polymerase, as well as the whole killed virus (Tsoukas, Raboud et al. 1998; Gorse, Simionescu et al. 2006; Ensoli, Bellino et al. 2010). Viral proteins have been delivered as recombinant proteins, DNA vaccines, on virus-like-particles or in viral vectors (Buonaguro, Tornesello et al. 2009; Rosenberg, Graham et al. 2010). CD8+ T cells with cytolytic activity appeared to control viral titers in both non-human primate

Immunotherapies and Vaccines 237

clinically beneficial especially in late stage disease (Arno, Ruiz et al. 1999; Davey, Chaitt et al. 1999; Levy, Capitant et al. 1999). Two rIL-2 trials called SILCAAT and ESPRIT is covered

The rIL-2, termed Proleukin, was given subcutaneously in both these studies. The SILCAAT trial was designed for late stage HIV infected volunteers as defined by CD4+T cells between 50 and 299 cells/mm3 and the ESPRIT trial was designed for early stage infected individuals, defined by CD4+T cells counts above 300 cells /mm3 (Committee 2009). The endpoints for both trials were effect of treatment on disease progression and death. HAART was administered alone or in combination with Proleukin. There was a significant increase in CD4+T cell numbers in patients treated with the combination of HAART/Proleukin compared to HAART alone. Over a period of 7-8 years, there was an increase in cell numbers. Nonetheless, this increase did not translate to clinical benefits since there was not a reduction in incidence to AIDS or length of time to AIDS in individuals who received HAART/Proleukin versus HAART alone. Further analysis of these results showed that these up-regulated CD4+T cells following HAART/Proleukin treatment were different from CD4+T cells activated by HAART alone. The HAART/Proleukin treatment resulted in increased numbers of naïve and central memory CD4+T cells. Treatment with HAART alone resulted in increase of effector memory CD4+T cells. Fewer regulatory T cells were present with the HAART alone versus HAART/Proleukin treatment. Furthermore, Proleukin treatment resulted in toxicity in some individuals. Even though the Proleukin treatment increased CD4+T cell numbers, a surrogate maker for vaccine efficacy, the level of toxicity of this drug does not justify its use (Peters and Samuel 2010). Similar to the Remune trials, the use of Proleukin increased CD4+T cells a surrogate marker for vaccine efficacy. The discovery of cytokines linked to HIV immune dysfunction continue. Therefore, the use of interleukins as

therapies both directly and as adjuvants need to be carefully considered (Clerici 2010).

CD4+T cells may not lead to better disease outcome.

In addition to cytokines, other immune system components were investigated as immunotherapies. One such immune component is serum Gc factor the precursor for macrophage activating factor (MAF) (Mosser 2003). During HIV infection, gp120 prevents deglycosylation of Gc factor affecting production of MAF and results in lack of macrophage activation (Nobuto Yamamoto 2009). The use of serum Gc factor as a therapy is a potent macrophage activator and has no side effects in humans (Mosser 2003; Yamamoto, Ushijima et al. 2009). Another immune component being used is dendritic cells (DC). Dendritic cells (DC) are potent professional antigen presenting cells and are ideal for priming T cells for cytotoxic activity (Van Gulck 2010). DC primed with HIV specific antigens stimulates T cells that can destroy HIV infected cells. In addition to DC primed T cells, direct use of T cells as therapy is being investigated. The use of genetically engineered T cells with modified CCR5 receptors demonstrate that these strategies increase CD4+T cells with engineered T cells/HAART compared to HAART alone (Gulick, Lalezari et al. 2008). As seen in previous studies, the populations of CD4+T cells increased need to be investigated as increase in

Pharmacological compounds are another area of HIV therapeutic development. New targets of HV therapeutics is covered in a review by Jiang, Yan; Liu, Xinyong; De Clercq, Erik (Jiang 2011).These compounds target different stages of HIV replication cycle including viral entry, reverse transcription and viral exit. Most of the compounds initially developed were targeted at reverse transcriptase and fall under the categories of NRTI and NNRTI. Additional compounds have been developed that target other viral proteins and parts of the replication cycle. There are now compounds that target the HIV receptor CD4, the co-

by the report of Peters B. and Samuel M(Peters and Samuel 2010) .

models, as well as infected individuals with HIV (O'Connell, Bailey et al. 2009). In an effort to induce more cytotoxic T lytic (CTL) responses viral vectors, such as canarypox virus and DNA vaccination, were implemented (Kutscher, Allgayer et al. 2010; Rosario, Bridgeman et al. 2010). One example of the use of recombinant canarypox virus vaccine to deliver viral proteins is the vaccine VCP1452 (ALVAC1452). ALVAC1452 was used to carry the HIV-1 genes: gag, pol, env and nef.

#### **2.3 Remune**

Remune, is a whole killed virus with a clade A envelope and a clade G gag depleted of gp120 administered in conjunction with incomplete Freund's adjuvant. This vaccine was shown to be safe and resulted in a maintenance of CD4+T cells in volunteers in a two year follow up study (Sukeepaisarncharoen, Churdboonchart et al. 2001). Remune clinical trial is covered in detail in the article by Fernandez –Cruz et al (Fernandez-Cruz, Navarro et al. 2003). The gp120 component of the virus was removed in an effort to present the more conserved antigens to induce T cell responses. In phase II clinical trials this vaccine was combined with HAART, where Remune was administered intramuscularly every 3 months. Trial participants had a mean CD4+T cell count of 586 cells /mm3 and a mean viral load of 953 RNA copies /ml. Peripheral blood mononuclear cells (PBMC) were collected from trial participants. Following stimulation with Env gp120 depleted virus or recombinant Gag p24, PBMCs proliferate was observed in vaccine volunteers compared to a negative control group that was vaccinated against Candida. In addition, there was proliferation following HIV-specific antigen proliferation in isolated CD8+T, CD4+T and NK cells. Predominantly, memory CD4+T and CD8+T proliferated to HIV antigen stimulation.

Remune / HAART combination therapy entered phase III trials, but it was terminated after an intermediate evaluation of the data showed no significant benefit of Remune to the individuals receiving the therapeutic vaccine (Moss, Wallace et al. 1999). Additional studies using only Remune elicited significant increases in CD4+ T cells and reduced viral loads following vaccination (Fernandez-Cruz, Navarro et al. 2003). However, Remune /ALVAC did not elicit significant increases in the cytotoxic T cell activity or enhanced CD4+ T cell compared to ALVAC alone. (Angel, Routy et al. 2011). Remune did not meet criteria for evaluation by the FDA as an Immunotherapy. Although these studies with Remune did not result in licensing, these studies contributed to the knowledge of the field with a better appreciation that increases in specific T cell responses measured by *in vitro* assays do not always correlate with the efficacy of vaccination. Since increase in CD4+ T cells numbers do not correlate with better prognosis better surrogates or immune markers of vaccine efficacy are needed.

#### **2.4 Proleukin (recombinant IL-2/rIL-2)**

Another form of immunotherapy being investigated for HIV is the use of components of the immune system, including cytokines, innate cells, or T cells (Pett 2009). Investigation of cytokine levels identified IL-2 to be reduced in HIV infected individuals with greatest reduction seen in individuals who progress to disease. (Lane and Fauci 1985; Kannanganat, Kapogiannis et al. 2007). IL-2 is known to be a T cell derived cytokine needed for stimulation of cell proliferation and enhancement of cytolytic activity (Malek and Castro 2010). In the early 20th century, recombinant IL-2 (rIL-2) was administered to individuals with high and low CD4+T cell counts and low viral loads either intravenously or subcutaneously. IL-2 administration resulted in an increased numbers of CD4+T cell counts, which may be

models, as well as infected individuals with HIV (O'Connell, Bailey et al. 2009). In an effort to induce more cytotoxic T lytic (CTL) responses viral vectors, such as canarypox virus and DNA vaccination, were implemented (Kutscher, Allgayer et al. 2010; Rosario, Bridgeman et al. 2010). One example of the use of recombinant canarypox virus vaccine to deliver viral proteins is the vaccine VCP1452 (ALVAC1452). ALVAC1452 was used to carry the HIV-1

Remune, is a whole killed virus with a clade A envelope and a clade G gag depleted of gp120 administered in conjunction with incomplete Freund's adjuvant. This vaccine was shown to be safe and resulted in a maintenance of CD4+T cells in volunteers in a two year follow up study (Sukeepaisarncharoen, Churdboonchart et al. 2001). Remune clinical trial is covered in detail in the article by Fernandez –Cruz et al (Fernandez-Cruz, Navarro et al. 2003). The gp120 component of the virus was removed in an effort to present the more conserved antigens to induce T cell responses. In phase II clinical trials this vaccine was combined with HAART, where Remune was administered intramuscularly every 3 months. Trial participants had a mean CD4+T cell count of 586 cells /mm3 and a mean viral load of 953 RNA copies /ml. Peripheral blood mononuclear cells (PBMC) were collected from trial participants. Following stimulation with Env gp120 depleted virus or recombinant Gag p24, PBMCs proliferate was observed in vaccine volunteers compared to a negative control group that was vaccinated against Candida. In addition, there was proliferation following HIV-specific antigen proliferation in isolated CD8+T, CD4+T and NK cells. Predominantly,

Remune / HAART combination therapy entered phase III trials, but it was terminated after an intermediate evaluation of the data showed no significant benefit of Remune to the individuals receiving the therapeutic vaccine (Moss, Wallace et al. 1999). Additional studies using only Remune elicited significant increases in CD4+ T cells and reduced viral loads following vaccination (Fernandez-Cruz, Navarro et al. 2003). However, Remune /ALVAC did not elicit significant increases in the cytotoxic T cell activity or enhanced CD4+ T cell compared to ALVAC alone. (Angel, Routy et al. 2011). Remune did not meet criteria for evaluation by the FDA as an Immunotherapy. Although these studies with Remune did not result in licensing, these studies contributed to the knowledge of the field with a better appreciation that increases in specific T cell responses measured by *in vitro* assays do not always correlate with the efficacy of vaccination. Since increase in CD4+ T cells numbers do not correlate with better prognosis better surrogates or immune markers of vaccine efficacy

Another form of immunotherapy being investigated for HIV is the use of components of the immune system, including cytokines, innate cells, or T cells (Pett 2009). Investigation of cytokine levels identified IL-2 to be reduced in HIV infected individuals with greatest reduction seen in individuals who progress to disease. (Lane and Fauci 1985; Kannanganat, Kapogiannis et al. 2007). IL-2 is known to be a T cell derived cytokine needed for stimulation of cell proliferation and enhancement of cytolytic activity (Malek and Castro 2010). In the early 20th century, recombinant IL-2 (rIL-2) was administered to individuals with high and low CD4+T cell counts and low viral loads either intravenously or subcutaneously. IL-2 administration resulted in an increased numbers of CD4+T cell counts, which may be

memory CD4+T and CD8+T proliferated to HIV antigen stimulation.

genes: gag, pol, env and nef.

**2.3 Remune** 

are needed.

**2.4 Proleukin (recombinant IL-2/rIL-2)** 

clinically beneficial especially in late stage disease (Arno, Ruiz et al. 1999; Davey, Chaitt et al. 1999; Levy, Capitant et al. 1999). Two rIL-2 trials called SILCAAT and ESPRIT is covered by the report of Peters B. and Samuel M(Peters and Samuel 2010) .

The rIL-2, termed Proleukin, was given subcutaneously in both these studies. The SILCAAT trial was designed for late stage HIV infected volunteers as defined by CD4+T cells between 50 and 299 cells/mm3 and the ESPRIT trial was designed for early stage infected individuals, defined by CD4+T cells counts above 300 cells /mm3 (Committee 2009). The endpoints for both trials were effect of treatment on disease progression and death. HAART was administered alone or in combination with Proleukin. There was a significant increase in CD4+T cell numbers in patients treated with the combination of HAART/Proleukin compared to HAART alone. Over a period of 7-8 years, there was an increase in cell numbers. Nonetheless, this increase did not translate to clinical benefits since there was not a reduction in incidence to AIDS or length of time to AIDS in individuals who received HAART/Proleukin versus HAART alone. Further analysis of these results showed that these up-regulated CD4+T cells following HAART/Proleukin treatment were different from CD4+T cells activated by HAART alone. The HAART/Proleukin treatment resulted in increased numbers of naïve and central memory CD4+T cells. Treatment with HAART alone resulted in increase of effector memory CD4+T cells. Fewer regulatory T cells were present with the HAART alone versus HAART/Proleukin treatment. Furthermore, Proleukin treatment resulted in toxicity in some individuals. Even though the Proleukin treatment increased CD4+T cell numbers, a surrogate maker for vaccine efficacy, the level of toxicity of this drug does not justify its use (Peters and Samuel 2010). Similar to the Remune trials, the use of Proleukin increased CD4+T cells a surrogate marker for vaccine efficacy. The discovery of cytokines linked to HIV immune dysfunction continue. Therefore, the use of interleukins as therapies both directly and as adjuvants need to be carefully considered (Clerici 2010).

In addition to cytokines, other immune system components were investigated as immunotherapies. One such immune component is serum Gc factor the precursor for macrophage activating factor (MAF) (Mosser 2003). During HIV infection, gp120 prevents deglycosylation of Gc factor affecting production of MAF and results in lack of macrophage activation (Nobuto Yamamoto 2009). The use of serum Gc factor as a therapy is a potent macrophage activator and has no side effects in humans (Mosser 2003; Yamamoto, Ushijima et al. 2009). Another immune component being used is dendritic cells (DC). Dendritic cells (DC) are potent professional antigen presenting cells and are ideal for priming T cells for cytotoxic activity (Van Gulck 2010). DC primed with HIV specific antigens stimulates T cells that can destroy HIV infected cells. In addition to DC primed T cells, direct use of T cells as therapy is being investigated. The use of genetically engineered T cells with modified CCR5 receptors demonstrate that these strategies increase CD4+T cells with engineered T cells/HAART compared to HAART alone (Gulick, Lalezari et al. 2008). As seen in previous studies, the populations of CD4+T cells increased need to be investigated as increase in CD4+T cells may not lead to better disease outcome.

Pharmacological compounds are another area of HIV therapeutic development. New targets of HV therapeutics is covered in a review by Jiang, Yan; Liu, Xinyong; De Clercq, Erik (Jiang 2011).These compounds target different stages of HIV replication cycle including viral entry, reverse transcription and viral exit. Most of the compounds initially developed were targeted at reverse transcriptase and fall under the categories of NRTI and NNRTI. Additional compounds have been developed that target other viral proteins and parts of the replication cycle. There are now compounds that target the HIV receptor CD4, the co-

Immunotherapies and Vaccines 239

TORO 2 enrolled 504 volunteers with patients having had treatment with one or more of the same groups of antiretroviral (NTRI, NNRTI and PI). Volunteers in both studies had viral loads greater than 5000 copies/ml of plasma. Patients were treated with 90mg of T20 with HAART therapy or therapy alone. After 48 weeks, there was a significant drop in viral loads in the T20 plus antiretroviral therapy versus the antiretroviral treatment alone. The volunteers in the T20/HAART arm of the study had a 2-fold increase in CD4+ T cells counts from baseline compared to HAART alone that lasted greater than 96 weeks. At that point all individuals in the study were placed on T20/HAART. However the responses of the individuals originally on HAART alone never reached the levels of the individuals who received T20/HAART. This outcome indicated that T20 should be given early in treatment. T20 is now called Enfuvirtide and has been licensed by the FDA as the first fusion inhibitor

This positive step forward in the immunotherapy field fuels the continuous research to find better and safer HIV immunotherapies. Other therapies being investigated include HIV frameshift efficiency modulators, DNAzymes and the theory of alloimmunity. The first two therapies are still early in development. Alloimmunity was observed in a NHP study where animals were protected from virus challenge. After investigation it was concluded that the protection observed because the virus was grown in human PBMCs (Langlois, Weinhold et al. 1992). The use of alloimmunity in the field of HIV vaccines has expanded since the initial finding. Thomas Lehner et al published a report covering discussions during a NIH workshop on the use of alloimmunity as a strategy for HIV vaccines (Lehner, Shearer et al.

Some of the earliest HIV/AIDS vaccines were based upon the envelope protein of the virus (Lasky, Groopman et al. 1986; Arthur, Pyle et al. 1987; Redfield, Birx et al. 1991). Soluble portion of envelope (gp120) or the entire gp160 envelope protein was used as recombinant proteins to vaccinate humans and induce a humoral response (Wintsch, Chaignat et al. 1991; Pincus, Messer et al. 1993). HIV envelope subunit vaccines elicited neutralizing antibody responses following vaccination without toxic side effect in human volunteers. The first phase III clinical trial in pursuit of an effective HIV vaccine was done by VaxGen using their vaccine called AIDSVAX and consisted of a bivalent subunit recombinant gp120 envelope

After showing protection in chimpanzee after homologous and heterologous challenges, the Vaxgen vaccine moved into clinical trials. The primate study was not very well powered and complete protection was not achieved in a suboptimal HIV animal model (Berman, Gregory et al. 1990). The initial VaxGen studies were done with a monovalent vaccine either from the HIV strain MN or IIIB (Migasena, Suntharasamai et al. 2000). Both these envelopes were from lab adapted virus strains. The recombinant proteins envelope MN and IIIB gp120s were produced in engineered bacteria. The phase I and II clinical trials showed that AIDSVAX was well tolerated with irritation just at site of injection. Six individuals acquired HIV during the trial. The vaccine that was sent into phase III clinical trial was bivalent with both clade B envelopes MN and IIIB in an effort to deal with virus diversity. The vaccine trials took place in the US with a total of 5000 at-risk women and homosexual men and

to be used for HIV therapy.

2000)

**3. Prophylaxis** 

proteins (Francis DP 1998).

**3.1 Vaxgen (AIDSVAX B/B and B/E) vaccine clinical trial** 

receptors CCR5 and CXCR4, integration of virus into the host genome, and viral membrane fusion (Latinovic, Le et al. ; Ferain, Hoveyda et al. 2011).

#### **2.5 Enfurvirtide (T20)**

One of the initial fusion proteins to enter clinical trials was called T20 (Kilby 1999). Following receptor/co-receptor binding, the viral envelope mediates fusion of the cell and viral membrane via the gp41 domain of the HIV envelope. The gp41 heptad repeat sequence is responsible for membrane fusion and is highly conserved between viruses of all clades. A therapy targeted at the fusion domain should overcome the challenge of virus diversity. Initial peptides DP107 and DP178 corresponding to the heptad repeat sequences were found to inhibit viral infectivity in cell culture (Wild, Oas et al. 1992; Wild, Shugars et al. 1994). To further support the theory that disruption of the fusion domain leads to lack of virus infectivity, mutations in the supercoil/heptad repeat region was performed. These mutant viruses could not infect permissive cells. The next step in development of this drug was determining the concentration needed in vivo to inhibit membrane fusion. Based on the DP178 peptide a 36 amino acid peptide was formulated and called T20. This peptide was confirmed to have inhibitory activity *in vitro*. After *in vitro* studies, dosing and safety studies were performed using T20 followed by clinical trials using this fusion inhibitor (Wild, Greenwell et al. 1993).

Sixteen HIV-infected adult volunteers were given T20 intravenously for 14 days (Kilby, Hopkins et al. 1998). Subjects were chosen based upon CD4+ T cell counts greater or equal to 100 cells/mm3 and viral loads of 10,000 or more copies of RNA/ml of plasma. All participants were newly infected and not on therapy or were using antiretroviral therapy but ceased treatment for the trial. People were given 3mg to 100mg intravenous doses. No participants reported any adverse effects or toxicity. A few individuals had elevated temperatures and mild headaches. The drug had a half-life of 1.83 hours. Overall, there was a significant decrease in RNA plasma levels, with the fusion peptide can decrease viral infectivity and indirectly reduce viral load.

In a follow up study, increasing doses of T20 over time were used (Kilby, Lalezari et al. 2002). Volunteers with viral loads greater than 5000 copies of RNA/ml of plasma were placed on T20 therapy. Volunteers received either intermittent injection or were fitted with a device to allow for continuous drug infusion instead of intravenous dosing. The trial took place over 28 days of outpatient treatment. Adverse effects were seen in some individuals fitted with the infusion device. The pump used in treatment had frequent alarming caused tender nodules under the skin when infusion took place. Because of this side effect some individuals were taken off the pump and changed to the injection arm of the study. In the case of toxicity one individual withdrew from the study on that basis. Following 28 days of treatment, there was a dose-dependent decline in RNA levels. The best results were seen in individuals receiving intermediate dose of 30mg of T20. Potential T20 resistant-viruses showed the development of multiple point mutations in the present of T20 treatment. Therefore, several conclusion were made: 1) a more user friendly outpatient devise is needed for administering treatment, 2) understanding and characterizing possible resistant virus need to be carefully monitored and 3) correct combination of other antiretroviral therapies and fusion inhibitors need to be considered.

T20 was moved to a large phase III clinical trial performed in the United States (TORO1) and Europe/Australia (TORO2) (Joly, Jidar et al. 2010) . TORO1 consisted of 491 individuals who had more than 6 months of therapy including NRTI, NNRTI or protease inhibitors.

receptors CCR5 and CXCR4, integration of virus into the host genome, and viral membrane

One of the initial fusion proteins to enter clinical trials was called T20 (Kilby 1999). Following receptor/co-receptor binding, the viral envelope mediates fusion of the cell and viral membrane via the gp41 domain of the HIV envelope. The gp41 heptad repeat sequence is responsible for membrane fusion and is highly conserved between viruses of all clades. A therapy targeted at the fusion domain should overcome the challenge of virus diversity. Initial peptides DP107 and DP178 corresponding to the heptad repeat sequences were found to inhibit viral infectivity in cell culture (Wild, Oas et al. 1992; Wild, Shugars et al. 1994). To further support the theory that disruption of the fusion domain leads to lack of virus infectivity, mutations in the supercoil/heptad repeat region was performed. These mutant viruses could not infect permissive cells. The next step in development of this drug was determining the concentration needed in vivo to inhibit membrane fusion. Based on the DP178 peptide a 36 amino acid peptide was formulated and called T20. This peptide was confirmed to have inhibitory activity *in vitro*. After *in vitro* studies, dosing and safety studies were performed using T20 followed by clinical trials using this fusion inhibitor (Wild,

Sixteen HIV-infected adult volunteers were given T20 intravenously for 14 days (Kilby, Hopkins et al. 1998). Subjects were chosen based upon CD4+ T cell counts greater or equal to 100 cells/mm3 and viral loads of 10,000 or more copies of RNA/ml of plasma. All participants were newly infected and not on therapy or were using antiretroviral therapy but ceased treatment for the trial. People were given 3mg to 100mg intravenous doses. No participants reported any adverse effects or toxicity. A few individuals had elevated temperatures and mild headaches. The drug had a half-life of 1.83 hours. Overall, there was a significant decrease in RNA plasma levels, with the fusion peptide can decrease viral infectivity and

In a follow up study, increasing doses of T20 over time were used (Kilby, Lalezari et al. 2002). Volunteers with viral loads greater than 5000 copies of RNA/ml of plasma were placed on T20 therapy. Volunteers received either intermittent injection or were fitted with a device to allow for continuous drug infusion instead of intravenous dosing. The trial took place over 28 days of outpatient treatment. Adverse effects were seen in some individuals fitted with the infusion device. The pump used in treatment had frequent alarming caused tender nodules under the skin when infusion took place. Because of this side effect some individuals were taken off the pump and changed to the injection arm of the study. In the case of toxicity one individual withdrew from the study on that basis. Following 28 days of treatment, there was a dose-dependent decline in RNA levels. The best results were seen in individuals receiving intermediate dose of 30mg of T20. Potential T20 resistant-viruses showed the development of multiple point mutations in the present of T20 treatment. Therefore, several conclusion were made: 1) a more user friendly outpatient devise is needed for administering treatment, 2) understanding and characterizing possible resistant virus need to be carefully monitored and 3) correct combination of other antiretroviral

T20 was moved to a large phase III clinical trial performed in the United States (TORO1) and Europe/Australia (TORO2) (Joly, Jidar et al. 2010) . TORO1 consisted of 491 individuals who had more than 6 months of therapy including NRTI, NNRTI or protease inhibitors.

fusion (Latinovic, Le et al. ; Ferain, Hoveyda et al. 2011).

**2.5 Enfurvirtide (T20)** 

Greenwell et al. 1993).

indirectly reduce viral load.

therapies and fusion inhibitors need to be considered.

TORO 2 enrolled 504 volunteers with patients having had treatment with one or more of the same groups of antiretroviral (NTRI, NNRTI and PI). Volunteers in both studies had viral loads greater than 5000 copies/ml of plasma. Patients were treated with 90mg of T20 with HAART therapy or therapy alone. After 48 weeks, there was a significant drop in viral loads in the T20 plus antiretroviral therapy versus the antiretroviral treatment alone. The volunteers in the T20/HAART arm of the study had a 2-fold increase in CD4+ T cells counts from baseline compared to HAART alone that lasted greater than 96 weeks. At that point all individuals in the study were placed on T20/HAART. However the responses of the individuals originally on HAART alone never reached the levels of the individuals who received T20/HAART. This outcome indicated that T20 should be given early in treatment. T20 is now called Enfuvirtide and has been licensed by the FDA as the first fusion inhibitor to be used for HIV therapy.

This positive step forward in the immunotherapy field fuels the continuous research to find better and safer HIV immunotherapies. Other therapies being investigated include HIV frameshift efficiency modulators, DNAzymes and the theory of alloimmunity. The first two therapies are still early in development. Alloimmunity was observed in a NHP study where animals were protected from virus challenge. After investigation it was concluded that the protection observed because the virus was grown in human PBMCs (Langlois, Weinhold et al. 1992). The use of alloimmunity in the field of HIV vaccines has expanded since the initial finding. Thomas Lehner et al published a report covering discussions during a NIH workshop on the use of alloimmunity as a strategy for HIV vaccines (Lehner, Shearer et al. 2000)

## **3. Prophylaxis**

Some of the earliest HIV/AIDS vaccines were based upon the envelope protein of the virus (Lasky, Groopman et al. 1986; Arthur, Pyle et al. 1987; Redfield, Birx et al. 1991). Soluble portion of envelope (gp120) or the entire gp160 envelope protein was used as recombinant proteins to vaccinate humans and induce a humoral response (Wintsch, Chaignat et al. 1991; Pincus, Messer et al. 1993). HIV envelope subunit vaccines elicited neutralizing antibody responses following vaccination without toxic side effect in human volunteers. The first phase III clinical trial in pursuit of an effective HIV vaccine was done by VaxGen using their vaccine called AIDSVAX and consisted of a bivalent subunit recombinant gp120 envelope proteins (Francis DP 1998).

### **3.1 Vaxgen (AIDSVAX B/B and B/E) vaccine clinical trial**

After showing protection in chimpanzee after homologous and heterologous challenges, the Vaxgen vaccine moved into clinical trials. The primate study was not very well powered and complete protection was not achieved in a suboptimal HIV animal model (Berman, Gregory et al. 1990). The initial VaxGen studies were done with a monovalent vaccine either from the HIV strain MN or IIIB (Migasena, Suntharasamai et al. 2000). Both these envelopes were from lab adapted virus strains. The recombinant proteins envelope MN and IIIB gp120s were produced in engineered bacteria. The phase I and II clinical trials showed that AIDSVAX was well tolerated with irritation just at site of injection. Six individuals acquired HIV during the trial. The vaccine that was sent into phase III clinical trial was bivalent with both clade B envelopes MN and IIIB in an effort to deal with virus diversity. The vaccine trials took place in the US with a total of 5000 at-risk women and homosexual men and

Immunotherapies and Vaccines 241

regimens that had proved successful in primates to stimulate a cellular response is a DNA prime followed by protein or viral vector boost (Barnett, Burke et al. 2010; Jaoko, Karita et al.

The Merck vaccine consisted of three recombinant adenoviral vectors of different serotype expressing the genes *gag, pol and nef* (Shiver, Fu et al. 2002). Preclinical trials in macaques vaccinated with the modified Ad5 expressing gag, pol and nef showed immunogenicity. When monkeys were challenge with SHIV and SIVmac239, animals had reduced viral loads. Further analysis identified the animal's HLA type as a major factor in the outcome of vaccination and efficacy seen. With reduced viral loads in the primate model after challenge the vaccine was moved into clinical trials. During clinical trials phase I and II the vaccine was safe and induce cellular immune responses measured by interferon gamma enzymelinked assay. Interferon gamma enzyme-linked assay (INF gamma ELISPOT) used is the standardize assay for cellular responses in a vaccine setting. STEP phase III trial enrolled a total of 3,000 healthy individuals. The endpoints for the STEP trial as with the VaxGen study were infection and viral load. Each volunteer received three injections of the three genes and received vaccinations two and three 6 months apart from each other. The STEP trial was stop after analysis of data collected from the ongoing study. The study was stopped due to the results pointing to increase rate of infectivity in vaccine groups. This outcome was unexpected as the volunteers in the vaccine arm of the study had quality cellular responses. Quality cellular responses were defined by moderate to high total INF gamma producing cells ELISPOT and the CD8+T cells generated were polyfunctional by ICS staining after stimulation with HIV antigens. The polyfunctional nature of the CD8+T cells was thought to be needed for the ideal response to viral load (Betts and Harari 2008; Hanke 2008). However, the STEP results had an additional element that complicated the outcome. Volunteers who had pre-existing immunity to Ad5 had a trend for higher rates of infectivity (Sekaly 2008).The failure of the STEP trial brought into scrutiny both the use of viral vectors and cellular responses as a correlate of protection. To overcome the hurdle of pre-existing immunity ways to modifying vector delivery and engineering vectors has been under

After the failure of the STEP vaccine trial, the results of the next phase III vaccine trial was of great interest. The vaccine components had a potential for inducing both cellular and humoral responses. Inducing both humoral and cellular responses to HIV antigens had been investigated as early as 1986 when recombinant vaccinia virus was used to delivery envelope gp41 and gpIIO induced both humoural and cellular responses in macaques

The ALVAC and AIDSVAX clinical trial in Thailand enrolled 16402 healthy men and women between the ages of 18-30 years into the study(Rerks-Ngarm, Pitisuttithum et al. 2009). The ALVAC vaccine contains a clade E envelope and a gag/pol from clade B. The AIDSVAX vaccine is the B/E vaccine covered in the previous section. The vaccine trial covered multiple centers and the individuals were randomized into placebo or vaccine groups. Vaccine groups received four injection of the canarypox virus vector vaccine ALVAC, followed by two boost injections with the AIDSVAX B/E recombinant gp120. The endpoints for the trials were HIV infection and early viral loads after the first 6 months and

2010; Keefer, Frey et al. 2011).

**3.2 Merck clinical trial** 

investigation.

(Zarling, Morton et al. 1986).

**3.3 ALVAC and AIDSVAX clinical trial** 

parallel studies were conducted in Thailand with intravenous drug users (Francis DP 1998). The vaccines were tailored to the trial sites. In the US, the vaccine consisted of envelopes from clade B. The Thailand vaccine was made from envelopes from clade B and E. Trials were powered to determine efficacy and were scheduled for three years allowing for longterm follow up. The endpoints designated for the trials were infection as measured by seroconversion and viral load as measured by polymerase chain reaction. In 2003, VaxGen reported the failure of its vaccine trial (Profile 2003). There was no significant decrease in infection in individuals who received the vaccine when all individuals were considered. However, the company reported that the vaccine was more immunogenic and produced higher levels of antibody responses in Black and Asian volunteers. Because of this finding, the AIDSVAX was included in a future study in combination with ALVAC-HIV-vCP1521 in Thailand.

Since the beginning of the VaxGen trials, HIV prophylaxis vaccines designed to elicit humoral response have increased in sophistication. Vaccine designs used to induce effective neutralizing antibodies is covered in a review by Vaine et al.(Vaine, Lu et al. 2009). Vaccine designs include use of envelopes with variable loops deleted, glycosylation mutated, eptitope grafting, envelope trimers and centralized sequences. Our lab has been involved in studies to increase antibody response to envelope. The initial studies performed used the molecular adjuvant in combination with sgp120 to increase antibody titers (Green, Montefiori et al. 2003). Results from that study showed that DNA vaccination with sgp120 linked to three copies of C3d efficiently increase antibody tiers in rabbits compared to non-C3d constructs. This work was expanded to link C3d3 to a more native envelope structure. Trimerize envelopes stabilized by the bacteriophage fibritin and linked to C3d showed better cross neutralizing titers to primary isolates compared to trimers without C3d. Both groups of mice vaccinated with envelope trimers with and without C3d induced high antienvelope responses (Bower, Yang et al. 2004). Our lab has also used virus-like particles (VLP) in an effort to present envelope in its native form(Young, Smith et al. 2004). Responses of mice vaccinated with the HIV VLP when compared to soluble gp120 or trimers had higher envelope titers and broader immune responses (McBurney, Young et al. 2007). The VLP induced both mucosal and systemic responses.

In parallel with developing vaccines to eliciting antibody responses vaccines aimed at eliciting T cell responses were being developed (Egan, Pavlat et al. 1995). Both animal and human studies have indicated that CD8+T cells were linked to reduce viral load and led to development of vaccines aimed at producing the ideal cellular responses (Asquith and McLean 2007). Several viral vectors have been used to elicit cellular responses to HIV proteins including poxvirus vectors, vaccinia virus vectors, adenovirus vectors, alphaviruses vectors, avipoxvirus vectors, poliovirus vectors and rhabdovirus vectors (Polo and Dubensky 2002). Viral vectors allow for 1) high production levels of antigens directly into cells, 2) potential adjuvant effect on the immune response given the viral nature of the system and 3) the particular characteristic allows for efficient uptake of vaccine by professional antigen presenting cells to stimulate the immune response. Viral vectors can be mucosal adjuvants. Mucosal stimulation is an asset in HIV vaccine development as most infection takes place via the mucosa (Chenine, Siddappa et al. 2010). There is a major disadvantage to viral vectors, the possibility of pre-existing immunity may lead to adverse effects and reduced immune response directed at the vaccine antigens (Gudmundsdotter, Nilsson et al. 2009; Pine, Kublin et al. 2011). Other strategies that have been investigated for inducing a cellular response include using various vaccine regimens. One of the vaccine

parallel studies were conducted in Thailand with intravenous drug users (Francis DP 1998). The vaccines were tailored to the trial sites. In the US, the vaccine consisted of envelopes from clade B. The Thailand vaccine was made from envelopes from clade B and E. Trials were powered to determine efficacy and were scheduled for three years allowing for longterm follow up. The endpoints designated for the trials were infection as measured by seroconversion and viral load as measured by polymerase chain reaction. In 2003, VaxGen reported the failure of its vaccine trial (Profile 2003). There was no significant decrease in infection in individuals who received the vaccine when all individuals were considered. However, the company reported that the vaccine was more immunogenic and produced higher levels of antibody responses in Black and Asian volunteers. Because of this finding, the AIDSVAX was included in a future study in combination with ALVAC-HIV-vCP1521 in

Since the beginning of the VaxGen trials, HIV prophylaxis vaccines designed to elicit humoral response have increased in sophistication. Vaccine designs used to induce effective neutralizing antibodies is covered in a review by Vaine et al.(Vaine, Lu et al. 2009). Vaccine designs include use of envelopes with variable loops deleted, glycosylation mutated, eptitope grafting, envelope trimers and centralized sequences. Our lab has been involved in studies to increase antibody response to envelope. The initial studies performed used the molecular adjuvant in combination with sgp120 to increase antibody titers (Green, Montefiori et al. 2003). Results from that study showed that DNA vaccination with sgp120 linked to three copies of C3d efficiently increase antibody tiers in rabbits compared to non-C3d constructs. This work was expanded to link C3d3 to a more native envelope structure. Trimerize envelopes stabilized by the bacteriophage fibritin and linked to C3d showed better cross neutralizing titers to primary isolates compared to trimers without C3d. Both groups of mice vaccinated with envelope trimers with and without C3d induced high antienvelope responses (Bower, Yang et al. 2004). Our lab has also used virus-like particles (VLP) in an effort to present envelope in its native form(Young, Smith et al. 2004). Responses of mice vaccinated with the HIV VLP when compared to soluble gp120 or trimers had higher envelope titers and broader immune responses (McBurney, Young et al. 2007). The

In parallel with developing vaccines to eliciting antibody responses vaccines aimed at eliciting T cell responses were being developed (Egan, Pavlat et al. 1995). Both animal and human studies have indicated that CD8+T cells were linked to reduce viral load and led to development of vaccines aimed at producing the ideal cellular responses (Asquith and McLean 2007). Several viral vectors have been used to elicit cellular responses to HIV proteins including poxvirus vectors, vaccinia virus vectors, adenovirus vectors, alphaviruses vectors, avipoxvirus vectors, poliovirus vectors and rhabdovirus vectors (Polo and Dubensky 2002). Viral vectors allow for 1) high production levels of antigens directly into cells, 2) potential adjuvant effect on the immune response given the viral nature of the system and 3) the particular characteristic allows for efficient uptake of vaccine by professional antigen presenting cells to stimulate the immune response. Viral vectors can be mucosal adjuvants. Mucosal stimulation is an asset in HIV vaccine development as most infection takes place via the mucosa (Chenine, Siddappa et al. 2010). There is a major disadvantage to viral vectors, the possibility of pre-existing immunity may lead to adverse effects and reduced immune response directed at the vaccine antigens (Gudmundsdotter, Nilsson et al. 2009; Pine, Kublin et al. 2011). Other strategies that have been investigated for inducing a cellular response include using various vaccine regimens. One of the vaccine

VLP induced both mucosal and systemic responses.

Thailand.

regimens that had proved successful in primates to stimulate a cellular response is a DNA prime followed by protein or viral vector boost (Barnett, Burke et al. 2010; Jaoko, Karita et al. 2010; Keefer, Frey et al. 2011).

## **3.2 Merck clinical trial**

The Merck vaccine consisted of three recombinant adenoviral vectors of different serotype expressing the genes *gag, pol and nef* (Shiver, Fu et al. 2002). Preclinical trials in macaques vaccinated with the modified Ad5 expressing gag, pol and nef showed immunogenicity. When monkeys were challenge with SHIV and SIVmac239, animals had reduced viral loads. Further analysis identified the animal's HLA type as a major factor in the outcome of vaccination and efficacy seen. With reduced viral loads in the primate model after challenge the vaccine was moved into clinical trials. During clinical trials phase I and II the vaccine was safe and induce cellular immune responses measured by interferon gamma enzymelinked assay. Interferon gamma enzyme-linked assay (INF gamma ELISPOT) used is the standardize assay for cellular responses in a vaccine setting. STEP phase III trial enrolled a total of 3,000 healthy individuals. The endpoints for the STEP trial as with the VaxGen study were infection and viral load. Each volunteer received three injections of the three genes and received vaccinations two and three 6 months apart from each other. The STEP trial was stop after analysis of data collected from the ongoing study. The study was stopped due to the results pointing to increase rate of infectivity in vaccine groups. This outcome was unexpected as the volunteers in the vaccine arm of the study had quality cellular responses. Quality cellular responses were defined by moderate to high total INF gamma producing cells ELISPOT and the CD8+T cells generated were polyfunctional by ICS staining after stimulation with HIV antigens. The polyfunctional nature of the CD8+T cells was thought to be needed for the ideal response to viral load (Betts and Harari 2008; Hanke 2008). However, the STEP results had an additional element that complicated the outcome. Volunteers who had pre-existing immunity to Ad5 had a trend for higher rates of infectivity (Sekaly 2008).The failure of the STEP trial brought into scrutiny both the use of viral vectors and cellular responses as a correlate of protection. To overcome the hurdle of pre-existing immunity ways to modifying vector delivery and engineering vectors has been under investigation.

After the failure of the STEP vaccine trial, the results of the next phase III vaccine trial was of great interest. The vaccine components had a potential for inducing both cellular and humoral responses. Inducing both humoral and cellular responses to HIV antigens had been investigated as early as 1986 when recombinant vaccinia virus was used to delivery envelope gp41 and gpIIO induced both humoural and cellular responses in macaques (Zarling, Morton et al. 1986).

#### **3.3 ALVAC and AIDSVAX clinical trial**

The ALVAC and AIDSVAX clinical trial in Thailand enrolled 16402 healthy men and women between the ages of 18-30 years into the study(Rerks-Ngarm, Pitisuttithum et al. 2009). The ALVAC vaccine contains a clade E envelope and a gag/pol from clade B. The AIDSVAX vaccine is the B/E vaccine covered in the previous section. The vaccine trial covered multiple centers and the individuals were randomized into placebo or vaccine groups. Vaccine groups received four injection of the canarypox virus vector vaccine ALVAC, followed by two boost injections with the AIDSVAX B/E recombinant gp120. The endpoints for the trials were HIV infection and early viral loads after the first 6 months and

Immunotherapies and Vaccines 243

Infectious Diseases (NIAID) are ran by different clinical networks. The clinical networks include: 1) AIDS clinical trials groups, 2) HIV prevention trials network, 3) HIV vaccine trials Network 4) International maternal pediatric Adolescent AIDS Clinical trials, 5) International network of strategic initiatives in global HIV trials and 6) Microbicide trials network volunteers. Ongoing clinical trials can be found at the AIDSinfo website

In therapeutic research, studies are expanding to include drugs targeted at eliminating viral reservoirs (Huelsmann, Hofmann et al. 2011; Kovochich, Marsden et al. 2011). Follow up studies of individuals on HAART are being done to evaluating health and treatment efficacy (Mahdavi, Malyuta et al. 2010; Torti, d'Arminio-Monforte et al. 2011). Findings from these evaluations will be use to better treatments and evaluate possible side effects of long term drug use (Boyd and Hill 2010; Kranzer, Lewis et al. 2010; Shapiro, Hughes et al. 2010; Shrestha, Sudenga et al. 2010). Another area of HIV therapy receiving increase attention is the use and effectiveness of HAART in individuals with cancer and bacterial/virus coinfections such as tuberculosis, HPV (Crane, Sirivichayakul et al. 2010; Hermans, Kiragga et

New strategies or modification of old strategies are being used for vaccine development as more knowledge of the virus and the immune response to the virus continue to be dissected. Table 3 shows vaccine strategies used over the years and their limitations. One example is seen in a paper by Somogyi, E., J. Xu, et al where a VLP is used to present 15 antigens(Somogyi, Xu et al. 2011). While this vaccine is design for therapeutic purposes the platform could be applied to prophylaxis vaccines as well. As with therapeutics, clinical trials for preventative vaccines are continuous taking place. The trials that are currently being done by HIV vaccine network can be found at

Restricted specificity of neutralizing antibodies, absence

No neutralizing antibodies for patient isolates of HIV-1;

Dissemination in immunosuppressed vaccines

Limited immunogenicity in humans at achievable

Limited experience in humans

(http://www.aidsinfo.nih.gov/Vaccines ).

al. 2010; Minkoff, Zhong et al. 2010).

http://www.hvtn.org/science/trials.html.

Inactivated viruses with

Recombinant envelope

Alphaviruses, adeno-

Envelope subunit

Letvin (Letvin 2002)

Live, recombinant vectors:

adjuvants

Poxviruses Vaccinia MVA, NYVAC Canary pox

protein

**Vaccine Design Limitations** 

Live, attenuated virus Pathogenicity in vaccines

of CTLs

dosages

immunogens No elicitation of neutralizing antibodies

associated virus Limited experience in humans

Gene-deleted adenovirus Pre-existing immunity to adenovirus may limit immunogenicity

Table 3. HIV-1 vaccine design adapted from" Strategies for an HIV vaccine" by Norman L.

Plasmid DNA Limited immunogenicity in humans

absence of CTLs

every 6 months thereafter for 3 years. Measurement of cellular immunogenicity was done by interferon gamma ELISPOT and intracellular cytokine staining (ICS) for antigens gag and envelope. Humoral responses were measured for binding antibodies to various gp120 envelopes and p24 (gag core). T cell responses via ELISPOT showed a 19.7 % in vaccinated individuals 6 months after the final vaccination. In addition greater cytokine responses were measured in the CD4+ T cells of vaccinated individuals. Binding antibodies to the envelopes MN and A244 present in the vaccine were similar and had a GMT-1 of 31,207 and 14588 respectively. There were only mild to moderate adverse effects mainly at the site of injection as in preliminary studies. When it came to trial endpoints there was no significant difference in viral loads of individuals who got infected whether or not they got the vaccine. Nonetheless there was a silver lining, the study recorded a 31.3% protection rate using a 95%confidence interval. This outcome resulted in ripples across the world. This was the first time any efficacy was reported in an HIV vaccine trial. However, a study did not result in the elucidation of a correlate of protection for HIV. The only immune parameter measured that should any potential as a correlate of protection was antibody binding to envelopes. This vaccine trial infused a new hope into the HIV vaccine field, showing that protection from infection was possible.

Besides establishing correlates of protection the HIV vaccine field has other hurdles to overcome. These challenges include (Moutsopoulos, Nares et al. 2007)vaccine design to overcoming variability and induce the appropriate immune response at the mucosal surface. The two main strategies being used to overcome virus variability are using centralized sequence usually based on envelopes of one or multiple clades, mosaic antigens and polyvalent vaccines consisting of multiple genes of HIV from one or multiple clades (McBurney and Ross 2008) (Santra, Korber et al. 2008; McElrath and Haynes 2010). Our lab have used consensus envelopes in an effort to expand HIV immune responses breath when compared to monovalent vaccines or a polyvalent primary envelope VLP mixture (McBurney and Ross 2009). In a review by Gao F et al. the use of centralized envelopes (consensus, center of the tree and ancestral) to induce HIV specific immune responses is covered (Gao 2007). The use of the centralized immunogens resulted in a superior breadth of cellular and humoral immune response (Kothe, Li et al. 2006; Liao, Sutherland et al. 2006; Kothe, Decker et al. 2007; Santra, Korber et al. 2008). In regard to generating mucosal immunity different vaccine strategies including vaccination at oral or vaginal in primates and use of adjuvants are being investigated to establish protective immune response at the mucosa (Peters, Peng et al. 2003; Duerr 2010; Sui, Zhu et al. 2010). To better direct the mucosal vaccine development investigation of the immune environment during infection and what is needed to prevent infection is being done (Gurney, Elliott et al. 2005; Moutsopoulos, Nares et al. 2007; Burgener, Boutilier et al. 2008; Schulbin, Bode et al. 2008).

#### **4. Moving forward**

Collaborative efforts between basic research, pharmacology, vaccinology and immunology are moving the HIV search for treatment forward. Trials aimed at investigating new drugs and therapeutic vaccines are being done worldwide (Choudhary and Margolis 2011). WHO and world governments continue to devote money to clinical trials for potential preventative measures in an effort to curb the HIV/AIDS global epidemic. The phase II and III clinical trials of preventative vaccines sponsored by National Institute of Allergy and

every 6 months thereafter for 3 years. Measurement of cellular immunogenicity was done by interferon gamma ELISPOT and intracellular cytokine staining (ICS) for antigens gag and envelope. Humoral responses were measured for binding antibodies to various gp120 envelopes and p24 (gag core). T cell responses via ELISPOT showed a 19.7 % in vaccinated individuals 6 months after the final vaccination. In addition greater cytokine responses were measured in the CD4+ T cells of vaccinated individuals. Binding antibodies to the envelopes MN and A244 present in the vaccine were similar and had a GMT-1 of 31,207 and 14588 respectively. There were only mild to moderate adverse effects mainly at the site of injection as in preliminary studies. When it came to trial endpoints there was no significant difference in viral loads of individuals who got infected whether or not they got the vaccine. Nonetheless there was a silver lining, the study recorded a 31.3% protection rate using a 95%confidence interval. This outcome resulted in ripples across the world. This was the first time any efficacy was reported in an HIV vaccine trial. However, a study did not result in the elucidation of a correlate of protection for HIV. The only immune parameter measured that should any potential as a correlate of protection was antibody binding to envelopes. This vaccine trial infused a new hope into the HIV vaccine field, showing that

Besides establishing correlates of protection the HIV vaccine field has other hurdles to overcome. These challenges include (Moutsopoulos, Nares et al. 2007)vaccine design to overcoming variability and induce the appropriate immune response at the mucosal surface. The two main strategies being used to overcome virus variability are using centralized sequence usually based on envelopes of one or multiple clades, mosaic antigens and polyvalent vaccines consisting of multiple genes of HIV from one or multiple clades (McBurney and Ross 2008) (Santra, Korber et al. 2008; McElrath and Haynes 2010). Our lab have used consensus envelopes in an effort to expand HIV immune responses breath when compared to monovalent vaccines or a polyvalent primary envelope VLP mixture (McBurney and Ross 2009). In a review by Gao F et al. the use of centralized envelopes (consensus, center of the tree and ancestral) to induce HIV specific immune responses is covered (Gao 2007). The use of the centralized immunogens resulted in a superior breadth of cellular and humoral immune response (Kothe, Li et al. 2006; Liao, Sutherland et al. 2006; Kothe, Decker et al. 2007; Santra, Korber et al. 2008). In regard to generating mucosal immunity different vaccine strategies including vaccination at oral or vaginal in primates and use of adjuvants are being investigated to establish protective immune response at the mucosa (Peters, Peng et al. 2003; Duerr 2010; Sui, Zhu et al. 2010). To better direct the mucosal vaccine development investigation of the immune environment during infection and what is needed to prevent infection is being done (Gurney, Elliott et al. 2005; Moutsopoulos, Nares et al. 2007; Burgener, Boutilier et al.

Collaborative efforts between basic research, pharmacology, vaccinology and immunology are moving the HIV search for treatment forward. Trials aimed at investigating new drugs and therapeutic vaccines are being done worldwide (Choudhary and Margolis 2011). WHO and world governments continue to devote money to clinical trials for potential preventative measures in an effort to curb the HIV/AIDS global epidemic. The phase II and III clinical trials of preventative vaccines sponsored by National Institute of Allergy and

protection from infection was possible.

2008; Schulbin, Bode et al. 2008).

**4. Moving forward** 

Infectious Diseases (NIAID) are ran by different clinical networks. The clinical networks include: 1) AIDS clinical trials groups, 2) HIV prevention trials network, 3) HIV vaccine trials Network 4) International maternal pediatric Adolescent AIDS Clinical trials, 5) International network of strategic initiatives in global HIV trials and 6) Microbicide trials network volunteers. Ongoing clinical trials can be found at the AIDSinfo website (http://www.aidsinfo.nih.gov/Vaccines ).

In therapeutic research, studies are expanding to include drugs targeted at eliminating viral reservoirs (Huelsmann, Hofmann et al. 2011; Kovochich, Marsden et al. 2011). Follow up studies of individuals on HAART are being done to evaluating health and treatment efficacy (Mahdavi, Malyuta et al. 2010; Torti, d'Arminio-Monforte et al. 2011). Findings from these evaluations will be use to better treatments and evaluate possible side effects of long term drug use (Boyd and Hill 2010; Kranzer, Lewis et al. 2010; Shapiro, Hughes et al. 2010; Shrestha, Sudenga et al. 2010). Another area of HIV therapy receiving increase attention is the use and effectiveness of HAART in individuals with cancer and bacterial/virus coinfections such as tuberculosis, HPV (Crane, Sirivichayakul et al. 2010; Hermans, Kiragga et al. 2010; Minkoff, Zhong et al. 2010).

New strategies or modification of old strategies are being used for vaccine development as more knowledge of the virus and the immune response to the virus continue to be dissected. Table 3 shows vaccine strategies used over the years and their limitations. One example is seen in a paper by Somogyi, E., J. Xu, et al where a VLP is used to present 15 antigens(Somogyi, Xu et al. 2011). While this vaccine is design for therapeutic purposes the platform could be applied to prophylaxis vaccines as well. As with therapeutics, clinical trials for preventative vaccines are continuous taking place. The trials that are currently being done by HIV vaccine network can be found at http://www.hvtn.org/science/trials.html.


Table 3. HIV-1 vaccine design adapted from" Strategies for an HIV vaccine" by Norman L. Letvin (Letvin 2002)

Immunotherapies and Vaccines 245

Barre-Sinoussi, F., J. C. Chermann, et al. (1983). "Isolation of a T-Lymphotropic Retrovirus

Berman, P. W., T. J. Gregory, et al. (1990). "Protection of chimpanzees from infection by HIV-

Betts, M. R. and A. Harari (2008). "Phenotype and function of protective T cell immune

Bower, J. F., X. Yang, et al. (2004). "Elicitation of Neutralizing Antibodies with DNA

PharmacoEconomics 28: 17-34 10.2165/11587420-000000000-000000000. Brinckmann, S., K. da Costa, et al. (2011). "Rational design of HIV vaccines and microbicides:

Broder, S. (2010). "The development of antiretroviral therapy and its impact on the HIV-

Brown, R. J. P., P. J. Peters, et al. (2011). "Inter-compartment recombination of HIV-1

Buonaguro, L., M. L. Tornesello, et al. (2009). "Short Communication: Limited Induction of

Burgener, A., J. Boutilier, et al. (2008). "Identification of Differentially Expressed Proteins in

Carter, C. C., A. Onafuwa-Nuga, et al. (2010). "HIV-1 infects multipotent progenitor cells

Cecchinato, V., C. J. Trindade, et al. (2008). "Altered balance between Th17 and Th1 cells at

Chenine, A. L., N. B. Siddappa, et al. (2010). "Relative Transmissibility of an R5 Clade C

Chirenje, Z. M., J. Marrazzo, et al. (2010). "Antiretroviral-based HIV prevention strategies for

Choudhary, S. K. and D. M. Margolis (2011). "Curing HIV: Pharmacologic Approaches to

women." Expert Review of Anti-infective Therapy 8(10): 1177-1186.

220(4599): 868-871.

345(6276): 622-625.

310.1097/COH.1090b1013e3282fbaa1081.

Translational Medicine 9(1): 40.

Research 7(10): 4446-4454.

451.

418.

1/AIDS pandemic." Antiviral Research 85(1): 1-18.

Research and Human Retroviruses 25(8): 819-822.

infected macaques." Mucosal Immunol 1(4): 279-288.

Routes." Journal of Infectious Diseases 201(8): 1155-1163.

to entry inhibitors." J. Virol.: JVI.00131-00111.

from a Patient at Risk for Acquired Immune Deficiency Syndrome (AIDS)." Science

1 after vaccination with recombinant glycoprotein gp120 but not gp160." Nature

responses in HIV." Current Opinion in HIV and AIDS 3(3): 349-355

Vaccines Expressing Soluble Stabilized Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Trimers Conjugated to C3d." J. Virol. 78(9): 4710-4719. Boyd, M. A. and A. M. Hill (2010). "Clinical Management of Treatment-Experienced,

HIV/AIDS Patients in the Combination Antiretroviral Therapy Era."

report of the EUROPRISE network annual conference 2010." Journal of

contributes to env intra-host diversity and modulates viral tropism and senstivity

IL-10 in PBMCs from HIV-Infected Subjects Treated with HIV-VLPs." AIDS

the Cervical Mucosa of HIV-1-Resistant Sex Workers." Journal of Proteome

causing cell death and establishing latent cellular reservoirs." Nat Med 16(4): 446-

mucosal sites predicts AIDS progression in simian immunodeficiency virus-

Simian- Human Immunodeficiency Virus Across Different Mucosae in Macaques Parallels the Relative Risks of Sexual HIV-1 Transmission in Humans via Different

Target HIV-1 Latency." Annual Review of Pharmacology and Toxicology 51(1): 397-

Besides the strategies seen in the table prophylaxis treatment being investigated are topical microbicides, combination therapy of vaccines and microbicides and the use of antivirals as preventative treatment (PrEP) (Chirenje, Marrazzo et al. 2010; Mayer and Venkatesh 2010; Brinckmann, da Costa et al. 2011; Oh, Price et al. 2011). In areas where women are prohibited from use of condoms the use of effective microbicides would allow these women to protect themselves from infection. In the case of microbicides time of application is an essential part of evaluation.

The use of antiviral as post exposure treatment for healthcare workers is also under investigation. PrEP has been expanded to other individuals, including men who have sex with men. A national clinical trial was completed and results of trial were reported this year (2011). The PrEP was safe and resulted in partial efficiency in reducing HIV acquisition (DK Smith 2011). One concern of PrEP is the development of drug resistant viruses due to therapy prior to infection (Abbas, Hood et al. 2011).

The war against HIV continues to be fought with scientific innovation together with continued funding from both government and private agencies. With such continued efforts the road to epidemic control and /eradication may be closer than it was over twenty- five years ago.

## **5. References**


Besides the strategies seen in the table prophylaxis treatment being investigated are topical microbicides, combination therapy of vaccines and microbicides and the use of antivirals as preventative treatment (PrEP) (Chirenje, Marrazzo et al. 2010; Mayer and Venkatesh 2010; Brinckmann, da Costa et al. 2011; Oh, Price et al. 2011). In areas where women are prohibited from use of condoms the use of effective microbicides would allow these women to protect themselves from infection. In the case of microbicides time of application is an

The use of antiviral as post exposure treatment for healthcare workers is also under investigation. PrEP has been expanded to other individuals, including men who have sex with men. A national clinical trial was completed and results of trial were reported this year (2011). The PrEP was safe and resulted in partial efficiency in reducing HIV acquisition (DK Smith 2011). One concern of PrEP is the development of drug resistant viruses due to

The war against HIV continues to be fought with scientific innovation together with continued funding from both government and private agencies. With such continued efforts the road to epidemic control and /eradication may be closer than it was over twenty- five

Aaron N. Endsley, N. N. S., Rodney J.Y. Ho (2008). "Combining Drug and Immune Therapy:

Abbas, U. L., G. Hood, et al. (2011). "Factors Influencing the Emergence and Spread of HIV

Angel, J. B. a., J.-P. b. Routy, et al. (2011). "A randomized controlled trial of HIV therapeutic vaccination using ALVAC with or without Remune." AIDS 25(6): 731-739. Arno, A., L. Ruiz, et al. (1999). "Efficacy of Low-Dose Subcutaneous Interleukin-2 to Treat

Arthur, L. O., S. W. Pyle, et al. (1987). "Serological responses in chimpanzees inoculated with

Asquith, B. and A. R. McLean (2007). "In vivo CD8+ T cell control of immunodeficiency

Baenziger, S., M. Heikenwalder, et al. (2009). "Triggering TLR7 in mice induces immune

Barnett, S. W., B. Burke, et al. (2010). "Antibody-Mediated Protection against Mucosal

Proceedings of the National Academy of Sciences 84(23): 8583-8587.

A Potential Solution to Drug Resistance and Challenges of HIV Vaccines?" Current

Drug Resistance Arising from Rollout of Antiretroviral Pre-Exposure Prophylaxis

Advanced Human Immunodeficiency Virus Type 1 in Persons with '©Ω250/μL CD4 T Cells and Undetectable Plasma Virus Load." Journal of Infectious Diseases

human immunodeficiency virus glycoprotein (gp120) subunit vaccine."

virus infection in humans and macaques." Proceedings of the National Academy of

activation and lymphoid system disruption, resembling HIV-mediated pathology."

Simian-Human Immunodeficiency Virus Challenge of Macaques Immunized with Alphavirus Replicon Particles and Boosted with Trimeric Envelope Glycoprotein in

essential part of evaluation.

years ago.

**5. References** 

therapy prior to infection (Abbas, Hood et al. 2011).

HIV Research 6: 401-410.

180(1): 56-60.

(PrEP)." PLoS ONE 6(4): e18165.

Sciences 104(15): 6365-6370.

MF59 Adjuvant." J. Virol. 84(12): 5975-5985.

Blood 113(2): 377-388.


Immunotherapies and Vaccines 247

Douek, D. C., J. M. Brenchley, et al. (2002). "HIV preferentially infects HIV-specific CD4+ T

Duerr, A. (2010). "Update on mucosal HIV vaccine vectors." Current Opinion in HIV and

Egan, M. A., W. A. Pavlat, et al. (1995). "Induction of Human Immunodeficiency Virus Type

1 (HIV-1)-Specific Cytolytic T Lymphocyte Responses in Seronegative Adults by a Nonreplicating, Host-Range-Restricted Canarypox Vector (ALVAC) Carrying the

immunodeficiency virus type 1 integrase on viral replication." J. Virol. 69(5): 2729-

Immune Activation and Loss of Regulatory T-Cells and Improves Immune

mechanism to inhibit HIV replication." Journal of Pharmacology and Experimental

implications for therapy.(LEADING ARTICLE)(Report)." Journal of HIV Therapy

(Remune®) as a therapeutic vaccine in the treatment of HIV infection." Expert

(Remune®) as a therapeutic vaccine in the treatment of HIV infection." Expert

bystander cells and not in productively infected cells of HIV- and SIV-infected

for lifelong persistence of HIV-1, even in patients on effective combination

Issues in HIV Vaccine Clinical Trials: Lessons From a Workshop." JAIDS Journal of

Pitisuttitham P, Matthews T, Schwartz DH, Berman PW (1998). "Advancing AIDSVAX to phase 3. Safety, immunogenicity, and plans for phase 3." AIDS Res

Ensoli, B., S. Bellino, et al. (2010). "Therapeutic Immunization with HIV-1 Tat Reduces

Fauci, A. S., D. Mavilio, et al. (2005). "NK cells in HIV infection: Paradigm for protection or

Ferain, T., H. R. Hoveyda, et al. (2011). "Agonist-induced internalization of CCR5 as a

Fernandez, S., A. Lim, et al. (2009). "Immune activation and the pathogenesis of HIV disease:

Fernandez-Cruz, E., J. Navarro, et al. (2003). "The potential role of the HIV-1 immunogen

Fernandez-Cruz, E., J. n. Navarro, et al. (2003). "The potential role of the HIV-1 immunogen

Finkel, T. H., G. Tudor-Williams, et al. (1995). "Apoptosis occurs predominantly in

Finzi, D., J. Blankson, et al. (1999). "Latent infection of CD4+ T cells provides a mechanism

Follmann, D. P., A. M. D. P. M. P. H. H. Duerr, et al. (2007). "Endpoints and Regulatory

Francis DP, G. T., McElrath MJ, Belshe RB, Gorse GJ, Migasena S, Kitayaporn D,

Acquired Immune Deficiency Syndromes 44(1): 49-60.

Function in Subjects on HAART." PLoS ONE 5(11): e13540.

targets for ambush." Nat Rev Immunol 5(11): 835-843.

AIDS 5(5): 397-403 310.1097/COH.1090b1013e32833d32832e32839.

HIV-1MN env Gene." Journal of Infectious Diseases 171(6): 1623-1627. Emini, E. A., W. A. Schleif, et al. (1992). "Prevention of HIV-1 infection in chimpanzees by gpl20 V3 domain-specific monoclonal antibody." Nature 355(6362): 728-730. Engelman, A., G. Englund, et al. (1995). "Multiple effects of mutations in human

cells." Nature 417(6884): 95-98.

2736.

Therapeutics.

14(3): 52(55).

Review of Vaccines 2(6): 739-752.

Review of Vaccines 2(6): 739-752.

therapy." Nat Med 5(5): 512-517.

lymph nodes." Nat Med 1(2): 129-134.

Hum Retroviruses. 14(Suppl 3): S325-331.


Chouquet, C., B. Autran, et al. (2002). "Correlation between breadth of memory HIV-specific

Chun, T.-W., L. Carruth, et al. (1997). "Quantification of latent tissue reservoirs and total

Chun, T.-W., D. C. Nickle, et al. (2008). "Persistence of HIV in Gut-Associated Lymphoid

Ciccone, E. J., J. H. Greenwald, et al. (2011). "CD4+ T Cells, Including Th17 and Cycling

Clerici, M. a. b. (2010). "Beyond IL-17: new cytokines in the pathogenesis of HIV infection."

Committee, T. I. E. S. G. a. S. S. (2009). "Interleukin-2 Therapy in Patients with HIV

Crabtree-Ramirez, B., A. Villasis-Keever, et al. (2010). "Effectiveness of Highly Active

Crane, M., S. Sirivichayakul, et al. (2010). "No Increase in Hepatitis B Virus (HBV)-Specific

Cuevas, M. T., A. Fernandez-Garcia, et al. (2010). "Short Communication: Biological and

D'Orsogna, L. J. a., R. G. b. Krueger, et al. (2007). "Circulating memory B-cell subpopulations

Davenport, M. P., R. M. Ribeiro, et al. (2004). "Predicting the Impact of a Nonsterilizing Vaccine against Human Immunodeficiency Virus." J. Virol. 78(20): 11340-11351. Davey, R. T., D. G. Chaitt, et al. (1999). "A Randomized Trial of High-versus Low-Dose

De Clercq, E. (2010). "Antiretroviral drugs." Current Opinion in Pharmacology 10(5): 507-

Delelis, O., K. Carayon, et al. (2008). "Integrase and integration: biochemical activities of

DK Smith, M., RM Grant, MD, PJ Weidle, PharmD, A Lansky, PhD, J Mermin, MD, KA

Centers for Disease Control and Prevention (CDC). 60: 65-68.

Infection." New England Journal of Medicine 361(16): 1548-1559.

Highly Active Antiretroviral Therapy." J. Virol. 84(6): 2657-2665.

body viral load in HIV-1 infection." Nature 387(6629): 183-188.

2399-2407.

197(5): 714-720.

1747-1752.

849-858.

515.

Progressors." J. Virol.: JVI.02643-02610.

Human Retroviruses 26(9): 1019-1025.

HIV-1 integrase." Retrovirology 5(1): 114.

Current Opinion in HIV & AIDS 5(2): 184-188.

Research and Human Retroviruses 26(4): 373-378.

cytotoxic T cells, viral load and disease progression in HIV infection." AIDS 16(18):

Tissue despite Long-Term Antiretroviral Therapy." Journal of Infectious Diseases

Subsets, are Intact in the Gut Mucosa of HIV-1 Infected Long-Term Non-

Antiretroviral Therapy (HAART) Among HIV-Infected Patients in Mexico." AIDS

CD8+ T Cells in Patients with HIV-1-HBV Coinfections following HBV-Active

Genetic Characterization of HIV Type 1 Subtype B and Nonsubtype B Transmitted Viruses: Usefulness for Vaccine Candidate Assessment." AIDS Research and

are affected differently by HIV infection and antiretroviral therapy." AIDS 21(13):

Subcutaneous Interleukin-2 Outpatient Therapy for Early Human Immunodeficiency Virus Type 1 Infection." Journal of Infectious Diseases 179(4):

Fenton, MD, PhD, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, CDC. (2011). Interim guidance: preexposure prophylaxis for the prevention of HIV infection in men who have sex with men. MMWR Weekly,


Immunotherapies and Vaccines 249

Jiang, Y. L., Xinyong; De Clercq, Erik (2011). "New Therapeutic Approaches Targeted at the

John, M., C. B. Moore, et al. (2001). "Chronic hyperlactatemia in HIV-infected patients taking

Joly, V., K. Jidar, et al. (2010). "Enfuvirtide: from basic investigations to current clinical use."

Jose Esparza, W. L. H., Saladin Osmanov (1996). "HIV Vaccine Development." AIDS

Kannanganat, S., B. G. Kapogiannis, et al. (2007). "Human Immunodeficiency Virus Type 1

Keefer, M. C., S. E. Frey, et al. (2011). "A phase I trial of preventive HIV vaccination with

Kelley, C. F., J. D. Barbour, et al. (2007). "The Relation Between Symptoms, Viral Load, and

Kilby, J. M., S. Hopkins, et al. (1998). "Potent suppression of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entry." Nat Med 4(11): 1302-1307. Kilby, J. M., J. P. Lalezari, et al. (2002). "The Safety, Plasma Pharmacokinetics, and Antiviral

Kolte, L., J. C. Gaardbo, et al. (2011). "Dysregulation of CD4+CD25+CD127lowFOXP3+ regulatory T cells in HIV-infected pregnant women." Blood 117(6): 1861-1868. Kothe, D. L., J. M. Decker, et al. (2007). "Antigenicity and immunogenicity of HIV-1 consensus subtype B envelope glycoproteins." Virology 360(1): 218-234. Kothe, D. L., Y. Li, et al. (2006). "Ancestral and consensus envelope immunogens for HIV-1

Kovochich, M., M. D. Marsden, et al. (2011). "Activation of Latent HIV Using Drug-Loaded

Kranzer, K., J. J. Lewis, et al. (2010). "Treatment Interruption in a Primary Care

Kronenberg, A., H. M. Riehle, et al. (2001). "Liver failure after long-term nucleoside

Kuhrt, D., S. A. Faith, et al. (2010). "Evidence of Early B-Cell Dysregulation in Simian

Antiretroviral Therapy Program in South Africa: Cohort Analysis of Trends and Risk Factors." JAIDS Journal of Acquired Immune Deficiency Syndromes 55(3): e17-

Immunodeficiency Virus Infection: Rapid Depletion of Naive and Memory B-Cell Subsets with Delayed Reconstitution of the Naive B-Cell Population." J. Virol. 84(5):

antiretroviral therapy." AIDS 15(6): 717-723.

Cytokines." J. Virol. 81(21): 12071-12076.

subtype C." Virology 352(2): 438-449.

Nanoparticles." PLoS ONE 6(4): e18270.

e23 10.1097/QAI.1090b1013e3181f1275fd.

antiretroviral therapy." The Lancet 358(9283): 759-760.

uninfected subjects." Vaccine 29(10): 1948-1958.

10(suppl): S123-S132.

1170.

18(10): 685-693.

2466-2476.

Expert Opinion on Pharmacotherapy 11(16): 2701-2713.

Late Stages of the HIV-1 Replication Cycle." Current Medicinal Chemistry 18: 16-28.

Controllers but Not Noncontrollers Maintain CD4 T Cells Coexpressing Three

heterologous poxviral-vectors containing matching HIV-1 inserts in healthy HIV-

Viral Load Set Point in Primary HIV Infection." JAIDS Journal of Acquired Immune Deficiency Syndromes 45(4): 445-448 410.1097/QAI.1090b1013e318074ef318076e. Kilby, J. M. (1999). "Therapeutic potential of blocking HIV entry into cells: focus on

membrane fusion inhibitors." Expert Opinion on Investigational Drugs 8(8): 1157-

Activity of Subcutaneous Enfuvirtide (T-20), a Peptide Inhibitor of gp41-Mediated Virus Fusion, in HIV-Infected Adults." AIDS Research and Human Retroviruses


Gallo, R. C., P. S. Sarin, et al. (1983). "Isolation of Human T-Cell Leukemia Virus in Acquired

Gao, F. L., Hua-Xin; Hahn, Beatrice H.; Letvin, Norman L.; Korber, Bette T.; Haynes, Barton

Gorse, G. J., R. E. Simionescu, et al. (2006). "Cellular Immune Responses in Asymptomatic

Green, T. D., D. C. Montefiori, et al. (2003). "Enhancement of Antibodies to the Human

Greenbaum, A. H., L. E. Wilson, et al. (2008). "Effect of age and HAART regimen on clinical

Gudmundsdotter, L., C. Nilsson, et al. (2009). "Recombinant Modified Vaccinia Ankara

Gulick, R. M., J. Lalezari, et al. (2008). "Maraviroc for Previously Treated Patients with R5 HIV-1 Infection." New England Journal of Medicine 359(14): 1429-1441. Gulick, R. M., J. W. Mellors, et al. (1997). "Treatment with Indinavir, Zidovudine, and

Antiretroviral Therapy." New England Journal of Medicine 337(11): 734-739. Gurney, K. B., J. Elliott, et al. (2005). "Binding and Transfer of Human Immunodeficiency Virus by DC-SIGN+ Cells in Human Rectal Mucosa." J. Virol. 79(9): 5762-5773. Gurunathan, S., R. E. Habib, et al. (2009). "Use of predictive markers of HIV disease

Hanke, T. (2008). "STEP trial and HIV-1 vaccines inducing T-cell responses." Expert Review

Hartigan-O'Connor, D. J., L. A. Hirao, et al. (2011). "Th17 cells and regulatory T cells in elite

Hermans, S. M., A. N. Kiragga, et al. (2010). "Incident Tuberculosis during Antiretroviral

Hofer, U. and R. Speck (2009). "Disturbance of the gut-associated lymphoid tissue is

Huelsmann, P., A. Hofmann, et al. (2011). "A suicide gene approach using the human proapoptotic protein tBid inhibits HIV-1 replication." BMC Biotechnology 11(1): 4. Jaoko, W., E. Karita, et al. (2010). "Safety and Immunogenicity Study of Multiclade HIV-1

control over HIV and SIV." Current Opinion in HIV and AIDS 6(3): 221-227

Therapy Contributes to Suboptimal Immune Reconstitution in a Large Urban HIV

associated with disease progression in chronic HIV infection." Seminars in

Adenoviral Vector Vaccine Alone or as Boost following a Multiclade HIV-1 DNA

despite pre-existing vaccinia immunity." Vaccine 27(33): 4468-4474.

progression in vaccine trials." Vaccine 27(14): 1997-2015.

210.1097/COH.1090b1013e32834577b32834573.

Immunopathology 31(2): 257-266.

Vaccine in Africa." PLoS ONE 5(9): e12873.

Clinic in Sub-Saharan Africa." PLoS ONE 5(5): e10527.

F. (2007). "Centralized HIV-1 Envelope Immunogens and Neutralizing Antibodies."

Human Immunodeficiency Virus Type 1 (HIV-1) Infection and Effects of Vaccination with Recombinant Envelope Glycoprotein of HIV-1." Clin. Vaccine

Immunodeficiency Virus Type 1 Envelope by Using the Molecular Adjuvant C3d."

response in an urban cohort of HIV-infected individuals." AIDS 22(17): 2331-2339

(MVA) effectively boosts DNA-primed HIV-specific immune responses in humans

Lamivudine in Adults with Human Immunodeficiency Virus Infection and Prior

Immune Deficiency Syndrome (AIDS)." Science 220(4599): 865-867.

Current HIV Research 5: 572-577.

2310.1097/QAD.2330b2013e32831883f32831889.

Immunol. 13(1): 26-32.

J. Virol. 77(3): 2046-2055.

of Vaccines 7: 303-309.


Immunotherapies and Vaccines 251

Mahdavi, S., R. Malyuta, et al. (2010). "Treatment and disease progression in a birth cohort of vertically HIV-1 infected children in Ukraine." BMC Pediatrics 10(1): 85. Malek, T. R. and I. Castro (2010). "Interleukin-2 Receptor Signaling: At the Interface between

Maloy, K. J. and M. C. Kullberg (2008). "IL-23 and Th17 cytokines in intestinal homeostasis."

Mayer, K. H. and K. K. Venkatesh (2010). "Chemoprophylaxis for HIV Prevention: New

McBurney, S. P. and T. M. Ross (2008). "Viral sequence diversity: challenges for AIDS

McBurney, S. P. and T. M. Ross (2009). "Human immunodeficiency virus-like particles with

McBurney, S. P., K. R. Young, et al. (2007). "Membrane embedded HIV-1 envelope on the

McElrath, M. J. and B. F. Haynes (2010). "Induction of Immunity to Human Immunodeficiency Virus Type-1 by Vaccination." Immunity 33(4): 542-554. Michel, P., A. T. Balde, et al. (2000). "Reduced Immune Activation and T Cell Apoptosis in

Miedema, F. (2008). "A brief history of HIV vaccine research: stepping back to the drawing

Migasena, S., P. Suntharasamai, et al. (2000). "AIDSVAX® (MN) in Bangkok Injecting Drug

Minkoff, H., Y. Zhong, et al. (2010). "Influence of Adherent and Effective Antiretroviral

Mitsuya, H., K. J. Weinhold, et al. (1985). "3'-Azido-3'-deoxythymidine (BW A509U): an

Montagnier, L. (2010). "25†years after HIV discovery: Prospects for cure and vaccine."

Proceedings of the National Academy of Sciences 82(20): 7096-7100.

Syndromes 55: S122-S127 110.1097/QAI.1090b1013e3181fbcb1094c.

vaccine designs." Expert Review of Vaccines 7(9): 1405-1417.

Opportunities and New Questions." JAIDS Journal of Acquired Immune Deficiency

consensus envelopes elicited broader cell-mediated peripheral and mucosal immune responses than polyvalent and monovalent Env vaccines." Vaccine 27(32):

surface of a virus-like particle elicits broader immune responses than soluble

Human Immunodeficiency Virus Type 2 Compared with Type 1: Correlation of T Cell Apoptosis with Œ≤2 Microglobulin Concentration and Disease Evolution."

Users: A Report on Safety and Immunogenicity, Including Macrophage-Tropic Virus Neutralization." AIDS Research and Human Retroviruses 16(7): 655-663. Milush, J. M., K. D. Mir, et al. (2011). "Lack of clinical AIDS in SIV-infected sooty mangabeys

with significant CD4+ T cell loss is associated with double-negative T cells." The

Therapy Use on Human Papillomavirus Infection and Squamous Intraepithelial Lesions in Human Immunodeficiency Virus—Positive Women." Journal of

antiviral agent that inhibits the infectivity and cytopathic effect of human Tlymphotropic virus type III/lymphadenopathy-associated virus in vitro."

Tolerance and Immunity." Immunity 33(2): 153-165.

Mucosal Immunol 1(5): 339-349.

envelopes." Virology 358(2): 334-346.

Journal of Infectious Diseases 181(1): 64-75.

Journal of Clinical Investigation 121(3): 1102-1110.

board?" AIDS 22(14): 1699-1703.

Infectious Diseases 201(5): 681-690.

Virology 397(2): 248-254.

4337-4349.


Kutscher, S., S. Allgayer, et al. (2010). "MVA-nef induces HIV-1-specific polyfunctional and

Lane, H. C. and A. S. Fauci (1985). "Immunologic Abnormalities in the Acquired Immunodeficiency Syndrome." Annual Review of Immunology 3(1): 477-500. Langlois, A. J., K. J. Weinhold, et al. (1992). "Detection of Anti-Human Cell Antibodies in

Lasky, L., J. Groopman, et al. (1986). "Neutralization of the AIDS retrovirus by antibodies to

Latinovic, O. a., N. a. Le, et al. "Synergistic inhibition of R5 HIV-1 by maraviroc and CCR5

Lavreys, L., J. M. Baeten, et al. (2006). "Higher Set Point Plasma Viral Load and More-Severe

1–Infected African Women." Clinical Infectious Diseases 42(9): 1333-1339. Lehner, T., G. M. Shearer, et al. (2000). "Alloimmunization as a Strategy for Vaccine Design against HIV/AIDS." AIDS Research and Human Retroviruses 16(4): 309-313. Lerner, P., M. Guadalupe, et al. (2011). "The Gut Mucosal Viral Reservoir in HIV-Infected

a recombinant envelope glycoprotein." Science 233(4760): 209-212.

immune assays." Gene Ther 17(11): 1372-1383.

and Human Retroviruses 8(9): 1641-1652.

AIDS.

110(1): 15-27.

253.

Lancet 353(9168): 1923-1929.

Invest. 95(5): 2061-2066.

viruses." Virology 353(2): 268-282.

proliferative T-cell responses revealed by the combination of short- and long-term

Sera from Macaques Immunized with Whole Inactivated Virus." AIDS Research

antibody HGS004 in primary cells: implications for treatment and prevention."

Acute HIV Type 1 (HIV-1) Illness Predict Mortality among High-Risk HIV-

Patients Is Not the Major Source of Rebound Plasma Viremia following Interruption of Highly Active Antiretroviral Therapy." J. Virol. 85(10): 4772-4782. Letang, E., J. M. Miro, et al. (2011). "Incidence and Predictors of Immune Reconstitution

Inflammatory Syndrome in a Rural Area of Mozambique." PLoS ONE 6(2): e16946.

interleukin-2 in asymptomatic HIV-1 infection: a randomised controlled trial." The

induces antibodies that neutralize subsets of subtype B and C HIV-1 primary

presenting as chylothorax in a patient with HIV and Mycobacterium tuberculosis

AIDSVAX(TM) B/E, HIV gp120 Vaccine - Genentech, HIV gp120 Vaccine AIDSVAX - VaxGen, HIV Vaccine AIDSVAX - VaxGen." Drugs in R&D 4(4): 249-

"CD8 naive T cell counts decrease progressively in HIV-infected adults." J Clin

Preventive Vaccine Trials." JAIDS Journal of Acquired Immune Deficiency

Letvin, N. L. (2002). "Strategies for an HIV vaccine." The Journal of Clinical Investigation

Levy, Y., C. Capitant, et al. (1999). "Comparison of subcutaneous and intravenous

Liao, H.-X., L. L. Sutherland, et al. (2006). "A group M consensus envelope glycoprotein

Lin, J.-N., C.-H. Lai, et al. (2010). "Immune reconstitution inflammatory syndrome

Ltd., A. I. (2003). "HIV gp120 Vaccine - VaxGen: AIDSVAX(TM), AIDSVAX(TM) B/B,

M Roederer, J. G. D., M T Anderson, P A Raju, L A Herzenberg, and L A Herzenberg (1995).

MacLachlan, E., K. H. Mayer, et al. (2009). "The Potential Role of Biomarkers in HIV

Syndromes 51(5): 536-545 510.1097/QAI.1090b1013e3181adcbbe.

coinfection: a case report." BMC Infectious Diseases 10(1): 321.


Immunotherapies and Vaccines 253

Peto, T. (1996). "Surrogate markers in HIV disease." Journal of Antimicrobial Chemotherapy

Pett, S. L. (2009). "Immunotherapies in HIV-1 infection." Current Opinion in HIV & AIDS

Pincus, S. H., K. G. Messer, et al. (1993). "Differences in the antibody response to human

Polo, J. M. and T. W. Dubensky (2002). "Virus-based vectors for human vaccine

Prince, A. M., H. Reesink, et al. (1991). "Prevention of HIV Infection by Passive

Profile, A. R. D. (2003). "HIV gp120 Vaccine - VaxGen: AIDSVAX(TM), AIDSVAX(TM) B/B,

Putkonen, P., R. Thorstensson, et al. (1991). "Prevention of HIV-2 and SIVsm infection by passive immunization in cynomolgus monkeys." Nature 352(6334): 436-438. Ranasinghe, C., F. Eyers, et al. (2011). "A comparative analysis of HIV-specific

poxvirus-poxvirus prime boost immunisations." Vaccine 29(16): 3008-3020. Redfield, R. R., D. L. Birx, et al. (1991). "A Phase I Evaluation of the Safety and

Rerks-Ngarm, S., P. Pitisuttithum, et al. (2009). "Vaccination with ALVAC and AIDSVAX to

Robertson, D. L., J. P. Anderson, et al. (2000). "HIV-1 Nomenclature Proposal." Science

Rodes, B., C. Toro, et al. (2004). "Differences in disease progression in a cohort of long-term

Rosario, M., A. Bridgeman, et al. (2010). "Long peptides induce polyfunctional T cells

Rosenberg, E. S., B. S. Graham, et al. (2010). "Safety and Immunogenicity of Therapeutic

in macaques." European Journal of Immunology 40(7): 1973-1984.

Acute/Early HIV-1 Infection." PLoS ONE 5(5): e10555.

Immunology 170(10): 5176-5187.

Candidate in Humans." PLoS ONE 6(4): e18526.

applications." Drug Discovery Today 7(13): 719-727.

37(suppl B): 161-170.

Retroviruses 7(12): 971-973.

324(24): 1677-1684.

2209-2220.

1116.

288(5463): 55.

4(3): 188-193.

253.

Immunization with Recombinant Listeria monocytogenes HIV Gag." The Journal of

immunodeficiency virus-1 envelope glycoprotein (gp160) in infected laboratory workers and vaccinees." The Journal of Clinical Investigation 91(5): 1987-1996. Pine, S. O., J. G. Kublin, et al. (2011). "Pre-Existing Adenovirus Immunity Modifies a

Complex Mixed Th1 and Th2 Cytokine Response to an Ad5/HIV-1 Vaccine

Immunization with HIV Immunoglobulin." AIDS Research and Human

AIDSVAX(TM) B/E, HIV gp120 Vaccine - Genentech, HIV gp120 Vaccine AIDSVAX - VaxGen, HIV Vaccine AIDSVAX - VaxGen." Drugs in R&D 4(4): 249-

mucosal/systemic T cell immunity and avidity following rDNA/rFPV and

Immunogenicity of Vaccination with Recombinant gp160 in Patients with Early Human Immunodeficiency Virus Infection." New England Journal of Medicine

Prevent HIV-1 Infection in Thailand." New England Journal of Medicine 361(23):

non-progressors after more than 16 years of HIV-1 infection." AIDS 18(8): 1109-

against conserved regions of HIV-1 with superior breadth to single-gene vaccines

DNA Vaccination in Individuals Treated with Antiretroviral Therapy during


Monteiro, J. P., L. C. J. Alcantara, et al. (2009). "Genetic variability of human

Moss, R. B., M. R. Wallace, et al. (1999). "Phenotypic Analysis of Human Immunodeficiency

Mosser, D. M. (2003). "The many faces of macrophage activation." Journal of Leukocyte

Moutsopoulos, N. M., S. Nares, et al. (2007). "Tonsil Epithelial Factors May Influence

Nanjundappa, R. H., R. Wang, et al. (2011). "GP120-specific exosome-targeted T cell-based

Nkolola, J. P., H. Peng, et al. (2010). "Breadth of Neutralizing Antibodies Elicited by Stable,

Nobuto Yamamoto, N. U., and Yoshihiko Koga (2009). "Immunotherapy of HIV-Infected

O'Connell, K. A., J. R. Bailey, et al. (2009). "Elucidating the elite: mechanisms of control in HIV-1 infection." Trends in Pharmacological Sciences 30(12): 631-637. Oh, C., J. Price, et al. (2011). "Inhibition of HIV-1 infection by aqueous extracts of Prunella

Okulicz, J. F. and O. Lambotte (2011). "Epidemiology and clinical characteristics of elite

Paci, P., F. Martini, et al. (2011). "Timely HAART initiation may pave the way for a better

Pantaleo, G. and R. A. Koup (2004). "Correlates of immune protection in HIV-1 infection:

Paroli, M., A. Propato, et al. (2001). "The immunology of HIV-infected long-term non-

Peters, B. S. (2000). "HIV immunotherapeutic vaccines." Antiviral Chemistry &

Peters, B. S. and M. Samuel (2010). "Implications of the SILCAAT and ESPRIT trials and the

Peters, C., X. Peng, et al. (2003). "The Induction of HIV Gag-Specific CD8+ T Cells in the

progressors--a current view." Immunology Letters 79(1-2): 127-129.

HIV-1 Immunogen." Journal of Infectious Diseases 180(3): 641-648.

genotypes." Journal of Medical Virology 81(3): 391-399.

Biology 73(2): 209-212.

Vaccine 29(19): 3538-3547.

810.

15(1): 15(13).

Pigs." J. Virol. 84(7): 3270-3279.

Journal of Medical Virology 81: 16-26.

vulgaris L." Virology Journal 8(1): 188.

Chemotherapy 11(5): 311-320.

110.1097/COH.1090b1013e328344f328335e.

viral control." BMC Infectious Diseases 11(1): 56.

Journal of Pathology 171(2): 571-579.

immunodeficiency virus-1 in Bahia state, Northeast, Brazil: High diversity of HIV

Virus (HIV) Type 1 Cell-Mediated Immune Responses after Treatment with an

Oropharyngeal Human Immunodeficiency Virus Transmission." The American

vaccine capable of stimulating DC- and CD4+ T-independent CTL responses."

Homogeneous Clade A and Clade C HIV-1 gp140 Envelope Trimers in Guinea

Patients With Gc Protein-Derived Macrophage Activating Factor (GcMAF)."

controllers." Current Opinion in HIV and AIDS 6(3): 163-168

what we know, what we don't know, what we should know." Nat Med 10(8): 806-

future for HIV immunotherapy.(LEADING ARTICLE)(human immunodeficiency virus)(enhanced suppression of the platelet IIb/IIIa receptor with integrilin therapy)(study of interleukin-2 in people with low CD4+ T cell counts on active anti-human immunodeficiency virus therapy)(Report)." Journal of HIV Therapy

Spleen and Gut-Associated Lymphoid Tissue by Parenteral or Mucosal

Immunization with Recombinant Listeria monocytogenes HIV Gag." The Journal of Immunology 170(10): 5176-5187.


Immunotherapies and Vaccines 255

Sundling, C., S. O'Dell, et al. (2010). "Immunization with Wild-Type or CD4-Binding-

Tappuni, A. R. (2011). "Immune Reconstitution Inflammatory Syndrome." Advances in

Torti, C., A. d'Arminio-Monforte, et al. (2011). "Long-term CD4+ T-cell count evolution after

Tsoukas, C. M., J. Raboud, et al. (1998). "Active Immunization of Patients with HIV

Vaine, M., S. Lu, et al. (2009). "Progress on the Induction of Neutralizing Antibodies Against

Van Gulck, E. F. V. T., Viggo; N. Berneman, Zwi; Vanham, Guido (2010). "Role of Dendritic

Virgin, H. W. and B. D. Walker (2010). "Immunology and the elusive AIDS vaccine." Nature

Von Kleist, M., S. Menz, et al. (2011). "HIV Quasispecies Dynamics during Pro-Active

Wild, C., T. Greenwell, et al. (1993). "A Synthetic Peptide from HIV-1 gp41 Is a Potent

Wild, C., T. Oas, et al. (1992). "A Synthetic Peptide Inhibitor of Human Immunodeficiency

Wild, C. T., D. C. Shugars, et al. (1994). "Peptides Corresponding to a Predictive α-Helical

Wilson, N. A., B. F. Keele, et al. (2009). "Vaccine-Induced Cellular Responses Control Simian

Wintsch, J., C.-L. Chaignat, et al. (1991). "Safety and Immunogenicity of a Genetically

Wong, J. K., M. Hezareh, et al. (1997). "Recovery of Replication-Competent HIV Despite Prolonged Suppression of Plasma Viremia." Science 278(5341): 1291-1295.

Cells in HIV-Immunotherapy." Current HIV Research 8(4): 310-322.

84(18): 9086-9095.

Dental Research 23(1): 90-96.

Retroviruses 14(6): 483-490.

Retroviruses 9(11): 1051-1053.

States of America 91(21): 9770-9774.

200900001.

464(7286): 224-231.

89(21): 10537-10541.

Diseases 163(2): 219-225.

6508-6521.

only NRTI." BMC Infectious Diseases 11(1): 23.

in Latent Reservoirs." PLoS ONE 6(3): e18204.

Defective HIV-1 Env Trimers Reduces Viremia Equivalently following Heterologous Challenge with Simian-Human Immunodeficiency Virus." J. Virol.

switching from regimens including HIV nucleoside reverse transcriptase inhibitors (NRTI) plus protease inhibitors to regimens containing NRTI plus non-NRTI or

Infection: A Study of the Effect of VaxSyn, a Recombinant HIV Envelope Subunit Vaccine, on Progression of Immunodeficiency." AIDS Research and Human

HIV Type 1 (HIV-1)." BioDrugs 23(3): 137-153 110.2165/00063030-200923030-

Treatment Switching: Impact on Multi-Drug Resistance and Resistance Archiving

Inhibitor of Virus-Mediated Cell—Cell Fusion." AIDS Research and Human

Virus Replication: Correlation between Solution Structure and Viral Inhibition." Proceedings of the National Academy of Sciences of the United States of America

Domain of Human Immunodeficiency Virus Type 1 gp41 are Potent Inhibitors of Virus Infection." Proceedings of the National Academy of Sciences of the United

Immunodeficiency Virus Replication after Heterologous Challenge." J. Virol. 83(13):

Engineered Human Immunodeficiency Virus Vaccine." Journal of Infectious


Sabado, R. L., M. O'Brien, et al. (2010). "Evidence of dysregulation of dendritic cells in

Salgado, M., N. I. Rallun, et al. (2011). "Long-term non-progressors display a greater number

Salgado, M., N. I. RallÛn, et al. (2011). "Long-term non-progressors display a greater

Santra, S., B. T. Korber, et al. (2008). "A centralized gene-based HIV-1 vaccine elicits broad

Schulbin, H., H. Bode, et al. (2008). "Cytokine Expression in the Colonic Mucosa of Human

Antiretroviral Therapy." Antimicrob. Agents Chemother. 52(9): 3377-3384. Sekaly, R.-P. (2008). "The failed HIV Merck vaccine study: a step back or a launching point

Shapiro, R. L., M. D. Hughes, et al. (2010). "Antiretroviral Regimens in Pregnancy and

Shiver, J. W., T.-M. Fu, et al. (2002). "Replication-incompetent adenoviral vaccine vector

Shrestha, S., S. Sudenga, et al. (2010). "The impact of highly active antiretroviral therapy on

Siliciano, J. D. and R. F. Siliciano (2004). "A long-term latent reservoir for HIV-1: discovery and clinical implications." Journal of Antimicrobial Chemotherapy 54(1): 6-9. Sistigu, A., L. Bracci, et al. (2011). "Strong CD8+ T cell antigenicity and immunogenicity of

Somogyi, E., J. Xu, et al. (2011). "A plasmid DNA immunogen expressing fifteen protein

Streeck, H. and D. F. Nixon (2010). "T Cell Immunity in Acute HIV-1 Infection." Journal of

Sui, Y., Q. Zhu, et al. (2010). "Innate and adaptive immune correlates of vaccine and

Sundling, C., M. N. E. Forsell, et al. (2010). "Soluble HIV-1 Env trimers in adjuvant elicit

Proceedings of the National Academy of Sciences 107(21): 9843-9848. Sukeepaisarncharoen, W., V. Churdboonchart, et al. (2001). "Long-term follow-up of HIV-1-

activation/maturation of dendritic cells." Vaccine 29(18): 3465-3475.

positive adolescents." BMC Infectious Diseases 10(1): 295.

Infectious Diseases 202(Supplement 2): S302-S308.

Experimental Medicine 207(9): 2003-2017.

HIV." Vaccine 29(4): 744-753.

2(5): 391-398.

of Th17 cells than HIV-infected typical progressors." Clinical Immunology 139(2):

number of Th17 cells than HIV-infected typical progressors." Clinical Immunology

cross-clade cellular immune responses in rhesus monkeys." Proceedings of the

Immunodeficiency Virus-Infected Individuals before and during 9 Months of

for future vaccine development?" The Journal of Experimental Medicine 205(1): 7-

Breast-Feeding in Botswana." New England Journal of Medicine 362(24): 2282-2294.

elicits effective anti-immunodeficiency-virus immunity." Nature 415(6869): 331-335.

prevalence and incidence of cervical human papillomavirus infections in HIV-

large foreign proteins incorporated in HIV-1 VLPs able to induce a Nef-dependent

antigens and complex virus-like particles (VLP+) mimicking naturally occurring

adjuvant-induced control of mucosal transmission of SIV in macaques."

infected Thai patients immunized with Remune monotherapy." HIV Clinical Trials

potent and diverse functional B cell responses in primates." The Journal of

primary HIV infection." Blood 116(19): 3839-3852.

National Academy of Sciences 105(30): 10489-10494.

110-114.

12.

139(2): 110-114.


**10** 

*USA* 

**HIV Envelope-Specific Antibody** 

*Vaccine Branch, National Cancer Institute, Bethesda,* 

Egidio Brocca-Cofano, Peng Xiao and Marjorie Robert-Guroff

Following transmission, the human immunodeficiency virus (HIV) initiates persistent infection by integrating into the genome of host cells. To date it has not been possible to clear these cells by anti-viral or immune therapy, during either active or latent infection. This makes design of an efficacious HIV vaccine exceedingly difficult, since complete prevention of infection, or "sterilizing immunity", is required. As cellular immunity targets already infected cells, humoral immune responses which can prevent initial infection by means of anti-envelope neutralizing antibodies have been a prime focus of vaccine development. In proof of concept studies, passive administration of potent neutralizing antibodies has prevented infection of non-human primates by intravenous and mucosal routes (Mascola et al., 1999; Baba et al., 2000; Mascola et al., 2000; Parren et al., 2001), validating the research focus on neutralizing antibody induction. However, the task of designing an envelope vaccine is complicated by the extreme variability among HIV isolates, the propensity for neutralization escape resulting from immune pressure exerted by induced antibodies, conformational features of the HIV envelope which make immunogen design difficult, and additional envelope characteristics which effectively hide areas of vulnerability which might ordinarily be antibody targets. There are several excellent recent reviews covering the issue of broadly neutralizing antibodies (Mascola & Montefiori, 2010; Walker & Burton, 2010; Zolla-Pazner & Cardozo, 2010; McElrath & Haynes, 2010) and it is not the intent of this review to reproduce that information. Rather, we will briefly summarize some of the salient issues and approaches, and then discuss more extensively

Broadly neutralizing antibodies are difficult to elicit by vaccination. But the HIV envelope protein is quite immunogenic, and an array of non-neutralizing antibodies is induced by both natural infection and vaccination. Through use of sensitive new methods, these antibodies have exhibited several functional activities associated with protection. In serum these include antibody-dependent cellular cytotoxicity (ADCC) (Weinhold, 1990) and antibody-dependent cell mediated viral inhibition (ADCVI) (Forthal et al., 2006). Secretory antibodies have also been associated with protection via mechanisms such as transcytosis inhibition (Bomsel et al., 1998). Augmented by high avidity and recall memory responses which improve their efficacy, these antibody activities can contribute in varying degrees to vaccine-induced protective efficacy. The main thrust of this review will be to examine these

**1. Introduction** 

non-neutralizing anti-envelope antibodies.

**and Vaccine Efficacy** 


## **HIV Envelope-Specific Antibody and Vaccine Efficacy**

Egidio Brocca-Cofano, Peng Xiao and Marjorie Robert-Guroff *Vaccine Branch, National Cancer Institute, Bethesda, USA* 

## **1. Introduction**

256 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

World Health Organization, W. (2006). ANTIRETROVIRAL THERAPY FOR HIV Infection

World Health Organization , W. (2009). "Global summary of the AIDS epidemic." Retrieved

Yamamoto, N., N. Ushijima, et al. (2009). "Immunotherapy of HIV-infected patients with Gc

Young, K. R., J. M. Smith, et al. (2004). "Characterization of a DNA vaccine expressing a human immunodeficiency virus-like particle." Virology 327(2): 262-272. Zarling, J. M., W. Morton, et al. (1986). "T-cell responses to human AIDS virus in macaques immunized with recombinant vaccinia viruses." Nature 323(6086): 344-346.

Programme, World Health Organization 1-134.

Virology 81(1): 16-26.

in adults and adolescents: Recommendations for a public health approach W. H. A.

02.11.2011, 2011, from http://www.who.int/hiv/data/2009\_global\_summary.png.

protein-derived macrophage activating factor (GcMAF)." Journal of Medical

Following transmission, the human immunodeficiency virus (HIV) initiates persistent infection by integrating into the genome of host cells. To date it has not been possible to clear these cells by anti-viral or immune therapy, during either active or latent infection. This makes design of an efficacious HIV vaccine exceedingly difficult, since complete prevention of infection, or "sterilizing immunity", is required. As cellular immunity targets already infected cells, humoral immune responses which can prevent initial infection by means of anti-envelope neutralizing antibodies have been a prime focus of vaccine development. In proof of concept studies, passive administration of potent neutralizing antibodies has prevented infection of non-human primates by intravenous and mucosal routes (Mascola et al., 1999; Baba et al., 2000; Mascola et al., 2000; Parren et al., 2001), validating the research focus on neutralizing antibody induction. However, the task of designing an envelope vaccine is complicated by the extreme variability among HIV isolates, the propensity for neutralization escape resulting from immune pressure exerted by induced antibodies, conformational features of the HIV envelope which make immunogen design difficult, and additional envelope characteristics which effectively hide areas of vulnerability which might ordinarily be antibody targets. There are several excellent recent reviews covering the issue of broadly neutralizing antibodies (Mascola & Montefiori, 2010; Walker & Burton, 2010; Zolla-Pazner & Cardozo, 2010; McElrath & Haynes, 2010) and it is not the intent of this review to reproduce that information. Rather, we will briefly summarize some of the salient issues and approaches, and then discuss more extensively non-neutralizing anti-envelope antibodies.

Broadly neutralizing antibodies are difficult to elicit by vaccination. But the HIV envelope protein is quite immunogenic, and an array of non-neutralizing antibodies is induced by both natural infection and vaccination. Through use of sensitive new methods, these antibodies have exhibited several functional activities associated with protection. In serum these include antibody-dependent cellular cytotoxicity (ADCC) (Weinhold, 1990) and antibody-dependent cell mediated viral inhibition (ADCVI) (Forthal et al., 2006). Secretory antibodies have also been associated with protection via mechanisms such as transcytosis inhibition (Bomsel et al., 1998). Augmented by high avidity and recall memory responses which improve their efficacy, these antibody activities can contribute in varying degrees to vaccine-induced protective efficacy. The main thrust of this review will be to examine these

HIV Envelope-Specific Antibody and Vaccine Efficacy 259

While a significant percentage of HIV-infected individuals with chronic disease have a degree of neutralizing antibody breadth, a much smaller percentage are able to neutralize across all HIV clades (Simek et al., 2009). Long-term non-progressors have rather poor neutralizing antibody responses. Rather potent neutralizing activity seems to require a lengthy time period of sustained viremia and development of strong binding avidity, suggesting that antigen persistence and antibody maturation are needed for development of a broad response (Sather et al., 2009). Neutralizing antibodies develop very slowly in HIVinfected individuals. Antibodies with specificity for gp41 appear first at around 13 days post-infection and anti-gp120 antibodies at around day 28 (Tomaras et al., 2008). However, neutralizing antibodies appear later, usually months after infection, and thus do not appear to control viremia (Aasa-Chapman et al., 2004; Gray et al., 2007). This slow development reflects, at least in part, the same obstacles facing vaccine-induction of a neutralizing antibody response: conformational and carbohydrate masking of critical epitopes; homology of some epitopes with self proteins, leading to polyreactive antibodies that are subject to immune tolerance; and envelope variability leading to immune selective pressure and viral escape (McElrath and Haynes, 2010). Later in the course of disease, loss of CD4 help and B cell dysfunction exacerbates the poor neutralizing antibody development (Alter and Moody,

In addition to improved envelope design based on increasing knowledge of the structure of the HIV envelope, other approaches have attempted to better expose critical conserved epitopes on envelope immunogens. These have included deletion of variable loops to expose otherwise hidden regions of the envelope; alteration of glycosylation patterns to prevent masking; preparation of trimeric forms of the envelope to mimic the natural structure on the surface of the virion, and introduction of critical epitopes on other scaffolds for better presentation to the immune system (Hu and Stamatatos, 2007). These alterations have had varying degrees of success, although none has induced the breadth and potency of neutralizing antibody response needed for a highly effective vaccine. It is hoped that continued improvements in envelope immunogens fostered by greater knowledge of envelope structure and understanding of the natural process of broadly neutralizing

The RV144 phase III trial in Thailand which assessed an ALVAC–recombinant prime/Env protein boost regimen, showed only modest efficacy, protecting 31% of vaccinated individuals in the intent-to-treat group (Rerks-Ngarm et al., 2009). Nevertheless, this outcome provided the first evidence that development of a safe and effective preventive HIV vaccine is possible. This study also highlighted the need to better understand immune correlates of protection associated with decreased HIV acquisition. The RV144 vaccine components have induced a broad constellation of immune responses, including T-cell–line adapted neutralizing antibody (Nitayaphan et al., 2004), antibody-dependent cell-mediated cytotoxicity (Karnasuta et al., 2005), and CD4+ and CD8+ T cell responses, but clear immune correlates have not been defined. Currently in order to combat the extensive genomic diversity of HIV both strong cellular and humoral immune responses are believed necessary

**2.1 Development of neutralizing antibodies in natural infection** 

2010).

**2.2 Improved envelope immunogen design** 

antibody induction will achieve the desired goal.

**3. Non-neutralizing antibodies** 

non-neutralizing antibody responses in HIV and SIV infection and following vaccination, to describe how they may contribute to protection, and to summarize their potential utility amidst the array of additional immune protective mechanisms available to the host, including innate, cellular, and mucosal immunity.

## **2. Neutralizing antibodies**

The variability of the HIV envelope is notorious. The envelope exhibits a 30% difference in amino acid sequence between the 9 clades designated A – K, omitting E and I (Korber et al., 2001). Envelope diversity within clades can be as high as 20% (Mascola & Montefiori, 2010). Therefore the goal of eliciting anti-envelope antibodies able to broadly recognize and protect against this spectrum of isolates is daunting. In fact it is difficult to induce broadly neutralizing antibodies by vaccination. Most that arise are relatively weak and only able to neutralize the most sensitive or easy to neutralize "Tier I" isolates (Mascola et al., 2005; Seaman et al., 2010). Yet recent publications document development of cross-reactive neutralizing antibodies during HIV infection (Sather et al., 2009) with 20 to 34% of HIV-infected individuals possessing significant breadth (Simek et al., 2009; Doria-Rose et al., 2010). That vaccine induction of broadly neutralizing antibodies is a realistic goal is also illustrated by the isolation of a handful of naturally elicited antibodies that recognize a wide spectrum of HIV isolates. These include b12, an monoclonal antibody selected by random reassortment of a phage library which recognizes the CD4 binding site of the HIV envelope (Burton et al., 1994); 2G12, a monoclonal antibody that recognizes carbohydrate moieties (Trkola et al., 1996) on the silent face of the envelope; and monoclonal antibodies 2F5 and 4E10 that target the membrane-proximal external region (MPER) of the viral envelope transmembrane protein (Muster et al., 1993; Zwick et al., 2001). Additionally, the V3 region of the external envelope protein gp120, originally believed to elicit only type-specific neutralizing antibodies, has been shown to elicit broader responses. A panel of V3 monoclonal antibodies including 447-52D was recently reported to exhibit significant cross-clade neutralizing activity (Hioe et al., 2010). The basis for this breadth may be the conserved structural elements in the V3 loop which provide for its essential function as part of the binding region to chemokine co-receptors and which outweigh the inherent variability of the amino acid sequence in importance (Almond et al., 2010; Jiang et al., 2010). Portions of the V3 and V2 loops comprise a novel quaternary epitope, recognized only as part of the native trimer. Monoclonal antibody 2909, the first such described human antibody, is potent but relatively strain specific (Gorny et al., 2005). In contrast, the recently identified monoclonals PG9 and PG16 (Walker et al., 2009) also recognize quarternary epitopes, but differ by exhibiting great neutralization breadth, attributed to dependence on an asparagine-linked carbohydrate moiety at residue 160 in the V2 loop. The 2909 antibody recognizes a lysine at this position (Zolla-Pazner & Cardozo, 2010). New methods of high-throughput monoclonal antibody cloning and screening have facilitated isolation of several additional broadly neutralizing antibodies (Scheid et al., 2009; Corti et al., 2010). To date VRC01, a CD4 binding site antibody, has shown the greatest

breadth, neutralizing 91% of tested HIV isolates, representative of all major HIV clades (Wu et al., 2010). Structural knowledge of the HIV envelope, computer-assisted protein design, and state-of-the-art methods for memory B cell sorting and single cell PCR facilitated its isolation. It is hoped that similar new methodologies can lead to design of an appropriate vaccine component able to elicit neutralizing antibody breadth.

non-neutralizing antibody responses in HIV and SIV infection and following vaccination, to describe how they may contribute to protection, and to summarize their potential utility amidst the array of additional immune protective mechanisms available to the host,

The variability of the HIV envelope is notorious. The envelope exhibits a 30% difference in amino acid sequence between the 9 clades designated A – K, omitting E and I (Korber et al., 2001). Envelope diversity within clades can be as high as 20% (Mascola & Montefiori, 2010). Therefore the goal of eliciting anti-envelope antibodies able to broadly recognize and protect against this spectrum of isolates is daunting. In fact it is difficult to induce broadly neutralizing antibodies by vaccination. Most that arise are relatively weak and only able to neutralize the most sensitive or easy to neutralize "Tier I" isolates (Mascola et al., 2005; Seaman et al., 2010). Yet recent publications document development of cross-reactive neutralizing antibodies during HIV infection (Sather et al., 2009) with 20 to 34% of HIV-infected individuals possessing significant breadth (Simek et al., 2009; Doria-Rose et al., 2010). That vaccine induction of broadly neutralizing antibodies is a realistic goal is also illustrated by the isolation of a handful of naturally elicited antibodies that recognize a wide spectrum of HIV isolates. These include b12, an monoclonal antibody selected by random reassortment of a phage library which recognizes the CD4 binding site of the HIV envelope (Burton et al., 1994); 2G12, a monoclonal antibody that recognizes carbohydrate moieties (Trkola et al., 1996) on the silent face of the envelope; and monoclonal antibodies 2F5 and 4E10 that target the membrane-proximal external region (MPER) of the viral envelope transmembrane protein (Muster et al., 1993; Zwick et al., 2001). Additionally, the V3 region of the external envelope protein gp120, originally believed to elicit only type-specific neutralizing antibodies, has been shown to elicit broader responses. A panel of V3 monoclonal antibodies including 447-52D was recently reported to exhibit significant cross-clade neutralizing activity (Hioe et al., 2010). The basis for this breadth may be the conserved structural elements in the V3 loop which provide for its essential function as part of the binding region to chemokine co-receptors and which outweigh the inherent variability of the amino acid sequence in importance (Almond et al., 2010; Jiang et al., 2010). Portions of the V3 and V2 loops comprise a novel quaternary epitope, recognized only as part of the native trimer. Monoclonal antibody 2909, the first such described human antibody, is potent but relatively strain specific (Gorny et al., 2005). In contrast, the recently identified monoclonals PG9 and PG16 (Walker et al., 2009) also recognize quarternary epitopes, but differ by exhibiting great neutralization breadth, attributed to dependence on an asparagine-linked carbohydrate moiety at residue 160 in the V2 loop. The 2909 antibody recognizes a lysine at this position (Zolla-Pazner & Cardozo, 2010). New methods of high-throughput monoclonal antibody cloning and screening have facilitated isolation of several additional broadly neutralizing antibodies (Scheid et al., 2009; Corti et al., 2010). To date VRC01, a CD4 binding site antibody, has shown the greatest breadth, neutralizing 91% of tested HIV isolates, representative of all major HIV clades (Wu et al., 2010). Structural knowledge of the HIV envelope, computer-assisted protein design, and state-of-the-art methods for memory B cell sorting and single cell PCR facilitated its isolation. It is hoped that similar new methodologies can lead to design of an appropriate

including innate, cellular, and mucosal immunity.

vaccine component able to elicit neutralizing antibody breadth.

**2. Neutralizing antibodies** 

#### **2.1 Development of neutralizing antibodies in natural infection**

While a significant percentage of HIV-infected individuals with chronic disease have a degree of neutralizing antibody breadth, a much smaller percentage are able to neutralize across all HIV clades (Simek et al., 2009). Long-term non-progressors have rather poor neutralizing antibody responses. Rather potent neutralizing activity seems to require a lengthy time period of sustained viremia and development of strong binding avidity, suggesting that antigen persistence and antibody maturation are needed for development of a broad response (Sather et al., 2009). Neutralizing antibodies develop very slowly in HIVinfected individuals. Antibodies with specificity for gp41 appear first at around 13 days post-infection and anti-gp120 antibodies at around day 28 (Tomaras et al., 2008). However, neutralizing antibodies appear later, usually months after infection, and thus do not appear to control viremia (Aasa-Chapman et al., 2004; Gray et al., 2007). This slow development reflects, at least in part, the same obstacles facing vaccine-induction of a neutralizing antibody response: conformational and carbohydrate masking of critical epitopes; homology of some epitopes with self proteins, leading to polyreactive antibodies that are subject to immune tolerance; and envelope variability leading to immune selective pressure and viral escape (McElrath and Haynes, 2010). Later in the course of disease, loss of CD4 help and B cell dysfunction exacerbates the poor neutralizing antibody development (Alter and Moody, 2010).

#### **2.2 Improved envelope immunogen design**

In addition to improved envelope design based on increasing knowledge of the structure of the HIV envelope, other approaches have attempted to better expose critical conserved epitopes on envelope immunogens. These have included deletion of variable loops to expose otherwise hidden regions of the envelope; alteration of glycosylation patterns to prevent masking; preparation of trimeric forms of the envelope to mimic the natural structure on the surface of the virion, and introduction of critical epitopes on other scaffolds for better presentation to the immune system (Hu and Stamatatos, 2007). These alterations have had varying degrees of success, although none has induced the breadth and potency of neutralizing antibody response needed for a highly effective vaccine. It is hoped that continued improvements in envelope immunogens fostered by greater knowledge of envelope structure and understanding of the natural process of broadly neutralizing antibody induction will achieve the desired goal.

## **3. Non-neutralizing antibodies**

The RV144 phase III trial in Thailand which assessed an ALVAC–recombinant prime/Env protein boost regimen, showed only modest efficacy, protecting 31% of vaccinated individuals in the intent-to-treat group (Rerks-Ngarm et al., 2009). Nevertheless, this outcome provided the first evidence that development of a safe and effective preventive HIV vaccine is possible. This study also highlighted the need to better understand immune correlates of protection associated with decreased HIV acquisition. The RV144 vaccine components have induced a broad constellation of immune responses, including T-cell–line adapted neutralizing antibody (Nitayaphan et al., 2004), antibody-dependent cell-mediated cytotoxicity (Karnasuta et al., 2005), and CD4+ and CD8+ T cell responses, but clear immune correlates have not been defined. Currently in order to combat the extensive genomic diversity of HIV both strong cellular and humoral immune responses are believed necessary

HIV Envelope-Specific Antibody and Vaccine Efficacy 261

Traditionally, ADCC killing was assessed using assays in which target cells were labeled with radioactive isotopes such as 51Chromium. Disadvantages of this method include difficulty labeling certain cell types, low assay sensitivity and high spontaneous chromium release resulting in high background values (Volgmann et al., 1989). Several flow cytometrybased alternatives have recently circumvented the problems associated with radioactive labeling of target cells in cytotoxicity assays (Wilkinson et al., 2001; Gomez-Roman et al., 2006a; Stratov et al., 2008; Chung et al., 2009). These assays have provided greater ease of use and importantly greater sensitivity, facilitating investigation of the role of ADCC

Over the past 20 years, the dogma that T cells and neutralizing antibodies are protective immune correlates for many vaccines led to lack of interest in the ADCC mechanism. However the gradual accumulation of evidence from natural infection and vaccine studies supporting a protective role for ADCC has stimulated studies of this immune response. A number of early studies documented the induction of ADCC antibodies during HIV infection (Lyerly et al., 1987; Rook et al., 1987; Ojo-amaize et al., 1987). A potential role for ADCC in modulating the course of HIV infection was eventually suggested based on studies showing an inverse association between ADCC antibody levels and clinical stage of the disease. Baum et al, (1996) presented strong evidence that higher titers of antibodies mediating ADCC correlated with a successful host defense against HIV-1, and Forthal et al., (2001a) reported an inverse association between ADCC activity and plasma viremia. Higher ADCC activity has also been correlated with slower disease progression in children (Ljunggren et al., 1990; Broliden et al., 1993). More recently, ADCC activity has been demonstrated in cervical lavage fluids of HIV-infected women (Battle-Miller et al., 2002), and associated with lower genital HIV RNA loads (Nag et al., 2004). Elite controllers also have higher ADCC antibody titers than viremic individuals, whereas neutralizing antibody activity tends to be higher in viremic individuals (Lambotte et al., 2009). Nevertheless, not all studies have concluded that ADCC plays a role in protective efficacy (Dalgleish et al., 1990; Lifson et al., 1991; Chuenchitra et al., 2003). The conflicting results reflect the complexity of the virus-host interaction and elements that contribute to the ADCC response, including the integrity of the host immune system, extent of viremia, level and affinity of

Non-human primate studies have stimulated interest in the ADCC mechanism and its role in protective efficacy. ADCC activity has been correlated with delayed disease progression in SIV infected macaques (Banks et al., 2002). Additional convincing evidence has come from pre-clinical vaccine studies. A replicating adenovirus type 5 host range mutant (Ad5hr)-SIV recombinant prime/SIV gp120 protein boost regimen was shown to elicit potent protection against an intrarectal SIVmac251 challenge (Patterson et al., 2004). The vaccine did not induce antibodies able to neutralize primary SIVmac251, however, the reduced acute viremia was significantly correlated with anti-envelope binding antibodies that mediated ADCC against SIVmac251-infected cells (Gomez-Roman et al., 2005). Subsequent studies, one involving a comparison of Ad5hr-SIV recombinant priming via the upper respiratory tract versus the oral route followed by SIV gp120 boosting and SIVmac251

activity in natural infection and vaccine-induced protection.

**3.1.1 HIV-specific ADCC responses in natural infection** 

antibodies induced, and functionality of effector cells.

**3.1.2 ADCC responses in non-human primate models** 

for a successful vaccine (Amanna & Slifka, 2010; Benmira et al., 2010). However, as strong cellular immunity was not elicited in the majority of RV144 vaccinees, humoral immunity is believed to have contributed to the protection against HIV acquisition. As the vaccine regimen did not elicit antibodies able to neutralize primary HIV isolates, the focus of research has shifted to the potential for non-neutralizing antibodies to mediate protection. Neutralizing antibodies are able to prevent infection of susceptible cells; however, once a cell is infected it is difficult to imagine a role for neutralizing antibodies (Battle-Miller et al., 2002). Other antibody functions such as ADCC and ADCVI working together with innate effector cells provide a means to target and kill virus infected cells (Fig. 1A). Such mechanisms could control or possibly eradicate the small foci of infected cells that form in the lamina propria after viral transmission and prior to systemic spread of the virus (Fig. 1B; Haase, 2005).

#### **3.1 ADCC**

ADCC bridges innate and adaptive immunity. It involves effector cells able to mediate cell lysis, target cells expressing cell surface antigen, and specific antibody that recognizes the cell surface antigen and activates effector cells via interaction with Fc receptors. The interaction between the Fc domain of the antibody and the corresponding receptor on effector cells triggers a series of events that lead to the destruction of the infected cell via cytotoxic granules (perforin, granzyme) or a death-receptor-dependent pathway (Fas/Fas ligand; TNF/TNFR) (de Saint Basile et al., 2010; Chavez-Galan et al., 2009).

Most ADCC responses described in the literature are directed against the envelope protein (Env) (Ahmad & Menezes, 1996; Baum et al., 1996; Alsmadi & Tilley, 1998), although Nef (Yamada et al., 2004) and Tat (Florese et al., 2009) have also been shown to be ADCC targets. Additionally, a recent study in chronically infected subjects reported that Pol is an ADCC target, but this Pol-specific ADCC activity did not correlate with delayed HIV progression (Isitman et al., 2010). Moreover, the *pol* gene encodes internal proteins, so it is possible that the Pol-specific ADCC activity observed was targeting by-stander cells that had scavenged dead-cell debris. Despite the potential efficacy of ADCC, little is known about specific epitopes recognized by antibodies able to mediate ADCC. Epitopes recognized by both anti-Env and anti-Nef antibodies that mediate ADCC have been described (Alsmadi et al., 1997; Yamada et al., 2004; Los Alamos National Laboratory Molecular Immunology Database). As discussed below, anti-Env antibodies that mediate ADCC have been associated with protection, however, whether anti-Nef or anti-Tat antibodies have an impact on natural infection is not known.

Effector cells that mediate ADCC are not major histocompatibility complex restricted, and multiple subpopulations of peripheral blood mononuclear cells (PBMCs) are involved in mediating ADCC function. NK cells, γδ T cells, neutrophils, monocytes, and macrophages all express the Fc receptor that can engage antibodies (Forthal & Moog, 2009). A large number of these cells are always present in peripheral tissues, in contrast to memory B and T cells in lymphoid tissue which require activation for neutralizing antibody or T cell functions. Since HIV infection rapidly spreads during the first 2 weeks after transmission, the significant time advantage provided, for example, by pre-existing vaccine-elicited antibody and Fc receptor-bearing cells, may facilitate better control of viremia. IgG1 and IgG3 are the most common IgG isotypes to mediate ADCC via strong interaction with the Fc-binding receptor CD16/FcγRIII expressed mainly on NK cells (Niwa et al., 2005).

for a successful vaccine (Amanna & Slifka, 2010; Benmira et al., 2010). However, as strong cellular immunity was not elicited in the majority of RV144 vaccinees, humoral immunity is believed to have contributed to the protection against HIV acquisition. As the vaccine regimen did not elicit antibodies able to neutralize primary HIV isolates, the focus of research has shifted to the potential for non-neutralizing antibodies to mediate protection. Neutralizing antibodies are able to prevent infection of susceptible cells; however, once a cell is infected it is difficult to imagine a role for neutralizing antibodies (Battle-Miller et al., 2002). Other antibody functions such as ADCC and ADCVI working together with innate effector cells provide a means to target and kill virus infected cells (Fig. 1A). Such mechanisms could control or possibly eradicate the small foci of infected cells that form in the lamina propria after viral transmission and prior to systemic spread of the virus (Fig. 1B;

ADCC bridges innate and adaptive immunity. It involves effector cells able to mediate cell lysis, target cells expressing cell surface antigen, and specific antibody that recognizes the cell surface antigen and activates effector cells via interaction with Fc receptors. The interaction between the Fc domain of the antibody and the corresponding receptor on effector cells triggers a series of events that lead to the destruction of the infected cell via cytotoxic granules (perforin, granzyme) or a death-receptor-dependent pathway (Fas/Fas

Most ADCC responses described in the literature are directed against the envelope protein (Env) (Ahmad & Menezes, 1996; Baum et al., 1996; Alsmadi & Tilley, 1998), although Nef (Yamada et al., 2004) and Tat (Florese et al., 2009) have also been shown to be ADCC targets. Additionally, a recent study in chronically infected subjects reported that Pol is an ADCC target, but this Pol-specific ADCC activity did not correlate with delayed HIV progression (Isitman et al., 2010). Moreover, the *pol* gene encodes internal proteins, so it is possible that the Pol-specific ADCC activity observed was targeting by-stander cells that had scavenged dead-cell debris. Despite the potential efficacy of ADCC, little is known about specific epitopes recognized by antibodies able to mediate ADCC. Epitopes recognized by both anti-Env and anti-Nef antibodies that mediate ADCC have been described (Alsmadi et al., 1997; Yamada et al., 2004; Los Alamos National Laboratory Molecular Immunology Database). As discussed below, anti-Env antibodies that mediate ADCC have been associated with protection, however, whether anti-Nef or anti-Tat antibodies have an impact on natural

Effector cells that mediate ADCC are not major histocompatibility complex restricted, and multiple subpopulations of peripheral blood mononuclear cells (PBMCs) are involved in mediating ADCC function. NK cells, γδ T cells, neutrophils, monocytes, and macrophages all express the Fc receptor that can engage antibodies (Forthal & Moog, 2009). A large number of these cells are always present in peripheral tissues, in contrast to memory B and T cells in lymphoid tissue which require activation for neutralizing antibody or T cell functions. Since HIV infection rapidly spreads during the first 2 weeks after transmission, the significant time advantage provided, for example, by pre-existing vaccine-elicited antibody and Fc receptor-bearing cells, may facilitate better control of viremia. IgG1 and IgG3 are the most common IgG isotypes to mediate ADCC via strong interaction with the

Fc-binding receptor CD16/FcγRIII expressed mainly on NK cells (Niwa et al., 2005).

ligand; TNF/TNFR) (de Saint Basile et al., 2010; Chavez-Galan et al., 2009).

Haase, 2005).

infection is not known.

**3.1 ADCC** 

Traditionally, ADCC killing was assessed using assays in which target cells were labeled with radioactive isotopes such as 51Chromium. Disadvantages of this method include difficulty labeling certain cell types, low assay sensitivity and high spontaneous chromium release resulting in high background values (Volgmann et al., 1989). Several flow cytometrybased alternatives have recently circumvented the problems associated with radioactive labeling of target cells in cytotoxicity assays (Wilkinson et al., 2001; Gomez-Roman et al., 2006a; Stratov et al., 2008; Chung et al., 2009). These assays have provided greater ease of use and importantly greater sensitivity, facilitating investigation of the role of ADCC activity in natural infection and vaccine-induced protection.

#### **3.1.1 HIV-specific ADCC responses in natural infection**

Over the past 20 years, the dogma that T cells and neutralizing antibodies are protective immune correlates for many vaccines led to lack of interest in the ADCC mechanism. However the gradual accumulation of evidence from natural infection and vaccine studies supporting a protective role for ADCC has stimulated studies of this immune response. A number of early studies documented the induction of ADCC antibodies during HIV infection (Lyerly et al., 1987; Rook et al., 1987; Ojo-amaize et al., 1987). A potential role for ADCC in modulating the course of HIV infection was eventually suggested based on studies showing an inverse association between ADCC antibody levels and clinical stage of the disease. Baum et al, (1996) presented strong evidence that higher titers of antibodies mediating ADCC correlated with a successful host defense against HIV-1, and Forthal et al., (2001a) reported an inverse association between ADCC activity and plasma viremia. Higher ADCC activity has also been correlated with slower disease progression in children (Ljunggren et al., 1990; Broliden et al., 1993). More recently, ADCC activity has been demonstrated in cervical lavage fluids of HIV-infected women (Battle-Miller et al., 2002), and associated with lower genital HIV RNA loads (Nag et al., 2004). Elite controllers also have higher ADCC antibody titers than viremic individuals, whereas neutralizing antibody activity tends to be higher in viremic individuals (Lambotte et al., 2009). Nevertheless, not all studies have concluded that ADCC plays a role in protective efficacy (Dalgleish et al., 1990; Lifson et al., 1991; Chuenchitra et al., 2003). The conflicting results reflect the complexity of the virus-host interaction and elements that contribute to the ADCC response, including the integrity of the host immune system, extent of viremia, level and affinity of antibodies induced, and functionality of effector cells.

#### **3.1.2 ADCC responses in non-human primate models**

Non-human primate studies have stimulated interest in the ADCC mechanism and its role in protective efficacy. ADCC activity has been correlated with delayed disease progression in SIV infected macaques (Banks et al., 2002). Additional convincing evidence has come from pre-clinical vaccine studies. A replicating adenovirus type 5 host range mutant (Ad5hr)-SIV recombinant prime/SIV gp120 protein boost regimen was shown to elicit potent protection against an intrarectal SIVmac251 challenge (Patterson et al., 2004). The vaccine did not induce antibodies able to neutralize primary SIVmac251, however, the reduced acute viremia was significantly correlated with anti-envelope binding antibodies that mediated ADCC against SIVmac251-infected cells (Gomez-Roman et al., 2005). Subsequent studies, one involving a comparison of Ad5hr-SIV recombinant priming via the upper respiratory tract versus the oral route followed by SIV gp120 boosting and SIVmac251

HIV Envelope-Specific Antibody and Vaccine Efficacy 263

Fig. 1. **Control of viral infection by non-neutralizing antibodies. A. ADCC and ADCVI. (**1)Fc receptors on effector cells recognize the Fc domain of antibody bound to antigen on infected cells, inducing release of cytotoxic granules and cell lysis via ADCC. (2) Activation of effector cells may lead to production of chemokines and cytokines and viral inhibition by

transmission may occur by transcytosis of cell-free (1) or cell-associated virus (2). T cells, effector cells (macrophages, monocytes, NK cells, γδ T-cells, neutrophils) and plasma cells are present in the lamina propria. Mucosal antibodies, secreted by plasma cells (3), may block infection by neutralizing virus (4), or by blocking transcytosis of cell-associated (5) or cell-free virus (6). Antibodies can mediate ADCC and ADCVI to eradicate or control infected

ADCVI. **B. A mucosal surface with a single layer of columnar epithelium**. Viral

cell foci, blocking dissemination of virus to lymph nodes (7).

challenge, and another involving a comparison of Ad5hr-recombinant priming with and without subsequent envelope protein boosting followed by challenge with the chimeric virus SHIV89.6P, again showed significant correlations of ADCC activity with reduced acute viremia (Hidajat et al., 2009; Xiao et al., 2010). The latter study also revealed a correlation of ADCC activity with reduced chronic viremia. The importance of antibody maturation in induction of the functional antibody responses was indicated by the significant correlation of ADCC-mediating antibodies with binding antibody avidity.

The non-human primate model provides a good mimic for exploration of vaccine strategies and immune mechanisms. For example, passive antibody transfer studies can directly explore the ability of antibodies to mediate protection. Binley et al. (2000) observed that passive infusion of IgG to rapid and normal progressor SIVmac251 infected animals caused small and transient reductions in plasma viremia by a mechanism that was inconsistent with virus neutralization but which could have been effector cell mediated, implicating ADCC. In contrast, infusion of IgG possessing high titers of anti-Env antibodies able to mediate ADCC had no effect on viral loads following two sequential oral challenges of neonatel macaques with SIVmac251 (Florese et al., 2006). In both these studies, high viral loads may have contributed to negligible protective effects. Further, the neonatal macaque study may have been compromised by low levels and poorly functioning effector cells in the baby animals. An improved experimental design using repetitive low dose challenge might yield more significant results.

Passive transfer of monoclonal antibodies has proven more informative in elucidating mechanisms of antibody-mediated protection. An elegant study by Hessell et al. (2007) using the neutralizing b12 monoclonal antibody and a mutant unable to bind the Fc receptor and complement, showed that protection from a SHIVSF162P3 challenge mediated by the b12 antibody was in part due to Fc-mediated effects. A follow-on study using low-titered b12, mutant antibody, and low-dose repeated SHIVSF162P3 challenge, supported a contribution of effector function to the delayed acquisition observed (Hessell et al., 2009).

As illustrated by the b12 monoclonal, neutralizing antibodies may also mediate ADCC activity via their Fc domain. However, all ADCC mediating antibodies do not necessarily possess neutralizing activity. A neutralizing antibody must target a specific region of the viral envelope, whereas antibodies that mediate ADCC are required only to recognize an exposed target epitope on the surface of the infected cell. Antibodies elicited in chimpanzees to an HIV clade B immunization regimen were able to mediate ADCC killing of clade A, B, C, and AE env-expressing target cells (Gomez-Roman et al., 2006). Therefore, in addition to the ability to rapidly respond, the ADCC effector mechanism can provide the breadth of antibody recognition believed necessary for protective efficacy.

The extent to which the ADCC mechanism contributes to vaccine-induced protection is not yet clarified. A definitive conclusion will perhaps come from immune correlates identified in human clinical vaccine trials. To date only a few such trials have evaluated ADCC activity. Goepfert et al. (2007) reported that Env-specific ADCC activity, correlated with binding antibodies, was detected in most individuals that received a candidate AIDS vaccine containing gp120. Further, as mentioned above, a phase II human trial in Thailand has shown induction of ADCC mediating antibodies (Karnasuta et al., 2005). The role of these antibodies in the protection against HIV acquisition seen in the RV144 trial which used similar immunogens is currently being actively explored.

challenge, and another involving a comparison of Ad5hr-recombinant priming with and without subsequent envelope protein boosting followed by challenge with the chimeric virus SHIV89.6P, again showed significant correlations of ADCC activity with reduced acute viremia (Hidajat et al., 2009; Xiao et al., 2010). The latter study also revealed a correlation of ADCC activity with reduced chronic viremia. The importance of antibody maturation in induction of the functional antibody responses was indicated by the significant correlation

The non-human primate model provides a good mimic for exploration of vaccine strategies and immune mechanisms. For example, passive antibody transfer studies can directly explore the ability of antibodies to mediate protection. Binley et al. (2000) observed that passive infusion of IgG to rapid and normal progressor SIVmac251 infected animals caused small and transient reductions in plasma viremia by a mechanism that was inconsistent with virus neutralization but which could have been effector cell mediated, implicating ADCC. In contrast, infusion of IgG possessing high titers of anti-Env antibodies able to mediate ADCC had no effect on viral loads following two sequential oral challenges of neonatel macaques with SIVmac251 (Florese et al., 2006). In both these studies, high viral loads may have contributed to negligible protective effects. Further, the neonatal macaque study may have been compromised by low levels and poorly functioning effector cells in the baby animals. An improved experimental design using repetitive low dose challenge might yield more

Passive transfer of monoclonal antibodies has proven more informative in elucidating mechanisms of antibody-mediated protection. An elegant study by Hessell et al. (2007) using the neutralizing b12 monoclonal antibody and a mutant unable to bind the Fc receptor and complement, showed that protection from a SHIVSF162P3 challenge mediated by the b12 antibody was in part due to Fc-mediated effects. A follow-on study using low-titered b12, mutant antibody, and low-dose repeated SHIVSF162P3 challenge, supported a contribution of

As illustrated by the b12 monoclonal, neutralizing antibodies may also mediate ADCC activity via their Fc domain. However, all ADCC mediating antibodies do not necessarily possess neutralizing activity. A neutralizing antibody must target a specific region of the viral envelope, whereas antibodies that mediate ADCC are required only to recognize an exposed target epitope on the surface of the infected cell. Antibodies elicited in chimpanzees to an HIV clade B immunization regimen were able to mediate ADCC killing of clade A, B, C, and AE env-expressing target cells (Gomez-Roman et al., 2006). Therefore, in addition to the ability to rapidly respond, the ADCC effector mechanism can provide the breadth of

The extent to which the ADCC mechanism contributes to vaccine-induced protection is not yet clarified. A definitive conclusion will perhaps come from immune correlates identified in human clinical vaccine trials. To date only a few such trials have evaluated ADCC activity. Goepfert et al. (2007) reported that Env-specific ADCC activity, correlated with binding antibodies, was detected in most individuals that received a candidate AIDS vaccine containing gp120. Further, as mentioned above, a phase II human trial in Thailand has shown induction of ADCC mediating antibodies (Karnasuta et al., 2005). The role of these antibodies in the protection against HIV acquisition seen in the RV144 trial which used

effector function to the delayed acquisition observed (Hessell et al., 2009).

antibody recognition believed necessary for protective efficacy.

similar immunogens is currently being actively explored.

of ADCC-mediating antibodies with binding antibody avidity.

significant results.

Fig. 1. **Control of viral infection by non-neutralizing antibodies. A. ADCC and ADCVI. (**1)Fc receptors on effector cells recognize the Fc domain of antibody bound to antigen on infected cells, inducing release of cytotoxic granules and cell lysis via ADCC. (2) Activation of effector cells may lead to production of chemokines and cytokines and viral inhibition by ADCVI. **B. A mucosal surface with a single layer of columnar epithelium**. Viral transmission may occur by transcytosis of cell-free (1) or cell-associated virus (2). T cells, effector cells (macrophages, monocytes, NK cells, γδ T-cells, neutrophils) and plasma cells are present in the lamina propria. Mucosal antibodies, secreted by plasma cells (3), may block infection by neutralizing virus (4), or by blocking transcytosis of cell-associated (5) or cell-free virus (6). Antibodies can mediate ADCC and ADCVI to eradicate or control infected cell foci, blocking dissemination of virus to lymph nodes (7).

HIV Envelope-Specific Antibody and Vaccine Efficacy 265

reduced chronic phase viremia (Xiao et al., 2010) suggesting a broader role for ADCVI in controlling viral replication over the course of disease rather than impacting only early posttransmission viral spread. In this same study, a negative correlation between ADCVI activity 4 weeks post-challenge and neutralizing antibody titer 8 weeks post challenge was observed which became progressively weaker over time, and disappeared by 24 weeks postchallenge. The ADCVI assay evaluates viral inhibition in the presence of serum plus effector cells, and subtracts inhibition observed with serum in the absence of effector cells. This latter inhibition is attributed to neutralizing antibody. Therefore, the inverse correlation between the two activities might indicate that both neutralizing and non-neutralizing antibodies were mediating ADCVI. Neutralizing monoclonal antibodies are known to mediate ADCVI activity (Hessell et al., 2007). Development of *de novo* neutralizing antibody depends on the presence of sufficient viral antigen to drive the antibody response. The inverse relationship between ADCVI and the more slowly developing neutralizing antibody may reflect control of viremia by ADCVI and/or other immune mechanisms at the expense of strong neutralizing antibody induction due to a reduced viral burden. The complexity of the *in vivo* situation makes the relationships between functional antibody activities and viral burden

Mucosal surfaces are the major site for HIV entry. Therefore, an effective HIV vaccine may require the presence of antibodies able to prevent infection at mucosal sites. IgA antibodies are the most prevalent at mucosal surfaces, and might contribute to protection by one or more mechanisms including classical neutralization, but also non-neutralizing activities such as immune exclusion involving mucus entrapment and clearance, ADCC discussed above, and inhibition of HIV transcytosis across the epithelial cell barrier (Fig. 1B; Kozlowski and Neutra, 2003). Study of HIV-exposed but uninfected individuals (so called highly-exposed, seronegative; or HEPS), has shown the presence of functional HIV-specific IgA at mucosal surfaces of these individuals (Miyazawa et al., 2009; Lopalco, 2004),

Several vaccine approaches have been evaluated in non-human primates for the ability to elicit viral-specific IgA antibodies at genital/rectal sites. These have included tonsillar immunizations with replication-defective SIV (Vagenas et al., 2009), administration of DNA vaccines intranasally or rectally, followed by boosting with MVA recombinants (Bertley et al., 2004; Wang et al., 2004), intradermal or intramuscular administrations of DNA vaccines together with GM-CSF DNA or CCL27 DNA as adjuvants (Lai et al., 2007; Kraynyak et al., 2010) vaginal delivery of trimeric HIV envelope together with Carbopol gel (Cranage et al., 2011), upper respiratory track immunization with replication-competent Ad-recombinants followed by intramuscular boosting with envelope protein (Florese et al., 2009; Hidajat et al., 2009; Xiao et al., 2010), and intramuscular plus intranasal immunization with a gp41 subunit vaccine delivered on virosomes (Bomsel et al., 2011). These have had varying degrees of success in consistently eliciting mucosal IgA antibodies. Only a few studies, however, have investigated functionality of vaccine-elicited IgA as discussed below. Immune exclusion is difficult to assess in vitro due to the necessity for a mucus barrier, but neutralizing, ADCC, and ADCVI activities can be evaluated. Transcytosis inhibition seems especially relevant for

difficult to resolve.

mucosal protection.

**4. Secretory antibody** 

implicating the antibody in resistance to HIV infection.

### **3.2 ADCVI**

Like ADCC, ADCVI requires antibody that forms a bridge between an infected target cell and an FcγR-bearing effector cell (Forthal et al., 2001). However, ADCVI is a broader activity not restricted to target cell lysis, as with ADCC. Rather it encompasses several mechanisms by which viral replication following target cell infection is inhibited. These may include ADCC activity, but also noncytolytic mechanisms of virus control, such as secretion of inhibitory chemokines (Fig. 1A), or FcγR-mediated phagocytosis of immune complexes. The readout in ADCVI assays is the percentage of virus inhibition due to effector cells together with a test antibody relative to a negative control antibody. This biological endpoint allows assessment of ADCVI against any lentiviral strain able to infect cells. As the ADCVI assay uses heat-inactivated serum, complement activities do not play a role (Forthal & Landucci, 1998). Overall, ADCVI is a measure of the combined ability of antibody and effector cells to inhibit the spread of virus infection (Forthal et al., 2001; Forthal & Moog, 2009). Both polyclonal and monoclonal antibodies can mediate ADCVI. Intact IgG, not just the F(ab')2 portion is required (Forthal et al., 2006), emphasizing the importance of Fc-Fc receptor interactions in mediating the functional activity.

#### **3.2.1 ADCVI during HIV infection**

ADCVI has been associated with reduction in viremia during HIV infection. In HIV-infected individuals, systemic non-neutralizing antibodies appear early during acute infection, generally before a neutralizing antibody response (Sawyer et al., 1990). Not surprisingly, in individuals with acute HIV infection, non-neutralizing ADCVI antibodies appeared as early as the first week after onset of symptoms or the first month after HIV exposure (Forthal et al., 2001). ADCVI activity became more potent as the viral load fell (in the absence of antiretroviral therapy), resulting in an inverse relationship between ADCVI activity and acute plasma viremia, suggesting a protective effect. Importantly, ADCVI antibodies appeared to be broadly reactive with different HIV strains. The demonstration of an association between non-neutralizing but functional antibodies able to mediate ADCVI activities and protection is noteworthy and timely, in view of the recent outcome of the RV144 phase IIb vaccine trial in Thailand as discussed above. A previous study of serum samples from the Vax004 trial which evaluated gp120 vaccines similar to those used for boosting in RV144 revealed an inverse correlation between the HIV infection rate of vaccinated individuals and vaccine-elicited ADCVI antibody activity (Forthal et al., 2007). Although this trial did not result in protection, the results support the hypothesis that similar functional antibody activities may have contributed to protection in the RV144 trial. Taken together, these observations have renewed interest in defining the mechanisms of FcγR-mediated protection by ADCC and ADCVI.

#### **3.2.2 ADCVI in rhesus macaque models**

In support of a protective role for ADCVI, significant correlations between ADCVI activity mediated by vaccine-induced antibodies and decreased acute viremia have been reported in both SIV and SHIV rhesus macaque models (Florese et al., 2009; Hidajat et al., 2009; Xiao et al., 2010). Further, passive infusion of anti-SIV immune serum with strong ADCVI activity to newborn rhesus macaques prevented infection from an oral SIVmac251 challenge (Forthal et al., 2006). A recent study showed that vaccine-elicited antibody mediated ADCVI activity that was recalled 4 weeks post-challenge. This post-challenge activity was correlated with

Like ADCC, ADCVI requires antibody that forms a bridge between an infected target cell and an FcγR-bearing effector cell (Forthal et al., 2001). However, ADCVI is a broader activity not restricted to target cell lysis, as with ADCC. Rather it encompasses several mechanisms by which viral replication following target cell infection is inhibited. These may include ADCC activity, but also noncytolytic mechanisms of virus control, such as secretion of inhibitory chemokines (Fig. 1A), or FcγR-mediated phagocytosis of immune complexes. The readout in ADCVI assays is the percentage of virus inhibition due to effector cells together with a test antibody relative to a negative control antibody. This biological endpoint allows assessment of ADCVI against any lentiviral strain able to infect cells. As the ADCVI assay uses heat-inactivated serum, complement activities do not play a role (Forthal & Landucci, 1998). Overall, ADCVI is a measure of the combined ability of antibody and effector cells to inhibit the spread of virus infection (Forthal et al., 2001; Forthal & Moog, 2009). Both polyclonal and monoclonal antibodies can mediate ADCVI. Intact IgG, not just the F(ab')2 portion is required (Forthal et al., 2006), emphasizing the importance of Fc-Fc receptor

ADCVI has been associated with reduction in viremia during HIV infection. In HIV-infected individuals, systemic non-neutralizing antibodies appear early during acute infection, generally before a neutralizing antibody response (Sawyer et al., 1990). Not surprisingly, in individuals with acute HIV infection, non-neutralizing ADCVI antibodies appeared as early as the first week after onset of symptoms or the first month after HIV exposure (Forthal et al., 2001). ADCVI activity became more potent as the viral load fell (in the absence of antiretroviral therapy), resulting in an inverse relationship between ADCVI activity and acute plasma viremia, suggesting a protective effect. Importantly, ADCVI antibodies appeared to be broadly reactive with different HIV strains. The demonstration of an association between non-neutralizing but functional antibodies able to mediate ADCVI activities and protection is noteworthy and timely, in view of the recent outcome of the RV144 phase IIb vaccine trial in Thailand as discussed above. A previous study of serum samples from the Vax004 trial which evaluated gp120 vaccines similar to those used for boosting in RV144 revealed an inverse correlation between the HIV infection rate of vaccinated individuals and vaccine-elicited ADCVI antibody activity (Forthal et al., 2007). Although this trial did not result in protection, the results support the hypothesis that similar functional antibody activities may have contributed to protection in the RV144 trial. Taken together, these observations have renewed interest in defining the mechanisms of

In support of a protective role for ADCVI, significant correlations between ADCVI activity mediated by vaccine-induced antibodies and decreased acute viremia have been reported in both SIV and SHIV rhesus macaque models (Florese et al., 2009; Hidajat et al., 2009; Xiao et al., 2010). Further, passive infusion of anti-SIV immune serum with strong ADCVI activity to newborn rhesus macaques prevented infection from an oral SIVmac251 challenge (Forthal et al., 2006). A recent study showed that vaccine-elicited antibody mediated ADCVI activity that was recalled 4 weeks post-challenge. This post-challenge activity was correlated with

**3.2 ADCVI** 

interactions in mediating the functional activity.

FcγR-mediated protection by ADCC and ADCVI.

**3.2.2 ADCVI in rhesus macaque models** 

**3.2.1 ADCVI during HIV infection** 

reduced chronic phase viremia (Xiao et al., 2010) suggesting a broader role for ADCVI in controlling viral replication over the course of disease rather than impacting only early posttransmission viral spread. In this same study, a negative correlation between ADCVI activity 4 weeks post-challenge and neutralizing antibody titer 8 weeks post challenge was observed which became progressively weaker over time, and disappeared by 24 weeks postchallenge. The ADCVI assay evaluates viral inhibition in the presence of serum plus effector cells, and subtracts inhibition observed with serum in the absence of effector cells. This latter inhibition is attributed to neutralizing antibody. Therefore, the inverse correlation between the two activities might indicate that both neutralizing and non-neutralizing antibodies were mediating ADCVI. Neutralizing monoclonal antibodies are known to mediate ADCVI activity (Hessell et al., 2007). Development of *de novo* neutralizing antibody depends on the presence of sufficient viral antigen to drive the antibody response. The inverse relationship between ADCVI and the more slowly developing neutralizing antibody may reflect control of viremia by ADCVI and/or other immune mechanisms at the expense of strong neutralizing antibody induction due to a reduced viral burden. The complexity of the *in vivo* situation makes the relationships between functional antibody activities and viral burden difficult to resolve.

#### **4. Secretory antibody**

Mucosal surfaces are the major site for HIV entry. Therefore, an effective HIV vaccine may require the presence of antibodies able to prevent infection at mucosal sites. IgA antibodies are the most prevalent at mucosal surfaces, and might contribute to protection by one or more mechanisms including classical neutralization, but also non-neutralizing activities such as immune exclusion involving mucus entrapment and clearance, ADCC discussed above, and inhibition of HIV transcytosis across the epithelial cell barrier (Fig. 1B; Kozlowski and Neutra, 2003). Study of HIV-exposed but uninfected individuals (so called highly-exposed, seronegative; or HEPS), has shown the presence of functional HIV-specific IgA at mucosal surfaces of these individuals (Miyazawa et al., 2009; Lopalco, 2004), implicating the antibody in resistance to HIV infection.

Several vaccine approaches have been evaluated in non-human primates for the ability to elicit viral-specific IgA antibodies at genital/rectal sites. These have included tonsillar immunizations with replication-defective SIV (Vagenas et al., 2009), administration of DNA vaccines intranasally or rectally, followed by boosting with MVA recombinants (Bertley et al., 2004; Wang et al., 2004), intradermal or intramuscular administrations of DNA vaccines together with GM-CSF DNA or CCL27 DNA as adjuvants (Lai et al., 2007; Kraynyak et al., 2010) vaginal delivery of trimeric HIV envelope together with Carbopol gel (Cranage et al., 2011), upper respiratory track immunization with replication-competent Ad-recombinants followed by intramuscular boosting with envelope protein (Florese et al., 2009; Hidajat et al., 2009; Xiao et al., 2010), and intramuscular plus intranasal immunization with a gp41 subunit vaccine delivered on virosomes (Bomsel et al., 2011). These have had varying degrees of success in consistently eliciting mucosal IgA antibodies. Only a few studies, however, have investigated functionality of vaccine-elicited IgA as discussed below. Immune exclusion is difficult to assess in vitro due to the necessity for a mucus barrier, but neutralizing, ADCC, and ADCVI activities can be evaluated. Transcytosis inhibition seems especially relevant for mucosal protection.

HIV Envelope-Specific Antibody and Vaccine Efficacy 267

gp41-specific vaginal IgA that mediated transcytosis inhibition, and vaginal IgG that had neutralizing and/or ADCC activity. Both the transcytosis inhibition and ADCC activity were significantly inversely correlated with acute viremia. Of particular interest, sera from these macaques lacked anti-HIV activity in neutralization, ADCC, and transcytosis inhibition assays, suggesting that the IgG with protective activity was locally produced. A

In addition to functionality, the overall quality of an antibody response largely determines its effectiveness. Antibody avidity, a measure of the strength of the binding interaction between an antigen with multiple antigenic determinants and multivalent antibodies (Siegrist et al., 2004), is one characteristic which contributes to efficacy. It develops in germinal centers following somatic hypermutation of immunoglobulin genes and selection of B cells for high affinity binding to antigen (Berek et al., 1991; French et al., 1989; Griffiths et al., 1984). Thus, this antibody maturation process is dependent on both time and antigen exposure. The importance of antibody avidity has been shown in **s**tudies associating low antibody avidity with poor protective efficacy of an RSV vaccine (Delgado et al., 2009). In contrast, high-avidity neutralizing (Barnett et al., 2010) and non-neutralizing (Zhao et al., 2009; Xiao et al., 2010) HIV-1 Env-specific antibodies have been inversely correlated with reduced SHIV viremia following challenge. Importantly, in the Xiao et al. (2010) study, significant correlations were seen between antibody avidity and both functional antibody activities: ADCC and ADCVI, both also correlated with reduced viremia. The results overall suggest that antibody maturation following vaccination is associated with better functional

A critical feature of protective humoral immunity is memory. The success of vaccination depends on the differentiation of naïve B cells into plasma cells and memory B cells. Plasma cells are terminally differentiated and continuously secrete antibody without requiring further antigenic stimulation. In contrast, memory B cells represent an important second line of immune defense that is initiated if pre-existing antibody levels are too low to prevent infection or if an invading pathogen is able to circumvent the pre-existing antibody response. Memory B cells do not actively secrete antibody but instead maintain their immunoglobulin in the membrane-bound form, which together with Igα and Igβ form the antigen-specific B cell receptor. Following exposure to the initial antigen these cells become fully activated, proliferate, and differentiate into antibody secreting cells (ASC) (Ahmed & Gray, 1996; McHeyzer-Williams & McHeyzer-Williams, 2005; Pierce & Liu, 2010). Little is understood about the regulation of vaccine-induced humoral immunity. Differentiation of memory B cells into short-lived plasma cells is dependent on the presence of antigen (Dorner & Radbruch, 2007; Cagigi, et al., 2008). In contrast, long-lived antibody responses generated by viral infections or vaccinations are not dependent on the continuous presence of memory B cells but are rather produced by long-lived plasma cells that reside in the bone marrow and do not require antigen for continued production of antibody (Dorner & Radbruch, 2007; Radbruch et al., 2006). In fact, vaccine-induced B cell memory is maintained for more than 50 years after smallpox vaccination (Crotty et al., 2003), whereas antibody

similar suggestion was reported in the study of Xiao et al. (2010).

**6. The role of memory B cells in vaccine-mediated immunity** 

**5. Antibody avidity** 

antibody activity.

#### **4.1 Transcytosis inhibition**

HIV-1 transmission mainly occurs through exposure of mucosal surfaces to HIV-infected fluids, such as semen, cervicovaginal fluid, saliva, colostrum, and breast milk (Pope and Haase, 2003). A key entry event is translocation of virus across the epithelium. In rectal, intestinal, colonic, and endocervical mucosa, the epithelium is made up of a single layer of polarized, columnar epithelial cells with tight junctions separating the cells into the apical domain, which faces the lumen, and the basolateral domain, which faces the serosal side and the internal milieu (Bomsel, 1997). In contrast, ectocervical and vaginal epithelium is composed of pluristratified epithelial cells that lack a polarized plasma membrane and tight junctions, allowing intraepithelial dendritic cells and Langerhans cells to diffuse into the epithelium (Bomsel and Alfsen, 2003). Depending on the site of infection, several mechanisms for HIV-1 transmission across mucosal epithelia have been proposed, including columnar epithelial cell transcytosis, direct infection of epithelial cells, and dendritic/Langerhans cell transport (Bomsel & David, 2002; Shattock et al., 2000).

The major type of HIV transcytosis is cell-associated (Bomsel & Alfsen, 2003), generated by cell-cell contact of virally-infected cells with apical epithelial cell surfaces. It is a rapid, efficient, and nondegradative process in which virus is transported from the apical to the basolateral surface of polarized epithelial cells. Cell-free virus transcytosis is also possible but inefficient (Bobardt et al., 2007; Bomsel, 1997). Rather than fusion and infection, interactions between viral components, including gp41 (Alfsen et al., 2001), gp120 (Bobardt et al., 2007), and gp160 (Hocini et al., 1997), and host epithelial cell surface molecules, such as glycosphingolipid galactosyl-ceramide (GalCer) (Alfsen & Bomsel, 2002; Meng et al., 2002), an important component of endocytotic "raft" membrane microdomains, the coreceptor CCR5 (Bomsel et al., 2007), and the heparin sulfate proteoglycan attachment receptor, agrin (Alfsen et al., 2005), lead to transcytosis of the virus across the epithelial barrier and its trapping by submucosal dendritic cells which disseminate it to target CD4+ T cells.

Immunoglobulin A (IgA) and immunoglobulin G (IgG) anti-HIV antibodies have been detected in nearly all external secretions. Although mucosal IgG may interfere with viral infection in tissues underlying mucosal epithelia and secondary lymphoid tissues, mucosal IgA is thought to best protect mucosal surfaces (Pope and Haase, 2003). HIV-1 entry via transcytosis *in vitro* can be inhibited by dimeric IgA (dIgA) isolated from HIV-1-infected subjects (Bomsel et al., 1998), secretory IgA specific for gp41 (Alfsen et al., 2001), and mucosal and serum IgA from HIV-1-exposed seronegative individuals (Devito et al., 2000). Recently, transcytosis inhibition of both SIV and SHIV by vaccine-elicited mucosal antibodies has been evaluated in pre-clinical studies in non-human primates. In rhesus macaques, mucosal priming with replication-competent Ad-HIV or SIV recombinants followed by intramuscular boosting with envelope protein elicited antibodies in rectal secretions able to inhibit SIV and SHIV transcytosis *in vitro (*Hidajat et al., 2009; Xiao et al., 2010). Importantly, a significant correlation between transcytosis inhibition and reduced chronic viremia was seen in the study by Xiao et al. (2010) suggesting that mucosal IgA present in the submucosa may play a role in viremia control during the course of infection. However, the strongest evidence to date for a contribution of transcytosis inhibition to vaccine-elicited protection was recently reported by Bomsel et al. (2011). Following intramuscular plus intranasal immunization with gp41 subunit immunogens on virosomes, 4 out of 5 rhesus macaques were protected from SHIVSF162P3 acquisition following repetitive low-dose challenge, whereas all controls became infected. The protected macaques had gp41-specific vaginal IgA that mediated transcytosis inhibition, and vaginal IgG that had neutralizing and/or ADCC activity. Both the transcytosis inhibition and ADCC activity were significantly inversely correlated with acute viremia. Of particular interest, sera from these macaques lacked anti-HIV activity in neutralization, ADCC, and transcytosis inhibition assays, suggesting that the IgG with protective activity was locally produced. A similar suggestion was reported in the study of Xiao et al. (2010).

## **5. Antibody avidity**

266 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

HIV-1 transmission mainly occurs through exposure of mucosal surfaces to HIV-infected fluids, such as semen, cervicovaginal fluid, saliva, colostrum, and breast milk (Pope and Haase, 2003). A key entry event is translocation of virus across the epithelium. In rectal, intestinal, colonic, and endocervical mucosa, the epithelium is made up of a single layer of polarized, columnar epithelial cells with tight junctions separating the cells into the apical domain, which faces the lumen, and the basolateral domain, which faces the serosal side and the internal milieu (Bomsel, 1997). In contrast, ectocervical and vaginal epithelium is composed of pluristratified epithelial cells that lack a polarized plasma membrane and tight junctions, allowing intraepithelial dendritic cells and Langerhans cells to diffuse into the epithelium (Bomsel and Alfsen, 2003). Depending on the site of infection, several mechanisms for HIV-1 transmission across mucosal epithelia have been proposed, including columnar epithelial cell transcytosis, direct infection of epithelial cells, and

dendritic/Langerhans cell transport (Bomsel & David, 2002; Shattock et al., 2000).

The major type of HIV transcytosis is cell-associated (Bomsel & Alfsen, 2003), generated by cell-cell contact of virally-infected cells with apical epithelial cell surfaces. It is a rapid, efficient, and nondegradative process in which virus is transported from the apical to the basolateral surface of polarized epithelial cells. Cell-free virus transcytosis is also possible but inefficient (Bobardt et al., 2007; Bomsel, 1997). Rather than fusion and infection, interactions between viral components, including gp41 (Alfsen et al., 2001), gp120 (Bobardt et al., 2007), and gp160 (Hocini et al., 1997), and host epithelial cell surface molecules, such as glycosphingolipid galactosyl-ceramide (GalCer) (Alfsen & Bomsel, 2002; Meng et al., 2002), an important component of endocytotic "raft" membrane microdomains, the coreceptor CCR5 (Bomsel et al., 2007), and the heparin sulfate proteoglycan attachment receptor, agrin (Alfsen et al., 2005), lead to transcytosis of the virus across the epithelial barrier and its trapping by submucosal dendritic cells which disseminate it to target CD4+ T

Immunoglobulin A (IgA) and immunoglobulin G (IgG) anti-HIV antibodies have been detected in nearly all external secretions. Although mucosal IgG may interfere with viral infection in tissues underlying mucosal epithelia and secondary lymphoid tissues, mucosal IgA is thought to best protect mucosal surfaces (Pope and Haase, 2003). HIV-1 entry via transcytosis *in vitro* can be inhibited by dimeric IgA (dIgA) isolated from HIV-1-infected subjects (Bomsel et al., 1998), secretory IgA specific for gp41 (Alfsen et al., 2001), and mucosal and serum IgA from HIV-1-exposed seronegative individuals (Devito et al., 2000). Recently, transcytosis inhibition of both SIV and SHIV by vaccine-elicited mucosal antibodies has been evaluated in pre-clinical studies in non-human primates. In rhesus macaques, mucosal priming with replication-competent Ad-HIV or SIV recombinants followed by intramuscular boosting with envelope protein elicited antibodies in rectal secretions able to inhibit SIV and SHIV transcytosis *in vitro (*Hidajat et al., 2009; Xiao et al., 2010). Importantly, a significant correlation between transcytosis inhibition and reduced chronic viremia was seen in the study by Xiao et al. (2010) suggesting that mucosal IgA present in the submucosa may play a role in viremia control during the course of infection. However, the strongest evidence to date for a contribution of transcytosis inhibition to vaccine-elicited protection was recently reported by Bomsel et al. (2011). Following intramuscular plus intranasal immunization with gp41 subunit immunogens on virosomes, 4 out of 5 rhesus macaques were protected from SHIVSF162P3 acquisition following repetitive low-dose challenge, whereas all controls became infected. The protected macaques had

**4.1 Transcytosis inhibition** 

cells.

In addition to functionality, the overall quality of an antibody response largely determines its effectiveness. Antibody avidity, a measure of the strength of the binding interaction between an antigen with multiple antigenic determinants and multivalent antibodies (Siegrist et al., 2004), is one characteristic which contributes to efficacy. It develops in germinal centers following somatic hypermutation of immunoglobulin genes and selection of B cells for high affinity binding to antigen (Berek et al., 1991; French et al., 1989; Griffiths et al., 1984). Thus, this antibody maturation process is dependent on both time and antigen exposure. The importance of antibody avidity has been shown in **s**tudies associating low antibody avidity with poor protective efficacy of an RSV vaccine (Delgado et al., 2009). In contrast, high-avidity neutralizing (Barnett et al., 2010) and non-neutralizing (Zhao et al., 2009; Xiao et al., 2010) HIV-1 Env-specific antibodies have been inversely correlated with reduced SHIV viremia following challenge. Importantly, in the Xiao et al. (2010) study, significant correlations were seen between antibody avidity and both functional antibody activities: ADCC and ADCVI, both also correlated with reduced viremia. The results overall suggest that antibody maturation following vaccination is associated with better functional antibody activity.

## **6. The role of memory B cells in vaccine-mediated immunity**

A critical feature of protective humoral immunity is memory. The success of vaccination depends on the differentiation of naïve B cells into plasma cells and memory B cells. Plasma cells are terminally differentiated and continuously secrete antibody without requiring further antigenic stimulation. In contrast, memory B cells represent an important second line of immune defense that is initiated if pre-existing antibody levels are too low to prevent infection or if an invading pathogen is able to circumvent the pre-existing antibody response. Memory B cells do not actively secrete antibody but instead maintain their immunoglobulin in the membrane-bound form, which together with Igα and Igβ form the antigen-specific B cell receptor. Following exposure to the initial antigen these cells become fully activated, proliferate, and differentiate into antibody secreting cells (ASC) (Ahmed & Gray, 1996; McHeyzer-Williams & McHeyzer-Williams, 2005; Pierce & Liu, 2010). Little is understood about the regulation of vaccine-induced humoral immunity. Differentiation of memory B cells into short-lived plasma cells is dependent on the presence of antigen (Dorner & Radbruch, 2007; Cagigi, et al., 2008). In contrast, long-lived antibody responses generated by viral infections or vaccinations are not dependent on the continuous presence of memory B cells but are rather produced by long-lived plasma cells that reside in the bone marrow and do not require antigen for continued production of antibody (Dorner & Radbruch, 2007; Radbruch et al., 2006). In fact, vaccine-induced B cell memory is maintained for more than 50 years after smallpox vaccination (Crotty et al., 2003), whereas antibody

HIV Envelope-Specific Antibody and Vaccine Efficacy 269

replication. Mucosal antibodies that block viral entry through mechanisms such as transcytosis inhibition help control viral transmission and spread. As summarized here, maturation of vaccine-induced antibody responses is necessary for optimal function. Both antibody avidity and memory are directly associated with functional activity and control of viremia. An HIV/AIDS vaccine should be able to induce both cellular and humoral immunity. Regarding the latter, the success of a vaccine will depend on stimulating the production of mature high-titered antibodies with sufficiently broad reactivity to protect against HIV and SIV encounters. The path to induction of protective anti-envelope antibodies will come from understanding the B cell regulatory pathway of specific antibody production and from design of optimal immunogens. Coordination between human vaccine clinical trials and nonhuman primate vaccine challenge studies is essential to advance new

This work was supported by the Intramural Research Program of the National Institutes of

Aasa-Chapman, M.M., Hayman, A., Newton, P., Cornforth, D., Williams, I., Borrow, P.,

Ahmad, A., & Menezes, J. (1996). Antibody-dependent cellular cytotoxicity in HIV

Ahmed, R., & Gray, D. (1996). Immunological memory and protective immunity:

Alfsen, A. & Bomsel, M. (2002). HIV-1 gp41 envelope residues 650-685 exposed on native

Alfsen, A., Iniguez, P., Bouguyon, E., & Bomsel, M. (2001). Secretory IgA specific for a

Alfsen, A., Yu, H., Magerus-Chatinet, A., Schmitt, A., & Bomsel, M. (2005). HIV-1-infected

Almond, D., Kimura, T., Kong, X., Swetnam, J., Zolla-Pazner, S., & Cardozo, T. (2010).

Alsmadi, O., & Tilley, S.A. (1998). Antibody-dependent cellular cytotoxicity directed against

HIV type 1's V3 loop. *AIDS Research and Human Retroviruses*, 26, 717-723. Alsmadi, O., Herz, R., Murphy, E., Pinter, A., & Tilley, S.A. (1997). A novel antibody-

Balfe, P., & McKnight, A. (2004). Development of the antibody response in acute

virus act as a lectin to bind epithelial cell galactosyl ceramide. *Journal of Biological* 

conserved epitope on gp41 envelope glycoprotein inhibits epithelial transcytosis of

blood mononuclear cells form an integrin- and agrin-dependent viral synapse to induce efficient HIV-1 transcytosis across epithelial cell monolayer. *Molecular* 

Structural conservation predominates over sequence variability in the crown of

dependent cellular cytotoxicity epitope in gp120 is identified by two monoclonal antibodies isolated from a long-term survivor of human immunodeficiency virus

cells expressing human immunodeficiency virus type 1 envelope of primary or

vaccine concepts and accelerate the pace of HIV-1 vaccine efficacy trials.

**8. Acknowledgement** 

**9. References** 

Health, National Cancer Institute.

HIV-1 infection. *AIDS* 18, 371-381.

*Chemistry* 277, 25649-25659.

*Biology of the Cell* 16, 4267-4279.

infections. *FASEB Journal* 10, 258-266.

understanding their relation. *Science* 272, 54-60.

HIV-1. *Journal of Immunology* 166, 6257-6265.

type 1 infection. *Journal of Virology* 71, 925-933.

responses to tetanus toxoid and diphtheria vaccines have half-lives of 11 and 19 years, respectively (Amanna et al., 2007).

Memory B cell and serum antibody levels do not always correlate. This lack of correlation implies that the serum antibody level is maintained by long-lived plasma cells in the bone marrow and not by memory B cells circulating in the blood. However, in one of the first studies to examine the frequency of specific memory B cells in humans (Bonsignori et al., 2009), plasma antibody and memory B cell responses to HIV-1 envelope were compared in a group of chronic HIV-1 infected individuals and in volunteers vaccinated in the VAX004 clinical trial (Gilbert et al., 2005). A significant correlation between blood anti-Env memory B cells levels and plasma anti-Env antibody titers was found in both chronic HIV-1 infection and after vaccination with rgp120, suggesting that plasma antibody was maintained predominantly by short-lived memory B cells. Additionally, the half-life of anti-Env antibodies was shorter than those for influenza and tetanus toxoid, demonstrating that the HIV-1 envelope does not elicit long-lived B cell memory to the degree of other antigens. This outcome is not surprising for the HIV-infected cohort, as B-cell dysfunction, including loss of memory B cell subsets has been well-documented in HIV and SIV infection (Cagigi et al., 2008; Kuhrt et al., 2010a; Shen & Tomaras, 2011). However, the reasons for impaired memory induction in vaccinees is not well understood, and may include immune suppression due to binding of gp120 to CD4 or binding of carbohydrates to mannose receptors on dendritic cells and B cells (Bonsignori et al., 2009).

SIV and SHIV non-human primate models have been very valuable in HIV vaccine development. Human memory B cells have been extensively studied (Bonsignori et al., 2009; Crotty et al., 2004; Bernasconi et al., 2002), but only recently have rhesus macaque memory B cell studies been undertaken (Douagi et al., 2010; Kuhrt et al., 2010). We have recently shown induction of SIV and HIV Env-specific IgG and IgA ASC in rhesus macaques following priming with replicating Ad-SIV or HIV recombinants and boosting with SIV or HIV envelope protein (Brocca-Cofano et al., 2011). Env-specific IgG and IgA specific activities were correlated with several antibody activities, including ADCC, ADCVI, and/or transcytosis inhibition, indicating that maturation of antibody responses is critical for improved functionality. Further, IgG and IgA memory B cells post challenge were inversely correlated with chronic viremia indicating that vaccine-induced memory B cells were recalled and influenced disease outcome. That memory B cells should exhibit a protective role is not surprising in view of the reported association between loss of memory B cells and rapid disease progression in both HIV and SIV infection (Titanji et al., 2006; Titanji et al., 2010). Our induction of strong anti-envelope memory B cell responses by vaccination (Brocca-Cofano et al., 2011) may reflect use of a replicating vector to prime immune responses followed by envelope boosting. The combined approach may have provided both the antigen persistence and time necessary to allow antibody maturation.

#### **7. Conclusion**

Antibodies are key to host defense and critical for HIV vaccine design. Antibodies that recognize conserved epitopes and broadly neutralize virus can prevent infection. Once infection has occurred, other antibodies that interact with viral antigens expressed on the infected cell surface are needed to eliminate initial foci, or control subsequent systemic spread of the virus. Fc receptor-bearing effector cells, such as NK cells, can mediate killing of infected cells by ADCC and/or ADCVI activities. The latter can also inhibit viral replication. Mucosal antibodies that block viral entry through mechanisms such as transcytosis inhibition help control viral transmission and spread. As summarized here, maturation of vaccine-induced antibody responses is necessary for optimal function. Both antibody avidity and memory are directly associated with functional activity and control of viremia. An HIV/AIDS vaccine should be able to induce both cellular and humoral immunity. Regarding the latter, the success of a vaccine will depend on stimulating the production of mature high-titered antibodies with sufficiently broad reactivity to protect against HIV and SIV encounters. The path to induction of protective anti-envelope antibodies will come from understanding the B cell regulatory pathway of specific antibody production and from design of optimal immunogens. Coordination between human vaccine clinical trials and nonhuman primate vaccine challenge studies is essential to advance new vaccine concepts and accelerate the pace of HIV-1 vaccine efficacy trials.

### **8. Acknowledgement**

This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute.

#### **9. References**

268 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

responses to tetanus toxoid and diphtheria vaccines have half-lives of 11 and 19 years,

Memory B cell and serum antibody levels do not always correlate. This lack of correlation implies that the serum antibody level is maintained by long-lived plasma cells in the bone marrow and not by memory B cells circulating in the blood. However, in one of the first studies to examine the frequency of specific memory B cells in humans (Bonsignori et al., 2009), plasma antibody and memory B cell responses to HIV-1 envelope were compared in a group of chronic HIV-1 infected individuals and in volunteers vaccinated in the VAX004 clinical trial (Gilbert et al., 2005). A significant correlation between blood anti-Env memory B cells levels and plasma anti-Env antibody titers was found in both chronic HIV-1 infection and after vaccination with rgp120, suggesting that plasma antibody was maintained predominantly by short-lived memory B cells. Additionally, the half-life of anti-Env antibodies was shorter than those for influenza and tetanus toxoid, demonstrating that the HIV-1 envelope does not elicit long-lived B cell memory to the degree of other antigens. This outcome is not surprising for the HIV-infected cohort, as B-cell dysfunction, including loss of memory B cell subsets has been well-documented in HIV and SIV infection (Cagigi et al., 2008; Kuhrt et al., 2010a; Shen & Tomaras, 2011). However, the reasons for impaired memory induction in vaccinees is not well understood, and may include immune suppression due to binding of gp120 to CD4 or binding of carbohydrates to mannose

SIV and SHIV non-human primate models have been very valuable in HIV vaccine development. Human memory B cells have been extensively studied (Bonsignori et al., 2009; Crotty et al., 2004; Bernasconi et al., 2002), but only recently have rhesus macaque memory B cell studies been undertaken (Douagi et al., 2010; Kuhrt et al., 2010). We have recently shown induction of SIV and HIV Env-specific IgG and IgA ASC in rhesus macaques following priming with replicating Ad-SIV or HIV recombinants and boosting with SIV or HIV envelope protein (Brocca-Cofano et al., 2011). Env-specific IgG and IgA specific activities were correlated with several antibody activities, including ADCC, ADCVI, and/or transcytosis inhibition, indicating that maturation of antibody responses is critical for improved functionality. Further, IgG and IgA memory B cells post challenge were inversely correlated with chronic viremia indicating that vaccine-induced memory B cells were recalled and influenced disease outcome. That memory B cells should exhibit a protective role is not surprising in view of the reported association between loss of memory B cells and rapid disease progression in both HIV and SIV infection (Titanji et al., 2006; Titanji et al., 2010). Our induction of strong anti-envelope memory B cell responses by vaccination (Brocca-Cofano et al., 2011) may reflect use of a replicating vector to prime immune responses followed by envelope boosting. The combined approach may have provided both

respectively (Amanna et al., 2007).

receptors on dendritic cells and B cells (Bonsignori et al., 2009).

the antigen persistence and time necessary to allow antibody maturation.

Antibodies are key to host defense and critical for HIV vaccine design. Antibodies that recognize conserved epitopes and broadly neutralize virus can prevent infection. Once infection has occurred, other antibodies that interact with viral antigens expressed on the infected cell surface are needed to eliminate initial foci, or control subsequent systemic spread of the virus. Fc receptor-bearing effector cells, such as NK cells, can mediate killing of infected cells by ADCC and/or ADCVI activities. The latter can also inhibit viral

**7. Conclusion** 


HIV Envelope-Specific Antibody and Vaccine Efficacy 271

through primary genital epithelial cells. *Journal of Virology* 81, 395-405. Bomsel, M. (1997). Transcytosis of infectious human immunodeficiency virus across a tight

Bomsel, M., & Alfsen, A. (2003). Entry of viruses through the epithelial barrier: pathogenic

Bomsel, M., & David, V. (2002). Mucosal gatekeepers: selecting HIV viruses for early

Bomsel, M., Heyman, M., Hocini, H., Lagaye, S., Belec, L., Dupont, C., & Desgranges, C.

Bonsignori, M., Moody, M.A., Parks, R.J., Holl, T.M., Kelsoe, G., Hicks, C.B., Vandergrift, N.,

Broliden, K., Sievers, E., Tovo, P.A., Moschese, V., Scarlatti, G., Broliden, P.A., Fundaro, C.,

Burton, D.R., Pyati, J., Koduri, R., Sharp, S.J., Thornton, G.B., Parren, P.W.H.I., Sawyer,

Cagigi, A., Nilsson, A., De Milito, A., & Chiodi, F. (2008). B cell immunopathology during HIV-1 infection: lessons to learn for HIV-1 vaccine design. *Vaccine* 26, 3016-3025. Chavez-Galan, L., Arenas-Del Angel, M.C., Zenteno, E., Chavez, R., & Lascurain, R. (2009).

Chuenchitra, T., Wasi, C., Louisirirojchanakul, S., Nitayaphan, S., Sutthent, R., Cox, J.H., de

vaccination or HIV-1 infection. *Journal of Immunology* 183, 2708-2717. Brocca-Cofano, E., McKinnon, K., Demberg, T., Venzon, D., Hidajat, R., Xiao, P., Daltabuit-

barriers by anti-HIV envelope protein dIgA or IgM. *Immunity* 9, 277-287. Bomsel, M., Pastori, C., Tudor, D., Alberti, C., Garcia, S., Ferrari, D., Lazzarin, A., & Lopalco,

inhibit HIV-1 transport across human epithelial cells. *AIDS* 21:13-22. Bomsel, M., Tudor, D., Drillet, A., Alfsen, A., Ganor, Y., Roger, M., Mouz, N., Amacker, M.,

against vaginal SHIV challenges. *Immunity* 34, 269-280.

(1998). Intracellular neutralization of HIV transcytosis across tight epithelial

L. (2007). Natural mucosal antibodies reactive with first extracellular loop of CCR5

Chalifour, A., Diomede, L., Devillier, G., Cong, Z., Wei, Q., Gao, H., Qin, C., Yang, G., Zurbriggen, R., Lopalco, L., & Fleury, S. (2011). Immunization with HIV-1 gp41 subunit virosomes induces mucosal antibodies protecting nonhuman primates

Tomaras, G.D., & Haynes, B.F. (2009). HIV-1 envelope induces memory B cell responses that correlate with plasma antibody levels after envelope gp120 protein

Test, M., Patterson, L.J., & Robert-Guroff, M. (2011). Vaccine-elicited SIV and HIV envelope-specific IgA and IgG memory B cells in rhesus macaque peripheral blood correlate with functional antibody responses and reduced viremia. *Vaccine* In press.

& Rossi, P. (1993). Antibody-dependent cellular cytotoxicity and neutralizing activity in sera of HIV-1-infected mothers and their children. *Clinical and* 

L.S.W., Hendry, R.M., Dunlop, N., Nara, P.L., Lamacchia, M., Garratty, E., Stiehm, E.R., Bryson, Y.J., Cao, Y., Moore, J.P., Ho, D.D., & Barbas, C.F., III. (1994). Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal

Cell death mechanisms induced by cytotoxic lymphocytes. *Cellular & Molecular* 

Souza, M.S., Brown, A.E., Birx, D.L., & Polonis, V.R. (2003). Longitudinal study of

human epithelial cell line barrier. *Nature Medicine* 3, 42-47.

infection. *Nature Medicine* 8, 114-116.

*Experimental Immunology* 93, 56-64.

antibody. *Science* 266, 1024-1027.

*Immunology* 6, 15-25.

trickery. *Nature Reviews Molecular and Cellular Biology* 4, 57-68.

serum into simian immunodeficiency virus-infected rhesus macaques undergoing a rapid disease course has minimal effect on plasma viremia. *Virology* 270, 237-249. Bobardt, M.D., Chatterji, U., Selvarajah, S., Van der Schueren, B., David, G., Kahn, B., &

Gallay, P.A. (2007). Cell-free human immunodeficiency virus type 1 transcytosis

laboratory-adapted strains by human and chimpanzee monoclonal antibodies of different epitope specificities. *Journal of Virology* 72, 286-293.


Alter, G. & Moody, A. (2010). The humoral response to HIV-1: new insights, renewed focus.

Amanna, I.J., Carlson, N.E., & Slifka, M.K. (2007). Duration of humoral immunity to

Amanna, I.J., & Slifka, M.K. (2010). Contributions of humoral and cellular immunity to

Baba, T.W., Liska, V., Hofmann-Lehmann, R., Vlasak, J., Xu, W., Ayehunie, S., Cavacini,

Banks, N.D., Kinsey, N., Clements, J., & Hildreth, J.E.K. (2002). Sustained antibody-

Barnett, S.W., Burke, B., Sun, Y., Kan, E., Legg, H., Lian, Y., Bost, K., Zhou, F., Goodsell, A.,

Battle-Miller, K., Eby, C.A., Landay, A.L., Cohen, M.H., Sha, B.E., & Baum, L.L. (2002).

Baum, L.L., Cassutt, K.J., Knigge, K., Khattri, R., Margolick, J., Rinaldo, C., Kleeberger, C.A.,

Benmira, S., Bhattacharya, V., & Schmid, M.L. (2010). An effective HIV vaccine: A combination of humoral and cellular immunity? *Current HIV Research* 8, 441-449. Berek, C., Berger, A., & Apel, M. (1991). Maturation of the immune response in germinal

Bernasconi, N.L., Traggiai, E., & Lanzavecchia, A. (2002). Maintenance of serological memory by polyclonal activation of human memory B cells. *Science* 298, 2199-2202.

Bertley, F.M.N., Kozlowski, P.A., Wang, S., Chappelle, J., Patel, J., Sonuyi, O., Mazzara, G.,

Binley, J.M., Clas, B., Gettie, A., Vesanen, M., Montefiori, D.C., Sawyer, L., Booth, J., Lewis,

Montefiori, D., Carville, A., Mansfield, K.G., & Aldovini, A. (2004). Control of simian/human immunodeficiency virus viremia and disease progression after IL-2 augmented DNA-modified vaccinia virus Ankara nasal vaccination in nonhuman

M., Marx, P.A., Bonhoeffer, S., & Moore, J.P. (2000). Passive infusion of immune

different epitope specificities. *Journal of Virology* 72, 286-293.

vaccine-induced protection in humans. *Virology* 411, 206-215.

*The Journal of Infectious Diseases* 202, S315-S322.

MF59 adjuvant. *Journal of Virology* 84, 5975-5985.

*Journal of Immunology* 157, 2168-2173.

primates. *Journal of Immunology* 172, 3745-3757.

centers. *Cell* 67, 1121-1129.

206.

1205.

439-447.

laboratory-adapted strains by human and chimpanzee monoclonal antibodies of

common viral and vaccine antigens. *New England Journal of Medicine* 357, 1903-1815.

L.A., Posner, M.R., Katinger, H., Stiegler, G., Bernacky, B.J., Rizvi T.A., Schmidt, R., Hill, L.R., Keeling, M.E., Lu, Y., Wright, J.E., Chou, T-C., & Ruprecht, R.M. (2000). Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection. *Nature Medicine* 6, 200-

dependent cell-mediated cytotoxicity (ADCC) in SIV-infected macaques correlates with delayed progression to AIDS. *AIDS Research and Human Retroviruses* 18, 1197-

Zur Megede, J., Polo, J., Donnelly, J., Ulmer, J., Otten, G.R., Miller, C.J., Vajdy, M., & Srivastava, I.K. (2010). Antibody-mediated protection against mucosal simianhuman immunodeficiency virus challenge of macaques immunized with alphavirus replicon particles and boosted with trimeric envelope glycoprotein in

Antibody-dependent cell-mediated cytotoxicity in cervical lavage fluids of human immunodeficiency virus type 1--infected women. *Journal of Infectious Diseases* 185,

Nishanian, P., Henrard, D.R., & Phair, J. (1996). HIV-1 gp120-specific antibodydependent cell-mediated cytotoxicity correlates with rate of disease progression. serum into simian immunodeficiency virus-infected rhesus macaques undergoing a rapid disease course has minimal effect on plasma viremia. *Virology* 270, 237-249.


HIV Envelope-Specific Antibody and Vaccine Efficacy 273

Douagi, I., Forsell, M.N.E., Sundling, C., O'Dell, S., Feng, Y., Dosenovic, P., Li, Y., Seder, R.,

Florese, R.H., Demberg, T., Xiao, P., Kuller, L., Larsen, K., Summers, L.E., Venzon, D.,

Florese, R.H., Van Rompay, K.K., Aldrich, K., Forthal, D.N., Landucci ,G., Mahalanabis, M.,

against oral SIVmac251 challenge. *Journal of Immunology* 177, 4028-4036. Forthal, D.N., Gilbert, P.B., Landucci, G., & Phan, T. (2007). Recombinant gp120 vaccine-

Forthal, D.N., & Landucci, G. (1998). In vitro reduction of virus infectivity by antibodydependent cell-mediated immunity. *Journal of Immunological Methods* 220, 129-138. Forthal, D.N., Landucci, G., Cole, K.S., Marthas, M., Becerra, J.C., & Van Rompay, K. (2006).

Forthal, D.N., Landucci, G., & Daar, E.S. (2001). Antibody from patients with acute human

the presence of natural-killer effector cells. *Journal of Virology* 75, 6953-6961. Forthal, D.N., Landucci, G., & Keenan, B. (2001a). Relationship between antibody-dependent

Forthal, D.N., & Moog, C. (2009). Fc receptor-mediated antiviral antibodies. *Current Opinion* 

French, D.L., Laskov, R., Scharff M.D. (1989). The role of somatic hypermutation in the

Gilbert, P.B., Peterson, M.L., Follmann, D., Hudgens, M.G., Francis, D.P., Gurwith, M.,

Goepfert, P.A., Tomaras, G.D., Horton, H., Montefiori, D., Ferrari, G., Deers, M., Voss, G.,

1695.

*Immunology* 182, 3718-3727.

*Immunology* 178, 6596-6603.

*in HIV AIDS* 4, 388-393.

cells. *Journal of Virology* 80, 9217-9225.

*Research and Human Retroviruses* 17, 553-561.

generation of antibody diversity. *Science* 244, 1152-1157.

uninfected human volunteers. *Vaccine* 25, 510-518.

preventive vaccine trial. *Journal of Infectious Diseases* 191, 666-677.

Loré, K., Mascola, J.R., Wyatt, R.T., & Hedestam, G.B.K. (2010) Influence of novel CD4 binding-defective HIV-1 envelope glycoprotein immunogens on neutralizing antibody and T-cell responses in nonhuman primates. *Journal of Virology* 84, 1683-

Cafaro, A., Ensoli, B., & Robert-Guroff, M. (2009). Contribution of nonneutralizing vaccine-elicited antibody activities to improved protective efficacy in rhesus macaques immunized with Tat/Env compared with multigenic vaccines. *Journal of* 

Haigwood, N., Venzon, D., Kalyanaraman, V.S., Marthas, M.L., & Robert-Guroff, M. (2006). Evaluation of passively transferred, nonneutralizing antibody-dependent cellular cytotoxicity-mediating IgG in protection of neonatal rhesus macaques

induced antibodies inhibit clinical strains of HIV-1 in the presence of Fc receptorbearing effector cells and correlate inversely with HIV infection rate. *Journal of* 

Rhesus macaque polyclonal and monoclonal antibodies inhibit simian immunodeficiency virus in the presence of human or autologous rhesus effector

immunodeficiency virus (HIV) infection inhibits primary strains of HIV type 1 in

cellular cytotoxicity, plasma HIV type 1 RNA, and CD4+ lymphocyte count. *AIDS* 

Heyward, W.L., Jobes, D.V., Popovic, V., Self, S.G., Sinangil, F., Burke, D., & Berman, P.W. (2005). Correlation between immunologic responses to a recombinant glycoprotein 120 vaccine and incidence of HIV-1 infection in a phase 3 HIV-1

Koutsoukos, M., Pedneault, L., Vandepapeliere, P., McElrath, M.J., Spearman, P., Fuchs, J.D., Koblin, B.A., Blattner, W.A., Frey, S., Baden, L.R., Harro, C., Evans, T., & the NIAID HIV Vaccine Trials Network. (2007). Durable HIV-1 antibody and Tcell responses elicited by an adjuvanted multi-protein recombinant vaccine in

humoral immune responses in HIV type 1 subtype CRF01\_AE (E)-infected Thai patients with different rates of disease progression. *AIDS Research and Human Retroviruses* 19, 293-305.


Chung, A.W., Rollman, E., Center, R.J., Kent, S.J., & Stratov, I. (2009). Rapid degranulation of

Corti, D., Langedijk, J.P., Hinz, A., Seaman, M.S., Vanzetta, F., Fernandez-Rodriguez, B.M.,

Cranage, M.P., Fraser, C.A., Cope, A., McKay, P.F., Seaman, M.S., Cole, T., Nahmoud, A.N.,

Crotty, S., Aubert, R.D., Glidewell, J., & Ahmed, R. (2004). Tracking human antigen-specific

Crotty, S., Felgner, P., Davies, H., Glidewell, J., Villarreal, L., & Ahmed, R. (2003). Cutting

Dalgleish, A., Sinclair, A., Steel, M., Beatson, D., Ludlam, C., & Habeshaw, J. (1990). Failure

Delgado, M.F., Coviello, S., Monsalvo, A.C., Melendi, G.A., Hernandez, J.Z., Batalle, J.P.,

De Saint Basile, G., Menasche, G., & Fischer, A. (2010). Molecular mechanisms of biogenesis and exocytosis of cytotoxic granules. *Nature Reviews Immunology* 11, 568–579. Devito, C., Broliden, K., Kaul, R., Svensson, L., Johansen, K., Kiama., P, Kimani, J., Lopalco,

Doria-Rose, N.A., Klein, R.M., Daniels, M.G., O'Dell, S., Nason, M., Lagedes, A.,

Dorner, T. & Radbruch, A. (2007). Antibodies and B cell memory in viral immunity.

haemophiliac cohort. *Clinical and Experimental Immunology* 81, 5-10.

across human epithelial cells. *Journal of Immunology* 165, 5170-5176.

variables. *Journal of Virology* 84, 1631-1636.

*Retroviruses* 19, 293-305.

individuals. *PLoS One* 5, e8805.

*Immunological Methods* 286, 111-122.

*Immunology* 171, 4969-4973.

*Medicine* 15, 34-41.

*Immunity* 27, 384-392.

adjuvanted formulation. *Vaccine* 29, 1421-1430.

1202-1210.

humoral immune responses in HIV type 1 subtype CRF01\_AE (E)-infected Thai patients with different rates of disease progression. *AIDS Research and Human* 

NK cells following activation by HIV-specific antibodies. *Journal of Immunology* 182,

Silacci, C., Pinna, D., Jarrossay, D., Balla-Jhagjhoorsingh, S., Willems, B., Zekveld, M.J., Dreja, H., O'Sullivan, E., Pade, C., Orkin C., Jeffs, S.A., Montefiori, D.C., Davis, D., Weissenhorn, W., McKnight, A., Heeney, J.L., Sallusto, F., Sattentau, Q.J., Weiss, R.A., & Lanzavecchia, A. (2010). Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected

Hall, J., Giles, E., Voss, G., Page, M., Almond, N., & Shattock, R.J. (2011). Antibody responses after intravaginal immunization with trimeric HIV-1CN54 clade C gp140 in Carbopol gel are augmented by systemic priming or boosting with an

memory B cells: a sensitive and generalized ELISPOT system. *Journal of* 

edge: long-term B cell memory in humans after smallpox vaccination. *Journal of* 

of ADCC to predict HIV-associated disease progression or outcome in a

Diaz, L., Trento, A., Chang, H.Y., Mitzner, W., Ravetch, J., Melero, J.A., Irusta, P.M., & Polack, F.P. (2009). Lack of antibody affinity maturation due to poor Toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease. *Nature* 

L., Piconi, S., Bwayo, J.J., Plummer, F., Clerici, M., & Hinkula, J. (2000). Mucosal and plasma IgA from HIV-1-exposed uninfected individuals inhibit HIV-1 transcytosis

Bhattacharya, T., Migueles, S.A., Wyatt, R.T., Korber, B.T., Mascola, J.R., & Connors, M. (2010). Breadth of human immunodeficiency virus-specific neutralizing activity in sera: clustering analysis and association with clinical


HIV Envelope-Specific Antibody and Vaccine Efficacy 275

Hu, S-L. & Stamatatos, L. (2007). Prospects of HIV Env modification as an approach to HIV

Isitman, G., Chung, A.W., Navis, M., Kent, S.J., & Stratov, I. (2010). Pol as a target for

Jiang, X., Burke, V., Totrov, M., Williams, C., Cardozo, T., Gorny, M.K., Zolla-Pazner, S., &

Karnasuta, C., Paris, R.M., Cox, J.H., Nitayaphan, S., Pitisuttithum, P., Thogncharoen, P.,

Korber, B., Gaschen, B., Yusim K., Thakallapally, R., Kesmir, C., & Detours, V. (2001).

Kozlowski, P.A. & Neutra, M.R. (2003). The role of nucosal immunity in prevention of HIV

Kraynyak, K.A., Kutzler, M.A., Cisper, N.J., Khan, A.S., Draghia-Akli, R., Sardesal, N.Y.,

Kuhrt, D., Faith, S., Hattemer, A., Leone A., Sodora, D., Picker, L., Borghesi, L., & Cole, K.S.

Kuhrt, D., Faith, S.A., Leone, A., Rohankedkar, M., Sodora, D.L., Picker, L.J., & Cole, K.S.

Lambotte, O., Ferrari, G., Moog, C., Yates, N..L, Liao, H-X., Parks, R.J., Hicks, C.B., Owzard,

Lifson, A.R., Buchbinder, S.R., Sheppard, H.W., Mawle, A.C., Wilber, J.C., Stanley, M., Hart

responses in HIV-1 elite controllers. *AIDS* 23, 897-906.

antibody dependent cellular cytotoxicity responses in HIV-1 infection. *Virology* 412,

Kong, X. (2010). Conserved structural elements in the V3 crown of HIV-1 gp120.

Brown, A.E., Gurunathan, S., Tartaglia, J., Heyward, W.L., McNeil, J.G., Birx, D.L., de Souza, M.S., & TAVEG Thai AIDS Vaccine Evaluation Group, Thailand. (2005). Antibody-dependent cell-mediated cytotoxic responses in participants enrolled in a phase I/II ALVAC-HIV/AIDSVAX B/E prime-boost HIV-1 vaccine trial in

Evolutionary and immunological implications of contemporary HIV-1 variation.

Lewis, M.G., Yan, J., & Weiner, D.B. (2010). Systemic immunization with CCL27/CTACK modulates immune responses at mucosal sites in mice and

(2010). Naive and memory B cells in the rhesus macaque can be differentiated by surface expression of CD27 and have differential responses to CD40 ligation.

(2010a). Evidence of early B-cell dysregulation in simian immunodeficiency virus infection: rapid depletion of naïve and memory B-cell subsets with delayed reconstitution of the naïve B-cell population. *Journal of Virology* 84, 2466-2476. Lai, L., Vodros, D., Kozlowski, P.A., Montefiori, D.C., Wilson, R.L., Akerstrom, V.L.,

Chennareddi, L., Yu, T., Kannanganat, S., Ofielu, L., Villinger, F., Wyatt, L.S., Moss, B., Amara, R.R., & Robinson, H.L. (2007). GM-CSF DNA: an adjuvant for higher avidity IgG, rectal IgA, and increased protection against the acute phase of a SHIV-89.6P challenge by a DNA/MVA immunodeficiency virus vaccine. *Virology* 369,

K., Tomaras, G.D., Montefiori, D.C., Haynes, B.F., & Delfraissy, J-F. (2009). Heterogeneous neutralizing antibody and antibody-dependent cell cytotoxicity

C.E., Hessol, N.A., & Holmberg, S.D. (1991). Long-term human immunodeficiency virus infection in asymptomatic homosexual and bisexual men with normal CD4+ lymphocyte counts: immunologic and virologic characteristics. *Journal of Infectious* 

vaccine design. *Current HIV Research* 5, 507-513.

*Nature Structural & Molecular Biology* 17, 955-962.

transmission. *Current Molecular Medicine* 3, 217-228.

*Journal of Immunological Methods* 363, 166-176.

Thailand. *Vaccine* 23, 2522-2529.

*British Medical Bulletin* 58, 19-42.

macaques. *Vaccine* 28, 1942-1951.

110-116.

153-167.

*Diseases* 163, 959-965.


Gomez-Roman, V.R. , Florese, R.H., Patterson, L.J., Peng, B., Venzon, D., Aldrich, K., &

Gomez-Roman, V.R., Florese, R.H., Peng, B., Montefiori, D., Kalyanaraman, V.S., Venzon,

Gomez-Roman, V.R., Patterson, L.J., Venzon, D., Liewehr, D., Aldrich, K., Florese, R., &

macaques challenged with SIVmac251. *Journal of Immunology* 174, 2185-2189. Gorny, M.K., Stamatatos, L., Volsky, B., Revesz, K., Williams, C., Wang, X., Cohen, S.,

Gray, E.S., Moore, P.L., Choge, I.A., Decker, J.M., Bibollet-Ruche, F., Li, H., Leseka, N.,

Griffiths, G.M., Berek, C., Kaartinen, M., & Milstein, C. (1984). Somatic mutation and the maturation of immune response to 2-phenyl oxazolone. *Nature* 312, 271-275. Haase, A.T. (2005). Perils at mucosal front lines for HIV and SIV and their hosts. *Nature* 

Hessell A.J., Hangartner, L., Hunter, M., Havenith, C.E.G., Beurskens, F.J., Bakker, J.M.,

Hessell, A.J., Poignard, P., Hunter, M., Hangartner, L., Tehrani, D.M., Bleeker, W.K., Parren,

Hidajat, R., Xiao, P., Zhou, Q., Venzon, D., Summers, L.E., Kalyanaraman, V.S., Montefiori,

Hioe, C.E., Wrin, T., Seaman, M.S., Yu, X., Wood, B., Self, S., Williams, C., Gorny, M.K., &

Hocini, H., Belec, L., Iscaki, S., Garin, B., Pillot, J., Becquart, P., & Bomsel, M. (1997). High-

activities against multiple HIV-1 subtypes. *PLoS One* 5, e10254.

type 1 subtype C infection. *Journal of Virology* 81, 6187-6196.

antibody protection against HIV. *Nature* 449, 101-105.

macaques. *Journal of Virology* 83, 791-801.

*Immunological Methods* 308, 53-67.

*Journal of Virology* 79, 5232-5237.

*Reviews Immunology* 5, 783-792.

15, 951-955,

13,1179-1185.

*Acquired Immune Deficiency Syndromes* 43, 270-277.

Robert-Guroff, M. (2006). A simplified method for the rapid fluorometric assessment of antibody-dependent cell-mediated cytotoxicity. *Journal of* 

D., Srivastava, I., Barnett, S.W., & Robert-Guroff, M. (2006). An adenovirus-based HIV subtype-B prime/boost vaccine regimen elicits antibodies mediating broad antibody-dependent cellular cytotoxicity against non-subtype-B strains. *Journal of* 

Robert-Guroff, M. (2005). Vaccine-elicited antibodies mediate antibody-dependent cellular cytotoxicity correlated with significantly reduced acute viremia in rhesus

Staudinger, R., & Zolla-Pazner, S. (2005). Identification of a new quaternary neutralizing epitope on human immunodeficiency virus type 1 virus particles.

Treurnicht, F., Mlisana, K., Shaw, G.M., Karim, S.S., Williamson, C., & Morris, L. (2007). Neutralizing antibody responses in acute human immunodeficiency virus

Lanigan, C.M.S., Landucci, G., Forthal, D.N., Parren, P.W.H.I., Marx, P.A., & Burton, D.R. (2007). Fc receptor but not complement binding is important in

P.W.H.I., Marx, P.A., & Burton, D.R. (2009). Effective, low-titer antibody protection against low-dose repeated mucosal SHIV challenge in macaques. *Nature Medicine*

D.C., & Robert-Guroff, M. (2009). Correlation of vaccine-elicited systemic and mucosal nonneutralizing antibody activities with reduced acute viremia following intrarectal simian immunodeficiency virus SIVmac251 challenge of rhesus

Zolla-Pazner, S. Anti-V3 monoclonal antibodies display broad neutralizing

level ability of secretory IgA to block HIV type 1 transcytosis: contrasting secretory IgA and IgG responses to glycoprotein 160. *AIDS Research and Human Retroviruses*


HIV Envelope-Specific Antibody and Vaccine Efficacy 277

Niwa, R., Natsume, A., Uehara, A., Wakitani, M., Iida, S., Uchida, K., Satoh, M., & Shitara, K.

Ojo-Amaize, E.A., Nishanian, P., Keith, D.E., Jr, Houghton, R.L., Heitjan, D.F., Fahey, J.L., &

Parren, P.W.H.I., Marx, P.A., Hessell, A.J., Luckay, A., Harouse J., Cheng-Mayer, C., Moore,

giving complete neutralization in vitro. *Journal of Virology* 75, 8340-8347. Patterson, L.J., Malkevitch, N., Venzon, D., Pinczewski, J., Gómez-Román, V.R., Wang, L.,

Pierce, S.K., & Liu, W. (2010). The tipping points in the initiation of B cell signalling: how small changes make big differences. *Nature Reviews Immunology* 10, 767-777. Pope, M., & Haase, A.T. (2003). Transmission, acute HIV-1 infection and the quest for

Radbruch, A., Muehlinghaus, G., Luger, E.O., Inamine, A., Smith, K.G., Dörner, T., & Hiepe,

Rerks-Ngarm, S., Pitisuttithum, P., Nitayaphan, S., Kaewkungwal, J., Chiu, J., Paris, R.,

Rook, A.H., Lane, H.C., Folks, T., McCoy, S., Alter, H., & Fauci, A.S. (1987). Sera from

Sather, D.N., Armann, J., Ching, L.K., Mavrantoni, A., Sellhorn, G., Caldwell, Z., Yu, X.,

immunodeficiency virus type 1 infection. *Journal of Virology* 83, 757-769. Sawyer, L.A., Katzenstein, D.A., Hendry, R.M., Boone, E.J., Vujcic, L.K., Williams, C.C.,

F. (2006).Competence and competition: the challenge of becoming a long-lived

Premsri, N., Namwat, C., de Souza, M., Adams, E., Benenson, M., Gurunathan, S., Tartaglia, J., McNeil, J.G., Francis, D.P., Stablein, D., Birx, D.L., Chunsuttiwat, S., Khamboonruang, C., Thongcharoen, P., Robb, M.L., Michael, N.L., Kunasol, P., & Kim, J.H. for the NOPH-TAVEG Investigators. (2009). Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. *New England Journal of* 

HTLV-III/LAV antibody-positive individuals mediate antibody-dependent cellular cytotoxicity against HTLV-III/LAV-infected T cells. *Journal of Immunology* 138,

Wood, B., Self, S., Kalams, S., & Stamatatos, L. (2009). Factors associated with the development of cross-reactive neutralizing antibodies during human

Zeger, S.L., Saah, A.J., Rinaldo, C.R., Jr., Phair, J.P., Giorgi, J.V., & Quinnan, G.V., Jr.

706.

*Immunological Methods* 306, 151-160.

boosting. *Journal of Virology* 78, 2212-2221.

strategies to prevent infection. *Nature Medicine* 9, 847-852.

plasma cell. *Nature Reviews Immunology* 6, 741-750.

*of Immunology* 139, 2458-2463.

*Medicine* 361 2209-2220.

1064-1067.

R., Bussaratid, V., Singharaj, P., el Habib, R., Gurunathan, S., Heyward, W., Birx, D., McNeil, J., & Brown, A.E., for the Thai AIDS Vaccine Evaluation Group. (2004). Safety and immunogenicity of an HIV subtype B and E prime-boost vaccine combination in HIV-negative Thai adults. *The Journal of Infectious Diseases* 190, 702-

(2005). IgG subclass-independent improvement of antibody-dependent cellular cytotoxicity by fucose removal from Asn297-linked oligosaccharides. *Journal of* 

Giorgi, J.V. (1987). Antibodies to human immunodeficiency virus in human sera induce cell-mediated lysis of human immunodeficiency virus-infected cells. *Journal* 

J.P., & Burton, D.R. (2001). Antibody protect smacaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels

Kalyanaraman, V.S., Markham, P.D., Robey, F.A., & Robert-Guroff, M. (2004). Protection against mucosal simian immunodeficiency virus SIV(mac251) challenge by using replicating adenovirus-SIV multigene vaccine priming and subunit


Ljunggren, K., Moschese, V., Broliden, P.A., Giaquinto, C., Quinti, I., Fenyö, E.M., Wahren,

Lopalco, L. (2004). Humoral immunity in HIV-1 exposure: cause or effect of HIV resistance?

Lyerly, H.K., Reed, D.L., Matthers, T.J., Langlois, A.J., Ahearne, P.A., Petteway, S.R., Jr., &

Mascola, J.R., D'Souza, P., Gilbert, P., Hahn, B.H., Haigwood, N.L., Morris, L., Petropoulos,

Mascola, J.R., Lewis, M.G., Stiegler, G., Harris, D., VanCott, T.C., Hayes, D., Louder, M.K.,

Mascola, J.R. & Montefiori, D.C. (2010). The role of antibodies in HIV vaccines. *Annual* 

Mascola, J.R., Stiegler, G., VanCott, T.C., Katinger, H., Carpenter, C.B., Hanson, C.E., Beary,

by passive infusion of neutralizing antibodies. *Nature Medicine* 5, 207-210. McElrath, M.J. & Haynes, B.F. (2010). Induction of immunity to human immunodeficiency

McHeyzer-Williams, L.J., & McHeyzer-Williams, M.G. (2005). Antigen-specific memory B

Meng, G., Wei, X., Wu, X., Sellers, M.T., Decker, J.M., Moldoveanu, Z., Orenstein, J.M.,

Miyazawa, M., Lopalco, L., Mazzotta, F., Caputo, S.L., Veas, F., & Clerici, M., for the ESN

Muster, T., Steindl, F., Purtscher, M., Trkola, A., Klima, A., Himmler, G., Ruker, F., &

Nag, P., Kim, J., Sapiega, V., Landay, A.L., Bremer, J.W., Mestecky, J., Reichelderfer, P.,

Nitayaphan, S., Pitisuttithum, P., Karnasuta, C., Eamsila, C., de Souza, M., Morgan, P.,

immunodeficiency virus type 1. *Journal of Virology* 67, 6642-6647.

*Current HIV Research* 2, 127-139.

*Virology* 79, 10103-10107.

*Medicine* 8, 150-156.

individuals. *AIDS* 23, 161-175.

*Journal of Virology* 73, 4009-4018.

*Review of Immunology* 28, 412-444.

virus type-1 by vaccination. *Immunity* 33, 542-554.

loads. *Journal of Infectious Diseases* 190, 1970-1978.

cell development. *Annual Review of Immunology* 23, 487-513.

409-422.

B., Rossi, P., and Jondal, M. (1990). Antibodies mediating cellular cytotoxicity and neutralization correlate with a better clinical stage in children born to human immunodeficiency virus-infected mothers. *Journal of Infectious Diseases* 161,198-202.

Weinhold, K.J. (1987). Anti-GP 120 antibodies from HIV seropositive individuals mediate broadly reactive anti-HIV ADCC. *AIDS Research and Human Retroviruses* 3,

C.J., Polonis, V.R., Sarzotti M., & Montefiori, D.C. (2005). Recommendations for the design and use of standard virus panels to assess neutralizing antibody responses elicited by candidate human immunodeficiency virus type 1 vaccines. *Journal of* 

Brown, C.R., Sapan, C.V., Frankel S.S., Lu, Y., Robb, M.L., Katinger, H., & Birx, D.L. (1999). Protection of macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies.

H., Hayes, D., Frankel, S.S., Birx, D.L., & Lewis, M.G. (2000). Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus

Graham, M.F., Kappes, J.C., Mestecky, J., Shaw, G.M., & Smith, P.D. (2002). Primary intestinal epithelial cells selectively transfer R5 HIV-1 to CCR5+ cells. *Nature* 

Study Group. (2009). The "immunologic advantage" of HIV-exposed seronegative

Katinger, H. (1993). A conserved neutralizing epitope on gp41 of human

Kovacs, A., Cohn, J., Weiser, B., & Baum, L.L. (2004). Women with cervicovaginal antibody-dependent cell-mediated cytotoxicity have lower genital HIV-1 RNA

Polonis, V., Benenson, M., VanCott, T., Ratto-Kim, S., Kim, J., Thapinta, D., Garner,

R., Bussaratid, V., Singharaj, P., el Habib, R., Gurunathan, S., Heyward, W., Birx, D., McNeil, J., & Brown, A.E., for the Thai AIDS Vaccine Evaluation Group. (2004). Safety and immunogenicity of an HIV subtype B and E prime-boost vaccine combination in HIV-negative Thai adults. *The Journal of Infectious Diseases* 190, 702- 706.


HIV Envelope-Specific Antibody and Vaccine Efficacy 279

Trkola, A., Purtscher, M., Muster, T., Ballaun, C., Buchacher, A., Sullivan, N., Srinivasan, K.,

Vagenas, P., Williams, V.G., Piatak, M., Jr., Bess, J.W., Jr., Lifson, J.D., Blanchard, J.L., Gettie,

Volgmann, T., Klein-Struckmeier, A., & Mohr, H. (1989). A fluorescence-based assay for

Walker, L.M. & Burton, D.R. (2010). Rational antibody-based HIV-1 vaccine design: current approaches and future directions. *Current Opinion in Immunology* 22, 358-366. Walker, L.M., Phogat, S.K., Chan-Hui, P-Y., Wagner, D., Phung, P., Goss, J.L., Wrin, T.,

Wang, S., Bertley, F.M.N., Kozlowski, P.A., Herrmann, L., Manson, K., Mazzara, G., Piatak,

Weinhold, K.J. (1990). Nonrestricted forms of anti-HIV-1 cytotoxicity. In: *Techniques in HIV* 

Wilkinson, R.W., Lee-MacAry, A.E., Davies, D., Snary, D., & Ross, E.L. (2001). Antibody-

Wu, X., Yang, Z., Li, Y., Hogerkorp, C-M., Schief, W.R., Seaman, M.S., Zhou, T., Schmidt,

Xiao, P., Zhao, J., Patterson, L.J., Brocca-Cofano, E., Venzon, D., Kozlowski, P.A., Hidajat, R.,

SHIV89.6P challenge in rhesus macaques*. Journal of Virology* 84, 7161-7173. Yamada, T., Watanabe, N., Nakamura, T., & Iwamoto, A. (2004). Antibody-dependent

Zhao, J., Lai, L., Amara, R.R., Montefiori, D.C., Villinger, F., Chennareddi, L., Wyatt, L.S.,

fluorophores. *Journal of Immunological Methods* 258, 183-191.

surface. *Journal of Immunology* 172 2401-2406.

immunodeficiency virus type 1. *Journal of Virology* 70, 1100-1108.

*Deficiency Syndromes* 52, 433-442.

*Methods* 119, 45-51.

*Science* 326, 285-289.

*Retroviruses* 20, 846-859.

88-8, New York.

856-861.

Sodroski, J. Moore, J.P., & Katinger, H. (1996). Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human

A., & Robbiani, M. (2009). Tonsillar application of AT-2 SIV affords partial protection against rectal challenge with SIVmac239. *Journal of Acquired Immune* 

quantitation of lymphokine-activated killer cell activity. *Journal of Immunological* 

Simek, M.D., Fling, S., Mitchan, J.L., Lehrman, J.K., Priddy, F.H., Olsen, O.A., Frey, S.M., Hammond, P.W., Protocol G Principal Investigators, Kaminsky, S., Zamb, T., Moyle, M., Koff, W.C., Poignard, P., & Burton, D.R. (2009). Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target.

M., Johnson, R.P., Carville, A., Mansfield, K., & Aldovini, A. (2004). An SHIV DNA/MVA rectal vaccination in macaques provides systemic and mucosal virusspecific responses and protection against AIDS. *AIDS Research and Human* 

*Research,* Aldovini, A & Walker, BD, pp. 187 – 199. Stockton Press, ISBN 0-935859-

dependent cell-mediated cytotoxicity: a flow cytometry-based assay using

S.D., Wu, L., Xu, L., Longo, N.S., McKee, K., O'Dell, S., Louder, M.K., Wycuff, D.L., Feng, Y., Nason, M., Doria-Rose, N., Connors, M., Kwong, P.D., Roederer, M., Wyatt, R.T., Nabel, G.J., & Mascola, J.R. (2010). Rational design of envelope identifies broadly neutralizing human monoclonal antiobides to HIV-1. *Science* 329,

Demberg, T., & Robert-Guroff, M. (2010). Multiple vaccine-elicited nonneutralizing antienvelope antibody activities contribute to protective efficacy by reducing both acute and chronic viremia following simian/human immunodeficiency virus

cellular cytotoxicity via humoral immune epitope of Nef protein expressed on cell

Moss, B., & Robinson, H.L. (2009). Preclinical studies of human immunodeficiency

(1990). Possible beneficial effects of neutralizing antibodies and antibodydependent, cell-mediated cytotoxicity in human immunodeficiency virus infection. *AIDS Research and Human Retroviruses* 6, 341-356.


Scheid, J.F., Mouquet, H., Feldhahn, N., Seaman, M.S., Velinzon, K., Pietzsch, J., Ott, R.G.,

Seaman, M.S., Janes, H., Hawkins, N., Grandpre, L.E., Devoy, C., Giri, A., Coffey, R.T.,

assessment of neutralizing antibodies. *Journal of Virology* 84, 1439-1452. Shattock, R.J., Griffin, G.E., & Gorodeski, G.I. (2000). In vitro models of mucosal HIV

Shen, X., & Tomaras, G.D. (2011). Alterations of the B-cell response by HIV-1 replication.

Siegrist, C.A., Pihlgren, M., Tougne, C., Efler, S.M., Morris, M.L., AlAdhami, M.J., Cameron,

Stratov, I., Chung, A., & Kent, S.J. (2008). Robust NK cell-mediated human

Titanji, K., De Milito, A., Cagigi, A., Thorstensson, R., Grutzmeier, S., Atlas, A., Hejdeman,

Titanji, K., Velu, V., Chennareddi, L., Vijay-Kumar, M., Gewirtz, A.T., Freeman, G.J., &

Tomaras, G.D., Yates, N.L., Liu, P., Qin, L., Fouda, G.G., Chavez, L.L., Decamp, A.C., Parks,

process of human anti-hepatitis B vaccine response. *Vaccine* 23, 615-622. Simek, M.D., Rida, W., Priddy, F.H., Pung, P., Carrow, E., Laufer, D.S., Lehrman, J.K., Boaz,

D.W., Cooper, C.L., Heathcote, J., Davis, H.L., & Lambert, P.H. (2004). Coadministration of CpG oligonucleotides enhances the late affinity maturation

M., Tarragona-Fiol, T., Miiro, G., Birungi, J., Pozniak, A., McPhee, D.A., Manigart, O., Karita, E., Inwoley, A., Jaoko, W., DeHovitz, J., Bekker, L-G., Pitisuttithum, P., Paris, R., Walker, L.M., Poignard, P., Wrin, T., Fast, P.E., Burton, D.R., & Koff, W.C.

immunodeficiency virus (HIV)-specific antibody-dependent responses in HIV

B., Kroon, F.P., Lopalco, L., Nilsson, A., & Chiodi, F. (2006). Loss of memory B cells impairs maintenance of long-term serologic memory during HIV-1 infection. *Blood*

Amara, R.R. (2010). Acute depletion of activated memory B cells involves the PD-1 pathway in rapidly progressing SIV-infected macaques. *Journal of Clinical* 

R.J., Ashley, V.C., Lucas, J.T., Cohen, M., Eron, J., Hicks, C.B., Liao, H-X., Self, S.G., Landucci, G., Forthal, D.N., Weinhold, K.J., Keele, B.F., Hahn, B.H., Greenberg, M.L., Morris, L., Abdool Karim, S.S., Blattner, W.A., Montefiori, D.C., Shaw, G.M., Perelson, A.S., & Haynes, B.F. (2008). Initial B-cell responses to transmitted human immunodeficiency virus type 1: Virion-binding immunoglobulin M (IgM) and IgG antibodies followed by plasma anti-gp41 antibodies with ineffective control of

*AIDS Research and Human Retroviruses* 6, 341-356.

transmission. *Nature Medicine* 6,607-608.

(2009). *Journal of Virology* 83, 7337-7348.

108, 1580-1587.

*Investigation* 120, 3878-3890.

infected subjects. *Journal of Virology* 82, 5450–5459.

initial viremia. *Journal of Virology* 82, 12449-12463.

*Current HIV/AIDS Reports* 8, 23-30.

*Nature* 458, 636-640.

(1990). Possible beneficial effects of neutralizing antibodies and antibodydependent, cell-mediated cytotoxicity in human immunodeficiency virus infection.

Anthony, R.M., Zebroski ,H., Hurley, A., Phogat, A., Chakrabarti, B., Li, Y., Connors, M., Pereyra ,F., Walker, B.D., Wardemann, H., Ho, D., Wyatt, R.T., Mascola, J.R., Ravetch, J.V., & Nussenzweig, M.C. (2009). Broad diversity of neutralizing antibodies isolated from memory B cells in HIV-infected individuals.

Harris, L., Wood, B., Daniels, M.G., Bhattacharya, T., Lapedes, A., Polonis, V.R., McCutchan, F.E., Gilbert, P.B., Self, S.G., Korber, B.T., Montefiori, D.C., & Mascola, J.R.. (2010). Tiered categorization of a diverse panel of HIV-1 Env pseudoviruses for


virus/AIDS vaccines: inverse correlation between avidity of anti-Env antibodies and peak postchallenge viremia. *Journal of Virology* 83, 4102-4111.

**11** 

*USA* 

**in HIV Infection** 

Vishwanath Venketaraman et al.\*

**Role of Cytokines and Chemokines** 

*College of Osteopathic Medicine of the Pacific, Western University of Health Sciences,* 

Human immunodeficiency virus (HIV) is the cause of acquired immunodeficiency syndrome (AIDS). Blood monocytes and resident macrophages are important *in vivo* cell targets for HIV infection and their role in AIDS pathogenesis are well documented. These cells of innate immune defenses usually survive HIV infection, serve as a major virus reservoir, and function as immunoregulatory cells through secretion of several pro-inflammatory cytokines and chemokines in response to HIV infection, thereby recruiting and activating new target cells for the virus, including CD4+ T cells. This review describes the alterations in the synthesis of host cytokines and chemokines following HIV infection thereby favoring successful survival of the virus inside the host and enhancing the susceptibility of the host to opportunistic infections.

HIV infects immune cells of the macrophage and T-cell lineage. Entry into these cells requires CD4 as a receptor in addition to a co-receptor which most frequently is either chemokine receptor CCR5 or CXCR4 (Gorry & Ancuta, 2011). Binding and entry into human cells requires the two HIV envelope glycoproteins gp120 and gp41. Gp41 possesses a transmembrane domain and is associated with the viral envelope while Gp120 is present in association with Gp41 but does not insert into or contact the viral membrane (Tagliamonte *et al*; 2010). These two viral glycoproteins are present in HIV as tetramers. Therefore, three Gp41 molecules associate within the viral membrane, while three molecules of Gp120 associate with Gp41 (Tagliamonte *et al*; 2010). To facilitate HIV-1 entry into human cells, Gp120 binds to human cellular CD4 with high affinity. Binding causes a conformational change in Gp120 that reveals a co-receptor binding site. Binding to one of the chemokine

Devin Morris2, Clare Donohou5, Andrea Sipin4, Steven Kung4, Hyoung Oh2, Mesharee Franklin2, John P. Murad3, Fadi T. Khasawneh3, Beatrice Saviola1, Timothy Guilford6 and Clare Donahue6

*1College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, USA*

**1. Introduction** 

 \*

<sup>5</sup>*Pitzer College, USA* 6*Your Energy Systems, USA*

**2. HIV and chemokine receptors** 

<sup>2</sup>*Graduate College of Biomedical Sciences, USA*

<sup>3</sup>*College of Pharmacy, Western University of Health Sciences, USA* 4*California State Polytechnic University, USA*


## **Role of Cytokines and Chemokines in HIV Infection**

Vishwanath Venketaraman et al.\*

*College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, USA* 

## **1. Introduction**

280 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

Zolla-Pazner, S. & Cardozo, T. (2010). Structure-function relationships of HIV-1 envelope

Zwick, M.B., Labrijn, A.F., Wang, M., Spenlehauer, C., Saphire, E.O., Binley, J.M., Moore,

and peak postchallenge viremia. *Journal of Virology* 83, 4102-4111.

527-535.

10892-10905.

virus/AIDS vaccines: inverse correlation between avidity of anti-Env antibodies

sequence-variable regions refocus vaccine design. *Nature Reviews Immunology* 10,

J.P., Stiegler, G., Katinger, H., Burton, D.R., & Parren, P.W.H.I. (2001). Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41. *Journal of Virology* 75,

> Human immunodeficiency virus (HIV) is the cause of acquired immunodeficiency syndrome (AIDS). Blood monocytes and resident macrophages are important *in vivo* cell targets for HIV infection and their role in AIDS pathogenesis are well documented. These cells of innate immune defenses usually survive HIV infection, serve as a major virus reservoir, and function as immunoregulatory cells through secretion of several pro-inflammatory cytokines and chemokines in response to HIV infection, thereby recruiting and activating new target cells for the virus, including CD4+ T cells. This review describes the alterations in the synthesis of host cytokines and chemokines following HIV infection thereby favoring successful survival of the virus inside the host and enhancing the susceptibility of the host to opportunistic infections.

## **2. HIV and chemokine receptors**

HIV infects immune cells of the macrophage and T-cell lineage. Entry into these cells requires CD4 as a receptor in addition to a co-receptor which most frequently is either chemokine receptor CCR5 or CXCR4 (Gorry & Ancuta, 2011). Binding and entry into human cells requires the two HIV envelope glycoproteins gp120 and gp41. Gp41 possesses a transmembrane domain and is associated with the viral envelope while Gp120 is present in association with Gp41 but does not insert into or contact the viral membrane (Tagliamonte *et al*; 2010). These two viral glycoproteins are present in HIV as tetramers. Therefore, three Gp41 molecules associate within the viral membrane, while three molecules of Gp120 associate with Gp41 (Tagliamonte *et al*; 2010). To facilitate HIV-1 entry into human cells, Gp120 binds to human cellular CD4 with high affinity. Binding causes a conformational change in Gp120 that reveals a co-receptor binding site. Binding to one of the chemokine

John P. Murad3, Fadi T. Khasawneh3, Beatrice Saviola1, Timothy Guilford6 and Clare Donahue6 *1College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, USA*

<sup>2</sup>*Graduate College of Biomedical Sciences, USA*

<sup>\*</sup> Devin Morris2, Clare Donohou5, Andrea Sipin4, Steven Kung4, Hyoung Oh2, Mesharee Franklin2,

<sup>3</sup>*College of Pharmacy, Western University of Health Sciences, USA* 4*California State Polytechnic University, USA*

<sup>5</sup>*Pitzer College, USA*

<sup>6</sup>*Your Energy Systems, USA*

Role of Cytokines and Chemokines in HIV Infection 283

CCL2 or monocyte chemotactic protein-1 (MCP-1), of the C-C chemokine family, is a cytokine with the ability to influence both innate and adaptive immune responses (Daly *et al*; 2003). This chemokine is produced by a variety of different cell types including endothelial cells, fibroblasts, epithelial cells, smooth muscle cells, mesangial cells, astrocytic cells, and microglial cells. However, despite the wide range of cell types that have the ability to manufacture CCL2, the majority of CCL2 is produced by macrophages and monocytes

Although technically a chemokine, CCL2 is often classified as an inflammatory cytokine due to its ability to attract various leukocytes (monocytes, memory T cells, basophils, natural killer (NK) cells etc.) to sites of trauma, bacterial and mycobacterial infection, toxin exposure, and ischemia (Daly *et al*; 2003, Deshmane *et al*; 2009, Mahad *et al*; 2003,, Charo *et al*; 2006). Besides attracting various leukocytes, CCL2 also specifically regulates the infiltration of monocytes, memory T lymphocytes, and NK cells (Deshmane *et al*; 2009). In addition, CCL2 has been found to have a profound effect on the differentiation of naïve helper T cells (Daly *et al*; 2003). Interestingly, studies have found that CCL2 expression tends to lead to the development of a Th2 immune response. Taking this tendency into account, it seems likely that CCL2 concentrations in HIV patients, which Weiss *et al*. (Weiss *et al*; 1997) found were correlated with viral load, can be linked to the Th1 to Th2 cytokine response switch often

CCL2 has also been found to play other roles in HIV pathogenesis. Eugenin *et al.* (Eugenin *et al*; 2006) noted that CCL2 in the central nervous system (CNS) attracts HIV-infected leukocytes into the brain thereby increasing the rate of HIV-1-infected cell-dispersal and causing the eventual impairment of the blood-brain-barrier. In fact, multiple studies indicate that CCL2 is largely responsible for the development of HIV encephalitis (HIVE), HIV-1 associated dementia (HAD), and NeuroAIDS (Deshmane *et al*; 2009, Eugenin *et al*; 2006).

Under normal conditions, the immune system utilizes a Th1 subset response to viral infections. Activated antigen presenting cells (APC) secrete interleukin-12 (IL-12) which causes Th cell differentiation into the Th1 subset of cells (Clerci et al; 1993). These Th1 cells then secrete a characteristic Th1 profile of cytokines consisting of interleukin-2 (IL-2), interferon-gamma (IFN-), and tumor necrosis factor-beta (TNF-β). IL-2 induces proliferation of naïve Th cells (T0), amplifying the Th response. IFN- induces further IL-12 production in activated APCs, amplifying the Th1 response, and suppressing any Th2 response. IFN- also plays an important role in the activation of cytotoxic TC cells which

In individuals infected with HIV, the normal Th1 response to viral infection is shifted to a Th2 response (Klein et al; 1997, Osakwe et al; 2010). Measurement of the serum cytokine levels of HIV infected patients has revealed an increase in Th2 cytokines as well as a decrease in Th1 cytokines (Klein et al; 1997, Osakwe et al; 2010). Assays have shown elevated serum IL-4 levels in HIV seropositive individuals (Clerci et al; 1993). IL-4 in the presence of proliferating T0 cells leads to their differentiation into the Th2 subset. Th2 cells promote B-cell proliferation, class switching, and eosinophil activation (Clerci et al; 1993). This Th2 response is not appropriate for control of intracellular pathogens such as HIV, and

**3. Chemokine ligand-2 (CCL2)** 

observed in HIV-1-infected patients (Deshmane *et al*; 2009).

**4. HIV and the Th1 to Th2 Cytokine shift** 

so allows it to persist and spread in CD4+ T-cells.

destroy virally infected cells.

(Deshmane *et al*; 2009).

receptors is then facilitated which in turn induces a conformational change in the glycoprotein gp41 N-terminus (Tagliamonte *et al*; 2010). A fusion peptide portion of gp41 inserts into the host cell membrane and lowers energy that is required for fusion of the host and viral membranes (Tagliamonte *et al*; 2010). The viral core is then translocated into the cytoplasm of the host cell.

HIV-1 viral variants can in general use either the CCR5 or CXCR4 co-receptor for entry into human cells (Gorry & Ancuta, 2011). They may also at times use a variety of other chemokine receptors for entry (Gorry *et al*; 2007). The normal function of chemokine receptors is to bind chemokines that target immune cells to areas of inflammation within the human body. Certain HIV-1 viruses may have an increased ability to either bind the CCR5 receptor and are known as R5 viruses, bind to CXCR4 and are known as X4 viruses, or bind with mixed affinity to either receptor. This differential affinity lies within the specific alterations in amino acid sequence of the gp120 glycoprotein (Gorry & Ancuta, 2011). Although not correlating completely, macrophage tropic HIV-1 viruses generally are R5 and T-cell tropic viruses are X4 viruses (Gorry & Ancuta, 2011). Early during infection R5 viruses predominate, and it appears that there is some mechanism which selects these viruses during the transmission process (Grivel *et al*; 2010). For example, an HIV-1 naive individual may be exposed to both R5 and X4 virus particles from an infected individual, but only become infected with the R5 viral particles. There may be multiple factors which affect this process, including co-receptor availability and pH at the sites of infection. Acidic pH may act to disrupt the cationic charge present in gp120 proteins which bind to CXCR4 preferentially (Kwong *et al*; 2010, Edo-Matas *et al*; 2010). R5 viruses are also prominent during chronic infection. X4 viruses or R5X4 viruses which have mixed affinity can arise later during infection and often their presence precedes disease progression and immune cell depletion (Mariani, 2010).

Deletion of the CCR5 receptor can in many cases abrogate infection with HIV-1 completely. It has previously been identified that individuals homozygous for a 32 base pair deletion within the *CCR5* gene resulting in a nonfunctional CCR5 molecule are resistant to infection with the HIV-1 virus, though there have been some instances where homozygous *CCR532* individuals were infected with X4 HIV-1 (Samson, 1996). Additionally, people who carry one allele of *CCR532* have a slower progression of the disease. This knowledge has led to the development of treatments for HIV-1 infection. Transplantation of stem cells from individuals homozygous for *CCR532* into CCR5 HIV-1 positive individuals resulted in clearing of the virus from the infected patients (Hutter, 2009). Monoclonal antibodies against CCR5 to inhibit binding of HIV-1 to this co-receptor are a potential therapeutic to prevent viral entry and replication (Tenorio, 2011, Suleiman, 2010). In addition there are plans to use an individual's native stem cells as a target to disrupt the *CCR5* receptor gene which can then be transplanted back into the HIV infected patient to effect elimination of the HIV-1 virus from the body (Cannon and June, 2011). Pitfalls of these therapies include the problem that the CCR5 chemokine receptor has a native function within the body, and that disrupting this receptor may cause unforeseen deficits in the immune system. In fact, lack of the CCR5 receptor gene has been associated with increased risk of severe infection with other viruses such as the West Nile Virus, and certain flaviviruses (Lin *et al*; 2008, Kindberg *et al*; 2008). Notwithstanding the previously mentioned caveat, interference with the CCR5 receptor may indeed be a promising target to treat those infected with HIV-1 as well as prevent infection for those exposed to the virus via sexual activity, needle sharing, or accidental hospital transmission.

## **3. Chemokine ligand-2 (CCL2)**

282 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

receptors is then facilitated which in turn induces a conformational change in the glycoprotein gp41 N-terminus (Tagliamonte *et al*; 2010). A fusion peptide portion of gp41 inserts into the host cell membrane and lowers energy that is required for fusion of the host and viral membranes (Tagliamonte *et al*; 2010). The viral core is then translocated into the

HIV-1 viral variants can in general use either the CCR5 or CXCR4 co-receptor for entry into human cells (Gorry & Ancuta, 2011). They may also at times use a variety of other chemokine receptors for entry (Gorry *et al*; 2007). The normal function of chemokine receptors is to bind chemokines that target immune cells to areas of inflammation within the human body. Certain HIV-1 viruses may have an increased ability to either bind the CCR5 receptor and are known as R5 viruses, bind to CXCR4 and are known as X4 viruses, or bind with mixed affinity to either receptor. This differential affinity lies within the specific alterations in amino acid sequence of the gp120 glycoprotein (Gorry & Ancuta, 2011). Although not correlating completely, macrophage tropic HIV-1 viruses generally are R5 and T-cell tropic viruses are X4 viruses (Gorry & Ancuta, 2011). Early during infection R5 viruses predominate, and it appears that there is some mechanism which selects these viruses during the transmission process (Grivel *et al*; 2010). For example, an HIV-1 naive individual may be exposed to both R5 and X4 virus particles from an infected individual, but only become infected with the R5 viral particles. There may be multiple factors which affect this process, including co-receptor availability and pH at the sites of infection. Acidic pH may act to disrupt the cationic charge present in gp120 proteins which bind to CXCR4 preferentially (Kwong *et al*; 2010, Edo-Matas *et al*; 2010). R5 viruses are also prominent during chronic infection. X4 viruses or R5X4 viruses which have mixed affinity can arise later during infection and often their presence precedes

Deletion of the CCR5 receptor can in many cases abrogate infection with HIV-1 completely. It has previously been identified that individuals homozygous for a 32 base pair deletion within the *CCR5* gene resulting in a nonfunctional CCR5 molecule are resistant to infection with the HIV-1 virus, though there have been some instances where homozygous *CCR532* individuals were infected with X4 HIV-1 (Samson, 1996). Additionally, people who carry one allele of *CCR532* have a slower progression of the disease. This knowledge has led to the development of treatments for HIV-1 infection. Transplantation of stem cells from individuals homozygous for *CCR532* into CCR5 HIV-1 positive individuals resulted in clearing of the virus from the infected patients (Hutter, 2009). Monoclonal antibodies against CCR5 to inhibit binding of HIV-1 to this co-receptor are a potential therapeutic to prevent viral entry and replication (Tenorio, 2011, Suleiman, 2010). In addition there are plans to use an individual's native stem cells as a target to disrupt the *CCR5* receptor gene which can then be transplanted back into the HIV infected patient to effect elimination of the HIV-1 virus from the body (Cannon and June, 2011). Pitfalls of these therapies include the problem that the CCR5 chemokine receptor has a native function within the body, and that disrupting this receptor may cause unforeseen deficits in the immune system. In fact, lack of the CCR5 receptor gene has been associated with increased risk of severe infection with other viruses such as the West Nile Virus, and certain flaviviruses (Lin *et al*; 2008, Kindberg *et al*; 2008). Notwithstanding the previously mentioned caveat, interference with the CCR5 receptor may indeed be a promising target to treat those infected with HIV-1 as well as prevent infection for those exposed to the virus via sexual activity, needle sharing, or

disease progression and immune cell depletion (Mariani, 2010).

cytoplasm of the host cell.

accidental hospital transmission.

CCL2 or monocyte chemotactic protein-1 (MCP-1), of the C-C chemokine family, is a cytokine with the ability to influence both innate and adaptive immune responses (Daly *et al*; 2003). This chemokine is produced by a variety of different cell types including endothelial cells, fibroblasts, epithelial cells, smooth muscle cells, mesangial cells, astrocytic cells, and microglial cells. However, despite the wide range of cell types that have the ability to manufacture CCL2, the majority of CCL2 is produced by macrophages and monocytes (Deshmane *et al*; 2009).

Although technically a chemokine, CCL2 is often classified as an inflammatory cytokine due to its ability to attract various leukocytes (monocytes, memory T cells, basophils, natural killer (NK) cells etc.) to sites of trauma, bacterial and mycobacterial infection, toxin exposure, and ischemia (Daly *et al*; 2003, Deshmane *et al*; 2009, Mahad *et al*; 2003,, Charo *et al*; 2006). Besides attracting various leukocytes, CCL2 also specifically regulates the infiltration of monocytes, memory T lymphocytes, and NK cells (Deshmane *et al*; 2009). In addition, CCL2 has been found to have a profound effect on the differentiation of naïve helper T cells (Daly *et al*; 2003). Interestingly, studies have found that CCL2 expression tends to lead to the development of a Th2 immune response. Taking this tendency into account, it seems likely that CCL2 concentrations in HIV patients, which Weiss *et al*. (Weiss *et al*; 1997) found were correlated with viral load, can be linked to the Th1 to Th2 cytokine response switch often observed in HIV-1-infected patients (Deshmane *et al*; 2009).

CCL2 has also been found to play other roles in HIV pathogenesis. Eugenin *et al.* (Eugenin *et al*; 2006) noted that CCL2 in the central nervous system (CNS) attracts HIV-infected leukocytes into the brain thereby increasing the rate of HIV-1-infected cell-dispersal and causing the eventual impairment of the blood-brain-barrier. In fact, multiple studies indicate that CCL2 is largely responsible for the development of HIV encephalitis (HIVE), HIV-1 associated dementia (HAD), and NeuroAIDS (Deshmane *et al*; 2009, Eugenin *et al*; 2006).

## **4. HIV and the Th1 to Th2 Cytokine shift**

Under normal conditions, the immune system utilizes a Th1 subset response to viral infections. Activated antigen presenting cells (APC) secrete interleukin-12 (IL-12) which causes Th cell differentiation into the Th1 subset of cells (Clerci et al; 1993). These Th1 cells then secrete a characteristic Th1 profile of cytokines consisting of interleukin-2 (IL-2), interferon-gamma (IFN-), and tumor necrosis factor-beta (TNF-β). IL-2 induces proliferation of naïve Th cells (T0), amplifying the Th response. IFN- induces further IL-12 production in activated APCs, amplifying the Th1 response, and suppressing any Th2 response. IFN- also plays an important role in the activation of cytotoxic TC cells which destroy virally infected cells.

In individuals infected with HIV, the normal Th1 response to viral infection is shifted to a Th2 response (Klein et al; 1997, Osakwe et al; 2010). Measurement of the serum cytokine levels of HIV infected patients has revealed an increase in Th2 cytokines as well as a decrease in Th1 cytokines (Klein et al; 1997, Osakwe et al; 2010). Assays have shown elevated serum IL-4 levels in HIV seropositive individuals (Clerci et al; 1993). IL-4 in the presence of proliferating T0 cells leads to their differentiation into the Th2 subset. Th2 cells promote B-cell proliferation, class switching, and eosinophil activation (Clerci et al; 1993). This Th2 response is not appropriate for control of intracellular pathogens such as HIV, and so allows it to persist and spread in CD4+ T-cells.

Role of Cytokines and Chemokines in HIV Infection 285

undefined (Fantuzzi, 2003). IL-1 consists of two distinct ligands (IL-1α and IL-1β) with two indistinguishable biological activities that signal through the IL-1 receptor (IL-1R1) ( Bujak and Frangogiannis, 2009). Both IL-1α and IL-1β can also bind the IL-1 receptor accessory protein (IL-1RAcP). Once bound to the receptor, the complex transduces a signal that initiates a wide variety of inflammatory genes by activating the NF-κB system. The NF-κB transcribed genes can produce a variety of inflammatory products including chemokines, pro-inflammatory cytokines, such as TNF-, IL-6 or IL-8 (Nambu and Nakae, 2010), adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) (Marui et al; 1993), colony-stimulating factors, and mesenchymal growth factor genes (Bujak and Frangogiannis, 2009). In addition, expression of inducible nitric oxide synthase, type 2 cyclooxygenase (COX)-2, and type 2 phospholipase A2 is exquisitely sensitive to IL-1 (Bujak and Frangogiannis, 2009). IL-1 has also been associated with augmentation of the mast cell activation and Th2 cytokine secretion, suggesting involvement of IL-1 in allergic diseases

Knockouts of IL-1 have been used to study acute and chronic neurodegenerative conditions, in which a role for IL-1 has been well established (Fantuzzi, 2003). For example, in rodent studies the presence of IL-1 after occlusion of the middle cerebral artery will increase the ischemic damage area. It has been shown that caspase-1 cleaves the inactive pre-form to the active mature form of IL-1β, which contributes to the damage from ischemia (Bujak and Frangogiannis, 2009). In resting cells, procaspase-1 is bound to an inhibitory molecule that prevents its activation. After damage to cells, conversion of procaspase-1 to caspase-1 is triggered by a molecular complex termed the "IL-1β inflammasome" (Martinon, 2002). Macrophages and dendritic cells produce IL-1, IL-12 and other cytokines that permit CD4 cells to reach the level of maturation needed to produce IL-2, which is needed for selfreplication of the CD4 cells and for the growth and function of CD8 cells (Levy, 2007). Thus

Elevation of IL-1 and TNF-α has been demonstrated in the serum of some patients with HIV-1 (Lepe-Zuniga et al; 1987). High levels of IL-1 (Lepe-Zuniga et al; 1987, Weiss et al; 1989, Molina et al; 1989, Roux-Lombard et al; 1989, Emilie et al; 1990) and TNF-α ( Roux-Lombard et al; 1989) are produced in the supernatant of cultured peripheral blood monocyte early in the onset of HIV disease. The levels of TNF-α and IL-1 in the serum were positively correlated in symptomatic versus asymptomatic individuals ( Lepe-Zuniga et al; 1987). HIV virus is present in mononuclear phagocytes and in the blood and brain of AIDS patients. Production of IL-1 and TNF-α from mononuclear phagocytes after stimulation with HIV-1 may contribute to some of the symptoms of AIDS such as fever, cachexia and aseptic

Chronic infection and viral latency are typical of HIV-1 infection. Stimulation with IL-1β as well as TNF-α can stimulate viral replication in chronically infected cells (Devadas et al; 2004). Monocytes are also major reservoirs for HIV-1 in infected tissue and vectors for virus transmission to target cells, as well as sources of potent cytokines that can affect cell function and virus replication (Devadas et al; 2004). It is thought that stimulation of viral replication in chronically infected cells is due to activation of NF-B. In addition to IL-1 and TNF-α, contact with macrophages as well as a number of stressors can stimulate NF-B, including phorbol esters, radical oxygen intermediates, and UV irradiation (Devadas et al; 2004). Clinical manifestations of AIDS include both immunologic and neurologic disorders. In the brain it has been shown that IL-1 induces activation and proliferation of astrocytes, while TNF-α contributes to necrosis of cerebral blood vessels and possibly to demyelination

such as allergic asthma (Dinarello, 1999).

meningitis (Merrill et al; 1989).

IL-1 plays a role in maintaining normal immune function.

#### **5. TNF- and HIV infection**

It has also been shown that HIV infection induces increased production of TNF- by macrophages. TNF- stimulates the production of free radicals. Moreover, enhanced levels of free radicals are likely to increase TNF- in various cells*.* TNF- consists of 233 amino acids and is expressed on all somatic cells, particularly on the cell membrane where it becomes hydrolyzed to its soluble form. TNF- is considered as one of the most highly studied pro-inflammatory cytokines because it plays a critical role in the origin and progression of diseases such as HIV-1 (Bahia and Silakari, 2010). The immuno-regulatory response of the host influences the pathogenesis of HIV-1 infection, triggering monocytes, macrophages, and natural killer cells to produce TNF- (Alfano and Poli, 2005). As a result, there is a positive correlation between HIV-1 viremia and TNF- levels in serum of HIV-1 infected patients. This relationship suggests that reducing TNF- levels may also reduce occurrence of HIV-1 viremia. In excess, TNF- may cause severe inflammatory damage and toxicity, making control of its production and secretion highly important. Regulating its release serves as a potential means of therapy for HIV-1 and other diseases. TNF- can also induce other pro-inflammatory cytokines such as IL-6 and IL-8, which aid in the upregulation of viral replication (Fernandez-Ortega et al; 2004). Studies have also shown the ability of TNF- to stimulate production of anti-inflammatory cytokine IL-10, preventing further inflammation by causing TNF- inhibition (Leghmari et al; 2008). TNF- is secreted during the early phase of acute inflammatory diseases. Its pathogenic role in HIV-1 infection involves activation of nuclear factor kB (NF-B), stimulating apoptosis of T lymphocytes. Tissue and plasma samples of hosts express high levels of TNF-, contributing to fever, anorexia, and other symptoms of HIV/AIDS. TNF- must be targeted at an appropriate time during production to prevent progression to the chronic stage. Local effect of the cytokine may be beneficial to the host, so monitoring its development is critical. Highly active antiretroviral therapy helps to reduce mortality rates, and development of potent antiretroviral drugs blocking HIV transcription continues to be successful. However, drug resistance and toxicity remains a challenge in this field of medicine (Fernandez-Ortega et al; 2004).

#### **6. Interleukin 1 (IL-1) and HIV infection**

HIV infection and its viral proteins can disturb the production of cytokines and disrupt their usual interactions resulting in disruption of the normal immune function. IL-1 and TNF-α are produced by activation of mononuclear phagocytes as well as microglia in the brain in response to normal immune stimuli such as immune complexes, lipopolysaccharides and phorbol esters (Burchett et al; 1998). It has been reported that IL-1 and TNF-α will be produced by either the binding of gp120 to the CD4 molecules on mononuclear phagocytes or infection with HIV (Merrill et al; 1989, Cheung et al; 2008).

IL-1 is the first discovered and most studied member of the cytokine family (Fantuzzi, 2003). IL-1 is a pro-inflammatory cytokine that plays a fundamental role in host defense by inducing acute and chronic inflammation through activation of the innate and acquired immune systems (Nambu and Nakae, 2010). IL-1 has been described as the prototypic proinflammatory cytokine as it was originally described as the first "endogenous" pyrogen due to its fever-inducing properties in both rabbits and humans (Dinarello, 1999). However, in spite of much research in the area of fever induction, the role of IL-1 in this area is still

It has also been shown that HIV infection induces increased production of TNF- by macrophages. TNF- stimulates the production of free radicals. Moreover, enhanced levels of free radicals are likely to increase TNF- in various cells*.* TNF- consists of 233 amino acids and is expressed on all somatic cells, particularly on the cell membrane where it becomes hydrolyzed to its soluble form. TNF- is considered as one of the most highly studied pro-inflammatory cytokines because it plays a critical role in the origin and progression of diseases such as HIV-1 (Bahia and Silakari, 2010). The immuno-regulatory response of the host influences the pathogenesis of HIV-1 infection, triggering monocytes, macrophages, and natural killer cells to produce TNF- (Alfano and Poli, 2005). As a result, there is a positive correlation between HIV-1 viremia and TNF- levels in serum of HIV-1 infected patients. This relationship suggests that reducing TNF- levels may also reduce occurrence of HIV-1 viremia. In excess, TNF- may cause severe inflammatory damage and toxicity, making control of its production and secretion highly important. Regulating its release serves as a potential means of therapy for HIV-1 and other diseases. TNF- can also induce other pro-inflammatory cytokines such as IL-6 and IL-8, which aid in the upregulation of viral replication (Fernandez-Ortega et al; 2004). Studies have also shown the ability of TNF- to stimulate production of anti-inflammatory cytokine IL-10, preventing further inflammation by causing TNF- inhibition (Leghmari et al; 2008). TNF- is secreted during the early phase of acute inflammatory diseases. Its pathogenic role in HIV-1 infection involves activation of nuclear factor kB (NF-B), stimulating apoptosis of T lymphocytes. Tissue and plasma samples of hosts express high levels of TNF-, contributing to fever, anorexia, and other symptoms of HIV/AIDS. TNF- must be targeted at an appropriate time during production to prevent progression to the chronic stage. Local effect of the cytokine may be beneficial to the host, so monitoring its development is critical. Highly active antiretroviral therapy helps to reduce mortality rates, and development of potent antiretroviral drugs blocking HIV transcription continues to be successful. However, drug resistance and toxicity remains a challenge in this field of medicine (Fernandez-Ortega et al;

HIV infection and its viral proteins can disturb the production of cytokines and disrupt their usual interactions resulting in disruption of the normal immune function. IL-1 and TNF-α are produced by activation of mononuclear phagocytes as well as microglia in the brain in response to normal immune stimuli such as immune complexes, lipopolysaccharides and phorbol esters (Burchett et al; 1998). It has been reported that IL-1 and TNF-α will be produced by either the binding of gp120 to the CD4 molecules on mononuclear phagocytes

IL-1 is the first discovered and most studied member of the cytokine family (Fantuzzi, 2003). IL-1 is a pro-inflammatory cytokine that plays a fundamental role in host defense by inducing acute and chronic inflammation through activation of the innate and acquired immune systems (Nambu and Nakae, 2010). IL-1 has been described as the prototypic proinflammatory cytokine as it was originally described as the first "endogenous" pyrogen due to its fever-inducing properties in both rabbits and humans (Dinarello, 1999). However, in spite of much research in the area of fever induction, the role of IL-1 in this area is still

**5. TNF- and HIV infection** 

2004).

**6. Interleukin 1 (IL-1) and HIV infection** 

or infection with HIV (Merrill et al; 1989, Cheung et al; 2008).

undefined (Fantuzzi, 2003). IL-1 consists of two distinct ligands (IL-1α and IL-1β) with two indistinguishable biological activities that signal through the IL-1 receptor (IL-1R1) ( Bujak and Frangogiannis, 2009). Both IL-1α and IL-1β can also bind the IL-1 receptor accessory protein (IL-1RAcP). Once bound to the receptor, the complex transduces a signal that initiates a wide variety of inflammatory genes by activating the NF-κB system. The NF-κB transcribed genes can produce a variety of inflammatory products including chemokines, pro-inflammatory cytokines, such as TNF-, IL-6 or IL-8 (Nambu and Nakae, 2010), adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) (Marui et al; 1993), colony-stimulating factors, and mesenchymal growth factor genes (Bujak and Frangogiannis, 2009). In addition, expression of inducible nitric oxide synthase, type 2 cyclooxygenase (COX)-2, and type 2 phospholipase A2 is exquisitely sensitive to IL-1 (Bujak and Frangogiannis, 2009). IL-1 has also been associated with augmentation of the mast cell activation and Th2 cytokine secretion, suggesting involvement of IL-1 in allergic diseases such as allergic asthma (Dinarello, 1999).

Knockouts of IL-1 have been used to study acute and chronic neurodegenerative conditions, in which a role for IL-1 has been well established (Fantuzzi, 2003). For example, in rodent studies the presence of IL-1 after occlusion of the middle cerebral artery will increase the ischemic damage area. It has been shown that caspase-1 cleaves the inactive pre-form to the active mature form of IL-1β, which contributes to the damage from ischemia (Bujak and Frangogiannis, 2009). In resting cells, procaspase-1 is bound to an inhibitory molecule that prevents its activation. After damage to cells, conversion of procaspase-1 to caspase-1 is triggered by a molecular complex termed the "IL-1β inflammasome" (Martinon, 2002).

Macrophages and dendritic cells produce IL-1, IL-12 and other cytokines that permit CD4 cells to reach the level of maturation needed to produce IL-2, which is needed for selfreplication of the CD4 cells and for the growth and function of CD8 cells (Levy, 2007). Thus IL-1 plays a role in maintaining normal immune function.

Elevation of IL-1 and TNF-α has been demonstrated in the serum of some patients with HIV-1 (Lepe-Zuniga et al; 1987). High levels of IL-1 (Lepe-Zuniga et al; 1987, Weiss et al; 1989, Molina et al; 1989, Roux-Lombard et al; 1989, Emilie et al; 1990) and TNF-α ( Roux-Lombard et al; 1989) are produced in the supernatant of cultured peripheral blood monocyte early in the onset of HIV disease. The levels of TNF-α and IL-1 in the serum were positively correlated in symptomatic versus asymptomatic individuals ( Lepe-Zuniga et al; 1987). HIV virus is present in mononuclear phagocytes and in the blood and brain of AIDS patients. Production of IL-1 and TNF-α from mononuclear phagocytes after stimulation with HIV-1 may contribute to some of the symptoms of AIDS such as fever, cachexia and aseptic meningitis (Merrill et al; 1989).

Chronic infection and viral latency are typical of HIV-1 infection. Stimulation with IL-1β as well as TNF-α can stimulate viral replication in chronically infected cells (Devadas et al; 2004). Monocytes are also major reservoirs for HIV-1 in infected tissue and vectors for virus transmission to target cells, as well as sources of potent cytokines that can affect cell function and virus replication (Devadas et al; 2004). It is thought that stimulation of viral replication in chronically infected cells is due to activation of NF-B. In addition to IL-1 and TNF-α, contact with macrophages as well as a number of stressors can stimulate NF-B, including phorbol esters, radical oxygen intermediates, and UV irradiation (Devadas et al; 2004).

Clinical manifestations of AIDS include both immunologic and neurologic disorders. In the brain it has been shown that IL-1 induces activation and proliferation of astrocytes, while TNF-α contributes to necrosis of cerebral blood vessels and possibly to demyelination

Role of Cytokines and Chemokines in HIV Infection 287

are protease inhibitors (Xing et al; 1998). They activate target genes involved in differentiation, survival, apoptosis and proliferation. The members of this cytokine family have pro- as well as anti-inammatory properties and are major players in haematopoiesis, as well as in acute-phase and immune responses of the organism. IL-6-type cytokines bind to plasma membrane receptor complexes containing the common signal transducing receptor chain gp 130 (glycoprotein 130). Signal transduction involves the activation of JAK (Janus kinase) tyrosine kinase family members, leading to the activation of transcription factors of the STAT (signal transducers and activators of transcription) family. Another major signaling pathway for IL-6-type cytokines is the MAPK (mitogen-activated protein kinase) cascade (Heinrich et al; 2003). IL-6 was originally identified as β2 (IFN-β2), IL-1 inducible 26kD protein and as a factor that induces the differentiation of B cells to antibody

The induction of IL-6 by live HIV preparations occurred in the absence of T cells and could be neutralized by human anti-HIV serum indicating that HIV was responsible for this IL-6 inducing activity. It has been demonstrated that IL-6 can be produced by a variety of cells upon various kinds of stimulation: T cells infected with HTLV-1, fibroblasts stimulated with polyI:C, IL-1, platelet-derived growth factor, TNF-, FCS, or LPS, and monocyte/macrophages stimulated with LPS. Monocyte/ macrophages, one of the target cells of HIV, produced IL-6 upon stimulation with both live and inactivated HIV (Nakajima et al; 1989). A study of women treated for cervical intraepithelial lesions showed that after treatment, there were increased levels of genital HIV, TNF-α, IL-6, and other activation markers in cervicovaginal lavage (Spear et al; 2008). In univariate analysis, genital tract HIV RNA was significantly associated with plasma HIV RNA and several of the cytokines, while in multivariate analysis, genital tract HIV RNA was significantly associated only with plasma HIV RNA and IL-6 (Spear et al; 2008). Another study was done to determine the effect of HIV on thymic stromal cells and the production of cytokines important in thymocyte development, three types of adherent thymic cultures were established and studied: thymic epithelial cells (TECs), macrophage-enriched, and mixed cultures of macrophages and TECs (M phi/TEC). M phi/TEC and macrophage-enriched cultures were infected by both HIV strains without cytopathic changes. The TECs grew well in culture exposed to HIV-1 strains HIV-1IIIB and HIV-1Ba-L for at least 6 weeks and showed no evidence of infection, cytopathology, or changes in cytokine production with HIV. Only cultures containing macrophages (M phi/TEC or macrophage enriched) showed changes in cytokine (IL-1 alpha, IL-1 beta, and IL-6) production with HIV. Unstimulated macrophageenriched cultures produced small amounts of IL-6 that were increased by HIV 20-fold

There are many studies showing the increase of IL-6 expression within HIV infected cells but not many studies suggesting what IL-6 does to HIV. In a study done by Miles in 1990, it was found that IL-6 might actually be a growth factor for the HIV virus (Miles et al; 1990). There was a proliferative response of the AIDS-Kaposi sarcoma (AIDS-KS) cells to high concentrations of hrIL-6 and the detection of IL-6-rRNA in the areas of the skin involved with Kaposi sarcoma. AIDS-KS cells synthesized, released, and responded to biologically active IL-6. AIDS-KS cells, in which IL-6 protein translation arrest was induced by an IL-6 anti sense oligodeoxynucleotide, did not proliferate optimally unless exogenous hrIL-6 was

producing plasma cells (Hibi et al; 1996).

(Sandborg et al; 1994).

added (Miles et al; 1990).

(Merrill et al; 1989). A feedback process has been described in which HIV-1 and TNF-α can each induce expression of the other. It has been proposed that IL-1 will participate in this feedback loop by inducing TNF-α or by direct T-cell activation, which is needed for HIV-1 replication (Merrill et al; 1989).

IL-1 has been implicated in the pathogenesis of HIV associated dementia (HAD) (Kaul et al; 2001). Both IL-1β and TNF-α are highly expressed in the central nervous system (CNS) of individuals with HAD, correlate with neuronal injury and are implicated in the pathogenesis of HAD (Epstein and Gendelman, 1993,, Brabers and Nottet, 2006). HIV-1, recombinant gp120, and viral transactivator Tat can activate astrocytes to secrete proinflammatory cytokines TNF-α, IL-6, and IL-1β (Corasaniti et al; 2001), which may contribute to the inflammatory environment in the brain (Herbein and Varin, 2010). Microglia and macrophages in the brain can release IL-1β after stimulation with HIV-1 envelope protein gp120 (Merrill et al; 1992, Wahl et al; 1989), which is elevated in brain during HIV (Tyor et al; 1992) and has been shown to be elevated in the cerebral spinal fluid during HIV infection (Gallo et al; 1989).

About 25% of subjects with HIV will develop dementia, particularly HIV encephalitis (HIVE), which can occur in spite of the use of HAART (Levy, 2007). Macrophage inflammatory products including IL-1β have been demonstrated in HIV related encephalitis in mouse and human brain tissue (Persidsky et al; 1997). It has been suggested that the release of neurotoxins, including L-cysteine, from macrophages in the brain is mediated by IL-1 following stimulation of the macrophages by the HIV membrane protein gp120.

L-Cysteine can be released from human monocyte derived macrophages stimulated by either gp120 (Lipton, 1998), or by IL-1 (Yeh et al; 2000). It has been suggested that cytokines including IL-1 may mediate the neurotoxic actions of gp120 (Yeh et al; 2000). Cysteine can act as an endogenous neurotoxin (Olney et al; 1990), which under both physiologic and pathophysiologic conditions stimulates *N*-methyl-D-aspartate subtype of glutamate receptor (NMDARs) and leads to neuronal apoptosis (Yeh et al; 2000). Thus, immune activation of macrophages in the brain without direct HIV infection may lead to neural damage (Yeh et al; 2000).

Autopsy evaluation of brain tissue from HIVE cases shows increased IL-1β in the frontal white matter of all 11 of the brains evaluated (67). Additionally, IL-1β, but not TNF expression was detected in HIVp24-positive cells in the HIVE patients, which indicates that IL-1β is induced by HIV-1 infection. The authors concluded that a macrophage/microglia lineage is the main cell type to release cytokines in HIVE, and IL-1β expression by HIV-1 infected cells may be one of the important factors for induction of HIVE (67).

## **7. Interleukin-6 (IL-6)**

The family of IL-6-type cytokines comprises IL-6, IL-11, LIF (leukaemia inhibitory factor), OSM (oncostatin M), CNTF (ciliary neurotrophic factor), CT-1 (cardiotrophin-1) and CLC (cardiotrophin-like cytokine) (Heinrich et al; 2003). IL-6 is a pleiotropic cytokine that is commonly produced at local tissue sites and released into circulation in almost all situations of homeostatic perturbation typically including endotoxemia, endotoxic lung, trauma, and acute infections. In addition to its critical participation in the generation of immunity against chronic intracellular infections, circulating IL-6, together with other alarm cytokines TNF and IL-1, is known to be required for the induction of acute phase reactions composed of fever, corticosterone release, and hepatic production of acute phase proteins many of which

(Merrill et al; 1989). A feedback process has been described in which HIV-1 and TNF-α can each induce expression of the other. It has been proposed that IL-1 will participate in this feedback loop by inducing TNF-α or by direct T-cell activation, which is needed for HIV-1

IL-1 has been implicated in the pathogenesis of HIV associated dementia (HAD) (Kaul et al; 2001). Both IL-1β and TNF-α are highly expressed in the central nervous system (CNS) of individuals with HAD, correlate with neuronal injury and are implicated in the pathogenesis of HAD (Epstein and Gendelman, 1993,, Brabers and Nottet, 2006). HIV-1, recombinant gp120, and viral transactivator Tat can activate astrocytes to secrete proinflammatory cytokines TNF-α, IL-6, and IL-1β (Corasaniti et al; 2001), which may contribute to the inflammatory environment in the brain (Herbein and Varin, 2010). Microglia and macrophages in the brain can release IL-1β after stimulation with HIV-1 envelope protein gp120 (Merrill et al; 1992, Wahl et al; 1989), which is elevated in brain during HIV (Tyor et al; 1992) and has been shown to be elevated in the cerebral spinal fluid

About 25% of subjects with HIV will develop dementia, particularly HIV encephalitis (HIVE), which can occur in spite of the use of HAART (Levy, 2007). Macrophage inflammatory products including IL-1β have been demonstrated in HIV related encephalitis in mouse and human brain tissue (Persidsky et al; 1997). It has been suggested that the release of neurotoxins, including L-cysteine, from macrophages in the brain is mediated by

L-Cysteine can be released from human monocyte derived macrophages stimulated by either gp120 (Lipton, 1998), or by IL-1 (Yeh et al; 2000). It has been suggested that cytokines including IL-1 may mediate the neurotoxic actions of gp120 (Yeh et al; 2000). Cysteine can act as an endogenous neurotoxin (Olney et al; 1990), which under both physiologic and pathophysiologic conditions stimulates *N*-methyl-D-aspartate subtype of glutamate receptor (NMDARs) and leads to neuronal apoptosis (Yeh et al; 2000). Thus, immune activation of macrophages in the brain without direct HIV infection may lead to neural damage (Yeh et

Autopsy evaluation of brain tissue from HIVE cases shows increased IL-1β in the frontal white matter of all 11 of the brains evaluated (67). Additionally, IL-1β, but not TNF expression was detected in HIVp24-positive cells in the HIVE patients, which indicates that IL-1β is induced by HIV-1 infection. The authors concluded that a macrophage/microglia lineage is the main cell type to release cytokines in HIVE, and IL-1β expression by HIV-1-

The family of IL-6-type cytokines comprises IL-6, IL-11, LIF (leukaemia inhibitory factor), OSM (oncostatin M), CNTF (ciliary neurotrophic factor), CT-1 (cardiotrophin-1) and CLC (cardiotrophin-like cytokine) (Heinrich et al; 2003). IL-6 is a pleiotropic cytokine that is commonly produced at local tissue sites and released into circulation in almost all situations of homeostatic perturbation typically including endotoxemia, endotoxic lung, trauma, and acute infections. In addition to its critical participation in the generation of immunity against chronic intracellular infections, circulating IL-6, together with other alarm cytokines TNF and IL-1, is known to be required for the induction of acute phase reactions composed of fever, corticosterone release, and hepatic production of acute phase proteins many of which

infected cells may be one of the important factors for induction of HIVE (67).

IL-1 following stimulation of the macrophages by the HIV membrane protein gp120.

replication (Merrill et al; 1989).

during HIV infection (Gallo et al; 1989).

al; 2000).

**7. Interleukin-6 (IL-6)** 

are protease inhibitors (Xing et al; 1998). They activate target genes involved in differentiation, survival, apoptosis and proliferation. The members of this cytokine family have pro- as well as anti-inammatory properties and are major players in haematopoiesis, as well as in acute-phase and immune responses of the organism. IL-6-type cytokines bind to plasma membrane receptor complexes containing the common signal transducing receptor chain gp 130 (glycoprotein 130). Signal transduction involves the activation of JAK (Janus kinase) tyrosine kinase family members, leading to the activation of transcription factors of the STAT (signal transducers and activators of transcription) family. Another major signaling pathway for IL-6-type cytokines is the MAPK (mitogen-activated protein kinase) cascade (Heinrich et al; 2003). IL-6 was originally identified as β2 (IFN-β2), IL-1 inducible 26kD protein and as a factor that induces the differentiation of B cells to antibody producing plasma cells (Hibi et al; 1996).

The induction of IL-6 by live HIV preparations occurred in the absence of T cells and could be neutralized by human anti-HIV serum indicating that HIV was responsible for this IL-6 inducing activity. It has been demonstrated that IL-6 can be produced by a variety of cells upon various kinds of stimulation: T cells infected with HTLV-1, fibroblasts stimulated with polyI:C, IL-1, platelet-derived growth factor, TNF-, FCS, or LPS, and monocyte/macrophages stimulated with LPS. Monocyte/ macrophages, one of the target cells of HIV, produced IL-6 upon stimulation with both live and inactivated HIV (Nakajima et al; 1989). A study of women treated for cervical intraepithelial lesions showed that after treatment, there were increased levels of genital HIV, TNF-α, IL-6, and other activation markers in cervicovaginal lavage (Spear et al; 2008). In univariate analysis, genital tract HIV RNA was significantly associated with plasma HIV RNA and several of the cytokines, while in multivariate analysis, genital tract HIV RNA was significantly associated only with plasma HIV RNA and IL-6 (Spear et al; 2008). Another study was done to determine the effect of HIV on thymic stromal cells and the production of cytokines important in thymocyte development, three types of adherent thymic cultures were established and studied: thymic epithelial cells (TECs), macrophage-enriched, and mixed cultures of macrophages and TECs (M phi/TEC). M phi/TEC and macrophage-enriched cultures were infected by both HIV strains without cytopathic changes. The TECs grew well in culture exposed to HIV-1 strains HIV-1IIIB and HIV-1Ba-L for at least 6 weeks and showed no evidence of infection, cytopathology, or changes in cytokine production with HIV. Only cultures containing macrophages (M phi/TEC or macrophage enriched) showed changes in cytokine (IL-1 alpha, IL-1 beta, and IL-6) production with HIV. Unstimulated macrophageenriched cultures produced small amounts of IL-6 that were increased by HIV 20-fold (Sandborg et al; 1994).

There are many studies showing the increase of IL-6 expression within HIV infected cells but not many studies suggesting what IL-6 does to HIV. In a study done by Miles in 1990, it was found that IL-6 might actually be a growth factor for the HIV virus (Miles et al; 1990). There was a proliferative response of the AIDS-Kaposi sarcoma (AIDS-KS) cells to high concentrations of hrIL-6 and the detection of IL-6-rRNA in the areas of the skin involved with Kaposi sarcoma. AIDS-KS cells synthesized, released, and responded to biologically active IL-6. AIDS-KS cells, in which IL-6 protein translation arrest was induced by an IL-6 anti sense oligodeoxynucleotide, did not proliferate optimally unless exogenous hrIL-6 was added (Miles et al; 1990).

Role of Cytokines and Chemokines in HIV Infection 289

progresses, decreased IFN-γ leads to decrease in IL-12 which leads to decreased CD4+ and

A decrease of IL-12 concentration increases the probability for opportunistic infections. Taoufik *et al*; 1997 and Mirani *et al*; 2002, showed IL-12 mRNA was diminished while IL-10 production was up-regulated in the presence of *Staphylococcus aureus* and HIV gp120, further inhibiting IL-12 cytokine production. Even though IL-12 is potent, Villinger and Ansari 2010, noted that when IL-12 therapy was administered in the late stages of HIV, it

In addition to the Th1 subset response mediation mentioned earlier, IFN- normally acts on APCs to enhance their expression of major histocompatibility complex II (MHC-II), thereby enhancing their antigen presentation ability (Li et al; 2011). HIV transactivator protein (TAT) interferes with the intracellular signaling normally performed by the IFN- bound IFN- receptor (Cheng et al; 2009). In so doing, the TAT protein lowers the antigen presentation capacity of dendritic cells and macrophages, further limiting the immune

TGF-β cytokine family are closely related polypeptides which include tissue growth factors that have a diverse range of proteins that regulate many physiological processes including embryonic development, homeostasis, wound healing, chemotaxis, cell cycle control, cell proliferation, differentiation, apoptosis, adhesion, and migration (Leask and Abraham, 2004). TGF-β is one of the most immunosuppressive substances produced in the body and yet may inhibit or stimulate cell growth, depending on the cell type and culture conditions (Liu and Gaston Pravia, 2010). TGF-β is produced in many immune cells including lymphocytes, macrophages and dendritic cells (Liu and Gaston Pravia, 2010). Receptors for TGF-β have been found on all cell lines tested, allowing this cytokine to have effects on almost any tissue in the body (Leask and Abraham, 2004). It has also been shown to play a central role in tissue fibrosis (Leask and Abraham, 2004). Because of the multifunctional role played by TGF-β, it plays a central role in the pathogenesis of many diseases.(Leask and

There are three forms of TGF-β (TGF-β1, TGF-β2, and TGF-β3) in mammalian cells. TGF-βs are synthesized using inactive precursors and cannot bind receptors until they are activated. After release of TGF-β from cells they associate with latency-associated protein and form a small inactive complex. In the extracellular matrix, this complex is bound by latent TGF-βbinding protein (LTBP), a component of the extracellular matrix that is necessary for the secretion and storage of TGF-β (Letterio and Roberts, 1998). Intracellular activity of TGF-β is mediated by the actions of Smad transcription factors as well as independent factors ( Letterio and Roberts, 1998). Active Smad complexes bind to DNA weakly and high affinity binding is achieved by the association of Smad proteins with a large number of transcription factor partners (Massague, 1992). The variations of Smad proteins in transcriptional regulations and the diversity of Smad-independent pathways allow the pleiotropic actions

CD8+ T cell response.

Abraham, 2004).

of TGF-β ( Letterio and Roberts, 1998).

failed to restore normal levels of CD4 T cells and IFN-.

**10. Additional effects of HIV on IFN- signaling** 

response to the invading virus (Salgame et al; 2009).

**11. The transforming growth factor β (TGF-β)** 

#### **8. Interleukin-17 (IL-17)**

IL-17 is an inflammatory cytokine that is exclusively produced by a recently discovered subset of CD4+ T helper (Th) cells, referred to as Th17 cells (Crome et al; 2009). This cytokine has been found to help regulate the inflammatory response by activating fibroblasts, recruiting neutrophils, and acting on macrophages to promote both their recruitment and survival (Crome et al; 2009, Chang et al; 2007). In addition, IL-17 is thought to play a significant role in activating and inducing anti-microbial peptides and proinflammatory cytokines like IL-6, CCL2, and TNF- (Crome et al; 2009, Chang et al; 2007). Furthermore, high levels of this cytokine have been linked to a number of inflammatory diseases including rheumatoid arthritis, multiple sclerosis (MS), and asthma. Low levels, on the other hand, are thought to cause both impaired host defense against mycobacterial infection and decreased antibacterial immunity (Crome et al; 2009, Brenchley et al; 2008).

Studies of the effects of HIV on IL-17 concentrations using flow cytometry have found that HIV-infected patients have significantly increased levels of IL-17 (Giorgio, 2003). Venketaraman *et al.* (unpublished data) was also able to show increased levels of IL-17 in HIV-infected blood plasma using ELISA assays. However, Brenchley *et al.*; 2008 noted that there were significantly fewer IL-17 producing Th17 cells in the gastrointestinal tract of HIVinfected patients. In fact, the study indicated that Th17 cells were preferentially targeted during HIV infection.

The decrease of IL-17 concentrations at the mucosal wall of the gastrointestinal tract could greatly increase the probability of bacterial infections, which could in turn have significant implications for the speed of HIV pathogenesis (Brenchley et al; 2008). As Levy et al; 2009 noted, chronic immune activation increases the production of pro-inflammatory cytokines (IL-6, IL-17, TNF-, etc.). This up-regulation of pro-inflammatory cytokines often leads to the rapid loss of CD4+ T cells via apoptosis. Decreased IL-17 concentrations due to HIV infection can therefore ultimately lead to the general advancement of HIV by creating an environment favorable to opportunistic infection and chronic immune activation (Maek-A-Nantawat et al; 2007).

### **9. Interleukin-12 (IL-12)**

IL-12 is a heterodimeric pro-inflammatory cytokine that is produced by dendritic cells and phagocytes during an infection (Giorgio, 2003). It is a cytokine identified as a master switch for leading the naïve CD4+ T cells towards the Th1 pathway and also activating NK cells (Villinger and Ansari, 2010). Not only does it directly induce T, NK, and NKT cell cytotoxicity, IL-12 also promotes macrophage activity via T- and NK-cell-produced IFN-γ (Giorgio, 2003, Egilmez et al; 2011). The pathway is antagonized in the presence of IL-10 (Villinger and Ansari, 2010).

IL-12 plays important roles in protecting the body from various microbial infections such as parasites, bacteria, and viruses (Yang et al; 2010). With mutations in genes of the IL-12, the cells are susceptible to intracellular pathogens such as tuberculosis, leprosy, HIV-1, hepatitis and malaria (Vannberg et al; 2011). One of the characteristics of HIV infection is the gradual deterioration of cellular effector responses. Studies has concluded that CD4+ and CD8+ T cell responses were enhanced *ex vivo* by the addition of IL-12, but that capacity to respond is decreased in patients with marked CD4 loss (Villinger and Ansari, 2010). Louis *et al;*. 2010, also added that IL-12 production required the presence of IFN-γ. Therefore, as HIV

IL-17 is an inflammatory cytokine that is exclusively produced by a recently discovered subset of CD4+ T helper (Th) cells, referred to as Th17 cells (Crome et al; 2009). This cytokine has been found to help regulate the inflammatory response by activating fibroblasts, recruiting neutrophils, and acting on macrophages to promote both their recruitment and survival (Crome et al; 2009, Chang et al; 2007). In addition, IL-17 is thought to play a significant role in activating and inducing anti-microbial peptides and proinflammatory cytokines like IL-6, CCL2, and TNF- (Crome et al; 2009, Chang et al; 2007). Furthermore, high levels of this cytokine have been linked to a number of inflammatory diseases including rheumatoid arthritis, multiple sclerosis (MS), and asthma. Low levels, on the other hand, are thought to cause both impaired host defense against mycobacterial infection and decreased antibacterial immunity (Crome et al; 2009, Brenchley et al; 2008). Studies of the effects of HIV on IL-17 concentrations using flow cytometry have found that HIV-infected patients have significantly increased levels of IL-17 (Giorgio, 2003). Venketaraman *et al.* (unpublished data) was also able to show increased levels of IL-17 in HIV-infected blood plasma using ELISA assays. However, Brenchley *et al.*; 2008 noted that there were significantly fewer IL-17 producing Th17 cells in the gastrointestinal tract of HIVinfected patients. In fact, the study indicated that Th17 cells were preferentially targeted

The decrease of IL-17 concentrations at the mucosal wall of the gastrointestinal tract could greatly increase the probability of bacterial infections, which could in turn have significant implications for the speed of HIV pathogenesis (Brenchley et al; 2008). As Levy et al; 2009 noted, chronic immune activation increases the production of pro-inflammatory cytokines (IL-6, IL-17, TNF-, etc.). This up-regulation of pro-inflammatory cytokines often leads to the rapid loss of CD4+ T cells via apoptosis. Decreased IL-17 concentrations due to HIV infection can therefore ultimately lead to the general advancement of HIV by creating an environment favorable to opportunistic infection and chronic immune activation (Maek-A-

IL-12 is a heterodimeric pro-inflammatory cytokine that is produced by dendritic cells and phagocytes during an infection (Giorgio, 2003). It is a cytokine identified as a master switch for leading the naïve CD4+ T cells towards the Th1 pathway and also activating NK cells (Villinger and Ansari, 2010). Not only does it directly induce T, NK, and NKT cell cytotoxicity, IL-12 also promotes macrophage activity via T- and NK-cell-produced IFN-γ (Giorgio, 2003, Egilmez et al; 2011). The pathway is antagonized in the presence of IL-10

IL-12 plays important roles in protecting the body from various microbial infections such as parasites, bacteria, and viruses (Yang et al; 2010). With mutations in genes of the IL-12, the cells are susceptible to intracellular pathogens such as tuberculosis, leprosy, HIV-1, hepatitis and malaria (Vannberg et al; 2011). One of the characteristics of HIV infection is the gradual deterioration of cellular effector responses. Studies has concluded that CD4+ and CD8+ T cell responses were enhanced *ex vivo* by the addition of IL-12, but that capacity to respond is decreased in patients with marked CD4 loss (Villinger and Ansari, 2010). Louis *et al;*. 2010, also added that IL-12 production required the presence of IFN-γ. Therefore, as HIV

**8. Interleukin-17 (IL-17)** 

during HIV infection.

Nantawat et al; 2007).

**9. Interleukin-12 (IL-12)** 

(Villinger and Ansari, 2010).

progresses, decreased IFN-γ leads to decrease in IL-12 which leads to decreased CD4+ and CD8+ T cell response.

A decrease of IL-12 concentration increases the probability for opportunistic infections. Taoufik *et al*; 1997 and Mirani *et al*; 2002, showed IL-12 mRNA was diminished while IL-10 production was up-regulated in the presence of *Staphylococcus aureus* and HIV gp120, further inhibiting IL-12 cytokine production. Even though IL-12 is potent, Villinger and Ansari 2010, noted that when IL-12 therapy was administered in the late stages of HIV, it failed to restore normal levels of CD4 T cells and IFN-.

## **10. Additional effects of HIV on IFN- signaling**

In addition to the Th1 subset response mediation mentioned earlier, IFN- normally acts on APCs to enhance their expression of major histocompatibility complex II (MHC-II), thereby enhancing their antigen presentation ability (Li et al; 2011). HIV transactivator protein (TAT) interferes with the intracellular signaling normally performed by the IFN- bound IFN- receptor (Cheng et al; 2009). In so doing, the TAT protein lowers the antigen presentation capacity of dendritic cells and macrophages, further limiting the immune response to the invading virus (Salgame et al; 2009).

## **11. The transforming growth factor β (TGF-β)**

TGF-β cytokine family are closely related polypeptides which include tissue growth factors that have a diverse range of proteins that regulate many physiological processes including embryonic development, homeostasis, wound healing, chemotaxis, cell cycle control, cell proliferation, differentiation, apoptosis, adhesion, and migration (Leask and Abraham, 2004). TGF-β is one of the most immunosuppressive substances produced in the body and yet may inhibit or stimulate cell growth, depending on the cell type and culture conditions (Liu and Gaston Pravia, 2010). TGF-β is produced in many immune cells including lymphocytes, macrophages and dendritic cells (Liu and Gaston Pravia, 2010). Receptors for TGF-β have been found on all cell lines tested, allowing this cytokine to have effects on almost any tissue in the body (Leask and Abraham, 2004). It has also been shown to play a central role in tissue fibrosis (Leask and Abraham, 2004). Because of the multifunctional role played by TGF-β, it plays a central role in the pathogenesis of many diseases.(Leask and Abraham, 2004).

There are three forms of TGF-β (TGF-β1, TGF-β2, and TGF-β3) in mammalian cells. TGF-βs are synthesized using inactive precursors and cannot bind receptors until they are activated. After release of TGF-β from cells they associate with latency-associated protein and form a small inactive complex. In the extracellular matrix, this complex is bound by latent TGF-βbinding protein (LTBP), a component of the extracellular matrix that is necessary for the secretion and storage of TGF-β (Letterio and Roberts, 1998). Intracellular activity of TGF-β is mediated by the actions of Smad transcription factors as well as independent factors ( Letterio and Roberts, 1998). Active Smad complexes bind to DNA weakly and high affinity binding is achieved by the association of Smad proteins with a large number of transcription factor partners (Massague, 1992). The variations of Smad proteins in transcriptional regulations and the diversity of Smad-independent pathways allow the pleiotropic actions of TGF-β ( Letterio and Roberts, 1998).

Role of Cytokines and Chemokines in HIV Infection 291

reduced effector function (Fogle et al; 2010, Bucci et al; 1998, Tompkins and Tompkins 2008). IL-10 and TGF- overlap with each other in many of their biological effects including

IL-10 is an anti-inflammatory cytokine that essentially plays two regulatory roles in innate and adaptive immunity. It suppresses the up-regulation of various genes in macrophages and dendritic cells that are normally stimulated via toll-like receptors and promotes the proliferation of cytotoxic T cells, activates B cells, and induces the upregulation of specific genes in toll-like receptor activated phagocytic and dendritic cells (Trincheri, 2007). In addition, a critical function of IL-10 is its ability to inhibit pro-inflammatory cytokines such as TNF-α, IFN-γ, IL-1, IL-6, IL-2, and IL-12 (Trincheri, 2007). IL-10 decreases the production of pro-inflammatory cytokines by limiting the major histocompatibility class II and CD80/CD 86 expressed on monocytes and macrophage (Wang et al; 2005). IL-10 was believed to be produced by CD4+ Th2 cells; however, studies have shown that it is secreted by both Th1 and Th2 cells (Brockman et al; 2009). Also, cells from the myeloid lineage which include macrophages and dendritic cells also produce cytokine IL-10 (Hedrich and Bream, 2010). Furthermore, IL-10 is regulated both at the transcriptional and post-translational level

There are several speculations of the role of IL-10 in HIV pathogenesis and the subject has been a popular interest in many studies. Ji *et al*., 2005 reported that CD14+ monocytes are the main cells producing cytokine IL-10 in PBMCs after HIV-1 infection via interactions independent of CD4+ molecules, thus, concluding that IL-10 production is dependent on the presence of CD14+ monocytes. Moreover, as the patient progresses to advanced stages of HIV disease, the frequency of IL-10 producing cells increases significantly (118). On the other hand, Naicker *et al*., 2009, stated that different stages of the HIV disease will govern what role IL-10 will play in infected individuals. For instance, in acute HIV-1 individuals, IL-10 may promote viral replication by inhibiting effector immune response from both arms of the innate and adaptive immunity (Naicker et al; 2009). Furthermore, it was proposed in a chronic phase, that IL-10 resembled a protective role by reducing immune activation, inhibiting virus replication in macrophages, and the increase in production of IL-10 levels

It has been shown that HIV infection induces increased production of free radicals by macrophages. Free radical formation occurs as a byproduct of oxidative stress. Oxidative stress occurs when there is a disproportion between the reactive oxygen elements in the body versus the ability of the body to properly eliminate these reactive species. The presence of free radicals has been implicated in disturbing and damaging a number of biological processes (Karthikeyan et al; 2010). With regards to HIV infection the increase of oxidative stress has been seen to influence components in antioxidant defense in physiological antioxidants such as glutathione which are seen to decrease dramatically in HIV patients (Pace and Leaf, 1995). In addition to glutathione, vitamin A, C, and E at high doses as well as improving low levels of selenium were associated with assisting the prevention of HIV infection progression by working as antioxidants to remove free radicals

inhibition of T cell proliferation and IFN- production (Othieno et al; 1999).

and is involved in various signaling pathways (Couper et al; 2008).

lowered plasma viral load and increased CD4+ cell count (Naicker et al; 2009).

**12. Interleukin-10 (IL-10)** 

**13. HIV and free radicals** 

HIV infection leads to a variety of disturbances in cytokine expression that can lead to a state of chronic activation of B cells and release of cytokines that may actually play an important role in the pathogenesis of HIV infection (Li and Flavell, 2008). Early HIV-1 infection is associated with a massive oligoclonal expansion of CD8 T cells (Massague and Gomis, 2006), however despite the high number of circulating CD8+ T cells the cytotoxic T lymphocyte (CTL) response is highly variable among HIV-1 infected individuals (Poli and Fauci, 1993). It has also been shown that the immune dysfunction in the initial phase of HIV infection exceeds CD4+ T cell infection and loss (Pantaleo et al; 1994 ). It appears that the immunosuppression effect occurs almost immediately upon infection (Garba et al; 2002). The result is diminished T cell response to antigen stimulation and persistence of HIV replication (Pantaleo and Fauci, 1995).

HIV-1 products such as TAT, induce the transcription of cytokines with immunosuppressive effects, including TGF-β (Cohen et al; 1999). It has been reported that extracellular TAT can be taken up by bystander cells and that it is possible that exogenous TAT, not associated with direct infection of a cell, can induce TGF-β transcription in immune competent cells (Pantaleo et al; 1993). Macrophages appear to be very sensitive to TAT and are affected by TAT concentrations 1,000-fold lower (500 pM) than those that affect T cells (Cohen et al; 1999). Macrophages stimulated by TAT either by infection or by the uptake of soluble TAT (sTat) induce Fas ligand (FasL), which in turn can trigger the apoptosis of antigen-reacting, Fas-expressing helper T cells. This mechanism would suppress T-cell dependent cellular and humoral immune responses to both HIV and other antigens (Cohen et al; 1999).

The transactivating effect of HIV-1 TAT is mediated by activator protein-1, which is the same multimolecular complex that is activated by TGF-β (Cohen et al; 1999). HIV-1 can induce both the transcription and secretion of TGF-β (Reinhold et al; 1999) and the induction of TGF-β can increase the apoptosis of NK cells (Poggi and Zocchi, 2006). TGF-β and Tat have been detected in the sera of early HIV-1 infected individuals at levels that were biologically active *in vitro* (Reinhold et al; 1999).

Some HIV-infected individuals have been shown to lose the ability of their cytotoxic T lymphocytes CTL (CD8+) to control infection in cells that carry HIV as well as other infectious agents (Pantaleo et al; 1993). About 25% of HIV infected individuals have been shown to produce TGF-β1 in response to stimulation with HIV proteins or peptides (Garba et al; 2002). It has been shown that the loss of CTL activity is related to the production of TGF-β1 in sufficient amounts to significantly reduce the IFN-γ response of CD8+ cells to both HIV and other viral proteins such as vaccinia virus (Garba et al; 2002).

It has been established that feline CD4+CD25+ T regulatory (T reg) cells share phenotypic and functional characteristics with human and murine T reg cells (Vahlenkamp et al; 2004). Early in the infection with feline immunodeficiency virus (FIV), CD4+CD25+ T reg cells exhibit increased expression of a membrane TGF- β (mTGF-β) (Mexas et al; 2008). The appearance of TGF-β+CD4+CD25+ lymphocytes within the lymph node of FIV+ cats occurs in both acute and chronic FIV, even though mTGF-β does not appear in the blood (Fogle et al; 2010). There is also evidence of increased expression of TGF-βRII, the receptor of TGF-β1, on CD8+ lymphocytes in FIV+ cats that would make the CD8+ lymphocytes much more sensitive to TGF-β inhibition (Fogle et al; 2010). In FIV lentiviral infection, during both the acute and chronic stages of infection, CD4+CD25+ Tregs suppress CD8+ responses and the CD4+CD25+ Tregs use mTGF-β to suppress IFN-γ expression resulting in suppression of CD8+ lymphocyte function (Fogle et al; 2010). These findings help explain the paradox of chronic HIV-1 infection, in which CD8+ T cells display an activated phenotype but exhibit reduced effector function (Fogle et al; 2010, Bucci et al; 1998, Tompkins and Tompkins 2008). IL-10 and TGF- overlap with each other in many of their biological effects including

## **12. Interleukin-10 (IL-10)**

290 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

HIV infection leads to a variety of disturbances in cytokine expression that can lead to a state of chronic activation of B cells and release of cytokines that may actually play an important role in the pathogenesis of HIV infection (Li and Flavell, 2008). Early HIV-1 infection is associated with a massive oligoclonal expansion of CD8 T cells (Massague and Gomis, 2006), however despite the high number of circulating CD8+ T cells the cytotoxic T lymphocyte (CTL) response is highly variable among HIV-1 infected individuals (Poli and Fauci, 1993). It has also been shown that the immune dysfunction in the initial phase of HIV infection exceeds CD4+ T cell infection and loss (Pantaleo et al; 1994 ). It appears that the immunosuppression effect occurs almost immediately upon infection (Garba et al; 2002). The result is diminished T cell response to antigen stimulation and persistence of HIV

HIV-1 products such as TAT, induce the transcription of cytokines with immunosuppressive effects, including TGF-β (Cohen et al; 1999). It has been reported that extracellular TAT can be taken up by bystander cells and that it is possible that exogenous TAT, not associated with direct infection of a cell, can induce TGF-β transcription in immune competent cells (Pantaleo et al; 1993). Macrophages appear to be very sensitive to TAT and are affected by TAT concentrations 1,000-fold lower (500 pM) than those that affect T cells (Cohen et al; 1999). Macrophages stimulated by TAT either by infection or by the uptake of soluble TAT (sTat) induce Fas ligand (FasL), which in turn can trigger the apoptosis of antigen-reacting, Fas-expressing helper T cells. This mechanism would suppress T-cell dependent cellular and

The transactivating effect of HIV-1 TAT is mediated by activator protein-1, which is the same multimolecular complex that is activated by TGF-β (Cohen et al; 1999). HIV-1 can induce both the transcription and secretion of TGF-β (Reinhold et al; 1999) and the induction of TGF-β can increase the apoptosis of NK cells (Poggi and Zocchi, 2006). TGF-β and Tat have been detected in the sera of early HIV-1 infected individuals at levels that were

Some HIV-infected individuals have been shown to lose the ability of their cytotoxic T lymphocytes CTL (CD8+) to control infection in cells that carry HIV as well as other infectious agents (Pantaleo et al; 1993). About 25% of HIV infected individuals have been shown to produce TGF-β1 in response to stimulation with HIV proteins or peptides (Garba et al; 2002). It has been shown that the loss of CTL activity is related to the production of TGF-β1 in sufficient amounts to significantly reduce the IFN-γ response of CD8+ cells to

It has been established that feline CD4+CD25+ T regulatory (T reg) cells share phenotypic and functional characteristics with human and murine T reg cells (Vahlenkamp et al; 2004). Early in the infection with feline immunodeficiency virus (FIV), CD4+CD25+ T reg cells exhibit increased expression of a membrane TGF- β (mTGF-β) (Mexas et al; 2008). The appearance of TGF-β+CD4+CD25+ lymphocytes within the lymph node of FIV+ cats occurs in both acute and chronic FIV, even though mTGF-β does not appear in the blood (Fogle et al; 2010). There is also evidence of increased expression of TGF-βRII, the receptor of TGF-β1, on CD8+ lymphocytes in FIV+ cats that would make the CD8+ lymphocytes much more sensitive to TGF-β inhibition (Fogle et al; 2010). In FIV lentiviral infection, during both the acute and chronic stages of infection, CD4+CD25+ Tregs suppress CD8+ responses and the CD4+CD25+ Tregs use mTGF-β to suppress IFN-γ expression resulting in suppression of CD8+ lymphocyte function (Fogle et al; 2010). These findings help explain the paradox of chronic HIV-1 infection, in which CD8+ T cells display an activated phenotype but exhibit

humoral immune responses to both HIV and other antigens (Cohen et al; 1999).

both HIV and other viral proteins such as vaccinia virus (Garba et al; 2002).

replication (Pantaleo and Fauci, 1995).

biologically active *in vitro* (Reinhold et al; 1999).

IL-10 is an anti-inflammatory cytokine that essentially plays two regulatory roles in innate and adaptive immunity. It suppresses the up-regulation of various genes in macrophages and dendritic cells that are normally stimulated via toll-like receptors and promotes the proliferation of cytotoxic T cells, activates B cells, and induces the upregulation of specific genes in toll-like receptor activated phagocytic and dendritic cells (Trincheri, 2007). In addition, a critical function of IL-10 is its ability to inhibit pro-inflammatory cytokines such as TNF-α, IFN-γ, IL-1, IL-6, IL-2, and IL-12 (Trincheri, 2007). IL-10 decreases the production of pro-inflammatory cytokines by limiting the major histocompatibility class II and CD80/CD 86 expressed on monocytes and macrophage (Wang et al; 2005). IL-10 was believed to be produced by CD4+ Th2 cells; however, studies have shown that it is secreted by both Th1 and Th2 cells (Brockman et al; 2009). Also, cells from the myeloid lineage which include macrophages and dendritic cells also produce cytokine IL-10 (Hedrich and Bream, 2010). Furthermore, IL-10 is regulated both at the transcriptional and post-translational level and is involved in various signaling pathways (Couper et al; 2008).

inhibition of T cell proliferation and IFN- production (Othieno et al; 1999).

There are several speculations of the role of IL-10 in HIV pathogenesis and the subject has been a popular interest in many studies. Ji *et al*., 2005 reported that CD14+ monocytes are the main cells producing cytokine IL-10 in PBMCs after HIV-1 infection via interactions independent of CD4+ molecules, thus, concluding that IL-10 production is dependent on the presence of CD14+ monocytes. Moreover, as the patient progresses to advanced stages of HIV disease, the frequency of IL-10 producing cells increases significantly (118). On the other hand, Naicker *et al*., 2009, stated that different stages of the HIV disease will govern what role IL-10 will play in infected individuals. For instance, in acute HIV-1 individuals, IL-10 may promote viral replication by inhibiting effector immune response from both arms of the innate and adaptive immunity (Naicker et al; 2009). Furthermore, it was proposed in a chronic phase, that IL-10 resembled a protective role by reducing immune activation, inhibiting virus replication in macrophages, and the increase in production of IL-10 levels lowered plasma viral load and increased CD4+ cell count (Naicker et al; 2009).

## **13. HIV and free radicals**

It has been shown that HIV infection induces increased production of free radicals by macrophages. Free radical formation occurs as a byproduct of oxidative stress. Oxidative stress occurs when there is a disproportion between the reactive oxygen elements in the body versus the ability of the body to properly eliminate these reactive species. The presence of free radicals has been implicated in disturbing and damaging a number of biological processes (Karthikeyan et al; 2010). With regards to HIV infection the increase of oxidative stress has been seen to influence components in antioxidant defense in physiological antioxidants such as glutathione which are seen to decrease dramatically in HIV patients (Pace and Leaf, 1995). In addition to glutathione, vitamin A, C, and E at high doses as well as improving low levels of selenium were associated with assisting the prevention of HIV infection progression by working as antioxidants to remove free radicals

Role of Cytokines and Chemokines in HIV Infection 293

lesions was seen to be reduced in response to antioxidant supplement and vitamin treatments, which correlates to free radical influence in DNA damage, and potential

Both HIV-1 and HIV-2 cause AIDS, but HIV-1 is found worldwide, whereas HIV-2 is found primarily in West Africa. Chemokine receptors, such as CXCR4 and CCR5 proteins, are required for the entry of HIV into CD4-positive cells. After establishing infection, HIV alters the synthesis of host cytokines and chemokines and kills CD4+ T lymphocytes thereby resulting in the loss of cell-mediated immunity and a high probability that the host will

Alfano, M and Poli, G. 2005. Role of cytokines and chemokines in the regulation of innate

Bahia, M.S and Silakari, O. 2010. Tumor Necrosis Factor Alpha Converting Enzyme: An

Bautista AP. 2001 Free radicals, chemokines, and cell injury in HIV-1 and SIV infections and alcoholic hepatitis. Free Radical Biology and Medicine*.* 31(12):1527-1532. Brabers NA and Nottet HS. 2006. Role of the pro-inflammatory cytokines TNF-alpha and IL-

Brach MA, de Vos S, Arnold C, Gruss HJ, Mertelsmann R and Herrmann F. 1992.

Brenchley, Jason M., Paiardini, M., Knox, Kenneth S., Asher, Ava I., Cervasi, B., Asher, Tedi

Brockman MA, Kwon DS, Tighe DP, Pavlik DF, Rosato PC, Sela J, Porichis F, Le Gall S,

Bucci JG, Gebhard DH, Childers TA, English RV, Tompkins MB and Tompkins WA. 1998.

Bujak M and Frangogiannis NG. 2009. The role of IL-1 in the pathogenesis of heart disease.

Burchett SK, Weaver WM, Westall JA, Larsen A, Kronheim S and Wilson CB. 1998.

mononuclear phagocytes. Journal of immunology. 140(10):3473-81.

and reversibly inhibits virus-specific T cells. Blood. 114(2):346-56.

chain. The Journal of infectious diseases.178(4):968-77.

Arch Immunol Ther Exp (Warsz). 57(3):165-76.

chi B and NF-IL6. Eur J Immunol*.* Oct 22(10):2705-2711.

Encouraging Target for Various Inflammatory Disorders. Chemical Biology & Drug

1beta in HIV-associated dementia. European journal of clinical investigation.

Leukotriene B4 transcriptionally activates interleukin-6 expression involving NK-

E., Scheinberg, P., Price, David A., Hage, Chadi A., Kholi, Lisa M., Khoruts, A., Frank, I., Else, J., Schacker, T., Silvestri, G and Daniel C. Douek. 2008. Differential Th17 CD4 T-cell depletion in pathogenic and nonpathogenic lentiviral infections.

Waring MT, Moss K, Jessen H, Pereyra F, Kavanagh DG, Walker BD and Kaufmann DE. 2009. IL-10 is up-regulated in multiple cell types during viremic HIV infection

The CD8+ cell phenotype mediating antiviral activity in feline immunodeficiency virus-infected cats is characterized by reduced surface expression of the CD8 beta

Regulation of tumor necrosis factor/cachectin and IL-1 secretion in human

immunity and HIV infection. Molecular Immunology 42: 161-182.

progression of HIV infection (Olinski et al; 2002).

**14. Conclusion** 

**15. References** 

develop opportunistic infections.

Design. 75: 415-443.

Blood 112(7): 2826-2835.

36(7):447-58.

(Garland and Fawzi, 1999). The aforementioned studies may provide a low cost method for improving the prognosis of HIV infected patients in high risk, underprivileged areas of the world.

Chronic oxidative stress is often associated with HIV infection and research indicates a benefit for increased antioxidant vitamins and supplements in reduction of DNA base damage, which in turn can slow progression of infection (Jaruga et al; 2002). Neutrophils from asymptomatic HIV patients show increased oxygen radical production which can be modified by treating with N-acetyl cysteine, a compound used as an antioxidant (Smietana et al; 2008). The role of free radical oxidative stress on DNA damage is correlated with stimulated DNA repair mechanisms which activate enzymes associated with initiation of apoptosis such as poly ADP-ribose transferase and p53. Reduced NAD/NADH production would lower ATP synthesis that in turn correlates with a deficiency in glutathione; which as mentioned is an endogenous antioxidant important in resolving imbalance of free radicals (Dobmeyer et al; 1997).

The progression of HIV is correlated with a decreased immunity. One way in which this decreased immunity progresses is by free radical overload of monocytes and granulocytes which leads to deficiency of antioxidant mechanisms which may lead to the loss of CD4 cells often seen in the progression of HIV (Dobmeyer et al; 1997). The decreased immunity may also be related to the reactive oxygen species and free radical presence which is higher in HIV infected patients. With HIV infection progression there is an increased production of reactive oxygen species which leads to the theory of free radical mediated apoptosis of lymphocytes which reduces the ability for immune response to progressive HIV infections (Dobmeyer et al; 1997). With regards to CD4 cell counts the apoptosis of lymphocytes by free radicals leads to progression of immunodeficiency and makes for a quicker transition from HIV infection to AIDS (Bautisita, 2001). It has been published that during HIV-1 infection, hematopoietic cells are exposed to high amounts of free radicals. Subsequently there is a reduction of leukocytopoiesis and increase susceptibility to further infections (Masutani, 2000). Furthermore, there is a link to lipid peroxidation observed in patients with HIV or AIDS to a deficiency of antioxidants which leads to free radical proliferation (Favier et al; 1994 ).

Rate of viral replication is a key process to the proliferation of HIV infection. The conditions in which viruses such as HIV will proliferate seem to correlate with the presence of oxidative stress/free radicals *in vitro* (Fuchs et al; 1991). There tends to be an increase in nuclear transcription factor and inflammatory cytokine activation of the immune system (Brach et al; 1992). The progression of the virus/infection will then allow for opportunistic infections which then would also promote more oxidative stress due to increased free radical elements, again improving viral replication and weakening antioxidant defense (Knysz, 2007).

Damage or altering of the DNA repair machinery is an important aspect of the progression of HIV infection pathogenesis (Olinski et al; 2002). There is a slow and deliberate degradation of cellular components such as membrane blebbing, chromatin condensation, and DNA cleavage ability. Additionally, there is evidence that shows oxidative DNA damage will lead to the apoptotic cell death in HIV infected patients. There appears to be an increase in oxidatively modified DNA bases in HIV infected patients leading to what is known as pyrimidine and purine derived lesions. One specific lesion labeled, 8-OH-Gua was found in isolated lymphocytes of HIV patients. The presence of this lesion leads to transversions of DNA base pairs unless repaired before replication. The number of these lesions was seen to be reduced in response to antioxidant supplement and vitamin treatments, which correlates to free radical influence in DNA damage, and potential progression of HIV infection (Olinski et al; 2002).

## **14. Conclusion**

292 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

(Garland and Fawzi, 1999). The aforementioned studies may provide a low cost method for improving the prognosis of HIV infected patients in high risk, underprivileged areas of the

Chronic oxidative stress is often associated with HIV infection and research indicates a benefit for increased antioxidant vitamins and supplements in reduction of DNA base damage, which in turn can slow progression of infection (Jaruga et al; 2002). Neutrophils from asymptomatic HIV patients show increased oxygen radical production which can be modified by treating with N-acetyl cysteine, a compound used as an antioxidant (Smietana et al; 2008). The role of free radical oxidative stress on DNA damage is correlated with stimulated DNA repair mechanisms which activate enzymes associated with initiation of apoptosis such as poly ADP-ribose transferase and p53. Reduced NAD/NADH production would lower ATP synthesis that in turn correlates with a deficiency in glutathione; which as mentioned is an endogenous antioxidant important in resolving imbalance of free radicals

The progression of HIV is correlated with a decreased immunity. One way in which this decreased immunity progresses is by free radical overload of monocytes and granulocytes which leads to deficiency of antioxidant mechanisms which may lead to the loss of CD4 cells often seen in the progression of HIV (Dobmeyer et al; 1997). The decreased immunity may also be related to the reactive oxygen species and free radical presence which is higher in HIV infected patients. With HIV infection progression there is an increased production of reactive oxygen species which leads to the theory of free radical mediated apoptosis of lymphocytes which reduces the ability for immune response to progressive HIV infections (Dobmeyer et al; 1997). With regards to CD4 cell counts the apoptosis of lymphocytes by free radicals leads to progression of immunodeficiency and makes for a quicker transition from HIV infection to AIDS (Bautisita, 2001). It has been published that during HIV-1 infection, hematopoietic cells are exposed to high amounts of free radicals. Subsequently there is a reduction of leukocytopoiesis and increase susceptibility to further infections (Masutani, 2000). Furthermore, there is a link to lipid peroxidation observed in patients with HIV or AIDS to a deficiency of antioxidants which leads to free radical proliferation (Favier

Rate of viral replication is a key process to the proliferation of HIV infection. The conditions in which viruses such as HIV will proliferate seem to correlate with the presence of oxidative stress/free radicals *in vitro* (Fuchs et al; 1991). There tends to be an increase in nuclear transcription factor and inflammatory cytokine activation of the immune system (Brach et al; 1992). The progression of the virus/infection will then allow for opportunistic infections which then would also promote more oxidative stress due to increased free radical elements, again improving viral replication and weakening antioxidant defense

Damage or altering of the DNA repair machinery is an important aspect of the progression of HIV infection pathogenesis (Olinski et al; 2002). There is a slow and deliberate degradation of cellular components such as membrane blebbing, chromatin condensation, and DNA cleavage ability. Additionally, there is evidence that shows oxidative DNA damage will lead to the apoptotic cell death in HIV infected patients. There appears to be an increase in oxidatively modified DNA bases in HIV infected patients leading to what is known as pyrimidine and purine derived lesions. One specific lesion labeled, 8-OH-Gua was found in isolated lymphocytes of HIV patients. The presence of this lesion leads to transversions of DNA base pairs unless repaired before replication. The number of these

world.

(Dobmeyer et al; 1997).

et al; 1994 ).

(Knysz, 2007).

Both HIV-1 and HIV-2 cause AIDS, but HIV-1 is found worldwide, whereas HIV-2 is found primarily in West Africa. Chemokine receptors, such as CXCR4 and CCR5 proteins, are required for the entry of HIV into CD4-positive cells. After establishing infection, HIV alters the synthesis of host cytokines and chemokines and kills CD4+ T lymphocytes thereby resulting in the loss of cell-mediated immunity and a high probability that the host will develop opportunistic infections.

## **15. References**


Role of Cytokines and Chemokines in HIV Infection 295

Egilmez NK, Harden JL, Virtuoso LP, Schwendener RA and Kilinc MO. 2011. Nitric oxide

Emilie D, Peuchmaur M, Maillot MC, Crevon MC, Brousse N, Delfraissy JF, et al. 1990.

Epstein LG and Gendelman HE. 1993. Human immunodeficiency virus type 1 infection of the nervous system: pathogenetic mechanisms. Annals of neurology. 33(5):429-36. Eugenin, Eliseo A., Osiecki, K., Lopez, L., Goldstein, H., Calderon, Tina M and Joan W.

Favier A, Sappey C, Leclerc P, Faure P and Micoud M. 1994. Antioxidant status and lipid peroxidation in patients infected with HIV. Chem Biol Interact*.* 91(2-3):165-180. Fernandez-Ortega, C., Dubed, M., Ramos, T., Navea, L., Alvarez, G., Lobaina, L., Lopez, L.,

Fogle JE, Mexas AM, Tompkins WA and Tompkins MB. 2010. CD4(+)CD25(+) T regulatory

Fuchs J, Ochsendorf F, Schofer H, Milbradt R and Rubsamen-Waigmann H. 1991. Oxidative

Gallo P, Frei K, Rordorf C, Lazdins J, Tavolato B and Fontana A. 1989. Human

Garland M and Fawzi WW. 1999. Antioxidants and progression of human immunodeficiency virus (HIV) disease. Nutrition Research*.*19(8):1259-1276. Gorry, P.R. and Ancuta, P. 2011. Coreptors and HIV-1 Pathogenesis. Curr HIV/AIDS Rep.

Gorry, P.R., et al. 2007. Changes in region of gp120 contribute to unusually broad co-

Grivel, J., et al. 2010. Selective transmission of R5 HIV-1 variants: where is the gatekeeper?

Hedrich, C. M., & Bream and J. H. 2010. Cell type-specific regulation of IL-10 expression in

Heinrich, Peter C., Iris Behrmann, Serge Haan, Heike M. Hermanns, Gerhard Müller-Newen

Journal of Transitional Medicine. 9 (Suppl 1): S6.

inflammation and disease. Immunol Res*.* 47(1-3): 185-206.

and Its Regulation. Institut Für Biochemie. 374: 1-20.

imbalance in HIV infected patients. Med Hypotheses*.* 36(1):60-64.

nodes. The Journal of clinical investigation. 86(1):148-59.

NeuroAIDS. The Journal of Neuroscience. 26(4): 1098-1106. Fantuzzi G. 2003. Cytokine knockouts. Totawa, NJ: Humana Press; p. 471.

Immunotherapy 2011; 1-7.

Communications. 325: 1075-1081.

retroviruses. 26(2):201-16.

168(5):2247-54.

8(1):45-53.

163-178.

short-circuits interleukin-12-mediated tumor regression. Cancer Immunology,

Production of interleukins in human immunodeficiency virus-1-replicating lymph

Berman. 2006. CCL2/monocyte chemoattractant protein-1 mediates enhanced transmigration of human immunodeficiency virus (HIV)-infected leukocytes across the blood-brain-barrier: a potential mechanism of HIV-CNS invasion and

Casilla, D and Rodriguez, L. 2004. Non-induced leukocyte extract reduces HIV replication and TNF secretion. Biochemical and Biophysical Research

cells inhibit CD8(+) IFN-gamma production during acute and chronic FIV infection utilizing a membrane TGF-beta-dependent mechanism. AIDS research and human

immunodeficiency virus type 1 (HIV-1) infection of the central nervous system: an evaluation of cytokines in cerebrospinal fluid. J Neuroimmunol. 23(2):109-16. Garba ML, Pilcher CD, Bingham AL, Eron J and Frelinger JA. 2002. HIV antigens can induce

TGF-beta(1)-producing immunoregulatory CD8+ T cells. Journal of immunology.

receptor usage of an HIV-1 isolate from CCR5 Delta32 heterozygote. Virology. 362:

and Fred Schaper. 2003. Principles of Interleukin (IL)-6-type Cytokine Signalling


Cannon, P and June, C. 2011. Chemokine receptor 5 knockout strategies. Current Opinion in

Chang, Seon H., and Chen Dong. 2007. A novel heterodimeric cytokine consisting of IL-17 and IL-17F regulates inflammatory responses. *Cell Research* 17: 435-440. Charo, Israel F. and Richard M. Ransohoff. 2006. The many roles of chemokines and

Cheng SM, Li JCB, Lin SS, Lee DCW, Liu L, Chen Z, and Lau ASY. 2009. HIV-1

Cheung R, Ravyn V, Wang L, Ptasznik A and Collman RG. 2008. Signaling mechanism of

Clerici M, Frances TH, Venzon D J, Blatt S, Hendrix CW, Wynn TA and Shearer GM. 1993.

Immunodeficiency Virus-seropositive Individuals. J Clin Invest*.* 91:759-765. Cohen SS, Li C, Ding L, Cao Y, Pardee AB, Shevach EM, et al. 1999. Pronounced acute

Couper, K. N., Blount, D. G and Riley, E. M. 2008. IL-10: the master regulator of immunity to

Crome, S.Q., Wang, A.Y and M.K. Levings. 2009. Translational Mini-Review Series on Th17

Daly, Christine and Barrett J. Rollins. 2003. Monocyte Chemoattractant Protein-1 (CCL2) in

Deshmane, Satish L., Kremlev, S., Amini, S and Bassel E. Sawaya. 2009. Monocyte

Devadas K, Hardegen NJ, Wahl LM, Hewlett IK, Clouse KA, Yamada KM, et al. 2004.

Dinarello CA. 1999. Cytokines as endogenous pyrogens. The Journal of infectious

Dobmeyer TS, Findhammer S, Dobmeyer JM, et al. 1997. Ex vivo induction of apoptosis in

Edo-Matas D., van Dort KA, Setiawan LC, Schuitemaker H, Kootstra NA. 2011. Comparison

human Immunodeficiency type 1 variants. Virology. 412(2):269-77.

dysregulation of IFN-g signaling. Blood. 13: 5192-5201.

Journal of immunology. 180(10):6675-84.

journal of pharmacology. 134(6):1344-50.

infection. J Immunol*.* 180(9): 5771-5777.

Journal of Translational Immunology. 159: 109-119.

controversies. Microcirculation. 10: 247-257.

infection. Free Radic Biol Med*.* 22(5):775-785.

Research. 29(6): 313-326.

diseases.179 Suppl 2:S294-304.

173(11):6735-44.

chemokine receptors in inflammation. The New England Journal of Medicine. 345:

transactivator protein induction of suppressor cytokine signaling-2 contributes to

HIV-1 gp120 and virion-induced IL-1beta release in primary human macrophages.

Changes in Interleukin-2 and Interleukin-4 Production in Asymptomatic Human

immunosuppression in vivo mediated by HIV Tat challenge. Proceedings of the National Academy of Sciences of the United States of America. 96(19):10842-7. Corasaniti MT, Bilotta A, Strongoli MC, Navarra M, Bagetta G and Di Renzo G. 2001. HIV-1

coat protein gp120 stimulates interleukin-1beta secretion from human neuroblastoma cells: evidence for a role in the mechanism of cell death. British

Cells: Function and regulation of human T helper 17 cells in health and disease. The

Inflammatory Disease and Adaptive Immunity: Therapeutic opportunities and

chemoattractant protein-1 (MCP-1): an overview. Journal of Interferon & Cytokine

Mechanisms for macrophage-mediated HIV-1 induction. Journal of immunology.

lymphocytes is mediated by oxidative stress: role for lymphocyte loss in HIV

of in vivo and in vitro evolution of CCR5 to CXCR4 co-receptor use of primary

HIV and AIDS. 6: 74-79.

610-621.


Role of Cytokines and Chemokines in HIV Infection 297

Lipton SA. 1998. Neuronal injury associated with HIV-1: approaches to treatment. Annu Rev

Liu RM and Gaston Pravia KA. 2010. Oxidative stress and glutathione in TGF-beta-mediated

Louis S., Dutertre CA., Vimeux L., Fery L., Henno L., Dioccous S., Kahi S., Deveau C., Meyer

Maek-A-Nantawat, W, Buranapraditkun, S, Klaewsongkram, J and Kiat Ruxrungthum.

Mahad, Don J and Richard M. Ransohoff. 2003. The role of MCP-1 (CCL2) and CCR2 in

Marui N, Offermann MK, Swerlick R, Kunsch C, Rosen CA, Ahmad M, et al. 1993. Vascular

Massague J and Gomis RR. 2006. The logic of TGF-beta signaling. FEBS letters. 580(12):2811-

Mariani, S.A. 2010. Asymmetric HIV-1 co-receptor use and replication in CD4+ T

Masutani H. 2000. Oxidative stress response and signaling in hematological malignancies

Merrill JE, Koyanagi Y and Chen IS. 1989. Interleukin-1 and tumor necrosis factor alpha can

Merrill JE, Koyanagi Y, Zack J, Thomas L, Martin F and Chen IS. 1992. Induction of

Mexas AM, Fogle JE, Tompkins WA and Tompkins MB. 2008. CD4+CD25+ regulatory T

Miles, Steven A., Ahmad R. Rezai, Jesus F. Salazar-Gonzalez, Meta Vander Meyden, Ronald

be induced from mononuclear phagocytes by human immunodeficiency virus type

interleukin-1 and tumor necrosis factor alpha in brain cultures by human

cells are infected and activated during acute FIV infection. Vet Immunol

H. Stevens, Diane M. Logan, Ronald T. Mitsuyasu, Tetsuya Taga, Toshio Hirano, Tadamitsu Kishimoto and Otoniel Matinez-Maza. 1990. AIDS Kaposi Sarcoma-

lymphocytes. Journal of Translational Medicine. 9 (Suppl 1):S8.

1 binding to the CD4 receptor. Journal of virology. 63(10):4404-8.

immunodeficiency virus type 1. Journal of virology. 66(4):2217-25.

endothelial cells. The Journal of clinical investigation. 92(4):1866-74. Martinon F, Burns K and Tschopp J. 2002. The inflammasome: a molecular platform

Massague J. 1992.Receptors for the TGF-beta family. Cell. 69(7):1067-70.

and HIV infection. Int J Hematol*.* 71(1):25-32.

fibrogenesis. Free radical biology & medicine. 48(1):1-15.

2008. Genetic deficiency of chemokine receptor CCR5 is a strong risk factor for symptomatic West Nile virus infection: a meta-analysis of 4 cohorts in the US

L., Goujard C and Hosmalin A. 2010. IL-23 and IL-12p70 production by monocytes and dendritic cells in primary HIV-1 infection. Journal of Leukocyte Biology.

2007. Increased interleukin-17 production both in helper T cell subset Th17 and CD4-negative T cells in human immunodeficiency virus infection. Viral

multiple sclerosis and experimental autoimmune encephalomyelitis (EAE).

cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular

triggering activation of inflammatory caspases and processing of proIL-beta.

Li MO and Flavell RA. 2008. TGF-beta: a master of all T cell trades. Cell. 134(3):392-404. Lim JK, Louie CY, Glaser C, Jean C, Johnson B, Johnson H, McDermott DH, Murphy PM.

epidemic. J Infect Dis. 197:262-5.

Pharmacol Toxicol. 38:159-77.

Immunology. 20(1): 66-75.

Molecular cell. 10(2):417-26.

Immunopathol. 126 (3-4):263-72.

Seminars in Immunology. 15: 23-32.

87(4):645.

20.


Herbein G and Varin A. 2010. The macrophage in HIV-1 infection: from activation to

Hibi M K. Nakajima and T. Hirano. 1996. IL-6 Cytokine Family and Signal Transduction: a Model of the Cytokine System. Journal of Molecular Medicine. 74.1: 1-12. Hutter G. 2009. Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell

Jaruga P, Jaruga B, Gackowski D, et al. 2002. Supplementation with antioxidant vitamins

Ji, J., Sahu, G. K., Braciale, V. L and Cloyd, M. W. 2005. HIV-1 induces IL-10 production in human monocytes via a CD4-independent pathway. Int Immunol. 17(6): 729-736. Karthikeyan R, Manivasagam T, Anantharaman P, Balasubramanian T and Somasundaram

Kaul M, Garden GA and Lipton SA. 2001.Pathways to neuronal injury and apoptosis in

Kindberg, E., Mickiene A, Ax C, Akerlind B, Vene S, Lindquist L, Lundkvist A, Svensson L.

Kwong P.D., Wyatt R, Sattentau QJ, Sodroski J, Hendrickson WA. 2000. Oligomeric

Klein SA, Dobmeyer JM, Dobmeyer TS, Pape M, Ottmann OG, Helm EB, Hoelzer D and

Knysz B, Szetela B and Gladysz A. 2007. Pathogenesis of HIV-1 infection - chosen aspects.

Leask A and Abraham DJ. 2004. TGF-beta signaling and the fibrotic response. The FASEB

Leghmari, K., Bennasser, Y., Tkaczuk, J and Bahraoui, E. 2008. HIV-1 Tat protein induces IL-

Lepe-Zuniga JL, Mansell PW and Hersh EM. 1987. Idiopathic production of interleukin-1 in

Letterio JJ and Roberts AB. 1998. Regulation of immune responses by TGF-beta. Annual

Levy and Jay A. 2009. HIV pathogenesis: 25 years of progress and persistent challenges.

Li JCB, Au K, Fang J, Yim HCH, Chow K, Ho P and Lau ASY. 2011. HIV-1 trans-activator

PKC- and p38 MAP kinase. Cellular Immunology. 253: 45-53.

Levy J. 2007. HIV and the pathogenesis of AIDS. Washington, DC: ASM Press.

prevents oxidative modification of DNA in lymphocytes of HIV-infected patients.

S. 2010. Chemopreventive effect of Padina boergesenii extracts on ferric nitrilotriacetate (Fe-NTA)-induced oxidative damage in Wistar rats. Journal of

2008. A deletion in the chemokine receptor 5 (CCR5) gene is associated with

modeling and electrostatic analysis of the gp120 envelope glycoprotein of human

Rossol R, 1997. Demonstration of the Th1 to Th2 cytokine shift during the course of HIV-1 infection using cytoplasmic cytokine detection on single cell level by flow

journal : official publication of the Federation of American Societies for

10 production by an alternative TNF-a-independent pathway in monocytes: Role of

acquired immune deficiency syndrome. Journal of clinical microbiology. 25(9):1695-

protein dysregulates IFN-g signaling and contributes to the suppression of

deactivation? Retrovirology. 7:33.

Free Radic Biol Med*.* 32(5):414-420.

Applied Phycology*.* 1-7

transplantation. N. Engl J Med. 360: 692-698.

HIV-associated dementia. Nature. 410(6831):988-94.

tickborne encephalitis. J Infect Dis. 197:266-9.

immunodeficiency virus. J Virol. 74:1961-1972.

cytometry. AIDS. 11:1111-1118

HIV & AIDS Review*.* 6(1):7-11.

700.

AIDS. 23:147-160.

Experimental Biology. 18(7):816-27.

review of immunology. 16:137-61.

autophagy induction. AIDS*.* 25:15-25.

Li MO and Flavell RA. 2008. TGF-beta: a master of all T cell trades. Cell. 134(3):392-404.


Role of Cytokines and Chemokines in HIV Infection 299

Poli G and Fauci AS. 1993. Cytokine modulation of HIV expression. Semin Immunol.

Reinhold D, Wrenger S, Kahne T and Ansorge S. 1999. HIV-1 Tat: immunosuppression via

Salgame P, Guan MX, Agahtehrani A and Henderson EE. 1998. Infection of T Cell Subsets

Samson, M. 1996. Resistance to HIV-1 infection in Caucasian individuals bearing mutant

Sandborg, Christy I., Karen L. Imfeld, Frank Zaldivar and Monique A. Berman. 1994. HIV

Smietana M, Clayette P, Mialocq P, Vasseur J-J and Oiry J. 2008. Synthesis of new N-

Spear, Gregory T., M. Reza Zariffard, Hua Y. Chen, Joshua J. Anzinger, Kathryn Anastos,

Rwandan Women. AIDS Research and Human Retroviruses*.* 24.7: 973-76. Suleiman, J. 2010. Vicriviroc in combination therapy with an optimized regimen for

Tagliamonte, M, Tornesello ML, Buonaguro FM, Buonaguro L. 2011. Conformational HIV-1

Taoufik Y, Lantz O, Wallon C, Charles A, Dussaix E and Delfraissy JF. 1997. Human

Tompkins MB and Tompkins WA. 2008. Lentivirus-induced immune dysregulation. Vet

Trinchieri Giorgio. 2003. Interleukin-12 and the regulation of innate resistance and adaptive

Trinchieri, G. 2007. Interleukin-10 production by effector T cells: Th1 cells show self control.

Tyor WR, Glass JD, Griffin JW, Becker PS, McArthur JC, Bezman L, et al. 1992.Cytokine

Vahlenkamp TW, Tompkins MB and Tompkins WA. 2004. Feline immunodeficiency virus

CD4+CD25+ T regulatory cells. Journal of immunology. 172(8):4752-61. Vannberg FO, Chapman SJ and Hill AVS. 2011. Human genetic susceptibility to intracellular

expression in the brain during the acquired immunodeficiency syndrome. Annals

infection phenotypically and functionally activates immunosuppressive

monocytes: an indirect interleukin-10-mediated effect. Blood. 89: 2842. Tenorio, A. R. 2011. The monoclonal CCR5 antibody PRO-140: the promise of once-weekly

alleles of the CCR-5 chemokine receptor gene. Nature. 382: 722-725.

by HIV-1 and the Effects of Interleukin-12. J. Interferon and Cytokine Res*.* 18:521-

Type 1 Induction of Interleukin 1 and 6 Production by Human Thymic Cells. AIDS

isobutyryl-l-cysteine/MEA conjugates: Evaluation of their free radical-scavenging activities and anti-HIV properties in human macrophages. Bioorganic Chemistry*.* 

John Rusine, John Gatabazi, Audrey L. French, Mardge Cohen and Alan L. Landay. 2008. Positive Association between HIV RNA and IL-6 in the Genital Tract of

treatment-experienced subjects: 48-week results of the VICTOR-E1 phase 2 trial. J

envelope on particulate structures: a tool for chemokine coreceptor binding studies.

immunodeficiency virus gp120 inhibits interleukin-12 secretion by human

TGF-beta1 induction. Immunology today. 20(8):384-5.

Research and Human Retroviruses*.* 10.10: 1221-229.

Journal of Translational Medicine. 9 (Suppl 1):S1.

HIV therapy. Curr HIV/AIDS Rep. 8(1):1-3.

immunity. Nature Reviews Immunology. 3: 133.

pathogens. Immunological Reviews. 240:105.

Immunol Immunopathol. 123(1-2):45-55.

J Exp Med. 204(2): 239-243.

of neurology. 31(4):349-60.

5(3):165-73.

36(3):133-140.

Infect Dis. 201:590-9.

528.

Derived Cells Produce and Respond to Interleukin 6. Proceedings of the National Academy of Sciences*.* 87.11: 4068-072.


Mirani M, Elenkov I, Volpi S, Hiroi N, Chrousos GP and Kino T. 2002. HIV-1 protein Vpr

immunodeficiency virus. The Journal of clinical investigation. 84(3):733-7. Naicker, D. D., Werner, L., Kormuth, E., Passmore, J. A., Mlisana, K., Karim, S. A., et al.

Nakajima K, Martínez-Maza O, Hirano T, Breen EC, Nishanian PG, Salazar-Gonzalez JF,

Olinski R, Gackowski D, Foksinski M, Rozalski R, Roszkowski K and Jaruga P. 2002.

Olney JW, Zorumski C, Price MT and Labruyere J. 1990. L-cysteine, a bicarbonate-sensitive

Osakwe CE, Bleotu C, Chifiriuc MC, Crancea C, Otelea D, Paraschiv S, Petrea S, Dinu M,

Othieno C, Hirsch CS, Hamilton BD, Wilkinson K, Ellner JJ and Toossi Z. 1999. Interaction of

Pace GW and Leaf CD. 1995. The role of oxidative stress in HIV disease. Free Radic Biol

Pantaleo G, Graziosi C, Demarest JF, Butini L, Montroni M, Fox CH, et al. 1993. HIV

Pantaleo G, Demarest JF, Soudeyns H, Graziosi C, Denis F, Adelsberger JW, et al. 1994.

Pantaleo G and Fauci AS. 1995. New concepts in the immunopathogenesis of HIV infection.

Persidsky Y, Buttini M, Limoges J, Bock P and Gendelman HE. 1997. An analysis of HIV-1-

Poggi A and Zocchi MR. 2006. HIV-1 Tat triggers TGF-beta production and NK cell

primary immune response to HIV. Nature. 370(6489):463-7.

HIV-1 encephalitis. Journal of neurovirology. 3(6):401-16.

endogenous excitotoxin. Science (New York, NY. 248(4955):596-9.

primary HIV-1 pathogenesis. J Infect Dis*.* 200(3): 448-452.

beta 2) production by HIV. J Immunol.142(2):531-6 Nambu A and Nakae S. 2010. IL-1 and Allergy. Allergol Int. 59(2):125-35.

interleukin-10. Infect Immun. 67(11):5730-5.

stage of disease. Nature. 362(6418):355-8.

Annual review of immunology. 13:487-512.

immunology. 13(2-4):369-72.

33(2):192-200.

69(1):24-34.

Med*.* 19(4):523-528.

Academy of Sciences*.* 87.11: 4068-072.

Derived Cells Produce and Respond to Interleukin 6. Proceedings of the National

suppresses IL-12 production from human monocytes by enhancing glucocorticoid action: potential implications of Vpr coactivator activity for the innate and cellular immunity deficits observed in HIV-1 infection. Journal of Immunology.169:6361. Molina JM, Scadden DT, Byrn R, Dinarello CA and Groopman JE. 1989. Production of tumor

necrosis factor alpha and interleukin 1 beta by monocytic cells infected with human

2009. Interleukin-10 promoter polymorphisms influence HIV-1 susceptibility and

Fahey JL and Kishimoto T. 1989. Induction of IL-6 (B cell stimulatory factor-2/IFN-

Oxidative DNA damage: assessment of the role in carcinogenesis, atherosclerosis, and acquired immunodeficiency syndrome. Free Radical Biology and Medicine*.* 

Baicus C, Streinu-Cercel A and Lazar V. 2010. TH1/TH2 Cytokine Levels as an Indicator for Disease Progression in Human Immunodeficiency Virus Type 1 Infection and Response to Antiretroviral Therapy. Roum Arch Microbiol Immunol*.* 

Mycobacterium tuberculosis-induced transforming growth factor beta1 and

infection is active and progressive in lymphoid tissue during the clinically latent

Major expansion of CD8+ T cells with a predominant V beta usage during the

associated inflammatory products in brain tissue of humans and SCID mice with

apoptosis that is prevented by pertussis toxin B. Clinical & developmental


**12** 

*Brazil* 

**(HIV-1) Infection** 

*State University of Londrina* 

Maria Angelica Ehara Watanabe

**The Role of Genetic Polymorphisms in the** 

Edna Maria Vissoci Reiche, Marla Karine Amarante and

**Chemokine and Their Receptors and Cytokines** 

The natural history and pathogenic processes of human immunodeficiency virus type 1 (HIV-1) infection are complex and variable, and depend on many viral and host factors and their interactions (Pantaleo et al., 1997). Individuals are not equal susceptible to the infection and have differences in their viral set points, rates of decline of CD4+ T cells, levels of viremia, emergence of citotoxic T lymphocyte (CTL) escape mutants, and development of opportunistic infections resulting in varying incubation periods of the virus (Kaur & Mehra, 2009). HIV-1 infected individuals present different rates of disease progression; while a majority of individual progress to acquired immunodeficiency syndrome (AIDS) after the infection, most of them can be turned aviremic even the absence of antiretroviral therapy (ARV) up to ten years and are called typical progressors. Most importantly, ~5% to 10% of persistently infected individuals show no signs of disease progression for over 12 years and remain asymptomatic and aviremic and are classified as long term nonprogressors (LTNPs). On the other hand, rapid progressors are individuals that rapidly progress to AIDS within four years after primary HIV-1 infection and some individuals have been known to progress to AIDS and

death within a year after primary infection (Fauci et al., 1996; Rosenberg & Fauci, 1991).

Genetic factors may be one of the host factors responsible for the susceptibility to infection and disease progression. However, no single gene or polymorphism is likely to be responsible for these effects. Brass et al. (2008) have reported that HIV-1 uses at least 250 host-derived dependency factors for gaining entry into target cells and completing its life cycle. Hence, multiple genetic factors are expected to be involved in susceptibility, disease pathogenesis, and progression following HIV-1 infection. Some of these genes that have been established with HIV/AIDS conclusively involve are: (1) genes influencing viral entry by altering the expression on cell surface the levels of chemokine receptors and their ligands as well cytokines (Seisdedos & Parmentier, 2006; Reiche et al., 2007); (2) genes involved in anti-HIV immune response including the antiviral Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G) gene family on chromosome 22q13 (An et al., 2009); the virus restriction factor Tripartite Interaction Motif 5 (TRIM5) on

**1. Introduction** 

**in the Human Immunodeficiency Virus Type 1** 


## **The Role of Genetic Polymorphisms in the Chemokine and Their Receptors and Cytokines in the Human Immunodeficiency Virus Type 1 (HIV-1) Infection**

Edna Maria Vissoci Reiche, Marla Karine Amarante and Maria Angelica Ehara Watanabe *State University of Londrina Brazil* 

#### **1. Introduction**

300 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

Villinger F and Ansari AA. 2010. Role of IL-12 in HIV infection and vaccine. European

Wahl LM, Corcoran ML, Pyle SW, Arthur LO, Harel-Bellan A and Farrar WL. 1989. Human

Wang, Z. Y., Sato, H., Kusam, S., Sehra, S., Toney, L. M and Dent, A. L. 2005. Regulation of IL-10 gene expression in Th2 cells by Jun proteins. J Immunol. 174(4): 2098-2105. Weiss L, Haeffner-Cavaillon N, Laude M, Gilquin J and Kazatchkine MD. 1989. HIV

Weiss L, Si-Mohamed A, Giral P, Castiel P, Ledur A, Blondin C, Kazatchkine MD and N

Xing HQ, Hayakawa H, Izumo K, Kubota R, Gelpi E, Budka H, et al. In vivo expression of proinflammatory cytokines in HIV encephalitis: an analysis of 11 autopsy cases. Xing, Z., J. Gauldie, G. Cox, H. Baumann, M. Jordana, X. F. Lei and M. K. Achong. 1998. IL-6

Yeh MW, Kaul M, Zheng J, Nottet HS, Thylin M and Gendelman HE, et al. 2000. Cytokine-

neurotoxic levels of L-cysteine. Journal of immunology. 164(8):4265-70.

Sciences of the United States of America. 86(2):621-5.

immunodeficiency virus glycoprotein (gp120) induction of monocyte arachidonic acid metabolites and interleukin 1. Proceedings of the National Academy of

infection is associated with the spontaneous production of interleukin-1 (IL-1) in vivo and with an abnormal release of IL-1 alpha in vitro. AIDS (London, England).

Haeffner-Cavaillon. 1997. Plasma levels of monocyte chemoattractant protein-1 but not those of macrophage inhibitory protein-1 and RANTES correlate with virus load in human immunodeficiency virus infection. Journal of Infectious Diseases.

Is an Anti-inflammatory Cytokine Required for Controlling Local or Systemic Acute Inflammatory Responses. Journal of Clinical Investigation*.* 101.2: 311-20. Yang H, Wei J, Zhang H, Song W, Wei W, Zhang L, Qian K and He S. 2010.Upregulation of

Toll-like Receptor (TLR) expression and release of cytokines from mast cells by IL-

stimulated, but not HIV-infected, human monocyte-derived macrophages produce

Cytokine Network. 21:215.

3(11):695-9.

176: 1621-1624.

12. Cell Physiol Biochem. 26(3):337-46.

The natural history and pathogenic processes of human immunodeficiency virus type 1 (HIV-1) infection are complex and variable, and depend on many viral and host factors and their interactions (Pantaleo et al., 1997). Individuals are not equal susceptible to the infection and have differences in their viral set points, rates of decline of CD4+ T cells, levels of viremia, emergence of citotoxic T lymphocyte (CTL) escape mutants, and development of opportunistic infections resulting in varying incubation periods of the virus (Kaur & Mehra, 2009). HIV-1 infected individuals present different rates of disease progression; while a majority of individual progress to acquired immunodeficiency syndrome (AIDS) after the infection, most of them can be turned aviremic even the absence of antiretroviral therapy (ARV) up to ten years and are called typical progressors. Most importantly, ~5% to 10% of persistently infected individuals show no signs of disease progression for over 12 years and remain asymptomatic and aviremic and are classified as long term nonprogressors (LTNPs). On the other hand, rapid progressors are individuals that rapidly progress to AIDS within four years after primary HIV-1 infection and some individuals have been known to progress to AIDS and death within a year after primary infection (Fauci et al., 1996; Rosenberg & Fauci, 1991).

Genetic factors may be one of the host factors responsible for the susceptibility to infection and disease progression. However, no single gene or polymorphism is likely to be responsible for these effects. Brass et al. (2008) have reported that HIV-1 uses at least 250 host-derived dependency factors for gaining entry into target cells and completing its life cycle. Hence, multiple genetic factors are expected to be involved in susceptibility, disease pathogenesis, and progression following HIV-1 infection. Some of these genes that have been established with HIV/AIDS conclusively involve are: (1) genes influencing viral entry by altering the expression on cell surface the levels of chemokine receptors and their ligands as well cytokines (Seisdedos & Parmentier, 2006; Reiche et al., 2007); (2) genes involved in anti-HIV immune response including the antiviral Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G) gene family on chromosome 22q13 (An et al., 2009); the virus restriction factor Tripartite Interaction Motif 5 (TRIM5) on

The Role of Genetic Polymorphisms in the Chemokine and

with those HIV-1-infected subjects without these mutations.

**2.2 CCL5** 

**2.3 CCL3L1, CCL4L1** 

to HIV-1 infection (Rathore et al., 2009).

Their Receptors and Cytokines in the Human Immunodeficiency Virus Type 1 (HIV-1) Infection 303

The CCL5 (RANTES) gene is located on chromosome 17 which encodes a chemokine ligand for CCR1, CCR3, and CCR5. It suppresses infection for R5 strains of HIV-1 by blocking CCR5 (Paxton et al., 1996; Arenzana-Seisdedos et al., 1996). Some polymorphisms in *CCL5* promoter region protect HIV-1 infected subjects against disease progression as a result of increased CCL5 synthesis (Liu et al., 1999; Wichukchinda et al., 2006). Three single nucleotide polymorphisms (SNPs) in this gene (-28CG, -403GA, and in 1.1C) were reported for their roles in progression to AIDS in HIV-1 infected individuals. The variant alleles -28G and -403A were found to be associated with delayed progression to AIDS in Japanese population by increasing levels of CCL5 transcription and by reducing rates of CD4+ T-cell depletion (Liu et al., 1999). Other study also suggested that polymorphisms in the CCL5 promoter gene can influence the risks for HIV-1 infection and disease progression by increasing the CCL5 levels (McDermott et al., 2000; Koning et al., 2003). In the other hand, the SNP In 1.1C, nested within an intronic regulatory sequence element, accelerated the progression to AIDS in African Americans and European Americans by downregulating the CCL5 gene transcription (An et al., 2002). Study by Wichukchinda et al. (2006) demonstrated the mutation's protective effect by comparing disease progression in seroconverters carrying either the CCL5-*28G* or the CCL5 *In.1.1C* polymorphism. Individuals carrying the CCL5 *In.1.1C* allele progressed significantly faster to AIDS compared with those carrying the CCL5 - *28G* allele, along

Human CC chemokine ligand 3 like 1 gene (CCL3L1) is a natural ligand of HIV-1 coreceptor CCR5 and a potent HIV-1 supressive chemokine that can physically block the entry of HIV-1. The CCL3L1 gene is located on human chromosome 17q11.2 and shares 96.0% amino acid homology with CCL3. The copy number of the CCL3L1 gene varies among different individuals and populations groups (Kaur & Mehra, 2009). CCL3L1 and CCL4L1 genes harbor several SNPs and hotspots for duplication, resulting in distinct haplotypes and copy number variations, respectively, in different individuals (Modi et al., 2006). The copy numbers are highest in Africans, followed by Asians, Amerindians Central and South Asians, Middle East individuals and Europeans. Variations in a copy numbers of CCL3L1 alter the expression of this potent CCR5 ligand and might influence the entry of HIV-1 into host cells. Copy number variations in CCL3L1 have been associated with susceptibility to HIV-1 infection (Gonzalez et al., 2005). However, the results are contradictory in different populations. A study carried out in the Japanese population (Nakajima et al., 2007) has shown that HIV-1 infected individuals have lower copy number than healthy controls. On the contrary, studies in North Indian population showed that the copy number variation in CCL3L1 gene has no effect on acquisition in HIV-1 infected individuals compared with healthy controls (Nakajima et al., 2008). Another study showed an association of CCR5- 59029 A/G and CCL3L1 copy number polymorphism with HIV-1 transmission and progression among HIV-1 seropositive and repeatedly sexually exposed HIV-1 seronegative North Indians individuals, suggesting that these polymorphisms appeared to have synergistic or interactive effects and are expected to be involved in the host innate resistance

chromosome 11p15; (3) the human leukocyte antigens (HLA) polymorphic loci and their associated genes including HCP5, RNF39, and ZNRD1 on the short arm of human chromosome 6; (4) Dendritic Cell Specific Intracellular adhesion molecule-3-Grabbing Nonintegrin (DC-SIGN) on chromosome 19q13; (5) interferon regulatory factor 1; (6) killer cell immunoglobulin-like receptor (KIR) KIR3DSI on chromosome 19q13; and (7) Ly6 family of G [glycosylphosphatidylinositol (GPI)-anchored proteins] (Kaur & Mehra, 2009).

This chapter reviews the most important genetic polymorphisms already described in the chemokine, cytokine and their receptors, and their role on the host susceptibility or resistance to HIV-1 infection, on the clinical course of the disease and on the response to the ARV. For this purpose, *in vitro* and *in vivo* studies for inclusion were identified by a systematic search through PubMed for English-language literature, included original and review articles published up to 2010. These data could contribute to identify some genetic biomarkers for infection, transmission, disease progression or ARV therapeutic failure.

#### **2. Genetic polymorphisms in CC chemokines and their receptors**

Chemokines are low-molecular-weight potent chemoattractants produced by a variety of cell types that include T cells, macrophages, natural killer (NK) cells, B cells, fibroblasts, and mast cells. These are involved in cell trafficking and immunomodulation of inflammation and immune responses. The chemokines are subdivided into CC, CXC, and CX3C subfamilies, according to the number of cysteine residues in the molecule. Members of CC chemokines are CCL3 [macrophage inflammatory protein-1 (MIP-1)], CCL4 [macrophage inflammatory protein 1β (MIP-1)], and CCL5 [regulated upon activation normally Texpressed (RANTES)]. They are natural ligands for CC chemokine receptor R 5 (CCR5). One member of CXC chemokines is CXC ligand 12 (CXCL12), previously named stromal cellderived factor 1 (SDF1), a natural ligand for CXC receptor 4 (CXCR4). In certain instances, the chemokine receptors serve as entry portals for pathogens to gain entry into target cells and establish infection. Based on cell tropism, HIV-1 isolates are classified into two main groups. The vast majority of primary HIV-1 isolates is, predominantly, tropic for CCR5 and gradually tends to become CXCR4 tropic during late infection. All non-syncytium-inducing (NSI) strains of HIV-1 require CCR5 to gain entry into target cells and are also named R5 strains, while the syncytium-inducing (SI) strains use CXCR4 to enter into host cells and are also named as X4 strains (Berger et al., 1999). Functional genetic polymorphisms are known to occur in these proteins that affect their levels of expression and therefore might modulate their molecular interactions.

#### **2.1 CCL3, CCL4 and CCL18**

The genes coding for CCL3 (MIP-1), CCL4 (MIP-1), and CCL18 [also referred to as small inducible cytokine subfamily A member 18 or pulmonary and activation-regulated chemokine (PARC)], are clustered together within a 47-kb region on chromosome 17q12. These are potent chemokines produced by macrophages, NK cells, fibroblasts, and T cells. Of these, CCL2, CCL3 and CCL4 are natural ligands for the primary HIV-1 coreceptor CCR5, and their genetic polymorphisms have been implicated in HIV-1 acquisition and disease progression, although these associations are complicated because of strong linkage disequilibrium between them (Modi et al., 2006).

## **2.2 CCL5**

302 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

chromosome 11p15; (3) the human leukocyte antigens (HLA) polymorphic loci and their associated genes including HCP5, RNF39, and ZNRD1 on the short arm of human chromosome 6; (4) Dendritic Cell Specific Intracellular adhesion molecule-3-Grabbing Nonintegrin (DC-SIGN) on chromosome 19q13; (5) interferon regulatory factor 1; (6) killer cell immunoglobulin-like receptor (KIR) KIR3DSI on chromosome 19q13; and (7) Ly6 family of

This chapter reviews the most important genetic polymorphisms already described in the chemokine, cytokine and their receptors, and their role on the host susceptibility or resistance to HIV-1 infection, on the clinical course of the disease and on the response to the ARV. For this purpose, *in vitro* and *in vivo* studies for inclusion were identified by a systematic search through PubMed for English-language literature, included original and review articles published up to 2010. These data could contribute to identify some genetic biomarkers for infection, transmission, disease progression or ARV therapeutic

Chemokines are low-molecular-weight potent chemoattractants produced by a variety of cell types that include T cells, macrophages, natural killer (NK) cells, B cells, fibroblasts, and mast cells. These are involved in cell trafficking and immunomodulation of inflammation and immune responses. The chemokines are subdivided into CC, CXC, and CX3C subfamilies, according to the number of cysteine residues in the molecule. Members of CC chemokines are CCL3 [macrophage inflammatory protein-1 (MIP-1)], CCL4 [macrophage inflammatory protein 1β (MIP-1)], and CCL5 [regulated upon activation normally Texpressed (RANTES)]. They are natural ligands for CC chemokine receptor R 5 (CCR5). One member of CXC chemokines is CXC ligand 12 (CXCL12), previously named stromal cellderived factor 1 (SDF1), a natural ligand for CXC receptor 4 (CXCR4). In certain instances, the chemokine receptors serve as entry portals for pathogens to gain entry into target cells and establish infection. Based on cell tropism, HIV-1 isolates are classified into two main groups. The vast majority of primary HIV-1 isolates is, predominantly, tropic for CCR5 and gradually tends to become CXCR4 tropic during late infection. All non-syncytium-inducing (NSI) strains of HIV-1 require CCR5 to gain entry into target cells and are also named R5 strains, while the syncytium-inducing (SI) strains use CXCR4 to enter into host cells and are also named as X4 strains (Berger et al., 1999). Functional genetic polymorphisms are known to occur in these proteins that affect their levels of expression and therefore might modulate

The genes coding for CCL3 (MIP-1), CCL4 (MIP-1), and CCL18 [also referred to as small inducible cytokine subfamily A member 18 or pulmonary and activation-regulated chemokine (PARC)], are clustered together within a 47-kb region on chromosome 17q12. These are potent chemokines produced by macrophages, NK cells, fibroblasts, and T cells. Of these, CCL2, CCL3 and CCL4 are natural ligands for the primary HIV-1 coreceptor CCR5, and their genetic polymorphisms have been implicated in HIV-1 acquisition and disease progression, although these associations are complicated because of strong linkage

G [glycosylphosphatidylinositol (GPI)-anchored proteins] (Kaur & Mehra, 2009).

**2. Genetic polymorphisms in CC chemokines and their receptors** 

failure.

their molecular interactions.

**2.1 CCL3, CCL4 and CCL18** 

disequilibrium between them (Modi et al., 2006).

The CCL5 (RANTES) gene is located on chromosome 17 which encodes a chemokine ligand for CCR1, CCR3, and CCR5. It suppresses infection for R5 strains of HIV-1 by blocking CCR5 (Paxton et al., 1996; Arenzana-Seisdedos et al., 1996). Some polymorphisms in *CCL5* promoter region protect HIV-1 infected subjects against disease progression as a result of increased CCL5 synthesis (Liu et al., 1999; Wichukchinda et al., 2006). Three single nucleotide polymorphisms (SNPs) in this gene (-28CG, -403GA, and in 1.1C) were reported for their roles in progression to AIDS in HIV-1 infected individuals. The variant alleles -28G and -403A were found to be associated with delayed progression to AIDS in Japanese population by increasing levels of CCL5 transcription and by reducing rates of CD4+ T-cell depletion (Liu et al., 1999). Other study also suggested that polymorphisms in the CCL5 promoter gene can influence the risks for HIV-1 infection and disease progression by increasing the CCL5 levels (McDermott et al., 2000; Koning et al., 2003). In the other hand, the SNP In 1.1C, nested within an intronic regulatory sequence element, accelerated the progression to AIDS in African Americans and European Americans by downregulating the CCL5 gene transcription (An et al., 2002). Study by Wichukchinda et al. (2006) demonstrated the mutation's protective effect by comparing disease progression in seroconverters carrying either the CCL5-*28G* or the CCL5 *In.1.1C* polymorphism. Individuals carrying the CCL5 *In.1.1C* allele progressed significantly faster to AIDS compared with those carrying the CCL5 - *28G* allele, along with those HIV-1-infected subjects without these mutations.

## **2.3 CCL3L1, CCL4L1**

Human CC chemokine ligand 3 like 1 gene (CCL3L1) is a natural ligand of HIV-1 coreceptor CCR5 and a potent HIV-1 supressive chemokine that can physically block the entry of HIV-1. The CCL3L1 gene is located on human chromosome 17q11.2 and shares 96.0% amino acid homology with CCL3. The copy number of the CCL3L1 gene varies among different individuals and populations groups (Kaur & Mehra, 2009). CCL3L1 and CCL4L1 genes harbor several SNPs and hotspots for duplication, resulting in distinct haplotypes and copy number variations, respectively, in different individuals (Modi et al., 2006). The copy numbers are highest in Africans, followed by Asians, Amerindians Central and South Asians, Middle East individuals and Europeans. Variations in a copy numbers of CCL3L1 alter the expression of this potent CCR5 ligand and might influence the entry of HIV-1 into host cells. Copy number variations in CCL3L1 have been associated with susceptibility to HIV-1 infection (Gonzalez et al., 2005). However, the results are contradictory in different populations. A study carried out in the Japanese population (Nakajima et al., 2007) has shown that HIV-1 infected individuals have lower copy number than healthy controls. On the contrary, studies in North Indian population showed that the copy number variation in CCL3L1 gene has no effect on acquisition in HIV-1 infected individuals compared with healthy controls (Nakajima et al., 2008). Another study showed an association of CCR5- 59029 A/G and CCL3L1 copy number polymorphism with HIV-1 transmission and progression among HIV-1 seropositive and repeatedly sexually exposed HIV-1 seronegative North Indians individuals, suggesting that these polymorphisms appeared to have synergistic or interactive effects and are expected to be involved in the host innate resistance to HIV-1 infection (Rathore et al., 2009).

The Role of Genetic Polymorphisms in the Chemokine and

coreceptor CCR5.

(Chatterjee, 2010).

et al., 2008).

Their Receptors and Cytokines in the Human Immunodeficiency Virus Type 1 (HIV-1) Infection 305

CCR5-32 delay HIV-1 replication and the virus-mediated destruction of the CD4+/ CCR5+ T-cell lymphocyte population (Ioannidis et al., 2001). The observation that this naturallyoccurring genetic mutation can slow or delay the onset of AIDS in patient populations was the basis of therapeutic interventions targeting the interaction between the virus and the

Several other mutations in the coding region of the *CCR5* gene have been identified (Carrington et al., 1997). Ten common SNPs within the 1,000 base-pairs region upstream of CCR5-coding exons that exhibit promoter and regulatory activity have been described, possibly affecting the levels of CCR5 expression (Carrington et al., 1999; Martin et al., 1998; Kostrikis et al., 1998; Quillent et al., 1998; Piacentini et al., 2009). These polymorphisms are identified as CCR5P1 to CCR5P10 and the most common of them are CCR5P1 and CCR5P4

The CCR5P1/P1 promoter allele was the first genetic variant in the CCR5 promoter to be associated with rapid progression of AIDS, although variants of other genes have been described more recently to be AIDS-accelerating (Carrington et al., 1997; Faure et al., 2000). The hypothesis that the genetic effect is mediated by an increase in available CCR5 portals is also supported by the epidemiologic pattern. The strongest acceleration mediated by the CCR5P1/P1 genotype occurs in the first five years of infection, a period when R5 (NSI) virus

The A/G polymorphism at base-pair 59029 in the CCR5 promoter was identified and appears to affect the rate of progression to AIDS in HIV-1 infected homosexuals. The CCR5 59029 G/G genotype appears to be more protective than CCR5 59029 A/A, and this effect may be the result of a reduced CCR5 mRNA production. The A allele exhibits a 50.0% higher expression of CCR5 *in vitro* and confers faster disease progression than the G allele (Passam et al., 1999). These results indicated that this site in the CCR5 promoter is important and may be a useful target for treatment of the HIV-1 infection (McDermott et al., 1998). It is estimated that homozygozity for the CCR5P1/P1 promoter allele was responsible for the development of the disease in 10.0% to 17.0% of the patients who developed AIDS within 3.5 years of HIV-1 infection, irrespective of the CCR5-32 and CCR2-64I (defined in this chapter in the next sections) genotypes. The frequency of this susceptible genotype in

Homozygosity for CCR5-59356-T, a polymorphism more frequent in the African-American rather than in Caucasian or Hispanic populations, has been strongly associated with an increased rate of perinatal HIV-1 transmission (Kostrikis et al., 1999). Additionally, the *59353C* allele is found in higher frequency in some progressors compared with LTNPs (Jang

Complete linkage disequilibrium between CCR5P1 and CCR5-2459A sites and the CCR5P1 haplotype was shown to be associated with rapid progression to AIDS endpoints in both African-American and Caucasians cohorts. This effect was recessive in Caucasians and dominant in African-Americans, probably due to the presence of modulating genes or as yet unidentified polymorphisms with different frequencies among the racial groups (An et al., 2000). This same study described that both CCR5P1 homozygous and heterozygous African-Americans showed a trend towards more rapid progression to AIDS endpoints. Similar to the recessive effect of the CCR5P1 allele in Caucasians, which was strongest in the first 4-6 years following seroconversion, the dominant effect of the CCR5P1 allele in African-Americans was also evident in the first 4 years. This result is consistent with both the

strains predominate in 90.0-95.0% of patients (Schuitemaker et al., 1992).

the general population is only 7.0% to 13.0% (Martin et al., 1998).

### **2.4 CCR5**

CCR5 is normally expressed at very low levels on the surface of naïve CD4+ T cells and at higher levels in activated CD4+ memory T cells as well as in monocytes and macrophages (Potter et al., 2007). Multiple polymorphic variations have been described in the *CCR5* gene that is located on chromosome 3p21. CCR5-32 polymorphism is the first and most well characterized host restriction allele associated with AIDS. This natural knockout deletion of 32 base-pair creates a premature stop codon resulting in truncated protein product, a shortened protein which remains intracellular and fails to reach the cell surface in individuals homozygous for the variant. The first study described a frequency of approximately 0.100 for the null-mutant allele of the *CCR5* gene in the Caucasian population. Heterozygotes for the allele have reduced levels of quantifiable CCR5 receptors in the cell surface and were present at similar frequencies among infected and uninfected cohort controls. Among HIV-1 infected homosexual cohorts, heterozygosity correlated well with decreased disease progression. However, no correlation was apparent among the haemophilic population (Dean et al., 1996).

A second study identified the same mutant allele of CCR5 in two homozygous individuals who had been repeatedly exposed to the HIV-1 but remained uninfected (Liu et al., 1997). The results also showed that the mutation makes the CCR5 protein incapable of mediating infection by HIV-1 *in vitro*. A third study suggested that heterozygosity also provides some protection from HIV-1 infection (Samson et al., 1996) and the discrepancy regarding the protection from HIV-1 infection was most likely due to the difference between the populations evaluated. The first study (Dean et al., 1996) compared large and matched cohorts of individuals, whereas the third (Samson et al., 1996) examined only non-cohort population matched by geographical location and the use of a French surname.

Even though homozygosity for the CCR5-32 results in near-total protection for the HIV-1 infection, subjects can still be infected with T-tropic or SI strains of the virus, which use the CXCR4 coreceptor for cell entry (Dean et al., 1996; Samson et al., 1996; Zimmerman et al., 1997; O'Brien et al., 1997; Theodorou et al., 1997). Studies of HIV-1 infected homozygous for the CCR5-32 mutation have been reported, but are rare (O'Brien et al., 1997; Theodorou et al., 1997; Balotta et al., 1997; Biti et al., 1997), probably due to a T-tropic virus, strain which only uses CXCR4 as coreceptor for cell entry.

Heterozygosity for the CCR5-32 is significantly higher in cohorts of HIV-1 infected LTNPs compared to HIV-1 infected typical progressors (Cohen et al., 1997; Zimmerman et al., 1997; Eugen-Olsen et al., 1997). Although the heterozygosity was not related to the complete protection against HIV-1 infection (Dean et al., 1996; Samson et al., 1996), it may confer partial protection against disease progression or death in HIV-1 infected individuals (Zimmerman et al., 1997; Smith et al., 1997; Martin et al., 1998; de Roda et al., 1997; Meyer et al., 1999; Ionnadis et al., 1998). Presumably, heterozygosity limits the number of coreceptors available for HIV-1 binding. Indeed, CCR5 density of the surface of the CD4+ T cell has been correlated with viral load in persons with untreated HIV-1 infection (Reynes et al., 2000). Studies incorporating viral phenotype have suggested that the protective effect of CCR5-32 heterozygosity against disease progression is lost when the infection virus is T-tropic (Michael et al., 1997).

An international meta-analysis showed that HIV-1 infected subjects heterozygous for the CCR5-32 displayed lower HIV-1 RNA level than wild type patients. This result appears to be supported by the simple explanation that the fewer available CCR5 portals on cells of

CCR5 is normally expressed at very low levels on the surface of naïve CD4+ T cells and at higher levels in activated CD4+ memory T cells as well as in monocytes and macrophages (Potter et al., 2007). Multiple polymorphic variations have been described in the *CCR5* gene that is located on chromosome 3p21. CCR5-32 polymorphism is the first and most well characterized host restriction allele associated with AIDS. This natural knockout deletion of 32 base-pair creates a premature stop codon resulting in truncated protein product, a shortened protein which remains intracellular and fails to reach the cell surface in individuals homozygous for the variant. The first study described a frequency of approximately 0.100 for the null-mutant allele of the *CCR5* gene in the Caucasian population. Heterozygotes for the allele have reduced levels of quantifiable CCR5 receptors in the cell surface and were present at similar frequencies among infected and uninfected cohort controls. Among HIV-1 infected homosexual cohorts, heterozygosity correlated well with decreased disease progression. However, no correlation was apparent among the

A second study identified the same mutant allele of CCR5 in two homozygous individuals who had been repeatedly exposed to the HIV-1 but remained uninfected (Liu et al., 1997). The results also showed that the mutation makes the CCR5 protein incapable of mediating infection by HIV-1 *in vitro*. A third study suggested that heterozygosity also provides some protection from HIV-1 infection (Samson et al., 1996) and the discrepancy regarding the protection from HIV-1 infection was most likely due to the difference between the populations evaluated. The first study (Dean et al., 1996) compared large and matched cohorts of individuals, whereas the third (Samson et al., 1996) examined only non-cohort

Even though homozygosity for the CCR5-32 results in near-total protection for the HIV-1 infection, subjects can still be infected with T-tropic or SI strains of the virus, which use the CXCR4 coreceptor for cell entry (Dean et al., 1996; Samson et al., 1996; Zimmerman et al., 1997; O'Brien et al., 1997; Theodorou et al., 1997). Studies of HIV-1 infected homozygous for the CCR5-32 mutation have been reported, but are rare (O'Brien et al., 1997; Theodorou et al., 1997; Balotta et al., 1997; Biti et al., 1997), probably due to a T-tropic virus, strain which

Heterozygosity for the CCR5-32 is significantly higher in cohorts of HIV-1 infected LTNPs compared to HIV-1 infected typical progressors (Cohen et al., 1997; Zimmerman et al., 1997; Eugen-Olsen et al., 1997). Although the heterozygosity was not related to the complete protection against HIV-1 infection (Dean et al., 1996; Samson et al., 1996), it may confer partial protection against disease progression or death in HIV-1 infected individuals (Zimmerman et al., 1997; Smith et al., 1997; Martin et al., 1998; de Roda et al., 1997; Meyer et al., 1999; Ionnadis et al., 1998). Presumably, heterozygosity limits the number of coreceptors available for HIV-1 binding. Indeed, CCR5 density of the surface of the CD4+ T cell has been correlated with viral load in persons with untreated HIV-1 infection (Reynes et al., 2000). Studies incorporating viral phenotype have suggested that the protective effect of CCR5-32 heterozygosity against disease progression is lost when the infection virus is T-tropic

An international meta-analysis showed that HIV-1 infected subjects heterozygous for the CCR5-32 displayed lower HIV-1 RNA level than wild type patients. This result appears to be supported by the simple explanation that the fewer available CCR5 portals on cells of

population matched by geographical location and the use of a French surname.

**2.4 CCR5** 

haemophilic population (Dean et al., 1996).

only uses CXCR4 as coreceptor for cell entry.

(Michael et al., 1997).

CCR5-32 delay HIV-1 replication and the virus-mediated destruction of the CD4+/ CCR5+ T-cell lymphocyte population (Ioannidis et al., 2001). The observation that this naturallyoccurring genetic mutation can slow or delay the onset of AIDS in patient populations was the basis of therapeutic interventions targeting the interaction between the virus and the coreceptor CCR5.

Several other mutations in the coding region of the *CCR5* gene have been identified (Carrington et al., 1997). Ten common SNPs within the 1,000 base-pairs region upstream of CCR5-coding exons that exhibit promoter and regulatory activity have been described, possibly affecting the levels of CCR5 expression (Carrington et al., 1999; Martin et al., 1998; Kostrikis et al., 1998; Quillent et al., 1998; Piacentini et al., 2009). These polymorphisms are identified as CCR5P1 to CCR5P10 and the most common of them are CCR5P1 and CCR5P4 (Chatterjee, 2010).

The CCR5P1/P1 promoter allele was the first genetic variant in the CCR5 promoter to be associated with rapid progression of AIDS, although variants of other genes have been described more recently to be AIDS-accelerating (Carrington et al., 1997; Faure et al., 2000). The hypothesis that the genetic effect is mediated by an increase in available CCR5 portals is also supported by the epidemiologic pattern. The strongest acceleration mediated by the CCR5P1/P1 genotype occurs in the first five years of infection, a period when R5 (NSI) virus strains predominate in 90.0-95.0% of patients (Schuitemaker et al., 1992).

The A/G polymorphism at base-pair 59029 in the CCR5 promoter was identified and appears to affect the rate of progression to AIDS in HIV-1 infected homosexuals. The CCR5 59029 G/G genotype appears to be more protective than CCR5 59029 A/A, and this effect may be the result of a reduced CCR5 mRNA production. The A allele exhibits a 50.0% higher expression of CCR5 *in vitro* and confers faster disease progression than the G allele (Passam et al., 1999). These results indicated that this site in the CCR5 promoter is important and may be a useful target for treatment of the HIV-1 infection (McDermott et al., 1998).

It is estimated that homozygozity for the CCR5P1/P1 promoter allele was responsible for the development of the disease in 10.0% to 17.0% of the patients who developed AIDS within 3.5 years of HIV-1 infection, irrespective of the CCR5-32 and CCR2-64I (defined in this chapter in the next sections) genotypes. The frequency of this susceptible genotype in the general population is only 7.0% to 13.0% (Martin et al., 1998).

Homozygosity for CCR5-59356-T, a polymorphism more frequent in the African-American rather than in Caucasian or Hispanic populations, has been strongly associated with an increased rate of perinatal HIV-1 transmission (Kostrikis et al., 1999). Additionally, the *59353C* allele is found in higher frequency in some progressors compared with LTNPs (Jang et al., 2008).

Complete linkage disequilibrium between CCR5P1 and CCR5-2459A sites and the CCR5P1 haplotype was shown to be associated with rapid progression to AIDS endpoints in both African-American and Caucasians cohorts. This effect was recessive in Caucasians and dominant in African-Americans, probably due to the presence of modulating genes or as yet unidentified polymorphisms with different frequencies among the racial groups (An et al., 2000). This same study described that both CCR5P1 homozygous and heterozygous African-Americans showed a trend towards more rapid progression to AIDS endpoints. Similar to the recessive effect of the CCR5P1 allele in Caucasians, which was strongest in the first 4-6 years following seroconversion, the dominant effect of the CCR5P1 allele in African-Americans was also evident in the first 4 years. This result is consistent with both the

The Role of Genetic Polymorphisms in the Chemokine and

early in the course of infection (Mulherin et al., 2003).

association purposes (Kaur & Mehra, 2009).

difficult to test *in vivo* (Bleul et al., 1996).

**3.1 CXCL12 (SDF1)** 

Their Receptors and Cytokines in the Human Immunodeficiency Virus Type 1 (HIV-1) Infection 307

varies from 10.0% to 25.0% in both African-Americans and Caucasians, and in all other ethnic groups studied. The protective allele A occurs at a population frequency of 15.0%-17.0% in Chinese, 12.0% in the North Indians (Kaur et al., 2007), and 3.0%-15.0% in the South Indian populations (Ramana et al., 2001). Studies of HIV-1 infected commercial sex workers in Nairobi and Kenya, suggested that the presence of the mutation helped to explain slow progression in 21.0% to 46.0% of slow progressors (Anzala et al., 1998). The effect of the CCR5-32 allele on HIV-1 disease progression was also different from the effect of the CCR2 allele. The protection against AIDS provided by CCR5-32 was continuous during the course of infection, while the protection provided by CCR2-64I was the greatest

A meta-analysis study found that in the absence of highly active antiretroviral therapy (HAART), both CCR5-32 and CCR2-64I carriers progressed to AIDS at a 25.0% slower rate than individuals who lacked either of these protective alleles. They also progressed more slowly to death, approximately 35.0% and 25.0% slower, respectively (Ioannidis et al., 2001). Mabuka et al. (2009) verified that the presence of the CCR2-64I allele was associated with reduced viral load and with protection against early HIV-1 transmission among pregnant women who received short course zidovudine. In Kenya and other African countries, where approximately one quarter of individuals carry the variant allele, understanding this genetic mutation may help explain disparities in transmission risk and rates of disease progression

Because the genetic loci for *CCR5* and *CCR2* are in strong linkage disequilibrium, their combined analysis is greatly helpful to define CCR2-CCR5 extended haplotypes for disease

CXCL12 (SDF1) is a highly potent chemokine, the natural ligand for the CXCR4, and a potent entry inhibitor for T-tropic (X4 or SI) HIV-1 strains that generally emerge during the late-stage of HIV-1 infection (Bleul et al., 1996). The CXCL12-CXCR4 interactions are essential for the homing and retention of hematopoietic progenitor cells in the bone marrow and have been shown to control the navigation of progenitor cells between the bone marrow and the blood. The gene for CXCL12 (SDF1) is ~10kb long and located on human chromosome 10q11.1. It exists in two isoforms, and β, obtained as a consequence of alterative splicing. A common G to A transition, initially referred to as SDF1-3'A and currently named CXCL12-3'A, was described at an evolutionarily conserved sequence of the 3'UTR of the β transcript gene. The polymorphism at the 801 position (G801A or CXCL12'A) has been shown to have a recessive protective effect against HIV-1 infection. Homozygotes for the CXCL12-3'A variant showed a remarkable level of protection against AIDS, supporting the hypothesis that the CXCL12-3'A variant restricts the emergency of X4 HIV-1 strains, with overproduction of CXCL12 in local compartments, which binds to and blocks the CXCR4 receptors required for X4 viruses to emerge and multiply (Winkler et al., 1998). However, direct evidence for an effect of CXCL12-3'A on the synthesis, quantity or half life of the ligand has not been obtained *in vitro* (Arya et al., 1999). Because CXCL12 expression is limited to stromal cells and other tissues that are not easy to quantify, this hypothesis is

and could contribute to vaccine development and other prevention interventions.

**3. Genetic polymorphisms in CXC chemokines and their receptors** 

function of CCR5 as the coreceptor for early transmissible M-tropic HIV-1 strains and also with the studies of CCR5-32 showing an early effect (Dean et al., 1996).

Unlikely the CCR5-32 mutation which is found only in people of Northern European descent, the CCR5P1 allele has a frequency larger than 40.0% both in Caucasians, Asians and populations of African descent, suggesting that the CCR5P1 allele may have a more general effect on AIDS pathogenesis worldwide (An et al., 2000).

Another SNP in *CCR5* gene is a TA substitution (named CCR5-m303A polymorphism) and results in a Threonine to Alanine transition at position 303. It encodes a truncated protein and abolishes the coreceptor activity of CCR5. This polymorphism shows a weak association with delayed progression to AIDS. When is present in heterozygous state with CCR5-32, produces a phenotype of resistance to HIV-1 in primary isolates *in vitro* (Quillent et al., 1998).

Based on a unique constellation of additional multisite polymorphisms in CCR5 regulatory 5' region, a number of CCR5 haplotypes have been identified (Gonzalez et al.,1999; Martin et al., 1998). They have been designed based on the nucleotide position in the 5' untranslated region (UTR) and are referred to as human haplogroups A to G. Of these, HHE has been associated with accelerated disease progression in Caucasians (Gonzalez et al., 1999) and Thais (Nguyen et al., 2004) but not in African Americans. The HHC and HHD haplotypes showed positive association with fast progression to AIDS in African Americans (Kostrikis et al., 1999; Nguyen et al., 2004). In the Indian population, HHE has been implicated with susceptibility to infection and development of AIDS (Kaur et al., 2007). Whether this haplotype also influences disease progression is not clear because long-term follow-up is desirable to reach such a conclusion (Kaur & Mehra, 2009).

#### **2.5 CCR2**

Although the HIV-1 virus does not directly use CCR2 for host cell entry, and CCR2 is considered a minor coreceptor for HIV-1 infection, a Valine to Isoleucine substitution at position 64 in the first transmembrane domain of CCR2 (named CCR2-64I, V64I or G190A) has been associated with delayed progression to AIDS (Smith et al., 1997). HIV-1 infected individuals heterozygous or homozygous for this mutation appear to progress to AIDS or death more slowly. However, this mutation results in normal levels of expression of the CCR2 receptor and has not been shown to affect the susceptibility to HIV-1 infection (Smith et al., 1997; Martin et al., 1998; de Roda et al., 1997; Kostrikis et al., 1998; Mummidi et al., 1998; Mulherin et al., 2003).

Even though the change of the Valine to Isoleucine in a position buried in one of the seven transmembrane segments of this receptor could be considered innocuous, the epidemiological effect on AIDS progression was surprising. It has been shown that the CCR2-64I protein product can preferentially dimerizes with the CXCR4 polypeptide, sequestering it in the endoplasmic reticulum, while the CCR2 peptides cannot. Such differential intracellular kinetics between CCR2 allele products and primary HIV-1 coreceptors *in vivo* could reduce the rate of disease progression by limiting the number of available CXCR4 coreceptors, therefore also reducing indirectly the rate of viral replication (O'Brien and Moore, 2000). However, this effect on disease progression has not confirmed (Michael et al., 1997; Eugen-Olsen et al., 1998).

The distribution of CCR2-64I varies among different ethnic groups. Unlike the CCR5-32 mutation, which is found primarily in Caucasians, the frequency of the CCR2-64I allele varies from 10.0% to 25.0% in both African-Americans and Caucasians, and in all other ethnic groups studied. The protective allele A occurs at a population frequency of 15.0%-17.0% in Chinese, 12.0% in the North Indians (Kaur et al., 2007), and 3.0%-15.0% in the South Indian populations (Ramana et al., 2001). Studies of HIV-1 infected commercial sex workers in Nairobi and Kenya, suggested that the presence of the mutation helped to explain slow progression in 21.0% to 46.0% of slow progressors (Anzala et al., 1998). The effect of the CCR5-32 allele on HIV-1 disease progression was also different from the effect of the CCR2 allele. The protection against AIDS provided by CCR5-32 was continuous during the course of infection, while the protection provided by CCR2-64I was the greatest early in the course of infection (Mulherin et al., 2003).

A meta-analysis study found that in the absence of highly active antiretroviral therapy (HAART), both CCR5-32 and CCR2-64I carriers progressed to AIDS at a 25.0% slower rate than individuals who lacked either of these protective alleles. They also progressed more slowly to death, approximately 35.0% and 25.0% slower, respectively (Ioannidis et al., 2001).

Mabuka et al. (2009) verified that the presence of the CCR2-64I allele was associated with reduced viral load and with protection against early HIV-1 transmission among pregnant women who received short course zidovudine. In Kenya and other African countries, where approximately one quarter of individuals carry the variant allele, understanding this genetic mutation may help explain disparities in transmission risk and rates of disease progression and could contribute to vaccine development and other prevention interventions.

Because the genetic loci for *CCR5* and *CCR2* are in strong linkage disequilibrium, their combined analysis is greatly helpful to define CCR2-CCR5 extended haplotypes for disease association purposes (Kaur & Mehra, 2009).

## **3. Genetic polymorphisms in CXC chemokines and their receptors**

## **3.1 CXCL12 (SDF1)**

306 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

function of CCR5 as the coreceptor for early transmissible M-tropic HIV-1 strains and also

Unlikely the CCR5-32 mutation which is found only in people of Northern European descent, the CCR5P1 allele has a frequency larger than 40.0% both in Caucasians, Asians and populations of African descent, suggesting that the CCR5P1 allele may have a more general

Another SNP in *CCR5* gene is a TA substitution (named CCR5-m303A polymorphism) and results in a Threonine to Alanine transition at position 303. It encodes a truncated protein and abolishes the coreceptor activity of CCR5. This polymorphism shows a weak association with delayed progression to AIDS. When is present in heterozygous state with CCR5-32, produces a phenotype of resistance to HIV-1 in primary isolates *in vitro* (Quillent

Based on a unique constellation of additional multisite polymorphisms in CCR5 regulatory 5' region, a number of CCR5 haplotypes have been identified (Gonzalez et al.,1999; Martin et al., 1998). They have been designed based on the nucleotide position in the 5' untranslated region (UTR) and are referred to as human haplogroups A to G. Of these, HHE has been associated with accelerated disease progression in Caucasians (Gonzalez et al., 1999) and Thais (Nguyen et al., 2004) but not in African Americans. The HHC and HHD haplotypes showed positive association with fast progression to AIDS in African Americans (Kostrikis et al., 1999; Nguyen et al., 2004). In the Indian population, HHE has been implicated with susceptibility to infection and development of AIDS (Kaur et al., 2007). Whether this haplotype also influences disease progression is not clear because long-term follow-up is

Although the HIV-1 virus does not directly use CCR2 for host cell entry, and CCR2 is considered a minor coreceptor for HIV-1 infection, a Valine to Isoleucine substitution at position 64 in the first transmembrane domain of CCR2 (named CCR2-64I, V64I or G190A) has been associated with delayed progression to AIDS (Smith et al., 1997). HIV-1 infected individuals heterozygous or homozygous for this mutation appear to progress to AIDS or death more slowly. However, this mutation results in normal levels of expression of the CCR2 receptor and has not been shown to affect the susceptibility to HIV-1 infection (Smith et al., 1997; Martin et al., 1998; de Roda et al., 1997; Kostrikis et al., 1998; Mummidi et al.,

Even though the change of the Valine to Isoleucine in a position buried in one of the seven transmembrane segments of this receptor could be considered innocuous, the epidemiological effect on AIDS progression was surprising. It has been shown that the CCR2-64I protein product can preferentially dimerizes with the CXCR4 polypeptide, sequestering it in the endoplasmic reticulum, while the CCR2 peptides cannot. Such differential intracellular kinetics between CCR2 allele products and primary HIV-1 coreceptors *in vivo* could reduce the rate of disease progression by limiting the number of available CXCR4 coreceptors, therefore also reducing indirectly the rate of viral replication (O'Brien and Moore, 2000). However, this effect on disease progression has not confirmed

The distribution of CCR2-64I varies among different ethnic groups. Unlike the CCR5-32 mutation, which is found primarily in Caucasians, the frequency of the CCR2-64I allele

with the studies of CCR5-32 showing an early effect (Dean et al., 1996).

effect on AIDS pathogenesis worldwide (An et al., 2000).

desirable to reach such a conclusion (Kaur & Mehra, 2009).

et al., 1998).

**2.5 CCR2** 

1998; Mulherin et al., 2003).

(Michael et al., 1997; Eugen-Olsen et al., 1998).

CXCL12 (SDF1) is a highly potent chemokine, the natural ligand for the CXCR4, and a potent entry inhibitor for T-tropic (X4 or SI) HIV-1 strains that generally emerge during the late-stage of HIV-1 infection (Bleul et al., 1996). The CXCL12-CXCR4 interactions are essential for the homing and retention of hematopoietic progenitor cells in the bone marrow and have been shown to control the navigation of progenitor cells between the bone marrow and the blood. The gene for CXCL12 (SDF1) is ~10kb long and located on human chromosome 10q11.1. It exists in two isoforms, and β, obtained as a consequence of alterative splicing. A common G to A transition, initially referred to as SDF1-3'A and currently named CXCL12-3'A, was described at an evolutionarily conserved sequence of the 3'UTR of the β transcript gene. The polymorphism at the 801 position (G801A or CXCL12'A) has been shown to have a recessive protective effect against HIV-1 infection. Homozygotes for the CXCL12-3'A variant showed a remarkable level of protection against AIDS, supporting the hypothesis that the CXCL12-3'A variant restricts the emergency of X4 HIV-1 strains, with overproduction of CXCL12 in local compartments, which binds to and blocks the CXCR4 receptors required for X4 viruses to emerge and multiply (Winkler et al., 1998). However, direct evidence for an effect of CXCL12-3'A on the synthesis, quantity or half life of the ligand has not been obtained *in vitro* (Arya et al., 1999). Because CXCL12 expression is limited to stromal cells and other tissues that are not easy to quantify, this hypothesis is difficult to test *in vivo* (Bleul et al., 1996).

The Role of Genetic Polymorphisms in the Chemokine and

persons with and withoutthe polymorphism.

HIV-1 infection in high-risk Uighurs individuals.

groups (Qijian et al., 2010).

**3.2 CXCR1, CXCR2** 

Mehra, 2009).

**3.3 CXCR4** 

Moore, 2000).

Their Receptors and Cytokines in the Human Immunodeficiency Virus Type 1 (HIV-1) Infection 309

variants slow AIDS by limiting the number of CCR5 coreceptors that mediate the replication and spread of primary, early stage R5 HIV-1 strains, while the CXCL12-3'A variant restricts the emergence of X4 HIV-1 strains and the ensuing AIDS-accelerating process (O'Brien and

However, several studies show a lack of relationship between CXCL12 3'A and HIV-1 disease non-progression (Vidal et al., 2005a; Ioannidis et al., 2001; Tresoldi et al., 2002). For instance, in their international meta-analysis, Ioannidis et al. (2001) measured the effects of subjects homozygous for the CXCL12 3'A polymorphism by reviewing studies that prospectively followed HIV-1 infected patients from seroconversion to AIDS diagnosis and death. Results showed that being homozygous for the polymorphism had no effect on disease progression and there was no significant difference in HIV-1 RNA levels among

Recently, Tan et al. (2010) showed that the allelic frequency of CCR5-32, CCR5m303A, CCR2- 64I and CXCL12-3'A in HIV-1 infected and uninfected high-risk Uighurs individuals, the largest population of minority in China, was 4.4%, 2.7%, 25.7% and 57.4%, respectively. While there was no significant difference in the frequency of CCR5-32, CCR2-64I and CXCL12-3'A between HIV-1 seropositive and seronegative group, the frequency of CCR5m3030A in HIV-1 seropositive group was significantly higher than that in seronegative group. Furthermore, a woman who carried homozygous CCR5-32 was positive for HIV-1 infection. Therefore, these data suggest that the CCR5-32 CCR2-64I and CXCL1´3'A alleles may have limited effect on protecting from HIV-1 infection and CCR5m303A variant may be associated with the risk for

The distribution of CCR5-32, CCR2-64I, and CXCL12 3'A alleles was evaluated in Guangxi Province Zhuang population, the largest minority ethnic population with over 15 million people, mainly located in Guangxi Province, China. The CCR5-32 was absent, and CCR2- 64I and CXCL12 3'A alleles were relatively common and seem not to confer protection against HIV-1 infection in this population. The results suggest that the Zhuang people may have a similar genetic susceptibility to HIV-1 infection with most other Chinese ethnic

CXCR1 (IL-8RA) and CXCR2 (IL-8RB) are receptors for IL-8, a proinflammatory cytokine involved in chemoattraction and activation of neutrophils. CXCR1 and CXCR2 genes are located o chromosome 2q35 and several polymorphisms have been described including SNPs T92G (CXCR1 -300) and C1003T (CXCR1-142) that result in a CXCR1 haplotype Ha. A genetic study on French cohort composing of rapid and slow progressors HIV-1 infected individual identified a strong association of CXCR1 haplotype Ha with protection against rapid progression to AIDS (Vasilescu et al., 2007). It was suggested that the inhibitory effect of CXCR1 Ha could be mediated by suppressing CD4+ and CXCR4 expression (Kaur &

The highly conserved *CXCR4* gene is an obvious target as CXCR4 serves a coreceptor for X4 (SI) strains of HIV-1 to gain entry into cells. This gene is located on chromosome 2 and the screening of entire transcription unit resulted in the detection of two rare polymorphisms.

Global, regional, and ethnic distributions of frequencies of CXCL12 genotypes and of the CXCL12-3'A allele vary significantly, ranging from 0.029-0.091 in Africans, 0.056-0.150 in American Indians, 0.149-0.217 in Europeans, 0.09- 0.380 in North Asians, 0.06-0.43 in South Asians, and 0.536- 0.7145 in Oceanian population (Su et al., 1999). In other ethnic cohorts, the allelic frequency of CXCL12-3'A ranged from 0.100 to 0.332 (Passam et al., 2005; Williamson et al., 2000; Wang et al., 2003). The frequency of the CXCL12 polymorphisms was also investigated in various cohorts of HIV-1 exposed but uninfected and among HIV-1 infected individuals. The results showed that the CXCL12-3'A allele delayed progression to AIDS but not decreased the susceptibility to HIV-1 infection (Winkler et al., 1998). Another study showed that the CXCL12-3'A homozygous mutation did not influence the clinical course of asymptomatic patients. However, the lower number of deaths during the follow-up period among symptomatic patients who were homozygotes and heterozygotes for the CXCL12- 3'A allele, suggested that both genotypes could have a possible late-stage protective effect on the clinical outcome of HIV-1 patients after the AIDS diagnosis (Reiche et al., 2006). However, the disease-retarding role of homozygosity for the CXCL12-3'A allele has not been confirmed in other cohorts (Mummidi et al., 1998; Wang et al., 2003; van Rij et al., 1998; Magierowska et al., 1999; Rousset et al., 1999; Brambilla et al., 2000; Soriano et al., 2002).

Verma et al. (2007) observed a low frequency of CCR5-Δ32 (1.5%) and of CCR2-64I (9.1%) in healthy Northern Indians, suggesting high vulnerability of North Indians to HIV-1 infection. However, the allelic frequency of the CXCL12 3'A was high (20.4%) in the healthy HIV-1 seronegative Northern Indians included in their study, which was similar to that observed in South Indians (17.0%–35.0%) and South European populations (14.0%–33.0%) (Ramana et al., 2001).

Chaudhary et al. (2008) examined the SNP of CXCL12 3'A by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP), cloning and sequencing in individuals from Northen India and showed that the genotypic frequency of CXCL12 3'A/CXCL12 3'A in the 100 HIV-1 seronegative healthy individuals, in 150 HIV-1 seronegative individuals with high risk for sexually transmitted disease (STD), and in 100 HIV-1 seropositive patients were 4.0%, 18.0% and 7.0%, respectively. A significantly higher frequency of CXCL12 3'A/CXCL12 3'A was observed in high risk STD individuals as compared to HIV-1 seropositive (*p* = 0.014) and healthy HIV-1 seronegative tested individuals (*p* = 0.001), suggesting a protective role of CXCL12 3'A allele in HIV-1 infection. In this study it was observed a significant increase in the homozygous genotype for the mutant allele CXCL12 3'A in the high risk STD individuals as compared to both the healthy seronegative and HIV-1 seropositive individuals, suggesting a possible protective role of this allele in the homozygous state against HIV-1 infection.

The frequencies of CXCL12 3'A, CCR5-32, CCR5-m3030, and CCR2-64I allelic variants were investigated in unrelated healthy Bahraini individuals without any known history of HIV-1 infection or AIDS symptoms. The results showed that CCR2-64I allele (8.9%) and especially the CXCL12 3'A allele (26.5%) were predominant and may be associated with resistance to fast HIV-1 infection in this population, and thus their genotyping could be used for prognosis in HIV-1 infected individuals. No mutant alleles were detected for CCR5 m303A mutation and the frequency of 2.8% for CCR5-32 allele may be attributed to the admixture with people of European descent (Salem et al., 2009).

The epidemiological interaction of CCR5/CCR2 and CXCL12-3'A suggests that a functional interaction might explain the enhanced protection. One hypothesis is that CCR2 and CCR5 variants slow AIDS by limiting the number of CCR5 coreceptors that mediate the replication and spread of primary, early stage R5 HIV-1 strains, while the CXCL12-3'A variant restricts the emergence of X4 HIV-1 strains and the ensuing AIDS-accelerating process (O'Brien and Moore, 2000).

However, several studies show a lack of relationship between CXCL12 3'A and HIV-1 disease non-progression (Vidal et al., 2005a; Ioannidis et al., 2001; Tresoldi et al., 2002). For instance, in their international meta-analysis, Ioannidis et al. (2001) measured the effects of subjects homozygous for the CXCL12 3'A polymorphism by reviewing studies that prospectively followed HIV-1 infected patients from seroconversion to AIDS diagnosis and death. Results showed that being homozygous for the polymorphism had no effect on disease progression and there was no significant difference in HIV-1 RNA levels among persons with and withoutthe polymorphism.

Recently, Tan et al. (2010) showed that the allelic frequency of CCR5-32, CCR5m303A, CCR2- 64I and CXCL12-3'A in HIV-1 infected and uninfected high-risk Uighurs individuals, the largest population of minority in China, was 4.4%, 2.7%, 25.7% and 57.4%, respectively. While there was no significant difference in the frequency of CCR5-32, CCR2-64I and CXCL12-3'A between HIV-1 seropositive and seronegative group, the frequency of CCR5m3030A in HIV-1 seropositive group was significantly higher than that in seronegative group. Furthermore, a woman who carried homozygous CCR5-32 was positive for HIV-1 infection. Therefore, these data suggest that the CCR5-32 CCR2-64I and CXCL1´3'A alleles may have limited effect on protecting from HIV-1 infection and CCR5m303A variant may be associated with the risk for HIV-1 infection in high-risk Uighurs individuals.

The distribution of CCR5-32, CCR2-64I, and CXCL12 3'A alleles was evaluated in Guangxi Province Zhuang population, the largest minority ethnic population with over 15 million people, mainly located in Guangxi Province, China. The CCR5-32 was absent, and CCR2- 64I and CXCL12 3'A alleles were relatively common and seem not to confer protection against HIV-1 infection in this population. The results suggest that the Zhuang people may have a similar genetic susceptibility to HIV-1 infection with most other Chinese ethnic groups (Qijian et al., 2010).

## **3.2 CXCR1, CXCR2**

308 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

Global, regional, and ethnic distributions of frequencies of CXCL12 genotypes and of the CXCL12-3'A allele vary significantly, ranging from 0.029-0.091 in Africans, 0.056-0.150 in American Indians, 0.149-0.217 in Europeans, 0.09- 0.380 in North Asians, 0.06-0.43 in South Asians, and 0.536- 0.7145 in Oceanian population (Su et al., 1999). In other ethnic cohorts, the allelic frequency of CXCL12-3'A ranged from 0.100 to 0.332 (Passam et al., 2005; Williamson et al., 2000; Wang et al., 2003). The frequency of the CXCL12 polymorphisms was also investigated in various cohorts of HIV-1 exposed but uninfected and among HIV-1 infected individuals. The results showed that the CXCL12-3'A allele delayed progression to AIDS but not decreased the susceptibility to HIV-1 infection (Winkler et al., 1998). Another study showed that the CXCL12-3'A homozygous mutation did not influence the clinical course of asymptomatic patients. However, the lower number of deaths during the follow-up period among symptomatic patients who were homozygotes and heterozygotes for the CXCL12- 3'A allele, suggested that both genotypes could have a possible late-stage protective effect on the clinical outcome of HIV-1 patients after the AIDS diagnosis (Reiche et al., 2006). However, the disease-retarding role of homozygosity for the CXCL12-3'A allele has not been confirmed in other cohorts (Mummidi et al., 1998; Wang et al., 2003; van Rij et al., 1998; Magierowska et al., 1999; Rousset et al., 1999; Brambilla et al., 2000; Soriano et al., 2002). Verma et al. (2007) observed a low frequency of CCR5-Δ32 (1.5%) and of CCR2-64I (9.1%) in healthy Northern Indians, suggesting high vulnerability of North Indians to HIV-1 infection. However, the allelic frequency of the CXCL12 3'A was high (20.4%) in the healthy HIV-1 seronegative Northern Indians included in their study, which was similar to that observed in South Indians (17.0%–35.0%) and South European populations (14.0%–33.0%) (Ramana et

Chaudhary et al. (2008) examined the SNP of CXCL12 3'A by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP), cloning and sequencing in individuals from Northen India and showed that the genotypic frequency of CXCL12 3'A/CXCL12 3'A in the 100 HIV-1 seronegative healthy individuals, in 150 HIV-1 seronegative individuals with high risk for sexually transmitted disease (STD), and in 100 HIV-1 seropositive patients were 4.0%, 18.0% and 7.0%, respectively. A significantly higher frequency of CXCL12 3'A/CXCL12 3'A was observed in high risk STD individuals as compared to HIV-1 seropositive (*p* = 0.014) and healthy HIV-1 seronegative tested individuals (*p* = 0.001), suggesting a protective role of CXCL12 3'A allele in HIV-1 infection. In this study it was observed a significant increase in the homozygous genotype for the mutant allele CXCL12 3'A in the high risk STD individuals as compared to both the healthy seronegative and HIV-1 seropositive individuals, suggesting a possible protective role of

The frequencies of CXCL12 3'A, CCR5-32, CCR5-m3030, and CCR2-64I allelic variants were investigated in unrelated healthy Bahraini individuals without any known history of HIV-1 infection or AIDS symptoms. The results showed that CCR2-64I allele (8.9%) and especially the CXCL12 3'A allele (26.5%) were predominant and may be associated with resistance to fast HIV-1 infection in this population, and thus their genotyping could be used for prognosis in HIV-1 infected individuals. No mutant alleles were detected for CCR5 m303A mutation and the frequency of 2.8% for CCR5-32 allele may be attributed to the

The epidemiological interaction of CCR5/CCR2 and CXCL12-3'A suggests that a functional interaction might explain the enhanced protection. One hypothesis is that CCR2 and CCR5

this allele in the homozygous state against HIV-1 infection.

admixture with people of European descent (Salem et al., 2009).

al., 2001).

CXCR1 (IL-8RA) and CXCR2 (IL-8RB) are receptors for IL-8, a proinflammatory cytokine involved in chemoattraction and activation of neutrophils. CXCR1 and CXCR2 genes are located o chromosome 2q35 and several polymorphisms have been described including SNPs T92G (CXCR1 -300) and C1003T (CXCR1-142) that result in a CXCR1 haplotype Ha. A genetic study on French cohort composing of rapid and slow progressors HIV-1 infected individual identified a strong association of CXCR1 haplotype Ha with protection against rapid progression to AIDS (Vasilescu et al., 2007). It was suggested that the inhibitory effect of CXCR1 Ha could be mediated by suppressing CD4+ and CXCR4 expression (Kaur & Mehra, 2009).

#### **3.3 CXCR4**

The highly conserved *CXCR4* gene is an obvious target as CXCR4 serves a coreceptor for X4 (SI) strains of HIV-1 to gain entry into cells. This gene is located on chromosome 2 and the screening of entire transcription unit resulted in the detection of two rare polymorphisms.

The Role of Genetic Polymorphisms in the Chemokine and

not only in HIV-1 soropositive patients but also in healthy controls.

**5. Genetic polymorphism in cytokines and their receptors** 

**5.1 Interleukin-4 (IL-4) and IL-4 Receptor (IL-4R)** 

origin shared similar allele distributions.

population.

1997).

(Chatterjee et al., 2009).

Their Receptors and Cytokines in the Human Immunodeficiency Virus Type 1 (HIV-1) Infection 311

significantly more frequent in LTNPs than progressors, but not than healthy controls. It has been observed that there was a large discrepancy between these alleles among populations of the north and south. Populations in the same language-speaking family or with the same

Puissant et al. (2006) observed that some genetic polymorphisms had an impact on the evolution of plasma virus load and peripheral T lymphocyte counts in HIV-1 infected patients under HAART. After 1 year of HAART, patients with a virological response (undetectable plasma HIV-1 RNA) have a higher frequency of the homozygous CXCL12 3'A genotype than patients with other polymorphisms such as CCR5-32, CCR2-64I, CX3CR1- 249I, and CX3CR1-280M. Similarly, patients with a CD4+ T cell increase of over 200/mm3 from baseline after 1 year of HAART display higher frequencies of homozygous CXCL12 3'A and homozygous CX3CR1-280M genotypes than other patients. Moreover, the authors showed that CX3CR1-280M allele was associated with high peripheral CD4+ T cell counts

Qian et al. (2008) showed that the frequencies of CX3CR1-249I and 280M alleles varied substantially among different population and were independent risk factors for accelerating the progression to AIDS. Further, Parczewski et al. (2009) studied the influence of genetic variants for CCR5-32, CCR5 -G2459A, CCR2- G190A, CX3CR1- G744A, and CX3CR1- C838T in a cohort of 168 HIV-1 seropositive adults and 151 healthy newborns from northwestern Poland. The results showed that haplotypes containing CCR5-32, CCR2- G190A, and CX3CR1- G744A were significantly more common in the healthy newborns suggesting an association between these haplotypes and resistance to HIV-1 infection in this

Like chemokines, the role of cytokines in the modulation of HIV-1 infection and the rate of disease progression remains to be fully understood. Evidence of strong epidemiological associations between cytokines and HIV-1 disease progression has been limited and, in some cases, inconsistent across studies. Studies have shown that cytokines can have inhibitory, stimulatory or both effects on HIV-1 replication (Han et al., 1996; Naif et al.,

IL-4 is an important cytokine that induces differentiation of CD4+ Th cells. It also regulates the expression of the HIV-1 coreceptors CCR5 and CXCR4. IL-4 decreases the levels of CCR5 on the surfaces of CD4-bearing cells and increases CXCR4 levels on the same or other cells. *IL-4* gene is located on chromosome 11 and a SNP in the regulatory region of IL-4 gene (IL4-589 C/T), initially identified among HIV-1 seropositive Japanese individuals, has been reported to have a protective effect against transmission of HIV-1 through heterosexual contact. The IL4-R alpha I50V polymorphism in exon 5 of IL-4R gene affects the functional responsiveness of the gene (Risma et al., 2002). The SNP I50V was found to be associated with slow progression to AIDS in HIV-1 infected individuals (Soriano et al., 2005). However, another study suggested an association of IL-4R alpha I50V allele with increased likelihood of HIV-1 infection in North Indian population

One of these CXCR4 mutations was silent, and each was unique to two nonprogressors. However, no association with progression to AIDS was found (Cohen et al., 1998).

## **3.4 CXCR6**

The CXCR6 is a chemokine receptor that is known as a minor coreceptor in HIV-1 infection but could participate in disease progression through its role as a mediator of inflammation. Petit et al. (2008) described the effects of mutation of acidic extracellular CXCR6 residues on receptor function. Although most CXCR6 mutants examined were expressed at levels similar to wild-type CXCR6, the N-terminal E3Q mutant was poorly expressed, which may explain previously reported protective effects of a similar SNP, with respect to late-stage HIV-1 infection. In contrast to several other chemokine receptors, mutation of the CXCR6 Nterminal and inhibition of post-translational modifications of this region were without effect on receptor function. This data suggests a novel paradigm for the CXCR6:CXCL16 interaction, a finding which may impact the discovery of small-molecule antagonists of CXCR6.

Study by Limou et al. (2010) verified that the rs2234358 polymorphism in the CXCR6 gene was the strongest signal obtained for the genomewide association study comparing the 186 Genomics of Resistance to Immunodeficiency Virus (GRIV) LTNPs who were not elite controlls with 697 uninfected control subjects. This association was replicated in 3 additional independent European studies, reaching genomewide significance. This association with LTNPs is independent of the CCR2-CCR5 loci and the HCP5 polymorphisms. The statistical significance, the replication, and the magnitude of the association demonstrate that CXCR6 is likely involved in the molecular etiology of AIDS and, in particular, in LTNPs, emphasizing the power of extreme-phenotype cohorts.

## **4. Genetic polymorphisms in CX3C Chemokine Receptor (CX3CR)**

## **4.1 CX3CR1**

CX3CR1, a leukocyte chemotactic and adhesion receptor for the human chemokine fractalkine, has also been defined as a minor HIV-1 coreceptor, particularly expressed on brain. Mutations on the CX3CR1 gene, located at chromosome 3, have been described, such as V249I (substitution changed Valine to Isoleucine) and T280M (substitution changed Threonine to Methionine) (Faure et al., 2000), with frequency of 26.0% and 13.0%, respectively. The impact of CX3CR1 polymorphisms on HIV-1 pathogenesis is controversial, with conflicting reports of their role in disease progression in HIV-1 infected patients. Individuals homozygous for the 280M allele exhibited accelerated disease progression (Faure et al., 2000, 2003), with a small but statistically significant correlation with slightly earlier immunological and virologic failure (Brumme et al., 2003). However, further studies did not confirm this observation (McDermott et al., 2000; Kwa et al., 2003).

Polymorphisms in CCR2 and CX3CR1, which HIV-1 sometimes uses as coreceptors, have also been associated with slowing HIV-1 disease progression. For example, the *CCR2-64I* mutation has been shown to reduce CXCR4 expression on CD4+ T cells, thereby interfering with X4-tropic virus infection (Kalinkovich et al., 1999). In one study's cohort of HIV-1 positive Kenyan sex workers (Anzala et al., 1998), the frequency of being positive for *CCR2- 64I* was highest in LTNPs, which was three times greater than that for progressors. In a separate study, Vidal et al. (2005b) found that the *CX3CR1 V249I* polymorphism is significantly more frequent in LTNPs than progressors, but not than healthy controls. It has been observed that there was a large discrepancy between these alleles among populations of the north and south. Populations in the same language-speaking family or with the same origin shared similar allele distributions.

Puissant et al. (2006) observed that some genetic polymorphisms had an impact on the evolution of plasma virus load and peripheral T lymphocyte counts in HIV-1 infected patients under HAART. After 1 year of HAART, patients with a virological response (undetectable plasma HIV-1 RNA) have a higher frequency of the homozygous CXCL12 3'A genotype than patients with other polymorphisms such as CCR5-32, CCR2-64I, CX3CR1- 249I, and CX3CR1-280M. Similarly, patients with a CD4+ T cell increase of over 200/mm3 from baseline after 1 year of HAART display higher frequencies of homozygous CXCL12 3'A and homozygous CX3CR1-280M genotypes than other patients. Moreover, the authors showed that CX3CR1-280M allele was associated with high peripheral CD4+ T cell counts not only in HIV-1 soropositive patients but also in healthy controls.

Qian et al. (2008) showed that the frequencies of CX3CR1-249I and 280M alleles varied substantially among different population and were independent risk factors for accelerating the progression to AIDS. Further, Parczewski et al. (2009) studied the influence of genetic variants for CCR5-32, CCR5 -G2459A, CCR2- G190A, CX3CR1- G744A, and CX3CR1- C838T in a cohort of 168 HIV-1 seropositive adults and 151 healthy newborns from northwestern Poland. The results showed that haplotypes containing CCR5-32, CCR2- G190A, and CX3CR1- G744A were significantly more common in the healthy newborns suggesting an association between these haplotypes and resistance to HIV-1 infection in this population.

## **5. Genetic polymorphism in cytokines and their receptors**

Like chemokines, the role of cytokines in the modulation of HIV-1 infection and the rate of disease progression remains to be fully understood. Evidence of strong epidemiological associations between cytokines and HIV-1 disease progression has been limited and, in some cases, inconsistent across studies. Studies have shown that cytokines can have inhibitory, stimulatory or both effects on HIV-1 replication (Han et al., 1996; Naif et al., 1997).

## **5.1 Interleukin-4 (IL-4) and IL-4 Receptor (IL-4R)**

310 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

One of these CXCR4 mutations was silent, and each was unique to two nonprogressors.

The CXCR6 is a chemokine receptor that is known as a minor coreceptor in HIV-1 infection but could participate in disease progression through its role as a mediator of inflammation. Petit et al. (2008) described the effects of mutation of acidic extracellular CXCR6 residues on receptor function. Although most CXCR6 mutants examined were expressed at levels similar to wild-type CXCR6, the N-terminal E3Q mutant was poorly expressed, which may explain previously reported protective effects of a similar SNP, with respect to late-stage HIV-1 infection. In contrast to several other chemokine receptors, mutation of the CXCR6 Nterminal and inhibition of post-translational modifications of this region were without effect on receptor function. This data suggests a novel paradigm for the CXCR6:CXCL16 interaction, a finding which may impact the discovery of small-molecule antagonists of

Study by Limou et al. (2010) verified that the rs2234358 polymorphism in the CXCR6 gene was the strongest signal obtained for the genomewide association study comparing the 186 Genomics of Resistance to Immunodeficiency Virus (GRIV) LTNPs who were not elite controlls with 697 uninfected control subjects. This association was replicated in 3 additional independent European studies, reaching genomewide significance. This association with LTNPs is independent of the CCR2-CCR5 loci and the HCP5 polymorphisms. The statistical significance, the replication, and the magnitude of the association demonstrate that CXCR6 is likely involved in the molecular etiology of AIDS and, in particular, in LTNPs,

CX3CR1, a leukocyte chemotactic and adhesion receptor for the human chemokine fractalkine, has also been defined as a minor HIV-1 coreceptor, particularly expressed on brain. Mutations on the CX3CR1 gene, located at chromosome 3, have been described, such as V249I (substitution changed Valine to Isoleucine) and T280M (substitution changed Threonine to Methionine) (Faure et al., 2000), with frequency of 26.0% and 13.0%, respectively. The impact of CX3CR1 polymorphisms on HIV-1 pathogenesis is controversial, with conflicting reports of their role in disease progression in HIV-1 infected patients. Individuals homozygous for the 280M allele exhibited accelerated disease progression (Faure et al., 2000, 2003), with a small but statistically significant correlation with slightly earlier immunological and virologic failure (Brumme et al., 2003). However, further studies

Polymorphisms in CCR2 and CX3CR1, which HIV-1 sometimes uses as coreceptors, have also been associated with slowing HIV-1 disease progression. For example, the *CCR2-64I* mutation has been shown to reduce CXCR4 expression on CD4+ T cells, thereby interfering with X4-tropic virus infection (Kalinkovich et al., 1999). In one study's cohort of HIV-1 positive Kenyan sex workers (Anzala et al., 1998), the frequency of being positive for *CCR2- 64I* was highest in LTNPs, which was three times greater than that for progressors. In a separate study, Vidal et al. (2005b) found that the *CX3CR1 V249I* polymorphism is

emphasizing the power of extreme-phenotype cohorts.

**4. Genetic polymorphisms in CX3C Chemokine Receptor (CX3CR)** 

did not confirm this observation (McDermott et al., 2000; Kwa et al., 2003).

However, no association with progression to AIDS was found (Cohen et al., 1998).

**3.4 CXCR6** 

CXCR6.

**4.1 CX3CR1** 

IL-4 is an important cytokine that induces differentiation of CD4+ Th cells. It also regulates the expression of the HIV-1 coreceptors CCR5 and CXCR4. IL-4 decreases the levels of CCR5 on the surfaces of CD4-bearing cells and increases CXCR4 levels on the same or other cells. *IL-4* gene is located on chromosome 11 and a SNP in the regulatory region of IL-4 gene (IL4-589 C/T), initially identified among HIV-1 seropositive Japanese individuals, has been reported to have a protective effect against transmission of HIV-1 through heterosexual contact. The IL4-R alpha I50V polymorphism in exon 5 of IL-4R gene affects the functional responsiveness of the gene (Risma et al., 2002). The SNP I50V was found to be associated with slow progression to AIDS in HIV-1 infected individuals (Soriano et al., 2005). However, another study suggested an association of IL-4R alpha I50V allele with increased likelihood of HIV-1 infection in North Indian population (Chatterjee et al., 2009).

The Role of Genetic Polymorphisms in the Chemokine and

CD4+ T cell count (Vaamonde et al., 2006).

azidothymidine resistance mutations.

AIDS or therapeutic failure (Rigatto et al., 2008).

Their Receptors and Cytokines in the Human Immunodeficiency Virus Type 1 (HIV-1) Infection 313

(Dronda et al., 2002), hepatitis C virus coinfection (Greub et al., 2000), and lower baseline

Genetic polymorphisms could also explain the heterogeneity in sustaining viral suppression observed among patients receiving HAART (O'Brien et al., 2000). Approximately 10.0% of HIV-1 infected patients do not respond to HAART with a reduction of viral load, even if there is good compliance and no evidence of viral resistance (Lederberger et al., 1999). However, studies investigating the association between genetic polymorphisms and response to HAART have provided conflicting data. An improvement in the immunological and the virological responses in association with the CCR5-32, CCR2-64I, CXCL12-3'A, and CCR5-59029G/A polymorphisms has been reported (O'Brien et al., 2000; Guerin et al., 2000; Kasten et al., 2000; Yamashita et al., 2001). HIV-1 infected patients with wild type genotypes for the CCR5, CCR2, and 59029A alleles treated with HAART had the poorest response to therapy compared with patients with other genotypes combined with the CCR5-32, CCR-64I or 59029G alleles (O'Brien et al., 2000). Polymorphism within the *CX3CR1* gene was associated with accelerated virological and immunological therapy failure (Brumme et al., 2003). One report (Puissant et al., 2006) showed that, after one year of HAART, patients with undetectable plasma HIV-1 RNA levels have a higher frequency of the homozygous CXCL12-3'A genotype than other patients. Similarly, patients with a CD4+T cell count increase of over 200/mm3 from baseline after one year of HAART display higher frequencies of homozygous CXCL12-3'A and CX3CR1-280M genotypes than other patients. Another study that evaluated a subgroup of patients with baseline CD4+ T cell count of 201- 500 cells/mm3, showed that both the CXCL12-3'A and CCR2-64I alleles displayed a positive influence on clinical progression after HAART initiation. The CXCL12-3'A allele showed this effect through a more rapid CD4+ T lymphocyte restoration counts above the level of 500 cells/mm3, while CCR2-64I was associated with stronger viral suppression. Regarding the CCR5-32 and CCR5-59029G/A alleles, they had no effects on the response to HAART initiation (Passam et al., 2005). In contrast, other studies did not find such correlation (Brumme et al., 2001; Bratt et al., 1998; Wit et al., 2002). However, one of these studies (Bratt et al., 1998) did not exclude nonadherent patients, and analyzed their data on an intent-totreat basis, thus reducing the likelihood of finding a difference. In addition, 69.0% of their CCR5/32 heterozygous patients had SI virus isolates, and therefore, tended to have lower CD4+ T cell counts, higher plasma HIV-1 RNA levels, and a greater proportion of

Another study evaluated the CCR5-32, CXCL12-3'A, and CCR2-64I genetic polymorphisms in HIV-1 infected patients receiving HAART and the results showed that successful treatment was associated with heterozygosity for the CCR5-32, underscoring that the chemokine receptor polymorphisms have a modifying effect on the virological response to HAART. The course of mean viral load was significantly worse for patients without the CCR5-32 allele and the multivariate analysis demonstrated that heterozygosity for the CCR5-32 variant is an

The frequency of the CXCL12- 3'A, CCR2-64I, CCR5-32, and CCR5-Promoter-59029-A/G polymorphisms was also evaluated in 155 Brazilian HIV-1 infected on pre and post-HAART and their influence on CD4+ T cell counts. The results showed that the CD4+ T cell gain was influenced by carriage of one or more of these polymorphisms, highlighting the possibility that these genetic traits can be useful to identify patients at risk for faster progression to

independent prognostic factor for treatment outcome (Bogner et al., 2004).

## **5.2 Interleukin 10 (IL-10)**

The interleukin-10 (IL-10) is known to inhibit HIV-1 replication in macrophages *in vivo* (Kollmann et al., 1996). The gene encoding IL-10 is situated on chromosome 1 and a polymorphism with CA transition in the promoter region at position -592, named IL10- C592A, has been associated with diminished IL-10 production and accelerated progression to AIDS with a dominant effect (Winkler et al., 1998). This SNP is carried by 23.6% of the Caucasians, 40.0% of the African Americans, 33.0% of Hispanics, and 60.0% of the Asians. The molecular mechanism behind this SNP is not well understood but is has been predicted that IL-10 may control proliferation of HIV-1 by limiting the number of activated macrophages available for HIV-1 replication (Chatterjee, 2010).

## **5.3 Tumor Necrosis Factor Alpha (TNF-)**

TNF- is a pro-inflammatory cytokine and is known to be involved in the various immunogenetic events that influence HIV-1 infection. The gene encoding TNF- is located on chromosome 6 and four polymorphisms in the TNF- promoter have been identified, all with GA transitions; however, the information available of how TNF- genetic variants affect vulnerability to HIV-1 infection is inconsistent (reviewed by Chatterjee, 2010). Veloso et al. (2010) determined whether carriage of the TNF- -238G/A, -308G/A, and -863 C/A gene promoter SNPs influence the risk of HIV-1 infection and disease progression in Caucasian Spaniards. For this purpose, 239 heavily exposed but uninfected individuals (EU), 203 healthy controls (HC), 109 HIV-1 infected typical progressors (TP) and 75 HIV-1 infected LTNPs were evaluated. The results showed that the distribution of TNF- variants did not differ among HIV-1 infected compared with EU and among TP and LTNPs. The analysis in LTNPs subset indicated that TNF--238A variant allele was significantly overrepresented in patients who spontaneously controlled plasma viremia compared with those who had a detectable plasma viral load. Taken together, the results suggested that TNF- genetic variants were unrelated to disease progression in infected subjects but the - 238 G/A SNP may modulate the control of viremia in LTNPs.

#### **5.4 Interferon alpha receptor 1 (IFNAR1)**

Interferon alpha (IFN-) elicits a pleotropic antiviral response and form the first line of defense against HIV-1 infection. This cytokine acts through the IFN- receptor (IFNAR) that is composed of two subunits, IFNAR1 and IFNAR2, encoded by the gene located at chromosome 21q (Kim et al., 1997). Two SNPs in the *IFNAR1* gene, IFNAR1-18339GC (Valine to Leucine change in exon) and IFNAR1-30127CT (in intron), found in tight linkage disequilibrium were associated with susceptibility to HIV-1 infection (Diop et al., 2006).

## **6. Genetic polymorphism and the response to antiretroviral therapy**

The observations that genetic markers influence the natural history of HIV-1 and that the infection and the immunological and virologic responses to HAART is neither universal nor homogeneous (DeHovitz et al., 2000), lead to the thought that the response to treatment may also be genetically determined. Polymorphisms in the chemokine and chemokine receptor genes may affect response to HAART, in addition to other host factors governing poor immune response to HAART, such as increasing age (Viard et al., 2001), injection drug use

The interleukin-10 (IL-10) is known to inhibit HIV-1 replication in macrophages *in vivo* (Kollmann et al., 1996). The gene encoding IL-10 is situated on chromosome 1 and a polymorphism with CA transition in the promoter region at position -592, named IL10- C592A, has been associated with diminished IL-10 production and accelerated progression to AIDS with a dominant effect (Winkler et al., 1998). This SNP is carried by 23.6% of the Caucasians, 40.0% of the African Americans, 33.0% of Hispanics, and 60.0% of the Asians. The molecular mechanism behind this SNP is not well understood but is has been predicted that IL-10 may control proliferation of HIV-1 by limiting the number of activated

TNF- is a pro-inflammatory cytokine and is known to be involved in the various immunogenetic events that influence HIV-1 infection. The gene encoding TNF- is located on chromosome 6 and four polymorphisms in the TNF- promoter have been identified, all with GA transitions; however, the information available of how TNF- genetic variants affect vulnerability to HIV-1 infection is inconsistent (reviewed by Chatterjee, 2010). Veloso et al. (2010) determined whether carriage of the TNF- -238G/A, -308G/A, and -863 C/A gene promoter SNPs influence the risk of HIV-1 infection and disease progression in Caucasian Spaniards. For this purpose, 239 heavily exposed but uninfected individuals (EU), 203 healthy controls (HC), 109 HIV-1 infected typical progressors (TP) and 75 HIV-1 infected LTNPs were evaluated. The results showed that the distribution of TNF- variants did not differ among HIV-1 infected compared with EU and among TP and LTNPs. The analysis in LTNPs subset indicated that TNF--238A variant allele was significantly overrepresented in patients who spontaneously controlled plasma viremia compared with those who had a detectable plasma viral load. Taken together, the results suggested that TNF- genetic variants were unrelated to disease progression in infected subjects but the -

Interferon alpha (IFN-) elicits a pleotropic antiviral response and form the first line of defense against HIV-1 infection. This cytokine acts through the IFN- receptor (IFNAR) that is composed of two subunits, IFNAR1 and IFNAR2, encoded by the gene located at chromosome 21q (Kim et al., 1997). Two SNPs in the *IFNAR1* gene, IFNAR1-18339GC (Valine to Leucine change in exon) and IFNAR1-30127CT (in intron), found in tight linkage disequilibrium were associated with susceptibility to HIV-1 infection (Diop et al.,

The observations that genetic markers influence the natural history of HIV-1 and that the infection and the immunological and virologic responses to HAART is neither universal nor homogeneous (DeHovitz et al., 2000), lead to the thought that the response to treatment may also be genetically determined. Polymorphisms in the chemokine and chemokine receptor genes may affect response to HAART, in addition to other host factors governing poor immune response to HAART, such as increasing age (Viard et al., 2001), injection drug use

**6. Genetic polymorphism and the response to antiretroviral therapy** 

macrophages available for HIV-1 replication (Chatterjee, 2010).

238 G/A SNP may modulate the control of viremia in LTNPs.

**5.4 Interferon alpha receptor 1 (IFNAR1)** 

2006).

**5.3 Tumor Necrosis Factor Alpha (TNF-)** 

**5.2 Interleukin 10 (IL-10)** 

(Dronda et al., 2002), hepatitis C virus coinfection (Greub et al., 2000), and lower baseline CD4+ T cell count (Vaamonde et al., 2006).

Genetic polymorphisms could also explain the heterogeneity in sustaining viral suppression observed among patients receiving HAART (O'Brien et al., 2000). Approximately 10.0% of HIV-1 infected patients do not respond to HAART with a reduction of viral load, even if there is good compliance and no evidence of viral resistance (Lederberger et al., 1999).

However, studies investigating the association between genetic polymorphisms and response to HAART have provided conflicting data. An improvement in the immunological and the virological responses in association with the CCR5-32, CCR2-64I, CXCL12-3'A, and CCR5-59029G/A polymorphisms has been reported (O'Brien et al., 2000; Guerin et al., 2000; Kasten et al., 2000; Yamashita et al., 2001). HIV-1 infected patients with wild type genotypes for the CCR5, CCR2, and 59029A alleles treated with HAART had the poorest response to therapy compared with patients with other genotypes combined with the CCR5-32, CCR-64I or 59029G alleles (O'Brien et al., 2000). Polymorphism within the *CX3CR1* gene was associated with accelerated virological and immunological therapy failure (Brumme et al., 2003). One report (Puissant et al., 2006) showed that, after one year of HAART, patients with undetectable plasma HIV-1 RNA levels have a higher frequency of the homozygous CXCL12-3'A genotype than other patients. Similarly, patients with a CD4+T cell count increase of over 200/mm3 from baseline after one year of HAART display higher frequencies of homozygous CXCL12-3'A and CX3CR1-280M genotypes than other patients.

Another study that evaluated a subgroup of patients with baseline CD4+ T cell count of 201- 500 cells/mm3, showed that both the CXCL12-3'A and CCR2-64I alleles displayed a positive influence on clinical progression after HAART initiation. The CXCL12-3'A allele showed this effect through a more rapid CD4+ T lymphocyte restoration counts above the level of 500 cells/mm3, while CCR2-64I was associated with stronger viral suppression. Regarding the CCR5-32 and CCR5-59029G/A alleles, they had no effects on the response to HAART initiation (Passam et al., 2005). In contrast, other studies did not find such correlation (Brumme et al., 2001; Bratt et al., 1998; Wit et al., 2002). However, one of these studies (Bratt et al., 1998) did not exclude nonadherent patients, and analyzed their data on an intent-totreat basis, thus reducing the likelihood of finding a difference. In addition, 69.0% of their CCR5/32 heterozygous patients had SI virus isolates, and therefore, tended to have lower CD4+ T cell counts, higher plasma HIV-1 RNA levels, and a greater proportion of azidothymidine resistance mutations.

Another study evaluated the CCR5-32, CXCL12-3'A, and CCR2-64I genetic polymorphisms in HIV-1 infected patients receiving HAART and the results showed that successful treatment was associated with heterozygosity for the CCR5-32, underscoring that the chemokine receptor polymorphisms have a modifying effect on the virological response to HAART. The course of mean viral load was significantly worse for patients without the CCR5-32 allele and the multivariate analysis demonstrated that heterozygosity for the CCR5-32 variant is an independent prognostic factor for treatment outcome (Bogner et al., 2004).

The frequency of the CXCL12- 3'A, CCR2-64I, CCR5-32, and CCR5-Promoter-59029-A/G polymorphisms was also evaluated in 155 Brazilian HIV-1 infected on pre and post-HAART and their influence on CD4+ T cell counts. The results showed that the CD4+ T cell gain was influenced by carriage of one or more of these polymorphisms, highlighting the possibility that these genetic traits can be useful to identify patients at risk for faster progression to AIDS or therapeutic failure (Rigatto et al., 2008).

The Role of Genetic Polymorphisms in the Chemokine and

v. 351, pp. 1632–1633, ISSN 0099-5355.

6599, pp.400, ISSN 0028-0836.

pp. 252-253, ISSN 1078-8956.

1, p. 311-315, ISSN 0022-1899.

8075.

0269-9370.

ISSN 1044-5323.

Their Receptors and Cytokines in the Human Immunodeficiency Virus Type 1 (HIV-1) Infection 315

An, P.; Martin, M.P.; Nelson, G.W.; Carrington, M.; Smith, M.W.; Gong, K.; Vlahov, D.;

An, P.; Nelson, G.W.; Wang, L.; Donfield, S.; Goedert, J.J.; Phair, J.; Vlahov, D.; Buchbinder,

Anzala, A.O.; Ball, T.B.; Rostron, T.; O'Brien, S. J.; Plummer, F.A. & Rowland-Jones, S.L.

Arenzana-Seisdedos, F. & Parmentier, M. (2006). Genetics of resistance to HIV infection: role

Arenzana-Seisdedos, F.; Virelizier, J-L.; Rousset, D.; Clark-Lewis, I.; Loetscher, P.; Moser, B.

Arya, S.K.; Ginsberg, C.C.; Davis-Warren, A. & D'Costa, J. (1998). In vitro phenotype of

Balotta, C.; Bagnarelli, P.; Violin, M.; Ridolfo, A.L.; Zhou, D.; Berlusconi, A.; Corvasce, S.;

Biti, R.; Ffrench, R.; Young, J.; Bennets, B.; Steward, G. & Liang, T*.* (1997). HIV-1 infection in

Bleul, C.C.; Farzan, M.; Choe, H.; Parolin, C.; Clark-Lewis, I.; Sodoski, J.; Springer, T.A.

Brambilla, A.; Villa, C.; Rizzardi, G.; Veglia, F.; Ghezzi, S.; Lazzarin, A.; Cusini, M.;

Brass, A.L.; Dykxhoom, D.M.; Benita, Y.; Yan, N.; Engelman, A.; Xavier, R.J.; Lieberman, J. &

Bratt, G.; Karlsson, A.; Leandersson, A.C.; Albert, J.; Wahren, B. & Sandstrom, E. (1998).

blocks HIV-1 entry. *Nature*, v. 382, n. 6594, p. 829-833, ISSN 0028-0836. Bogner, J.; Lutz, B.; Klein, H.G.; Pollerer, C.; Troendle, U. & Goebel, F.D. (2004). Association

*HIV Medicine*, v. 5, n. 4, pp. 264-272, ISSN 1464-2662.

in vivo. *Journal of Human Virology*, v.2, p. 133-138, ISSN 1090-9508.

infected patient. *AIDS*, v. *11*, n.10, pp. F67-F71, ISSN 0269-9370.

*Academy of Sciences U S A*, v. 99, pp. 10002-10007, ISSN 0027-8424.

O'Brien, S.J. & Winkler, C.A. (2000). Influence of CCR5 promoter haplotypes on AIDS progression in African-Americans. *AIDS*, v. 14, n. 14, pp. 2117-2122, ISSN

S.; Farrar, W.L.; Modi, W.; O'Brien, S.J. & Winkler, C.A. (2002). Modulating influence on HIV/AIDS by interacting RANTES gene variants. *Proceedings National* 

(1998). CCR2–64I allele and genotype association with delayed AIDS progression in African women. University of Nairobi Collaboration for HIV Research. *The Lancet*,

of co-receptors and co-receptor ligands. *Seminars in Immunology*, v. 18, pp. 387-403,

& Baggiolini, M. (1996). HIV blocked by chemokine antagonist. *Nature*, v. 383, n.

SDF1 gene mutant that delay the onset of human immunodeficiency virus disease

Corbellino, M.; Clementi, M.; Clerici, M.; Moroni, M. & Galli, M. (1997). Homozygous delta 32 deletion of the CCR5-chemokine receptor gene in an HIV-1-

an individual homozygous for the CCR5 deletion allele. *Nature Medicine.*, v. 3, n.3,

(1996). The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and

of highly active antiretroviral therapy failure with chemokine receptor 5 wild type.

Muratori, S.; Santagostino, E.; Gringeri, A.; Louie, L.G.; Sheppard, H.W.; Poli, G.; Michael, N.L.; Pantaleo, G. & Vicenzi, E. (2000). Shorter survival of SDF1-3'A homozygotes linked to CD4+ T cell decrease in advanced human immunodeficiency virus type 1 infection*. The Journal of Infectious Diseases,* v. 182, n.

Elledge, S.J. (2008) Identification of host proteins required for HIV infection through a functional genomic screen. *Science*, v. 319, n.5868, pp.921-926, ISSN 0036-

Treatment history and baseline viral load, but not viral tropism or CCR-5 genotype,

The chemokine polymorphisms CXCR6-3E/K, In1.1T/C, H7 haplotype, CX3CR1-V249I, and CX3CR1-T280M have been shown to affect the course of HIV-1 infection. The influence on immunologic and virologic response to HAART in a group of 143 HIV-1 patients was studied by Passam et al. (2007). The results revealed an improved immunologic response to HAART in patients with the CX3CR1-249I or CX3CR1-280M allele. On the contrary, patients with initial viral load suppression due to HAART showed a faster virologic failure in the presence of the CXCR6-3K allele. The In1.1T/C polymorphism and H7 haplotype did not reveal any specific effect on HAART response.

## **7. Conclusion**

So far, the CCR5-32 allele remains the most important host factor known to be associated to the resistance to the HIV-1 infection. However, high frequencies of CCR5-32, CCR2-64I, and CXCL12-3'A alleles could be all the aftermath or adaptive episodes in which a pathogen exerted selective pressure favoring the survival of persons with these beneficial alleles.

Taken together, the reviewed studies suggest that the existence of genetic polymorphisms must be taken into account in the virological and immunological follow-up of HIV-1 infected patients under treatment with HAART, and that pharmacogenetics is very likely to influence the future individualization of HAART. The individual genetic conditions could be of interest not only in terms of disease progression, but also on the drug metabolism, and therapy response. The identification of genetic polymorphisms in the HIV-1 infected individuals could be useful to identify a possible genotype or genotype associations that could serve as a marker of either the disease progression in HIV-1 infected individuals or of a higher probability of HAART failure.

Genetic markers could also be useful to better characterize the genetic epidemiology of HIV-1 infection and to detect individuals at high risk of a faster disease progression. This information could lead to the use of different or more aggressive therapeutic strategies, the monitoring in shorter interval of time, or both procedures. Most of the studies reviewed here signaled that the chemokine receptor antagonists are important new antiviral drugs to combat the HIV-1 infection. HIV-1 infected patients in the advanced stages of the disease and/or with multiresistance to the antiretroviral agents currently available could benefit from these new therapeutic strategies.

It is possible that a multifaceted approach to antiretroviral therapy, which takes into account the genetic host factors and the use of combinations of inhibitors that target different steps of the viral life cycle, has the best potential for long-term control of HIV-1 infection. Such approach could lead to the optimal therapeutic effects of reducing viral loads and some immune restoration response, and also to an increase in the span of life of infected individuals. In this post-genomic era, the study of host factors and their genetic contribution to HIV-1 infection, addresses fundamental issues in our understanding of the pathogenesis of the infection and opens new opportunities for therapeutic intervention to be developed.

#### **8. References**

An, P.; Johnson, R.; Phair, J.; Kirk, G.D.; Yu, X-F.; Donfield, S.; Buchbinder, S.; Goedert, J.J. & Winkler, C.A. (2009). *APOBEC3B* deletion and risk of HIV-1 acquisition*. The Journal of Infectious Diseases*, v.200, pp.1054-1058, ISSN 0022-1899.

The chemokine polymorphisms CXCR6-3E/K, In1.1T/C, H7 haplotype, CX3CR1-V249I, and CX3CR1-T280M have been shown to affect the course of HIV-1 infection. The influence on immunologic and virologic response to HAART in a group of 143 HIV-1 patients was studied by Passam et al. (2007). The results revealed an improved immunologic response to HAART in patients with the CX3CR1-249I or CX3CR1-280M allele. On the contrary, patients with initial viral load suppression due to HAART showed a faster virologic failure in the presence of the CXCR6-3K allele. The In1.1T/C polymorphism and H7 haplotype did not

So far, the CCR5-32 allele remains the most important host factor known to be associated to the resistance to the HIV-1 infection. However, high frequencies of CCR5-32, CCR2-64I, and CXCL12-3'A alleles could be all the aftermath or adaptive episodes in which a pathogen exerted selective pressure favoring the survival of persons with these beneficial alleles. Taken together, the reviewed studies suggest that the existence of genetic polymorphisms must be taken into account in the virological and immunological follow-up of HIV-1 infected patients under treatment with HAART, and that pharmacogenetics is very likely to influence the future individualization of HAART. The individual genetic conditions could be of interest not only in terms of disease progression, but also on the drug metabolism, and therapy response. The identification of genetic polymorphisms in the HIV-1 infected individuals could be useful to identify a possible genotype or genotype associations that could serve as a marker of either the disease progression in HIV-1 infected individuals or of

Genetic markers could also be useful to better characterize the genetic epidemiology of HIV-1 infection and to detect individuals at high risk of a faster disease progression. This information could lead to the use of different or more aggressive therapeutic strategies, the monitoring in shorter interval of time, or both procedures. Most of the studies reviewed here signaled that the chemokine receptor antagonists are important new antiviral drugs to combat the HIV-1 infection. HIV-1 infected patients in the advanced stages of the disease and/or with multiresistance to the antiretroviral agents currently available could benefit

It is possible that a multifaceted approach to antiretroviral therapy, which takes into account the genetic host factors and the use of combinations of inhibitors that target different steps of the viral life cycle, has the best potential for long-term control of HIV-1 infection. Such approach could lead to the optimal therapeutic effects of reducing viral loads and some immune restoration response, and also to an increase in the span of life of infected individuals. In this post-genomic era, the study of host factors and their genetic contribution to HIV-1 infection, addresses fundamental issues in our understanding of the pathogenesis of the infection and opens new opportunities for therapeutic intervention to

An, P.; Johnson, R.; Phair, J.; Kirk, G.D.; Yu, X-F.; Donfield, S.; Buchbinder, S.; Goedert, J.J. &

*of Infectious Diseases*, v.200, pp.1054-1058, ISSN 0022-1899.

Winkler, C.A. (2009). *APOBEC3B* deletion and risk of HIV-1 acquisition*. The Journal* 

reveal any specific effect on HAART response.

a higher probability of HAART failure.

from these new therapeutic strategies.

be developed.

**8. References** 

**7. Conclusion** 


The Role of Genetic Polymorphisms in the Chemokine and

pp. 1527-1530, ISSN 0022-1899.

18, n. 2, pp. 110-116, ISSN 1077-9450.

n.3, pp. 335-337, ISSN 1525-4135.

9370.

8075.

0003-4819.

Their Receptors and Cytokines in the Human Immunodeficiency Virus Type 1 (HIV-1) Infection 317

Dean, M.; Carrington, M.; Winkler, C.; Huttley, G.A.; Smith, M.W.; Allikmets, R.; Goedert,

CKR5 structural gene. *Science*, v. 273, n.5823, pp. 1856-1862, ISSN 0036-8075. DeHovitz, J.A.; Kovacs, A.; Feldman, J.G.; Anastos, K.; Young, M.; Cohen, M.; Gange, S.J.;

Diop, G.; Hirtzig, T.; Do, H.; Coulonges, C.; Vasilescu, A.; Labib, T.; Spadoni, J.L.; Therwath,

J.J.; Buchbinder, S.P.; Vittinghoff, E.; Gomperts, E.; Donfield, S.; Vlahov, D.; Kaslow, R.; Saah, A.; Rinaldo, C.; Detels, R. & O'Brien, S.J. (1996). Hemophilia growth and development study, Multicenter AIDS Cohort Study, Multicenter Hemophilia Cohort Study, San Francisco City Cohort, Alive Study, O'Brien, S.J†. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the

Melnick, S. & Greenblatt, R.M*.* (2000). The relationship between virus load response to highly active antiretroviral therapy and change in CD4 cell counts: A report from the Women's interagency HIV study. *The Journal of Infectious Diseases,* v. 182, n. 5,

A.; Lathrop, M.; Matsuda, F. & Zagury, J.F. (2006). Exhaustive genotyping of the interferon alpha receptor 1 (IFNAR1) gene and association of an IFNAR1 protein variant with AIDS progression or susceptibility to HV-1 infection in a French AIDS cohort. *Biomedicine and Pharmacotherapy*, v.60, n.9, pp. 569-577, ISSN 0753-3322. Dronda, F.; Moreno, S.; Moreno, A.; Casado, J.L.; Perez-Elias, M.J. & Antela, A*.* (2002). Long-

term outcomes among antiretroviral-naive human immunodeficiency virusinfected patients with small increases in CD4+ cell counts after successful virologic suppression. *Clinical Infectious Diseases*, v. 35, n. 8, pp. 1005-1009, ISSN 1058-4838. Eugen-Olsen, J.; Iversen, A.K.; Benfield, T.L.; Koppelhus, U. & Garred, P. (1998). Chemokine

receptor CCR2b 64I polymorphism and its relation to CD4 T-cell counts and disease progression in a Danish cohort of HIV-infected individuals. Copenhagen AIDS cohort. *Journal of Acquired Immune Deficiency Syndrome and Human Retrovirology,* v.

Sorensen, A.M.; Katzenstein, T.; Dickmeiss, E.; Gerstoft, J.; Skinhoj, P.; Svejgaard, A.; Nielsen, J.O. & Hofmann, B. (1997). Heterozygosity for a deletion in the CKR-5 gene leads to prolonged AIDS-free survival and slower CD4 T-cell decline in a cohort of HIV-seropositive individuals. *AIDS* v. 11, n.3, pp. 305–310, ISSN 0269-

mechanisms of HIV infection. *Annals of Internal Medicine*, v. 124, pp. 654-663, ISSN

J.F.; McDermott, D.H.; Murphy, P.M.; Debré, P.; Théodorou, I. & Combadière, C. (2000). Rapid progression to AIDS in HIV+ individuals with a structural variant of the chemokine receptor CX3CR1. *Science,* v. 287, n. 5461, pp. 2274-2277, ISSN 0036-

SEROCO Study Group. (2003). Deleterious genetic influence of CX3CR1 genotypes on HIV-1 disease progression. *Journal of Acquired Immune Deficiency Syndrome*, v. 32,

McBride, M.; Cao, X.H.; Merrill, G.; O'Connell, P.; Bowden, D.W.; Freedman, B.I.;

Eugen-Olsen, J.; Iversen, A.K.; Garred, P.; Koppelhus, U.; Pedersen, C.; Benfield, T.L.;

Fauci, A.S.; Pantaleo, G.; Stanley, S. & Weissman, D. (1996). Immunopathogenic

Faure, S.; Meyer, L.; Costagliola, D.; Vaneensberghe, C.; Genin, E.; Autran, B.; Delfraissy,

Faure, S.; Meyer, L.; Genin, E.J.; Pellet, P.; Debre, P.; Theodorou, I. & Combadiere, C.

Gonzalez, E.; Bamshad, M.; Sato, N.; Mummidi, S.; Dhanda, R.; Catano, G.; Cabrera, S.;

influence prolonged antiviral efficacy of highly active antiretroviral treatment. *AIDS*, v. *12*, n.16, pp. 2193-2202, ISSN 0269-9370.


Brumme, Z.L.; Chan, K.J.; Dong, W.; Hogg, R.; O'Shaughnessy, M.V.; Montaner, J.S. &

Brumme, Z.L.; Dong, W.W.Y.; Chan, K.J.; Hogg, R.S.; Montaner, J.S.C.; O'Shaughnessy, M.V.

Carrington, M.; Kissner, T.; Gerrard, B.; Ivaniv, S.; O'Brien, S.J.; Dean, M.A. (1997). Novel

Carrington, M.; Nelson, G.W.; Martin, M.P.; Kissner, T.; Vlahov, D.; Goedert, J.J.; Kaslow, R.;

Chatterjee, A.; Rathone, A. & Dhole, T.N. (2009). Association of IL-4 589 C/T promoter and

Cohen, O.J.; Kinter, A. & Fauci, A.S. (1997). Host factors in the pathogenesis of HIV disease.

Cohen, O.J.; Paolucci, S.; Bende, S. M.; Daucher, M.; Moriuchi, H.; Moriuchi, M.; Cicala, C.;

Cohen, O.J.; Vaccarezza, M.; Lam, G.K.; Baird, B.; Wildt, K.; Murphy, P.M.; Zimmerman,

de Roda Husman, A.M.; Koot, M.; Cornelissen, M.; Keet, I.P.M.; Brouwer, M.; Broersen,

*Immunological Reviews,* v. 159, pp. 31-48, ISSN 0105-2896.

*Virology*, v.72, n. 7, pp. 6215-6217, ISSN 0022-538X.

100, n. 6, pp. 1581-1589, ISSN 0021-9738.

882-890, ISSN 0003-4819.

*AIDS*, v. *12*, n.16, pp. 2193-2202, ISSN 0269-9370.

15, n.17, pp. 2259-2266, ISSN 0269-9370.

pp. 1261-1267, ISSN 1434-5161.

196–201, ISSN 1386-6532.

0269-9370.

8075.

influence prolonged antiviral efficacy of highly active antiretroviral treatment.

Harrigan, P.R. (2001). CCR5Delta32 and promoter polymorphisms are not correlated with initial virological or immunological treatment response. *AIDS*, v.

& Harrigan, P.R. (2003). Influence of polymorphisms within the CX3CR1 and MDR-1 genes on initial antiretroviral therapy response. *AIDS*, v. 17, n.2, pp. 201-208, ISSN

alleles of the chemokine-receptor gene CCR5. *Journal of Human Genetics,* v. 61, n. 6,

Buchbinder, S.; Hoots, K. & O'Brien, S.J. (1999). HLA and HIV-1: heterozygosity advantage and B\*35-Cw\*04 disadvantage. *Science*, v. 283, p. 1748-1752, ISSN 0036-

Il-4RalphaI50V receptor polymorphism with susceptibility to HIV-1 infection in North Indians. *Journal of Medical Virology*, v.81, n.6, pp.959-956, ISSN 0146-6615. Chatterjee, K. (2010). Host genetic factors in susceptibility to HIV-1 infection and progression to AIDS. *Journal of Genetics*, v.89, n.1, pp.109-116, ISSN 0022-1333. Chaudhary, O.; Rajsekar, K.; Ahmed, I.; Verma, R.; Bala, M.; Bhasinc, R. & Luthra, K. (2008).

Polymorphic variants in DC-SIGN, DC-SIGNR and SDF-1 in high risk seronegative and HIV-1 patients in Northern Asian Indians. *Journal of Clinical Virology*, n. 43, pp.

Davey, R.T. Jr.; Baird. B. & Fauci, A.S. (1998). CXCR4 and CCR5 genetic polymorphisms in long-term nonprogressive human immunodeficiency virus infection: lack of association with mutations other than CCR5-Delta32. *Journal of* 

P.A.; Nutman, T.B.; Fox, C.H.; Hoover, S.; Adelsberger, K.J.; Baseler, M.; Arthos, J.; Davey, R.T.; Dewar, R.L.; Metcalf, J.; Schwartzentruber, D.J.; Orenstein, J.M.; Buchbinder, S.; Saah, A.J.; Detels, R.; Phair, J.; Rinaldo, C.; Margolick, J.B.; Pantaleo, G. & Fauci, A.S. (1997). Heterozygosity for a defective gene for CC chemokine receptor 5 is not the sole determinant for the immunologic and virologic phenotype of HIV-infected long-term nonprogressors. *The Journal of Clinical Investigation*, v.

S.M.; Bakker, M.; Roos, M.T.; Prins, M.; de Wolf, F.; Coutinho, R.A.; Miedema, F.; Goudsmit, J. & Schuitemaker, H. (1997). Association between CCR5 genotype and the clinical course of HIV-1 infection. *Annals of Internal Medicine*, v. 127, n. 10, pp.


The Role of Genetic Polymorphisms in the Chemokine and

n.3, pp. 153-156, ISSN 1744-3121.

v. 188, pp. 864–872, ISSN 0022-1899.

n.9156, pp. 863-868, ISSN 0099-5355.

286, ISSN 0378-1119.

2815.

Their Receptors and Cytokines in the Human Immunodeficiency Virus Type 1 (HIV-1) Infection 319

Kaur, G. & Mehra, N. (2009). Genetic determinants of HIV-1 infection and progression to

Kaur, G.; Singh, P.; Kumar, N.; Rapthap, C.C.; Sharma, G.; Vajpayee, M.; Wig, N.; Sharma,

Kaur, G.; Singh, P.; Rapthap, C.C.; Kumar, N.; Vajpayee, M.; Sharma, S.K.; Wanchu, A. &

Kolmann, T.R.; Pettoello-Mantovani, M.; Katopodis, N.F.; Hachamovitch, M.; Rubistein, A.;

Koning, F.A.; Kwa, D.; Boeser-Nunnink, B.; Dekker, J.; Vingerhoed, J.; Hiemstra, H. &

Kostrikis, L.G. ; Neumann, A.U. ; Thomson, B. ; Korber, B.T. ; McHardy, P. ; Karanicolas, R. ;

Kostrikis, L.G.; Huang, Y.; Moore, J.P.; Wolinsky, S.M.; Zhang, L.; Guo, Y.; Deutsch, L.;

Kwa, D.; Boeser-Nunnink, B. & Schuitemaker, H. (2003). Lack of evidence for an association

virus phenotype evolution. *AIDS*, v. 17, n. 5, pp. 759-761, ISSN 0269-9370. Ledergerber, B.; Egger, M.; Opravil, M.; Telenti, A.; Hirschel, B.; Battegay, M.; Vernazza, P.;

Limou, S.; Coulonges, C.; Herbeck, J.T.; van Manen, D.; An, P.; Le Clerc, S.; Delaneau, O.;

*Journal of Virology*, v. 73, n. 12, pp. 10264-10271, ISSN 0022-538X.

mutation. *Nature Medicine,* v. 4, n. 3, pp. 350-353, ISSN 1078-8956.

*Sciences USA,* v.93, n.7, pp. 3126-3131, ISSN 0027-8424.

AIDS: susceptibility to HIV infection. *Tissue Antigens*, v.73, pp.289-301, ISSN 0001-

S.K. & Mehra, N.K. (2007). Distribution of CCR2 polymorphism in HIV-1 infected and healthy subjects in North India. *International Journal of Immunogenetics,* v. 34,

Mehra, N.K. (2007). Polymorphism in the CCR5 gene promoter and HIV-1 infection in North Indians. *Human Immunology*, v. 68, n.5, pp. 454-461, ISSN 0198-8859. Kim, S.H.; Cohen, B.; Novick, D. & Rubinstein, M. (1997). Mamalian type I interferon

receptors consits of two subunits: IFNaR1 and IFNaR2. *Gene*, v. 196, n.1-2, pp.279-

Kim, A. & Goldstein, H. (1996). Inhibition of acute in vivo human immunodeficiency virus infection by human interleukin 10 treatment of SCID mice implanted with human fetal thymus and liver. *Proceedings of the National Academy of* 

Schuitemaker, H. (2003). Decreasing sensitivity to RANTES (regulated on activation, normally T cell-expressed and -secreted) neutralization of CC chemokine receptor 5-using, non-syncytium-inducing virus variants in the course of human immunodeficiency virus type 1 infection. *The Journal of Infectious Diseases,* 

Deustch, L. ; Lew, J.F. ; McIntoch, K. ; Pollack, H. ; Borkowsky, W. ; Spiegel, H.M. ; Palumbo, P. ; Oleske, J. ; Bardeguez, A. ; Luzuriaga, K. ; Sullivan, J. ; Wolinsky, S.M. ; Koup, R.A.; Ho, D.D. & Moore, J.P. (1999). A polymorphism in the regulatory region of the CC-chemokine receptor 5 gene influences perinatal transmission of human immunodeficiency virus type 1 to African-American infants

Phair, J.; Neumann, A.U.; Ho, D.D. (1998). A chemokine receptor CCR2 allele delays HIV-1 disease progression and is associated with a CCR5 promoter

between a polymorphism in CX3CR1 and the clinical course of HIV infection or

Sudre, P.; Flepp, M.; Furrer, H.; Francioli, P. & Weber, R. (1999). Clinical progression and virological failure on highly active antiretroviral therapy in HIV-1 patients: a prospective cohort study. Swiss HIV Cohort Study. *The Lancet,* v. 353,

Diop, G.; Taing, L.; Montes, M.; van't Wout, A.B., Gottlieb, G.S.; Therwath, A.; Rouzioux, C.; Delfraissy, J.F.; Lelièvre, J.D.; LévyM Y,L HercbergM S,L DinaM C,L.;

Anderson, S.A.; Walter, E.A.; Evans, J.S.; Stephan, K.T.; Clark, R.A.; Tyagi, S.; Ahuja, S.S.; Dolan, M.J. & Ahuja, S.K. Race-specific HIV-1 disease-modifying effects associated with CCR5 haplotypes. *Proceedings of the National Academy of Sciences USA*, v.96, pp12004-12009, ISSN 0027-8424.


Gonzalez, E.; Kulkarni, H.; Bolivar, H.; Mangano, A.; Sanchez, R.; Catano, G.; Invs., R.J.;

Greub, G.; Ledergerber, B.; Battegay, M.; Grob, P.; Perrin, L.; Furrer, H.; Burgisser, P.; Erb,

infected patients. *AIDS,* v. 14, n. 17, pp. 2788-2790, ISSN 0269-9370.

Han, X.; Becker, K.; Degen, H.J. Jablonowski, H. & Strohmeyer, G. (1996). Synergistic

Ioannidis, J.P.; O'Brien, T.R.; Rosenberg, P.S.; Contopoulos-Ioannidis, D.G. & Goedert, J.J*.* 

Ioannidis, J.P.A.; Rosenberg, P.S.; Goedert, J.J.; Ashton, L.J.; Benfield, T.L.; Buchbinder, S.P.;

Jang, D. H.; Choi, B.S. & Kim, S.S. (2008). The effects of RANTES/CCR5 promoter

Kalinkovich, A.; Weisman, Z. & Bentwich, Z. (1999). Chemokines and chemokine receptors:

Kasten, S.; Goldwich, A.; Schmitt, M.; Rascu, A.; Grunke, M.; Dechant, C.; Kalden, J.R. &

*Journal of Immunogenetics*, v. 35, pp. 101–105, ISSN 1744-3121.

role in HIV infection. *Immunological Letters*, v. 68, pp. 281–287.

*of Medical Research,* v. *5,* n. 8, pp. 323-328, ISSN: 0949-2321.

polymorphisms on HIV disease progression in HIV-infected Koreans. *International* 

Harrer. T. (2000). Positive influence of the Delta32CCR5 allele on response to highly active antiretroviral therapy (HAART) in HIV-1 infected patients. *European Journal* 

*USA*, v.96, pp12004-12009, ISSN 0027-8424.

1434-1440, ISSN 0036-8075.

ISSN 0014-2972.

536, ISSN 1078-8956.

135, pp. 782–795, ISSN 0003-4819.

Anderson, S.A.; Walter, E.A.; Evans, J.S.; Stephan, K.T.; Clark, R.A.; Tyagi, S.; Ahuja, S.S.; Dolan, M.J. & Ahuja, S.K. Race-specific HIV-1 disease-modifying effects associated with CCR5 haplotypes. *Proceedings of the National Academy of Sciences* 

Freedman, B.I.; Quinones, M.P.; Bamshad, M.J.; Murthy, K.K.; Rovin, B.H.; Bradley, W.; Clark, R.A.; Anderson, S.A.; O'Connell, R.J.; Agan B.K.; Ahuja, S.S.; Bologna, R.; Sen, L.; Dolan, M.J & Ahuja, S.K. (2005). The influence of CCL3L1 gene-containing segmental duplications on HIV-1/AIDS susceptibility. *Science*, v. 307, n. 5714, pp.

P.; Boggian, K.; Piffaretti, J.C.; Hirschel, B.; Janin, P.; Francioli, P.; Flepp, M. & Telenti, A*.* (2000). Clinical progression, survival, and immune recovery during antiretroviral therapy in patients with HIV-1 and hepatitis C virus coinfection: the Swiss HIV Cohort Study. *The Lancet*, v. *356*, n.9244, pp. 1800-1805, ISSN 0099-5355. Guerin, S.; Meyer, L.; Theodorou, I.; Boufassa, F.; Magierowska, M.; Goujard, C.; Rouzious,

C.; Debre, P.; Delfraissy, J.F.; SEROCO/HEMOCO Study Group. (2000). CCR5 delta32 deletion and response to highly active antiretroviral therapy in HIV-1-

stimulatory effects of tumor necrosis factor alpha and interferon gamma on replication of human immunodeficiency virus type 1 and on apoptosis of HIV-1 infected host cells. *European Journal of Clinical Investigation*, v.26, n.4, pp. 286-292,

(1998). Genetic effects on HIV disease progression. *Nature Medicine*, v. *4,* n.5, pp.

Coutinho, R.A.; Eugen-Olsen, J.; Gallart, T.; Katzenstein, T.L.; Kostrikis, L.G.; Kuipers, H.; Louie, L.G.; Mallal, S.A.; Margolick, J.B.; Martinez, O.P.; Meyer, L.; Michael, N.L.; Operskalski, E.; Pantaleo, G.; Rizzardi, G.P.; Schuitemaker, H.; Sheppard, H.; Stewart, G.J.; Theodorou, I.D.; Henrik, U.; Vicenzi, E.; Vlahov, D.; Wilkinson, D.; Workman, C.; Zagury, J-F. & O'Brien, T.R. (2001). Effects of CCR5- Delta32, CCR2–64I, and SDF-1 3'A alleles on HIV-1 disease progression: an international meta-analysis of individual-patient data. *Annals of Internal Medicine*, v.


The Role of Genetic Polymorphisms in the Chemokine and

n. 5, p. 624-626, ISSN 0269-9370.

ISSN 1078-8956.

ISSN 1434-5161.

0269-9370.

ISSN 1078-8956.

ISSN 0016-5751.

v.59, n.10, pp. 793-798, ISSN 0093-7711.

177, pp. 99-111, ISSN 0105-2896.

1424-8581.

0269-9370.

Their Receptors and Cytokines in the Human Immunodeficiency Virus Type 1 (HIV-1) Infection 321

Michael, N.L.; Louie, L.G.; Rohrbaugh, A.L.; Schultz, K.A.; Dayhoff, D.E.; Wang, C.E. &

Modi, W.S.; Lautenberger, J.; An, P.; Scott, K.; Goedert, J.J.; Kirk, G.D.; Buchbinder, S.; Phair,

Mulherin, S.A.; O'Brien, T.R.; Ioannidis, J.P.A.; Goedert, J.J.; , Buchbinder, S.P.; Coutinho,

Mummidi, S.; Ahuja, S.S.; Gonzalez, E.; Anderson, S.A.; Santiago, E.N.; Stephan, K.T.; Craig,

Naif, H.M.; Li, S.; Ho-Shon, M.; Mathijs, J.M.; Williamson, P. & Cunningham, A.L. (1997).

Nakajima, T.; Kaur, G.; Mehra, N. & Kimura, A. (2008). HIV-1/AIDS susceptibility and copy

Nakajima, T.; Ohtani, H.; Naruse, T.; Shibata, H.; Mimaya, J.I.; Terunuma, H. & Kimura, A.

Nguyen, L.; Li, M.; Chaowanachan, T.; Hu, D.J.; Vanichseni, S.; Mock, P.A.; van Griensven,

Thai injection drug users. *AIDS*, v.18, n.9, pp.1327-1333, ISSN 0269-9370. O' Brien, S.J.& Moore, J.P. (2000). The effect of genetic variation in chemokines and their

O' Brien, T.R.; McDermott, D.H.; Ioannidis, J.P.A.; Carrington, M.; Murphy, P.M.; Havlir,

polymorphism, and disease progression in 720 HIV-infected patients. *AIDS*, v. 13,

Sheppard, H.W. (1997). The role of CCR5 and CCR2 polymorphisms in HIV-1 transmission and disease progression. *Nature Medicine,* v.3, n.10, pp.1160-1162,

J.; Donfield, S.; O'Brien, S.J. & Winkler, C. (2006). Genetic variation in the CCL18- CCL3-CCL4 chemokine gene cluster influences HIV type transmission and AIDS disease progression. *American Journal of Human Genetics*, v. 79, n.1, pp; 120-128,

R.A.; Jamieson, B.D.; Meyers, L.; Michael, N.L.; Pantaleo, G.; Rizzardi, G.P.; Schuitemaker, H.; Shepaard, H.W.; Theodorou, I.D.; Vlahov, D. & Rosenberg, P.S. (2003). Effects of CCR5-Delta32 and CCR2-64I alleles on HIV-1 disease progression: the protection varies with duration of infection. *AIDS,* v. 17, n. 3, pp. 377-387, ISSN

F.E.; O'Connell, P.; Tryon, V.; Clark, R.A.; Dolan, M.J. & Ahuja, S.K. (1998). Genealogy of the CCR5 locus and chemokine system gene variants associated with altered rate if HIV-1 disease progression. *Nature Medicine*, v. 4, n.*7*, pp. 786-793,

The state of maturation of monocytes into macrophages determines the effects of IL-4 and IL-13 on HIV replication. *Journal of Immunology*, v. 158, n. 1, pp. 501-511,

number variations in CCL3L1, a gene enconding a natural ligand for HIV-1 coreceptor CCR5. *Cytogenetic and Genome Research,* v.123, n.1-4, pp. 156-160, ISSN

(2007). Copy number variations of CCL3L1 and long-term prognosis of HIV-1 infection in asymptomatic HIV-infected Japanese with hemophilia. *Immunogenetics*,

F.; Martin, M.; Sangkum, U.; Choopanya, K.; Tappero, J.W.; Lal, R.B. & Yang, C. (2004). CCR5 promoter haplotypes associated with HIV-1 discordant progression in

receptors on HIV transmission and progression to AIDS. *Immunological Reviews*, v.

D.V. & Richman, D.D. (2000). Effect of chemokine receptor gene polymorphisms on the response to potent antiretroviral therapy. *AIDS*, v. 14, n. 7, p. 821-826, ISSN

Phair, J.; Donfield, S.; Goedert, J.J.; Buchbinder, S.; EstaquierM J,L; Schächter, F.; Gut, I.; Froguel, P.; Mullins, J.I.; Schuitemaker, H.; Winkler, C. & Zagury, J.F. (2010). Multiple-cohort genetic association study reveals CXCR6 as a new chemokine receptor involved in long-term nonprogression to AIDS. *The Journal of Infectious Diseases*, v. 202, n. 6, pp. 908-915, ISSN 0022-1899.


Liu, H.; Chao, D.; Nakayama, E.E.; Taguchi, H.; Goto, M.; Xin, X.; Takamatsu, J.K.; Saito, H.;

Liu, S-L.; Schacker, T.; Musey, L.; Shriner, D.; McElrath, M.J.; Corey, L. & Mullins, J.I. (1997).

Mabuka, J.M.; Mackelprang, R.D.; Lohman-Payne, B.; Majiwa, M.; Bosire, R.; John-Stewart,

Magierowska, M.; Theodorou, I.; Debré, P.; Sanson, F.; Autran, B.; Rivière, Y.; Charron, D.;

Martin, M.P.; Dean, M.; Smith, M.W.; Winkler, C.; Gerrard, B.; Michael, N.L.; Lee, B.; Doms,

McDermott, D.H.; Beecroft, M.J.; Kleeberger, C.A.; Al-Sharif, F.M.; Ollier, W.E.; Zimmerman,

McDermott, D.H.; Colla, J.S.; Kleeberger, C.A.; Plankey, M.; Rosenberg, P.S.; Smith, E.D.;

Meyer, L.; Magierowska, M.; Hubert, J-B.; Theodorou, I.; van Rij, R.; Prins, M.; de Roda, H.;

*Diseases*, v. 202, n. 6, pp. 908-915, ISSN 0022-1899.

*Virology,* v. 71, n. 6, pp. 4284-4295, ISSN 0022-538X.

*Blood*, v. 93, n. 3, p. 936-941, ISSN 0006-4971.

2, pp. 235-237, ISSN 1525-4135.

pp. 2671–2678, ISSN 0269-9370.

pp.866-870, ISSN 0099-5355.

ISSN 0036-8075.

8424.

Phair, J.; Donfield, S.; Goedert, J.J.; Buchbinder, S.; EstaquierM J,L; Schächter, F.; Gut, I.; Froguel, P.; Mullins, J.I.; Schuitemaker, H.; Winkler, C. & Zagury, J.F. (2010). Multiple-cohort genetic association study reveals CXCR6 as a new chemokine receptor involved in long-term nonprogression to AIDS. *The Journal of Infectious* 

Ishikawa, Y.; Akaza, T.; Juji, T.; Takebe, Y.; Ohishi, T.; Fukutake, K.; Maruyama, Y.; Yashiki, S.; Sonoda, S.; Nakamura, T.; Nagai, Y.; Iwamoto, A. & Shioda, T. (1999). Polymorphism in RANTES chemokine promoter affects HIV-1 disease progression. *Proceedings National Academy of Sciences USA*, v. 96, n. 8, pp. 4581–4585, ISSN 0027-

Divergent patterns of progression to AIDS after infection from the same source: human immunodeficiency virus type 1 evolution and antiviral responses. *Journal of* 

G.; Rowland-Jones, S.; Overbaugh, J. & Farquhar, C. (2009). CCR2-64I polymorphism is associated with lower maternal HIV-1 viral load and reduced vertical HIV-1 transmission. *Journal of Acquired Immune Deficiency Syndrome*, v. 51, n.

French ALT and IMMUNOCO Study Groups & Costagliola, D. (1999). Combined genotypes of CCR5, CCR2, SDF1, and HLA genes can predict the long-term nonprogressor status in human immunodeficiency virus-1-infected individuals.

R.W.; Margolick, J.; Buchbinder, S.; Goedert, J.J.; O'Brien, T.R.; Hilgartner, M.W.; Vlahov, D.; O'Brien, S.J. & Carrington, M. (1998). Genetic acceleration of AIDS progression by a promoter variant of CCR5. *Science,* v. 282, n. 5395, pp. 1907-1911,

P.A.; Boatin, B.A.; Leitman, S.F.; Detels, R.; Hajeer, A.H. & Murphy, P.M. (2000). Chemokine RANTES promoter polymorphism affects risk of both HIV infection and disease progression in the Multicenter AIDS Cohort Study. *AIDS,* v. 14, n. 17,

Zimmerman, P.A.; Combadiere, C.; Leitman, S.F.; Kaslow, R.A.; Goedert, J.J.; Berger, E.A.; O'Brien, T.R. & Murphy, P.M. (2000). Genetic polymorphism in CX3CR1 and risk of HIV disease. *Science,* v. *290*, n.5499, pp. 2031, ISSN 0036-8075. McDermott, D.H.; Zimmernan, P.A.; Guignard, F.; Kleeberger, C.A.; Leitman, S.F. &

Murphy, P.M. (1998). CCR5 promoter polymorphism and HIV-1 disease progression. Multicenter AIDS Cohort Study (MACS). *The Lancet,* v. 352, n.9131,

Coutinho, R. & Schuitemaker, H. (1999). CC-chemokine receptor variants, SDF-1

polymorphism, and disease progression in 720 HIV-infected patients. *AIDS*, v. 13, n. 5, p. 624-626, ISSN 0269-9370.


The Role of Genetic Polymorphisms in the Chemokine and

793, ISSN 1107-3756.

932, ISSN 0022-1899.

ISSN 1570-162X.

v. 5, pp. 2382-2390.

v.169, n.3, pp. 1604-1610, ISSN 0016-5751.

*Retroviruses*, v. 25, n.10, pp 973-977.

gene. *The Lancet*, v. 351, pp. 14-18, ISSN 0099-5355.

Their Receptors and Cytokines in the Human Immunodeficiency Virus Type 1 (HIV-1) Infection 323

Quillent, C.; Oberlin, E.; Braun, J.; Rousset, D.; Gonzales-Canali, G.; Métais, P.; Montagnier,

Ramana, G.V.; Vasanthi, A.; Khaja, M.; Su, B.; Govindaiah, V.; Jin, L.; Singh, L. &

Rathore, A.; Chatterjee, A.; Sivarama, P.; Yamamoto, N.; Singhal, P.K. & Dhole, T.N. (2009).

Reiche, E. M.; Ehara Watanabe, M.A.; Bonametti, A. M.; Kaminami Morimoto, H.; Akira

Reiche, E.M.V.; Bonametti, A.M.; Voltarelli, J.C.; Morimoto, H.K. & Watanabe, M.A.E. (2007).

Rigato, P.O.; Hong, M.A.; Casseb, J.; Ueda, M.; de Castro, I.; Benard, G. & Duarte, A.J. (2008).

Risma, K.A.; Wang, N.; Andrews, R.P.; Cunningham, C.M.; Ericksen, M.B.; Bernstein, J.A.;

Rosenberg, Z.F. & Fauci, A.S. (1991). Immunopathogenesis of HIV infection. *FASEB Journal*,

Salem, A.H.; Farid, E.; Fadel, R.; Abu-Hijleh, M.; Almawi, W.; Han, K.; Batzer, M.A. (2009).

Samsom, M.; Libert, F.; Doranz, B.J.; Rucker, J.; Liesnard, C.; Farber, C.M.; Saragosti, S.;

Pradesh, South India. *Journal of Genetics*, v. 80, n.3, pp. 137–140.

*and Human Retroviruses*, v.25, n.11, pp.1149-1156, ISSN 0889-2229.

L.; Virelizier, J-L.; Arenzana-Seisdedos, F. & Beretta, A. (1998). HIV-1 resistance phenotype conferred by combination of two separate inherited mutations of CCR5

Chakraborty, R. (2001). Distribution of HIV-1 resistance-conferring polymorphic alleles SDF-1-3'A, CCR2-64I and CCR5-Delta32 in diverse populations of Andhra

Association of CCR5-59020 A/G and CCL3L1 copy number polymorphism with HIV type 1 transmission/progression among HIV type 1-seropositive and repeatedly sexually exposed HIV type 1-seronegative North Indians. *AIDS Research* 

Morimoto, A.; Wiechmann, S.L.; Breganó, J.W.; Matsuo, T.; Vissoci Reiche, F.; Miranda, H.C.; Brajão Oliveira, K.; Vogler, I.H.; Siscar, A.R. (2006). The effect of stromal cell-derived factor 1 (SDF1/CXCL12) genetic polymorphism on HIV-1 disease progression. *International Journal of Molecular Medicine,* v. 18, n.4, pp. 785-

Genetic polymorphisms in the chemokine and chemokine receptors: impact on clinical course and therapy of the human immunodeficiency virus type 1 infection (HIV-1). *Current Medicinal Chemistry*, v.14, n. 12, pp. 1325-1334, ISSN 0929-8673. Reynes, J.; Portales, P.; Segondy, M.; Baillat, V.; André, P.; Réant, B.; Avinens, O.; Couderc,

G.; Benkirane, M.; Clot, J.; Eliaou, J.F. & Corbeau, P. (2000). CD4+ T cell surface CCR5 density as a determining factor of virus load in persons infected with human immunodeficiency virus type 1. *The Journal of Infectious Diseases,* v. 181, n.3, pp. 927-

Better CD4+ T cell recovery in Brazilian HIV-infected individuals under HAART due to cumulative carriage of SDF-1-3'A, CCR2-V64I, CCR5-32 and CCR5 promoter 59029A/G polymorphisms. *Current HIV Research*, v. 6, n. 5, pp. 466-473,

Chakraborty, R & Hershey, G.K. (2002). V75R567 Il-4 receptor alpha is associated with allergic asthma and enhanced IL-4 receptor function. *Journal of Immunology*,

Distribution of four HIV type 1-resistance polymorphisms (CCR5-Delta32, CCR5 m303, CCR2-64I, and SDF1-3'A) in the Bahraini population. *AIDS Res Hum* 

Lapoumeroulie, C.; Cognaux, J.; Forceille, C.; Muyldermans, G.; Verhofstede, C.; Burtonboy, G.; Georges, M.; Imai, T.; Rana, S.; Yi, Y.; Smyth, R.J.; Collman, R.G.;


O'Brien, W.A.; Hartigan, P.M.; Daar, E.S.; Simberkoff, M.S. Hamilton, J.D. (1997). Changes in

AIDS. *Annals of Internal Medicine,* v. 126, n.12, pp. 939-945, ISSN 0003-4819. Pantaleo, G.; Demarest, J.F.; Schacker, T.; Vaccarezza, M.; Cohen, O.J.; Daucher, M.; Graziosi,

Parczewski, M.; Leszczyszyn-Pynka, M.; Kaczmarczyk, M.; Adler, G.; Binczak-Kuleta, A.;

Passam, A.M.; Sourvinos, G.; Krambovitis, E.; Miyakis, S.; Stavrianeas, N.; Zagoreos, I. &

Passam, A.M.; Zafiropoulos, A.; Miyakis, S.; Zagoreos, I.; Stavrianeas, N.G.; Krambovitis, E.

Paxton, W.A.; Martin, S.R.; Tse, D.; O'Brien, T.R.; Skurnick, J.; VanDevanter, N.; Padian, N.;

Petit, S.J.; Chayen, N.E. & Pease, J.E.(2008). Site-directed mutagenesis of the chemokine

Piacentini, L., Biasin, M., Fenizia, C. & Clerici, M. (2009). Genetic correlates of protection

Potter, S. J., Lacabaratz, C., Lambotte, O., Perez-Patrigeon, S., Vingert, B., Sinet, M., Colle, J.

Qian, Y.; Sun, H.; Lin, K.; Shi, L.; Shi, L. & Chu, J. (2008). Distribution of CCR5-Delta32,

*AIDS Research Human Retroviruses*, v. 24, n. 11, pp. 1391-1397.

study. *Journal of Virology,* v. 81, n.24, pp. 13904–13915, ISSN 0022-538X Puissant, B.; Roubinet, F.; Massip, P.; Sandres-Saune, K.; Apoil, P-A.; Abbal, M.; Pasquier, C.;

CXCL16. *European Journal of Immunology*, v. 38, n. 8, pp. 2337-2350.

*Academy of Sciences USA,* v. 94, n.1, pp. 254-258, ISSN 0027-8424.

*of Applied Genetics*, v. 50, n. 2, pp. 159-166, ISSN 1234-1983.

*Retroviruses*, v. 23, n. 8, pp.1026-1032, ISSN 0889-2229.

v. 34, pp. 302-309, ISSN 1386-6532.

1078-8956.

124, ISSN 0954-6820.

n. 2, pp. 153-162, ISSN 0889-2229.

plasma HIV RNA levels and CD4+ lymphocyte counts predict both response to antiretroviral therapy and therapeutic failure. VA Cooperative Study Group on

C.; Schnittman, S.S.; Quinn, T.C.; Shaw, G.M.; Perrin, L.; Tambussi, G.; Lazzarin, A.; Sekaly, R.P.; Soudeyns, H.; Corey, L. & Fauci, A.S. (1997). The qualitative nature of the primary immune response to HIV infection is a prognosticator of disease progression independent of the initial level of plasma viremia. *Proceedings National* 

Loniewska, B.; Boron-Kaczmarska, A. & Ciechanowicz, A. (2009). Sequence variants of chemokine receptor genes and susceptibility to HIV-1 infection. *Journal* 

Spandidos, D.A. (2007). Polymorphisms of CX(3)CR1 and CXCR6 receptors in relation to HAART therapy of HIV type 1 patients. *AIDS Research and Human* 

& Spandidos, D.A. (2005). CCR2-64I and CXCL12 3'A alleles confer a favorable prognosis to AIDS patients undergoing HAART therapy. *Journal of Clinical Virology,*

Braun, J.F.; Kotler, D.P.; Wolinsky, S.M. & Koup RA. (1996). Relative resistance to HIV-1 infection of CD4 lymphocytes from persons who remain uninfected despite multiple high-risk sexual exposure. *Nature Medicine,* v.2, n.4, pp. 412-417, ISSN

receptor CXCR6 suggests a novel paradigm for interactions with the ligand

against HIV infection: the ally within. *Journal of Internal Medicine,* v. 265, pp. 110–

H., Urrutia, A., Scott-Algara, D.; Boufassa, F.; Delfraissy, J.F.; Thèze, J.; Venet, A. & Chakrabarti, L.A. (2007). Preserved central memory and activated effector memory CD4+ T-cell subsets in human immunodeficiency virus controllers: an ANRS EP36

Izopet, J. & Blancher, A. (2006). Analysis of CCR5, CCR2, CX3CR1, and SDF1 polymorphisms in HIV-positive treated patients: impact on response to HAART and o peripheral T lymphocyte counts. *AIDS Research and Human Retroviruses*, v. 22,

CCR2-64I, SDF1-3'A, CX3CR1-249I, and CX3CR1-280M in Chinese populations.


The Role of Genetic Polymorphisms in the Chemokine and

0022-1899.

0889-2229.

variants and *CCR5-*

ISSN 1386-6532.

2350.

n.8, pp. 1290-1294, ISSN 0022-1899.

*Syndrome,* v. 40, n.5, pp. 527–531, ISSN 1525-4135.

Their Receptors and Cytokines in the Human Immunodeficiency Virus Type 1 (HIV-1) Infection 325

Tresoldi, E.; Romiti, M.L.; Boniotto, M.; Crovella, S.; Salvatori, F.; Palomba, E.; Pastore, A.;

Vaamonde, C.M.; Hoover, D.R.; Anastos, K.; Tan, T.; Shi, Q.; Gao, W.; Kovacs, A.; Cohen,

van Rij, R.P.; Broersen, S.; Goudsmit, J.; Coutinho, R.A. & Schuitemaker, H. (1998). The role

Vasilescu, A.; Terashima, Y.; Enbomoto, M.; Heath, S.; Poonpiriya, V.; Gatanaga, H.; Do, H.;

*National Academy of Sciences USA*, v. 104, n.9, pp. 3354-3359, ISSN 0027-8424. Veloso, S.; Olona, M.; García, F.; Domingo, P.; Alonso-Villaverde, C.; Broch, M.; Peraire, J.;

A.; Tasias, M.; Gatell, J.M.; Richart, C. & Vidal, F. (2010). Effect of *TNF-*

Verma, R.; Guptya, R.B.; Singh, K.; Bhasin, R.; Shukla, A.A.; Chauhan, S.S. & Luthra, K.

Viard, J.P.; Mocroft, A.; Chiesi, A.; Kirk, O.; Roge, B.; Panos, G.; Vetter, N.; Bruun , J.N.;

Vidal, F., Peraire, J., Domingo, P., Broch, M., Knobel, H., Pedrol, E., Dalmau, D., Vilades, C.

*Acquired Immune Deficiency Syndrome,* v. 40, pp. 276–279, ISSN 1525-4135. Vidal, F., Vilades, C., Domingo, P., Broch, M., Pedrol, E., Dalmau, D., Knobel, H., Peraire, J.,

HIV-1 infection. *AIDS*, v. 12, n. 9, pp. F-85-F90, ISSN 0269-9370.

Cancrini, C.; Martino, M.; Plebani, A.; Castelli, G.; Rossi, P.; Tovo, P.A.; Amoroso, A.; & Scarlatti, G. in Cooperation with the European Shared Cost Project Group and the Italian Register for GIV Infection in Children. (2002). Prognostic value of the stromal cell-derived factor 1 3'A mutation in pediatric human immunodeficiency virus type 1 infection. *The Journal of Infectious Diseases,* v. 185, pp. 696–700, ISSN

M.; De Hovitz J. & Glesby, M.J. (2006). Factors associated with poor immunologic response to virologic suppression by highly active antiretroviral therapy in HIVinfected women. *AIDS Research and Human Retroviruses*, v. 22, n.3, pp. 222-231, ISSN

of a stromal cell-derived factor-1 chemokine gene variant in the clinical course of

Diop, G.; Hirtzig, T.; Auewarakul, P.; Lauhakirti, D.; Sura, T.; Charneau, P.; Marullo, S.; Therwath, A.; Oka, S.; Kanegasaki, S.; Lathrop, M.; Matsushima, K.; Zagury, J.F. & Matsuda, F. (2007). A haplotype of the human CXCR1 gene protective against rapid disease progression in HIV-1+ patients. *Proceedings of the* 

Viladés, C.; Plana, M.; Pedrol, E.; López-Dupla, M.; Aguilar, C.; Gutiérrez, M.; Leon,

progression in Caucasian Spaniards. *BMC Medical Genetics*, v.11, pp.63, ISSN 1471-

(2007). Distribution of CR5Δ32, CCR2-64I and SDF1-3'A and plasma levels of SDF-1 in HIV-1 seronegative North Indians. *Journal of Clinical Virology*, v. 38, pp. 198–203,

Jonson, M. & Lundgren, J.D.(2001*)* Influence of age on CD4 cell recovery in human immunodeficiency virus-infected patients receiving highly active antiretroviral therapy: evidence from the Euro SIDA study. *The Journal of Infectious Diseases*, v. 183

& Sambeat, MA. (2005a). Lack of association of SDF-1 3'A variant allele with longterm nonprogressive HIV-1 infection is extended beyond 16 years. *Journal of* 

Gutierrez, C.; Sambeat, M.A.; Fontanet, A.; Deig, E.; Cairó, M.; Montero, M.; Richart, C.; Mallal, S. & Chemokines LTNP Study Group. (2005b). Spanish HIV-1 infected long-term nonprogressors of more than 15 years have an increased frequency of the CX3CR1 249I variant allele. *Journal of Acquired Immune Deficiency* 

*32* on the vulnerability to HIV-1 infections and disease

genetic

Doms, R.W.; Vassart, G. & Parmentier, M. (1996). Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. *Nature*, v. 382, n. 6593, pp. 722-726, ISSN 0028-0836.


Schuitemaker, H.; Koot, M.; Kootstra, N.A.; Dercksen, M.W.; de Goede, R.E.Y.; van

Smith, M.W.; Carrington, M.; Winkler, C.; Lomb, D.; Dean, M.; Huttley, G. & O'Brien, S.J.

Smith, M.W.; Dean, M.; Carrington, M.; Winkler, C.; Huttley, G.A.; Lomb, D.A.; Goedert, J.J.;

Soriano, A.; Lozano, F.; Oliva, H.; Garcia, F.; Nomdedeu, M.; De Lazzari, E.; Rodríguez, C.;

Soriano, A.; Martínez, C.; García, F.; Plana, M.; Palou, E.; Lejeune, M.; Aróstegui, J.I.; De

Su, B.; Jin, L.; Hu, F.; Xiao, J.; Luo, J.; Lu, D.; Zhang, W.; Chu, J.; Du, R.; Geng, Z.; Qiu, X.;

Su, Q.; Mai, Z.; Zang, N.; Wu, S.; Xiao, X. & Liang, H. (2010). Distribution of CCR5-delta-32,

Tan, X.H.; Zhang, J.Y.; Di, C.H.; Hu, A.R.; Yang, L.; Qu, S.; Zhao, R.L.; Yang, P.R. & Guo, S.X.

Theodorou, I.; Meyer, L.; Magierowska, M.; Katlama, C. & Rouzioux, C. (1997). HIV-1

gene. *Nature*, v. 382, n. 6593, pp. 722-726, ISSN 0028-0836.

ALIVE Study. *Science,* v*. 277*, pp. 959–965, ISSN 0036-8075.

*Immunogenetics,* v.57, n.9, pp.644-654, ISSN 0093-7711.

*Genetics*, v. 65, n. 4, pp. 1047-1053, ISSN 1434-5161.

*Genetics and Evolution*, v. 10, n. 2, pp. 268-272.

*Lancet,* v. 349, n.9060, pp. 1219-1220, ISSN 0099-5355.

*Association of Physicians in AIDS Care*, v. 9, n. 3, pp. 145-149.

1354-1360, ISSN 0022-538X.

1052-1053, ISSN 1078-8956.

1899.

Doms, R.W.; Vassart, G. & Parmentier, M. (1996). Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor

Steenwijk, R.P.; Lange, J.M.; Schattenkerk, J.K.M.; Miedema, F. & Tersmette, M. (1992). Biological phenotype of human immunodeficiency virus type 1 clones at different stages of infection: progression of disease is associated with a shift from monocytotropic to T-cell-tropic virus populations. *Journal of Virology*, n. 66, n.3, pp.

(1997). CCR2 chemokine receptor and AIDS progression. *Nature Medicine*, v.3, pp.

O'Brien, T.R.; Jacobson, L.P.; Kaslow, R.; Buchbinder, S.; Vittinghoff, E.; Vlahov, D.; Hoots, K.; Hilgartner, M.W. & O'Brien, S.J. (1997). Contrasting genetic influence of CCR2 and CCR5 variants on HIV-1 infection and disease progression. Hemophilia Growth and Development Study (HGDS), Multicenter AIDS Cohort Study (MACS), Multicenter Hemophilia Cohort Study (MHCS), San Francisco City Cohort (SFCC),

Barrasa, A.; Lorenzo, J.I.; Del Romero, J.; Plana, M.; Miró, J.M.; Gatell, J.M.; Vives, J. & Gallart, T. (2005). Polymorphisms in the interleukin-4 receptor alpha chain gene influence susceptibility to HIV-1 infection and its progression to AIDS.

Lazzari, E.; Rodriguez, C.; Barrasa, A.; Lorenzo, J.I.; Alcamí, J.; Romero, J.D.; Miró, J.M.; Gatell, J.M. & Gallart, T. (2002). Plasma stromal cell-derived factor (SDF)-1 levels, SDF1-3'A genotype, and expression of CXCR4 on T lymphocytes: their impact on resistance to in human immunodeficiency virus type 1 infection and its progression. *The Journal of Infectious Diseases*, v. 186, n. 7, pp. 922-931, ISSN 0022-

Xue, J.; Tan, J.; O'Brien, S.J. & Chakraborty, R. (1999). Distribution of two HIV-1 resistant polymorphisms (SDF1-3'A and CCR2-64I) in East Asia and world populations and its implication in AIDS epidemiology. *American Journal of Human* 

CCR2-64I, and SDF1-3'A in Guangxi Zhuang population. *Journal of the International* 

(2009). Distribution of CCR5-Delta32, CCR5m303A, CCR2-64I and SDF1-3'A in HIV-1 infected and uninfected high-risk Uighurs in Xinjiang, China. *Infection,* 

infection in an individual homozygous for CCR5 delta 32. Seroco Study Group. *The* 


**13** 

*Sweden* 

**CXCL8 Regulation and Function in HIV** 

Per-Erik Olsson, Hazem Khalaf and Jana Jass

**Infections and Potential Treatment Strategies** 

*Örebro Life Science Center, School of Science and Technology, Örebro University* 

Interleukin-8 (CXCL8) is a chemokine that was originally identified as a key factor in neutrophil recruitment and activation. Numerous cell types produce CXCL8, including immune cells, mucosal epithelial cells, endothelial cells and smooth muscle cells (Garcia-Vicuna et al., 2004). CXCL8 is one of the important inflammatory mediators responsible for the recruitment of neutrophils and T-cells to the site of infection, therefore it is an attractive target for therapy against diseases that affect immune cells such as HIV. HIV directly targets the host's immune system and thus reduces the ability of the innate and adaptive immune system to fight disease. As a chemokine, CXCL8 is a potential target for controling HIV infection by reducing the migration of T-cells to the site of infection. It is therefore necessary to identify the signaling pathways involved in CXCL8 regulation in order to develop viable CXCL8 based treatment strategies. In line with this, we have recent shown that CXCL8 activation in Jurkat T-cells is not primarily under the control of NFB, but that the AP-1 signaling pathway appears to be central for the regulation of CXCL8 (Khalaf et al., 2010). An understanding of the regulation and function of cytokines and chemokines, while complex, remains important for the development of new strategies in the development of HIV treatments. It is interesting to note that several lactobacilli strains are able to modulate CXCL8 expression and release (Anukam et al., 2009; Zhang et al., 2005). Disturbance of the lactobacilli flora in the vaginal tract has been shown to increase the risk of infections and acquisition of HIV type 1 (Taha et al., 1998). Several studies have shown that treatment with certain *Lactobacillus* species and strains have positive effects on women with HIV infections and have been successful in trials to counteract vaginal infections (Hummelen et al., 2010;

In the present chapter we give a background to CXCL8 regulation and function as well as its involvement in HIV etiology. We also provide an overview of the available information on the possible uses of *Lactobacillus* species in treatment of infections with special emphasis on HIV. In addition, the effects obtained by using lactobacilli treatment together with expression of adhesion inhibitors will be discussed. The aim is to give the reader an overview of the role of CXCL8 in HIV infections and combine this with information on how lactobacilli treatment influences the chemokine levels and evaluate these systems potential

**1. Introduction** 

Spear et al., 2007).

in the treatment of HIV patients.


## **CXCL8 Regulation and Function in HIV Infections and Potential Treatment Strategies**

Per-Erik Olsson, Hazem Khalaf and Jana Jass *Örebro Life Science Center, School of Science and Technology, Örebro University Sweden* 

## **1. Introduction**

326 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

Wang, F-S.; Hong, W-G.; Cao, Y.; Liu, M-X.; Jin, L.; Hu, L-P.; Wang, Z.; Feng, T-J.; Hou, J.;

Williamson, C.; Loubser, S.A.; Brice, B.; Joubert, G.; Smit, T.; Thomas, R.; Visagie , M.;

Winkler, C.; Modi, W.; Smith, M.W.; Nelson, G.W.; Wu, X.; Carrington, M.; Dean, M.; Honjo,

Wit, F.W.; van Rij. R.P.; Weverling, G.J.; Jange, J.M. & Schuitemaker, H. (2002). CC

Yamashita, T.E.;, Phair, J.P.; Muñoz, A.; Margolick, J.B.; Detels, R.; O'Brien, S.J.; Mellors,

Zimmerman, P.; Buckler-White, A.; Alkhatib, G.; Spalding, T.; Kubofcik, J.; Combadiere, C.;

quantified risk. *Molecular Medicine*, v. *3*, n. 1, pp. 23-26, ISSN 1077-1551.

*Infectious Diseases,* v. 186, n.12, pp. 1726-1732, ISSN 0022-1899.

*Immune Deficiency Syndrome*, v. 32, n. 2, pp. 124-130, ISSN 1525-4135. Wichukchinda, N., Nakayama, E. E., Rojanawiwat, A., Pathipvanich, P., Auwanit, W.,

females. *AIDS,* v. 20, pp.189–196, ISSN 0269-9370.

pp. 389-393, ISSN 0036-8075.

v. 15, p. 735-746, ISSN 0269-9370.

populations. *AIDS*, v. 14, n. 4, p. 449-451, ISSN 0269-9370.

Zhang, B.; Shi, M.; Xu, D-P.; Lei, Z-Y.; Wang, B.; Liu, Z-D.; Ye, J-J.; Peng, L.; Qiu, Y. & Winkler, C. (2003). Population survey of CCR5-∆32, CCR m3030, CCR2b 64I, and SDF1 3'A allele frequencies in indigenous Chinese healthy individuals, and HIV-1 infected and HIV-1-unifected individuals in HIV-1 risk groups. *Journal of Acquired* 

Vongsheree, S., Ariyoshi, K., Sawanpanyalert, P. & Shioda, T. (2006). Protective effects of IL4–589T and RANTES-28G on HIV-1 disease progression in infected Thai

Cooper M, Ryst E van der. (2000). Allelic frequencies of host genetic variants influencing susceptibility to HIV-1 infection and disease in South African

T.; Tashiro, K.; Yabe, D.; Buchbinder, S.; Vittinghoff, E.; Goedert, J.J.; O'Brien, T.R.; Jacobson, L.P.; Detels, R. Donfield, S.; Willoughby, A.; Gomperts, E.; Vlahov, D.; Phair, J. & O'Brien, S.J. (1998). Genetic restriction of AIDS pathogenesis by an SDF-1 chemokine gene variant. ALIVE study, Hemophilia Growth and Development Study (HGDS), Multicenter AIDS Cohort Study (MACS), Multicenter Hemophilia Cohort Study (MHCS), San Francisco City Cohort (SFCS). *Science*, v. 279, n. 5349,

chemokine receptor 5 delta32 and CC chemokine receptor 2 64I polymorphisms do not influence the virologic and immunologic response to antiretroviral combination therapy in human immunodeficiency virus type 1-infected patients. *The Journal of* 

J.W.; Wolinsky, S.M. & Jacobson, L.P. (2001). Immunologic and virologic response to highly active antiretroviral therapy in the Multicenter AIDS Cohort Study. *AIDS*,

Weissman, D.; Cohen, O.; Rubbert, A.; Lam, G.; Vaccarezza, M.; Kennedy, P.E.; Kumaraswami, V.; Giorgi, J.V.; Detels, R.; Hunter, J.; Choper, M.; Berger, E.A.; Fauci, A.S.; Nutman, T.B. & Murphy, P.M. (1997). Inherited resistance to HIV-1 conferred by an inactivating mutation in CC chemokine receptor 5: studies in populations with contrasting clinical phenotypes, defined racial background, and Interleukin-8 (CXCL8) is a chemokine that was originally identified as a key factor in neutrophil recruitment and activation. Numerous cell types produce CXCL8, including immune cells, mucosal epithelial cells, endothelial cells and smooth muscle cells (Garcia-Vicuna et al., 2004). CXCL8 is one of the important inflammatory mediators responsible for the recruitment of neutrophils and T-cells to the site of infection, therefore it is an attractive target for therapy against diseases that affect immune cells such as HIV. HIV directly targets the host's immune system and thus reduces the ability of the innate and adaptive immune system to fight disease. As a chemokine, CXCL8 is a potential target for controling HIV infection by reducing the migration of T-cells to the site of infection. It is therefore necessary to identify the signaling pathways involved in CXCL8 regulation in order to develop viable CXCL8 based treatment strategies. In line with this, we have recent shown that CXCL8 activation in Jurkat T-cells is not primarily under the control of NFB, but that the AP-1 signaling pathway appears to be central for the regulation of CXCL8 (Khalaf et al., 2010). An understanding of the regulation and function of cytokines and chemokines, while complex, remains important for the development of new strategies in the development of HIV treatments. It is interesting to note that several lactobacilli strains are able to modulate CXCL8 expression and release (Anukam et al., 2009; Zhang et al., 2005). Disturbance of the lactobacilli flora in the vaginal tract has been shown to increase the risk of infections and acquisition of HIV type 1 (Taha et al., 1998). Several studies have shown that treatment with certain *Lactobacillus* species and strains have positive effects on women with HIV infections and have been successful in trials to counteract vaginal infections (Hummelen et al., 2010; Spear et al., 2007).

In the present chapter we give a background to CXCL8 regulation and function as well as its involvement in HIV etiology. We also provide an overview of the available information on the possible uses of *Lactobacillus* species in treatment of infections with special emphasis on HIV. In addition, the effects obtained by using lactobacilli treatment together with expression of adhesion inhibitors will be discussed. The aim is to give the reader an overview of the role of CXCL8 in HIV infections and combine this with information on how lactobacilli treatment influences the chemokine levels and evaluate these systems potential in the treatment of HIV patients.

CXCL8 Regulation and Function in HIV Infections and Potential Treatment Strategies 329

2003). While inflammatory responses are mainly induced through signals transduced via TLRs on epithelial cell surface, there is evidence indicating involvement of other membrane receptors, including Dectin-1, in the enhancement of inflammatory responses (Gantner et al.,

Fig. 1. Inflammation induced by LPS, which binds to TLR4 and signals for MyD88 activation that in turn starts a phosphorylation cascade leading to NF-κB activation and nuclear

Recognition of toxins by epithelial- and immune cells occurs via TLRs that activate specific intracellular signaling pathways and result in the transcription of essential proteins for survival. Currently, more than 10 TLRs have been identified and recognize; among others, zymosan (TLR1/TLR2), LPS-LTA (TLR4-TLR2/TLR6) and dsRNA (TLR3) (Xu et al., 2001). NF-B has a central role in the induction of inflammatory responses by regulating a wide range of genes. Several stress factors can activate NF-B, including bacterial toxins, cytokines, reactive oxygen species and UV light. Bacterial toxins, such as LPS and peptidoglycan, activate NF-B via TLRs whereas cytokines, TNF, IL-1, signal via TNF-R1 and IL-1R on the membrane surface (Dinarello, 2000). LPS activates NF-B following

2003).

translocation.

**1.4 NF-B and MAPK signaling pathways** 

#### **1.1 Inflammatory responses**

Pro-inflammatory cytokines, such as TNF, IL-1 and IL-6, as well as the chemokine CXCL8 promote inflammation, whereas anti-inflammatory cytokines, including IL-4, IL-10 and IL-13, suppress the activity of pro-inflammatory cytokines (Dinarello, 2000). Gene-expression and release of cytokines, such as TNF, IL-1 and IL-6, and cell adhesion molecules, including ICAM-1, P selectin, and E selectin, are indicators of induced inflammatory responses (Pearson et al., 2003). NF-B has been proposed as the main transcriptional regulator of cytokine expression, adhesion factors and anti-apoptotic factors (McKay & Cidlowski, 1999). It has been suggested that inflammatory cytokines, such as TNF, are involved in the induction of reactive oxygen intermediates (O2- ) that cause DNA damage (Shoji et al., 1995). Recent observations have shown that elevated levels of IL-6 and C-reactive protein are associated with the development of atherosclerosis and type II diabetes (Libby et al., 2002). Elevated cytokine levels are also associated with cellular senescence and may be involved in telomere shortening and continuous cell divisions (Itahana et al., 2001).

#### **1.2 T-cell derived inflammatory responses**

Optimal T-cell activation is achieved following antigen binding to the T-cell receptor (TCR), together with co-stimulatory signals followed by cytokine expression (Gonzalo et al., 2001). T-cells produce a broad range of pro- and anti-inflammatory cytokines, including IL-2, IL-6, IL-10 and TNF, in response to infections (Opal & DePalo, 2000). IL-10 is another important anti-inflammatory cytokine expressed by activated T-cells, providing control of intestinal inflammatory responses, but is also important for normal T-cell function (Asseman et al., 1999). Chemokine expression (CXCL8) by CD8+ T-cells is crucial in immune responses by inducing cytokines, such as TNF and IFN-γ by CD4+ cells, and antibody secretion by B-cells (Kim et al., 1998). Recent findings provide evidence indicating expression of several types of Toll-like receptors (TLRs), including TLR4, on T-cells (Caramalho et al., 2003). These results demonstrate a specific role of T-cells in the induction of inflammatory responses by direct recognition of antigens, independent of antigen-presenting cells.

#### **1.3 Epithelial cell derived inflammatory responses**

Wounds, chemical irritation or infection may cause inflammation of the epithelial surface. Acute inflammation induces expression of pro-inflammatory cytokines and chemokines that attract immune cells, such as neutrophils, followed by the production of anti-inflammatory cytokines thereby initiating the healing process (Philip et al., 2004). The balance between pro- and anti-inflammatory cytokines determines the severity of an infection. However, prolonged inflammatory responses are prevented by the expression of IL-1ra, glucocorticoids and IL-10, which also improves cell survival (Berg et al., 1995; van der Poll et al., 1997; Walley et al., 1996). Lipopolysaccharides (LPS) are well-known bacterial toxins and potent inducers of inflammatory responses. It has been suggested that epithelial cells are refractory to LPS since they do not express surface CD14, an important signaling protein that functions by docking the LPS/LBP (Lipid binding protein)- complex with TLR4 to initiate intracellular signaling cascades (Svanborg et al., 1999). However, in the presence of serum cells, epithelial cells respond to LPS, demonstrating the presence of a second soluble form of CD14 (sCD14) (Noel et al., 1995; Riedel et al., 2006). Furthermore, CD14 independent pathways are also present in epithelial cells that can be triggered by substances such as peptidoglycan and result in the release of pro-inflammatory cytokines (Sato et al.,

Pro-inflammatory cytokines, such as TNF, IL-1 and IL-6, as well as the chemokine CXCL8 promote inflammation, whereas anti-inflammatory cytokines, including IL-4, IL-10 and IL-13, suppress the activity of pro-inflammatory cytokines (Dinarello, 2000). Gene-expression and release of cytokines, such as TNF, IL-1 and IL-6, and cell adhesion molecules, including ICAM-1, P selectin, and E selectin, are indicators of induced inflammatory responses (Pearson et al., 2003). NF-B has been proposed as the main transcriptional regulator of cytokine expression, adhesion factors and anti-apoptotic factors (McKay & Cidlowski, 1999). It has been suggested that inflammatory cytokines, such as TNF, are involved in the

Recent observations have shown that elevated levels of IL-6 and C-reactive protein are associated with the development of atherosclerosis and type II diabetes (Libby et al., 2002). Elevated cytokine levels are also associated with cellular senescence and may be involved in

Optimal T-cell activation is achieved following antigen binding to the T-cell receptor (TCR), together with co-stimulatory signals followed by cytokine expression (Gonzalo et al., 2001). T-cells produce a broad range of pro- and anti-inflammatory cytokines, including IL-2, IL-6, IL-10 and TNF, in response to infections (Opal & DePalo, 2000). IL-10 is another important anti-inflammatory cytokine expressed by activated T-cells, providing control of intestinal inflammatory responses, but is also important for normal T-cell function (Asseman et al., 1999). Chemokine expression (CXCL8) by CD8+ T-cells is crucial in immune responses by inducing cytokines, such as TNF and IFN-γ by CD4+ cells, and antibody secretion by B-cells (Kim et al., 1998). Recent findings provide evidence indicating expression of several types of Toll-like receptors (TLRs), including TLR4, on T-cells (Caramalho et al., 2003). These results demonstrate a specific role of T-cells in the induction of inflammatory responses by direct

Wounds, chemical irritation or infection may cause inflammation of the epithelial surface. Acute inflammation induces expression of pro-inflammatory cytokines and chemokines that attract immune cells, such as neutrophils, followed by the production of anti-inflammatory cytokines thereby initiating the healing process (Philip et al., 2004). The balance between pro- and anti-inflammatory cytokines determines the severity of an infection. However, prolonged inflammatory responses are prevented by the expression of IL-1ra, glucocorticoids and IL-10, which also improves cell survival (Berg et al., 1995; van der Poll et al., 1997; Walley et al., 1996). Lipopolysaccharides (LPS) are well-known bacterial toxins and potent inducers of inflammatory responses. It has been suggested that epithelial cells are refractory to LPS since they do not express surface CD14, an important signaling protein that functions by docking the LPS/LBP (Lipid binding protein)- complex with TLR4 to initiate intracellular signaling cascades (Svanborg et al., 1999). However, in the presence of serum cells, epithelial cells respond to LPS, demonstrating the presence of a second soluble form of CD14 (sCD14) (Noel et al., 1995; Riedel et al., 2006). Furthermore, CD14 independent pathways are also present in epithelial cells that can be triggered by substances such as peptidoglycan and result in the release of pro-inflammatory cytokines (Sato et al.,

telomere shortening and continuous cell divisions (Itahana et al., 2001).

recognition of antigens, independent of antigen-presenting cells.

**1.3 Epithelial cell derived inflammatory responses** 

) that cause DNA damage (Shoji et al., 1995).

**1.1 Inflammatory responses** 

induction of reactive oxygen intermediates (O2-

**1.2 T-cell derived inflammatory responses** 

2003). While inflammatory responses are mainly induced through signals transduced via TLRs on epithelial cell surface, there is evidence indicating involvement of other membrane receptors, including Dectin-1, in the enhancement of inflammatory responses (Gantner et al., 2003).

Fig. 1. Inflammation induced by LPS, which binds to TLR4 and signals for MyD88 activation that in turn starts a phosphorylation cascade leading to NF-κB activation and nuclear translocation.

## **1.4 NF-B and MAPK signaling pathways**

Recognition of toxins by epithelial- and immune cells occurs via TLRs that activate specific intracellular signaling pathways and result in the transcription of essential proteins for survival. Currently, more than 10 TLRs have been identified and recognize; among others, zymosan (TLR1/TLR2), LPS-LTA (TLR4-TLR2/TLR6) and dsRNA (TLR3) (Xu et al., 2001). NF-B has a central role in the induction of inflammatory responses by regulating a wide range of genes. Several stress factors can activate NF-B, including bacterial toxins, cytokines, reactive oxygen species and UV light. Bacterial toxins, such as LPS and peptidoglycan, activate NF-B via TLRs whereas cytokines, TNF, IL-1, signal via TNF-R1 and IL-1R on the membrane surface (Dinarello, 2000). LPS activates NF-B following

CXCL8 Regulation and Function in HIV Infections and Potential Treatment Strategies 331

Fig. 2. T-cells recognize antigens via TCR and co-stimulatory receptors (CD28), leading to a downstream signaling cascade involving PKC activation and several transcription factors

In order to understand how CXCL8 contributes to the etiology of HIV, it is important to characterize the cellular signal transduction pathways regulating CXCL8. There are over 50 chemokines identified and almost 18 chemokine receptors have been characterized (Alfano & Poli, 2005). Chemokines are divided into four categories according to the location of two cysteine residues at the N-terminal region. These include C, CC, CXC and CX3C chemokines (Zlotnik & Yoshie, 2000). Chemokine receptors have been designated the same nomenclature as for their respective chemokine. Besides several immune cells, many other cell types have been described to express chemokine receptors, including endothelial cells, fibroblasts and smooth muscle cells (Garcia-Vicuna et al., 2004). This indicates that chemokines are not only implicated in the regulation of cell trafficking but also in cell

Several chemokines share the same receptor but possess different binding affinities to each one. We have previously shown an association between IL-6 release and NF-B activity while CXCL8 release was more closely correlated with activator protein (AP)-1 activity (Khalaf et al., 2010). Blocking NF-B activation resulted in a complete inhibition of IL-6 while the CXCL8 levels remained elevated as shown both at the protein and mRNA levels.

including NF-B, AP-1 and NFAT.

**2. CXCL8 regulation and signal transduction** 

proliferation and gene regulation (Wong & Fish, 2003).

binding to a LPS-binding protein, which facilitates LPS binding to CD14. Once bound to CD14, the LPS-binding protein dissociates, and the LPS-CD14 complex associates with TLR4 with the help of the extracellular protein MD2. The binding of LPS-CD14 complex leads to the activation and binding of a cytoplasmic signaling molecule called myeloid differentiation factor 88 (MyD88). Binding of MyD88 leads to the activation of interleukin-1 receptor-associated kinase (IRAK), which phosphorylates TNF-receptor-associated factor (TRAF) 6. TRAF6 leads to the activation of TGF-beta activated kinase (TAK) 1, which activates NF-κB-inducible kinase (NIK). IκB kinase (IKK), activated by NIK, phosphorylates the inhibitory protein IB followed by subsequent degradation in the proteasome. This leads to NF-B activation, which translocates into the nucleus and initiates transcription of a wide range of inflammatory genes (Fig. 1) (Ali & Mann, 2004).

An additional important signaling pathway involved in the induction of cytokines and inflammation is the mitogen-activated protein kinase (MAPK) pathway. Several stress factors can induce the activation of MAPK pathway, however only pro-inflammatory cytokines and growth factors can induce inflammation, apoptosis or differentiation through either p38 MAPK or c-Jun NH2-terminal kinase (JNK). There are three major groups of MAPKs; p38 Map kinase family, extracellular signal-regulated kinase (Erk) family and JNK family (McCarroll et al., 2003). A series of phosphorylation steps are the key events leading to MAPK activation and gene-expression (Faure et al., 1994). Furthermore, activation of the Raf/MAPK pathway has been shown to stimulate transcription of, among others, cytokines through AP-1, NF-IL6 and NF-B (Bruder & Kovesdi, 1997).

Since T-cells lack TLRs, antigens are recognized by the TCR with the help of co-stimulatory receptors, such as CD28. This results in the activation of resting T-cells and a signaling cascade that activates phospholipase C (PLC) and cleavage of phosphatidylinositol bisphosphate (PIP2) to inositol (1,4,5)-trisphosphate (IP3) and diacylglycerol (DAG) (Teixeira et al., 2003). IP3 mobilize Ca2+ from intracellular stores and together with DAG activates PKC (Gajewski et al., 1994; Werlen et al., 1998). In addition, Ca2+ also activates calcineurin/calmodulin and RasGRP, which is a Ras activator and is directly connected to the MAPK pathway (Dower et al., 2000). Recent reports have revealed the importance of three additional intracellular proteins, namely CARMA1, Bcl10 and MALT1 (CBM) in the induction of NF-B (Scharschmidt et al., 2004). Down regulation of Bcl10 was shown to result in inhibition of NF-B, transduced via TCR/CD28 and PKC. It was suggested that Bcl10 is initially activated by TCR/PKC but further activation (>1h) promotes its degradation. Furthermore, deletion of any component of the CBM complex impairs antigen receptor activation of NF-B (Gaide et al., 2002; Narayan et al., 2006). CARMA1 has been shown to be required for NF-B activation through Akt signaling, in cooperation with PKC following short-term exposure (30min) of Jurkat T-cells with PMA (Narayan et al., 2006). These studies indicate that PKC is crucial for NF-B activation, following short-term exposure; from signals transduced via TCR and co-stimulatory receptors, such as CD28, and that the CBM complex proteins play a key role in these signaling processes (Fig. 2).

NF-κB is an important transcription factor complex involved in almost every aspect of cell regulation including apoptosis, differentiation, proliferation and initiation of immune responses (Barnes & Adcock, 1997; Makarov, 2000; Tergaonkar, 2006). NF-κB is an attractive therapeutic target since it is constitutively active in many human malignancies (Dolcet et al., 2005).

binding to a LPS-binding protein, which facilitates LPS binding to CD14. Once bound to CD14, the LPS-binding protein dissociates, and the LPS-CD14 complex associates with TLR4 with the help of the extracellular protein MD2. The binding of LPS-CD14 complex leads to the activation and binding of a cytoplasmic signaling molecule called myeloid differentiation factor 88 (MyD88). Binding of MyD88 leads to the activation of interleukin-1 receptor-associated kinase (IRAK), which phosphorylates TNF-receptor-associated factor (TRAF) 6. TRAF6 leads to the activation of TGF-beta activated kinase (TAK) 1, which activates NF-κB-inducible kinase (NIK). IκB kinase (IKK), activated by NIK, phosphorylates the inhibitory protein IB followed by subsequent degradation in the proteasome. This leads to NF-B activation, which translocates into the nucleus and initiates transcription of a wide

An additional important signaling pathway involved in the induction of cytokines and inflammation is the mitogen-activated protein kinase (MAPK) pathway. Several stress factors can induce the activation of MAPK pathway, however only pro-inflammatory cytokines and growth factors can induce inflammation, apoptosis or differentiation through either p38 MAPK or c-Jun NH2-terminal kinase (JNK). There are three major groups of MAPKs; p38 Map kinase family, extracellular signal-regulated kinase (Erk) family and JNK family (McCarroll et al., 2003). A series of phosphorylation steps are the key events leading to MAPK activation and gene-expression (Faure et al., 1994). Furthermore, activation of the Raf/MAPK pathway has been shown to stimulate transcription of, among others, cytokines

Since T-cells lack TLRs, antigens are recognized by the TCR with the help of co-stimulatory receptors, such as CD28. This results in the activation of resting T-cells and a signaling cascade that activates phospholipase C (PLC) and cleavage of phosphatidylinositol bisphosphate (PIP2) to inositol (1,4,5)-trisphosphate (IP3) and diacylglycerol (DAG) (Teixeira et al., 2003). IP3 mobilize Ca2+ from intracellular stores and together with DAG activates PKC (Gajewski et al., 1994; Werlen et al., 1998). In addition, Ca2+ also activates calcineurin/calmodulin and RasGRP, which is a Ras activator and is directly connected to the MAPK pathway (Dower et al., 2000). Recent reports have revealed the importance of three additional intracellular proteins, namely CARMA1, Bcl10 and MALT1 (CBM) in the induction of NF-B (Scharschmidt et al., 2004). Down regulation of Bcl10 was shown to result in inhibition of NF-B, transduced via TCR/CD28 and PKC. It was suggested that Bcl10 is initially activated by TCR/PKC but further activation (>1h) promotes its degradation. Furthermore, deletion of any component of the CBM complex impairs antigen receptor activation of NF-B (Gaide et al., 2002; Narayan et al., 2006). CARMA1 has been shown to be required for NF-B activation through Akt signaling, in cooperation with PKC following short-term exposure (30min) of Jurkat T-cells with PMA (Narayan et al., 2006). These studies indicate that PKC is crucial for NF-B activation, following short-term exposure; from signals transduced via TCR and co-stimulatory receptors, such as CD28, and

that the CBM complex proteins play a key role in these signaling processes (Fig. 2).

2005).

NF-κB is an important transcription factor complex involved in almost every aspect of cell regulation including apoptosis, differentiation, proliferation and initiation of immune responses (Barnes & Adcock, 1997; Makarov, 2000; Tergaonkar, 2006). NF-κB is an attractive therapeutic target since it is constitutively active in many human malignancies (Dolcet et al.,

range of inflammatory genes (Fig. 1) (Ali & Mann, 2004).

through AP-1, NF-IL6 and NF-B (Bruder & Kovesdi, 1997).

Fig. 2. T-cells recognize antigens via TCR and co-stimulatory receptors (CD28), leading to a downstream signaling cascade involving PKC activation and several transcription factors including NF-B, AP-1 and NFAT.

## **2. CXCL8 regulation and signal transduction**

In order to understand how CXCL8 contributes to the etiology of HIV, it is important to characterize the cellular signal transduction pathways regulating CXCL8. There are over 50 chemokines identified and almost 18 chemokine receptors have been characterized (Alfano & Poli, 2005). Chemokines are divided into four categories according to the location of two cysteine residues at the N-terminal region. These include C, CC, CXC and CX3C chemokines (Zlotnik & Yoshie, 2000). Chemokine receptors have been designated the same nomenclature as for their respective chemokine. Besides several immune cells, many other cell types have been described to express chemokine receptors, including endothelial cells, fibroblasts and smooth muscle cells (Garcia-Vicuna et al., 2004). This indicates that chemokines are not only implicated in the regulation of cell trafficking but also in cell proliferation and gene regulation (Wong & Fish, 2003).

Several chemokines share the same receptor but possess different binding affinities to each one. We have previously shown an association between IL-6 release and NF-B activity while CXCL8 release was more closely correlated with activator protein (AP)-1 activity (Khalaf et al., 2010). Blocking NF-B activation resulted in a complete inhibition of IL-6 while the CXCL8 levels remained elevated as shown both at the protein and mRNA levels.

CXCL8 Regulation and Function in HIV Infections and Potential Treatment Strategies 333

demonstrated a significant reduction of CXCR1 and CXCR2 following exposure of neutrophils with LPS or TNF that was shown to act through activation of serine proteinases. This may indicate that a mechanism used by microorganisms to evade the immune system is by reducing neutrophil migration towards the infected site. CXCR1 was further shown to be expressed on cytotoxic CD8+ effector T-cells, indicating the vital physiological role of CXCL8 in recruiting lymphocytes and therefore acting as an important link between innate

Fig. 3. Intracellular signaling cascade leading to *cxcl8* gene expression. NF-B (p50, p65) and AP-1 (jun, fos) are the main regulators at the transcriptional level, while p38 serves to

CXCL8 has been implicated in many cellular responses, such as HIV pathogenesis, angiogenesis and cell growth and survival. HIV-infected individuals have elevated CXCL8 levels that, due to the potent chemo-attractant characteristics of CXCL8, can result in the recruitment of target cells, leading to a progressive infection and HIV-1 replication (Ott et al., 1998). However, it has also been suggested that CXCL8 is involved in decreased replication of HIV-1 during the early stages of infection (Rollenhagen & Asin, 2010). In addition, CXCL8 can act as a potent anti-apoptotic agent, inducing the expression of prosurvival proteins, including Bcl-2 and Bcl-xL (Li et al., 2003). While the role of CXCL8 remains complex it remains an interesting candidate as a suitable therapeutic target in HIV

and adaptive immunity (Takata et al., 2004).

stabilize the mRNA molecules.

treatment.

**3. Targeting CXCL8 in HIV treatment strategies** 

Our results indicate that in Jurkat T-cells, IL-6 is regulated through NF-B while CXCL8 regulation is independent of NF-B and is closely associated with AP-1 activation. The interplay between immune cells and the expression levels of different cytokines/chemokines is an important factor for consideration.

The gram-negative derived endotoxin, LPS, is a known factor reported to induce CXCL8 expression and release. However, pre-treatment with anti-inflammatory cytokines, including IL-4, IL-10 and TGF-1, resulted in a significant reduction in CXCL8 expression (Ehrlich et al., 1998). Thus, the balance between pro- and anti-inflammatory cytokines is a determinant factor for immune cell activation as well as the expression levels of cytokines and chemokines released by different cells. Maintaining this balance is therefore of great importance, however there is a need to improve our understanding about the regulatory mechanisms controlling the expression of inflammatory mediators and their effect(s) on different immune cells.

The main regulators of *cxcl8* gene expression are NF-B and the MAP kinases JNK, ERK and p38 leading to the assembly and activation of the transcription factor AP-1. NF-B is required for CXCL8 release in most cell types, while optimal induction is achieved following binding of additional transcription factor including MAP kinases and C/EBP (Hoffmann et al., 2002). However, the ratio of activation between NF-B to MAPK and other transcription factors in this regulatory mechanism seems to differ depending on the pressure caused by a specific stress factor and cell type. In airway epithelial cells NF-B, ERK and JNK were found to be essential for TNF-induced CXCL8 expression, while p38 acted as a posttranscriptional regulator (Li et al., 2002). Even though p38 is not required for *cxcl8* gene expression, it plays a major role in CXCL8 release by stabilizing its mRNA through protein kinase-2 (Hoffmann et al., 2002). A simplified representation of the signaling pathways involved in CXCL8 expression and regulation is shown in figure 3. Furthermore, reactive oxygen intermediates (ROI) are important regulators of cytokine and chemokine expression and has been shown to mediate a dose-dependent CXCL8 expression (DeForge et al., 1993). They further demonstrated that the effect of these potent immune regulators could be almost completely abolished by applying the OH-radical scavenger DMSO, which reduced CXCL8 expression by 90%.

There are two well-characterized receptors for CXCL8, namely CXC chemokine receptor (CXCR)-1 and -2. CXCL8 binds to these receptors with high affinity (Bertini et al., 2004), while CXCR1 is specific for CXCL8, (NAP)-2 and granulocyte chemotactic protein (GCP)-2, CXCR2 can bind additional chemokines, including CXCL1, 2, 3, 5, 6 and 7 (Acosta et al., 2008). Despite their structural similarities, these receptors possess different biological effects through distinct signaling pathways (Gabellini et al., 2009).

Signals transduced through CXCR1 stimulate neutrophil migration through epithelial layers, while CXCR2 signaling promotes angiogenesis (Sturm et al., 2005). The intracellular protein Bcl-10 has been proposed to play a critical role in the signaling pathway leading to CXCL8 expression and its enhancement of angiogenesis through CXCR2 (Karl et al., 2005). Both NF-B and C/EBP have been reported to be downstream targets of activation in the CXCR2 signaling cascade, ultimately leading to CXCL8 expression, creating a positive feedback loop (Acosta & Gil, 2009). This positive feedback loop leading to neutrophil activation and migration is regulated by internalization of CXCR1 and CXCR2 upon ligand binding. Furthermore, a second regulatory mechanism of CXCL8 receptor expression involves metalloproteinases as important regulatory factors (Khandaker et al., 1999). They

Our results indicate that in Jurkat T-cells, IL-6 is regulated through NF-B while CXCL8 regulation is independent of NF-B and is closely associated with AP-1 activation. The interplay between immune cells and the expression levels of different

The gram-negative derived endotoxin, LPS, is a known factor reported to induce CXCL8 expression and release. However, pre-treatment with anti-inflammatory cytokines, including IL-4, IL-10 and TGF-1, resulted in a significant reduction in CXCL8 expression (Ehrlich et al., 1998). Thus, the balance between pro- and anti-inflammatory cytokines is a determinant factor for immune cell activation as well as the expression levels of cytokines and chemokines released by different cells. Maintaining this balance is therefore of great importance, however there is a need to improve our understanding about the regulatory mechanisms controlling the expression of inflammatory mediators and their effect(s) on

The main regulators of *cxcl8* gene expression are NF-B and the MAP kinases JNK, ERK and p38 leading to the assembly and activation of the transcription factor AP-1. NF-B is required for CXCL8 release in most cell types, while optimal induction is achieved following binding of additional transcription factor including MAP kinases and C/EBP (Hoffmann et al., 2002). However, the ratio of activation between NF-B to MAPK and other transcription factors in this regulatory mechanism seems to differ depending on the pressure caused by a specific stress factor and cell type. In airway epithelial cells NF-B, ERK and JNK were found to be essential for TNF-induced CXCL8 expression, while p38 acted as a posttranscriptional regulator (Li et al., 2002). Even though p38 is not required for *cxcl8* gene expression, it plays a major role in CXCL8 release by stabilizing its mRNA through protein kinase-2 (Hoffmann et al., 2002). A simplified representation of the signaling pathways involved in CXCL8 expression and regulation is shown in figure 3. Furthermore, reactive oxygen intermediates (ROI) are important regulators of cytokine and chemokine expression and has been shown to mediate a dose-dependent CXCL8 expression (DeForge et al., 1993). They further demonstrated that the effect of these potent immune regulators could be almost completely abolished by applying the OH-radical scavenger DMSO, which reduced

There are two well-characterized receptors for CXCL8, namely CXC chemokine receptor (CXCR)-1 and -2. CXCL8 binds to these receptors with high affinity (Bertini et al., 2004), while CXCR1 is specific for CXCL8, (NAP)-2 and granulocyte chemotactic protein (GCP)-2, CXCR2 can bind additional chemokines, including CXCL1, 2, 3, 5, 6 and 7 (Acosta et al., 2008). Despite their structural similarities, these receptors possess different biological effects

Signals transduced through CXCR1 stimulate neutrophil migration through epithelial layers, while CXCR2 signaling promotes angiogenesis (Sturm et al., 2005). The intracellular protein Bcl-10 has been proposed to play a critical role in the signaling pathway leading to CXCL8 expression and its enhancement of angiogenesis through CXCR2 (Karl et al., 2005). Both NF-B and C/EBP have been reported to be downstream targets of activation in the CXCR2 signaling cascade, ultimately leading to CXCL8 expression, creating a positive feedback loop (Acosta & Gil, 2009). This positive feedback loop leading to neutrophil activation and migration is regulated by internalization of CXCR1 and CXCR2 upon ligand binding. Furthermore, a second regulatory mechanism of CXCL8 receptor expression involves metalloproteinases as important regulatory factors (Khandaker et al., 1999). They

cytokines/chemokines is an important factor for consideration.

different immune cells.

CXCL8 expression by 90%.

through distinct signaling pathways (Gabellini et al., 2009).

demonstrated a significant reduction of CXCR1 and CXCR2 following exposure of neutrophils with LPS or TNF that was shown to act through activation of serine proteinases. This may indicate that a mechanism used by microorganisms to evade the immune system is by reducing neutrophil migration towards the infected site. CXCR1 was further shown to be expressed on cytotoxic CD8+ effector T-cells, indicating the vital physiological role of CXCL8 in recruiting lymphocytes and therefore acting as an important link between innate and adaptive immunity (Takata et al., 2004).

Fig. 3. Intracellular signaling cascade leading to *cxcl8* gene expression. NF-B (p50, p65) and AP-1 (jun, fos) are the main regulators at the transcriptional level, while p38 serves to stabilize the mRNA molecules.

## **3. Targeting CXCL8 in HIV treatment strategies**

CXCL8 has been implicated in many cellular responses, such as HIV pathogenesis, angiogenesis and cell growth and survival. HIV-infected individuals have elevated CXCL8 levels that, due to the potent chemo-attractant characteristics of CXCL8, can result in the recruitment of target cells, leading to a progressive infection and HIV-1 replication (Ott et al., 1998). However, it has also been suggested that CXCL8 is involved in decreased replication of HIV-1 during the early stages of infection (Rollenhagen & Asin, 2010). In addition, CXCL8 can act as a potent anti-apoptotic agent, inducing the expression of prosurvival proteins, including Bcl-2 and Bcl-xL (Li et al., 2003). While the role of CXCL8 remains complex it remains an interesting candidate as a suitable therapeutic target in HIV treatment.

CXCL8 Regulation and Function in HIV Infections and Potential Treatment Strategies 335

although an initial virus infection is CCR5-dependent, a large subset will switch to using CXCR4 as a co-receptor. This is a major concern since maraviroc has little or no effect on CXCR4-dependent viruses (Kuritzkes, 2011). The interplay between different immune cells during an established HIV infection remains important to understand. Determination of cytokine expression by specific cells and the effect that these inflammatory mediators have on other cells to produce chemokines is also important. Further investigations are needed to evaluate the patterns of immune cell activation and cytokine/chemokine regulation in order

*Lactobacillus* spp are part of the healthy human microbiota, found primarily in the gastrointestinal and vaginal tracts. Certain *Lactobacillus* spp have been identified as health promoting probiotic bacteria by inhibiting pathogen colonization and modulating the immune response in the host (reviewed in Reid et al., 2003). Evidence of immune modulating properties exhibited by certain lactobacilli strains has been shown through their ability to alter cytokine expression in tissue cells infected by pathogens, *in vitro* and *in vivo*, and thus helping maintain homeostasis (Anukam et al., 2009; Frick et al., 2007; Moorthy et al., 2010; Nandakumar et al., 2009; van Hemert et al., 2010; Zhang et al., 2005). Altered cytokine responses, including TNF, NF-B, IL-6, IL-1, CXCL8, IL-10 and IL-12, are dependent on cell type (human mucosal epithelial cells, human mononuclear cells, T-cells, dendritic cells etc) and *Lactobacillus* species and strain (Nandakumar et al., 2009; van Hemert et al., 2010). From these reports, probiotic lactobacilli demonstrate a clear potential for both development of new strategies to reduce the risk of HIV infection and combat AIDS

Vaginal infections such as bacterial vaginosis (BV) and candidiasis have been correlated with an increased risk of HIV infection (Sha et al., 2005; St John et al., 2007). BV and candidiasis are characterized by microbiota that is comprised of a range of anaerobic bacteria or *Candida albicans*, respectively*,* while deficient in lactobacilli (Sha et al., 2005). Furthermore, women already infected with HIV that lacked vaginal lactobacilli and had BV or candidiasis had higher levels of HIV shedding in the genital tract (Coleman et al., 2007; Spinillo et al., 2005). Increased HIV transcripts in vaginal cells and viral shedding increases the risk of HIV transmission. Furthermore, vaginal secretions from women with BV increased HIV expression in chronically infected monocyte cell line *in vitro*, while secretions from women without BV had no effect on HIV expression (Spear et al., 2007). This is believed to be due to the higher levels of proinflammatory cytokines primarily those that function through NF-B (Al-Harthi et al., 1998). *C. albicans* infections increase vaginal CXCL8 levels and neutrophil presence while higher levels of vaginal lactobacilli reduced CXCL8 levels and other pro-inflammatory cytokines (Spear et al., 2008). CXCL8 is a potent chemokine that recruits neutrophils to the site of infections, thus by reducing chemokine levels, the target cells for HIV infection are limited. Using lactobacilli to modulate the CXCL8 levels and other pro-inflammatory signals may thus reduce the risk of HIV infection

Certain *Lactobacillus* spp have been shown to reduce pro-inflammatory cytokine release from stimulated cells. The probiotic *Lactobacillus rhamnosus* GG reduced the *cxcl8* expression and CXCL8 and CCL11 secretion in TNF or IL-1 - stimulated human intestinal epithelial cells

**4. Probiotic** *Lactobacillus* **as an alternative HIV treatment strategy** 

progression through their anti-infective and immune-modulating properties.

to find suitable therapeutic targets.

and reduce viral replication.

Cellular HIV infection involves interactions between glycoprotein gp120, CD4 and CC/CXC receptors (Suresh & Wanchu, 2006). It is therefore possible that an HIV infection can be interrupted and the progression of an established infection can be delayed by targeting CC and/or CXC chemokine receptors. There are two well characterized chemokine receptors by which HIV can bind, enter and infect monocytes, microglia and T-lymphocytes, namely CCR5 and CXCR4 (Ghafouri et al., 2006). CXCL8 dependent activation of CXCR1 has also been suggested to result in inhibited HIV infection and entry into cells (Richardson et al., 2003). Richardson and colleagues showed that CXCR1 activation and internalization resulted in a cross-phosphorylation and internalization of CCR5. Furthermore, C-terminal mutation of CXCR1 internalized both CCR5 and CXCR4 and thus inhibited HIV-1 infection and entry. Furthermore, since HIV-1 competes with CCL5 and CXCL8 for the chemokine receptor DARC (duffy antigen receptor for chemokines) the serum levels of these chemokines may affect the progression of HIV by binding to their respective receptors (He et al., 2008). HIV-1 binding to DARC was also shown to affect chemokine-induced inflammation.

Even though CXCL8 expression is impaired in HIV-infected cells, pro-inflammatory cytokines such as TNF can induce CXCL8 production and expression from other immune cells. It was recently demonstrated that HIV-infected macrophages secrete TNF and IL-1 that in turn act on astrocytes to induce CXCL8 production (Zheng et al., 2008). CXCL8 production was mediated through the MAPK-associated pathways, including p38, c-Jun Nterminal kinase (JNK) and extracellular signal-regulated kinases (ERK1/2). This inflammatory process plays an important role in the pathogenesis of HIV-associated dementia. Reduction of CXCL8 production can therefore be used to control immune cell migration into the central nervous system, which will reduce the overall inflammatory responses at this site. In addition, CXCL8 is co-localize with CD68/CD40 cells, and CD40 receptor expression on microglial cells act as potent inducers of CXCL8 expression, through AP-1 and NF-B, following ligation of its ligand CD40L, which is expressed on monocytes and T-lymphocytes (D'Aversa et al., 2008). Taken together, these studies show the importance of understanding the regulatory mechanisms leading to chemokine expression in T-cells and other immune cells in order to find a suitable target to control HIV replication and progression.

Antiretroviral drugs that target different structural properties in HIV have failed to eradicate the virus but rather suppressed its spreading and pathogenesis. These difficulties have been due to the variability and ability of HIV to internalize without being detected, followed by reactivation (Archer et al., 2009). These drugs are therefore applied in a combination to inhibit several steps of the viral life cycle, including proteases to inhibit maturation, reverse transcription inhibitors and inhibitors for HIV integration into the genome (Arhel & Kirchhoff, 2010). Other difficulties include drug-drug interactions and long-term toxicities (Tilton & Doms, 2010). However, as chemokine levels are severely altered in HIV infected individuals and are involved in HIV replication this has led to an interest in applying this knowledge to controling HIV infections (Llano & Esté, 2005). Alternative treatment methods have been developed, targeting host proteins/receptors that are used by the virus to enter and infect a cell, such as maraviroc, which is a CCR5 antagonist (Swenson et al., 2011). As viruses are divided into CCR5- and CXCR4-dependent (Pilcher et al., 2004), it is important to accurately determine this mechanism before applying a specific treatment to successfully reduce/inhibit HIV spread and pathogenesis. However,

Cellular HIV infection involves interactions between glycoprotein gp120, CD4 and CC/CXC receptors (Suresh & Wanchu, 2006). It is therefore possible that an HIV infection can be interrupted and the progression of an established infection can be delayed by targeting CC and/or CXC chemokine receptors. There are two well characterized chemokine receptors by which HIV can bind, enter and infect monocytes, microglia and T-lymphocytes, namely CCR5 and CXCR4 (Ghafouri et al., 2006). CXCL8 dependent activation of CXCR1 has also been suggested to result in inhibited HIV infection and entry into cells (Richardson et al., 2003). Richardson and colleagues showed that CXCR1 activation and internalization resulted in a cross-phosphorylation and internalization of CCR5. Furthermore, C-terminal mutation of CXCR1 internalized both CCR5 and CXCR4 and thus inhibited HIV-1 infection and entry. Furthermore, since HIV-1 competes with CCL5 and CXCL8 for the chemokine receptor DARC (duffy antigen receptor for chemokines) the serum levels of these chemokines may affect the progression of HIV by binding to their respective receptors (He et al., 2008). HIV-1 binding to DARC was also shown to affect chemokine-induced

Even though CXCL8 expression is impaired in HIV-infected cells, pro-inflammatory cytokines such as TNF can induce CXCL8 production and expression from other immune cells. It was recently demonstrated that HIV-infected macrophages secrete TNF and IL-1 that in turn act on astrocytes to induce CXCL8 production (Zheng et al., 2008). CXCL8 production was mediated through the MAPK-associated pathways, including p38, c-Jun Nterminal kinase (JNK) and extracellular signal-regulated kinases (ERK1/2). This inflammatory process plays an important role in the pathogenesis of HIV-associated dementia. Reduction of CXCL8 production can therefore be used to control immune cell migration into the central nervous system, which will reduce the overall inflammatory responses at this site. In addition, CXCL8 is co-localize with CD68/CD40 cells, and CD40 receptor expression on microglial cells act as potent inducers of CXCL8 expression, through AP-1 and NF-B, following ligation of its ligand CD40L, which is expressed on monocytes and T-lymphocytes (D'Aversa et al., 2008). Taken together, these studies show the importance of understanding the regulatory mechanisms leading to chemokine expression in T-cells and other immune cells in order to find a suitable target to control HIV replication

Antiretroviral drugs that target different structural properties in HIV have failed to eradicate the virus but rather suppressed its spreading and pathogenesis. These difficulties have been due to the variability and ability of HIV to internalize without being detected, followed by reactivation (Archer et al., 2009). These drugs are therefore applied in a combination to inhibit several steps of the viral life cycle, including proteases to inhibit maturation, reverse transcription inhibitors and inhibitors for HIV integration into the genome (Arhel & Kirchhoff, 2010). Other difficulties include drug-drug interactions and long-term toxicities (Tilton & Doms, 2010). However, as chemokine levels are severely altered in HIV infected individuals and are involved in HIV replication this has led to an interest in applying this knowledge to controling HIV infections (Llano & Esté, 2005). Alternative treatment methods have been developed, targeting host proteins/receptors that are used by the virus to enter and infect a cell, such as maraviroc, which is a CCR5 antagonist (Swenson et al., 2011). As viruses are divided into CCR5- and CXCR4-dependent (Pilcher et al., 2004), it is important to accurately determine this mechanism before applying a specific treatment to successfully reduce/inhibit HIV spread and pathogenesis. However,

inflammation.

and progression.

although an initial virus infection is CCR5-dependent, a large subset will switch to using CXCR4 as a co-receptor. This is a major concern since maraviroc has little or no effect on CXCR4-dependent viruses (Kuritzkes, 2011). The interplay between different immune cells during an established HIV infection remains important to understand. Determination of cytokine expression by specific cells and the effect that these inflammatory mediators have on other cells to produce chemokines is also important. Further investigations are needed to evaluate the patterns of immune cell activation and cytokine/chemokine regulation in order to find suitable therapeutic targets.

## **4. Probiotic** *Lactobacillus* **as an alternative HIV treatment strategy**

*Lactobacillus* spp are part of the healthy human microbiota, found primarily in the gastrointestinal and vaginal tracts. Certain *Lactobacillus* spp have been identified as health promoting probiotic bacteria by inhibiting pathogen colonization and modulating the immune response in the host (reviewed in Reid et al., 2003). Evidence of immune modulating properties exhibited by certain lactobacilli strains has been shown through their ability to alter cytokine expression in tissue cells infected by pathogens, *in vitro* and *in vivo*, and thus helping maintain homeostasis (Anukam et al., 2009; Frick et al., 2007; Moorthy et al., 2010; Nandakumar et al., 2009; van Hemert et al., 2010; Zhang et al., 2005). Altered cytokine responses, including TNF, NF-B, IL-6, IL-1, CXCL8, IL-10 and IL-12, are dependent on cell type (human mucosal epithelial cells, human mononuclear cells, T-cells, dendritic cells etc) and *Lactobacillus* species and strain (Nandakumar et al., 2009; van Hemert et al., 2010). From these reports, probiotic lactobacilli demonstrate a clear potential for both development of new strategies to reduce the risk of HIV infection and combat AIDS progression through their anti-infective and immune-modulating properties.

Vaginal infections such as bacterial vaginosis (BV) and candidiasis have been correlated with an increased risk of HIV infection (Sha et al., 2005; St John et al., 2007). BV and candidiasis are characterized by microbiota that is comprised of a range of anaerobic bacteria or *Candida albicans*, respectively*,* while deficient in lactobacilli (Sha et al., 2005). Furthermore, women already infected with HIV that lacked vaginal lactobacilli and had BV or candidiasis had higher levels of HIV shedding in the genital tract (Coleman et al., 2007; Spinillo et al., 2005). Increased HIV transcripts in vaginal cells and viral shedding increases the risk of HIV transmission. Furthermore, vaginal secretions from women with BV increased HIV expression in chronically infected monocyte cell line *in vitro*, while secretions from women without BV had no effect on HIV expression (Spear et al., 2007). This is believed to be due to the higher levels of proinflammatory cytokines primarily those that function through NF-B (Al-Harthi et al., 1998). *C. albicans* infections increase vaginal CXCL8 levels and neutrophil presence while higher levels of vaginal lactobacilli reduced CXCL8 levels and other pro-inflammatory cytokines (Spear et al., 2008). CXCL8 is a potent chemokine that recruits neutrophils to the site of infections, thus by reducing chemokine levels, the target cells for HIV infection are limited. Using lactobacilli to modulate the CXCL8 levels and other pro-inflammatory signals may thus reduce the risk of HIV infection and reduce viral replication.

Certain *Lactobacillus* spp have been shown to reduce pro-inflammatory cytokine release from stimulated cells. The probiotic *Lactobacillus rhamnosus* GG reduced the *cxcl8* expression and CXCL8 and CCL11 secretion in TNF or IL-1 - stimulated human intestinal epithelial cells

CXCL8 Regulation and Function in HIV Infections and Potential Treatment Strategies 337

targets immune cells and thereby interferes with the innate immune systems, it is of interest to develop methods to block or reduce the ability of HIV to infect immune cells. Therefore, in order to develop viable CXCL8 based treatment strategies it is important to identify the signaling pathways involved in CXCL8 regulation as well as to determine the function of

Certain lactobacilli have been shown to have immune modulating abilities. There is a clear potential for using probiotic lactobacilli to counter infections, including HIV, as they have both anti-infective and immune-modulating properties. From this aspect, the ideal probiotic *Lactobacillus* species/strain for therapeutic use is one that increases intracellular CXCL8, while maintains a low level of secreted pro-inflammatory cytokines, such as NF-kB, TNF, CXCL8 and IL-6, that promote HIV replication and recruit HIV-target cells. Combining the health promoting properties of lactobacilli with modulation of *cxcl8* expression and release

The present study was made possible by grants from The Knowledge Foundation, Sweden, Sparbanksstiftelsen Nya, Sweden and funding from the Faculty of Business, Science and

Acosta, J.C., O'Loghlen, A., Banito, A., Guijarro, M.V., Augert, A., Raguz, S., Fumagalli, M.,

Acosta, J.C. & Gil, J. (2009). A role for CXCR2 in senescence, but what about in cancer?

Al-Harthi, L., Spear, G.T., Hashemi, F.B., Landay, A., Sha, B.E. & Roebuck, K.A. (1998). A

Alfano, M. & Poli, G. (2005). Role of cytokines and chemokines in the regulation of innate

Ali, S. & Mann, D.A. (2004). Signal transduction via the NF-kappaB pathway: a targeted

Anukam, K.C., Hayes, K., Summers, K. & Reid, G. (2009). Probiotic Lactobacillus rhamnosus

with urinary tract infections: a two-case study. *Advances in urology*: 680363. Archer, J., Braverman, M.S., Taillon, B.E., Desany, B., James, I., Harrigan, P.R., Lewis, M. &

immunity and HIV infection. *Mol Immunol*, Vol. 42 (No. 2): 161-182.

receptor reinforces senescence. *Cell*, Vol. 133 (No. 6): 1006-1018.

Da Costa, M., Brown, C., Popov, N., Takatsu, Y., Melamed, J., d'Adda di Fagagna, F., Bernard, D., Hernando, E. & Gil, J. (2008). Chemokine signaling via the CXCR2

human immunodeficiency virus (HIV)-inducing factor from the female genital tract activates HIV-1 gene expression through the kappaB enhancer. *The Journal of* 

treatment modality for infection, inflammation and repair. *Cell Biochem Funct*, Vol.

GR-1 and Lactobacillus reuteri RC-14 may help downregulate TNF-Alpha, IL-6, IL-8, IL-10 and IL-12 (p70) in the neurogenic bladder of spinal cord injured patient

Robertson, D.L. (2009). Detection of low-frequency pretherapy chemokine (CXC motif) receptor 4 (CXCR4)-using HIV-1 with ultra-deep pyrosequencing. *AIDS*, Vol.

CXCL8 and its receptors in different physiological responses.

can be of great importance in fighting HIV infections.

*Cancer Res*, Vol. 69 (No. 6): 2167-2170.

*Infectious Diseases*, Vol. 178 (No. 5): 1343-1351.

Engineering, Örebro University, Sweden.

22 (No. 2): 67-79.

23 (No. 10): 1209-1218.

**7. Acknowledgement** 

**8. References** 

(Caco-2bbe) by blocking NF-B activation and nuclear translocation (Donato et al., 2010). In the same study, related bacteria *Lactobacillus farciminis* and *Lactobacillus plantarum* RO403 did not alter the CXCL8 or CCL11 levels in the stimulated cells. Others reported that *L. plantarum* 299v showed differential influence on expression and secretion of CXCL8 in HT-29 colonic epithelial cells that were treated with TNF. *L. plantarum* 299v enhanced the *cxcl8* mRNA above that of TNF treatment alone while decreasing CXCL8 secretion from HT-29 cells (McCracken et al., 2002). The *L. plantarum* 299v alone did not induce CXCL8. This is especially interesting since CXCL8 has been shown to decreases transcription of RS-Tropic HIV-1 in peripheral blood lymphocytes and decreases replication in ectocervical tissue explants (Rollenhagen & Asin, 2010; Tiemessen et al., 2000). However, another study had reported that increased levels of CXCL8 stimulated HIV-1 replication in T lymphocytes and macrophages, and this could be significantly inhibited using CXCL8 antibodies or blocking CXCR1 and CXCR2 receptors (Lane et al., 2001). High levels of secreted CXCL8 have been associated with chronic infections in HIV infected persons, thus recruiting and exposing the target cells for HIV infection. Modulation of CXCL8 suggests a potential role for certain strains of lactobacilli in reducing the risk for HIV infection and disease progression.

## **5. Genetically modified lactobacilli for HIV treatment**

Commensal *Lactobacillus* spp from the gastrointestinal and vaginal tract have been considered safe and thus have been used to develop genetically engineered lactobacilli as potential live antiviral-fusion delivery systems. Several investigators have genetically engineered a human isolate of *Lactobacillus jensenii* to secrete fusion inhibitors that target necessary receptors for HIV infection with the aim of being used as a vaginal topical treatment. Chang and colleagues have genetically engineered *L. jensenii* to produce a twodomain CD4 protein that bound the HIV-1 gp120 moderately inhibiting HIV binding and entry into Hela cells expressing CD4-CXCR4 *in vitro* (Chang et al., 2003). Similarly, other fusion inhibitors have been successfully expressed from *L. jensenii* such as the anti-HIV-1 chemokine RANTES and a mutated CCR5 antagonist that showed inhibition of infecting Tcells and macrophages in a concentration dependent manner (Vangelista et al., 2010). A recent patent has been filed for genetically engineered *L. reuteri* RC-14 to be used in treatment of HIV and AIDS after infection by secreting fusion inhibitors in the gastrointestinal tract to reduce or slow the progression of AIDS (Lemke 2010; Patent #US 2010/0143305 A1). One report showed that *L. rhamnosus* GR-1 and *L reuteri* RC-14 did not naturally have the ability to alter RANTES in yeast-infected epithelial cells and *L. rhamnosus* GG did not induce the expression of CCL5 (Martinez et al., 2009; Nandakumar et al., 2009). However, to the authors' knowledge, there has been no systematic evaluation of lactobacilli for inducing HIV-1 fusion inhibitors in cell. The combination of genetically engineered lactobacilli strains to express fusion inhibitor molecules, including CXCR1 and 2 and CXCL8 modulation may further reduce HIV infection and AIDS progression.

#### **6. Conclusions**

It is clear that cytokines and chemokines are important factors in HIV infection and disease progression, making them plausible targets for anti-HIV therapy and to slow the progression to AIDS. CXCL8 is an important factor to consider in HIV therapy, as it is responsible for the recruitment of neutorphils and T-cells to the site of infection. As HIV targets immune cells and thereby interferes with the innate immune systems, it is of interest to develop methods to block or reduce the ability of HIV to infect immune cells. Therefore, in order to develop viable CXCL8 based treatment strategies it is important to identify the signaling pathways involved in CXCL8 regulation as well as to determine the function of CXCL8 and its receptors in different physiological responses.

Certain lactobacilli have been shown to have immune modulating abilities. There is a clear potential for using probiotic lactobacilli to counter infections, including HIV, as they have both anti-infective and immune-modulating properties. From this aspect, the ideal probiotic *Lactobacillus* species/strain for therapeutic use is one that increases intracellular CXCL8, while maintains a low level of secreted pro-inflammatory cytokines, such as NF-kB, TNF, CXCL8 and IL-6, that promote HIV replication and recruit HIV-target cells. Combining the health promoting properties of lactobacilli with modulation of *cxcl8* expression and release can be of great importance in fighting HIV infections.

## **7. Acknowledgement**

The present study was made possible by grants from The Knowledge Foundation, Sweden, Sparbanksstiftelsen Nya, Sweden and funding from the Faculty of Business, Science and Engineering, Örebro University, Sweden.

## **8. References**

336 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

(Caco-2bbe) by blocking NF-B activation and nuclear translocation (Donato et al., 2010). In the same study, related bacteria *Lactobacillus farciminis* and *Lactobacillus plantarum* RO403 did not alter the CXCL8 or CCL11 levels in the stimulated cells. Others reported that *L. plantarum* 299v showed differential influence on expression and secretion of CXCL8 in HT-29 colonic epithelial cells that were treated with TNF. *L. plantarum* 299v enhanced the *cxcl8* mRNA above that of TNF treatment alone while decreasing CXCL8 secretion from HT-29 cells (McCracken et al., 2002). The *L. plantarum* 299v alone did not induce CXCL8. This is especially interesting since CXCL8 has been shown to decreases transcription of RS-Tropic HIV-1 in peripheral blood lymphocytes and decreases replication in ectocervical tissue explants (Rollenhagen & Asin, 2010; Tiemessen et al., 2000). However, another study had reported that increased levels of CXCL8 stimulated HIV-1 replication in T lymphocytes and macrophages, and this could be significantly inhibited using CXCL8 antibodies or blocking CXCR1 and CXCR2 receptors (Lane et al., 2001). High levels of secreted CXCL8 have been associated with chronic infections in HIV infected persons, thus recruiting and exposing the target cells for HIV infection. Modulation of CXCL8 suggests a potential role for certain

strains of lactobacilli in reducing the risk for HIV infection and disease progression.

Commensal *Lactobacillus* spp from the gastrointestinal and vaginal tract have been considered safe and thus have been used to develop genetically engineered lactobacilli as potential live antiviral-fusion delivery systems. Several investigators have genetically engineered a human isolate of *Lactobacillus jensenii* to secrete fusion inhibitors that target necessary receptors for HIV infection with the aim of being used as a vaginal topical treatment. Chang and colleagues have genetically engineered *L. jensenii* to produce a twodomain CD4 protein that bound the HIV-1 gp120 moderately inhibiting HIV binding and entry into Hela cells expressing CD4-CXCR4 *in vitro* (Chang et al., 2003). Similarly, other fusion inhibitors have been successfully expressed from *L. jensenii* such as the anti-HIV-1 chemokine RANTES and a mutated CCR5 antagonist that showed inhibition of infecting Tcells and macrophages in a concentration dependent manner (Vangelista et al., 2010). A recent patent has been filed for genetically engineered *L. reuteri* RC-14 to be used in treatment of HIV and AIDS after infection by secreting fusion inhibitors in the gastrointestinal tract to reduce or slow the progression of AIDS (Lemke 2010; Patent #US 2010/0143305 A1). One report showed that *L. rhamnosus* GR-1 and *L reuteri* RC-14 did not naturally have the ability to alter RANTES in yeast-infected epithelial cells and *L. rhamnosus* GG did not induce the expression of CCL5 (Martinez et al., 2009; Nandakumar et al., 2009). However, to the authors' knowledge, there has been no systematic evaluation of lactobacilli for inducing HIV-1 fusion inhibitors in cell. The combination of genetically engineered lactobacilli strains to express fusion inhibitor molecules, including CXCR1 and 2 and CXCL8

It is clear that cytokines and chemokines are important factors in HIV infection and disease progression, making them plausible targets for anti-HIV therapy and to slow the progression to AIDS. CXCL8 is an important factor to consider in HIV therapy, as it is responsible for the recruitment of neutorphils and T-cells to the site of infection. As HIV

**5. Genetically modified lactobacilli for HIV treatment** 

modulation may further reduce HIV infection and AIDS progression.

**6. Conclusions** 


CXCL8 Regulation and Function in HIV Infections and Potential Treatment Strategies 339

Dower, N.A., Stang, S.L., Bottorff, D.A., Ebinu, J.O., Dickie, P., Ostergaard, H.L. & Stone, J.C.

Ehrlich, L.C., Hu, S., Sheng, W.S., Sutton, R.L., Rockswold, G.L., Peterson, P.K. & Chao, C.C.

Faure, M., Voyno-Yasenetskaya, T.A. & Bourne, H.R. (1994). cAMP and beta gamma

Gabellini, C., Trisciuoglio, D., Desideri, M., Candiloro, A., Ragazzoni, Y., Orlandi, A., Zupi,

Gaide, O., Favier, B., Legler, D.F., Bonnet, D., Brissoni, B., Valitutti, S., Bron, C., Tschopp, J.

Gantner, B.N., Simmons, R.M., Canavera, S.J., Akira, S. & Underhill, D.M. (2003).

Garcia-Vicuna, R., Gomez-Gaviro, M.V., Dominguez-Luis, M.J., Pec, M.K., Gonzalez-Alvaro,

Ghafouri, M., Amini, S., Khalili, K. & Sawaya, B.E. (2006). HIV-1 associated dementia:

Gonzalo, J.A., Tian, J., Delaney, T., Corcoran, J., Rottman, J.B., Lora, J., Al-garawi, A.,

He, W., Neil, S., Kulkarni, H., Wright, E., Agan, B.K., Marconi, V.C., Dolan, M.J., Weiss, R.A.

Hoffmann, E., Dittrich-Breiholz, O., Holtmann, H. & Kracht, M. (2002). Multiple control of

interleukin-8 gene expression. *J Leukoc Biol*, Vol. 72 (No. 5): 847-855.

induced NF-kappa B activation. *Nat Immunol*, Vol. 3 (No. 9): 836-843. Gajewski, T.F., Qian, D., Fields, P. & Fitch, F.W. (1994). Anergic T-lymphocyte clones have

kinase pathway in COS-7 cells. *J Biol Chem*, Vol. 269 (No. 11): 7851-7854. Frick, J.S., Schenk, K., Quitadamo, M., Kahl, F., Koberle, M., Bohn, E., Aepfelbacher, M. &

11): 3288-3297.

*Nat Immunol*, Vol. 1 (No. 4): 317-321.

Vol. 160 (No. 4): 1944-1948.

*diseases*, Vol. 13 (No. 1): 83-90.

*Natl Acad Sci U S A*, Vol. 91 (No. 1): 38-42.

*Arthritis Rheum*, Vol. 50 (No. 12): 3866-3877.

symptoms and causes. *Retrovirology*, Vol. 3: 28.

susceptibility. *Cell Host Microbe*, Vol. 4 (No. 1): 52-62.

receptor 2. *J Exp Med*, Vol. 197 (No. 9): 1107-1117.

2618-2627.

597-604.

barrier dysfunction and pro-inflammatory signalling. *Microbiology*, Vol. 156 (No. Pt

(2000). RasGRP is essential for mouse thymocyte differentiation and TCR signaling.

(1998). Cytokine regulation of human microglial cell IL-8 production. *J Immunol*,

subunits of heterotrimeric G proteins stimulate the mitogen-activated protein

Autenrieth, I.B. (2007). Lactobacillus fermentum attenuates the proinflammatory effect of Yersinia enterocolitica on human epithelial cells. *Inflammatory bowel* 

G. & Del Bufalo, D. (2009). Functional activity of CXCL8 receptors, CXCR1 and CXCR2, on human malignant melanoma progression. *Eur J Cancer*, Vol. 45 (No. 14):

& Thome, M. (2002). CARMA1 is a critical lipid raft-associated regulator of TCR-

altered inositol phosphate, calcium, and tyrosine kinase signaling pathways. *Proc* 

Collaborative induction of inflammatory responses by dectin-1 and Toll-like

I., Alvaro-Gracia, J.M. & Diaz-Gonzalez, F. (2004). CC and CXC chemokine receptors mediate migration, proliferation, and matrix metalloproteinase production by fibroblast-like synoviocytes from rheumatoid arthritis patients.

Kroczek, R., Gutierrez-Ramos, J.C. & Coyle, A.J. (2001). ICOS is critical for T helper cell-mediated lung mucosal inflammatory responses. *Nat Immunol*, Vol. 2 (No. 7):

& Ahuja, S.K. (2008). Duffy antigen receptor for chemokines mediates transinfection of HIV-1 from red blood cells to target cells and affects HIV-AIDS


Arhel, N. & Kirchhoff, F. (2010). Host proteins involved in HIV infection: new therapeutic

Asseman, C., Mauze, S., Leach, M.W., Coffman, R.L. & Powrie, F. (1999). An essential role

Barnes, P.J. & Adcock, I.M. (1997). NF-kappa B: a pivotal role in asthma and a new target for

Berg, D.J., Leach, M.W., Kuhn, R., Rajewsky, K., Muller, W., Davidson, N.J. & Rennick, D.

Bertini, R., Allegretti, M., Bizzarri, C., Moriconi, A., Locati, M., Zampella, G., Cervellera,

Bruder, J.T. & Kovesdi, I. (1997). Adenovirus infection stimulates the Raf/MAPK signaling pathway and induces interleukin-8 expression. *J Virol*, Vol. 71 (No. 1): 398-404. Caramalho, I., Lopes-Carvalho, T., Ostler, D., Zelenay, S., Haury, M. & Demengeot, J. (2003).

Chang, T.L., Chang, C.H., Simpson, D.A., Xu, Q., Martin, P.K., Lagenaur, L.A., Schoolnik,

Coleman, J.S., Hitti, J., Bukusi, E.A., Mwachari, C., Muliro, A., Nguti, R., Gausman, R.,

D'Aversa, T.G., Eugenin, E.A. & Berman, J.W. (2008). CD40-CD40 ligand interactions in

DeForge, L.E., Preston, A.M., Takeuchi, E., Kenney, J., Boxer, L.A. & Remick, D.G. (1993).

Dolcet, X., Llobet, D., Pallares, J. & Matias-Guiu, X. (2005). NF-kB in development and progression of human cancer. *Virchows Arch*, Vol. 446 (No. 5): 475-482. Donato, K.A., Gareau, M.G., Wang, Y.J. & Sherman, P.M. (2010). Lactobacillus rhamnosus

Dinarello, C.A. (2000). Proinflammatory cytokines. *Chest*, Vol. 118 (No. 2): 503-508.

for interleukin 10 in the function of regulatory T cells that inhibit intestinal

(1995). Interleukin 10 but not interleukin 4 is a natural suppressant of cutaneous

M.N., Di Cioccio, V., Cesta, M.C., Galliera, E., Martinez, F.O., Di Bitondo, R., Troiani, G., Sabbatini, V., D'Anniballe, G., Anacardio, R., Cutrin, J.C., Cavalieri, B., Mainiero, F., Strippoli, R., Villa, P., Di Girolamo, M., Martin, F., Gentile, M., Santoni, A., Corda, D., Poli, G., Mantovani, A., Ghezzi, P. & Colotta, F. (2004). Noncompetitive allosteric inhibitors of the inflammatory chemokine receptors CXCR1 and CXCR2: prevention of reperfusion injury. *Proc Natl Acad Sci U S A*, Vol.

Regulatory T cells selectively express toll-like receptors and are activated by

G.K., Ho, D.D., Hillier, S.L., Holodniy, M., Lewicki, J.A. & Lee, P.P. (2003). Inhibition of HIV infectivity by a natural human isolate of Lactobacillus jensenii engineered to express functional two-domain CD4. *Proc Natl Acad Sci U S A*, Vol.

Jensen, S., Patton, D., Lockhart, D., Coombs, R. & Cohen, C.R. (2007). Infectious correlates of HIV-1 shedding in the female upper and lower genital tracts. *AIDS*,

human microglia induce CXCL8 (interleukin-8) secretion by a mechanism dependent on activation of ERK1/2 and nuclear translocation of nuclear factorkappaB (NFkappaB) and activator protein-1 (AP-1). *J Neurosci Res*, Vol. 86 (No. 3):

Regulation of interleukin 8 gene expression by oxidant stress. *J Biol Chem*, Vol. 268

GG attenuates interferon-{gamma} and tumour necrosis factor-alpha-induced

targets. *Biochim Biophys Acta*, Vol. 1802 (No. 3): 313-321.

inflammation. *J Exp Med*, Vol. 190 (No. 7): 995-1004.

therapy. *Trends Pharmacol Sci*, Vol. 18 (No. 2): 46-50.

101 (No. 32): 11791-11796.

100 (No. 20): 11672-11677.

Vol. 21 (No. 6): 755-759.

(No. 34): 25568-25576.

630-639.

inflammatory responses. *J Exp Med*, Vol. 182 (No. 1): 99-108.

lipopolysaccharide. *J Exp Med*, Vol. 197 (No. 4): 403-411.

barrier dysfunction and pro-inflammatory signalling. *Microbiology*, Vol. 156 (No. Pt 11): 3288-3297.


CXCL8 Regulation and Function in HIV Infections and Potential Treatment Strategies 341

McCarroll, J.A., Phillips, P.A., Park, S., Doherty, E., Pirola, R.C., Wilson, J.S. & Apte, M.V.

McCracken, V.J., Chun, T., Baldeon, M.E., Ahrne, S., Molin, G., Mackie, R.I. & Gaskins, H.R.

McKay, L.I. & Cidlowski, J.A. (1999). Molecular control of immune/inflammatory

Moorthy, G., Murali, M.R. & Niranjali Devaraj, S. (2010). Lactobacilli inhibit Shigella

Nandakumar, N.S., Pugazhendhi, S. & Ramakrishna, B.S. (2009). Effects of enteropathogenic

Narayan, P., Holt, B., Tosti, R. & Kane, L.P. (2006). CARMA1 is required for Akt-mediated NF-kappaB activation in T cells. *Mol Cell Biol*, Vol. 26 (No. 6): 2327-2336. Noel, R.F., Jr., Sato, T.T., Mendez, C., Johnson, M.C. & Pohlman, T.H. (1995). Activation of

Opal, S.M. & DePalo, V.A. (2000). Anti-inflammatory cytokines. *Chest*, Vol. 117 (No. 4): 1162-

Ott, M., Lovett, J.L., Mueller, L. & Verdin, E. (1998). Superinduction of IL-8 in T cells by HIV-

Pearson, T.A., Mensah, G.A., Alexander, R.W., Anderson, J.L., Cannon, R.O., 3rd, Criqui, M.,

the American Heart Association. *Circulation*, Vol. 107 (No. 3): 499-511. Philip, M., Rowley, D.A. & Schreiber, H. (2004). Inflammation as a tumor promoter in cancer

Pilcher, C.D., Eron, J.J., Jr., Galvin, S., Gay, C. & Cohen, M.S. (2004). Acute HIV revisited:

Reid, G., Jass, J., Sebulsky, M.T. & McCormick, J.K. (2003). Potential uses of probiotics in clinical practice. *Clinical microbiology reviews*, Vol. 16 (No. 4): 658-672. Richardson, R.M., Tokunaga, K., Marjoram, R., Sata, T. & Snyderman, R. (2003). Interleukin-

new opportunities for treatment and prevention. *J Clin Invest*, Vol. 113 (No. 7): 937-

8-mediated heterologous receptor internalization provides resistance to HIV-1

*Experimental biology and medicine*, Vol. 227 (No. 8): 665-670.

signaling pathways. *Endocr Rev*, Vol. 20 (No. 4): 435-459.

soluble CD14. *Infect Immun*, Vol. 63 (No. 10): 4046-4053.

induction. *Semin Cancer Biol*, Vol. 14 (No. 6): 433-439.

2): 150-160.

*Liver*, Vol. 42 (No. 1): 33-39.

130 (No. 2): 170-178.

1172.

945.

2872-2880.

(2003). Pancreatic stellate cell activation by ethanol and acetaldehyde: is it mediated by the mitogen-activated protein kinase signaling pathway? *Pancreas*, Vol. 27 (No.

(2002). TNF-alpha sensitizes HT-29 colonic epithelial cells to intestinal lactobacilli.

responses: interactions between nuclear factor-kappa B and steroid receptor-

dysenteriae 1 induced pro-inflammatory response and cytotoxicity in host cells via impediment of Shigella-host interactions. *Digestive and liver disease : official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the* 

bacteria & lactobacilli on chemokine secretion & Toll like receptor gene expression in two human colonic epithelial cell lines. *The Indian journal of medical research*, Vol.

human endothelial cells by viable or heat-killed gram-negative bacteria requires

1 Tat protein is mediated through NF-kappaB factors. *J Immunol*, Vol. 160 (No. 6):

Fadl, Y.Y., Fortmann, S.P., Hong, Y., Myers, G.L., Rifai, N., Smith, S.C., Jr., Taubert, K., Tracy, R.P. & Vinicor, F. (2003). Markers of inflammation and cardiovascular disease: application to clinical and public health practice: A statement for healthcare professionals from the Centers for Disease Control and Prevention and


Hummelen, R., Changalucha, J., Butamanya, N.L., Cook, A., Habbema, J.D. & Reid, G.

Itahana, K., Dimri, G. & Campisi, J. (2001). Regulation of cellular senescence by p53. *Eur J* 

Karl, E., Warner, K., Zeitlin, B., Kaneko, T., Wurtzel, L., Jin, T., Chang, J., Wang, S., Wang,

Khalaf, H., Jass, J. & Olsson, P.E. (2010). Differential cytokine regulation by NF-kappaB and

Khandaker, M.H., Mitchell, G., Xu, L., Andrews, J.D., Singh, R., Leung, H., Madrenas, J.,

Kim, J.J., Nottingham, L.K., Sin, J.I., Tsai, A., Morrison, L., Oh, J., Dang, K., Hu, Y.,

Kuritzkes, D.R. (2011). Genotypic tests for determining coreceptor usage of HIV-1. *J Infect* 

Lane, B.R., Lore, K., Bock, P.J., Andersson, J., Coffey, M.J., Strieter, R.M. & Markovitz, D.M.

Li, A., Dubey, S., Varney, M.L., Dave, B.J. & Singh, R.K. (2003). IL-8 directly enhanced

Li, J., Kartha, S., Iasvovskaia, S., Tan, A., Bhat, R.K., Manaligod, J.M., Page, K., Brasier, A.R.

Libby, P., Ridker, P.M. & Maseri, A. (2002). Inflammation and atherosclerosis. *Circulation*,

Llano, A. & Esté, J.A. (2005). Chemokines and other cytokines in human immunodeficiency

Makarov, S.S. (2000). NF-kappaB as a therapeutic target in chronic inflammation: recent

Martinez, R.C., Seney, S.L., Summers, K.L., Nomizo, A., De Martinis, E.C. & Reid, G. (2009).

Effect of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 on the ability of Candida albicans to infect cells and induce inflammation. *Microbiology and* 

and regulated angiogenesis. *J Immunol*, Vol. 170 (No. 6): 3369-3376.

virus type 1 (HIV-1) infection. *Inmunología*, Vol. 24: 246-260.

advances. *Mol Med Today*, Vol. 6 (No. 11): 441-448.

*immunology*, Vol. 53 (No. 9): 487-495.

245-248.

2185.

102 (No. 6): 1112-1124.

(No. 17): 8195-8202.

L690-699.

*Dis*, Vol. 203 (No. 2): 146-148.

Vol. 105 (No. 9): 1135-1143.

*Biochem*, Vol. 268 (No. 10): 2784-2791.

chemokines. *Cancer Res*, Vol. 65 (No. 12): 5063-5069.

AP-1 in Jurkat T-cells. *BMC Immunol*, Vol. 11: 26.

(2010). Lactobacillus rhamnosus GR-1 and L. reuteri RC-14 to prevent or cure bacterial vaginosis among women with HIV. *Int J Gynaecol Obstet*, Vol. 111 (No. 3):

C.Y., Strieter, R.M., Nunez, G., Polverini, P.J. & Nor, J.E. (2005). Bcl-2 acts in a proangiogenic signaling pathway through nuclear factor-kappaB and CXC

Ferguson, S.S., Feldman, R.D. & Kelvin, D.J. (1999). Metalloproteinases are involved in lipopolysaccharide- and tumor necrosis factor-alpha-mediated regulation of CXCR1 and CXCR2 chemokine receptor expression. *Blood*, Vol. 93 (No. 7): 2173-

Kazahaya, K., Bennett, M., Dentchev, T., Wilson, D.M., Chalian, A.A., Boyer, J.D., Agadjanyan, M.G. & Weiner, D.B. (1998). CD8 positive T cells influence antigenspecific immune responses through the expression of chemokines. *J Clin Invest*, Vol.

(2001). Interleukin-8 stimulates human immunodeficiency virus type 1 replication and is a potential new target for antiretroviral therapy. *Journal of virology*, Vol. 75

endothelial cell survival, proliferation, and matrix metalloproteinases production

& Hershenson, M.B. (2002). Regulation of human airway epithelial cell IL-8 expression by MAP kinases. *Am J Physiol Lung Cell Mol Physiol*, Vol. 283 (No. 4):


CXCL8 Regulation and Function in HIV Infections and Potential Treatment Strategies 343

Taha, T.E., Hoover, D.R., Dallabetta, G.A., Kumwenda, N.I., Mtimavalye, L.A., Yang, L.P.,

Takata, H., Tomiyama, H., Fujiwara, M., Kobayashi, N. & Takiguchi, M. (2004). Cutting

Teixeira, C., Stang, S.L., Zheng, Y., Beswick, N.S. & Stone, J.C. (2003). Integration of DAG

Tergaonkar, V. (2006). NFkappaB pathway: a good signaling paradigm and therapeutic

Tiemessen, C.T., Kilroe, B. & Martin, D.J. (2000). Interleukin-8 fails to induce human

Tilton, J.C. & Doms, R.W. (2010). Entry inhibitors in the treatment of HIV-1 infection.

van der Poll, T., Jansen, P.M., Montegut, W.J., Braxton, C.C., Calvano, S.E., Stackpole, S.A.,

van Hemert, S., Meijerink, M., Molenaar, D., Bron, P.A., de Vos, P., Kleerebezem, M., Wells,

Vangelista, L., Secchi, M., Liu, X., Bachi, A., Jia, L., Xu, Q. & Lusso, P. (2010). Engineering

Walley, K.R., Lukacs, N.W., Standiford, T.J., Strieter, R.M. & Kunkel, S.L. (1996). Balance of

Werlen, G., Jacinto, E., Xia, Y. & Karin, M. (1998). Calcineurin preferentially synergizes with

Wong, M.M. & Fish, E.N. (2003). Chemokines: attractive mediators of the immune response.

Xu, X.H., Shah, P.K., Faure, E., Equils, O., Thomas, L., Fishbein, M.C., Luthringer, D., Xu,

target. *Int J Biochem Cell Biol*, Vol. 38 (No. 10): 1647-1653.

endotoxemia. *J Immunol*, Vol. 158 (No. 4): 1971-1975.

of HIV. *AIDS*, Vol. 12 (No. 13): 1699-1706.

*Immunol*, Vol. 173 (No. 4): 2231-2235.

*Blood*, Vol. 102 (No. 4): 1414-1420.

*Antiviral Res*, Vol. 85 (No. 1): 91-100.

*BMC microbiology*, Vol. 10: 293.

*Immun*, Vol. 64 (No. 11): 4733-4738.

*Semin Immunol*, Vol. 15 (No. 1): 5-14.

3001.

3108.

11): 3101-3111.

Vol. 101 (No. 1): 140-146.

245.

of maraviroc in treatment-experienced patients. *J Infect Dis*, Vol. 203 (No. 2): 237-

Liomba, G.N., Broadhead, R.L., Chiphangwi, J.D. & Miotti, P.G. (1998). Bacterial vaginosis and disturbances of vaginal flora: association with increased acquisition

edge: expression of chemokine receptor CXCR1 on human effector CD8+ T cells. *J* 

signaling systems mediated by PKC-dependent phosphorylation of RasGRP3.

immunodeficiency virus-1 expression in chronically infected promonocytic U1 cells but differentially modulates induction by proinflammatory cytokines. *Immunology*,

Smith, S.R., Swanson, S.W., Hack, C.E., Lowry, S.F. & Moldawer, L.L. (1997). Effects of IL-10 on systemic inflammatory responses during sublethal primate

J.M. & Marco, M.L. (2010). Identification of Lactobacillus plantarum genes modulating the cytokine response of human peripheral blood mononuclear cells.

of Lactobacillus jensenii to secrete RANTES and a CCR5 antagonist analogue as live HIV-1 blockers. *Antimicrobial agents and chemotherapy*, Vol. 54 (No. 7): 2994-

inflammatory cytokines related to severity and mortality of murine sepsis. *Infect* 

PKC-theta to activate JNK and IL-2 promoter in T lymphocytes. *Embo J*, Vol. 17 (No.

X.P., Rajavashisth, T.B., Yano, J., Kaul, S. & Arditi, M. (2001). Toll-like receptor-4 is expressed by macrophages in murine and human lipid-rich atherosclerotic plaques and upregulated by oxidized LDL. *Circulation*, Vol. 104 (No. 25): 3103-

infectivity. Role of signal strength and receptor desensitization. *J Biol Chem*, Vol. 278 (No. 18): 15867-15873.


Riedel, C.U., Foata, F., Philippe, D., Adolfsson, O., Eikmanns, B.J. & Blum, S. (2006). Anti-

Rollenhagen, C. & Asin, S.N. (2010). IL-8 decreases HIV-1 transcription in peripheral blood

Sato, M., Sano, H., Iwaki, D., Kudo, K., Konishi, M., Takahashi, H., Takahashi, T., Imaizumi,

Scharschmidt, E., Wegener, E., Heissmeyer, V., Rao, A. & Krappmann, D. (2004).

Sha, B.E., Zariffard, M.R., Wang, Q.J., Chen, H.Y., Bremer, J., Cohen, M.H. & Spear, G.T.

Shoji, Y., Uedono, Y., Ishikura, H., Takeyama, N. & Tanaka, T. (1995). DNA damage induced

Spear, G.T., St John, E. & Zariffard, M.R. (2007). Bacterial vaginosis and human

Spear, G.T., Zariffard, M.R., Cohen, M.H. & Sha, B.E. (2008). Vaginal IL-8 levels are

infected women. *Journal of reproductive immunology*, Vol. 78 (No. 1): 76-79. Spinillo, A., Zara, F., Gardella, B., Preti, E., Mainini, R. & Maserati, R. (2005). The effect of

St John, E., Mares, D. & Spear, G.T. (2007). Bacterial vaginosis and host immunity. *Current* 

Sturm, A., Baumgart, D.C., d'Heureuse, J.H., Hotz, A., Wiedenmann, B. & Dignass, A.U.

Suresh, P. & Wanchu, A. (2006). Chemokines and chemokine receptors in HIV infection: role in pathogenesis and therapeutics. *J Postgrad Med*, Vol. 52 (No. 3): 210-217. Svanborg, C., Godaly, G. & Hedlund, M. (1999). Cytokine responses during mucosal

Swenson, L.C., Mo, T., Dong, W.W., Zhong, X., Woods, C.K., Jensen, M.A., Thielen, A.,

activation. *World J Gastroenterol*, Vol. 12 (No. 23): 3729-3735.

signaling. *Mol Cell Biol*, Vol. 24 (No. 9): 3860-3873.

radical formation. *Immunology*, Vol. 84 (No. 4): 543-548.

immunodeficiency virus infection. *AIDS Res Ther*, Vol. 4: 25.

*infectious diseases*, Vol. 191 (No. 1): 25-32.

*HIV/AIDS reports*, Vol. 4 (No. 1): 22-28.

dependent pathway. *Cytokine*, Vol. 29 (No. 1): 42-48.

(No. 18): 15867-15873.

(No. 5): 463-469.

425.

3): 774-779.

(No. 1): 99-105.

infectivity. Role of signal strength and receptor desensitization. *J Biol Chem*, Vol. 278

inflammatory effects of bifidobacteria by inhibition of LPS-induced NF-kappaB

lymphocytes and ectocervical tissue explants. *J Acquir Immune Defic Syndr*, Vol. 54

H., Asai, Y. & Kuroki, Y. (2003). Direct binding of Toll-like receptor 2 to zymosan, and zymosan-induced NF-kappa B activation and TNF-alpha secretion are downregulated by lung collectin surfactant protein A. *J Immunol*, Vol. 171 (No. 1): 417-

Degradation of Bcl10 induced by T-cell activation negatively regulates NF-kappa B

(2005). Female genital-tract HIV load correlates inversely with Lactobacillus species but positively with bacterial vaginosis and Mycoplasma hominis. *The Journal of* 

by tumour necrosis factor-alpha in L929 cells is mediated by mitochondrial oxygen

positively associated with Candida albicans and inversely with lactobacilli in HIV-

vaginal candidiasis on the shedding of human immunodeficiency virus in cervicovaginal secretions. *American journal of obstetrics and gynecology*, Vol. 192 (No.

(2005). CXCL8 modulates human intestinal epithelial cells through a CXCR1

infections: role in disease pathogenesis and host defence. *Curr Opin Microbiol*, Vol. 2

Chapman, D., Lewis, M., James, I., Heera, J., Valdez, H. & Harrigan, P.R. (2011). Deep sequencing to infer HIV-1 co-receptor usage: application to three clinical trials of maraviroc in treatment-experienced patients. *J Infect Dis*, Vol. 203 (No. 2): 237- 245.


**14** 

*Canada* 

**Emerging Roles of Prostaglandins** 

Nancy Dumais, Sandra C. Côté and Anne-Marie Ducharme

Prostaglandins (PG), generated by cyclooxygenase (COX), are a group of lipid mediators formed in response to various stimuli. They include PGD2, PGE2, PGF2alpha, and PGI2. Immediately after synthesis, they are released outside the cell and exert their actions by binding to a G-protein-coupled rhodopsin-type receptor on the surface of target cells. There are seven types of prostaglandin receptors: the PGD receptor, four subtypes of PGE receptor, the PGF receptor, and the PGI receptor. Prostaglandins are involved in host defense against various pathogens. Along with mediating inflammatory symptoms, PGs might suppress some innate immune factors, including nitric oxide (NO) production. These immunomodulatory molecules have been shown to participate in the regulation of virus replication and the modulation of inflammatory responses following infection. Moreover, virus infection also stimulates the expression of a number of proinflammatory gene products, including COX-2, inducible nitric oxide synthase (iNOS) as well as

An overproduction of PGE2 (as high as 10−4M) is seen in a number of disorders (e.g. allergy, hyper-IgE syndrome, Hodgkin lymphoma, trauma, sepsis, and transplantation), most of which are characterized by elevated Th2 and IgE responses. Elevated levels of PGE2 have also been reported in individuals infected with HIV-1 and it has been postulated that this may contribute to the immunosuppressive state seen in such virally infected patients. The mechanism(s) responsible for the enhanced prostaglandin formation is still undefined. The initial contact between the virus particle and its target cell might represent the crucial step leading to the production of PGE2 by macrophages. This concept is supported by the finding that a significant production of endogenous PGE2 is induced (20- to 40-fold increase) following incubation of primary human monocytes with the HIV-1 external envelope glycoprotein gp120. Given that pro-inflammatory molecules such as PGE2 are up-regulated during HIV-1 infection, an imbalance in PGJ2 production is observed in HIV+ individuals. This book chapter will focus on roles of prostaglandins in HIV-1 replication and their potential therapeutic implications. We propose to review mechanisms by which the proinflammatory prostaglandin PGE2 and the anti-inflammatory prostaglandin PGJ2 regulate HIV-1 transcription and replication. Specific attention will be placed on how prostaglandins affect the nuclear translocation of NF-B (nuclear factor kappa B), an essential transcription factor for HIV-1 transcription. In addition, signaling pathways as well as other transcription

**1. Introduction** 

proinflammatory cytokines.

**in HIV-1 Transcription** 

*Université de Sherbrooke* 


## **Emerging Roles of Prostaglandins in HIV-1 Transcription**

Nancy Dumais, Sandra C. Côté and Anne-Marie Ducharme *Université de Sherbrooke Canada* 

## **1. Introduction**

344 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

Zhang, L., Li, N., Caicedo, R. & Neu, J. (2005). Alive and dead Lactobacillus rhamnosus GG

Zheng, J.C., Huang, Y., Tang, K., Cui, M., Niemann, D., Lopez, A., Morgello, S. & Chen, S.

Zlotnik, A. & Yoshie, O. (2000). Chemokines: a new classification system and their role in

cells. *The Journal of nutrition*, Vol. 135 (No. 7): 1752-1756.

immunity. *Immunity*, Vol. 12 (No. 2): 121-127.

100-110.

decrease tumor necrosis factor-alpha-induced interleukin-8 production in Caco-2

(2008). HIV-1-infected and/or immune-activated macrophages regulate astrocyte CXCL8 production through IL-1beta and TNF-alpha: involvement of mitogenactivated protein kinases and protein kinase R. *J Neuroimmunol*, Vol. 200 (No. 1-2):

> Prostaglandins (PG), generated by cyclooxygenase (COX), are a group of lipid mediators formed in response to various stimuli. They include PGD2, PGE2, PGF2alpha, and PGI2. Immediately after synthesis, they are released outside the cell and exert their actions by binding to a G-protein-coupled rhodopsin-type receptor on the surface of target cells. There are seven types of prostaglandin receptors: the PGD receptor, four subtypes of PGE receptor, the PGF receptor, and the PGI receptor. Prostaglandins are involved in host defense against various pathogens. Along with mediating inflammatory symptoms, PGs might suppress some innate immune factors, including nitric oxide (NO) production. These immunomodulatory molecules have been shown to participate in the regulation of virus replication and the modulation of inflammatory responses following infection. Moreover, virus infection also stimulates the expression of a number of proinflammatory gene products, including COX-2, inducible nitric oxide synthase (iNOS) as well as proinflammatory cytokines.

> An overproduction of PGE2 (as high as 10−4M) is seen in a number of disorders (e.g. allergy, hyper-IgE syndrome, Hodgkin lymphoma, trauma, sepsis, and transplantation), most of which are characterized by elevated Th2 and IgE responses. Elevated levels of PGE2 have also been reported in individuals infected with HIV-1 and it has been postulated that this may contribute to the immunosuppressive state seen in such virally infected patients. The mechanism(s) responsible for the enhanced prostaglandin formation is still undefined. The initial contact between the virus particle and its target cell might represent the crucial step leading to the production of PGE2 by macrophages. This concept is supported by the finding that a significant production of endogenous PGE2 is induced (20- to 40-fold increase) following incubation of primary human monocytes with the HIV-1 external envelope glycoprotein gp120. Given that pro-inflammatory molecules such as PGE2 are up-regulated during HIV-1 infection, an imbalance in PGJ2 production is observed in HIV+ individuals.

> This book chapter will focus on roles of prostaglandins in HIV-1 replication and their potential therapeutic implications. We propose to review mechanisms by which the proinflammatory prostaglandin PGE2 and the anti-inflammatory prostaglandin PGJ2 regulate HIV-1 transcription and replication. Specific attention will be placed on how prostaglandins affect the nuclear translocation of NF-B (nuclear factor kappa B), an essential transcription factor for HIV-1 transcription. In addition, signaling pathways as well as other transcription

Emerging Roles of Prostaglandins in HIV-1 Transcription 347

PGE2, while lymphocytes do not secrete this major product of arachidonic acid metabolism (Frey et al., 1986; Heinen et al., 1986; Kurland & Bockman, 1978; Phipps et al., 1988). A marked increase in PGE2 production is generated in response to a variety of immunological stimuli including interleukin (IL)-1, tumor necrosis factor-α (TNF-α), antigen-antibody complexes, and lipopolysaccharide (Roper & Phipps, 1994) in addition to exposition to microorganisms. PGE2 has been implicated in decreasing T-cell proliferation, IL-2 production, and IL-2 receptor expression (Goodwin et al., 1977; Goodwin & Ceuppens, 1983; Rincon et al., 1988; Roper & Phipps, 1994; Walker et al., 1983). PGE2 shifts the balance of the cellular immune response away from T-helper type 1 (Th1) favouring a Th2 response which drives humoral responses toward the production of IgE (Fedyk & Phipps, 1996). However, other findings have depicted PGE2 as a pleiotropic molecule that can act both negatively or positively on the immune system (Phipps et al., 1991). Depending on the cell type, binding of PGE2 to one of its six described receptors (EP1, EP2, EP3I, EP3II, EP3III, and EP4) can lead to phospholipase C activation, phosphatidylinositol turnover increase, activation of adenylate cyclase through cholera toxin-sensitive Gs proteins and mobilization of intracellular Ca2+ concentration (Coleman et al., 1994). PGE2 facilitates expansion of the Th17 subset of T helper cells of both human and mouse through elevation of cAMP via PGE2 receptors EP2

The balance of opposing prostaglandins produced in tissues profoundly influences inflammatory responses (Harris et al., 2002). The J series of prostaglandins are the end product metabolites of PGD2 and are abundantly produced by mast cells, platelets, as well as alveolar macrophages (Ito et al., 1989; Straus & Glass, 2001). One of these molecules, 15-d-PGJ2, is a natural activator of the peroxisome proliferators-activated receptor-γ (PPAR-γ), a nuclear receptor family member that elicits anti-inflammatory activities in macrophages (Hinz et al., 2003; Hortelano et al., 2000; Jiang et al., 1998; Ricote et al., 1998), lymphocytes (Clark et al., 2000; Padilla et al., 2000; Yang et al., 2000), dendritic cells (Faveeuw et al., 2000), and endothelial cells (Imaizumi et al., 2003). Currently, the mechanisms regulating the antiinflammatory effects of 15-d-PGJ2 and other PPAR-γ agonists are poorly understood, but it has been suggested to involve the inhibition of the nuclear factor B (NF-B) signaling pathway (Daynes & Jones, 2002; Rossi et al., 2000). PPAR-γ is also expressed at high levels both in the colonic epithelium and intestinal epithelial cells (Lefebvre et al., 1998; Saez et al., 2004; Saez et al., 1998; Sarraf et al., 1998; Sarraf et al., 1999), where, depending on the model system studied, it can result in either an increase or a decrease in proliferation (Brockman et

An overproduction of PGE2 as high as 10−4M is seen in a number of disorders (e.g.allergy, hyper-IgE syndrome, Hodgkin lymphoma, trauma, sepsis, and transplantation), most of which are characterized by elevated Th2 and IgE responses (Fedyk & Phipps, 1996; Haraguchi et al., 1995a; Phipps et al., 1991; Roper & Phipps, 1994). Elevated levels of PGE2 have also been reported in individuals infected with HIV-1 (Abel et al., 1992; Foley et al., 1992; Griffin et al., 1994a; Ramis et al., 1992) and it has been postulated that this may contribute to the immunosuppressive state seen in such virally-infected patients (Hui et al., 1995). *In vitro*, peripheral blood monocytes and macrophages from AIDS patients exhibit abnormal production of cyclooxygenase products (Coffey et al., 1999; Fernandez-Cruz et al., 1989; Foley et al., 1992; Mastino et al., 1993; Ramis et al., 1991). The mechanism(s)

and EP4 (Sakata et al., 2010).

al., 1998; Lefebvre et al., 1998).

**2.2 Prostaglandins and HIV infection** 

factors that are activated or repressed by prostaglandins that regulate HIV-1 gene expression will be reviewed.

#### **2. Prostaglandins: Their synthesis and roles in the regulation of inflammation**

#### **2.1 Prostaglandins synthesis**

The initial reaction in prostaglandin production is phospholipase A2 (PLA2)-mediated liberation of a 20-carbon essential fatty acid, arachidonic acid, from membrane phospholipids. Cyclooxygenase (COX) is the rate-limiting enzymes catalysing oxidation of arachidonic acid to the hydroperoxyendoperoxide, prostaglandin G2 (PGG2). Subsequently, PGG2 is reduced to form the hydroxylendoperoxide, prostaglandin H2 (PGH2). Then, prostanoids including prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin F2alpha (PGF2α), prostacyclin (PGI2), and thromboxane A2 (TXA2) are formed by the action of discrete prostaglandins synthases (reviewed in Coleman et al., 1994). Figure 1 reviews the arachidonic cascade.

Fig. 1. A model for prostaglandins synthesis.

Prostaglandin E2 (PGE2), an oxygenated polyunsaturated fatty acid that contains a cyclopentane ring structure, is present in high concentrations in individuals infected with numerous pathogens (Abel et al., 1992; Ben-Hur et al., 1996; Farrell & Kirkpatrick, 1987; Foley et al., 1992; Griffin et al., 1994a; Henke et al., 1992; Kernacki & Berk, 1994; Midulla et al., 1989; Onta et al., 1993; Ramis et al., 1992; Rastogi et al., 1992; Reiner & Malemud, 1984; Sorrell et al., 1989; Wang & Chadee, 1992, 1995). PGE2 are molecules that have been shown to modulate the immune response both *in vitro* and *in vivo* (Goodwin & Webb, 1980). Macrophages, follicular dendritic cells, fibroblasts, and vascular endothelial cells synthesize

factors that are activated or repressed by prostaglandins that regulate HIV-1 gene

**2. Prostaglandins: Their synthesis and roles in the regulation of inflammation** 

The initial reaction in prostaglandin production is phospholipase A2 (PLA2)-mediated liberation of a 20-carbon essential fatty acid, arachidonic acid, from membrane phospholipids. Cyclooxygenase (COX) is the rate-limiting enzymes catalysing oxidation of arachidonic acid to the hydroperoxyendoperoxide, prostaglandin G2 (PGG2). Subsequently, PGG2 is reduced to form the hydroxylendoperoxide, prostaglandin H2 (PGH2). Then, prostanoids including prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin F2alpha (PGF2α), prostacyclin (PGI2), and thromboxane A2 (TXA2) are formed by the action of discrete prostaglandins

Prostaglandin E2 (PGE2), an oxygenated polyunsaturated fatty acid that contains a cyclopentane ring structure, is present in high concentrations in individuals infected with numerous pathogens (Abel et al., 1992; Ben-Hur et al., 1996; Farrell & Kirkpatrick, 1987; Foley et al., 1992; Griffin et al., 1994a; Henke et al., 1992; Kernacki & Berk, 1994; Midulla et al., 1989; Onta et al., 1993; Ramis et al., 1992; Rastogi et al., 1992; Reiner & Malemud, 1984; Sorrell et al., 1989; Wang & Chadee, 1992, 1995). PGE2 are molecules that have been shown to modulate the immune response both *in vitro* and *in vivo* (Goodwin & Webb, 1980). Macrophages, follicular dendritic cells, fibroblasts, and vascular endothelial cells synthesize

synthases (reviewed in Coleman et al., 1994). Figure 1 reviews the arachidonic cascade.

expression will be reviewed.

**2.1 Prostaglandins synthesis** 

Fig. 1. A model for prostaglandins synthesis.

PGE2, while lymphocytes do not secrete this major product of arachidonic acid metabolism (Frey et al., 1986; Heinen et al., 1986; Kurland & Bockman, 1978; Phipps et al., 1988). A marked increase in PGE2 production is generated in response to a variety of immunological stimuli including interleukin (IL)-1, tumor necrosis factor-α (TNF-α), antigen-antibody complexes, and lipopolysaccharide (Roper & Phipps, 1994) in addition to exposition to microorganisms. PGE2 has been implicated in decreasing T-cell proliferation, IL-2 production, and IL-2 receptor expression (Goodwin et al., 1977; Goodwin & Ceuppens, 1983; Rincon et al., 1988; Roper & Phipps, 1994; Walker et al., 1983). PGE2 shifts the balance of the cellular immune response away from T-helper type 1 (Th1) favouring a Th2 response which drives humoral responses toward the production of IgE (Fedyk & Phipps, 1996). However, other findings have depicted PGE2 as a pleiotropic molecule that can act both negatively or positively on the immune system (Phipps et al., 1991). Depending on the cell type, binding of PGE2 to one of its six described receptors (EP1, EP2, EP3I, EP3II, EP3III, and EP4) can lead to phospholipase C activation, phosphatidylinositol turnover increase, activation of adenylate cyclase through cholera toxin-sensitive Gs proteins and mobilization of intracellular Ca2+ concentration (Coleman et al., 1994). PGE2 facilitates expansion of the Th17 subset of T helper cells of both human and mouse through elevation of cAMP via PGE2 receptors EP2 and EP4 (Sakata et al., 2010).

The balance of opposing prostaglandins produced in tissues profoundly influences inflammatory responses (Harris et al., 2002). The J series of prostaglandins are the end product metabolites of PGD2 and are abundantly produced by mast cells, platelets, as well as alveolar macrophages (Ito et al., 1989; Straus & Glass, 2001). One of these molecules, 15-d-PGJ2, is a natural activator of the peroxisome proliferators-activated receptor-γ (PPAR-γ), a nuclear receptor family member that elicits anti-inflammatory activities in macrophages (Hinz et al., 2003; Hortelano et al., 2000; Jiang et al., 1998; Ricote et al., 1998), lymphocytes (Clark et al., 2000; Padilla et al., 2000; Yang et al., 2000), dendritic cells (Faveeuw et al., 2000), and endothelial cells (Imaizumi et al., 2003). Currently, the mechanisms regulating the antiinflammatory effects of 15-d-PGJ2 and other PPAR-γ agonists are poorly understood, but it has been suggested to involve the inhibition of the nuclear factor B (NF-B) signaling pathway (Daynes & Jones, 2002; Rossi et al., 2000). PPAR-γ is also expressed at high levels both in the colonic epithelium and intestinal epithelial cells (Lefebvre et al., 1998; Saez et al., 2004; Saez et al., 1998; Sarraf et al., 1998; Sarraf et al., 1999), where, depending on the model system studied, it can result in either an increase or a decrease in proliferation (Brockman et al., 1998; Lefebvre et al., 1998).

#### **2.2 Prostaglandins and HIV infection**

An overproduction of PGE2 as high as 10−4M is seen in a number of disorders (e.g.allergy, hyper-IgE syndrome, Hodgkin lymphoma, trauma, sepsis, and transplantation), most of which are characterized by elevated Th2 and IgE responses (Fedyk & Phipps, 1996; Haraguchi et al., 1995a; Phipps et al., 1991; Roper & Phipps, 1994). Elevated levels of PGE2 have also been reported in individuals infected with HIV-1 (Abel et al., 1992; Foley et al., 1992; Griffin et al., 1994a; Ramis et al., 1992) and it has been postulated that this may contribute to the immunosuppressive state seen in such virally-infected patients (Hui et al., 1995). *In vitro*, peripheral blood monocytes and macrophages from AIDS patients exhibit abnormal production of cyclooxygenase products (Coffey et al., 1999; Fernandez-Cruz et al., 1989; Foley et al., 1992; Mastino et al., 1993; Ramis et al., 1991). The mechanism(s)

Emerging Roles of Prostaglandins in HIV-1 Transcription 349

patients. The formation and production of elevated levels of inflammatory mediators such as PGE2 is a hallmark of the HIV-1 infection (Foley et al., 1992; Griffin et al., 1994a; Ramis et al., 1992). Prostaglandins play a role in disease exacerbation by directly altering T-cell functions or macrophage activation. Although it was thought that PGE2 is primarily an immunosuppressive molecule that acts as a down-regulator of many aspects of B- and T-cell function and proliferation, other findings support a role for PGE2 as a potentiator of immunoglobulin class switching and cytokines and cytokine receptors synthesis (Phipps et al., 1991). Moreover, knowing that PGE2 is a good inducer of cAMP and that a 4-fold increase in intracellular levels of cAMP is seen in asymptomatic HIV-1-seropositive subjects as compared with uninfected controls (Hofmann et al., 1993), it is thus of prime importance to study the putative effect of PGE2 on the regulatory elements of HIV-1 in T cells considered to be the major cellular reservoir for HIV-1 in the human peripheral blood. As shown in Fig 3, exogenous PGE2 could further increase the overall positive effect mediated by various HIV-1 LTR-activating agents confirming that PGE2 could be considered by itself

Fig. 3. Activation of HIV-1 LTR by several stimuli in the absence or presence of PGE2. 1G5 cells, a clonal cell line derived from Jurkat E6.1 cells which has been with stably transfected with a luciferase gene driven by the HIV-1 LTR (Aguilar-Cordova et al., 1994), were either left untreated (control) or treated with PHA (3 μg/ml), OKT3 (1 μg/ml), PMA (20 ng/ml), or TNF-α (2 ng/ml) in the absence or presence of 100 nM PGE2 for 8 h. Cell lysates were evaluated for luciferase activity and are expressed as fold induction relative to basal

Northern blot assays, flow cytometric analyses, and pharmacological studies showed that the EP4 gene is expressed on T lymphoid cells such as Molt-4, KM-3, IG5 and Jurkat E6.1 (Blaschke et al., 1996; De Vries et al., 1995; Dumais et al., 1998; Mori et al., 1996). It has been demonstrated that EP4 receptors are coupled to adenylate cyclase via a stimulatory G protein (Gs) and that such activation results in an enhancement of intracellular cAMP levels (Coleman et al., 1995; Nishigaki et al., 1995). Interestingly, PGE2 has been shown to lead to an increase in intracellular cAMP levels partly via the EP4 receptor (Rodbell, 1980), a finding which lend credence to the potential implication of the EP4 receptor in the PGE2-induced up-

as a potent inducer of HIV-1 LTR transcription in T cells.

luciferase activity in untreated control cells (considered as 1).

regulation of HIV-1 LTR activity.

responsible for the enhanced prostaglandin formation is still undefined. The initial contact between the virus particle and its target cell might represent the crucial step leading to the production of PGE2 by macrophages. Significant production of endogenous PGE2 is induced (20- to 40-fold increase) following incubation of primary human monocytes with the HIV-1 external envelope glycoprotein gp120 (Wahl et al., 1989). However, in sharp contrast with this report, a previous study has demonstrated that interaction between gp120 and THP-1, a human monocytoid cell line, does not increase exogenous production of PGE2 (Hui et al., 1995). It is important to specify that, unlike monocyte/macrophages, promonocytoid THP-1 cells are not at a terminal stage of differentiation. In addition, a monomer form of gp120 was used in this study which might not parallel physiological conditions where gp120 is under a multimeric form (Pinter et al., 1989).

## **3. PGE2 and HIV transcription**

### **3.1 Importance of NF-B in HIV-1 gene transcription**

HIV-1 gene expression is regulated in a cell type- and differentiation-dependent manner by the binding of both host and viral proteins to the long terminal repeat (LTR), which serves as the viral promoter. Host transcription factors such as the Sp family, NF-B family, activator protein 1 (AP-1) proteins, nuclear factor of activated T cells (NFAT), and CCAAT enhancer binding protein (C/EBP) family members play essential roles in the regulation of HIV-1 transcription by binding sites in the LTR that display different levels of sequence conservation (Fig 2). Viral proteins such as HIV Vpr and Tat also bind to the LTR to regulate transcription (Kilareski et al., 2009). Many of these host and viral proteins engage in extensive proteinprotein interactions, leading to a complex system of transcriptional regulation.

Fig. 2. A schematic representation of a typical HIV-1 LTR.

The transcription factor NF-B is known to play a central role in the activation of HIV-1 gene expression. The enhancer in the U3 region of LTR contains two NF-B binding sites (Siebenlist et al., 1994) that are critical for LTR promoter activity and important for optimal HIV-1 replication (Santoro et al., 2003; Siebenlist et al., 1994). NF-B is an inducible transcription factor that plays an important role in cellular gene expression associated with immune responses, inflammation and cell survival (Ghosh et al., 1998; Viatour et al., 2005). In the host cytoplasm, NF-B is a heterodimeric molecule (p50/p65) that forms an inactive complex with its inhibitor IB. Stimulation with inflammatory cytokines, such as TNF-α and IL-1β, viral and bacterial antigens, and stress-inducing agents leads to immediate phosphorylation and subsequent degradation of IB by the proteasome, resulting in the translocation of NF-B from the cytoplasm to nucleus.

#### **3.2 Activation of HIV-1 LTR activity by PGE2**

Immune and inflammatory responses are triggered by microorganisms such as bacteria, viruses, and protozoan, all known to be potential opportunistic pathogens in HIV-1-positive

responsible for the enhanced prostaglandin formation is still undefined. The initial contact between the virus particle and its target cell might represent the crucial step leading to the production of PGE2 by macrophages. Significant production of endogenous PGE2 is induced (20- to 40-fold increase) following incubation of primary human monocytes with the HIV-1 external envelope glycoprotein gp120 (Wahl et al., 1989). However, in sharp contrast with this report, a previous study has demonstrated that interaction between gp120 and THP-1, a human monocytoid cell line, does not increase exogenous production of PGE2 (Hui et al., 1995). It is important to specify that, unlike monocyte/macrophages, promonocytoid THP-1 cells are not at a terminal stage of differentiation. In addition, a monomer form of gp120 was used in this study which might not parallel physiological conditions where gp120 is under a

HIV-1 gene expression is regulated in a cell type- and differentiation-dependent manner by the binding of both host and viral proteins to the long terminal repeat (LTR), which serves as the viral promoter. Host transcription factors such as the Sp family, NF-B family, activator protein 1 (AP-1) proteins, nuclear factor of activated T cells (NFAT), and CCAAT enhancer binding protein (C/EBP) family members play essential roles in the regulation of HIV-1 transcription by binding sites in the LTR that display different levels of sequence conservation (Fig 2). Viral proteins such as HIV Vpr and Tat also bind to the LTR to regulate transcription (Kilareski et al., 2009). Many of these host and viral proteins engage in extensive protein-

The transcription factor NF-B is known to play a central role in the activation of HIV-1 gene expression. The enhancer in the U3 region of LTR contains two NF-B binding sites (Siebenlist et al., 1994) that are critical for LTR promoter activity and important for optimal HIV-1 replication (Santoro et al., 2003; Siebenlist et al., 1994). NF-B is an inducible transcription factor that plays an important role in cellular gene expression associated with immune responses, inflammation and cell survival (Ghosh et al., 1998; Viatour et al., 2005). In the host cytoplasm, NF-B is a heterodimeric molecule (p50/p65) that forms an inactive complex with its inhibitor IB. Stimulation with inflammatory cytokines, such as TNF-α and IL-1β, viral and bacterial antigens, and stress-inducing agents leads to immediate phosphorylation and subsequent degradation of IB by the proteasome, resulting in the

Immune and inflammatory responses are triggered by microorganisms such as bacteria, viruses, and protozoan, all known to be potential opportunistic pathogens in HIV-1-positive

protein interactions, leading to a complex system of transcriptional regulation.

multimeric form (Pinter et al., 1989).

**3. PGE2 and HIV transcription** 

**3.1 Importance of NF-B in HIV-1 gene transcription** 

Fig. 2. A schematic representation of a typical HIV-1 LTR.

translocation of NF-B from the cytoplasm to nucleus.

**3.2 Activation of HIV-1 LTR activity by PGE2**

patients. The formation and production of elevated levels of inflammatory mediators such as PGE2 is a hallmark of the HIV-1 infection (Foley et al., 1992; Griffin et al., 1994a; Ramis et al., 1992). Prostaglandins play a role in disease exacerbation by directly altering T-cell functions or macrophage activation. Although it was thought that PGE2 is primarily an immunosuppressive molecule that acts as a down-regulator of many aspects of B- and T-cell function and proliferation, other findings support a role for PGE2 as a potentiator of immunoglobulin class switching and cytokines and cytokine receptors synthesis (Phipps et al., 1991). Moreover, knowing that PGE2 is a good inducer of cAMP and that a 4-fold increase in intracellular levels of cAMP is seen in asymptomatic HIV-1-seropositive subjects as compared with uninfected controls (Hofmann et al., 1993), it is thus of prime importance to study the putative effect of PGE2 on the regulatory elements of HIV-1 in T cells considered to be the major cellular reservoir for HIV-1 in the human peripheral blood. As shown in Fig 3, exogenous PGE2 could further increase the overall positive effect mediated by various HIV-1 LTR-activating agents confirming that PGE2 could be considered by itself as a potent inducer of HIV-1 LTR transcription in T cells.

Fig. 3. Activation of HIV-1 LTR by several stimuli in the absence or presence of PGE2. 1G5 cells, a clonal cell line derived from Jurkat E6.1 cells which has been with stably transfected with a luciferase gene driven by the HIV-1 LTR (Aguilar-Cordova et al., 1994), were either left untreated (control) or treated with PHA (3 μg/ml), OKT3 (1 μg/ml), PMA (20 ng/ml), or TNF-α (2 ng/ml) in the absence or presence of 100 nM PGE2 for 8 h. Cell lysates were evaluated for luciferase activity and are expressed as fold induction relative to basal luciferase activity in untreated control cells (considered as 1).

Northern blot assays, flow cytometric analyses, and pharmacological studies showed that the EP4 gene is expressed on T lymphoid cells such as Molt-4, KM-3, IG5 and Jurkat E6.1 (Blaschke et al., 1996; De Vries et al., 1995; Dumais et al., 1998; Mori et al., 1996). It has been demonstrated that EP4 receptors are coupled to adenylate cyclase via a stimulatory G protein (Gs) and that such activation results in an enhancement of intracellular cAMP levels (Coleman et al., 1995; Nishigaki et al., 1995). Interestingly, PGE2 has been shown to lead to an increase in intracellular cAMP levels partly via the EP4 receptor (Rodbell, 1980), a finding which lend credence to the potential implication of the EP4 receptor in the PGE2-induced upregulation of HIV-1 LTR activity.

Emerging Roles of Prostaglandins in HIV-1 Transcription 351

dissociation of IB from NF-B (Nabel and Baltimore, 1987). Recent studies have revealed that NF-B is regulated through phosphorylation of the p65 subunit by PKA which is directly regulated by intracellular levels of cAMP (Zhong et al., 1997). Experiments in Jurkat E6.1 T cells performed with B-driven reporter gene constructs (pB-TATA-LUC and pNF- B-LUC) and HIV-1 LTR-based vectors (pLTR-LUC and pmBLTR-LUC), suggest that NF- B-binding regions and another element(s) in the HIV-1 LTR are involved in the activation of HIV-1 LTR-dependent transcription induced by PGE2 (fig 4). These results hence support the notion that PGE2 might be activating the transcription factor NF-B via cAMP/PKA and

Fig. 4. NF-B-dependent and -independent activation of HIV-1 LTR by PGE2. Jurkat E6.1 cells were transiently transfected with pLTR-LUC, pmBLTR-LUC, pNF-B-LUC or pB-TATA-LUC and were either left untreated or were treated for 8 h with TNF-α (2 ng/ml), PHA/PMA (3 μg/ml and 20 ng/ml, respectively), or PGE2 (100 nM). Results shown are expressed as fold induction relative to basal luciferase activity in untreated control cells

**3.2.2 Other transcription factors implicated in the PGE2-induced HIV-1 gene** 

PGE2 could have the capacity to modulate several signal transduction pathways through its effect on transcription factors regulated by cAMP such as the cAMP response-element binding factor, the activating protein-1 (Haraguchi et al., 1995b) and Sp1 (Rohlff et al., 1997). The involvement of these three transcription factors in the observed NF-B-independent activation of HIV-1 LTR mediated by PGE2 was further investigated. We have previously discussed that interaction of PGE2 with EP4 receptor subtype in human T cells can upregulate HIV-1 replication via both NF-B-dependent and –independent pathways (Dumais et al., 1998). In this section, we will address the functional role played by other transcription

Several signal transduction pathways have been shown to regulate the expression of target genes by inducing the phosphorylation of specific transcription factors (Hunter & Karin, 1992). The second messenger cAMP mediates the transcriptional induction of numerous genes through protein kinase A (PKA)-dependent phosphorylation of the CREB at Ser133 (Gonzalez et al., 1989). CREB is a stimulus-induced 43-kDa basic leucine zipper (b-ZIP) transcription factor that binds to an octanucleotide cAMP-responsive element (CRE) (i.e., TGANNTCA) both as a homodimer and as a heterodimer in

calcium signaling pathways in human T lymphoid cells.

factors in the PGE2-induced HIV-1 LTR activation.

(considered as 1).

**transcription** 

#### **3.2.1 NF-κB-dependent signaling pathways involved in activation of HIV-1 LTR by PGE2 in T cells**

The involvement of specific intracellular second messengers in PGE2-mediated upregulation of HIV-1 LTR activity has been dissected using several signal transduction inhibitors. Only exogeneous PGE2 plays a role in the activation of HIV-1 LTR-driven gene expression as shown with experiments using indomethacin, a potent inhibitor of the cyclooxygenase pathway (Dumais et al., 1998). Moreover, it was demonstrated that T cells had a limited capacity to metabolize arachidonic acid to prostaglandins (Auberger et al., 1989; Fu et al., 1990; Goldyne & Rea, 1987). Interaction between PGE2 and an adenylate cyclase-coupled stimulatory receptor leads to activation of adenylate cyclase, hydrolysis of ATP, enhanced turnover of intracellular cAMP and binding to PKA (Kammer, 1988). In T cells, PGE2-induced enhancement of HIV-1 LTR dependent activity requires the participation of adenylate cyclase, cAMP as well as protein kinase A (Dumais et al., 1998) and elevation of cAMP levels resulted in HIV-1 replication (Nokta & Pollard, 1992). It is also well known that cAMP-dependent pathways regulate the immune effector functions of lymphocytes and macrophages. For example, during immune response, cAMP exhibits positive regulatory effects at low concentrations whereas inhibitory effects are seen at high concentrations (Koh et al., 1995). Many of the earlier studies have shown that PGE2 interaction with T cells *in vitro* resulted in an elevation of the cAMP level (Rincon et al., 1988) and that such elevated intracellular cAMP levels were responsible for the proliferative disturbances in T cells (Baker et al., 1981; Lingk et al., 1990; Munoz et al., 1990). In T cells, experiments with the calcium chelator BAPTA/AM and the calcium inhibitor CAI are suggestive of the importance of Ca2+ in the PGE2-induced activation of HIV-1 transcription (Dumais et al., 1998). However, given that there is no published report indicating Ca2+ influx through the EP4 receptor, our results with BAPTA/AM and carboxyamido-triazole (CAI), two inhibitors of intracellular calcium mobilization, lead us to postulate that PGE2 could generate calcium release from intracellular storage organelles. Up-regulation of HIV-1 LTR requires the implication of cAMP and calcium, as well as the participation of the NF-B transcription factor.

Several agents known as potent activators of HIV-1 transcription (e.g. PMA, PHA, TNF-α, and anti-CD3 antibody) are all acting through a common mechanism, namely via the nuclear translocation of the transcription factor NF-B which binds to the enhancer region of the HIV-1 LTR (Nabel, 1991). This transcription factor is sequestered in the cytoplasm due to its physical association with the inhibitor named IB. NF-B is a pleiotropic transcription factor that controls the expression of a wide variety of genes, including cytokines such as IL-1, IL-2, IL-6, IL-8, interferon-β, and TNF-α, as well as known genes for some cell adhesion molecules including ICAM-1 and VCAM-1. Its importance in the regulation of HIV-1 gene expression has been stated in numerous studies (Siebenlist et al., 1994). Results from mobility shift assays suggest that the PGE2-mediated effect on HIV-1 LTR activity is due to activation of the transcription factor NF-B. This is in agreement with the previous demonstration that PGE2 activates NF-B in the macrophage-like cell line J774 (Muroi & Suzuki, 1993). The fact that we have noticed that both NF-B and Ca2+ are key elements in the PGE2 effect on HIV-1 transcription is of interest considering that calcineurin, a Ca2+/calmodulin-dependent serine/threonine protein phosphatase, has been reported to activate NFB through the inactivation of IB (Frantz et al., 1994). Moreover, researchers had earlier found that cAMP-mediated enhancement of PKA might be involved in the

The involvement of specific intracellular second messengers in PGE2-mediated upregulation of HIV-1 LTR activity has been dissected using several signal transduction inhibitors. Only exogeneous PGE2 plays a role in the activation of HIV-1 LTR-driven gene expression as shown with experiments using indomethacin, a potent inhibitor of the cyclooxygenase pathway (Dumais et al., 1998). Moreover, it was demonstrated that T cells had a limited capacity to metabolize arachidonic acid to prostaglandins (Auberger et al., 1989; Fu et al., 1990; Goldyne & Rea, 1987). Interaction between PGE2 and an adenylate cyclase-coupled stimulatory receptor leads to activation of adenylate cyclase, hydrolysis of ATP, enhanced turnover of intracellular cAMP and binding to PKA (Kammer, 1988). In T cells, PGE2-induced enhancement of HIV-1 LTR dependent activity requires the participation of adenylate cyclase, cAMP as well as protein kinase A (Dumais et al., 1998) and elevation of cAMP levels resulted in HIV-1 replication (Nokta & Pollard, 1992). It is also well known that cAMP-dependent pathways regulate the immune effector functions of lymphocytes and macrophages. For example, during immune response, cAMP exhibits positive regulatory effects at low concentrations whereas inhibitory effects are seen at high concentrations (Koh et al., 1995). Many of the earlier studies have shown that PGE2 interaction with T cells *in vitro* resulted in an elevation of the cAMP level (Rincon et al., 1988) and that such elevated intracellular cAMP levels were responsible for the proliferative disturbances in T cells (Baker et al., 1981; Lingk et al., 1990; Munoz et al., 1990). In T cells, experiments with the calcium chelator BAPTA/AM and the calcium inhibitor CAI are suggestive of the importance of Ca2+ in the PGE2-induced activation of HIV-1 transcription (Dumais et al., 1998). However, given that there is no published report indicating Ca2+ influx through the EP4 receptor, our results with BAPTA/AM and carboxyamido-triazole (CAI), two inhibitors of intracellular calcium mobilization, lead us to postulate that PGE2 could generate calcium release from intracellular storage organelles. Up-regulation of HIV-1 LTR requires the implication of cAMP and calcium, as well as the participation of the NF-B

Several agents known as potent activators of HIV-1 transcription (e.g. PMA, PHA, TNF-α, and anti-CD3 antibody) are all acting through a common mechanism, namely via the nuclear translocation of the transcription factor NF-B which binds to the enhancer region of the HIV-1 LTR (Nabel, 1991). This transcription factor is sequestered in the cytoplasm due to its physical association with the inhibitor named IB. NF-B is a pleiotropic transcription factor that controls the expression of a wide variety of genes, including cytokines such as IL-1, IL-2, IL-6, IL-8, interferon-β, and TNF-α, as well as known genes for some cell adhesion molecules including ICAM-1 and VCAM-1. Its importance in the regulation of HIV-1 gene expression has been stated in numerous studies (Siebenlist et al., 1994). Results from mobility shift assays suggest that the PGE2-mediated effect on HIV-1 LTR activity is due to activation of the transcription factor NF-B. This is in agreement with the previous demonstration that PGE2 activates NF-B in the macrophage-like cell line J774 (Muroi & Suzuki, 1993). The fact that we have noticed that both NF-B and Ca2+ are key elements in the PGE2 effect on HIV-1 transcription is of interest considering that calcineurin, a Ca2+/calmodulin-dependent serine/threonine protein phosphatase, has been reported to activate NFB through the inactivation of IB (Frantz et al., 1994). Moreover, researchers had earlier found that cAMP-mediated enhancement of PKA might be involved in the

**3.2.1 NF-κB-dependent signaling pathways involved in activation of HIV-1 LTR by** 

**PGE2 in T cells** 

transcription factor.

dissociation of IB from NF-B (Nabel and Baltimore, 1987). Recent studies have revealed that NF-B is regulated through phosphorylation of the p65 subunit by PKA which is directly regulated by intracellular levels of cAMP (Zhong et al., 1997). Experiments in Jurkat E6.1 T cells performed with B-driven reporter gene constructs (pB-TATA-LUC and pNF- B-LUC) and HIV-1 LTR-based vectors (pLTR-LUC and pmBLTR-LUC), suggest that NF- B-binding regions and another element(s) in the HIV-1 LTR are involved in the activation of HIV-1 LTR-dependent transcription induced by PGE2 (fig 4). These results hence support the notion that PGE2 might be activating the transcription factor NF-B via cAMP/PKA and calcium signaling pathways in human T lymphoid cells.

Fig. 4. NF-B-dependent and -independent activation of HIV-1 LTR by PGE2. Jurkat E6.1 cells were transiently transfected with pLTR-LUC, pmBLTR-LUC, pNF-B-LUC or pB-TATA-LUC and were either left untreated or were treated for 8 h with TNF-α (2 ng/ml), PHA/PMA (3 μg/ml and 20 ng/ml, respectively), or PGE2 (100 nM). Results shown are expressed as fold induction relative to basal luciferase activity in untreated control cells (considered as 1).

#### **3.2.2 Other transcription factors implicated in the PGE2-induced HIV-1 gene transcription**

PGE2 could have the capacity to modulate several signal transduction pathways through its effect on transcription factors regulated by cAMP such as the cAMP response-element binding factor, the activating protein-1 (Haraguchi et al., 1995b) and Sp1 (Rohlff et al., 1997). The involvement of these three transcription factors in the observed NF-B-independent activation of HIV-1 LTR mediated by PGE2 was further investigated. We have previously discussed that interaction of PGE2 with EP4 receptor subtype in human T cells can upregulate HIV-1 replication via both NF-B-dependent and –independent pathways (Dumais et al., 1998). In this section, we will address the functional role played by other transcription factors in the PGE2-induced HIV-1 LTR activation.

Several signal transduction pathways have been shown to regulate the expression of target genes by inducing the phosphorylation of specific transcription factors (Hunter & Karin, 1992). The second messenger cAMP mediates the transcriptional induction of numerous genes through protein kinase A (PKA)-dependent phosphorylation of the CREB at Ser133 (Gonzalez et al., 1989). CREB is a stimulus-induced 43-kDa basic leucine zipper (b-ZIP) transcription factor that binds to an octanucleotide cAMP-responsive element (CRE) (i.e., TGANNTCA) both as a homodimer and as a heterodimer in

Emerging Roles of Prostaglandins in HIV-1 Transcription 353

the NRE, suggesting that the COUP-TF binding domain is not participating in PGE2-

The C/EBP family of nuclear proteins is a member of a larger superfamily of transcription factors characterized by the b-ZIP motif that also includes the ATF/CREB family (Johnson, 1993). In a number of cell types, C/EBPβ has been shown to function as a cAMP-activated transcription factor (Metz & Ziff, 1991; Roesler et al., 1988; Tae et al., 1995). Treatment of Jurkat cells with PGE2 resulted in a noticeable induction of nuclear translocation and activation of C/EBPβ. Indeed, DNA mobility shift assays provided clear evidence that PGE2 and forskolin treatment of human T cells increases the level of specific protein-DNA complexes when the consensus C/EBP binding site is used as a molecular probe. Although treatment of Jurkat cells with PGE2 did not alter the protein level of C/EBPβ in whole cell extracts, there was a redistribution of this protein from the cytoplasm to the nucleus upon

It has been previously demonstrated that individual C/EBP proteins can homodimerize or heterodimerize with other members of the C/EBP family of b-ZIP domain proteins to elicit specific cAMP-mediated transcriptional stimulation or repression (Metz & Ziff, 1991; Vinson et al., 1989; Williams et al., 1991). Moreover, it is now believed that transcriptional regulation of genes containing the recognition sites of either C/EBP or ATF/CREB may result from heterodimeric formation between different members in each of the C/EBP and ATF/CREB families (Vallejo, 1994). This mechanism may be used to respond to complex signals and transcriptional cues through single sequence elements including a response to cAMP, despite the absence of active CRE, AP1, and AP2 consensus nucleotide sequences (Kagawa & Waterman, 1990; Lund et al., 1990; Pittman et al., 1995). The best example is provided by the CFTR gene promoter that is controlled by interactions between C/EBP and ATF/CREB family members with CREB1 and ATF1 binding to the inverted CCAAT element of this gene to finely regulate its transcription (Pittman et al., 1995). Although the absence of CRE certainly may not preclude ATF or CREB protein from targeting promoters devoid of such cis-acting elements, it is interesting to note that regulation of the somatostatin gene requires protein-protein interaction between C/EBP and ATF/CREB transcription factors to elicit a cAMP-dependent response through the CRE element (Vallejo, 1994). Inversely, C/EBP proteins have been shown to bind specifically to the phosphoenolpyruvate carboxykinase gene CRE with high affinity to promote cAMPmediated transcriptional activation (Park et al., 1993). In addition, previous studies identified C/EBP as an effector of cAMP-mediated transcription of the phosphoenolpyruvate carboxykinase gene through combined interactions with liverspecific transcription factors (Roesler, 2000; Roesler et al., 1995). Experiments conducted with a vector coding for LIP suggested that C/EBP was playing a crucial role in activation of HIV-1 LTR-driven gene expression that is seen following treatment of human T cells with PGE2 (Dumais et al., 2002). It should be noted that the β-isoform of C/EBP has been intimately linked with the cAMP signaling system, as exemplified by the reported capacity of cAMP to stimulate C/EBPβ gene expression (Park et al., 1993) and translocation of C/EBPβ from the cytosol to the nucleus (1991). Thus, we propose that the PGE2-dependent increase in HIV-1 LTR transcriptional activity is mediated in part by C/EBPβ. The dominant negative form of C/EBP, i.e., LIP, has less impact on PGE2 mediated induction of HIV-1 LTR-driven activity than mutating the C/EBP binding sites,

suggesting that factors in addition to C/EBP may be binding to C/EBP sites.

mediated effect.

exposure to PGE2 (Dumais et al., 2002).

conjunction with other members of the activation transcription factor (ATF)/CREB superfamily of transcription factors (Gonzalez & Montminy, 1989; Habener, 1990; Hoeffler et al., 1988). It is now believed that the transcriptional regulation of genes containing either CCAAT/enhancer binding protein (C/EBP) or ATF/CREB recognition sites may involve the heterodimerization between different members of the b-ZIP family. This is clearly illustrated by the demonstration that transcription of HIV-1 in monocytic cells is regulated by a synergistic interaction between ATF/CREB and C/EBP protein families (Ross et al., 2001). The C/EBP-related family of nuclear transcription factors constitutes a class of proteins characterized by their ability to bind the CCAAT consensus sequence, inducing either transcriptional activation or repression of target genes (Cao et al., 1991; Chodosh et al., 1988; Johnson & McKnight, 1989; Williams et al., 1991). Members of this family include C/EBPα, C/EBPβ (also termed LAP, NF-IL6α, IL-6DBP, AGP/EBP), C/EBPγ, C/EBPδ (NF-IL6) and C/EBPε (Mueller et al., 1990). Interestingly, regulatory sequences of HIV-1, which are located within the LTR, harbor three C/EBP sites that bind C/EBPβ (Tesmer et al., 1993) (Fig 2) and these sites are essential to initiate virus replication in cells of the monocyte/macrophage lineage (Henderson et al., 1995) and in endothelial cells as recently described (Lee et al., 2001). PKA and transcription factors of the ATF/CREB family may be critical for HIV-1 expression and regulation. In this regard, HIV-1 infection has been associated with sustained elevation of cAMP in T cell lines and in normal peripheral blood mononuclear lymphocytes (Nokta & Pollard, 1992). Moreover, HIV-1 replication has been shown to be modulated by intracellular levels of cAMP (Dumais et al., 1998; Nokta & Pollard, 1992). For example, activation of the cAMP/PKA pathway by cholera toxin enhances HIV-1 transcription in latently infected monocytoid U1 cells (Chowdhury et al., 1993). It is still unknown whether the HIV-1 genome, especially the LTR, possesses CRE sequences. However, the downstream sequence elements located in the U5 domain of HIV-1 LTR has been proposed to act as 12-Otetradecanoylphorbol-13-acetate/phorbol ester responsive element (TRE)-like CRE that bind both AP-1 and CREB/ATF, allowing the induction of HIV-1 LTR activity through both protein kinase C and PKA activation signals (Rabbi et al., 1998).

PGE2 can act as a potent activator of HIV-1 LTR-driven transcription through effects on both NF-κB-dependent and -independent signaling events (Dumais et al., 1998). More recently, calcium and the CREB transcription factor were also found to be essential second messengers in the PGE2-mediated up-regulation of LTR activity in T cells (our unpublished observation). Although the binding of a member of the CREB family to the HIV-1 LTR via the CRE consensus sequence has not yet been described, it has been postulated that CREB can act indirectly on the regulatory elements of this retrovirus. For example, it has been shown that CREB interacts with HIV-1 LTR through an association with transcription factors such as TFIID and TFIIB (Ferreri et al., 1994; Rohr et al., 1999; Xing et al., 1995) or with the adapter CBP; the latter is known to interact with the general transcription machinery (Nordheim, 1994). Recently, a recognition sequence for members of the ATF/CREB family was identified within the untranslated leader region of HIV-1 as a novel TRE-like CRE capable of binding both AP-1 and ATF/CREB (Rabbi et al., 1997). However, the U5 region of the HIV-1 LTR is absent from our molecular constructs, rejecting the possible implication of TRE-like CRE in the noticed PGE2-induced viral activation. A recent report has shown that dopamine treatment of HIV-1-infected T cells leads to the binding of CREB to the COUP-TF sequence that is located at the 5' end of the HIV-1 LTR in a region called the NRE (Rohr et al., 1999). The various LTR constructs used in our study do not bear

conjunction with other members of the activation transcription factor (ATF)/CREB superfamily of transcription factors (Gonzalez & Montminy, 1989; Habener, 1990; Hoeffler et al., 1988). It is now believed that the transcriptional regulation of genes containing either CCAAT/enhancer binding protein (C/EBP) or ATF/CREB recognition sites may involve the heterodimerization between different members of the b-ZIP family. This is clearly illustrated by the demonstration that transcription of HIV-1 in monocytic cells is regulated by a synergistic interaction between ATF/CREB and C/EBP protein families (Ross et al., 2001). The C/EBP-related family of nuclear transcription factors constitutes a class of proteins characterized by their ability to bind the CCAAT consensus sequence, inducing either transcriptional activation or repression of target genes (Cao et al., 1991; Chodosh et al., 1988; Johnson & McKnight, 1989; Williams et al., 1991). Members of this family include C/EBPα, C/EBPβ (also termed LAP, NF-IL6α, IL-6DBP, AGP/EBP), C/EBPγ, C/EBPδ (NF-IL6) and C/EBPε (Mueller et al., 1990). Interestingly, regulatory sequences of HIV-1, which are located within the LTR, harbor three C/EBP sites that bind C/EBPβ (Tesmer et al., 1993) (Fig 2) and these sites are essential to initiate virus replication in cells of the monocyte/macrophage lineage (Henderson et al., 1995) and in endothelial cells as recently described (Lee et al., 2001). PKA and transcription factors of the ATF/CREB family may be critical for HIV-1 expression and regulation. In this regard, HIV-1 infection has been associated with sustained elevation of cAMP in T cell lines and in normal peripheral blood mononuclear lymphocytes (Nokta & Pollard, 1992). Moreover, HIV-1 replication has been shown to be modulated by intracellular levels of cAMP (Dumais et al., 1998; Nokta & Pollard, 1992). For example, activation of the cAMP/PKA pathway by cholera toxin enhances HIV-1 transcription in latently infected monocytoid U1 cells (Chowdhury et al., 1993). It is still unknown whether the HIV-1 genome, especially the LTR, possesses CRE sequences. However, the downstream sequence elements located in the U5 domain of HIV-1 LTR has been proposed to act as 12-Otetradecanoylphorbol-13-acetate/phorbol ester responsive element (TRE)-like CRE that bind both AP-1 and CREB/ATF, allowing the induction of HIV-1 LTR activity through

both protein kinase C and PKA activation signals (Rabbi et al., 1998).

PGE2 can act as a potent activator of HIV-1 LTR-driven transcription through effects on both NF-κB-dependent and -independent signaling events (Dumais et al., 1998). More recently, calcium and the CREB transcription factor were also found to be essential second messengers in the PGE2-mediated up-regulation of LTR activity in T cells (our unpublished observation). Although the binding of a member of the CREB family to the HIV-1 LTR via the CRE consensus sequence has not yet been described, it has been postulated that CREB can act indirectly on the regulatory elements of this retrovirus. For example, it has been shown that CREB interacts with HIV-1 LTR through an association with transcription factors such as TFIID and TFIIB (Ferreri et al., 1994; Rohr et al., 1999; Xing et al., 1995) or with the adapter CBP; the latter is known to interact with the general transcription machinery (Nordheim, 1994). Recently, a recognition sequence for members of the ATF/CREB family was identified within the untranslated leader region of HIV-1 as a novel TRE-like CRE capable of binding both AP-1 and ATF/CREB (Rabbi et al., 1997). However, the U5 region of the HIV-1 LTR is absent from our molecular constructs, rejecting the possible implication of TRE-like CRE in the noticed PGE2-induced viral activation. A recent report has shown that dopamine treatment of HIV-1-infected T cells leads to the binding of CREB to the COUP-TF sequence that is located at the 5' end of the HIV-1 LTR in a region called the NRE (Rohr et al., 1999). The various LTR constructs used in our study do not bear the NRE, suggesting that the COUP-TF binding domain is not participating in PGE2 mediated effect.

The C/EBP family of nuclear proteins is a member of a larger superfamily of transcription factors characterized by the b-ZIP motif that also includes the ATF/CREB family (Johnson, 1993). In a number of cell types, C/EBPβ has been shown to function as a cAMP-activated transcription factor (Metz & Ziff, 1991; Roesler et al., 1988; Tae et al., 1995). Treatment of Jurkat cells with PGE2 resulted in a noticeable induction of nuclear translocation and activation of C/EBPβ. Indeed, DNA mobility shift assays provided clear evidence that PGE2 and forskolin treatment of human T cells increases the level of specific protein-DNA complexes when the consensus C/EBP binding site is used as a molecular probe. Although treatment of Jurkat cells with PGE2 did not alter the protein level of C/EBPβ in whole cell extracts, there was a redistribution of this protein from the cytoplasm to the nucleus upon exposure to PGE2 (Dumais et al., 2002).

It has been previously demonstrated that individual C/EBP proteins can homodimerize or heterodimerize with other members of the C/EBP family of b-ZIP domain proteins to elicit specific cAMP-mediated transcriptional stimulation or repression (Metz & Ziff, 1991; Vinson et al., 1989; Williams et al., 1991). Moreover, it is now believed that transcriptional regulation of genes containing the recognition sites of either C/EBP or ATF/CREB may result from heterodimeric formation between different members in each of the C/EBP and ATF/CREB families (Vallejo, 1994). This mechanism may be used to respond to complex signals and transcriptional cues through single sequence elements including a response to cAMP, despite the absence of active CRE, AP1, and AP2 consensus nucleotide sequences (Kagawa & Waterman, 1990; Lund et al., 1990; Pittman et al., 1995). The best example is provided by the CFTR gene promoter that is controlled by interactions between C/EBP and ATF/CREB family members with CREB1 and ATF1 binding to the inverted CCAAT element of this gene to finely regulate its transcription (Pittman et al., 1995). Although the absence of CRE certainly may not preclude ATF or CREB protein from targeting promoters devoid of such cis-acting elements, it is interesting to note that regulation of the somatostatin gene requires protein-protein interaction between C/EBP and ATF/CREB transcription factors to elicit a cAMP-dependent response through the CRE element (Vallejo, 1994). Inversely, C/EBP proteins have been shown to bind specifically to the phosphoenolpyruvate carboxykinase gene CRE with high affinity to promote cAMPmediated transcriptional activation (Park et al., 1993). In addition, previous studies identified C/EBP as an effector of cAMP-mediated transcription of the phosphoenolpyruvate carboxykinase gene through combined interactions with liverspecific transcription factors (Roesler, 2000; Roesler et al., 1995). Experiments conducted with a vector coding for LIP suggested that C/EBP was playing a crucial role in activation of HIV-1 LTR-driven gene expression that is seen following treatment of human T cells with PGE2 (Dumais et al., 2002). It should be noted that the β-isoform of C/EBP has been intimately linked with the cAMP signaling system, as exemplified by the reported capacity of cAMP to stimulate C/EBPβ gene expression (Park et al., 1993) and translocation of C/EBPβ from the cytosol to the nucleus (1991). Thus, we propose that the PGE2-dependent increase in HIV-1 LTR transcriptional activity is mediated in part by C/EBPβ. The dominant negative form of C/EBP, i.e., LIP, has less impact on PGE2 mediated induction of HIV-1 LTR-driven activity than mutating the C/EBP binding sites, suggesting that factors in addition to C/EBP may be binding to C/EBP sites.

Emerging Roles of Prostaglandins in HIV-1 Transcription 355

HIV-1 will help us to understand their mechanism of action and establish their therapeutic

Sexual transmission is the predominant mode for epidemic spread of HIV-1 infection worldwide. Because semen contains both free HIV-1 virions and HIV-1-infected cells (Bouhlal et al., 2002; Royce et al., 1997; Shepard et al., 2000), it can lead to both free and cellassociated viral transmission. The intestinal mucosa of the rectum, which serves as a site for virus entry, is known to play a fundamental role in early HIV-1 infection (Belyakov & Berzofsky, 2004; Kozlowski & Neutra, 2003; Neutra et al., 1996). In contrast, the mechanism of HIV-1 transmission across the epithelium is not well understood. While it is known that the penetration of HIV-1 may occur through lesions in the epithelium (Dickerson et al., 1996; Kozak et al., 1997), the existence of lesions is not required (Miller et al., 1990; Spira et al., 1996). Some studies suggest that HIV-1 can be carried to lymphocytes by dendritic cells (Geijtenbeek et al., 2000; Pohlmann et al., 2001). It has been proposed that HIV-1 can cross the epithelium barrier via epithelial cell infection. A quantitative analysis of enhanced green fluorescent protein-tagged HIV infection of cells derived from the female reproductive tract, brain and colon demonstrated that gp120-independent HIV infection occurs in intestinal epithelial cells (Zheng et al., 2006). These results clearly illustrate the importance of such cells in viral latency and transmission during mucosal HIV-1 infection. Earlier *in vitro* studies showed that HIV can infect human intestinal cell lines lacking CD4 (Fantini et al., 1991; Fantini et al., 1993). These studies also demonstrated that galactosylceramide (GalCer), which binds with high-affinity to gp120, can act as a CD4 surrogate HIV-1 receptor (Meng et al., 2002). In fact, Caco-2 cells, a human intestinal cell line, can be infected by HIV-1 via GalCer and CXCR4, one of the two known HIV chemokine coreceptors (Delezay et al., 1997; Fantini et al., 1993). Also, primary human intestinal cells are capable of selectively transferring R5 HIV-1 to CCR5+ cells (cells that express both GalCer and CCR5 on their cell surface) (Meng et al., 2002). Thus, they proposed that infection of epithelial cells might facilitate HIV-1 penetration into the epithelium barrier (Zheng et al., 2006). Following viral replication in the infected epithelial cells, newly formed HIV virions may be discharged into the basolateral side of the epithelium and exposed to immune cells present in the mucosal milieu. This process may ultimately lead to the dissemination of the virus throughout the body. Therefore, it is essential to understand the mechanisms by which HIV replication in epithelial cells can be modulated by the immune system molecules present in the mucosal milieu. Prostaglandins play key roles in inflammation. During the time course of inflammation, the prostaglandins profile shifts from the predominantly pro-inflammatory PGE2 to the anti-inflammatory PGJ2, which is the end product metabolite of PGD2 (Gilroy et al., 1999; Ianaro et al., 2001; Kapoor et al., 2005a; Kapoor et al., 2005b). Pro-inflammatory molecules such as PGE2 are up-regulated during HIV-1 infection (Griffin et al., 1994b; Ramis et al., 1991) leading to an imbalance in PGJ2 production. Given that the cyclopentone prostaglandin PGJ2 has potent anti-inflammatory properties, it is important to determine whether the addition of PGJ2 could inhibit HIV-1 transcription in intestinal epithelial cells. Cyclopentone prostaglandins such as PGA1 possess potent antiviral activity against a wide variety of viruses such as herpesviruses (Hughes-Fulford et al., 1992; Yamamoto et al., 1987), poxviruses (Santoro et al., 1982), paramyxoviruses (Amici et al., 1992; Santoro et al., 1980), orthomyxoviruses (Santoro et al., 1988), picornaviruses (Ankel et al., 1985), togaviruses

**3.3 15-d-PGJ2 and HIV-1 replication/production in intestinal epithelial cells** 

potential in the resolution of inflammation in HIV+ individuals.

We have identified C/EBP as a PGE2-activated transcriptional regulator of HIV-1 LTR in Jurkat cells and demonstrated that C/EBP binding sites are functionally important for virus transcription. We also suggest that functional and physical association between members of two important transcription factor families, i.e., C/EBP and CREB, are required for activation of HIV-1 transcription by PGE2 (Dumais et al., 2002). Our findings represent a further indication of the high complexity of the molecular mechanisms that regulate HIV-1 gene expression following treatment of human T cells with PGE2. Fig 5 reviews the effect of PGE2 on HIV-1 LTR transcription.

Fig. 5. A model of PGE2-induced HIV-1 LTR activation in T cells.

#### **3.2.3 Significance**

Results from several studies showed that PGE2 have a major impact in HIV-1 pathogenesis exacerbation. Knowing that HIV-infected individuals have a deficiency in the production of anti-inflammatory molecules, more knowledge are required to fully understand the potential benefit of the resolution of inflammation for people on HAART. Because they are clinically important molecules, a further understanding of the roles that prostaglandins played in host defense and HIV pathogenesis will have great impact on therapeutic research. Detailed characterization of prostaglandins interactions with cells infected with

We have identified C/EBP as a PGE2-activated transcriptional regulator of HIV-1 LTR in Jurkat cells and demonstrated that C/EBP binding sites are functionally important for virus transcription. We also suggest that functional and physical association between members of two important transcription factor families, i.e., C/EBP and CREB, are required for activation of HIV-1 transcription by PGE2 (Dumais et al., 2002). Our findings represent a further indication of the high complexity of the molecular mechanisms that regulate HIV-1 gene expression following treatment of human T cells with PGE2. Fig 5 reviews the effect of

PGE2 on HIV-1 LTR transcription.

Fig. 5. A model of PGE2-induced HIV-1 LTR activation in T cells.

Results from several studies showed that PGE2 have a major impact in HIV-1 pathogenesis exacerbation. Knowing that HIV-infected individuals have a deficiency in the production of anti-inflammatory molecules, more knowledge are required to fully understand the potential benefit of the resolution of inflammation for people on HAART. Because they are clinically important molecules, a further understanding of the roles that prostaglandins played in host defense and HIV pathogenesis will have great impact on therapeutic research. Detailed characterization of prostaglandins interactions with cells infected with

**3.2.3 Significance** 

HIV-1 will help us to understand their mechanism of action and establish their therapeutic potential in the resolution of inflammation in HIV+ individuals.

#### **3.3 15-d-PGJ2 and HIV-1 replication/production in intestinal epithelial cells**

Sexual transmission is the predominant mode for epidemic spread of HIV-1 infection worldwide. Because semen contains both free HIV-1 virions and HIV-1-infected cells (Bouhlal et al., 2002; Royce et al., 1997; Shepard et al., 2000), it can lead to both free and cellassociated viral transmission. The intestinal mucosa of the rectum, which serves as a site for virus entry, is known to play a fundamental role in early HIV-1 infection (Belyakov & Berzofsky, 2004; Kozlowski & Neutra, 2003; Neutra et al., 1996). In contrast, the mechanism of HIV-1 transmission across the epithelium is not well understood. While it is known that the penetration of HIV-1 may occur through lesions in the epithelium (Dickerson et al., 1996; Kozak et al., 1997), the existence of lesions is not required (Miller et al., 1990; Spira et al., 1996). Some studies suggest that HIV-1 can be carried to lymphocytes by dendritic cells (Geijtenbeek et al., 2000; Pohlmann et al., 2001). It has been proposed that HIV-1 can cross the epithelium barrier via epithelial cell infection. A quantitative analysis of enhanced green fluorescent protein-tagged HIV infection of cells derived from the female reproductive tract, brain and colon demonstrated that gp120-independent HIV infection occurs in intestinal epithelial cells (Zheng et al., 2006). These results clearly illustrate the importance of such cells in viral latency and transmission during mucosal HIV-1 infection. Earlier *in vitro* studies showed that HIV can infect human intestinal cell lines lacking CD4 (Fantini et al., 1991; Fantini et al., 1993). These studies also demonstrated that galactosylceramide (GalCer), which binds with high-affinity to gp120, can act as a CD4 surrogate HIV-1 receptor (Meng et al., 2002). In fact, Caco-2 cells, a human intestinal cell line, can be infected by HIV-1 via GalCer and CXCR4, one of the two known HIV chemokine coreceptors (Delezay et al., 1997; Fantini et al., 1993). Also, primary human intestinal cells are capable of selectively transferring R5 HIV-1 to CCR5+ cells (cells that express both GalCer and CCR5 on their cell surface) (Meng et al., 2002). Thus, they proposed that infection of epithelial cells might facilitate HIV-1 penetration into the epithelium barrier (Zheng et al., 2006). Following viral replication in the infected epithelial cells, newly formed HIV virions may be discharged into the basolateral side of the epithelium and exposed to immune cells present in the mucosal milieu. This process may ultimately lead to the dissemination of the virus throughout the body. Therefore, it is essential to understand the mechanisms by which HIV replication in epithelial cells can be modulated by the immune system molecules present in the mucosal milieu. Prostaglandins play key roles in inflammation. During the time course of inflammation, the prostaglandins profile shifts from the predominantly pro-inflammatory PGE2 to the anti-inflammatory PGJ2, which is the end product metabolite of PGD2 (Gilroy et al., 1999; Ianaro et al., 2001; Kapoor et al., 2005a; Kapoor et al., 2005b). Pro-inflammatory molecules such as PGE2 are up-regulated during HIV-1 infection (Griffin et al., 1994b; Ramis et al., 1991) leading to an imbalance in PGJ2 production. Given that the cyclopentone prostaglandin PGJ2 has potent anti-inflammatory properties, it is important to determine whether the addition of PGJ2 could inhibit HIV-1 transcription in intestinal epithelial cells. Cyclopentone prostaglandins such as PGA1 possess potent antiviral activity against a wide variety of viruses such as herpesviruses (Hughes-Fulford et al., 1992; Yamamoto et al., 1987), poxviruses (Santoro et al., 1982), paramyxoviruses (Amici et al., 1992; Santoro et al., 1980), orthomyxoviruses (Santoro et al., 1988), picornaviruses (Ankel et al., 1985), togaviruses

Emerging Roles of Prostaglandins in HIV-1 Transcription 357

**3.3.2 15-d-PGJ2-inhibition of HIV-1 LTR activation is linked to modification to NF-kB** 

et al., 2001) as well as IL-8 expression in endothelial cells (Jozkowicz et al., 2001).

The PPAR-γ-independent mechanism by which 15-d-PGJ2 mediates its anti-inflammatory effect can be dependent upon the inhibition of the NF-B signaling pathway (Daynes & Jones, 2002; Rossi et al., 2000). The NF-B binding sites within the HIV-1 promoter confer a high level of viral transcription in many cell types (Rabson & Lin, 2000). Previous studies have shown that cyclopentone prostaglandins via their ability to modulate NF-B activity, significantly alter HIV-1 replication in T cells, monocytes/macrophages and intestinal epithelial cells (Boisvert et al., 2008; Hayes et al., 2002; Rozera et al., 1996; Skolnik et al., 2002). In Caco-2 cells, the functional role of NF-B was determined using pΒ-TATA-luc or pNFB-luc expression plasmids (Fig 7). The results demonstrated that the NaBut-induced

Fig. 7. NF-B-dependent inhibition of HIV-1 LTR by 15-d-PGJ2. Caco-2 cells were transiently transfected with pB-TATA-luc, pmB-luc and pNF-B-luc. Then, cells were treated with 20 M 15-d-PGJ2 used in combination with 2 mM NaBut in a 24 h incubation period. Caco-2 cells were lysed and luciferase activity was monitored. Results shown are expressed as fold induction relative to basal luciferase activity in untreated control cells (considered as 1).

It has been shown previously that 15-d-PGJ2 exerts its effect(s) on cells by activating the PPARγ transcription factor via PPAR-γ, the natural ligand of PGJ2 (Schoonjans et al., 1996; Spiegelman, 1998). However, several studies have reported PPAR-γ independent effects of PGJ2 on transcriptional regulation via the modulation of NF-B (Rossi et al., 2000; Straus et al., 2000). In Caco-2, ciglitazone, a PPAR-γ agonist, failed to mimic the PGJ2-induced suppression of LTR activity, a result that suggests a PPAR-γ-independent mechanism, such as the NF-B pathway, may play a role in this effect in intestinal epithelial cells (Boisvert et al., 2008). This result is in contrast to those of Skolnik et al. in 2002 (Skolnik et al., 2002) that showed that ciglitazone was able to reduce the HIV-1 promoter activity in monocytes and in peripheral blood mononuclear cells (PBMCs). In contrast, ciglitazone induces luciferase activity in this experimental model. The induction of NF-B activity in colon cancer cells via p65 phosphorylation has been previously reported (Chen & Harrison, 2005), and this phenomenon may explain why we observed an increase in luciferase expression following the ciglitazone treatment of Caco-2 cells. The blockade of PPAR-γ receptor activation by using a specific human PPAR-γ antagonist (GW9662) confirms that 15-d-PGJ2 repress LTR activity by a mechanism independent of PPAR-γ (Boisvert et al., 2008). Similarly, expression of IL-1β in human chondrocytes is inhibited by 15-d-PGJ2 by a PPAR-γ-independent mechanism (Boyault

**signaling pathway** 

(Mastromarino et al., 1993), rhabdoviruses (Ankel et al., 1985; Santoro et al., 1983) and retroviruses (Hayes et al., 2002; Rozera et al., 1996; Skolnik et al., 2002). In macrophages, rosiglitazone, troglitazone, and PGJ2 as well as fenofibrate (a PPAR-α agonist) can inhibit HIV-1 replication in U1 cells (Skolnik et al., 2002), while PGA1 and PGA2 can inhibit HIV-1 replication in U937 cells and human monocyte-derived macrophages (Hayes et al., 2002). During acute HIV-1 infection in the well-characterized T cell line CEM-SS, treatment with cyclopentone prostaglandins such as PGA1 and PGJ2 profoundly alters viral replication (Rozera et al., 1996). Moreover, this antiviral effect does not seem to be mediated by alterations in the expression of α-, β-, or γ-interferon, TNF-α, TNF-β, IL-6 or IL-10 in HIVinfected CEM-SS but rather by a direct, as yet unidentified, mechanism (Rozera et al., 1996).

#### **3.3.1 15-d-PGJ2 inhibition of HIV-1 transcription and viral production**

The potent anti-inflammatory molecule 15-d-PGJ2 strongly suppresses HIV-1 replication and particle production in Caco-2 cells, a human intestinal cell line that mimics rectal epithelium susceptible to HIV-1 (Delezay et al., 1997; Fantini et al., 1991; Fantini et al., 1992; Fantini et al., 1993; Zheng et al., 2006). Prophylactic or co-treatment with 15-d-PGJ2 of intestinal epithelial cells significantly reduces HIV replication as well as p24 core antigen production (Boisvert et al., 2008). The 15-d-PGJ2-mediated suppression of HIV-1 replication is a result of the inhibition of promoter activity as shown by the utilization of a pLTR-luc reporter plasmid (Fig 6). This suppression of HIV-1 LTR activity is dose-dependant and is optimal 24h post-treatment. Moreover, 15-d-PGJ2 inhibition of sodium butyrate (NaBut)-induced LTR activity is not specific to Caco-2 cells but can be observed in other intestinal epithelial cell lines such as HT-29 and SW620. Sodium butyrate plays major roles in HIV infection. Indeed, urinary butyrate levels were increased in the AIDS patients with weight loss (2.83±0.67 μmol/l) relative to the controls (1.31±0.13 μmol/l, P<0.05), with the HIV+ patients (1.65±0.18 μmol/l) and AIDS patients without weight loss (1.90±0.22 μmol/l) falling in between (Stein et al., 1997). NaBut is a deacetylase inhibitor that has been shown to activate HIV-1 replication in cells of T-lymphoid and monocytoid origin (Golub et al., 1991). Thus, 15-d-PGJ2, without significantly changing cell viability or the cell cycle by blocking them in G1 phase or altering apoptosis, profoundly affects HIV-1 replication and gene expression in intestinal epithelial cell lines (Boisvert et al., 2008).

Fig. 6. 15-d-PGJ2-mediated negative effect on HIV-1 LTR activity. Caco-2, SW620 and HT-29 cells were transiently transfected with pLTR-luc and treated with 15-d- PGJ2 (20 M) used in combination with 2 mM NaBut in a 24 h incubation period. Results shown are expressed as fold induction relative to basal luciferase activity in untreated control cells (considered as 1).

(Mastromarino et al., 1993), rhabdoviruses (Ankel et al., 1985; Santoro et al., 1983) and retroviruses (Hayes et al., 2002; Rozera et al., 1996; Skolnik et al., 2002). In macrophages, rosiglitazone, troglitazone, and PGJ2 as well as fenofibrate (a PPAR-α agonist) can inhibit HIV-1 replication in U1 cells (Skolnik et al., 2002), while PGA1 and PGA2 can inhibit HIV-1 replication in U937 cells and human monocyte-derived macrophages (Hayes et al., 2002). During acute HIV-1 infection in the well-characterized T cell line CEM-SS, treatment with cyclopentone prostaglandins such as PGA1 and PGJ2 profoundly alters viral replication (Rozera et al., 1996). Moreover, this antiviral effect does not seem to be mediated by alterations in the expression of α-, β-, or γ-interferon, TNF-α, TNF-β, IL-6 or IL-10 in HIVinfected CEM-SS but rather by a direct, as yet unidentified, mechanism (Rozera et al., 1996).

The potent anti-inflammatory molecule 15-d-PGJ2 strongly suppresses HIV-1 replication and particle production in Caco-2 cells, a human intestinal cell line that mimics rectal epithelium susceptible to HIV-1 (Delezay et al., 1997; Fantini et al., 1991; Fantini et al., 1992; Fantini et al., 1993; Zheng et al., 2006). Prophylactic or co-treatment with 15-d-PGJ2 of intestinal epithelial cells significantly reduces HIV replication as well as p24 core antigen production (Boisvert et al., 2008). The 15-d-PGJ2-mediated suppression of HIV-1 replication is a result of the inhibition of promoter activity as shown by the utilization of a pLTR-luc reporter plasmid (Fig 6). This suppression of HIV-1 LTR activity is dose-dependant and is optimal 24h post-treatment. Moreover, 15-d-PGJ2 inhibition of sodium butyrate (NaBut)-induced LTR activity is not specific to Caco-2 cells but can be observed in other intestinal epithelial cell lines such as HT-29 and SW620. Sodium butyrate plays major roles in HIV infection. Indeed, urinary butyrate levels were increased in the AIDS patients with weight loss (2.83±0.67 μmol/l) relative to the controls (1.31±0.13 μmol/l, P<0.05), with the HIV+ patients (1.65±0.18 μmol/l) and AIDS patients without weight loss (1.90±0.22 μmol/l) falling in between (Stein et al., 1997). NaBut is a deacetylase inhibitor that has been shown to activate HIV-1 replication in cells of T-lymphoid and monocytoid origin (Golub et al., 1991). Thus, 15-d-PGJ2, without significantly changing cell viability or the cell cycle by blocking them in G1 phase or altering apoptosis, profoundly affects HIV-1 replication and gene expression in

Fig. 6. 15-d-PGJ2-mediated negative effect on HIV-1 LTR activity. Caco-2, SW620 and HT-29 cells were transiently transfected with pLTR-luc and treated with 15-d- PGJ2 (20 M) used in combination with 2 mM NaBut in a 24 h incubation period. Results shown are expressed as fold induction relative to basal luciferase activity in untreated control cells (considered as 1).

**3.3.1 15-d-PGJ2 inhibition of HIV-1 transcription and viral production** 

intestinal epithelial cell lines (Boisvert et al., 2008).

#### **3.3.2 15-d-PGJ2-inhibition of HIV-1 LTR activation is linked to modification to NF-kB signaling pathway**

It has been shown previously that 15-d-PGJ2 exerts its effect(s) on cells by activating the PPARγ transcription factor via PPAR-γ, the natural ligand of PGJ2 (Schoonjans et al., 1996; Spiegelman, 1998). However, several studies have reported PPAR-γ independent effects of PGJ2 on transcriptional regulation via the modulation of NF-B (Rossi et al., 2000; Straus et al., 2000). In Caco-2, ciglitazone, a PPAR-γ agonist, failed to mimic the PGJ2-induced suppression of LTR activity, a result that suggests a PPAR-γ-independent mechanism, such as the NF-B pathway, may play a role in this effect in intestinal epithelial cells (Boisvert et al., 2008). This result is in contrast to those of Skolnik et al. in 2002 (Skolnik et al., 2002) that showed that ciglitazone was able to reduce the HIV-1 promoter activity in monocytes and in peripheral blood mononuclear cells (PBMCs). In contrast, ciglitazone induces luciferase activity in this experimental model. The induction of NF-B activity in colon cancer cells via p65 phosphorylation has been previously reported (Chen & Harrison, 2005), and this phenomenon may explain why we observed an increase in luciferase expression following the ciglitazone treatment of Caco-2 cells. The blockade of PPAR-γ receptor activation by using a specific human PPAR-γ antagonist (GW9662) confirms that 15-d-PGJ2 repress LTR activity by a mechanism independent of PPAR-γ (Boisvert et al., 2008). Similarly, expression of IL-1β in human chondrocytes is inhibited by 15-d-PGJ2 by a PPAR-γ-independent mechanism (Boyault et al., 2001) as well as IL-8 expression in endothelial cells (Jozkowicz et al., 2001).

The PPAR-γ-independent mechanism by which 15-d-PGJ2 mediates its anti-inflammatory effect can be dependent upon the inhibition of the NF-B signaling pathway (Daynes & Jones, 2002; Rossi et al., 2000). The NF-B binding sites within the HIV-1 promoter confer a high level of viral transcription in many cell types (Rabson & Lin, 2000). Previous studies have shown that cyclopentone prostaglandins via their ability to modulate NF-B activity, significantly alter HIV-1 replication in T cells, monocytes/macrophages and intestinal epithelial cells (Boisvert et al., 2008; Hayes et al., 2002; Rozera et al., 1996; Skolnik et al., 2002). In Caco-2 cells, the functional role of NF-B was determined using pΒ-TATA-luc or pNFB-luc expression plasmids (Fig 7). The results demonstrated that the NaBut-induced

Fig. 7. NF-B-dependent inhibition of HIV-1 LTR by 15-d-PGJ2. Caco-2 cells were transiently transfected with pB-TATA-luc, pmB-luc and pNF-B-luc. Then, cells were treated with 20 M 15-d-PGJ2 used in combination with 2 mM NaBut in a 24 h incubation period. Caco-2 cells were lysed and luciferase activity was monitored. Results shown are expressed as fold induction relative to basal luciferase activity in untreated control cells (considered as 1).

Emerging Roles of Prostaglandins in HIV-1 Transcription 359

AIDS patients exhibit abnormal production of cyclooxygenase products. Prostaglandins are complex immunomodulatory molecules that shape, on one hand, the immune system and, on the other hand, have an influence on gene transcription by inducing or repressing several transcription factors in cells. Recent studies have led to a better understanding of the unique characteristics and importance of prostaglandins on HIV-1 transcription and

Fig. 9. A model for prostaglandins actions on HIV-1 pathogenesis.

therapeutic agent and its clinical application.

Because of their intrinsic intracellular obligatory parasitic form of life, viruses depend heavily on cell metabolic machinery for their replication. Thus, changes in cellular metabolism might influence the viral life cycle. The data reported in this chapter highlight the positive action of PGE2, a powerful cAMP-inducing agent, on the regulatory elements of HIV-1. PGE2 has now emerged as an immuno-activator that acts on the EP4 receptor that facilitates HIV-1 LTR activation. Elevated levels of PGE2 detected in HIV-1-infected persons or induced by opportunistic pathogens might actively participate to immunological disturbances associated with AIDS and modify the pathogenesis of this retroviral disease by inducing a higher viral load. High concentrations of PGE2 (up to 100 μM) found in seminal fluids of HIV-1-infected persons might directly enhance virus replication and facilitate viral transmission during sexual activities. Thus, analysis of the role of the PGE2 signaling may provide deeper insight into the pathological mechanisms underlying HIV/AIDS exacerbation, which should be fully taken into account in developing an EP4 antagonist as a

Accumulating data from several studies suggest that PGJ2 has intracellular effects that may suppress inflammation. They include inhibition of NF-B by multiple mechanisms such as IB kinase inhibition, blockade of NF-B nuclear binding and activation of PPAR-. The consequences of these activities are complex, but are likely to play a role in the prevention and/or resolution of inflammation. In this chapter, we showed the potentiality of the anti-

**4. Conclusion** 

replication (Fig 9).

luciferase activity of the pΒ-TATA-luc construct, which contains the HIV-1 enhancer, is abrogated by 15-d-PGJ2 in a dose-dependent manner. A control construct containing the HIV-1 enhancer with inactivated NF-B binding sites, pmB-luc, was used to show that NF- B is necessary for the NaBut activation of the HIV-1 LTR in Caco-2. Similar results to pΒ-TATA-luc were found using the pNFB-luc construct, which contains five consensus NF-κB binding sites. Together, these data suggest that NF-B is involved in the 15-d-PGJ2-mediated suppression of HIV-1 LTR activation in Caco-2 cells.

15-d-PGJ2 alters the stability of IBα proteins thereby altering NF-κB activation. Moreover in human bronchial epithelium, cyclopentone prostaglandin such has PGA1, has been shown to enhance the expression of IBα, a primary inhibitor of the pro-inflammatory transcription factor NF-B (Thomas et al., 1998). In intestinal epithelial cells, IKK activity was lower in Caco-2 cells treated with 15-d-PGJ2 and the inhibition of IKK activity was direct without increasing IBα mRNA expression. Another group (Scher & Pillinger, 2005) reported an inhibitory effect of 15-d-PGJ2 on NF-B activation and expression of pro-inflammatory genes such as COX-2, IL-1β and TNF-α. Interestingly in human chondrocytes, 15d-PGJ2, but not troglitazone, modulates IL-1β expression by inhibiting NF-B and AP-1 activation pathways, a mechanism independent of PPAR-γ as observed with 15-d-PGJ2 and NaBut-induced LTR activation. Moreover, it was shown by electrophoretic mobility shift assays that 15-d-PGJ2 represses the nuclear translocation of the ubiquitous transcription factor NF-B, which also results in the repression of HIV-1 transcription (Boisvert et al., 2008). Taken together results showed that the cyclopentone PGJ2 inhibits NaBut-induced NF-B binding activity in Caco-2 cells. This effect is caused by a reduction in the activity of IKK which results in reduced NF-B nuclear translocation but not alterations in IκBα gene expression (Fig 8).

Fig. 8. Effect of 15-d-PGJ2 on NF-B and impact on HIV-1 transcription.

## **4. Conclusion**

358 HIV and AIDS – Updates on Biology, Immunology, Epidemiology and Treatment Strategies

luciferase activity of the pΒ-TATA-luc construct, which contains the HIV-1 enhancer, is abrogated by 15-d-PGJ2 in a dose-dependent manner. A control construct containing the HIV-1 enhancer with inactivated NF-B binding sites, pmB-luc, was used to show that NF- B is necessary for the NaBut activation of the HIV-1 LTR in Caco-2. Similar results to pΒ-TATA-luc were found using the pNFB-luc construct, which contains five consensus NF-κB binding sites. Together, these data suggest that NF-B is involved in the 15-d-PGJ2-mediated

15-d-PGJ2 alters the stability of IBα proteins thereby altering NF-κB activation. Moreover in human bronchial epithelium, cyclopentone prostaglandin such has PGA1, has been shown to enhance the expression of IBα, a primary inhibitor of the pro-inflammatory transcription factor NF-B (Thomas et al., 1998). In intestinal epithelial cells, IKK activity was lower in Caco-2 cells treated with 15-d-PGJ2 and the inhibition of IKK activity was direct without increasing IBα mRNA expression. Another group (Scher & Pillinger, 2005) reported an inhibitory effect of 15-d-PGJ2 on NF-B activation and expression of pro-inflammatory genes such as COX-2, IL-1β and TNF-α. Interestingly in human chondrocytes, 15d-PGJ2, but not troglitazone, modulates IL-1β expression by inhibiting NF-B and AP-1 activation pathways, a mechanism independent of PPAR-γ as observed with 15-d-PGJ2 and NaBut-induced LTR activation. Moreover, it was shown by electrophoretic mobility shift assays that 15-d-PGJ2 represses the nuclear translocation of the ubiquitous transcription factor NF-B, which also results in the repression of HIV-1 transcription (Boisvert et al., 2008). Taken together results showed that the cyclopentone PGJ2 inhibits NaBut-induced NF-B binding activity in Caco-2 cells. This effect is caused by a reduction in the activity of IKK which results in reduced NF-B nuclear

suppression of HIV-1 LTR activation in Caco-2 cells.

translocation but not alterations in IκBα gene expression (Fig 8).

Fig. 8. Effect of 15-d-PGJ2 on NF-B and impact on HIV-1 transcription.

AIDS patients exhibit abnormal production of cyclooxygenase products. Prostaglandins are complex immunomodulatory molecules that shape, on one hand, the immune system and, on the other hand, have an influence on gene transcription by inducing or repressing several transcription factors in cells. Recent studies have led to a better understanding of the unique characteristics and importance of prostaglandins on HIV-1 transcription and replication (Fig 9).

Fig. 9. A model for prostaglandins actions on HIV-1 pathogenesis.

Because of their intrinsic intracellular obligatory parasitic form of life, viruses depend heavily on cell metabolic machinery for their replication. Thus, changes in cellular metabolism might influence the viral life cycle. The data reported in this chapter highlight the positive action of PGE2, a powerful cAMP-inducing agent, on the regulatory elements of HIV-1. PGE2 has now emerged as an immuno-activator that acts on the EP4 receptor that facilitates HIV-1 LTR activation. Elevated levels of PGE2 detected in HIV-1-infected persons or induced by opportunistic pathogens might actively participate to immunological disturbances associated with AIDS and modify the pathogenesis of this retroviral disease by inducing a higher viral load. High concentrations of PGE2 (up to 100 μM) found in seminal fluids of HIV-1-infected persons might directly enhance virus replication and facilitate viral transmission during sexual activities. Thus, analysis of the role of the PGE2 signaling may provide deeper insight into the pathological mechanisms underlying HIV/AIDS exacerbation, which should be fully taken into account in developing an EP4 antagonist as a therapeutic agent and its clinical application.

Accumulating data from several studies suggest that PGJ2 has intracellular effects that may suppress inflammation. They include inhibition of NF-B by multiple mechanisms such as IB kinase inhibition, blockade of NF-B nuclear binding and activation of PPAR-. The consequences of these activities are complex, but are likely to play a role in the prevention and/or resolution of inflammation. In this chapter, we showed the potentiality of the anti-

Emerging Roles of Prostaglandins in HIV-1 Transcription 361

Cao, Z., Umek, R.M., McKnight, S.L., 1991. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes & development 5, 1538-1552. Chen, F., Harrison, L.E., 2005. Ciglitazone induces early cellular proliferation and NF-

Chodosh, L.A., Baldwin, A.S., Carthew, R.W., Sharp, P.A., 1988. Human CCAAT-binding

Chowdhury, M.I., Koyanagi, Y., Horiuchi, S., Hazeki, O., Ui, M., Kitano, K., Golde, D.W.,

Clark, R.B., Bishop-Bailey, D., Estrada-Hernandez, T., Hla, T., Puddington, L., Padula, S.J.,

Coffey, M.J., Phare, S.M., Cinti, S., Peters-Golden, M., Kazanjian, P.H., 1999. Granulocyte-

Coleman, R.A., Eglen, R.M., Jones, R.L., Narumiya, S., Shimizu, T., Smith, W.L., Dahlen, S.E.,

Coleman, R.A., Smith, W.L., Narumiya, S., 1994. International Union of Pharmacology

Daynes, R.A., Jones, D.C., 2002. Emerging roles of PPARs in inflammation and immunity.

De Vries, G.W., Guarino, P., McLaughlin, A., Chen, J., Andrews, S., Woodward, D.F., 1995.

Delezay, O., Koch, N., Yahi, N., Hammache, D., Tourres, C., Tamalet, C., Fantini, J., 1997.

Dickerson, M.C., Johnston, J., Delea, T.E., White, A., Andrews, E., 1996. The causal role for

Int J Biochem Cell Biol 37, 645-654.

with TNF-alpha. Virology 194, 345-349.

1371.

23, 283-285.

Nat Rev Immunol 2, 748-759.

journal of pharmacology 115, 1231-1234.

epithelial cell line HT-29. AIDS 11, 1311-1318.

proteins have heterologous subunits. Cell 53, 11-24.

immunodeficiency syndrome. Blood 94, 3897-3905.

receptors and their subtypes. Pharmacol Rev 46, 205-229.

kappaB transcriptional activity in colon cancer cells through p65 phosphorylation.

Takada, K., Yamamoto, N., 1993. cAMP stimulates human immunodeficiency virus (HIV-1) from latently infected cells of monocyte-macrophage lineage: synergism

2000. The nuclear receptor PPAR gamma and immunoregulation: PPAR gamma mediates inhibition of helper T cell responses. Journal of immunology 164, 1364-

macrophage colony-stimulating factor upregulates reduced 5-lipoxygenase metabolism in peripheral blood monocytes and neutrophils in acquired

Drazen, J.M., Gardiner, P.J., Jackson, W.T., et al., 1995. Prostanoid and leukotriene receptors: a progress report from the IUPHAR working parties on classification and nomenclature. Advances in prostaglandin, thromboxane, and leukotriene research

classification of prostanoid receptors: properties, distribution, and structure of the

An EP receptor with a novel pharmacological profile in the T-cell line Jurkat. British

Co-expression of CXCR4/fusin and galactosylceramide in the human intestinal

genital ulcer disease as a risk factor for transmission of human immunodeficiency virus. An application of the Bradford Hill criteria. Sex Transm Dis 23, 429-440. Dumais, N., Barbeau, B., Olivier, M., Tremblay, M.J., 1998. Prostaglandin E2 Up-regulates

HIV-1 long terminal repeat-driven gene activity in T cells via NF-kappaBdependent and -independent signaling pathways. J Biol Chem 273, 27306-27314. Dumais, N., Bounou, S., Olivier, M., Tremblay, M.J., 2002. Prostaglandin E(2)-mediated

activation of HIV-1 long terminal repeat transcription in human T cells necessitates CCAAT/enhancer binding protein (C/EBP) binding sites in addition to cooperative interactions between C/EBPbeta and cyclic adenosine 5'-

monophosphate response element binding protein. J Immunol 168, 274-282.

inflammatory molecule, PGJ2, to modulate the NaBut effect on HIV-1 LTR in intestinal epithelial cells by a PPAR-γ-independent mechanism via the inhibition of NF-B translocation to the nucleus. These results suggest that such prostaglandins may have therapeutic value in the treatment of HIV-1 infected individuals where inhibition of NF-B activity may be required.

## **5. References**


inflammatory molecule, PGJ2, to modulate the NaBut effect on HIV-1 LTR in intestinal epithelial cells by a PPAR-γ-independent mechanism via the inhibition of NF-B translocation to the nucleus. These results suggest that such prostaglandins may have therapeutic value in the treatment of HIV-1 infected individuals where inhibition of NF-B

Abel, P.M., McSharry, C., Galloway, E., Ross, C., Severn, A., Toner, G., Gruer, L., Wilkinson,

Aguilar-Cordova, E., Chinen, J., Donehower, L., Lewis, D.E., Belmont, J.W., 1994. A sensitive

Amici, C., Sistonen, L., Santoro, M.G., Morimoto, R.I., 1992. Antiproliferative prostaglandins activate heat shock transcription factor. Proc Natl Acad Sci U S A 89, 6227-6231. Ankel, H., Mittnacht, S., Jacobsen, H., 1985. Antiviral activity of prostaglandin A on

Auberger, P., Didier, M., Didier, J., Aussel, C., Fehlmann, M., 1989. A chymotryptic-type

Baker, P.E., Fahey, J.V., Munck, A., 1981. Prostaglandin inhibition of T-cell proliferation is

Belyakov, I.M., Berzofsky, J.A., 2004. Immunobiology of mucosal HIV infection and the basis

Ben-Hur, T., Rosenthal, J., Itzik, A., Weidenfeld, J., 1996. Rescue of HSV-1 neurovirulence is

Bouhlal, H., Chomont, N., Haeffner-Cavaillon, N., Kazatchkine, M.D., Belec, L., Hocini, H.,

Boyault, S., Simonin, M.A., Bianchi, A., Compe, E., Liagre, B., Mainard, D., Becuwe, P.,

Brockman, J.A., Gupta, R.A., Dubois, R.N., 1998. Activation of PPARgamma leads to

NF-kappaB and AP-1 activation pathways. FEBS Lett 501, 24-30.

and neuroendocrine responses. Journal of neurovirology 2, 279-288. Blaschke, V., Jungermann, K., Puschel, G.P., 1996. Exclusive expression of the Gs-linked

effects. AIDS research and human retroviruses 10, 295-301.

production in Jurkat T cells. Cellular signalling 1, 289-294.

mediated at two levels. Cellular immunology 61, 52-61.

Gen Virol 66 ( Pt 11), 2355-2364.

cells. Virology 380, 1-11.

epithelial cells. J Immunol 169, 3301-3306.

Gastroenterology 115, 1049-1055.

P.C., 1992. Heterogeneity of peripheral blood monocyte populations in human immunodeficiency virus-1 seropositive patients. FEMS Microbiol Immunol 5, 317-

reporter cell line for HIV-1 tat activity, HIV-1 inhibitors, and T cell activation

encephalomyocarditis virus-infected cells: a unique effect unrelated to interferon. J

protease inhibitor decreases interleukin 2 synthesis and induces prostaglandin

for development of a new generation of mucosal AIDS vaccines. Immunity 20, 247-

associated with induction of brain interleukin-1 expression, prostaglandin synthesis

prostaglandin E2 receptor subtype 4 mRNA in mononuclear Jurkat and KM-3 cells and coexpression of subtype 4 and 2 mRNA in U-937 cells. FEBS letters 394, 39-43. Boisvert, M., Cote, S., Vargas, A., Pasvanis, S., Bounou, S., Barbeau, B., Dumais, N., 2008.

PGJ2 antagonizes NF-kappaB-induced HIV-1 LTR activation in colonic epithelial

2002. Opsonization of HIV-1 by semen complement enhances infection of human

Dauca, M., Netter, P., Terlain, B., Bordji, K., 2001. 15-Deoxy-delta12,14-PGJ2, but not troglitazone, modulates IL-1beta effects in human chondrocytes by inhibiting

inhibition of anchorage-independent growth of human colorectal cancer cells.

activity may be required.

**5. References** 

323.

253.


Emerging Roles of Prostaglandins in HIV-1 Transcription 363

Ghosh, S., May, M.J., Kopp, E.B., 1998. NF-kappa B and Rel proteins: evolutionarily

Gilroy, D.W., Colville-Nash, P.R., Willis, D., Chivers, J., Paul-Clark, M.J., Willoughby, D.A.,

Goldyne, M.E., Rea, L., 1987. Stimulated T cell and natural killer (NK) cell lines fail to

Golub, E.I., Li, G.R., Volsky, D.J., 1991. Induction of dormant HIV-1 by sodium butyrate:

Gonzalez, G.A., Montminy, M.R., 1989. Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59, 675-680. Gonzalez, G.A., Yamamoto, K.K., Fischer, W.H., Karr, D., Menzel, P., Biggs, W., 3rd, Vale,

Goodwin, J.S., Ceuppens, J., 1983. Regulation of the immune response by prostaglandins.

Goodwin, J.S., Webb, D.R., 1980. Regulation of the immune response by prostaglandins.

Griffin, D.E., Wesselingh, S.L., McArthur, J.C., 1994a. Elevated central nervous system

Griffin, J.W., Wesselingh, S.L., Griffin, D.E., Glass, J.D., McArthur, J.C., 1994b. Peripheral

Habener, J.F., 1990. Cyclic AMP response element binding proteins: a cornucopia of

Haraguchi, S., Good, R.A., Day, N.K., 1995a. Immunosuppressive retroviral peptides: cAMP

Haraguchi, S., Good, R.A., James-Yarish, M., Cianciolo, G.J., Day, N.K., 1995b. Induction of

Hayes, M.M., Lane, B.R., King, S.R., Markovitz, D.M., Coffey, M.J., 2002. Peroxisome

Heinen, E., Cormann, N., Braun, M., Kinet-Denoel, C., Vanderschelden, J., Simar, L.J., 1986.

of prostaglandin secretion. Ann Inst Pasteur Immunol 137D, 369-382.

synthesize leukotriene B4. Prostaglandins 34, 783-795.

cell. The Journal of experimental medicine 146, 1719-1734.

Clinical immunology and immunopathology 15, 106-122.

Journal of clinical immunology 3, 295-315.

Res Publ Assoc Res Nerv Ment Dis 72, 159-182.

transcription factors. Mol Endocrinol 4, 1087-1094.

and cytokine patterns. Immunology today 16, 595-603.

modulators of immunity. Trends in immunology 23, 144-150.

Neurol 35, 592-597.

16913-16919.

260.

668.

698-701.

conserved mediators of immune responses. Annual review of immunology 16, 225-

1999. Inducible cyclooxygenase may have anti-inflammatory properties. Nat Med 5,

involvement of the TATA box in the activation of the HIV-1 promoter. AIDS 5, 663-

W.W., Montminy, M.R., 1989. A cluster of phosphorylation sites on the cyclic AMPregulated nuclear factor CREB predicted by its sequence. Nature 337, 749-752. Goodwin, J.S., Bankhurst, A.D., Messner, R.P., 1977. Suppression of human T-cell

mitogenesis by prostaglandin. Existence of a prostaglandin-producing suppressor

prostaglandins in human immunodeficiency virus-associated dementia. Ann

nerve disorders in HIV infection. Similarities and contrasts with CNS disorders.

intracellular cAMP by a synthetic retroviral envelope peptide: a possible mechanism of immunopathogenesis in retroviral infections. Proceedings of the National Academy of Sciences of the United States of America 92, 5568-5571. Harris, S.G., Padilla, J., Koumas, L., Ray, D., Phipps, R.P., 2002. Prostaglandins as

proliferator-activated receptor gamma agonists inhibit HIV-1 replication in macrophages by transcriptional and post-transcriptional effects. J Biol Chem 277,

Isolation of follicular dendritic cells from human tonsils and adenoids. VI. Analysis


Fantini, J., Baghdiguian, S., Yahi, N., Chermann, J.C., 1991. Selected human

Fantini, J., Bolmont, C., Yahi, N., 1992. Tumor necrosis factor-alpha stimulates both apical

Fantini, J., Cook, D.G., Nathanson, N., Spitalnik, S.L., Gonzalez-Scarano, F., 1993. Infection

Farrell, J.P., Kirkpatrick, C.E., 1987. Experimental cutaneous leishmaniasis. II. A possible role

Faveeuw, C., Fougeray, S., Angeli, V., Fontaine, J., Chinetti, G., Gosset, P., Delerive, P.,

Fedyk, E.R., Phipps, R.P., 1996. Prostaglandin E2 receptors of the EP2 and EP4 subtypes

Fernandez-Cruz, E., Gelpi, E., Longo, N., Gonzalez, B., de la Morena, M.T., Montes, M.G.,

Ferreri, K., Gill, G., Montminy, M., 1994. The cAMP-regulated transcription factor CREB

Foley, P., Kazazi, F., Biti, R., Sorrell, T.C., Cunningham, A.L., 1992. HIV infection of

Frantz, B., Nordby, E.C., Bren, G., Steffan, N., Paya, C.V., Kincaid, R.L., Tocci, M.J., O'Keefe,

Fu, J.Y., Masferrer, J.L., Seibert, K., Raz, A., Needleman, P., 1990. The induction and

Geijtenbeek, T.B., Kwon, D.S., Torensma, R., van Vliet, S.J., van Duijnhoven, G.C., Middel, J.,

B/MAD3, an inhibitor of NF-kappa B. The EMBO journal 13, 861-870. Frey, J., Janes, M., Engelhardt, W., Afting, E.G., Geerds, C., Moller, B., 1986. Fc gamma-

Academy of Sciences of the United States of America 91, 1210-1213.

differentiated human colon epithelial cells. Virology 185, 904-907.

alternative gp120 receptor. Proc Natl Acad Sci U S A 90, 2700-2704.

Immunology letters 34, 85-90.

America 93, 10978-10983.

3, 91-96.

FEBS 158, 85-89.

mice. Journal of immunology 138, 902-907.

in murine dendritic cells. FEBS letters 486, 261-266.

production of eicosanoids. Immunology 75, 391-397.

The Journal of biological chemistry 265, 16737-16740.

enhances trans-infection of T cells. Cell 100, 587-597.

immunodeficiency virus replicates preferentially through the basolateral surface of

and basal production of HIV in polarized human intestinal HT29 cells.

of colonic epithelial cell lines by type 1 human immunodeficiency virus is associated with cell surface expression of galactosylceramide, a potential

for prostaglandins in exacerbation of disease in Leishmania major-infected BALB/c

Maliszewski, C., Capron, M., Staels, B., Moser, M., Trottein, F., 2000. Peroxisome proliferator-activated receptor gamma activators inhibit interleukin-12 production

regulate activation and differentiation of mouse B lymphocytes to IgE-secreting cells. Proceedings of the National Academy of Sciences of the United States of

Rosello, J., Ramis, I., Suarez, A., Fernandez, A., et al., 1989. Increased synthesis and production of prostaglandin E2 by monocytes from drug addicts with AIDS. AIDS

interacts with a component of the TFIID complex. Proceedings of the National

monocytes inhibits the T-lymphocyte proliferative response to recall antigens, via

S.J., O'Neill, E.A., 1994. Calcineurin acts in synergy with PMA to inactivate I kappa

receptor-mediated changes in the plasma membrane potential induce prostaglandin release from human fibroblasts. European journal of biochemistry /

suppression of prostaglandin H2 synthase (cyclooxygenase) in human monocytes.

Cornelissen, I.L., Nottet, H.S., KewalRamani, V.N., Littman, D.R., Figdor, C.G., van Kooyk, Y., 2000. DC-SIGN, a dendritic cell-specific HIV-1-binding protein that


Emerging Roles of Prostaglandins in HIV-1 Transcription 365

Kagawa, N., Waterman, M.R., 1990. cAMP-dependent transcription of the human CYP21B

Kapoor, M., Clarkson, A.N., Sutherland, B.A., Appleton, I., 2005a. The role of antioxidants in

Kapoor, M., Shaw, O., Appleton, I., 2005b. Possible anti-inflammatory role of COX-2-derived

Kernacki, K.A., Berk, R.S., 1994. Characterization of the inflammatory response induced by corneal infection with Pseudomonas aeruginosa. J Ocul Pharmacol 10, 281-288. Kilareski, E.M., Shah, S., Nonnemacher, M.R., Wigdahl, B., 2009. Regulation of HIV-1 transcription in cells of the monocyte-macrophage lineage. Retrovirology 6, 118. Koh, W.S., Yang, K.H., Kaminski, N.E., 1995. Cyclic AMP is an essential factor in immune responses. Biochemical and biophysical research communications 206, 703-709. Kozak, S.L., Platt, E.J., Madani, N., Ferro, F.E., Jr., Peden, K., Kabat, D., 1997. CD4, CXCR-4,

isolates of human immunodeficiency virus type 1. J Virol 71, 873-882. Kozlowski, P.A., Neutra, M.R., 2003. The role of mucosal immunity in prevention of HIV

Kurland, J.I., Bockman, R., 1978. Prostaglandin E production by human blood monocytes

Lee, E.S., Zhou, H., Henderson, A.J., 2001. Endothelial cells enhance human

Lefebvre, A.M., Chen, I., Desreumaux, P., Najib, J., Fruchart, J.C., Geboes, K., Briggs, M.,

Lingk, D.S., Chan, M.A., Gelfand, E.W., 1990. Increased cyclic adenosine monophosphate

Lund, J., Ahlgren, R., Wu, D.H., Kagimoto, M., Simpson, E.R., Waterman, M.R., 1990.

production by macrophages infected with HIV. Cell Immunol 152, 120-130. Mastromarino, P., Conti, C., Petruzziello, R., De Marco, A., Pica, F., Santoro, M.G., 1993.

dependent mechanism. Journal of virology 75, 9703-9712.

APCMin/+ mice. Nature medicine 4, 1053-1057.

immunology 145, 449-455.

of the immune response. Immunology today 9, 222-229.

Inflammopharmacology 12, 505-519.

transmission. Curr Mol Med 3, 217-228.

6, 461-466.

952-957.

222.

(P-450C21) gene requires a cis-regulatory element distinct from the consensus cAMP-regulatory element. The Journal of biological chemistry 265, 11299-11305. Kammer, G.M., 1988. The adenylate cyclase-cAMP-protein kinase A pathway and regulation

models of inflammation: emphasis on L-arginine and arachidonic acid metabolism.

prostaglandins: implications for inflammation research. Curr Opin Investig Drugs

and CCR-5 dependencies for infections by primary patient and laboratory-adapted

and mouse peritoneal macrophages. The Journal of experimental medicine 147,

immunodeficiency virus type 1 replication in macrophages through a C/EBP-

Heyman, R., Auwerx, J., 1998. Activation of the peroxisome proliferator-activated receptor gamma promotes the development of colon tumors in C57BL/6J-

levels block progression but not initiation of human T cell proliferation. Journal of

Transcriptional regulation of the bovine CYP17 (P-450(17)alpha) gene. Identification of two cAMP regulatory regions lacking the consensus cAMPresponsive element (CRE). The Journal of biological chemistry 265, 3304-3312. Mastino, A., Grelli, S., Piacentini, M., Oliverio, S., Favalli, C., Perno, C.F., Garci, E., 1993.

Correlation between induction of lymphocyte apoptosis and prostaglandin E2

Inhibition of Sindbis virus replication by cyclopentenone prostaglandins: a cellmediated event associated with heat-shock protein synthesis. Antiviral Res 20, 209-


Henderson, A.J., Zou, X., Calame, K.L., 1995. C/EBP proteins activate transcription from the

Henke, A., Spengler, H.P., Stelzner, A., Nain, M., Gemsa, D., 1992. Lipopolysaccharide

Hinz, B., Brune, K., Pahl, A., 2003. 15-Deoxy-Delta(12,14)-prostaglandin J2 inhibits the

Hoeffler, J.P., Meyer, T.E., Yun, Y., Jameson, J.L., Habener, J.F., 1988. Cyclic AMP-responsive

Hofmann, B., Nishanian, P., Nguyen, T., Liu, M., Fahey, J.L., 1993. Restoration of T-cell

Hortelano, S., Castrillo, A., Alvarez, A.M., Bosca, L., 2000. Contribution of cyclopentenone

apoptosis in activated macrophages. Journal of immunology 165, 6525-6531. Hughes-Fulford, M., McGrath, M.S., Hanks, D., Erickson, S., Pulliam, L., 1992. Effects of

Hui, R., Curtis, J.F., Sumner, M.T., Shears, S.B., Glasgow, W.C., Eling, T.E., 1995. Human

Ianaro, A., Ialenti, A., Maffia, P., Pisano, B., Di Rosa, M., 2001. Role of cyclopentenone

Imaizumi, T., Kumagai, M., Hatakeyama, M., Tamo, W., Yamashita, K., Tanji, K., Yoshida,

Jiang, C., Ting, A.T., Seed, B., 1998. PPAR-gamma agonists inhibit production of monocyte

Johnson, P.F., 1993. Identification of C/EBP basic region residues involved in DNA

Johnson, P.F., McKnight, S.L., 1989. Eukaryotic transcriptional regulatory proteins. Annu

Jozkowicz, A., Dulak, J., Prager, M., Nanobashvili, J., Nigisch, A., Winter, B., Weigel, G.,

virus replication. Antimicrob Agents Chemother 36, 2253-2258.

human monocytes/macrophages. Journal of virology 69, 8020-8026. Hunter, T., Karin, M., 1992. The regulation of transcription by phosphorylation. Cell 70, 375-

prostaglandins in rat carrageenin pleurisy. FEBS Lett 508, 61-66.

Prostaglandins, leukotrienes, and essential fatty acids 37, 219-234.

inflammatory cytokines. Nature 391, 82-86.

biology 13, 6919-6930.

66, 165-177.

Rev Biochem 58, 799-839.

macrophages/monocytes. Journal of virology 69, 5337-5344.

Immunol 143, 65-70.

1430-1433.

387.

communications 302, 415-420.

analogues. AIDS 7, 659-664.

human immunodeficiency virus type 1 long terminal repeat in

suppresses cytokine release from coxsackie virus-infected human monocytes. Res

expression of proinflammatory genes in human blood monocytes via a PPARgamma-independent mechanism. Biochemical and biophysical research

DNA-binding protein: structure based on a cloned placental cDNA. Science 242,

function in HIV infection by reduction of intracellular cAMP levels with adenosine

prostaglandins to the resolution of inflammation through the potentiation of

dimethyl prostaglandin A1 on herpes simplex virus and human immunodeficiency

immunodeficiency virus type 1 envelope protein does not stimulate either prostaglandin formation or the expression of prostaglandin H synthase in THP-1

H., Satoh, K., 2003. 15-Deoxy-delta 12,14-prostaglandin J2 inhibits the expression of granulocyte-macrophage colony-stimulating factor in endothelial cells stimulated with lipopolysaccharide. Prostaglandins & other lipid mediators 71, 293-299. Ito, S., Narumiya, S., Hayaishi, O., 1989. Prostaglandin D2: a biochemical perspective.

sequence recognition and half-site spacing preference. Molecular and cellular

Huk, I., 2001. Prostaglandin-J2 induces synthesis of interleukin-8 by endothelial cells in a PPAR-gamma-independent manner. Prostaglandins Other Lipid Mediat


Emerging Roles of Prostaglandins in HIV-1 Transcription 367

Phipps, R.P., Illig, K., Schad, V., Bhimani, K., 1988. Differential presentation of tolerogenic

Phipps, R.P., Stein, S.H., Roper, R.L., 1991. A new view of prostaglandin E regulation of the

Pinter, A., Honnen, W.J., Tilley, S.A., Bona, C., Zaghouani, H., Gorny, M.K., Zolla-Pazner, S.,

Pittman, N., Shue, G., LeLeiko, N.S., Walsh, M.J., 1995. Transcription of cystic fibrosis

Pohlmann, S., Baribaud, F., Doms, R.W., 2001. DC-SIGN and DC-SIGNR: helping hands for

Rabbi, M.F., al-Harthi, L., Saifuddin, M., Roebuck, K.A., 1998. The cAMP-dependent protein

Rabbi, M.F., Saifuddin, M., Gu, D.S., Kagnoff, M.F., Roebuck, K.A., 1997. U5 region of the

Rabson, A.B., Lin, H.C., 2000. NF-kappa B and HIV: linking viral and immune activation.

Ramis, I., Rosello-Catafau, J., Gelpi, E., 1992. In vivo transformation of arachidonic acid into

Ramis, I., Rosello-Catafau, J., Gomez, G., Zabay, J.M., Fernandez Cruz, E., Gelpi, E., 1991.

Reiner, N.E., Malemud, C.J., 1984. Arachidonic acid metabolism in murine leishmaniasis

Ricote, M., Li, A.C., Willson, T.M., Kelly, C.J., Glass, C.K., 1998. The peroxisome proliferator-

Rincon, M., Tugores, A., Lopez-Rivas, A., Silva, A., Alonso, M., De Landazuri, M.O., Lopez-

activity in spleen cells. Cellular immunology 88, 501-510.

immunodeficiency virus type 1. Journal of virology 63, 2674-2679.

tumor line. Journal of leukocyte biology 43, 271-278.

immune response. Immunology today 12, 349-352.

268, 613-619.

28848-28857.

233, 235-245.

391, 79-82.

Virology 245, 257-269.

Adv Pharmacol 48, 161-207.

chromatography 575, 143-146.

Microbiol Immunol 4, 273-279.

immunology 18, 1791-1796.

HIV. Trends Immunol 22, 643-646.

phosphoenolpyruvate carboxykinase (GTP). The Journal of biological chemistry

immunoglobulin in vivo by macrophages and by a lymphoid dendritic cell-like

1989. Oligomeric structure of gp41, the transmembrane protein of human

transmembrane conductance regulator requires a CCAAT-like element for both basal and cAMP-mediated regulation. The Journal of biological chemistry 270,

kinase A and protein kinase C-beta pathways synergistically interact to activate HIV-1 transcription in latently infected cells of monocyte/macrophage lineage.

human immunodeficiency virus type 1 long terminal repeat contains TRE-like cAMP-responsive elements that bind both AP-1 and CREB/ATF proteins. Virology

12-hydroxy-5,8,10,14-eicosatetraenoic acid by human nasal mucosa. Journal of

Cyclooxygenase and lipoxygenase arachidonic acid metabolism by monocytes from human immune deficiency virus-infected drug users. J Chromatogr 557, 507-513. Rastogi, N., Bachelet, M., Carvalho de Sousa, J.P., 1992. Intracellular growth of

Mycobacterium avium in human macrophages is linked to the increased synthesis of prostaglandin E2 and inhibition of the phagosome-lysosome fusions. FEMS

(Donovani): ex-vivo evidence for increased cyclooxygenase and 5-lipoxygenase

activated receptor-gamma is a negative regulator of macrophage activation. Nature

Botet, M., 1988. Prostaglandin E2 and the increase of intracellular cAMP inhibit the expression of interleukin 2 receptors in human T cells. European journal of


Meng, G., Wei, X., Wu, X., Sellers, M.T., Decker, J.M., Moldoveanu, Z., Orenstein, J.M.,

Metz, R., Ziff, E., 1991. cAMP stimulates the C/EBP-related transcription factor rNFIL-6 to

Midulla, F., Huang, Y.T., Gilbert, I.A., Cirino, N.M., McFadden, E.R., Jr., Panuska, J.R., 1989.

Miller, C.J., Alexander, N.J., Sutjipto, S., Joye, S.M., Hendrickx, A.G., Jennings, M., Marx,

Mori, K., Tanaka, I., Kotani, M., Miyaoka, F., Sando, T., Muro, S., Sasaki, Y., Nakagawa, O.,

Mueller, C.R., Maire, P., Schibler, U., 1990. DBP, a liver-enriched transcriptional activator, is

Munoz, E., Zubiaga, A.M., Merrow, M., Sauter, N.P., Huber, B.T., 1990. Cholera toxin

Muroi, M., Suzuki, T., 1993. Role of protein kinase A in LPS-induced activation of NF-kappa

Nabel, G., Baltimore, D., 1987. An inducible transcription factor activates expression of

Neutra, M.R., Frey, A., Kraehenbuhl, J.P., 1996. Epithelial M cells: gateways for mucosal

Nishigaki, N., Negishi, M., Honda, A., Sugimoto, Y., Namba, T., Narumiya, S., Ichikawa, A.,

Nokta, M.A., Pollard, R.B., 1992. Human immunodeficiency virus replication: modulation by cellular levels of cAMP. AIDS research and human retroviruses 8, 1255-1261.

Padilla, J., Kaur, K., Cao, H.J., Smith, T.J., Phipps, R.P., 2000. Peroxisome proliferator

Nordheim, A., 1994. Transcription factors. CREB takes CBP to tango. Nature 370, 177-178. Onta, T., Sashida, M., Fujii, N., Sugawara, S., Rikiishi, H., Kumagai, K., 1993. Induction of

human immunodeficiency virus in T cells. Nature 326, 711-713.

Nabel, G.J., 1991. HIV. Tampering with transcription. Nature 350, 658.

infection and immunization. Cell 86, 345-348.

cells EP4 subtype. FEBS letters 364, 339-341.

and alveolar macrophages. Am Rev Respir Dis 140, 771-777.

in rhesus macaques. J Med Primatol 19, 401-409.

molecular medicine 74, 333-336.

posttranscriptionally. Cell 61, 279-291.

150-156.

1754-1766.

103.

Graham, M.F., Kappes, J.C., Mestecky, J., Shaw, G.M., Smith, P.D., 2002. Primary intestinal epithelial cells selectively transfer R5 HIV-1 to CCR5+ cells. Nat Med 8,

trans-locate to the nucleus and induce c-fos transcription. Genes & development 5,

Respiratory syncytial virus infection of human cord and adult blood monocytes

P.A., 1990. Effect of virus dose and nonoxynol-9 on the genital transmission of SIV

Ogawa, Y., Usui, T., Ozaki, S., Ichikawa, A., Narumiya, S., Nakao, K., 1996. Gene expression of the human prostaglandin E receptor EP4 subtype: differential regulation in monocytoid and lymphoid lineage cells by phorbol ester. Journal of

expressed late in ontogeny and its tissue specificity is determined

discriminates between T helper 1 and 2 cells in T cell receptor-mediated activation: role of cAMP in T cell proliferation. The Journal of experimental medicine 172, 95-

B proteins of a mouse macrophage-like cell line, J774. Cellular signalling 5, 289-298.

1995. Identification of prostaglandin E receptor 'EP2' cloned from mastocytoma

acute arthritis in mice by peptidoglycan derived from gram-positive bacteria and its possible role in cytokine production. Microbiology and immunology 37, 573-582.

activator receptor-gamma agonists and 15-deoxy-Delta(12,14)(12,14)-PGJ(2) induce apoptosis in normal and malignant B-lineage cells. J Immunol 165, 6941-6948. Park, E.A., Gurney, A.L., Nizielski, S.E., Hakimi, P., Cao, Z., Moorman, A., Hanson, R.W.,

1993. Relative roles of CCAAT/enhancer-binding protein beta and cAMP regulatory element-binding protein in controlling transcription of the gene for phosphoenolpyruvate carboxykinase (GTP). The Journal of biological chemistry 268, 613-619.


Emerging Roles of Prostaglandins in HIV-1 Transcription 369

Santoro, M.G., Jaffe, B.M., Garaci, E., Esteban, M., 1982. Antiviral effect of prostaglandins of

Santoro, M.G., Rossi, A., Amici, C., 2003. NF-kappaB and virus infection: who controls

Sarraf, P., Mueller, E., Jones, D., King, F.J., DeAngelo, D.J., Partridge, J.B., Holden, S.A.,

Sarraf, P., Mueller, E., Smith, W.M., Wright, H.M., Kum, J.B., Aaltonen, L.A., de la Chapelle,

Scher, J.U., Pillinger, M.H., 2005. 15d-PGJ2: the anti-inflammatory prostaglandin? Clin

Schoonjans, K., Staels, B., Auwerx, J., 1996. Role of the peroxisome proliferator-activated

Shepard, R.N., Schock, J., Robertson, K., Shugars, D.C., Dyer, J., Vernazza, P., Hall, C.,

Skolnik, P.R., Rabbi, M.F., Mathys, J.M., Greenberg, A.S., 2002. Stimulation of peroxisome

Sorrell, T.C., Rochester, C.P., Breen, F.N., Muller, M., 1989. Eicosanoids produced during

Spiegelman, B.M., 1998. PPAR-gamma: adipogenic regulator and thiazolidinedione

Spira, A.I., Marx, P.A., Patterson, B.K., Mahoney, J., Koup, R.A., Wolinsky, S.M., Ho, D.D.,

Straus, D.S., Glass, C.K., 2001. Cyclopentenone prostaglandins: new insights on biological

Straus, D.S., Pascual, G., Li, M., Welch, J.S., Ricote, M., Hsiang, C.H., Sengchanthalangsy,

Tae, H.J., Zhang, S., Kim, K.H., 1995. cAMP activation of CAAT enhancer-binding protein-

Tesmer, V.M., Rajadhyaksha, A., Babin, J., Bina, M., 1993. NF-IL6-mediated transcriptional

species-dependent. Immunol Cell Biol 67 ( Pt 3), 169-176.

activities and cellular targets. Med Res Rev 21, 185-210.

associated with human colon cancer. Mol Cell 3, 799-804.

general virology 63, 435-440.

medicine 4, 1046-1052.

Immunol 114, 100-109.

Defic Syndr 31, 1-10.

Exp Med 183, 215-225.

4844-4849.

expression. J Lipid Res 37, 907-925.

B. Annu Rev Cell Biol 10, 405-455.

receptor. Diabetes 47, 507-514.

biological chemistry 270, 21487-21494.

whom. The EMBO journal 22, 2552-2560.

the A series: inhibition of vaccinia virus replication in cultured cells. The Journal of

Chen, L.B., Singer, S., Fletcher, C., Spiegelman, B.M., 1998. Differentiation and reversal of malignant changes in colon cancer through PPARgamma. Nature

A., Spiegelman, B.M., Eng, C., 1999. Loss-of-function mutations in PPAR gamma

receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene

Cohen, M.S., Fiscus, S.A., 2000. Quantitation of human immunodeficiency virus type 1 RNA in different biological compartments. J Clin Microbiol 38, 1414-1418. Siebenlist, U., Franzoso, G., Brown, K., 1994. Structure, regulation and function of NF-kappa

proliferator-activated receptors alpha and gamma blocks HIV-1 replication and TNFalpha production in acutely infected primary blood cells, chronically infected U1 cells, and alveolar macrophages from HIV-infected subjects. J Acquir Immune

interactions between Pseudomonas aeruginosa and alveolar macrophages are

1996. Cellular targets of infection and route of viral dissemination after an intravaginal inoculation of simian immunodeficiency virus into rhesus macaques. J

L.L., Ghosh, G., Glass, C.K., 2000. 15-deoxy-delta 12,14-prostaglandin J2 inhibits multiple steps in the NF-kappa B signaling pathway. Proc Natl Acad Sci U S A 97,

beta gene expression and promoter I of acetyl-CoA carboxylase. The Journal of

activation of the long terminal repeat of the human immunodeficiency virus type 1.


Rodbell, M., 1980. The role of hormone receptors and GTP-regulatory proteins in membrane

Roesler, W.J., Graham, J.G., Kolen, R., Klemm, D.J., McFie, P.J., 1995. The cAMP response

cAMP responsiveness. The Journal of biological chemistry 270, 8225-8232. Roesler, W.J., Vandenbark, G.R., Hanson, R.W., 1988. Cyclic AMP and the induction of eukaryotic gene transcription. The Journal of biological chemistry 263, 9063-9066. Rohlff, C., Ahmad, S., Borellini, F., Lei, J., Glazer, R.I., 1997. Modulation of transcription

Rohr, O., Schwartz, C., Aunis, D., Schaeffer, E., 1999. CREB and COUP-TF mediate

Roper, R.L., Phipps, R.P., 1994. Prostaglandin E2 regulation of the immune response. Advances in prostaglandin, thromboxane, and leukotriene research 22, 101-111. Ross, H.L., Nonnemacher, M.R., Hogan, T.H., Quiterio, S.J., Henderson, A., McAllister, J.J.,

Rossi, A., Kapahi, P., Natoli, G., Takahashi, T., Chen, Y., Karin, M., Santoro, M.G., 2000.

Royce, R.A., Sena, A., Cates, W., Jr., Cohen, M.S., 1997. Sexual transmission of HIV. N Engl J

Rozera, C., Carattoli, A., De Marco, A., Amici, C., Giorgi, C., Santoro, M.G., 1996. Inhibition

cells. Evidence for a transcriptional block. J Clin Invest 97, 1795-1803. Saez, E., Rosenfeld, J., Livolsi, A., Olson, P., Lombardo, E., Nelson, M., Banayo, E., Cardiff,

mammary gland tumor development. Genes & development 18, 528-540. Saez, E., Tontonoz, P., Nelson, M.C., Alvarez, J.G., Ming, U.T., Baird, S.M., Thomazy, V.A.,

Sakata, D., Yao, C., Narumiya, S., 2010. Prostaglandin E2, an immunoactivator. J Pharmacol

Santoro, M.G., Benedetto, A., Carruba, G., Garaci, E., Jaffe, B.M., 1980. Prostaglandin A

Santoro, M.G., Benedetto, A., Zaniratti, S., Garaci, E., Jaffe, B.M., 1983. The relationship

cell lines and in persistently infected cells. Prostaglandins 25, 353-364. Santoro, M.G., Favalli, C., Mastino, A., Jaffe, B.M., Esteban, M., Garaci, E., 1988. Antiviral

element binding protein synergizes with other transcription factors to mediate

factor Sp1 by cAMP-dependent protein kinase. The Journal of biological chemistry

transcriptional activation of the human immunodeficiency virus type 1 genome in Jurkat T cells in response to cyclic AMP and dopamine. Journal of cellular

Krebs, F.C., Wigdahl, B., 2001. Interaction between CCAAT/enhancer binding protein and cyclic AMP response element binding protein 1 regulates human immunodeficiency virus type 1 transcription in cells of the monocyte/macrophage

Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of IkappaB

of HIV-1 replication by cyclopentenone prostaglandins in acutely infected human

R.D., Izpisua-Belmonte, J.C., Evans, R.M., 2004. PPAR gamma signaling exacerbates

Evans, R.M., 1998. Activators of the nuclear receptor PPARgamma enhance colon

between prostaglandins and virus replication: endogenous prostaglandin synthesis during infection and the effect of exogenous PGA on virus production in different

activity of a synthetic analog of prostaglandin A in mice infected with influenza A

Roesler, W.J., 2000. What is a cAMP response unit? Mol Cell Endocrinol 162, 1-7.

transduction. Nature 284, 17-22.

272, 21137-21141.

biochemistry 75, 404-413.

kinase. Nature 403, 103-108.

virus. Arch Virol 99, 89-100.

Med 336, 1072-1078.

Sci 112, 1-5.

lineage. Journal of virology 75, 1842-1856.

polyp formation. Nature medicine 4, 1058-1061.

compounds as antiviral agents. Science 209, 1032-1034.


Proceedings of the National Academy of Sciences of the United States of America 90, 7298-7302.

**Part 3** 

**From the Clinic to the Patients: HIV and Clinical Manifestations** 

