**Use of Animal Models for Anti-HIV Drug Development**

Zandrea Ambrose *University of Pittsburgh, USA* 

#### **1. Introduction**

Animal models serve as important tools for preclinical testing of therapeutic regimens against human immunodeficiency virus (HIV-1), the primary etiologic agent that causes acquired immunodeficiency syndrome (AIDS). Infection and treatment of patients often cannot be controlled in clinical studies. In addition, performing certain procedures and sampling cannot be routinely performed in humans with ease and may be unethical. There are many different primate and murine models of HIV/AIDS, each with their advantages and disadvantages. Some models are appropriate in certain contexts but not others. Knowing how the different models work and their limitations will help guide the researcher to select the appropriate model to answer a specific question. Information gained from the use of preclinical testing of antiretroviral therapies will help identify and improve preventive, therapeutic, and eradication strategies against HIV/AIDS in humans.

#### **2. HIV-1 infection of nonhuman primates or humanized mice: Which is the better model to use?**

An animal model for human disease should mimic the infection of humans as closely as possible. The disease course in the model should be similar to or more accelerated than in humans. In the case of HIV-1, an animal model that progresses to AIDS over the period of many years will cost time and money in preclinical studies. The use of animals instead of humans usually means certain procedures can be performed more easily and/or ethically. For example, removing vital organs to study pathogenesis, drug penetration, immunity, or virology cannot be performed in humans but can be done after necropsy of an animal. Moreover, unlike in humans, the exact virus, timing of infection, and timing of treatment can be controlled in a model.

HIV-1 does not efficiently replicate in most animals, including nonhuman primates. This is due to differences in host cell factors present in different species that are required for infection or due to innate immunity that appears to have evolved in mammals to ward off infections. Thus, either modification to HIV-1 or to the animal must be made for significant viral replication to occur. This is important for assessing the efficacy of experimental interventions for inhibiting the virus rather than spontaneous control by the host immune system.

Use of Animal Models for Anti-HIV Drug Development 73

Fig. 1. Diagram of the genomes of HIV-1, SIV, and chimeric viruses used in macaques. White

The strengths of murine models for biomedical research are the small size of mice and their relatively low cost, making it feasible to have increased numbers of animals in experiments for greater statistical power. However, HIV-1 encounters multiple barriers in the infection of mouse cells, beginning with the inability of the virus to use the murine CD4 receptor and coreceptors. To overcome these issues, scientists reconstituted a partial human immune system in severe combined immunodeficient (SCID) mice lacking lymphocytes by engrafting them with human peripheral blood lymphocytes or human fetal thymus and liver (Mosier et al., 1988; Namikawa et al., 1990). However, while peripheral T cell subsets could be reconstituted temporarily in these SCID-hu mice, they were not detected to a great extent in tissues. In addition, other human immune cells did not develop and only transient

Due to the limitations of SCID-hu mice, new advances in humanized murine models have been made to better reconstitute a human immune system and to lead to sustained HIV-1 replication. First, the addition of the SCID mutation into the nonobese diabetic strain (NOD/SCID), which lacks the IL-2 receptor -chain, resulted in mice without T, B, and NK cells. With the implantation of human CD34+ hematopoietic stem cells into these mice, they developed human lymphocytes and dendritic cells in the blood and in multiple lymphoid tissues. Thus, HIV-1 infection could be sustained at high levels for more than 40 days (Watanabe et al., 2007). Similarly, transplantation of human fetal bone marrow (containing CD34+ cells), liver, and thymus (BLT) into NOD/SCID mice could also generate functional human T, B, and dendritic cells in both the periphery and tissues. These animals could

color denotes HIV-1 sequences, while gray shading denotes SIV sequences.

**2.2 Humanized mouse models** 

HIV-1 replication could be detected in vivo.

#### **2.1 Nonhuman primate models**

Nonhuman primates are genetically and anatomically most similar to humans and would be the obvious choice for an animal model to study HIV-1. While HIV-1 is believed to have arisen from cross-species transmission of simian immunodeficiency virus (SIV) strains from chimpanzees and gorillas to humans (Keele et al., 2006; Van Heuverswyn et al., 2006), these animals are not routinely used for HIV research. These great apes are both endangered and very large. And although HIV-1 has been used in the past to infect chimpanzees in captivity, it does not cause significant disease for more than a decade (Novembre et al., 1997). Thus, this is an impractical model for testing treatments and vaccines against the virus.

Macaques, a genus of Old World monkeys, are routinely bred at primate centers and have been extensively investigated as HIV models. HIV-1 inoculation of macaques does not lead to productive infection, mainly due to restriction by simian innate immune factors, such as APOBEC3G and TRIM5, that target HIV-1 (Mariani et al., 2003; Stremlau et al., 2004). SIV is a primate lentivirus that is similar to HIV-1 and highly homologous to HIV type 2 (HIV-2), which was isolated from rhesus macaques at a primate facility (Daniel et al., 1985; Kanki et al., 1985). SIV infection of macaques is believed to have been another cross-species transmission during captivity from infected African primates (Hirsch et al., 1989), leading to pathogenesis similar to AIDS but in a more accelerated time frame as compared to HIV-1 infection of humans. In addition, adaptive immune responses against SIV in macaques are similar to anti-HIV-1 responses seen in humans (Sato and Johnson, 2007; Valentine and Watkins, 2008). The most widely used species of macaques for HIV/AIDS models are rhesus (*Macaca mulatta*), cynomolgus (*M. fasicularis*), and pigtailed (*M. nemestrina*).

While SIV shares high structural and sequence identity to HIV-1, the differences are significant enough to limit the design of both vaccines and therapy against the human virus. Therefore, HIV-1 sequences have been added into the SIV genome to make chimeric viruses, called SHIVs, which can still replicate well within macaques (Fig. 1). The first examples of SHIVs were SIV strains that encoded HIV-1 envelope in place of SIV envelope, such that vaccines or drugs could target this entry protein (Li et al., 1992; Luciw et al., 1995; Reimann et al., 1996; Shibata et al., 1991). More recently, the reverse transcriptase (RT) coding region of SIVs have been replaced with that of HIV-1 to produce RT-SHIV that can be targeted by RT inhibitors (Ambrose et al., 2004; Uberla et al., 1995). Both types of SHIVs have been shown to infect macaques after mucosal exposure, simulating sexual transmission (Lu et al., 1996; Turville et al., 2008). Simian-tropic HIV-1 (stHIV-1) viruses have been made that contain minimal SIV sequences (capsid and Vif coding regions) to circumvent restriction from APOBEC3G and TRIM5, but they suffered significant decreases in replication in the host compared to SIV or the previously described SHIVs, likely due to differences in the accessory proteins of HIV-1 as compared to SIV (Hatziioannou et al., 2009; Igarashi et al., 2007).

Baboons and other species of Old World monkeys have also been used as nonhuman primate models for studying HIV infection and AIDS. Baboons could be productively infected with HIV-2 but showed little pathogenesis (Barnett et al., 1994). African green monkeys and sooty mangabeys are naturally infected with SIV but do not experience disease despite very high levels of viremia. These animals are studied for their differences to Asian macaques to understand chronic, pathogenic SIV/HIV infection (Paiardini et al., 2009).

Nonhuman primates are genetically and anatomically most similar to humans and would be the obvious choice for an animal model to study HIV-1. While HIV-1 is believed to have arisen from cross-species transmission of simian immunodeficiency virus (SIV) strains from chimpanzees and gorillas to humans (Keele et al., 2006; Van Heuverswyn et al., 2006), these animals are not routinely used for HIV research. These great apes are both endangered and very large. And although HIV-1 has been used in the past to infect chimpanzees in captivity, it does not cause significant disease for more than a decade (Novembre et al., 1997). Thus,

Macaques, a genus of Old World monkeys, are routinely bred at primate centers and have been extensively investigated as HIV models. HIV-1 inoculation of macaques does not lead to productive infection, mainly due to restriction by simian innate immune factors, such as APOBEC3G and TRIM5, that target HIV-1 (Mariani et al., 2003; Stremlau et al., 2004). SIV is a primate lentivirus that is similar to HIV-1 and highly homologous to HIV type 2 (HIV-2), which was isolated from rhesus macaques at a primate facility (Daniel et al., 1985; Kanki et al., 1985). SIV infection of macaques is believed to have been another cross-species transmission during captivity from infected African primates (Hirsch et al., 1989), leading to pathogenesis similar to AIDS but in a more accelerated time frame as compared to HIV-1 infection of humans. In addition, adaptive immune responses against SIV in macaques are similar to anti-HIV-1 responses seen in humans (Sato and Johnson, 2007; Valentine and Watkins, 2008). The most widely used species of macaques for HIV/AIDS models are rhesus

While SIV shares high structural and sequence identity to HIV-1, the differences are significant enough to limit the design of both vaccines and therapy against the human virus. Therefore, HIV-1 sequences have been added into the SIV genome to make chimeric viruses, called SHIVs, which can still replicate well within macaques (Fig. 1). The first examples of SHIVs were SIV strains that encoded HIV-1 envelope in place of SIV envelope, such that vaccines or drugs could target this entry protein (Li et al., 1992; Luciw et al., 1995; Reimann et al., 1996; Shibata et al., 1991). More recently, the reverse transcriptase (RT) coding region of SIVs have been replaced with that of HIV-1 to produce RT-SHIV that can be targeted by RT inhibitors (Ambrose et al., 2004; Uberla et al., 1995). Both types of SHIVs have been shown to infect macaques after mucosal exposure, simulating sexual transmission (Lu et al., 1996; Turville et al., 2008). Simian-tropic HIV-1 (stHIV-1) viruses have been made that contain minimal SIV sequences (capsid and Vif coding regions) to circumvent restriction from APOBEC3G and TRIM5, but they suffered significant decreases in replication in the host compared to SIV or the previously described SHIVs, likely due to differences in the accessory proteins of HIV-1 as compared to SIV (Hatziioannou et al., 2009; Igarashi et al.,

Baboons and other species of Old World monkeys have also been used as nonhuman primate models for studying HIV infection and AIDS. Baboons could be productively infected with HIV-2 but showed little pathogenesis (Barnett et al., 1994). African green monkeys and sooty mangabeys are naturally infected with SIV but do not experience disease despite very high levels of viremia. These animals are studied for their differences to Asian macaques to

understand chronic, pathogenic SIV/HIV infection (Paiardini et al., 2009).

this is an impractical model for testing treatments and vaccines against the virus.

(*Macaca mulatta*), cynomolgus (*M. fasicularis*), and pigtailed (*M. nemestrina*).

**2.1 Nonhuman primate models** 

2007).

Fig. 1. Diagram of the genomes of HIV-1, SIV, and chimeric viruses used in macaques. White color denotes HIV-1 sequences, while gray shading denotes SIV sequences.

#### **2.2 Humanized mouse models**

The strengths of murine models for biomedical research are the small size of mice and their relatively low cost, making it feasible to have increased numbers of animals in experiments for greater statistical power. However, HIV-1 encounters multiple barriers in the infection of mouse cells, beginning with the inability of the virus to use the murine CD4 receptor and coreceptors. To overcome these issues, scientists reconstituted a partial human immune system in severe combined immunodeficient (SCID) mice lacking lymphocytes by engrafting them with human peripheral blood lymphocytes or human fetal thymus and liver (Mosier et al., 1988; Namikawa et al., 1990). However, while peripheral T cell subsets could be reconstituted temporarily in these SCID-hu mice, they were not detected to a great extent in tissues. In addition, other human immune cells did not develop and only transient HIV-1 replication could be detected in vivo.

Due to the limitations of SCID-hu mice, new advances in humanized murine models have been made to better reconstitute a human immune system and to lead to sustained HIV-1 replication. First, the addition of the SCID mutation into the nonobese diabetic strain (NOD/SCID), which lacks the IL-2 receptor -chain, resulted in mice without T, B, and NK cells. With the implantation of human CD34+ hematopoietic stem cells into these mice, they developed human lymphocytes and dendritic cells in the blood and in multiple lymphoid tissues. Thus, HIV-1 infection could be sustained at high levels for more than 40 days (Watanabe et al., 2007). Similarly, transplantation of human fetal bone marrow (containing CD34+ cells), liver, and thymus (BLT) into NOD/SCID mice could also generate functional human T, B, and dendritic cells in both the periphery and tissues. These animals could

Use of Animal Models for Anti-HIV Drug Development 75

As there is no cure for HIV-1 yet, efforts have been made to develop and evaluate compounds that would prevent HIV-1 infection prior to or immediately after exposure to the virus. These differ from vaccines in that they are not designed to elicit antiviral immunity in advance of exposure, but rather would inhibit the virus before, during, or just after exposure to HIV-1 to avoid systemic infection. Pre-exposure prophylaxis would be initiated in high-risk individuals likely to be exposed to HIV-1, whereas post-exposure prophylaxis would be used in individuals who were believed to be recently exposed to the virus. Animal models have been used rather extensively over the past decade in this area of research with generally positive results. Unlike in clinical trials, the timing and adherence of

The majority of prevention therapy studies have focused on pre-exposure prophylaxis, or microbicides, against mucosal transmission of virus. This is particularly relevant in areas of the world where people, especially women, often are unable to control their partners' use of condoms during sexual intercourse. Thus, a compound that can be applied mucosally or taken orally could inhibit HIV-1 infection either by targeting the virus or targeting viral

Generally, compounds that inhibit HIV-1 are discovered and characterized *in vitro*. Before going to clinical trials for efficacy testing, tolerability and toxicity studies in animals or people are often performed. In the case of topically applied microbicides, this entails determining whether a drug causes disruption of the mucosal epithelial layer that forms an intact barrier against incoming pathogens. The need for toxicity testing was made dramatically clear in the case of nonoxynol-9 (N-9), which was halted in clinical trials as a potential anti-HIV topical microbicide due to toxic effects that made users more susceptible to HIV-1 infection. N-9, a nonionic detergent present in some contraceptive gels, was shown long ago to inhibit viral replication *in vitro* (Hicks et al., 1985). Several clinical studies of N-9 use in women during vaginal sexual intercourse suggested that it slightly increased the risk for HIV-1 seroconversion (Wilkinson et al., 2002). There were discrepancies on whether or not N-9 caused toxicity in the female genital tract, which may have been due to poor adherence and inappropriate application of the product. A careful study in the pigtailed

macaque model showed that N-9 caused genital tissue damage (Patton et al., 1999).

The female genital tract consists of stratified squamous epithelial cells (vagina and ectocervix) and simple columnar epithelium (endocervix), while the GI tract consists only of a single layer of columnar epithelial cells. Trials of N-9 for rectal use showed that histological abnormalities occurred in almost 90% of the subjects (Tabet et al., 1999) and caused rapid exfoliation of the rectal epithelium (Phillips et al., 2000). Similar results were also observed in pigtailed macaques (Patton et al., 2002), suggesting that safety testing is necessary for microbicides prior to initiating clinical studies to prevent enhanced HIV-1 transmission.

Inflammation and toxicity markers and their correlation, or lack thereof, with complete protection from transmission are still incompletely defined. Administration of a foreign

**3. Therapy for HIV-1 prevention in animal models** 

**3.1 Pre-exposure prophylaxis** 

treatment and the timing of virus challenge can be controlled in the model.

interaction with host cell factors necessary for viral replication.

**3.1.1 Toxicity of mucosal drug application** 

stably maintain HIV-1 replication after intrarectal or intravaginal challenge (Denton et al., 2008; Sun et al., 2007). However, NOD/SCID mice develop thymic lymphomas, resulting in a limited lifespan (Shultz et al., 1995).

Another humanized mouse model utilizes Rag2-/-C-/- double knockout mice, which also lack T, B, and NK cells. These mice can also be reconstituted with CD34+ hematopoietic stem cells, leading to development of human T, B, and dendritic cells in the blood and different lymphoid tissues (Traggiai et al., 2004). The animals could be infected with HIV-1 and had detectable viremia for more than 27 weeks (Baenziger et al., 2006). Like the BLT model, Rag2-/-C-/- mice also had CD4+ target cells in mucosal tissues and could be infected intrarectally or intravaginally (Berges et al., 2008).


Table 1. Advantages and disadvantages to simian and murine models of HIV/AIDS

#### **2.3 What virus should be used with which model?**

With a wide array of simian and murine models of HIV/AIDS, it is difficult to know which one to use to answer a scientific question. Each model has its advantages and disadvantages (Table 1). Depending on the question, one has to weigh the pros and cons that may affect the results in deciding which model and which virus to employ. For example, macaques are more anatomically similar to humans and studying drug penetration into tissues or inhibition of virus in tissues, such as microbicides, may be more relevant in the monkey model. However, at this time HIV-1 replication is very limited in simians, necessitating the use of SIV or SHIVs. Therefore, drugs targeting viral proteins or virus-host cell protein interactions may be limited with these viruses and may require the use of HIV-1 in mice. Although mice are smaller and cheaper than monkeys, humanized murine models require significant expertise and human donors for tissue implantation and stem cell reconstitution that may not be more cost-effective for the investigator. The rest of the chapter will discuss the use of both simian and murine models for preclinical studies of anti-HIV therapies.

stably maintain HIV-1 replication after intrarectal or intravaginal challenge (Denton et al., 2008; Sun et al., 2007). However, NOD/SCID mice develop thymic lymphomas, resulting in

Another humanized mouse model utilizes Rag2-/-C-/- double knockout mice, which also lack T, B, and NK cells. These mice can also be reconstituted with CD34+ hematopoietic stem cells, leading to development of human T, B, and dendritic cells in the blood and different lymphoid tissues (Traggiai et al., 2004). The animals could be infected with HIV-1 and had detectable viremia for more than 27 weeks (Baenziger et al., 2006). Like the BLT model, Rag2-/-C-/- mice also had CD4+ target cells in mucosal tissues and could be infected

**Advantages Disadvantages Advantages Disadvantages** 

Requires SIV or SHIV Can use different

Table 1. Advantages and disadvantages to simian and murine models of HIV/AIDS

With a wide array of simian and murine models of HIV/AIDS, it is difficult to know which one to use to answer a scientific question. Each model has its advantages and disadvantages (Table 1). Depending on the question, one has to weigh the pros and cons that may affect the results in deciding which model and which virus to employ. For example, macaques are more anatomically similar to humans and studying drug penetration into tissues or inhibition of virus in tissues, such as microbicides, may be more relevant in the monkey model. However, at this time HIV-1 replication is very limited in simians, necessitating the use of SIV or SHIVs. Therefore, drugs targeting viral proteins or virus-host cell protein interactions may be limited with these viruses and may require the use of HIV-1 in mice. Although mice are smaller and cheaper than monkeys, humanized murine models require significant expertise and human donors for tissue implantation and stem cell reconstitution that may not be more cost-effective for the investigator. The rest of the chapter will discuss the use of both simian and murine models for preclinical studies of anti-HIV therapies.

**Macaques Humanized CD34+ reconstituted mice** 

Target cells are

HIV-1 strains

Can create genetically identical animals with cells from same

use of less drugs

Lack of macrophages (Rag2/C-/-) and robust antiretroviral immunity

Small tissue/blood

Requires access to donor tissues and ability to perform transplants

erent than humans

Limited lifespan, especially BLT

samples

Less overall cost Anatomically diff-

human

donor

a limited lifespan (Shultz et al., 1995).

Similar viral pathogenesis to

Similar antiviral immune responses to

Long-term viral persistence during suppressive antiretroviral therapy

Access to large tissue/blood samples

humans

humans

intrarectally or intravaginally (Berges et al., 2008).

Genetically different than humans

Expensive and requires trained veterinary staff

more drugs

**2.3 What virus should be used with which model?** 

Large size requiring

Longer lifespan Small size allows the

#### **3. Therapy for HIV-1 prevention in animal models**

As there is no cure for HIV-1 yet, efforts have been made to develop and evaluate compounds that would prevent HIV-1 infection prior to or immediately after exposure to the virus. These differ from vaccines in that they are not designed to elicit antiviral immunity in advance of exposure, but rather would inhibit the virus before, during, or just after exposure to HIV-1 to avoid systemic infection. Pre-exposure prophylaxis would be initiated in high-risk individuals likely to be exposed to HIV-1, whereas post-exposure prophylaxis would be used in individuals who were believed to be recently exposed to the virus. Animal models have been used rather extensively over the past decade in this area of research with generally positive results. Unlike in clinical trials, the timing and adherence of treatment and the timing of virus challenge can be controlled in the model.

#### **3.1 Pre-exposure prophylaxis**

The majority of prevention therapy studies have focused on pre-exposure prophylaxis, or microbicides, against mucosal transmission of virus. This is particularly relevant in areas of the world where people, especially women, often are unable to control their partners' use of condoms during sexual intercourse. Thus, a compound that can be applied mucosally or taken orally could inhibit HIV-1 infection either by targeting the virus or targeting viral interaction with host cell factors necessary for viral replication.

#### **3.1.1 Toxicity of mucosal drug application**

Generally, compounds that inhibit HIV-1 are discovered and characterized *in vitro*. Before going to clinical trials for efficacy testing, tolerability and toxicity studies in animals or people are often performed. In the case of topically applied microbicides, this entails determining whether a drug causes disruption of the mucosal epithelial layer that forms an intact barrier against incoming pathogens. The need for toxicity testing was made dramatically clear in the case of nonoxynol-9 (N-9), which was halted in clinical trials as a potential anti-HIV topical microbicide due to toxic effects that made users more susceptible to HIV-1 infection. N-9, a nonionic detergent present in some contraceptive gels, was shown long ago to inhibit viral replication *in vitro* (Hicks et al., 1985). Several clinical studies of N-9 use in women during vaginal sexual intercourse suggested that it slightly increased the risk for HIV-1 seroconversion (Wilkinson et al., 2002). There were discrepancies on whether or not N-9 caused toxicity in the female genital tract, which may have been due to poor adherence and inappropriate application of the product. A careful study in the pigtailed macaque model showed that N-9 caused genital tissue damage (Patton et al., 1999).

The female genital tract consists of stratified squamous epithelial cells (vagina and ectocervix) and simple columnar epithelium (endocervix), while the GI tract consists only of a single layer of columnar epithelial cells. Trials of N-9 for rectal use showed that histological abnormalities occurred in almost 90% of the subjects (Tabet et al., 1999) and caused rapid exfoliation of the rectal epithelium (Phillips et al., 2000). Similar results were also observed in pigtailed macaques (Patton et al., 2002), suggesting that safety testing is necessary for microbicides prior to initiating clinical studies to prevent enhanced HIV-1 transmission.

Inflammation and toxicity markers and their correlation, or lack thereof, with complete protection from transmission are still incompletely defined. Administration of a foreign

Use of Animal Models for Anti-HIV Drug Development 77

prophylaxis. Tenofovir prevents HIV-1 replication, it is already approved for use in humans in the oral formulation, and high drug concentrations can be achieved in the male and female genital tracts (Kwara et al., 2008; Vourvahis et al., 2008). The use of a gel may not provide complete coverage of the mucosal surface, possibly leading to breakthrough infections. The CAPRISA 004 clinical trial recently showed a 39% overall reduction in HIV-1 incidence in women using a vaginal tenofovir gel (Abdool Karim et al., 2010). The iPrEx and TDF2 trials showed that oral tenofovir in combination with another RT inhibitor, emtracitabine (FTC), reduced transmission in men who have sex with men (Grant et al., 2010) or in heterosexual men and women (Roehr, 2011) by 44% and 63%, respectively. It is unclear whether the greater efficacy in the latter studies as compared to the CAPRISA 004 study was due to oral administration of drug or use of two drugs instead of tenofovir alone. In macaques, intermittent dosing (2 hours before and 24 hours after challenge) of tenofovir alone or with FTC was equally as effective as daily dosing during weekly repeated low-dose rectal challenges (Garcia-Lerma et al., 2008). Less frequent doses would reduce cost to the user and would not require strict daily adherence. However, breakthrough infections in 2 of 6 animals resulted in drug resistant viruses, which could potentially compromise future therapy. Interestingly, with the exception of tenofovir or tenofovir/FTC, no other microbicides have been successful in the clinic. It remains to be seen whether or not this is due to a specific property of tenofovir or RT inhibitors in general. Tenofovir acts specifically on viral reverse transcription, whereas most other microbicides tested clinically were nonspecific entry inhibitors. Also, tenofovir is the only drug that has been tested with repeated low-dose SIV oral, intravaginal, and intrarectal challenges in macaques (Garcia-Lerma et al., 2008; Parikh et al., 2009; Van Rompay et al., 2006). A nonspecific virus inactivating compound was tested in repeated high-dose intravaginal challenges 10-47 weeks apart, in which 90% of the animals were protected after the initial challenge (Ambrose et al., 2008). However, nearly all the animals became infected after the second challenge. It is unclear if noninfectious virus at the site of transmission elicits a detrimental immune response, inflammation, and/or an increase in target cells that can cause a subject to become more sensitized to infection.

Until recently, mucosal challenge of SIV or SHIV required high doses of virus to ensure that all or most of the control animals become infected. More and more investigators are using repeated low-dose challenges in animal studies to recapitulate more realistic levels of virus detected in semen of untreated HIV+ men. Semen viral RNA levels are often similar to or lower than in the blood (Gupta et al., 1997; Liuzzi et al., 1996), but are significantly lower than those of laboratory stocks used in high-dose challenges (Marthas et al., 2001). Also, high-risk individuals may be exposed multiple times prior to becoming infected. A direct comparison of a high-dose challenge and repeated low-dose rectal challenges in a small study on oral tenofovir pre-exposure prophylaxis showed that the low-dose model was actually somewhat more stringent (Subbarao et al., 2007). The disadvantage of the low-dose model is that multiple challenges are required to infect untreated control monkeys, and this number varies significantly among animals. This can affect statistics and often leads to

Most post-exposure prophylaxis research has focused on entry and reverse transcription inhibitors that target the earliest steps of HIV-1 replication. Tenofovir was the first

longer experiments, which can increase study costs.

**3.2 Post-exposure prophylaxis** 

compound is likely to induce innate immune responses. This is also the initial response of the body in countering HIV-1 immediately following exposure. Maintaining normal, intact tissue and normal microflora at mucosal sites during use of a topical microbicide will continue to be a challenge. And the use of oral drugs that can penetrate tissues also will need to be evaluated for safety and lack of mucosal toxicity.

#### **3.1.2 Therapeutic prevention studies**

Many compounds have been tested as pre-exposure prophylaxis in animal models prior to intravaginal, intrarectal, or oral exposure of virus. Such preventive drugs can be nonspecific to HIV-1 or specific antiretroviral compounds. Intravaginal or intrarectal transmission of HIV-1 occurs during sexual contact, while oral transmission may contribute to infection of infants during vaginal delivery. While both macaque and humanized mouse models have been useful for mucosal viral transmission, HIV-1 can only be used to infect humanized mice. Macaques can be infected effectively with chimeric SHIV viruses containing HIV-1 envelope or RT. And the anatomy of nonhuman primates, including the gastrointestinal and genital tracts, is more similar to that of humans.

The majority of such pre-exposure prophylaxis studies have been performed with various levels of success in female macaques for the prevention of vaginal transmission. Compounds tested in macaque models have aimed to interfere with nonspecific viral attachment (Ambrose et al., 2008; Boadi et al., 2005; Kenney et al., 2011; Kim et al., 2006; Lagenaur et al., 2011; Li et al., 2009; Manson et al., 2000; Tevi-Benissan et al., 2000; Tsai et al., 2004; Wyand et al., 1999), specific interactions of envelope with receptor/co-receptors (Kish-Catalone et al., 2007; Lederman et al., 2004; Mascola et al., 2000; Parren et al., 2001; Veazey et al., 2008; Veazey et al., 2010; Veazey et al., 2003a; Veazey et al., 2005a; Veazey et al., 2009; Veazey et al., 2003b; Veazey et al., 2005b), and reverse transcription (Kenney et al., 2011; Parikh et al., 2009; Stolte-Leeb et al., 2011; Turville et al., 2008). Another study investigated hormone treatment, which leads to thickening of the vaginal epithelium, to prevent vaginal transmission of SIV in macaques (Smith et al., 2000b). More recently, the humanized mouse model has been used to prevent intravaginal transmission, using drugs targeting RT (Denton et al., 2008; Denton et al., 2011), integrase (Neff et al., 2011a), the CCR5 co-receptor (Neff et al., 2011a; Neff et al., 2010), and viral protein expression (Wheeler et al., 2011).

Fewer studies have evaluated compounds that prevent intrarectal or oral transmission of HIV-1 or SHIVs. The FDA-approved RT inhibitor tenofovir was successful in preventing intrarectal transmission of SIV in macaques (Cranage et al., 2008) and of HIV-1 in humanized mice (Denton et al., 2010). Also, a novel RT inhibitor (Singer et al., 2011) and a nonspecific envelope attachment inhibitor (Tsai et al., 2003) were used to prevent intrarectal transmission of SHIVs in macaques. For prevention of oral viral transmission, only macaque models have been used. First, neutralizing antibodies were shown to be protective in neonates (Baba et al., 2000). More recently, subcutanously administered tenofovir was found to be somewhat protective against oral SIV challenge (Van Rompay et al., 2006; Van Rompay et al., 2001) while an oral tenofovir solution was ineffective (Van Rompay et al., 2006; Van Rompay et al., 2002b).

While most of these studies have focused on topical gels and novel compounds, oral FDAapproved antiretroviral compounds have been investigated recently as pre-exposure

compound is likely to induce innate immune responses. This is also the initial response of the body in countering HIV-1 immediately following exposure. Maintaining normal, intact tissue and normal microflora at mucosal sites during use of a topical microbicide will continue to be a challenge. And the use of oral drugs that can penetrate tissues also will

Many compounds have been tested as pre-exposure prophylaxis in animal models prior to intravaginal, intrarectal, or oral exposure of virus. Such preventive drugs can be nonspecific to HIV-1 or specific antiretroviral compounds. Intravaginal or intrarectal transmission of HIV-1 occurs during sexual contact, while oral transmission may contribute to infection of infants during vaginal delivery. While both macaque and humanized mouse models have been useful for mucosal viral transmission, HIV-1 can only be used to infect humanized mice. Macaques can be infected effectively with chimeric SHIV viruses containing HIV-1 envelope or RT. And the anatomy of nonhuman primates, including the gastrointestinal

The majority of such pre-exposure prophylaxis studies have been performed with various levels of success in female macaques for the prevention of vaginal transmission. Compounds tested in macaque models have aimed to interfere with nonspecific viral attachment (Ambrose et al., 2008; Boadi et al., 2005; Kenney et al., 2011; Kim et al., 2006; Lagenaur et al., 2011; Li et al., 2009; Manson et al., 2000; Tevi-Benissan et al., 2000; Tsai et al., 2004; Wyand et al., 1999), specific interactions of envelope with receptor/co-receptors (Kish-Catalone et al., 2007; Lederman et al., 2004; Mascola et al., 2000; Parren et al., 2001; Veazey et al., 2008; Veazey et al., 2010; Veazey et al., 2003a; Veazey et al., 2005a; Veazey et al., 2009; Veazey et al., 2003b; Veazey et al., 2005b), and reverse transcription (Kenney et al., 2011; Parikh et al., 2009; Stolte-Leeb et al., 2011; Turville et al., 2008). Another study investigated hormone treatment, which leads to thickening of the vaginal epithelium, to prevent vaginal transmission of SIV in macaques (Smith et al., 2000b). More recently, the humanized mouse model has been used to prevent intravaginal transmission, using drugs targeting RT (Denton et al., 2008; Denton et al., 2011), integrase (Neff et al., 2011a), the CCR5 co-receptor (Neff et al., 2011a; Neff et al., 2010), and viral protein expression (Wheeler et al., 2011).

Fewer studies have evaluated compounds that prevent intrarectal or oral transmission of HIV-1 or SHIVs. The FDA-approved RT inhibitor tenofovir was successful in preventing intrarectal transmission of SIV in macaques (Cranage et al., 2008) and of HIV-1 in humanized mice (Denton et al., 2010). Also, a novel RT inhibitor (Singer et al., 2011) and a nonspecific envelope attachment inhibitor (Tsai et al., 2003) were used to prevent intrarectal transmission of SHIVs in macaques. For prevention of oral viral transmission, only macaque models have been used. First, neutralizing antibodies were shown to be protective in neonates (Baba et al., 2000). More recently, subcutanously administered tenofovir was found to be somewhat protective against oral SIV challenge (Van Rompay et al., 2006; Van Rompay et al., 2001) while an oral tenofovir solution was ineffective (Van Rompay et al., 2006; Van

While most of these studies have focused on topical gels and novel compounds, oral FDAapproved antiretroviral compounds have been investigated recently as pre-exposure

need to be evaluated for safety and lack of mucosal toxicity.

and genital tracts, is more similar to that of humans.

**3.1.2 Therapeutic prevention studies** 

Rompay et al., 2002b).

prophylaxis. Tenofovir prevents HIV-1 replication, it is already approved for use in humans in the oral formulation, and high drug concentrations can be achieved in the male and female genital tracts (Kwara et al., 2008; Vourvahis et al., 2008). The use of a gel may not provide complete coverage of the mucosal surface, possibly leading to breakthrough infections. The CAPRISA 004 clinical trial recently showed a 39% overall reduction in HIV-1 incidence in women using a vaginal tenofovir gel (Abdool Karim et al., 2010). The iPrEx and TDF2 trials showed that oral tenofovir in combination with another RT inhibitor, emtracitabine (FTC), reduced transmission in men who have sex with men (Grant et al., 2010) or in heterosexual men and women (Roehr, 2011) by 44% and 63%, respectively. It is unclear whether the greater efficacy in the latter studies as compared to the CAPRISA 004 study was due to oral administration of drug or use of two drugs instead of tenofovir alone. In macaques, intermittent dosing (2 hours before and 24 hours after challenge) of tenofovir alone or with FTC was equally as effective as daily dosing during weekly repeated low-dose rectal challenges (Garcia-Lerma et al., 2008). Less frequent doses would reduce cost to the user and would not require strict daily adherence. However, breakthrough infections in 2 of 6 animals resulted in drug resistant viruses, which could potentially compromise future therapy.

Interestingly, with the exception of tenofovir or tenofovir/FTC, no other microbicides have been successful in the clinic. It remains to be seen whether or not this is due to a specific property of tenofovir or RT inhibitors in general. Tenofovir acts specifically on viral reverse transcription, whereas most other microbicides tested clinically were nonspecific entry inhibitors. Also, tenofovir is the only drug that has been tested with repeated low-dose SIV oral, intravaginal, and intrarectal challenges in macaques (Garcia-Lerma et al., 2008; Parikh et al., 2009; Van Rompay et al., 2006). A nonspecific virus inactivating compound was tested in repeated high-dose intravaginal challenges 10-47 weeks apart, in which 90% of the animals were protected after the initial challenge (Ambrose et al., 2008). However, nearly all the animals became infected after the second challenge. It is unclear if noninfectious virus at the site of transmission elicits a detrimental immune response, inflammation, and/or an increase in target cells that can cause a subject to become more sensitized to infection.

Until recently, mucosal challenge of SIV or SHIV required high doses of virus to ensure that all or most of the control animals become infected. More and more investigators are using repeated low-dose challenges in animal studies to recapitulate more realistic levels of virus detected in semen of untreated HIV+ men. Semen viral RNA levels are often similar to or lower than in the blood (Gupta et al., 1997; Liuzzi et al., 1996), but are significantly lower than those of laboratory stocks used in high-dose challenges (Marthas et al., 2001). Also, high-risk individuals may be exposed multiple times prior to becoming infected. A direct comparison of a high-dose challenge and repeated low-dose rectal challenges in a small study on oral tenofovir pre-exposure prophylaxis showed that the low-dose model was actually somewhat more stringent (Subbarao et al., 2007). The disadvantage of the low-dose model is that multiple challenges are required to infect untreated control monkeys, and this number varies significantly among animals. This can affect statistics and often leads to longer experiments, which can increase study costs.

#### **3.2 Post-exposure prophylaxis**

Most post-exposure prophylaxis research has focused on entry and reverse transcription inhibitors that target the earliest steps of HIV-1 replication. Tenofovir was the first

Use of Animal Models for Anti-HIV Drug Development 79

prevent *in utero* transmission of HIV-2 to the fetus/infant (Ho et al., 2000). SIV transmission in nonhuman primates via breastfeeding has been demonstrated and used to study tenofovir pharmacokinetics in breast milk (Van Rompay et al., 2005). However, this model has not be used in any therapeutic prevention studies. Oral inoculation macaque models have been used to study pre-exposure and post-exposure prophylaxis, as discussed above. It appears that the first few hours after virus exposure is critical for using therapy to stop infection. This is important to minimize the spread of virus to new target cells. While most research has focused on a single inhibitor or class of inhibitors, it remains to be seen if drug combinations will afford more time to an exposed individual to prevent fulminant infection. Treatment of adults and infants, even when initiated immediately after exposure, may be ineffective if they have been exposed to drug-resistant or highly pathogenic viruses. This could be alleviated with combination therapy. Also, post-exposure treatment has generally been administered daily for 4 weeks. To save money and to avoid prolonged side effects from the drugs, can the duration of therapy be shortened and remain effective? Finally, will breakthrough infections result in selection of drug-resistant viruses, which could lead to

**4. Animal models for new suppressive antiretrovirals, combinations, and** 

toxic properties, pharmacokinetics and bioavailability, and antiretroviral efficacy.

macaques as a potential method to prevent MTCT (Winters et al., 2010).

**4.2 Efficacy in viral inhibition** 

Animal models for antiretroviral research can be used to test novel compounds for their

While it is impossible to document or list all antiretroviral pharmacokinetics and toxicity research in animals here, a few examples of early and new studies should be noted. Nucleoside analogs that inhibit reverse transcription, such as ddC (Kelley et al., 1987) and d4T (Keller et al., 1995), were tested in mice and monkeys for toxicity and pharmacokinetics. The short-term and long-term effects of tenofovir in newborn/infant macaques showed bone and kidney toxicities (Van Rompay et al., 2004). Also, non-nucleoside RT inhibitors, such as efavirenz, were tested in animals for pharmacokinetics (Balani et al., 1999). Investigators also developed drugs to the other HIV-1 enzymes: protease and integrase. While many of the protease inhibitors had poor efficacy against SIV, their pharmacokinetics could be assessed in animals, such as that of nelfinavir (Kaldor et al., 1997). Many integrase inhibitors were screened by Merck, leading to testing of many potential new compounds for good bioavailability in nonhuman primates and mice (Gardelli et al., 2007; Pace et al., 2007). Even HIV-1 entry inhibitors, such as SCH-D, which would later be named vicriviroc, were tested in mice and monkeys for general pharmacokinetics (Tagat et al., 2004). Also, maraviroc was studied for intrapartum pharmacokinetics and dynamics in pregnant

The virus and model used to test antiretroviral drug efficacy are dependent on the drug target. For example, due to sequence differences in the Gag protein, the maturation inhibitor bevirimat does not inhibit SIV (Zhou et al., 2004). Therefore, proof of concept *in vivo* studies

virologic failure during subsequent antiretroviral treatment?

**4.1 Toxicity, pharmacokinetics, tissue distribution** 

**deliveries** 

successful drug used for post-exposure treatment in the macaque model against intravenous challenge of SIV (Tsai et al., 1995). Animals did not become infected when daily tenofovir treatment was initiated 4 or 24 hours post-challenge, continuing for 4 weeks. A similar study with AZT did not show protection 3 hours after intravenous SIV inoculation (Fazely et al., 1991). Later, vaginal transmission was largely prevented in macaques after tenofovir treatment began 12 or 36 hours post-challenge but not after 72 hours (Otten et al., 2000).

The other post-exposure prophylaxis method used in the macaque model has been passive transfer of neutralizing antibodies targeting HIV-1 envelope. Antibody transfer into macaques 6 hours post-infection with SHIV showed 75% protection, which declined to 50% after 24 hours (Nishimura et al., 2003). Multiple monoclonal antibodies were more successful than an individual antibody. Complete protection was also seen after antibody infusion 1 hour after oral SHIV challenge of neonates, which decreased after 12 or 24 hours (Ferrantelli et al., 2004).

While these post-exposure prophylaxis studies showed promise, highly pathogenic viruses may be more difficult to control. One study showed that a combination of neutralizing antibodies given to neonates at 1 hour and 8 days post-challenge led to 100% protection in animals infected with a SHIV, but did not protect animals from a more pathogenic virus (Hofmann-Lehmann et al., 2001). This same highly pathogenic virus also failed to be controlled by triple therapy of AZT, 3TC, and the protease inhibitor indinavir when initiated 4 hours after intravenous infection and continued for 14-28 days (Bourry et al., 2009; Le Grand et al., 2000). These studies noted that although most of the animals became infected, plasma viremia was reduced compared to the control animals, suggesting a partial protective effect of the treatment regimens.

#### **3.3 Mother-to-child transmission prevention**

Mother-to-child transmission (MTCT) of HIV-1 occurs via three mechanisms: *in utero* via virus transfer across the placenta, intrapartum presumably by oral inoculation of the infected mother's blood, and breastfeeding. An early clinical trial showed that AZT therapy of HIV-infected women during pregnancy and of infants in the first 6 weeks after birth led to a 67.5% reduction in MTCT (Connor et al., 1994).

In resource-rich nations, combination therapy is now the standard of care for pregnant women. However, the cost of multiple drugs and/or multiple doses of drugs poses a challenge in resource-limited countries. Thus, studies were conducted in which single doses of nevirapine were administered to women during labor (and sometimes to the infant) to prevent MTCT. Nevirapine, a non-nucleoside RT inhibitor, has less toxicity than AZT and has a long half-life (Musoke et al., 1999). Single-dose nevirapine treatment effectively reduced HIV-1 MTCT to a similar degree as multiple doses of AZT (Guay et al., 1999). Unfortunately, it was revealed that single-dose nevirapine caused the development of drugresistant virus in the mothers and in infants that subsequently became infected (Eshleman et al., 2001), which could be present at low frequencies for long periods of time (Flys et al., 2005; Palmer et al., 2006). Such drug resistance compromised subsequent treatment in the mothers (Jourdain et al., 2004).

Few studies on MTCT have been performed in animal models. One study using a pregnant macaque model of MTCT showed that short-term combination therapy could successfully

successful drug used for post-exposure treatment in the macaque model against intravenous challenge of SIV (Tsai et al., 1995). Animals did not become infected when daily tenofovir treatment was initiated 4 or 24 hours post-challenge, continuing for 4 weeks. A similar study with AZT did not show protection 3 hours after intravenous SIV inoculation (Fazely et al., 1991). Later, vaginal transmission was largely prevented in macaques after tenofovir treatment began 12 or 36 hours post-challenge but not after 72 hours (Otten et al., 2000).

The other post-exposure prophylaxis method used in the macaque model has been passive transfer of neutralizing antibodies targeting HIV-1 envelope. Antibody transfer into macaques 6 hours post-infection with SHIV showed 75% protection, which declined to 50% after 24 hours (Nishimura et al., 2003). Multiple monoclonal antibodies were more successful than an individual antibody. Complete protection was also seen after antibody infusion 1 hour after oral SHIV challenge of neonates, which decreased after 12 or 24 hours

While these post-exposure prophylaxis studies showed promise, highly pathogenic viruses may be more difficult to control. One study showed that a combination of neutralizing antibodies given to neonates at 1 hour and 8 days post-challenge led to 100% protection in animals infected with a SHIV, but did not protect animals from a more pathogenic virus (Hofmann-Lehmann et al., 2001). This same highly pathogenic virus also failed to be controlled by triple therapy of AZT, 3TC, and the protease inhibitor indinavir when initiated 4 hours after intravenous infection and continued for 14-28 days (Bourry et al., 2009; Le Grand et al., 2000). These studies noted that although most of the animals became infected, plasma viremia was reduced compared to the control animals, suggesting a partial

Mother-to-child transmission (MTCT) of HIV-1 occurs via three mechanisms: *in utero* via virus transfer across the placenta, intrapartum presumably by oral inoculation of the infected mother's blood, and breastfeeding. An early clinical trial showed that AZT therapy of HIV-infected women during pregnancy and of infants in the first 6 weeks after birth led

In resource-rich nations, combination therapy is now the standard of care for pregnant women. However, the cost of multiple drugs and/or multiple doses of drugs poses a challenge in resource-limited countries. Thus, studies were conducted in which single doses of nevirapine were administered to women during labor (and sometimes to the infant) to prevent MTCT. Nevirapine, a non-nucleoside RT inhibitor, has less toxicity than AZT and has a long half-life (Musoke et al., 1999). Single-dose nevirapine treatment effectively reduced HIV-1 MTCT to a similar degree as multiple doses of AZT (Guay et al., 1999). Unfortunately, it was revealed that single-dose nevirapine caused the development of drugresistant virus in the mothers and in infants that subsequently became infected (Eshleman et al., 2001), which could be present at low frequencies for long periods of time (Flys et al., 2005; Palmer et al., 2006). Such drug resistance compromised subsequent treatment in the

Few studies on MTCT have been performed in animal models. One study using a pregnant macaque model of MTCT showed that short-term combination therapy could successfully

(Ferrantelli et al., 2004).

protective effect of the treatment regimens.

**3.3 Mother-to-child transmission prevention**

to a 67.5% reduction in MTCT (Connor et al., 1994).

mothers (Jourdain et al., 2004).

prevent *in utero* transmission of HIV-2 to the fetus/infant (Ho et al., 2000). SIV transmission in nonhuman primates via breastfeeding has been demonstrated and used to study tenofovir pharmacokinetics in breast milk (Van Rompay et al., 2005). However, this model has not be used in any therapeutic prevention studies. Oral inoculation macaque models have been used to study pre-exposure and post-exposure prophylaxis, as discussed above.

It appears that the first few hours after virus exposure is critical for using therapy to stop infection. This is important to minimize the spread of virus to new target cells. While most research has focused on a single inhibitor or class of inhibitors, it remains to be seen if drug combinations will afford more time to an exposed individual to prevent fulminant infection. Treatment of adults and infants, even when initiated immediately after exposure, may be ineffective if they have been exposed to drug-resistant or highly pathogenic viruses. This could be alleviated with combination therapy. Also, post-exposure treatment has generally been administered daily for 4 weeks. To save money and to avoid prolonged side effects from the drugs, can the duration of therapy be shortened and remain effective? Finally, will breakthrough infections result in selection of drug-resistant viruses, which could lead to virologic failure during subsequent antiretroviral treatment?

#### **4. Animal models for new suppressive antiretrovirals, combinations, and deliveries**

Animal models for antiretroviral research can be used to test novel compounds for their toxic properties, pharmacokinetics and bioavailability, and antiretroviral efficacy.

#### **4.1 Toxicity, pharmacokinetics, tissue distribution**

While it is impossible to document or list all antiretroviral pharmacokinetics and toxicity research in animals here, a few examples of early and new studies should be noted. Nucleoside analogs that inhibit reverse transcription, such as ddC (Kelley et al., 1987) and d4T (Keller et al., 1995), were tested in mice and monkeys for toxicity and pharmacokinetics. The short-term and long-term effects of tenofovir in newborn/infant macaques showed bone and kidney toxicities (Van Rompay et al., 2004). Also, non-nucleoside RT inhibitors, such as efavirenz, were tested in animals for pharmacokinetics (Balani et al., 1999). Investigators also developed drugs to the other HIV-1 enzymes: protease and integrase. While many of the protease inhibitors had poor efficacy against SIV, their pharmacokinetics could be assessed in animals, such as that of nelfinavir (Kaldor et al., 1997). Many integrase inhibitors were screened by Merck, leading to testing of many potential new compounds for good bioavailability in nonhuman primates and mice (Gardelli et al., 2007; Pace et al., 2007). Even HIV-1 entry inhibitors, such as SCH-D, which would later be named vicriviroc, were tested in mice and monkeys for general pharmacokinetics (Tagat et al., 2004). Also, maraviroc was studied for intrapartum pharmacokinetics and dynamics in pregnant macaques as a potential method to prevent MTCT (Winters et al., 2010).

#### **4.2 Efficacy in viral inhibition**

The virus and model used to test antiretroviral drug efficacy are dependent on the drug target. For example, due to sequence differences in the Gag protein, the maturation inhibitor bevirimat does not inhibit SIV (Zhou et al., 2004). Therefore, proof of concept *in vivo* studies

Use of Animal Models for Anti-HIV Drug Development 81

RT-SHIVs were created to study non-nucleoside RT inhibitors, such as efavirenz, that are specific to HIV-1 but not to HIV-2 or SIV. Efavirenz monotherapy leads to the emergence of common resistance mutations that are seen in patients (Ambrose et al., 2007; Hofman et al., 2004). Efavirenz combined with tenofovir and FTC is a commonly prescribed combination therapy for HIV-infected individuals and was shown to be effective in reducing RT-SHIV plasma viremia in two species of macaques (Ambrose et al., 2007; North et al., 2005). Prior efavirenz resistance was shown to compromise combination therapy in one animal, leading to accumulation of FTC-resistant mutations in the virus and, ultimately, failure of therapy (Ambrose et al., 2007). This was similar to results described earlier in the single-dose nevirapine trials for prevention of MTCT. In addition, the RT-SHIV model allowed the study of viral subpopulation dynamics in animals, including those of drug-resistant viral

While resistance to clinically used antiretrovirals, such as FTC, tenofovir, and efavirenz, are well documented, compounds for pre-exposure prophylaxis have also been evaluated *in vivo* for the ability to select for drug-resistant viruses. As stated earlier, breakthrough infections after FTC and tenofovir prophylaxis was demonstrated in macaques (Garcia-Lerma et al., 2008). Similarly, mutations in the SHIV envelope were observed in virus from a macaque after ineffective use of the topical entry inhibitor PSC-RANTES that conferred little or no resistance to the compound (Dudley et al., 2009). Monitoring of potential drugresistant virus is critical in preclinical and clinical studies with new inhibitors, as resistance

One complication of HIV-1 infection is the presence of large numbers of infected cells within different tissues. Although discussed more in the next section, CD4+ target cells are present within lymphoid tissues, the brain, and multiple mucosal tissues. Drug penetration into these tissues is likely to be lower than in the blood, allowing viral replication to continue within tissues. Research on better drug delivery methods into different tissues has been ongoing. This will be especially important for methods to target drugs into mucosal tissues

While there may be many novel delivery methods in the pipeline, only a few have been tested *in vivo* to specifically prevent transmission of or reduce replication of HIV-1/2. One novel delivery method of antiretrovirals to tissues is the formation and administration of drug nanocomplexes. A protease inhibitor, indinavir, was formed into nanoparticles and delivered to HIV-2-infected macaques, leading to significantly increased lymph node lymphocyte drug concentrations as compared to animals given the drug orally (Kinman et al., 2003). Nanoparticles are often cleared from the blood and tissues by phagocytic cells, such as macrophages. As macrophages are infected by HIV-1, these cells have been proposed as a target for drug delivery and phagocytosis of drug by infected cells may be beneficial. A single intravenous dose of indinavir-containing nanoparticles in mice with HIV+ brain macrophages led to drug levels in the brain for up to 14 days and reduced virus replication in the brain (Dou et al., 2009). Nevertheless, optimization of nanocomplexes by coating their surfaces with polyethylene glycol resulted in sustained levels in the blood of

may negatively impact efficacy of future treatment regimens.

during pre-exposure prophylaxis and also persisting viral reservoirs.

mice and less clearing by macrophages (Levchenko et al., 2002).

variants (Shao et al., 2009).

**4.4 New delivery methods** 

were performed in a humanized mouse model to show drug inhibition against HIV-1 (Stoddart et al., 2007). Similarly, testing of a CCR5 inhibitor like maraviroc is best used with HIV-1 envelope. Therefore, nonhuman primate studies were performed with SHIV virus containing a CCR5-tropic envelope (Veazey et al., 2003a).

Many novel antiretroviral small molecule inhibitors have been characterized for efficacy against HIV, SIV, or SHIV in animal models, many leading to FDA approval or to more improved versions of the compounds (reviewed in Ambrose and KewalRamani, 2008; Van Rompay, 2010). For example, PMEA, a drug related to tenofovir, was found to inhibit SIV infection effectively in macaques (Balzarini et al., 1991). Unfortunately, PMEA was very toxic *in vivo*, which lead to the development of the related and less-toxic compound tenofovir (previously called PMPA), which has been studied extensively in monkeys for viral inhibition (Balzarini et al., 1991; Lifson et al., 2003; Smith et al., 2000a; Tsai et al., 1998). Another RT inhibitor, stavudine, was tested for antiretroviral properties in HIV-2-infected macaques (Watson et al., 1997). And after many years of screening compounds *in vitro*, a precursor to the first FDA-approved integrase inhibitor, raltegravir, was shown to be effective in viral inhibition in the macaque model (Hazuda et al., 2004).

While many exciting novel antiretroviral inhibitors are currently under investigation in *in vitro* models, general efficacy testing is not always performed in an animal model prior to clinical studies. This is due to the high costs associated with these experiments, particularly in macaques and humanized mice. The major advantage of an animal model is the ability to perform procedures that may exacerbate disease or that are highly invasive. These may not be ethical or easy to perform in humans. For example, treatment interruptions or intentional induction of drug resistance is likely to be harmful to an HIV-infected individual. However, understanding the effects of these procedures can lead to a better understanding of the development of drug resistance and its effects on subsequent treatment regimens. Also, obtaining organs, longitudinal blood or tissue samples, and specific time points after infection or therapy initiation cannot be acquired easily in people.

#### **4.3 Drug resistance**

Drug resistance in HIV-infected individuals arises from inadequate therapy, which could be the result of ineffective prescribed therapy or nonadherence. While most data on HIV-1 drug resistance have been obtained from clinical studies, the macaque model has been used to address various questions on the development and consequences of drug-resistant virus.

Because nucleoside analogs such as tenofovir and FTC inhibit SIV and have good pharmacokinetics in macaques, mutations to these drugs have been studied in the monkey model. The primary resistance mutation to FTC has been studied for its effects on *in vivo* viral virulence and fitness (Van Rompay et al., 2002a). Similarly, mutations conferring resistance to tenofovir were studied in SIV for their emergence and suppression *in vivo* during tenofovir monotherapy (Van Rompay et al., 2007). Resistant viruses were identified in macaques with breakthrough infections after intermittent tenofovir/FTC pre-exposure prophylaxis with intrarectal challenge (Garcia-Lerma et al., 2008). And FTC- and tenofovirresistant SHIV mutants were created and tested for their mucosal transmissibility (Cong et al., 2011), presumably to determine the effects of tenofovir and FTC pre-exposure prophylaxis on transmitted drug-resistant virus.

were performed in a humanized mouse model to show drug inhibition against HIV-1 (Stoddart et al., 2007). Similarly, testing of a CCR5 inhibitor like maraviroc is best used with HIV-1 envelope. Therefore, nonhuman primate studies were performed with SHIV virus

Many novel antiretroviral small molecule inhibitors have been characterized for efficacy against HIV, SIV, or SHIV in animal models, many leading to FDA approval or to more improved versions of the compounds (reviewed in Ambrose and KewalRamani, 2008; Van Rompay, 2010). For example, PMEA, a drug related to tenofovir, was found to inhibit SIV infection effectively in macaques (Balzarini et al., 1991). Unfortunately, PMEA was very toxic *in vivo*, which lead to the development of the related and less-toxic compound tenofovir (previously called PMPA), which has been studied extensively in monkeys for viral inhibition (Balzarini et al., 1991; Lifson et al., 2003; Smith et al., 2000a; Tsai et al., 1998). Another RT inhibitor, stavudine, was tested for antiretroviral properties in HIV-2-infected macaques (Watson et al., 1997). And after many years of screening compounds *in vitro*, a precursor to the first FDA-approved integrase inhibitor, raltegravir, was shown to be

While many exciting novel antiretroviral inhibitors are currently under investigation in *in vitro* models, general efficacy testing is not always performed in an animal model prior to clinical studies. This is due to the high costs associated with these experiments, particularly in macaques and humanized mice. The major advantage of an animal model is the ability to perform procedures that may exacerbate disease or that are highly invasive. These may not be ethical or easy to perform in humans. For example, treatment interruptions or intentional induction of drug resistance is likely to be harmful to an HIV-infected individual. However, understanding the effects of these procedures can lead to a better understanding of the development of drug resistance and its effects on subsequent treatment regimens. Also, obtaining organs, longitudinal blood or tissue samples, and specific time points after

Drug resistance in HIV-infected individuals arises from inadequate therapy, which could be the result of ineffective prescribed therapy or nonadherence. While most data on HIV-1 drug resistance have been obtained from clinical studies, the macaque model has been used to address various questions on the development and consequences of drug-resistant virus. Because nucleoside analogs such as tenofovir and FTC inhibit SIV and have good pharmacokinetics in macaques, mutations to these drugs have been studied in the monkey model. The primary resistance mutation to FTC has been studied for its effects on *in vivo* viral virulence and fitness (Van Rompay et al., 2002a). Similarly, mutations conferring resistance to tenofovir were studied in SIV for their emergence and suppression *in vivo* during tenofovir monotherapy (Van Rompay et al., 2007). Resistant viruses were identified in macaques with breakthrough infections after intermittent tenofovir/FTC pre-exposure prophylaxis with intrarectal challenge (Garcia-Lerma et al., 2008). And FTC- and tenofovirresistant SHIV mutants were created and tested for their mucosal transmissibility (Cong et al., 2011), presumably to determine the effects of tenofovir and FTC pre-exposure

containing a CCR5-tropic envelope (Veazey et al., 2003a).

effective in viral inhibition in the macaque model (Hazuda et al., 2004).

infection or therapy initiation cannot be acquired easily in people.

prophylaxis on transmitted drug-resistant virus.

**4.3 Drug resistance**

RT-SHIVs were created to study non-nucleoside RT inhibitors, such as efavirenz, that are specific to HIV-1 but not to HIV-2 or SIV. Efavirenz monotherapy leads to the emergence of common resistance mutations that are seen in patients (Ambrose et al., 2007; Hofman et al., 2004). Efavirenz combined with tenofovir and FTC is a commonly prescribed combination therapy for HIV-infected individuals and was shown to be effective in reducing RT-SHIV plasma viremia in two species of macaques (Ambrose et al., 2007; North et al., 2005). Prior efavirenz resistance was shown to compromise combination therapy in one animal, leading to accumulation of FTC-resistant mutations in the virus and, ultimately, failure of therapy (Ambrose et al., 2007). This was similar to results described earlier in the single-dose nevirapine trials for prevention of MTCT. In addition, the RT-SHIV model allowed the study of viral subpopulation dynamics in animals, including those of drug-resistant viral variants (Shao et al., 2009).

While resistance to clinically used antiretrovirals, such as FTC, tenofovir, and efavirenz, are well documented, compounds for pre-exposure prophylaxis have also been evaluated *in vivo* for the ability to select for drug-resistant viruses. As stated earlier, breakthrough infections after FTC and tenofovir prophylaxis was demonstrated in macaques (Garcia-Lerma et al., 2008). Similarly, mutations in the SHIV envelope were observed in virus from a macaque after ineffective use of the topical entry inhibitor PSC-RANTES that conferred little or no resistance to the compound (Dudley et al., 2009). Monitoring of potential drugresistant virus is critical in preclinical and clinical studies with new inhibitors, as resistance may negatively impact efficacy of future treatment regimens.

#### **4.4 New delivery methods**

One complication of HIV-1 infection is the presence of large numbers of infected cells within different tissues. Although discussed more in the next section, CD4+ target cells are present within lymphoid tissues, the brain, and multiple mucosal tissues. Drug penetration into these tissues is likely to be lower than in the blood, allowing viral replication to continue within tissues. Research on better drug delivery methods into different tissues has been ongoing. This will be especially important for methods to target drugs into mucosal tissues during pre-exposure prophylaxis and also persisting viral reservoirs.

While there may be many novel delivery methods in the pipeline, only a few have been tested *in vivo* to specifically prevent transmission of or reduce replication of HIV-1/2. One novel delivery method of antiretrovirals to tissues is the formation and administration of drug nanocomplexes. A protease inhibitor, indinavir, was formed into nanoparticles and delivered to HIV-2-infected macaques, leading to significantly increased lymph node lymphocyte drug concentrations as compared to animals given the drug orally (Kinman et al., 2003). Nanoparticles are often cleared from the blood and tissues by phagocytic cells, such as macrophages. As macrophages are infected by HIV-1, these cells have been proposed as a target for drug delivery and phagocytosis of drug by infected cells may be beneficial. A single intravenous dose of indinavir-containing nanoparticles in mice with HIV+ brain macrophages led to drug levels in the brain for up to 14 days and reduced virus replication in the brain (Dou et al., 2009). Nevertheless, optimization of nanocomplexes by coating their surfaces with polyethylene glycol resulted in sustained levels in the blood of mice and less clearing by macrophages (Levchenko et al., 2002).

Use of Animal Models for Anti-HIV Drug Development 83

as good as what is observed in humans on combination antiretroviral therapy. Standard assays detect down to 50 copies of viral RNA per milliliter of plasma, but even with typical three drug regimens it has been difficult to completely inhibit plasma viremia in some macaques to undetectable levels (Ambrose et al., 2007; Dinoso et al., 2009; Lugli et al., 2011; North et al., 2005). The ability to achieve complete suppression generally correlates with the level of plasma viremia prior to treatment, such that higher viral loads are more difficult to completely suppress (Kearney et al., 2011). In mouse models, combination therapy has only been evaluated in humanized Rag2-/-C-/- mice, which also showed incomplete viral suppression (Choudhary et al., 2009; Sango et al., 2010). These results may be due to reduced efficacy against SIV proteins and/or different drug pharmacokinetics in macaques or mice

In identifying and characterizing persistent viral reservoirs in an animal model, complete and sustained suppression is necessary. Recent reports suggest that ongoing viral replication does not occur during suppressive therapy in humans, based on a lack of viral evolution (Bailey et al., 2006; Kieffer et al., 2004; Nottet et al., 2009; Persaud et al., 2007). This was also observed in RT-SHIV-infected monkeys with sustained plasma virus suppression (Kearney et al., 2011). Continued virus replication in the presence of incomplete suppression will lead to infection of new target cells and re-seeding of reservoirs, which will interfere with identification of the actual reservoir that was established prior to initiation of therapy. This is especially important when looking at the reduction of infected cells by virus

Despite the lack of complete suppression in many animals, the macaque model has been used to try to identify and characterize viral reservoirs. In an SIV model with FTC and tenofovir therapy, it was shown that resting CD4+ T lymphocytes from lymph nodes but not thymocytes contribute to the reservoir (Shen et al., 2003). While viral DNA was detected in multiple tissues of RT-SHIV-infected animals with and without triple therapy, very little or no viral RNA was detected in these tissues during complete suppression (North et al., 2010; Ambrose et al., unpublished results). Lymphoid and gastrointestinal tissues showed the highest level of viral DNA in virally suppressed animals, suggesting that they consist of the majority of the viral reservoir. In a model of neuropathogenic AIDS, although viremia in plasma and cerebral spinal fluid was significantly reduced by therapy, levels of viral DNA in the brains of these animals were not significantly different than the untreated controls, suggesting that virally infected cells in the central nervous system can contribute to the

To target persisting HIV-infected cells with therapeutic drugs for their eradication, one needs to know where the reservoirs are located and what types of cells are infected. While more research needs to be performed to address the type and location of infected CD4+ cells that persist and if these vary at different anatomical locations, investigators are already

The majority of these strategies aim to stimulate latently infected, resting CD4+ cells during suppressive antiretroviral therapy. The purpose is to promote replication of virus in those cells without causing global immune activation and without leading to spread of infection to

**5.2 Preliminary studies using animal models for eradication strategies** 

testing novel therapies to selectively target infected cells.

as compared to humans.

eradication therapies, as discussed below.

reservoir (Zink et al., 2010).

Another newly developed technology is the silencing of protein expression by small interfering RNAs (siRNA). siRNA can be designed to target specific mRNA in cells for degradation, leading to loss of protein expression (Hammond et al., 2000; Zamore et al., 2000). Because siRNA can be designed specifically to silence almost any RNA, they have been proposed for treatment of various diseases, including HIV-1 infection. However, targeting siRNA to specific cell types, to infected cells, and into tissues has been challenging, as they do not easily cross the plasma membrane and are quickly degraded by nucleases. Therefore, different delivery methods have been explored for more optimal targeting of siRNA to HIV-infected cells. For example, siRNA were bound to an antibody fragment that recognized HIV-1 envelope. These antibody-siRNA molecules were able to specifically target and enter envelope-expressing cells in mice (Song et al., 2005). And many techniques for introducing siRNA into nanoparticles have been tested for better pharmacokinetics, but these have been mainly preclinical studies not involving HIV-1 (reviewed in Yuan et al., 2011).

More recently, another method of delivering siRNA has been tested in both humanized mouse models to inhibit HIV-1 infection. RNA aptamer-siRNA chimeras were generated such that the aptamer portion would selectively bind to a cell surface molecule, delivering the siRNA to target cells (McNamara et al., 2006). RNA aptamers are oligonucleotides that are designed to bind tightly to specific proteins (Ellington and Szostak, 1990; Tuerk and Gold, 1990). One group used a mixture of chimeric RNAs with a CD4-binding aptamer and with siRNA targeting mRNA encoding the HIV-1 co-receptor CCR5 and viral proteins as a topical vaginal microbicide in BLT mice (Wheeler et al., 2011). The drug was able reduce CCR5 expression *in vivo* and resulted in a significant reduction of virus transmission. Another group used chimeric RNAs with HIV-1 envelope-binding aptamers and siRNA targeting multiple HIV-1 mRNA (Neff et al., 2011b). They were injected into HIV-1-infected Rag2-/-C-/- humanized mice, leading to a significant reduction of both plasma viremia and CD4+ cell depletion.

The development of novel drugs and drug delivery methods is important for HIV-1 research. First, toxicity and adverse side effects of existing antiretrovirals are not trivial and should be improved to promote better adherence. Second, a lack of adherence will lead to drug resistance, which can affect multiple available treatment regimens. Third, improved prevention strategies will be necessary in the absence of a cure and in curbing the spread of HIV-1 infection among high-risk individuals. Lastly, as discussed below, targeting of new drugs to tissues to eliminate infected cells is an exciting possibility only recently realized.

#### **5. Animal models for targeting persisting viral reservoirs**

There is no cure for HIV-1. While HIV-infected individuals can suppress plasma viremia to undetectable levels with effective therapy, infected cells remain in the body and virus will return to the blood if therapy is halted or drug-resistance arises. The cells and tissues that harbor proviral DNA are considered viral reservoirs and are not completely characterized. Animal models allow investigators to more easily evaluate where infected cells are located and how to eradicate them than in humans.

#### **5.1 Evaluating viral reservoirs during antiretroviral therapy**

Because antiretroviral drugs were designed to target HIV-1, some of them are inefficient or unable to suppress SIV replication. In an animal model, antiretroviral suppression should be

Another newly developed technology is the silencing of protein expression by small interfering RNAs (siRNA). siRNA can be designed to target specific mRNA in cells for degradation, leading to loss of protein expression (Hammond et al., 2000; Zamore et al., 2000). Because siRNA can be designed specifically to silence almost any RNA, they have been proposed for treatment of various diseases, including HIV-1 infection. However, targeting siRNA to specific cell types, to infected cells, and into tissues has been challenging, as they do not easily cross the plasma membrane and are quickly degraded by nucleases. Therefore, different delivery methods have been explored for more optimal targeting of siRNA to HIV-infected cells. For example, siRNA were bound to an antibody fragment that recognized HIV-1 envelope. These antibody-siRNA molecules were able to specifically target and enter envelope-expressing cells in mice (Song et al., 2005). And many techniques for introducing siRNA into nanoparticles have been tested for better pharmacokinetics, but these have been mainly preclinical studies not involving HIV-1 (reviewed in Yuan et al., 2011).

More recently, another method of delivering siRNA has been tested in both humanized mouse models to inhibit HIV-1 infection. RNA aptamer-siRNA chimeras were generated such that the aptamer portion would selectively bind to a cell surface molecule, delivering the siRNA to target cells (McNamara et al., 2006). RNA aptamers are oligonucleotides that are designed to bind tightly to specific proteins (Ellington and Szostak, 1990; Tuerk and Gold, 1990). One group used a mixture of chimeric RNAs with a CD4-binding aptamer and with siRNA targeting mRNA encoding the HIV-1 co-receptor CCR5 and viral proteins as a topical vaginal microbicide in BLT mice (Wheeler et al., 2011). The drug was able reduce CCR5 expression *in vivo* and resulted in a significant reduction of virus transmission. Another group used chimeric RNAs with HIV-1 envelope-binding aptamers and siRNA targeting multiple HIV-1 mRNA (Neff et al., 2011b). They were injected into HIV-1-infected Rag2-/-C-/- humanized mice,

leading to a significant reduction of both plasma viremia and CD4+ cell depletion.

**5. Animal models for targeting persisting viral reservoirs** 

**5.1 Evaluating viral reservoirs during antiretroviral therapy**

and how to eradicate them than in humans.

The development of novel drugs and drug delivery methods is important for HIV-1 research. First, toxicity and adverse side effects of existing antiretrovirals are not trivial and should be improved to promote better adherence. Second, a lack of adherence will lead to drug resistance, which can affect multiple available treatment regimens. Third, improved prevention strategies will be necessary in the absence of a cure and in curbing the spread of HIV-1 infection among high-risk individuals. Lastly, as discussed below, targeting of new drugs to tissues to eliminate infected cells is an exciting possibility only recently realized.

There is no cure for HIV-1. While HIV-infected individuals can suppress plasma viremia to undetectable levels with effective therapy, infected cells remain in the body and virus will return to the blood if therapy is halted or drug-resistance arises. The cells and tissues that harbor proviral DNA are considered viral reservoirs and are not completely characterized. Animal models allow investigators to more easily evaluate where infected cells are located

Because antiretroviral drugs were designed to target HIV-1, some of them are inefficient or unable to suppress SIV replication. In an animal model, antiretroviral suppression should be as good as what is observed in humans on combination antiretroviral therapy. Standard assays detect down to 50 copies of viral RNA per milliliter of plasma, but even with typical three drug regimens it has been difficult to completely inhibit plasma viremia in some macaques to undetectable levels (Ambrose et al., 2007; Dinoso et al., 2009; Lugli et al., 2011; North et al., 2005). The ability to achieve complete suppression generally correlates with the level of plasma viremia prior to treatment, such that higher viral loads are more difficult to completely suppress (Kearney et al., 2011). In mouse models, combination therapy has only been evaluated in humanized Rag2-/-C-/- mice, which also showed incomplete viral suppression (Choudhary et al., 2009; Sango et al., 2010). These results may be due to reduced efficacy against SIV proteins and/or different drug pharmacokinetics in macaques or mice as compared to humans.

In identifying and characterizing persistent viral reservoirs in an animal model, complete and sustained suppression is necessary. Recent reports suggest that ongoing viral replication does not occur during suppressive therapy in humans, based on a lack of viral evolution (Bailey et al., 2006; Kieffer et al., 2004; Nottet et al., 2009; Persaud et al., 2007). This was also observed in RT-SHIV-infected monkeys with sustained plasma virus suppression (Kearney et al., 2011). Continued virus replication in the presence of incomplete suppression will lead to infection of new target cells and re-seeding of reservoirs, which will interfere with identification of the actual reservoir that was established prior to initiation of therapy. This is especially important when looking at the reduction of infected cells by virus eradication therapies, as discussed below.

Despite the lack of complete suppression in many animals, the macaque model has been used to try to identify and characterize viral reservoirs. In an SIV model with FTC and tenofovir therapy, it was shown that resting CD4+ T lymphocytes from lymph nodes but not thymocytes contribute to the reservoir (Shen et al., 2003). While viral DNA was detected in multiple tissues of RT-SHIV-infected animals with and without triple therapy, very little or no viral RNA was detected in these tissues during complete suppression (North et al., 2010; Ambrose et al., unpublished results). Lymphoid and gastrointestinal tissues showed the highest level of viral DNA in virally suppressed animals, suggesting that they consist of the majority of the viral reservoir. In a model of neuropathogenic AIDS, although viremia in plasma and cerebral spinal fluid was significantly reduced by therapy, levels of viral DNA in the brains of these animals were not significantly different than the untreated controls, suggesting that virally infected cells in the central nervous system can contribute to the reservoir (Zink et al., 2010).

#### **5.2 Preliminary studies using animal models for eradication strategies**

To target persisting HIV-infected cells with therapeutic drugs for their eradication, one needs to know where the reservoirs are located and what types of cells are infected. While more research needs to be performed to address the type and location of infected CD4+ cells that persist and if these vary at different anatomical locations, investigators are already testing novel therapies to selectively target infected cells.

The majority of these strategies aim to stimulate latently infected, resting CD4+ cells during suppressive antiretroviral therapy. The purpose is to promote replication of virus in those cells without causing global immune activation and without leading to spread of infection to

Use of Animal Models for Anti-HIV Drug Development 85

the genetic differences between HIV-1 and SIV, particularly the accessory proteins that appear to significantly contribute to pathogenesis. Not only will these findings make a better animal model, but virus-host cell protein interactions characterized by these studies may also lead to new therapeutic targets. Also, alterations of the current humanized mouse models to improve tissue immune cell reconstitution and anti-HIV adaptive immune

While much work has been accomplished in HIV-1 research, questions remain for pre- and post-exposure prophylaxis, antiretroviral pharmacokinetics and efficacy, drug resistance, new drug delivery technologies, and methods for virus eradication. Using the models described here can help answer many of these questions, if used appropriately. Animal models continue to improve therapeutics against HIV-1 and they will be critical for addressing these important issues, bridging basic research together with clinical trials for

Abdool Karim, Q., Abdool Karim, S.S., Frohlich, J.A., Grobler, A.C., Baxter, C., Mansoor,

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Bandarenko, N., Schmitz, J.L., Bosch, R.J., Landay, A.L.*, et al.* (2008). Valproic acid without intensified antiviral therapy has limited impact on persistent HIV infection

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HIV-1 prevention, treatment, and eradication.

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No.4, pp. 363-373.

**7. References** 

new cells. Histone deacetylase (HDAC) inhibitors and phorbol esters that stimulate protein kinase C (PKC) have received attention for their ability to activate latent HIV-1 expression *in vitro* (Colin and Van Lint, 2009). Clinical studies showed little or no decrease in latently infected cells or vDNA in most subjects by administration of a FDA-approved HDAC inhibitor, valproic acid, in the presence of suppressive ART (Archin et al., 2008; Sagot-Lerolle et al., 2008; Siliciano et al., 2007). This is most likely to be a result of incorrect HDAC(s) targeting and/or inadequate drug concentrations (Huber et al., 2011; Keedy et al., 2009).

Animal models will be useful to test efficacy and mechanisms of action of potential therapies to eliminate infected cells from the body. Very recently a few studies have described the use of novel strategies *in vivo* and *ex vivo* to target viral reservoirs. Lipid nanoparticles containing the PKC activator bryostatin minimally induced proviral expression from infected CD4+ cells isolated from SCID-hu mice with thymus/liver implants (Kovochich et al., 2011). Also, a gold-based compound, auranofin, was used to treat SIV-infected macaques already receiving triple therapy (Lewis et al., 2011). After a month of treatment, PBMC proviral DNA levels decreased in the animals and plasma viral RNA was reduced after interruption of treatment.

While new eradication treatments are promising for reducing or eliminating viral reservoirs, there are many issues and remaining questions that need to be addressed. First, an assumption is that latently infected cells will die after they are activated. However, to date this has not been demonstrated in animals or in people. And it remains to be seen if there are strategies that can selectively activate HIV- or SIV-infected cells but not uninfected cells. Second, it is important to identify models or treatment options that can achieve complete, sustained virus suppression as is observed in patients. If this is achieved, methods to measure decreases in viral reservoirs will need to be optimized, including assays to measure a single copy of plasma viral RNA, a single infected cell, and spliced vs. unspliced viral RNA.

Finally, there are two caveats to the mouse and macaque models for studying persisting viral reservoirs. While mice can be rather effectively repopulated with a largely functional immune system, their anatomy is largely different than that of humans. Identification of reservoirs in mice will need to be confirmed with what is already known in humans. And although monkeys are more similar anatomically to humans, HIV-1 replication is not maintained in these animals. It remains to be seen whether transcriptional activation of SIV is similar to or different than that of HIV-1. Compounds designed to target activation of the HIV-1 LTR may not work or may work differently against SIV LTRs. Characterization of the various models for investigation of virus reservoirs should, therefore, continue.

#### **6. Conclusion**

This chapter has highlighted the use of macaque and mouse models for antiretroviral therapies. These animal models have improved greatly since the development of SHIVs and the ability to reconstitute a functional human immune system in mice. Refinement of these models is ongoing to make them more closely resemble humans with regards to infection, pathogenesis, and antiretroviral immunity. For example, macaque models can be improved by increased understanding of macaque cell proteins that inhibit HIV-1 infection as well as the genetic differences between HIV-1 and SIV, particularly the accessory proteins that appear to significantly contribute to pathogenesis. Not only will these findings make a better animal model, but virus-host cell protein interactions characterized by these studies may also lead to new therapeutic targets. Also, alterations of the current humanized mouse models to improve tissue immune cell reconstitution and anti-HIV adaptive immune responses will better recapitulate HIV-1 infection of humans.

While much work has been accomplished in HIV-1 research, questions remain for pre- and post-exposure prophylaxis, antiretroviral pharmacokinetics and efficacy, drug resistance, new drug delivery technologies, and methods for virus eradication. Using the models described here can help answer many of these questions, if used appropriately. Animal models continue to improve therapeutics against HIV-1 and they will be critical for addressing these important issues, bridging basic research together with clinical trials for HIV-1 prevention, treatment, and eradication.

#### **7. References**

84 Antiviral Drugs – Aspects of Clinical Use and Recent Advances

new cells. Histone deacetylase (HDAC) inhibitors and phorbol esters that stimulate protein kinase C (PKC) have received attention for their ability to activate latent HIV-1 expression *in vitro* (Colin and Van Lint, 2009). Clinical studies showed little or no decrease in latently infected cells or vDNA in most subjects by administration of a FDA-approved HDAC inhibitor, valproic acid, in the presence of suppressive ART (Archin et al., 2008; Sagot-Lerolle et al., 2008; Siliciano et al., 2007). This is most likely to be a result of incorrect HDAC(s) targeting and/or inadequate drug concentrations (Huber et al., 2011; Keedy et al.,

Animal models will be useful to test efficacy and mechanisms of action of potential therapies to eliminate infected cells from the body. Very recently a few studies have described the use of novel strategies *in vivo* and *ex vivo* to target viral reservoirs. Lipid nanoparticles containing the PKC activator bryostatin minimally induced proviral expression from infected CD4+ cells isolated from SCID-hu mice with thymus/liver implants (Kovochich et al., 2011). Also, a gold-based compound, auranofin, was used to treat SIV-infected macaques already receiving triple therapy (Lewis et al., 2011). After a month of treatment, PBMC proviral DNA levels decreased in the animals and plasma viral

While new eradication treatments are promising for reducing or eliminating viral reservoirs, there are many issues and remaining questions that need to be addressed. First, an assumption is that latently infected cells will die after they are activated. However, to date this has not been demonstrated in animals or in people. And it remains to be seen if there are strategies that can selectively activate HIV- or SIV-infected cells but not uninfected cells. Second, it is important to identify models or treatment options that can achieve complete, sustained virus suppression as is observed in patients. If this is achieved, methods to measure decreases in viral reservoirs will need to be optimized, including assays to measure a single copy of plasma viral RNA, a single infected cell, and spliced vs. unspliced viral

Finally, there are two caveats to the mouse and macaque models for studying persisting viral reservoirs. While mice can be rather effectively repopulated with a largely functional immune system, their anatomy is largely different than that of humans. Identification of reservoirs in mice will need to be confirmed with what is already known in humans. And although monkeys are more similar anatomically to humans, HIV-1 replication is not maintained in these animals. It remains to be seen whether transcriptional activation of SIV is similar to or different than that of HIV-1. Compounds designed to target activation of the HIV-1 LTR may not work or may work differently against SIV LTRs. Characterization of the

This chapter has highlighted the use of macaque and mouse models for antiretroviral therapies. These animal models have improved greatly since the development of SHIVs and the ability to reconstitute a functional human immune system in mice. Refinement of these models is ongoing to make them more closely resemble humans with regards to infection, pathogenesis, and antiretroviral immunity. For example, macaque models can be improved by increased understanding of macaque cell proteins that inhibit HIV-1 infection as well as

various models for investigation of virus reservoirs should, therefore, continue.

RNA was reduced after interruption of treatment.

2009).

RNA.

**6. Conclusion** 


Use of Animal Models for Anti-HIV Drug Development 87

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**6** 

*Japan* 

**Discovery of Novel Antiviral Agents Directed** 

**Against the Influenza A Virus Nucleoprotein** 

The influenza virus types A, B, and C belong to the family *Orthomyxoviridae*. Influenza B and C viruses are predominantly human pathogens, whereas influenza A virus spreads not only in humans but also in many animals, including birds and pigs. This characteristic of influenza A viruses is a major cause of influenza pandemics in humans. Influenza A viruses are responsible for periodic widespread epidemics, or pandemics, which have taken the form of respiratory diseases with cold-like symptoms, but also sometimes serious disease with high mortality rates (Webster et al., 1992). Four outbreaks of influenza occurred in the last and current centuries: Spanish influenza (H1N1) in 1918, Asian influenza (H2N2) in 1957, Hong Kong influenza (H3N2) in 1968, and H1N1 influenza in 2009. Recently, an influenza pandemic was caused by swine influenza (H1N1) in 2009, and according to the European Centre for Disease Prevention and Control, so far this global pandemic brought on 1.2 million infections and took the lives of more than fourteen thousand people. A strong sense of fear pervaded, but many lessons were learned from this pandemic. For example, this pandemic illustrated once again that the available vaccines against influenza virus were not completely effective for the prevention of influenza outbreaks, due to the extraordinarily rapid mutation rate that influenza viruses possess (Steinhauer & Holland, 1987). In addition, we did not have enough efficient antiviral drugs to cover all of the people infected (Stiver, 2004). On the other hand, there had been avian influenza (H5N1) infections in humans several years ago in Southeast Asia (Tran et al., 2004), and, importantly, that virus caused a high mortality rate. Putting it all together, in the face of the persistent threat of human influenza A infections, and moreover, the possibility of outbreaks of an avian influenza (H5N1) pandemic (Ungchusak et al., 2005; Wang et al., 2008), there is much concern about the shortage of effective anti-influenza virus agents, and for this reason, the development of novel anti-influenza virus agents is being strongly

Influenza A virus is a negative-stranded RNA virus with an eight-segmented genome that encodes 12 different proteins, including polymerase basic (PB)1, PB1-F2, an N-terminally truncated version of the polypeptide (N40), the translation of which is directed by PB1 codon 40, PB2, polymerase acidic protein (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix protein (M)1, M2, nonstructural protein (NS)1 and NS2 (Wise

**1. Introduction** 

demanded.

*Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama,* 

Yoko Aida, Yutaka Sasaki and Kyoji Hagiwara

and viral activity of tenofovir in the male genital tract. *Journal of Acquired Immune Deficiency Syndromes*, Vol.47, No.3, pp. 329-333.


## **Discovery of Novel Antiviral Agents Directed Against the Influenza A Virus Nucleoprotein**

Yoko Aida, Yutaka Sasaki and Kyoji Hagiwara *Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama, Japan* 

#### **1. Introduction**

98 Antiviral Drugs – Aspects of Clinical Use and Recent Advances

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Varrone, J., Rabi, S.A.*, et al.* (2010). Simian immunodeficiency virus-infected macaques treated with highly active antiretroviral therapy have reduced central nervous system viral replication and inflammation but persistence of viral DNA. The influenza virus types A, B, and C belong to the family *Orthomyxoviridae*. Influenza B and C viruses are predominantly human pathogens, whereas influenza A virus spreads not only in humans but also in many animals, including birds and pigs. This characteristic of influenza A viruses is a major cause of influenza pandemics in humans. Influenza A viruses are responsible for periodic widespread epidemics, or pandemics, which have taken the form of respiratory diseases with cold-like symptoms, but also sometimes serious disease with high mortality rates (Webster et al., 1992). Four outbreaks of influenza occurred in the last and current centuries: Spanish influenza (H1N1) in 1918, Asian influenza (H2N2) in 1957, Hong Kong influenza (H3N2) in 1968, and H1N1 influenza in 2009. Recently, an influenza pandemic was caused by swine influenza (H1N1) in 2009, and according to the European Centre for Disease Prevention and Control, so far this global pandemic brought on 1.2 million infections and took the lives of more than fourteen thousand people. A strong sense of fear pervaded, but many lessons were learned from this pandemic. For example, this pandemic illustrated once again that the available vaccines against influenza virus were not completely effective for the prevention of influenza outbreaks, due to the extraordinarily rapid mutation rate that influenza viruses possess (Steinhauer & Holland, 1987). In addition, we did not have enough efficient antiviral drugs to cover all of the people infected (Stiver, 2004). On the other hand, there had been avian influenza (H5N1) infections in humans several years ago in Southeast Asia (Tran et al., 2004), and, importantly, that virus caused a high mortality rate. Putting it all together, in the face of the persistent threat of human influenza A infections, and moreover, the possibility of outbreaks of an avian influenza (H5N1) pandemic (Ungchusak et al., 2005; Wang et al., 2008), there is much concern about the shortage of effective anti-influenza virus agents, and for this reason, the development of novel anti-influenza virus agents is being strongly demanded.

Influenza A virus is a negative-stranded RNA virus with an eight-segmented genome that encodes 12 different proteins, including polymerase basic (PB)1, PB1-F2, an N-terminally truncated version of the polypeptide (N40), the translation of which is directed by PB1 codon 40, PB2, polymerase acidic protein (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix protein (M)1, M2, nonstructural protein (NS)1 and NS2 (Wise

Discovery of Novel Antiviral Agents Directed Against the Influenza A Virus Nucleoprotein 101

(a) Influenza virus particles (A/WSN/33) were isolated by sucrose density gradient ultracentrifugation, and purified particles were observed by electron microscope with uranyl acetate-staining. (b) Model of influenza virus particle. The particle contains eight segments that make up the RNA genome. There were three major proteins (HA, NA, and M2) on the surface of the viron. (c) Diagram of the viral ribonucleoprotein (vRNP). The vRNA is associated with NP, PB2, PB1, and PA, which are composed of

Influenza A virus is one of the rare RNA viruses that replicate in the nucleus. The design of effective anti-influenza virus therapeutics is based on detailed knowledge of the biology of the virus. Fig. 2 shows the viral life cycle of the influenza virus. Generally, the influenza virus is adsorbed to the host cell through the binding of HA glycoproteins with sialic acid groups as receptors on the host cell, which are distributed on membrane-bound proteins and lipids. Then, the influenza virus is taken up into host cells through receptor-mediated endocytosis (Matlin et al., 1981). In the acidic environment of the endosome, HA changes to the active form and promotes fusion of the viral envelope with the endosomal membrane (Stegmann et al., 1987; White et al., 1982). The acidification of the endosome is necessary not only for the membrane fusion of HA but also the activation of the M2 ion channel. The activation of the M2 ion channel leads to proton entry into the virions, which causes uncoating of the vRNPs (Bui et al., 1996; Pinto et al., 1992). All vRNA are associated with the NP, which is bound at a distance of 24 nucleotides (Compans et al., 1972; Ortega et al., 2000). It is also suggested that vRNA is associated with trimeric RNA polymerase, which are composed of vRNPs (Klumpp et al., 1997). Influenza virus transcription and replication are initiated after the transport of vRNPs into the nucleus. After uncoating, the vRNPs are transported into the nucleus through importin / transport systems, where they undergo transcription and replication (Herz et al., 1981; Martin & Helenius, 1991). Transcription of vRNA requires capped RNA primers snartched from cellular pre-mRNAs and premature poly(A) termination of transcripts (Bouloy et al., 1978; Plotch et al., 1979; Robertson et al.,

Fig. 1. Electron microscopy and model of influenza virus particles.

vRNPs.

**2. Viral life cycle** 

et al., 2009) (Fig. 1b). Nine of these proteins are incorporated into the virion. The influenza virus particles, which are ~100 nm in diameter, form by budding from the plasma membrane of infected cells (Fig. 1a)**.** On the surface of the virion, there are two main antigenic determinants, the spike glycoproteins HA and NA. There are considerable antigenic variations among influenza viruses consisting of 16 different types of HA (H1- H16) and 9 different types of NA (N1-N9) (Fouchier et al., 2005; Hinshaw et al., 1982; Kawaoka et al., 1990; Rohm et al., 1996; World Health Organization, 1980). In addition, another viral protein is inserted in the viral membrane: M2, a low-abundance ion channel involved in uncoating and HA maturation (Fig. 1b). Underlying the membrane is the matrix or M1 protein, the major structural component of the virion, which is thought to act as an adaptor between the lipid envelope and the ribonucleoprotein (RNP) complexes and is probably the mediator of virus budding (Gómez-Puertas et al., 2000). Inside the shell of M1 lie the viral RNP complexes (vRNPs), which comprise the genomic RNA segments in association with a trimeric RNA polymerase (PB1, PB2 and PA subunits) and the stoichiometric quantities of NP (Fig. 1c). Also found in the virion are small quantities of the NEP and NS2 proteins.

The surface proteins HA, NA, and M2 are the targets of vaccines and anti-viral drugs. Two classes of drugs including adamantanes (amantadine and rimantadine) and NA inhibitors (oseltamivir, zanamivir, peramivir, and laninamivir) are available for the treatment of the influenza infection (Kubo et al., 2010; Vanvoris et al., 1981; Wingfiel et al., 1969; Yamashita, 2011). Because the adamantanes exert several toxic effects on the central nervous system (Bryson et al., 1980; Keyser et al., 2000) and also because of the emergence of resistant variants (Bright et al., 2005, 2006), the use of these drugs is limited. Currently, the NA inhibitors are used widely for drug therapies to treat influenza patients because of high inhibitory effects and little toxicity. However, resistant strains, and especially oseltamivirresistant strains, have also been reported in recent years (Besselaar et al., 2008; Dharan et al., 2009; Hauge et al., 2009; Hurt et al., 2009). The fact that available drugs target only two steps of the viral life cycle, along with the appearance of oseltamivir-resistant influenza strains, strongly highlights the need for treatment alternatives or novel antiviral drugs targeting other proteins besides M2 or NA.

In contrast to the surface proteins, after entry into the cytoplasm, the vRNPs are imported into the nucleus for the production of viral messenger RNAs. RNA polymerases, such as PB1, PB2, and PA, transcribe and replicate the virus genome, while NP encapsidates the virus genome to form an RNP complex for the purposes of transcription and packaging. Thus, influenza virus transcription and replication are initiated after transport to the nucleus of vRNPs. A promising target for blocking influenza A viruses is the NP, which is expressed in the early stage of infection and plays important roles in numerous steps of viral replication. NP preserves viral genomic RNA (vRNA) stability and contains many functional domains in its sequence, such as a nuclear localization signal (NLS), an RNA binding site, an NP-NP binding site, and a PB2 binding domain. In addition, NP is relatively well conserved compared with viral surface spike protein. Here, we summarize current knowledge about influenza therapy, the functions of NP involved in the nuclear-import step of influenza virus replication, and how this could facilitate the discovery of a new small molecule involved in influenza replication.

(a) Influenza virus particles (A/WSN/33) were isolated by sucrose density gradient ultracentrifugation, and purified particles were observed by electron microscope with uranyl acetate-staining. (b) Model of influenza virus particle. The particle contains eight segments that make up the RNA genome. There were three major proteins (HA, NA, and M2) on the surface of the viron. (c) Diagram of the viral ribonucleoprotein (vRNP). The vRNA is associated with NP, PB2, PB1, and PA, which are composed of vRNPs.

Fig. 1. Electron microscopy and model of influenza virus particles.

#### **2. Viral life cycle**

100 Antiviral Drugs – Aspects of Clinical Use and Recent Advances

et al., 2009) (Fig. 1b). Nine of these proteins are incorporated into the virion. The influenza virus particles, which are ~100 nm in diameter, form by budding from the plasma membrane of infected cells (Fig. 1a)**.** On the surface of the virion, there are two main antigenic determinants, the spike glycoproteins HA and NA. There are considerable antigenic variations among influenza viruses consisting of 16 different types of HA (H1- H16) and 9 different types of NA (N1-N9) (Fouchier et al., 2005; Hinshaw et al., 1982; Kawaoka et al., 1990; Rohm et al., 1996; World Health Organization, 1980). In addition, another viral protein is inserted in the viral membrane: M2, a low-abundance ion channel involved in uncoating and HA maturation (Fig. 1b). Underlying the membrane is the matrix or M1 protein, the major structural component of the virion, which is thought to act as an adaptor between the lipid envelope and the ribonucleoprotein (RNP) complexes and is probably the mediator of virus budding (Gómez-Puertas et al., 2000). Inside the shell of M1 lie the viral RNP complexes (vRNPs), which comprise the genomic RNA segments in association with a trimeric RNA polymerase (PB1, PB2 and PA subunits) and the stoichiometric quantities of NP (Fig. 1c). Also found in the virion are small quantities of the

The surface proteins HA, NA, and M2 are the targets of vaccines and anti-viral drugs. Two classes of drugs including adamantanes (amantadine and rimantadine) and NA inhibitors (oseltamivir, zanamivir, peramivir, and laninamivir) are available for the treatment of the influenza infection (Kubo et al., 2010; Vanvoris et al., 1981; Wingfiel et al., 1969; Yamashita, 2011). Because the adamantanes exert several toxic effects on the central nervous system (Bryson et al., 1980; Keyser et al., 2000) and also because of the emergence of resistant variants (Bright et al., 2005, 2006), the use of these drugs is limited. Currently, the NA inhibitors are used widely for drug therapies to treat influenza patients because of high inhibitory effects and little toxicity. However, resistant strains, and especially oseltamivirresistant strains, have also been reported in recent years (Besselaar et al., 2008; Dharan et al., 2009; Hauge et al., 2009; Hurt et al., 2009). The fact that available drugs target only two steps of the viral life cycle, along with the appearance of oseltamivir-resistant influenza strains, strongly highlights the need for treatment alternatives or novel antiviral drugs targeting

In contrast to the surface proteins, after entry into the cytoplasm, the vRNPs are imported into the nucleus for the production of viral messenger RNAs. RNA polymerases, such as PB1, PB2, and PA, transcribe and replicate the virus genome, while NP encapsidates the virus genome to form an RNP complex for the purposes of transcription and packaging. Thus, influenza virus transcription and replication are initiated after transport to the nucleus of vRNPs. A promising target for blocking influenza A viruses is the NP, which is expressed in the early stage of infection and plays important roles in numerous steps of viral replication. NP preserves viral genomic RNA (vRNA) stability and contains many functional domains in its sequence, such as a nuclear localization signal (NLS), an RNA binding site, an NP-NP binding site, and a PB2 binding domain. In addition, NP is relatively well conserved compared with viral surface spike protein. Here, we summarize current knowledge about influenza therapy, the functions of NP involved in the nuclear-import step of influenza virus replication, and how this could facilitate the discovery of a new small

NEP and NS2 proteins.

other proteins besides M2 or NA.

molecule involved in influenza replication.

Influenza A virus is one of the rare RNA viruses that replicate in the nucleus. The design of effective anti-influenza virus therapeutics is based on detailed knowledge of the biology of the virus. Fig. 2 shows the viral life cycle of the influenza virus. Generally, the influenza virus is adsorbed to the host cell through the binding of HA glycoproteins with sialic acid groups as receptors on the host cell, which are distributed on membrane-bound proteins and lipids. Then, the influenza virus is taken up into host cells through receptor-mediated endocytosis (Matlin et al., 1981). In the acidic environment of the endosome, HA changes to the active form and promotes fusion of the viral envelope with the endosomal membrane (Stegmann et al., 1987; White et al., 1982). The acidification of the endosome is necessary not only for the membrane fusion of HA but also the activation of the M2 ion channel. The activation of the M2 ion channel leads to proton entry into the virions, which causes uncoating of the vRNPs (Bui et al., 1996; Pinto et al., 1992). All vRNA are associated with the NP, which is bound at a distance of 24 nucleotides (Compans et al., 1972; Ortega et al., 2000). It is also suggested that vRNA is associated with trimeric RNA polymerase, which are composed of vRNPs (Klumpp et al., 1997). Influenza virus transcription and replication are initiated after the transport of vRNPs into the nucleus. After uncoating, the vRNPs are transported into the nucleus through importin / transport systems, where they undergo transcription and replication (Herz et al., 1981; Martin & Helenius, 1991). Transcription of vRNA requires capped RNA primers snartched from cellular pre-mRNAs and premature poly(A) termination of transcripts (Bouloy et al., 1978; Plotch et al., 1979; Robertson et al.,

Discovery of Novel Antiviral Agents Directed Against the Influenza A Virus Nucleoprotein 103

binds to cholesterol and this allows M2 to alter membrane curvature at the site of virus budding (Rossman et al., 2010a, 2010b). Bud formation and bud release are the last steps of the viral life cycle. NA is responsible for cleaving terminal sialic acid residues from the ends of glycoconjugates on both the virus particle and the host cell in order to facilitate virus

The life-cycle of influenza virus is a major target for drug development. Accordingly, significant efforts have been made recently to identify molecules that inhibit the different stages of the influenza virus life cycle. As shown in Fig. 2, the current treatments for influenza infections target two steps of the replication cycle: uncoating and budding. Six drugs are currently available (Table 1): the adamantanes and neuraminidase inhibitors, including amantadine, rimantadine, zanamivir, oseltamivir, peramivir, and laninamivir (Kubo et al., 2010; Vanvoris et al., 1981; Wingfiel et al., 1969, Yamashita, 2011). The adamantanes block the function of the M2 ion channel, preventing acidification-triggered uncoating. The adamantanes were the first effective drugs licensed for influenza treatment (Davies et al., 1964; Dolin et al., 1982; Wang et al., 1993). Despite a degree of treatment effectiveness, however, both drugs induced significant adverse effects in the central nervous system, as well as the emergence of drug-resistant mutants (Bright et al., 2005, 2006; Bryson et al., 1980; Keyser et al., 2000). Recently, the vast majority of circulating seasonal influenza strains has been adamantanes-resistant (Bright et al., 2005, 2006). Neuraminidase inhibitors inhibit the release of virions by competitively inhibiting viral NA. Currently, zanamivir and oseltamivir are widely used to treat acute uncomplicated illness due to influenza A and B. Zanamivir mimics the natural substrate, which fits into the active site pocket of NA (Varghese et al., 1992, 1995; von Itzstein et al., 1993). Oseltamivir was developed through the modification of the sialic acid analogue framework (Kim et al., 1997). Many reports have shown that both drugs are highly efficient in the treatment of influenza (Cooper et al., 2003; Hayden et al., 1997; Monto et al., 1999; Nicholson et al., 2000). In recent years, peramivir and laninamivir, which also target NA, have been licensed as anti-influenza drugs (Kubo et al., 2010; Yamashita, 2011). For oseltamivir, the appearance of drug-resistant mutants has significantly increased in many countries (Besselaar et al., 2008; Dharan et al., 2009; Hauge et al., 2009; Hurt et al., 2009). The oseltamivir-resistant H275Y virus also displays reduced susceptibility to peramivir *in vitro* (Nguyen et al., 2010). On the other hand, no zanamivirresistant virus has emerged at present. However, because zanamivir requires treatment by

the intravenous route, it is not commonly used in clinical treatment.

As mentioned above, in addition to the fact that available drugs target only two steps of the viral life cycle, this appearance of oseltamivir-resistant influenza strains strongly highlights the need for treatment alternatives or novel antiviral drugs targeting other proteins besides M2 or NA. Potential targets for blocking influenza A virus replication are influenza virus RNA polymerases and NP, which is required to form the RNPs. Recently, favipiravir, a novel therapeutic drug targeting viral replication and translation, has been identified (Furuta et al., 2005). Favipiravir, developed by Furuta *et al*. at Toyama Chemical Co., Ltd., inhibits the replication and translation of influenza viruses in a GTP-competitive manner. In addition to these drugs, a few novel antiviral compounds, mycalamide analogs (Hagiwara et al., 2010), nucleozin (Kao et al., 2010) and nucleozin analog FA-2 (Su et al., 2010), were

release (Air & Laver, 1989).

**3. Influenza therapy** 

The crucial steps in Influenza virus multiplication are: 1) attachment of the virus to its target cell; 2) entry of the virus via receptor-mediated endocytosis; 3) fusion of endosome and viral membranes; 4) the RNPs release into the cytoplasm and 5) transport into nucleus; 6) virus transcription and replication in nucleus; 7) the RNPs construction; 8) virion assembly and 9) viral budding. Anti-influenza drugs target two different steps in the viral life-cycle; these steps are shown in boxes.

Fig. 2. Model of the life cycle of the influenza virus.

1981). In contrast, the replication of vRNA is performed in a primer-independent manner (Nagata et al., 2008). The newly synthesized vRNPs are exported from the nucleus to the cytoplasm in association with the viral proteins M1 and NS2 and the cellular protein chromosome region maintenance 1 (CRM1) (Cros & Palese, 2003; Neumann et al., 2004). These vRNPs are incorporated into budding virions. The vRNA is specifically packaged in preference to other cellular RNAs and the different vRNAs are present in an equimolar ratio within a population of virions (Palese, 1977). A mechanism for the specific packaging of vRNA is mediated by *cis*-acting packaging signals in the vRNAs. Specific packaging signals exist in the UTRs and coding regions at both the 5' and 3' ends of the vRNAs (de Wit et al., 2006; Fujii et al., 2003; Liang et al., 2008; Muramoto et al., 2006; Noda et al., 2006; Ozawa et al., 2007). The structure of eight separate segments is associated with inter-segment interactions (Muramoto et al., 2006). However, it remains uncertain whether there are specific interactions among the eight RNPs within the virions. The M1 and M2 proteins play central roles in the assembly and budding process. The M1 protein is associated with the cytoplasmic tail of HA and NA. This binding allows for M1 to associate with lipid raft membrane domains, triggering a conformational change that enables M1 polymerization at the site of virus budding (Barman et al., 2004; Gómez-Puertas et al., 2000; Ruigrok et al., 2001). The M2 protein is required for the membrane scission of the budding virions. M2 binds to cholesterol and this allows M2 to alter membrane curvature at the site of virus budding (Rossman et al., 2010a, 2010b). Bud formation and bud release are the last steps of the viral life cycle. NA is responsible for cleaving terminal sialic acid residues from the ends of glycoconjugates on both the virus particle and the host cell in order to facilitate virus release (Air & Laver, 1989).

#### **3. Influenza therapy**

102 Antiviral Drugs – Aspects of Clinical Use and Recent Advances

The crucial steps in Influenza virus multiplication are: 1) attachment of the virus to its target cell; 2) entry of the virus via receptor-mediated endocytosis; 3) fusion of endosome and viral membranes; 4) the RNPs release into the cytoplasm and 5) transport into nucleus; 6) virus transcription and replication in nucleus; 7) the RNPs construction; 8) virion assembly and 9) viral budding. Anti-influenza drugs target

1981). In contrast, the replication of vRNA is performed in a primer-independent manner (Nagata et al., 2008). The newly synthesized vRNPs are exported from the nucleus to the cytoplasm in association with the viral proteins M1 and NS2 and the cellular protein chromosome region maintenance 1 (CRM1) (Cros & Palese, 2003; Neumann et al., 2004). These vRNPs are incorporated into budding virions. The vRNA is specifically packaged in preference to other cellular RNAs and the different vRNAs are present in an equimolar ratio within a population of virions (Palese, 1977). A mechanism for the specific packaging of vRNA is mediated by *cis*-acting packaging signals in the vRNAs. Specific packaging signals exist in the UTRs and coding regions at both the 5' and 3' ends of the vRNAs (de Wit et al., 2006; Fujii et al., 2003; Liang et al., 2008; Muramoto et al., 2006; Noda et al., 2006; Ozawa et al., 2007). The structure of eight separate segments is associated with inter-segment interactions (Muramoto et al., 2006). However, it remains uncertain whether there are specific interactions among the eight RNPs within the virions. The M1 and M2 proteins play central roles in the assembly and budding process. The M1 protein is associated with the cytoplasmic tail of HA and NA. This binding allows for M1 to associate with lipid raft membrane domains, triggering a conformational change that enables M1 polymerization at the site of virus budding (Barman et al., 2004; Gómez-Puertas et al., 2000; Ruigrok et al., 2001). The M2 protein is required for the membrane scission of the budding virions. M2

two different steps in the viral life-cycle; these steps are shown in boxes.

Fig. 2. Model of the life cycle of the influenza virus.

The life-cycle of influenza virus is a major target for drug development. Accordingly, significant efforts have been made recently to identify molecules that inhibit the different stages of the influenza virus life cycle. As shown in Fig. 2, the current treatments for influenza infections target two steps of the replication cycle: uncoating and budding. Six drugs are currently available (Table 1): the adamantanes and neuraminidase inhibitors, including amantadine, rimantadine, zanamivir, oseltamivir, peramivir, and laninamivir (Kubo et al., 2010; Vanvoris et al., 1981; Wingfiel et al., 1969, Yamashita, 2011). The adamantanes block the function of the M2 ion channel, preventing acidification-triggered uncoating. The adamantanes were the first effective drugs licensed for influenza treatment (Davies et al., 1964; Dolin et al., 1982; Wang et al., 1993). Despite a degree of treatment effectiveness, however, both drugs induced significant adverse effects in the central nervous system, as well as the emergence of drug-resistant mutants (Bright et al., 2005, 2006; Bryson et al., 1980; Keyser et al., 2000). Recently, the vast majority of circulating seasonal influenza strains has been adamantanes-resistant (Bright et al., 2005, 2006). Neuraminidase inhibitors inhibit the release of virions by competitively inhibiting viral NA. Currently, zanamivir and oseltamivir are widely used to treat acute uncomplicated illness due to influenza A and B. Zanamivir mimics the natural substrate, which fits into the active site pocket of NA (Varghese et al., 1992, 1995; von Itzstein et al., 1993). Oseltamivir was developed through the modification of the sialic acid analogue framework (Kim et al., 1997). Many reports have shown that both drugs are highly efficient in the treatment of influenza (Cooper et al., 2003; Hayden et al., 1997; Monto et al., 1999; Nicholson et al., 2000). In recent years, peramivir and laninamivir, which also target NA, have been licensed as anti-influenza drugs (Kubo et al., 2010; Yamashita, 2011). For oseltamivir, the appearance of drug-resistant mutants has significantly increased in many countries (Besselaar et al., 2008; Dharan et al., 2009; Hauge et al., 2009; Hurt et al., 2009). The oseltamivir-resistant H275Y virus also displays reduced susceptibility to peramivir *in vitro* (Nguyen et al., 2010). On the other hand, no zanamivirresistant virus has emerged at present. However, because zanamivir requires treatment by the intravenous route, it is not commonly used in clinical treatment.

As mentioned above, in addition to the fact that available drugs target only two steps of the viral life cycle, this appearance of oseltamivir-resistant influenza strains strongly highlights the need for treatment alternatives or novel antiviral drugs targeting other proteins besides M2 or NA. Potential targets for blocking influenza A virus replication are influenza virus RNA polymerases and NP, which is required to form the RNPs. Recently, favipiravir, a novel therapeutic drug targeting viral replication and translation, has been identified (Furuta et al., 2005). Favipiravir, developed by Furuta *et al*. at Toyama Chemical Co., Ltd., inhibits the replication and translation of influenza viruses in a GTP-competitive manner. In addition to these drugs, a few novel antiviral compounds, mycalamide analogs (Hagiwara et al., 2010), nucleozin (Kao et al., 2010) and nucleozin analog FA-2 (Su et al., 2010), were

Discovery of Novel Antiviral Agents Directed Against the Influenza A Virus Nucleoprotein 105

The nucleoprotein has been proven to bind non-specifically to RNA at one in every 24 nucleotides (Compans et al., 1972; Ortega et al., 2000), and indeed, the protein has been shown to have an RNA-binding groove between the head and body domains. This groove is located exterior to the nucleoprotein oligomers. The surface of the groove is occupied by several basic residues, such as R65, R150, R152, R156, R174, R175, R195, R199, R213, R214, R221, R236, R355, K357, R361, and R391 (Ye et al., 2006), which can interact with nucleotides. These distributed basic residues are highly conserved, and therefore it is likely that the

The crystal structure of NP derived from H5N1 has also been reported. It forms a different trimer compared with H1N1; however, the tail-loop interactions were identical (Ng et al., 2008). Both papers suggest that the tail-loop binding pocket is a good target for the

Most RNA viruses that lack a DNA phase replicate in the cytoplasm (Cros & Palese, 2003). However, several negative-stranded RNA viruses, such as influenza, Thogoto, and Borna disease viruses, replicate their RNAs in the nucleus, taking advantage of the host cell's

NLS, nuclear localization signal; NAS, nuclear accumulation signal

compounds targeting these regions can inhibit viral multiplication.

Fig. 3. Summary of NP functions.

development of anti-influenza virus drugs.

**5. Nuclear transport of NP** 

recently reported as influenza inhibitors targeting NP. Moreover, another anti-influenza compound, 367, has been identified that targets the PB1 protein and influenza RNAdependent RNA polymerase activity (Su et al., 2010). These observations suggest that the drug targeting of components of the RNPs, such as PA, PB1, PB2, and NP, may provide a new strategy for influenza therapy.


Table 1. Available drugs against influenza virus infection.

#### **4. NP of influenza A virus**

#### **4.1. Function of NP**

The influenza virus NP, which is encoded by the fifth genome segment, is expressed in the early stage of infection. It is the major component of the RNP and contains many functional domains in its sequence, such as a NLS, an RNA binding site, an NP-NP binding site, and a PB2 binding domain (Albo et al., 1995; Biswas et al., 1998; Cros et al., 2005; Davey et al., 1985; Elton et al., 1999; Ketha & Atreya, 2008; Kobayashi et al., 1994; Neumann et al., 1997; Ozawa et al., 2007; Wang et al., 1997; Weber et al., 1998; Ye et al., 2006). The major functional domains of NP are summarized in Fig. 3. Thus, NP plays important roles in numerous stages of viral replication, such as in the nuclear transport of NP and RNPs, replication and transcription of genomic RNA, and nuclear export and packaging of RNPs. In addition to these, some domains that are important for the maintenance of three-dimensional structure, such as the tail-loop structure, pocket structure, and regulation of particle formation, as well as domains that are important for interactions with host proteins, have been reported (Fig. 3) (Digard et al., 1999; Momose et al., 2001; Ng et al., 2008, 2009; Noton et al., 2009; Wang et al., 1997; Ye et al., 2006). Moreover, phylogenetic analysis of viral strains isolated from different hosts revealed that the NP gene is relatively well conserved (Shu et al., 1993), especially in the functional domains (Heiny et al., 2007; Li et al., 2009; Ng et al., 2009). Thus, all of these functional domains could be considered potential targets for antiviral agents.

#### **4.2 Structure of NP**

The crystal structure of NP of influenza A virus H1N1 (Ye et al., 2006) is shown in Fig. 4. The N-terminal truncated NP derived from H1N1 forms trimers through NP-NP interactions using a tail-loop structure constructed from the segment at amino acid positions 402-428 (Fig. 3 & 4) and pocket structures constructed from segments at amino acid positions 160-167, 321-334, and 340-349 (Fig. 3, Ye et al., 2006).


NLS, nuclear localization signal; NAS, nuclear accumulation signal

Fig. 3. Summary of NP functions.

104 Antiviral Drugs – Aspects of Clinical Use and Recent Advances

recently reported as influenza inhibitors targeting NP. Moreover, another anti-influenza compound, 367, has been identified that targets the PB1 protein and influenza RNAdependent RNA polymerase activity (Su et al., 2010). These observations suggest that the drug targeting of components of the RNPs, such as PA, PB1, PB2, and NP, may provide a

Classification Generic Name Commercial Name

Rimantadine Flumadine

 Zanamivir Relenza Peramivir Rapiacta Laninamivir Inamivir

The influenza virus NP, which is encoded by the fifth genome segment, is expressed in the early stage of infection. It is the major component of the RNP and contains many functional domains in its sequence, such as a NLS, an RNA binding site, an NP-NP binding site, and a PB2 binding domain (Albo et al., 1995; Biswas et al., 1998; Cros et al., 2005; Davey et al., 1985; Elton et al., 1999; Ketha & Atreya, 2008; Kobayashi et al., 1994; Neumann et al., 1997; Ozawa et al., 2007; Wang et al., 1997; Weber et al., 1998; Ye et al., 2006). The major functional domains of NP are summarized in Fig. 3. Thus, NP plays important roles in numerous stages of viral replication, such as in the nuclear transport of NP and RNPs, replication and transcription of genomic RNA, and nuclear export and packaging of RNPs. In addition to these, some domains that are important for the maintenance of three-dimensional structure, such as the tail-loop structure, pocket structure, and regulation of particle formation, as well as domains that are important for interactions with host proteins, have been reported (Fig. 3) (Digard et al., 1999; Momose et al., 2001; Ng et al., 2008, 2009; Noton et al., 2009; Wang et al., 1997; Ye et al., 2006). Moreover, phylogenetic analysis of viral strains isolated from different hosts revealed that the NP gene is relatively well conserved (Shu et al., 1993), especially in the functional domains (Heiny et al., 2007; Li et al., 2009; Ng et al., 2009). Thus, all of these functional domains could be considered potential targets for antiviral

The crystal structure of NP of influenza A virus H1N1 (Ye et al., 2006) is shown in Fig. 4. The N-terminal truncated NP derived from H1N1 forms trimers through NP-NP interactions using a tail-loop structure constructed from the segment at amino acid positions 402-428 (Fig. 3 & 4) and pocket structures constructed from segments at amino acid

positions 160-167, 321-334, and 340-349 (Fig. 3, Ye et al., 2006).

M2 Channel Inhibitor Amantadine Symmetrel

Neuraminidase Inhibitor Oseltamivir Tamiflu

Table 1. Available drugs against influenza virus infection.

new strategy for influenza therapy.

**4. NP of influenza A virus** 

**4.1. Function of NP** 

agents.

**4.2 Structure of NP** 

The nucleoprotein has been proven to bind non-specifically to RNA at one in every 24 nucleotides (Compans et al., 1972; Ortega et al., 2000), and indeed, the protein has been shown to have an RNA-binding groove between the head and body domains. This groove is located exterior to the nucleoprotein oligomers. The surface of the groove is occupied by several basic residues, such as R65, R150, R152, R156, R174, R175, R195, R199, R213, R214, R221, R236, R355, K357, R361, and R391 (Ye et al., 2006), which can interact with nucleotides. These distributed basic residues are highly conserved, and therefore it is likely that the compounds targeting these regions can inhibit viral multiplication.

The crystal structure of NP derived from H5N1 has also been reported. It forms a different trimer compared with H1N1; however, the tail-loop interactions were identical (Ng et al., 2008). Both papers suggest that the tail-loop binding pocket is a good target for the development of anti-influenza virus drugs.

#### **5. Nuclear transport of NP**

Most RNA viruses that lack a DNA phase replicate in the cytoplasm (Cros & Palese, 2003). However, several negative-stranded RNA viruses, such as influenza, Thogoto, and Borna disease viruses, replicate their RNAs in the nucleus, taking advantage of the host cell's

Discovery of Novel Antiviral Agents Directed Against the Influenza A Virus Nucleoprotein 107

and NTF2 (Stewart, 2000). Imp functions as an adaptor molecule, binding Imp via its amino-terminally located Imp-binding (IBB) domain and binding an NLS-bearing protein via its two central region-located NLS-binding sites (Herold et al., 1998; Kobe, 1999) (Fig. 5). Imp is the transport receptor that carries the Imp-NLS complex from the cytoplasm to the nuclear side of the NPC. Once the heterotrimer consisting of Imp, Imp, and the NLSbearing protein reaches the nuclear face of the NPC, the GTP-bound form of Ran binds directly to Imp, releasing Imp and the NLS-bearing protein into the nucleoplasm. Ran, which is found in its GDP-bound form in the cytoplasm and in its GTP-bound form in the nucleus, is a major determinant of the directionality of transport across the nuclear

A challenge faced by influenza virus is that of the trafficking of viral components into the nucleus through the NPC (Fig. 5). The genomic RNAs of influenza virus associate with proteins to form large complexes called vRNPs, which exceed the size limit for passive diffusion through the NPCs. The vRNPs is believed to be 10–20 nm wide (Compans et al., 1972). The vRNA, coated by NPs (1NP for each 24 nucleotides) (Compans et al., 1972; Ortega et al., 2000), forms a loop (Fig. 1C & 6). The trimeric polymerase complex, PB1, PB2, and PA, binds to the partially complementary ends of the vRNA, giving rise to a complex panhandle

Fig. 5. Model of the nuclear transport through classical importin / import pathway and

To ensure efficient transport across the nuclear membrane, influenza virus uses NLSs exposed on PB2, PB1, PA and NP. These signals recruit cellular import complexes, which are responsible for the translocation of the vRNPs through the NPC. Although all of the

membrane.

structure (Martin-Benito et al., 2001).

NPC, nuclear pore complex

influenza virus.

The NP trimer was constructed according to NP-NP interactions using pocket structure (amino acid positions 160-167, 327-334 and 340-349) and tail-loop structure (amino acid positions 402-428). The positions of the three tail loops are highlighted in white and indicated by arrows. This structure is based on the available structural information for NP (PDB code: 2IQH).

Fig. 4. Crystal structure of influenza virus NP trimer analyzed by X-ray.

nuclear machinery. The cell nucleus is separated from the cytoplasm by a double-layer membrane contiguous with the ER called the nuclear envelope (Terry et al., 2007). This nuclear envelope is composed of two lipid bilayers, the outer and inner nuclear membranes (Gruenbaum et al., 2005). Viruses that replicate their genome in the host cell nucleus have evolved strategies for moving viral components across this membrane barrier. These membranes are separated by a lumen and joined at nuclear pore complexes (NPCs) (Fahrenkrog & Aebi, 2003) that serve as gates for traffic crossing the nuclear envelope. Entrance into and exit from the nucleus occurs via these NPCs, which are > 60 MDa macromolecular structures that form channels spanning the nuclear envelope (Cronshaw et al., 2002; Rout et al., 2000). Each NPC is equipped to facilitate both the import and export of proteins and RNAs (Dworetzky & Feldherr, 1988).

The movement of ions, metabolites and other small molecules through the NPC occurs via passive diffusion, but the translocation of cargos larger than ~40 kDa generally requires specific signals known as NLSs. The nuclear import of basic NLS-bearing proteins is mediated by specific soluble factors, including importin- (Imp) (Goldfarb et al., 2004), importin- (Imp) (Harel & Forbes, 2004), small GTPase Ran/TC4 (Quimby & Dasso, 2003), and NTF2 (Stewart, 2000). Imp functions as an adaptor molecule, binding Imp via its amino-terminally located Imp-binding (IBB) domain and binding an NLS-bearing protein via its two central region-located NLS-binding sites (Herold et al., 1998; Kobe, 1999) (Fig. 5). Imp is the transport receptor that carries the Imp-NLS complex from the cytoplasm to the nuclear side of the NPC. Once the heterotrimer consisting of Imp, Imp, and the NLSbearing protein reaches the nuclear face of the NPC, the GTP-bound form of Ran binds directly to Imp, releasing Imp and the NLS-bearing protein into the nucleoplasm. Ran, which is found in its GDP-bound form in the cytoplasm and in its GTP-bound form in the nucleus, is a major determinant of the directionality of transport across the nuclear membrane.

A challenge faced by influenza virus is that of the trafficking of viral components into the nucleus through the NPC (Fig. 5). The genomic RNAs of influenza virus associate with proteins to form large complexes called vRNPs, which exceed the size limit for passive diffusion through the NPCs. The vRNPs is believed to be 10–20 nm wide (Compans et al., 1972). The vRNA, coated by NPs (1NP for each 24 nucleotides) (Compans et al., 1972; Ortega et al., 2000), forms a loop (Fig. 1C & 6). The trimeric polymerase complex, PB1, PB2, and PA, binds to the partially complementary ends of the vRNA, giving rise to a complex panhandle structure (Martin-Benito et al., 2001).

#### NPC, nuclear pore complex

106 Antiviral Drugs – Aspects of Clinical Use and Recent Advances

The NP trimer was constructed according to NP-NP interactions using pocket structure (amino acid positions 160-167, 327-334 and 340-349) and tail-loop structure (amino acid positions 402-428). The positions of the three tail loops are highlighted in white and indicated by arrows. This structure is based

nuclear machinery. The cell nucleus is separated from the cytoplasm by a double-layer membrane contiguous with the ER called the nuclear envelope (Terry et al., 2007). This nuclear envelope is composed of two lipid bilayers, the outer and inner nuclear membranes (Gruenbaum et al., 2005). Viruses that replicate their genome in the host cell nucleus have evolved strategies for moving viral components across this membrane barrier. These membranes are separated by a lumen and joined at nuclear pore complexes (NPCs) (Fahrenkrog & Aebi, 2003) that serve as gates for traffic crossing the nuclear envelope. Entrance into and exit from the nucleus occurs via these NPCs, which are > 60 MDa macromolecular structures that form channels spanning the nuclear envelope (Cronshaw et al., 2002; Rout et al., 2000). Each NPC is equipped to facilitate both the import and export of

The movement of ions, metabolites and other small molecules through the NPC occurs via passive diffusion, but the translocation of cargos larger than ~40 kDa generally requires specific signals known as NLSs. The nuclear import of basic NLS-bearing proteins is mediated by specific soluble factors, including importin- (Imp) (Goldfarb et al., 2004), importin- (Imp) (Harel & Forbes, 2004), small GTPase Ran/TC4 (Quimby & Dasso, 2003),

on the available structural information for NP (PDB code: 2IQH).

proteins and RNAs (Dworetzky & Feldherr, 1988).

Fig. 4. Crystal structure of influenza virus NP trimer analyzed by X-ray.

Fig. 5. Model of the nuclear transport through classical importin / import pathway and influenza virus.

To ensure efficient transport across the nuclear membrane, influenza virus uses NLSs exposed on PB2, PB1, PA and NP. These signals recruit cellular import complexes, which are responsible for the translocation of the vRNPs through the NPC. Although all of the

Discovery of Novel Antiviral Agents Directed Against the Influenza A Virus Nucleoprotein 109

**NP-mRFP-Flag mRFP-Flag reference**

AB D C EFG AB D C EFG

Approximately 2,000 compounds are spotted, with duplicates, on a glass plate using a photo-cross

**NP**

Methods Targeting Positive Negative

replication 9 63

replication 1 <sup>8</sup>

derivatives to NP 4 0

array assay Binding to NP 72 6,800

Table 2. Screening for NP inhibitors from the NPDepo RIKEN Natural Products Depository.

artificial analog of mycalamide. Among them, two derivatives showed lower inhibitory effect compared with compound 1, whereas compound 4 (Fig. 7, Table 2) showed strong

Furthermore, surface plasmon resonance imaging experiments demonstrated that the binding activity of each compound to NP correlated with its antiviral activity. Finally, it was shown that these compounds bound NP within the N-terminal 110-amino acid region but their binding abilities were dramatically reduced when the N-terminal 13-amino acid tail was deleted, suggesting that the compounds might bind to this region, which mediates the nuclear transport of NP and its binding to viral RNA. These data suggest that compound binding to the N-terminal 13-amino acid tail region corresponding to an unconventional

Inhibition of viral

**NP-mRFP-Flag**

(not determined)

**mRFP compound Flag**

Fig. 6. Summary of compound screening using chemical array.

Plaque assay Inhibition of viral

activity reaching inhibition level up to 97% (Hagiwara et al., 2010).

NLS may inhibit viral replication by inhibiting the nuclear transport of NP.

Surface plasmon resonance Binding of four

**positive**

**positive**

linker.

Photo-crosslinked chemical

Plaque assay using derivatives

proteins of the vRNPs carry NLSs (Akkina et al., 1987; Jones et al., 1986; Nieto et al., 1994), the NP was shown to be sufficient to mediate the nuclear import of viral RNAs (O'Neill et al., 1995). As shown in Fig. 3, a more detailed analysis revealed the presence of three NLSs on the NP (Neumann et al., 1997; Wang et al., 1997), including an unconventional NLS at the very N-terminus, located between amino acids 3 and 13. A second NLS resides in the central part of the NP, located between amino acids 198 and 216. This bipartite signal appears to be weaker than the unconventional N-terminal NLS (Weber et al., 1998). Of these NLSs, the unconventional, N-terminal NLS of the NP is indispensable for the nuclear transport of NP and vRNPs (Cross et al., 2005; O'Neill et al., 1995). Alanine-substituted mutants of this unconventional NLS of the NP have shown that the amino acids at position 7 and 8 are critical for nuclear localization (Neumann et al., 1997). Furthermore, the mutation of amino acids at position 7 and 8 of NP leads to a reduction of viral growth compared with wild-type virus (Ozawa et al., 2007).

The transport of vRNPs of influenza A virus into the nucleus is performed through the classical nuclear import pathway, the imp/ transport system shown in Fig. 5. As an adaptor, imp binds with the NLSs of viral proteins and then NLS-impbinds to the receptor on impthrough the IBB domain on imp(Cross et al., 2005; O'Neill et al., 1995). This NLS-bearing protein/receptor complex is imported into the nucleus. The imp family comprises six members in humans. Based on structural similarity, the imp family is grouped into three subfamilies, imp1/Rch1 (Rch1), imp3/Qip1 (Qip1), and imp5/NPI-1 (NPI-1) (Goldfarb et al., 2004). Interestingly, NP binds to several types of human impincluding Rch1, Qip1, and NPI-1, and regulates not only the nuclear transport of vRNPs but also host cell tropism and the growth of influenza virus (Gabriel et al., 2011). Therefore, NP functions as the main regulator of vRNPs trafficking and is a potentially useful target for the development of novel compounds that inhibit influenza A virus replication.

#### **6. Screening of anti-viral drugs for NP**

Recently, we demonstrated that NP is a novel target for the development of new antiviral drugs against the influenza virus using screening of NP-binding compounds by photocross-linked chemical arrays. Chemical arrays represent one of the most promising and high-throughput approaches for screening ligands against proteins of interest (Kanoh et al., 2006), and several successful results from chemical arrays have been reported (Koehler et al., 2003; Kuruvilla et al., 2002; Miyazaki et al., 2008). The screening protocol using chemical array was shown in Fig. 6. Approximately 25,000 small-molecules have been developed at RIKEN were fixed on a glass plate by photo-cross linker. To firstly identify inhibitors of NP, a large-scale chemical array approach of 6,800 compounds from an RIKEN NPDepo chemical library was used that detected specific interactions of small molecules with NP.

Using purified, recombinant influenza virus (A/WSN/33) NP, which was fused to monomeric red fluorescent protein (mRFP), we succeeded in detecting 72 compounds as positive. Next, plaque assay was used to investigate whether the 72 compounds inhibited multiplication of the influenza virus (A/WSN/33). Among them, 9 compounds showed inhibitory activity against influenza virus multiplication (Table 2). Furthermore, to obtain the compound which shows high inhibition activity, we searched for the derivatives of compounds from RIKEN NPDepo and found three derivatives of compound 1, which is

proteins of the vRNPs carry NLSs (Akkina et al., 1987; Jones et al., 1986; Nieto et al., 1994), the NP was shown to be sufficient to mediate the nuclear import of viral RNAs (O'Neill et al., 1995). As shown in Fig. 3, a more detailed analysis revealed the presence of three NLSs on the NP (Neumann et al., 1997; Wang et al., 1997), including an unconventional NLS at the very N-terminus, located between amino acids 3 and 13. A second NLS resides in the central part of the NP, located between amino acids 198 and 216. This bipartite signal appears to be weaker than the unconventional N-terminal NLS (Weber et al., 1998). Of these NLSs, the unconventional, N-terminal NLS of the NP is indispensable for the nuclear transport of NP and vRNPs (Cross et al., 2005; O'Neill et al., 1995). Alanine-substituted mutants of this unconventional NLS of the NP have shown that the amino acids at position 7 and 8 are critical for nuclear localization (Neumann et al., 1997). Furthermore, the mutation of amino acids at position 7 and 8 of NP leads to a reduction of viral growth compared with wild-type

The transport of vRNPs of influenza A virus into the nucleus is performed through the classical nuclear import pathway, the imp/ transport system shown in Fig. 5. As an adaptor, imp binds with the NLSs of viral proteins and then NLS-impbinds to the receptor on impthrough the IBB domain on imp(Cross et al., 2005; O'Neill et al., 1995). This NLS-bearing protein/receptor complex is imported into the nucleus. The imp family comprises six members in humans. Based on structural similarity, the imp family is grouped into three subfamilies, imp1/Rch1 (Rch1), imp3/Qip1 (Qip1), and imp5/NPI-1 (NPI-1) (Goldfarb et al., 2004). Interestingly, NP binds to several types of human impincluding Rch1, Qip1, and NPI-1, and regulates not only the nuclear transport of vRNPs but also host cell tropism and the growth of influenza virus (Gabriel et al., 2011). Therefore, NP functions as the main regulator of vRNPs trafficking and is a potentially useful target for the development of novel compounds that inhibit influenza A virus

Recently, we demonstrated that NP is a novel target for the development of new antiviral drugs against the influenza virus using screening of NP-binding compounds by photocross-linked chemical arrays. Chemical arrays represent one of the most promising and high-throughput approaches for screening ligands against proteins of interest (Kanoh et al., 2006), and several successful results from chemical arrays have been reported (Koehler et al., 2003; Kuruvilla et al., 2002; Miyazaki et al., 2008). The screening protocol using chemical array was shown in Fig. 6. Approximately 25,000 small-molecules have been developed at RIKEN were fixed on a glass plate by photo-cross linker. To firstly identify inhibitors of NP, a large-scale chemical array approach of 6,800 compounds from an RIKEN NPDepo chemical library was used that detected specific interactions of small molecules with NP.

Using purified, recombinant influenza virus (A/WSN/33) NP, which was fused to monomeric red fluorescent protein (mRFP), we succeeded in detecting 72 compounds as positive. Next, plaque assay was used to investigate whether the 72 compounds inhibited multiplication of the influenza virus (A/WSN/33). Among them, 9 compounds showed inhibitory activity against influenza virus multiplication (Table 2). Furthermore, to obtain the compound which shows high inhibition activity, we searched for the derivatives of compounds from RIKEN NPDepo and found three derivatives of compound 1, which is

virus (Ozawa et al., 2007).

replication.

**6. Screening of anti-viral drugs for NP** 

Approximately 2,000 compounds are spotted, with duplicates, on a glass plate using a photo-cross linker.


Fig. 6. Summary of compound screening using chemical array.

Table 2. Screening for NP inhibitors from the NPDepo RIKEN Natural Products Depository.

artificial analog of mycalamide. Among them, two derivatives showed lower inhibitory effect compared with compound 1, whereas compound 4 (Fig. 7, Table 2) showed strong activity reaching inhibition level up to 97% (Hagiwara et al., 2010).

Furthermore, surface plasmon resonance imaging experiments demonstrated that the binding activity of each compound to NP correlated with its antiviral activity. Finally, it was shown that these compounds bound NP within the N-terminal 110-amino acid region but their binding abilities were dramatically reduced when the N-terminal 13-amino acid tail was deleted, suggesting that the compounds might bind to this region, which mediates the nuclear transport of NP and its binding to viral RNA. These data suggest that compound binding to the N-terminal 13-amino acid tail region corresponding to an unconventional NLS may inhibit viral replication by inhibiting the nuclear transport of NP.

Discovery of Novel Antiviral Agents Directed Against the Influenza A Virus Nucleoprotein 111

As described earlier, we first experimentally identified NP as a valuable drug target for inhibiting the influenza virus (Hagiwara et al., 2010). Interestingly, two other compounds that inhibit the function of NP have been reported. One compound, called nucleozin, was randomly screened from a commercial chemical library. Nucleozin inhibits nuclear localization of NP by inducing aggregation of NP (Kao et al., 2010). Furthermore, some nucleozin derivatives have been developed (Gerritz et al., 2011; Su et al., 2010) and the cocrystal structures of NP and these nucleozin derivatives were also reported (Gerritz et al., 2011). The crystal structures indicated that two derivatives bound to the NP dimer (Fig. 8) and might induce the aggregation of NP, thereby inhibiting nuclear transport of NP and

Another compound was screened *in silico* and found to inhibit construction of the NP trimer. The compound interacts with E339-R416 salt bridge in the tail-loop binding pocket and thereby inhibits functional oligomerization of NP, which would in turn inhibit the

Using three different methods (direct binding, random screening, and *in silico* screening), compounds that inhibit the function of NP have been obtained. The results strongly suggest

Influenza A viruses are responsible for seasonal epidemics and high mortality pandemics. Influenza A virus has a segmented genome of eight negative-strand RNA segments, which are packaged into virions as RNPs. In addition to RNA, RNP contains the viral NP and the three subunits of the RNA-dependent RNA polymerase, PB1, PB2, and PA. The NP is expressed in the early stage of infection and plays important roles in numerous steps of viral replication. NP also is relatively well conserved compared with viral surface spike proteins. Using three different methods, direct binding, random screening, and *in silico* screening, several small molecules that interact with NP and inhibit virus multiplication were discovered. Since there are currently only two types of drugs available for the treatment of influenza virus infection, M2 inhibitors and NA inhibitors, the discovery of a novel mechanism of inhibition of influenza virus replication may supply the field of drug

This study was supported in part by a Japan Advanced Molecular Imaging Program (J-AMP), by a RIKEN Program for Drug Discovery and Medical Technology Platforms, and by

Air, G.M. & Laver, W.G. (1989). The neuraminidase of influenza virus. *Proteins*, Vol.6, No.4,

that NP is a good target for the development of an anti-influenza virus drug.

**7. Alternative anti-influenza virus compounds targeting NP** 

thereby influenza virus multiplication.

**8. Conclusions** 

multiplication of the influenza virus (Shen et al., 2011).

development with an effective new strategy.

the Chemical Biology Research Project (RIKEN).

pp. 341-356, ISSN 0887-3585

**9. Acknowledgments** 

**10. References** 

Compound 1, which is artificial analog of mycalamide was finally selected as the anti-influenza virus agent. Compound 4 was the derivative of compound 1 that showed the strongest activity in inhibiting influenza virus multiplication.

Fig. 7. Chemical structure of three derivatives of compound 1.

The positions of the compounds are highlighted in white and indicated by arrows. The compounds induce the aggregation of NP. This structure is based on the available structural information for NP (PDB code: 3RO5).

Fig. 8. Structure of influenza virus NP bound to compounds.

#### **7. Alternative anti-influenza virus compounds targeting NP**

As described earlier, we first experimentally identified NP as a valuable drug target for inhibiting the influenza virus (Hagiwara et al., 2010). Interestingly, two other compounds that inhibit the function of NP have been reported. One compound, called nucleozin, was randomly screened from a commercial chemical library. Nucleozin inhibits nuclear localization of NP by inducing aggregation of NP (Kao et al., 2010). Furthermore, some nucleozin derivatives have been developed (Gerritz et al., 2011; Su et al., 2010) and the cocrystal structures of NP and these nucleozin derivatives were also reported (Gerritz et al., 2011). The crystal structures indicated that two derivatives bound to the NP dimer (Fig. 8) and might induce the aggregation of NP, thereby inhibiting nuclear transport of NP and thereby influenza virus multiplication.

Another compound was screened *in silico* and found to inhibit construction of the NP trimer. The compound interacts with E339-R416 salt bridge in the tail-loop binding pocket and thereby inhibits functional oligomerization of NP, which would in turn inhibit the multiplication of the influenza virus (Shen et al., 2011).

Using three different methods (direct binding, random screening, and *in silico* screening), compounds that inhibit the function of NP have been obtained. The results strongly suggest that NP is a good target for the development of an anti-influenza virus drug.

#### **8. Conclusions**

110 Antiviral Drugs – Aspects of Clinical Use and Recent Advances

Compound 1, which is artificial analog of mycalamide was finally selected as the anti-influenza virus agent. Compound 4 was the derivative of compound 1 that showed the strongest activity in inhibiting

Fig. 7. Chemical structure of three derivatives of compound 1.

The positions of the compounds are highlighted in white and indicated by arrows.

Fig. 8. Structure of influenza virus NP bound to compounds.

The compounds induce the aggregation of NP. This structure is based on the available structural

influenza virus multiplication.

information for NP (PDB code: 3RO5).

Influenza A viruses are responsible for seasonal epidemics and high mortality pandemics. Influenza A virus has a segmented genome of eight negative-strand RNA segments, which are packaged into virions as RNPs. In addition to RNA, RNP contains the viral NP and the three subunits of the RNA-dependent RNA polymerase, PB1, PB2, and PA. The NP is expressed in the early stage of infection and plays important roles in numerous steps of viral replication. NP also is relatively well conserved compared with viral surface spike proteins. Using three different methods, direct binding, random screening, and *in silico* screening, several small molecules that interact with NP and inhibit virus multiplication were discovered. Since there are currently only two types of drugs available for the treatment of influenza virus infection, M2 inhibitors and NA inhibitors, the discovery of a novel mechanism of inhibition of influenza virus replication may supply the field of drug development with an effective new strategy.

#### **9. Acknowledgments**

This study was supported in part by a Japan Advanced Molecular Imaging Program (J-AMP), by a RIKEN Program for Drug Discovery and Medical Technology Platforms, and by the Chemical Biology Research Project (RIKEN).

#### **10. References**

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**7** 

*Portugal* 

**Targeting Norovirus: Strategies for the** 

Gastroenteritis is globally responsible for great morbidity and mortality among all ages. In the developing countries, it still represents one of the top causes of death for children <5 years of age, resulting in 1,8 million fatalities every year (Boschi-Pinto et al., 2008; Bryce et al., 2005). Viruses are responsible for the majority of cases of gastroenteritis with rotaviruses

Noroviruses are today recognized as the leading cause of foodborne outbreaks and sporadic cases of gastroenteritis worldwide (Glass et al., 2009; Patel et al., 2009). Nowadays, they are even considered the second most important agent of severe childhood diarrhea after rotavirus (Koopmans, 2008; Patel et al., 2008; Ramani & Kang, 2009) but the importance of norovirus in this age group is expected to increase in the upcoming years, as a consequence

The recognition of the clinical importance of norovirus only began in the late 1990s when sensitive routine diagnostic methods became available. In fact, the first norovirus (the *Norwalk* virus) was discovered in 1972 (Kapikian et al., 1972) but more than twenty years were necessary to disclose the important role of these viruses as human pathogens. Noroviruses are now on the upswing and a fundamental question is being raised: are

Despite the increasing attention given to norovirus today and the significant morbidity and mortality associated with norovirus gastroenteritis, no specific antiviral drugs or vaccines are yet available for treatment or prevention of norovirus illness. Only recently, a recombinant intranasal vaccine has entered a phase I clinical trial (El-Kamary et al., 2010; Vinje, 2010)

This chapter will highlight the importance of finding specific antiviral therapy for norovirus infection and explore biological features of norovirus, pointing out directions to stop or

Norovirus gastroenteritis is generally acute and self-limited, but in infants, elderly, and immunocompromised individuals it may be more severe and prolonged since they are more

of the implementation of routine rotavirus vaccination (Koo et al., 2010).

**1. Introduction**

and noroviruses being the major pathogens.

norovirus really emerging? (Widdowson et al., 2005)

**1.1 Clinical disease, transmission and epidemiology** 

control this important human pathogen.

**Discovery of New Antiviral Drugs** 

Joana Rocha-Pereira and Maria São José Nascimento *Laboratório de Microbiologia, Departamento de Ciências Biológicas,* 

*Faculdade de Farmácia, Universidade do Porto, Porto,* 

