**31. Epidemiology of CCR5∆32 gene**

250 Immunodeficiency

B HIV strains in England.

**30. CCR5 gene mutation** 

individuals infected with subtypes D and C is faster than in those infected with subtypes A and A/G in Africa and that subtype D infection leads to faster rates of CD4 cell decline and subsequent virological failure compared to infection with subtype B and other non-subtype

A disproportionate transmission and distribution of HIV epidemic in the world has been observed with alarming rates in Sub Saharan Africa (SSA). Currently there is very little explanation for this observation of differences in susceptibility to HIV infection which could among other factors be attributable to variation in host genetics. Host genetic factors, including some polymorphisms in chemokine receptors and chemokine genes have been identified as having an impact on both HIV-1 infection and disease progression to death. Scientists have observed that despite multiple or repeated unprotected sexual exposures to HIV-1, some individuals remain HIV sero-negative. More so the discovery of long-term nonprogressors (LTNP) in HIV infection prompted further investigations to ascertain the role of host genetic factors in the progression of HIV infection to AIDS. Polymorphisms in these

Analysis of blood samples from infected persons and the repeatedly exposed but somehow uninfected have shown specific molecular differences within the coding and regulatory regions of chemokine receptors and coreceptors genes. There is a deletion of 32 base pairs from the coding region of CCR5 gene in the second ECL. In these individuals the 32-base deletion in the CCR5 gene results in a frame shift and truncation of the normal CCR5 protein which renders them uninfectable after exposure to CCR5 tropic HIV viruses. This aberrant protein has been associated with protection against HIV-1 infection in people who are homozygous for mutant genotypes. Thus individuals resistant to HIV infection inherit two mutated copies of the dysfunctional gene for CCR5 from either parent. Without functional coreceptors, HIV is not able to enter immune cells. Despite the strong protective effect conferred by congenital CCR5 deficiencies, a handful of infected *CCR5-Δ32* homozygotes have been reported, all invariably harboring CXCR4- dependent HIV-1 strains. The rare homozygous individuals that got infected by HIV have been shown to be through CXCR4 coreceptor mediated entry only. Interestingly it has been found that in such homozygous conditions there is no evidence of health/phenotypic impairment caused by the absence of functional CCR5 coreceptors. However, HIV-infected individuals who would have inherited a copy of the defective CCR5 gene from only one parent, heterozygotes for a Δ32 deletion (CCR5-wt/Δ32) are not protected against HIV-1 infection but are associated with a much slow progression to AIDS relative to those with two normal copies of the gene. Heterozygous individuals have lower plasma HIV RNA levels in the early years of infection, which gives them a medical advantage of delaying disease progression. Since these mutations do not account for all cases of resistance to HIV infection, scientists are

**29. Host coreceptor genes & insights on resistance to HIV infection** 

human chemokine receptor genes will therefore affect the evolution of HIV-1.

A general North to South downhill gradient in CCR5∆32 gene frequency has been observed. The highest frequency has been found in Northern and North-eastern Europe especially amongst the Finnish and Swedish populations. Data confirm the high frequency of CCR5- Delta 32 among northern European Caucasians, a gene frequency declining across Europe and Asia reflecting recent population admixture. The virtual absence of CCR5-Delta 32 deletion among native Africans, East Asians, and American-Indians is suggestive that the mutation arose in northern Europe in response to selective pressures due to other factors including infection epidemics. The CCR5∆32 has a prevalence rate of between 20-35% among the Jewish population. In Africa studies indicated that the allelic frequency of CCR5∆32 mutation is 0.1% in the black South African population.

#### **32. CCR2 –V641 mutation**

A CCR2 –V641 mutation from a conservative valine to isoleucine substitution in the transmembrane region has also been associated with some protective effects against HIV infection and reduction in progression of HIV to AIDS. The protective effect of CCR2 –V641 is believed is to be through regulation linkage disequilibrium in the regulatory region or promoter region of this gene. CCR2-V64I is common among Africans, yet this race is one of the most affected populations by the HIV pandemic. Hence, the protective effect of CCR2 – V641 against HIV infection remains controversial. Despite the availability of evidence linking some host genetic factors to protection against HIV infection, very little information is available regarding the role of CCR5-∆32 and CCR2-V64I polymorphism on HIV transmission.

#### **33. Coreceptor antagonists and antiretroviral therapy**

To successfully infect an immune cell HIV env gp 120 has to interact with the host cellular receptor CD4 and either a CCR5 or CXCR4. Both coreceptors are recognized as novel targets for anti-HIV-therapy consequently resulting in the development of a new class of antiretroviral drugs called coreceptor antagonists or blockers. Blocking these coreceptors would protect the host cell from viral entry and would reduce the viral transmission and pathogenesis. Interestingly coreceptor antagonists differ from the other antiretroviral agents that target HIV proteins in the sense that they bind and inhibit receptors encoded by the

host itself. Besides blocking replication of the virus in other cells, coreceptor antagonists may reduce the decline of immune system through other ways. It is known that HIV-1 env glycoproteins may trigger autophagy in uninfected CD4 positive T cells, leading to their apoptosis, and consequently increasing immune decline. These cells need to present, among other things, a CXCR4 coreceptor in their surface. The blockage of the interaction between glycoproteins and the coreceptor of uninfected cells could help to avoid immune impairment. CCR5 antagonists design and development are a step ahead relative to their CXCR4 counterparts. This unique new class of drugs not only increases the alternative therapeutic options for HIV treatment but also maximizes potency, minimizes toxicity and reduces the risk of drug resistance development. Range of coreceptor antagonists discovered to date includes modified chemokines monoclonal antibodies, peptides and small organic molecules.

Coreceptor Usage in HIV Infection 253

chemokine receptors. Although chemokines or derivative-molecules could be exploited as therapeutic agents against HIV, the risk of inducing inflammatory side-effects or of interfering with the physiology of the homeostatic chemokine system represents a potential limitation.

Another type of coreceptor antagonists are monoclonal antibodies. Examples of such agents are CCR5mAb004 of Human Genome Sciences (Rockville, MD, USA) and PRO 140 of Progenics Pharmaceuticals (Tarrytown, NY, USA). They are both mouse-derived but humanized monoclonal antibodies capable of blocking CCR5 without activating the signal transduction pathways. In phase I clinical trials CCR5mAb004 has been shown to be safe and well tolerated.

AMD3100 is a small-molecule CXCR4 antagonist with activity against X4 viruses' in vitro and antiviral activity in vivo, although it is no longer being pursued clinically because of pharmacology and toxicology considerations. More examples of small molecule chemokine receptor antagonists of CXCR4 coreceptor include AMD11070/ AMD070, AMD3465, ALX40- 4C, T22, T134 and T140. AMD11070 is known to specifically inhibit CXCR4 in a reversible way. Other CXCR4 selective inhibitor is AMD3100 from AnorMED. AMD3100 is highly specific for CXCR4 that blocks replication of X4 strains. However, clinical trials of AMD3100 have been stopped due to side effects of cardiotoxicity. A relatively safer derivative of AMD3100 known as AMD070 is currently undergoing clinical trials. Previously AMD3100 has proved to be a useful tool to probe interactions between env proteins and CXCR4 which is important to identify pathways by which HIV-1 may become resistant to this class of antiviral agents. To date none of CXCR4 blockers has successfully proceeded into clinical practice due to their severe side effects. Generally, CXCR4-based blocking agents are less attractive due to the crucial role of CXCR4 in many biological processes. However, agents that aim at downmodulating CXCR4 expression may provide some benefits to HIV-positive patients. Antagonism of CXCR4 has been shown to significantly improve survival from lethal infection through enhanced intraparenchymal migration of West Nile Virus-specific CD8+ T cells within the brain, leading to reduced viral loads and decreased immunopathology at affected sites.

The CCR5 antagonists act on a wide spectrum of viruses with affinity or tropism for this receptor. They are absorbed orally and have powerful antiviral activity through interaction with amino acids from the pocket formed by the transmembrane (TM) domains of CCR5. Mechanism of action is through allosteric effects, altering extracellular CCR5 conformation after binding to a hydrophobic pocket of CCR5, formed by transmembrane helices. Mutations within transmembrane domains are associated with impaired activity. Small molecule CCR5 antagonists such as maraviroc, vicriviroc, aplaviroc, TAK-779, and TAK-220) have been found to fit in the same binding pocket. Other small-molecule CCR5 antagonists include:

**36. Monoclonal antibodies as coreceptor antagonists** 

**37. Small molecule CXR4 chemokine receptor antagonists** 

**38. Small-molecule CCR5 antagonists** 

#### **34. Mechanism of action of coreceptor antagonists**

Coreceptor antagonists' antiviral properties are related to their ability to internalize the receptor, preventing it from being expressed on the cell surface thereby avoiding viral replication by reducing coreceptors availability to bind to HIV glycoproteins.

**Figure 4.** Mode of Action of Coreceptor antagonists

#### **35. Coreceptor antagonists in development**

#### **35.1. Modified chemokines**

Modified chemokines include RANTES, a natural chemokine ligand for CCR5 manufactured by RANTES engineering technology including other novel agents that have not been very successful and their future remains uncertain. Other possible antagonists are zinc finger nucleases that cause mutagenesis in the CCR5 gene by binding and cleaving the gene, producing abnormal proteins. Zinc fingers, along with RNA interference, ribozymes, intrakines, and intrabodies are HIV-1 vector-delivered genetic disruption mechanisms that target HIV-1 chemokine receptors. Although chemokines or derivative-molecules could be exploited as therapeutic agents against HIV, the risk of inducing inflammatory side-effects or of interfering with the physiology of the homeostatic chemokine system represents a potential limitation.

#### **36. Monoclonal antibodies as coreceptor antagonists**

252 Immunodeficiency

molecules.

host itself. Besides blocking replication of the virus in other cells, coreceptor antagonists may reduce the decline of immune system through other ways. It is known that HIV-1 env glycoproteins may trigger autophagy in uninfected CD4 positive T cells, leading to their apoptosis, and consequently increasing immune decline. These cells need to present, among other things, a CXCR4 coreceptor in their surface. The blockage of the interaction between glycoproteins and the coreceptor of uninfected cells could help to avoid immune impairment. CCR5 antagonists design and development are a step ahead relative to their CXCR4 counterparts. This unique new class of drugs not only increases the alternative therapeutic options for HIV treatment but also maximizes potency, minimizes toxicity and reduces the risk of drug resistance development. Range of coreceptor antagonists discovered to date includes modified chemokines monoclonal antibodies, peptides and small organic

Coreceptor antagonists' antiviral properties are related to their ability to internalize the receptor, preventing it from being expressed on the cell surface thereby avoiding viral

Modified chemokines include RANTES, a natural chemokine ligand for CCR5 manufactured by RANTES engineering technology including other novel agents that have not been very successful and their future remains uncertain. Other possible antagonists are zinc finger nucleases that cause mutagenesis in the CCR5 gene by binding and cleaving the gene, producing abnormal proteins. Zinc fingers, along with RNA interference, ribozymes, intrakines, and intrabodies are HIV-1 vector-delivered genetic disruption mechanisms that target HIV-1

replication by reducing coreceptors availability to bind to HIV glycoproteins.

**34. Mechanism of action of coreceptor antagonists** 

**Figure 4.** Mode of Action of Coreceptor antagonists

**35.1. Modified chemokines** 

**35. Coreceptor antagonists in development** 

Another type of coreceptor antagonists are monoclonal antibodies. Examples of such agents are CCR5mAb004 of Human Genome Sciences (Rockville, MD, USA) and PRO 140 of Progenics Pharmaceuticals (Tarrytown, NY, USA). They are both mouse-derived but humanized monoclonal antibodies capable of blocking CCR5 without activating the signal transduction pathways. In phase I clinical trials CCR5mAb004 has been shown to be safe and well tolerated.

#### **37. Small molecule CXR4 chemokine receptor antagonists**

AMD3100 is a small-molecule CXCR4 antagonist with activity against X4 viruses' in vitro and antiviral activity in vivo, although it is no longer being pursued clinically because of pharmacology and toxicology considerations. More examples of small molecule chemokine receptor antagonists of CXCR4 coreceptor include AMD11070/ AMD070, AMD3465, ALX40- 4C, T22, T134 and T140. AMD11070 is known to specifically inhibit CXCR4 in a reversible way. Other CXCR4 selective inhibitor is AMD3100 from AnorMED. AMD3100 is highly specific for CXCR4 that blocks replication of X4 strains. However, clinical trials of AMD3100 have been stopped due to side effects of cardiotoxicity. A relatively safer derivative of AMD3100 known as AMD070 is currently undergoing clinical trials. Previously AMD3100 has proved to be a useful tool to probe interactions between env proteins and CXCR4 which is important to identify pathways by which HIV-1 may become resistant to this class of antiviral agents. To date none of CXCR4 blockers has successfully proceeded into clinical practice due to their severe side effects. Generally, CXCR4-based blocking agents are less attractive due to the crucial role of CXCR4 in many biological processes. However, agents that aim at downmodulating CXCR4 expression may provide some benefits to HIV-positive patients. Antagonism of CXCR4 has been shown to significantly improve survival from lethal infection through enhanced intraparenchymal migration of West Nile Virus-specific CD8+ T cells within the brain, leading to reduced viral loads and decreased immunopathology at affected sites.

#### **38. Small-molecule CCR5 antagonists**

The CCR5 antagonists act on a wide spectrum of viruses with affinity or tropism for this receptor. They are absorbed orally and have powerful antiviral activity through interaction with amino acids from the pocket formed by the transmembrane (TM) domains of CCR5. Mechanism of action is through allosteric effects, altering extracellular CCR5 conformation after binding to a hydrophobic pocket of CCR5, formed by transmembrane helices. Mutations within transmembrane domains are associated with impaired activity. Small molecule CCR5 antagonists such as maraviroc, vicriviroc, aplaviroc, TAK-779, and TAK-220) have been found to fit in the same binding pocket. Other small-molecule CCR5 antagonists include: Tak-652, spirodiketopiperazine derivatives such as E913 and SCH-C/SCH-351125. Spirodiketopiperazine derivatives have been associated with severe hepatotoxicity, leading to the cancellation of their development. SCH351125 or SCH-C was the first CCR5 antagonist to enter clinical efficacy studies but has been dropped out due to variable antiviral effect and provocation of prolonged QTc interval. To counter these setbacks Vicriviroc was developed which was much more potent than SCH-C, safer and better oral bioavailability.

Coreceptor Usage in HIV Infection 255

headache, asthenia, dizziness, gingivitis and nausea. It is known that before starting on a coreceptor antagonist, patients need to determine their viral tropism. The introduction of the CCR5 antagonist, maraviroc for HIV-1 therapy has increased the research interest in the epidemiology of tropism and its relationship with HIV-1 subtype. A greater understanding of the tropism of non-B subtypes is key for the optimal use of CCR5 antagonists in the treatment of these infections in the developing world and HIV-1 prevention strategies. There is no food effect as it can be taken with or without food and can be stored at room temperature. Hence it is very suitable for resource poor settings which also bear the brunt of

The CCR5-to-CXCR4 switch represents a concern because while CCR5 inhibitors suppress R5 viruses they may allow the emergence of CXCR4-tropic viruses. Efforts to develop HIV entry inhibitors are hampered by problems associated with rapid evolution of the virus, leading to drug resistance. Blocking only one of the pathways for HIV entry into cells has resulted in the opening of the other pathways potentially accelerating disease progression by promoting the evolution of more virulent CXCR4-dependent variants. In this view, AnorMED Company has announced the discovery and development of a joint CXCR4/CCR5

There have been speculations that new, safe and effective chemokine receptor inhibitors should facilitate CCR5 and CXCR4 internalization independent of the cellular signaling. The most anticipated eventual combination of CXCR4 and CCR5 inhibitors would be beneficial,

antagonist although little is known about the binding mechanism at this time.

the HIV scourge with predominant R5 variants.

**Figure 6.** Mode of action of maraviroc, adopted from Qian K, 2008.

**40. Challenges of CCR5 antagonists** 

**41. Future of coreceptor antagonists** 

Two agents, maraviroc and vicriviroc, are the agents within this family that have passed the final stages of clinical development. These agents have their scientific names ending in "– viroc", to denote their action of "viral receptor occupancy". Since these drugs show activity against R5 viruses only, viral tropism testing should be made before their prescription and eventually during treatment in order to exclude the presence of X4 viruses. They are less expensive to produce and have good oral bioavailability. Their half maximal inhibitory concentrations (IC50) are in the nanomolar ranges.
