Since the identification of CCR5 and CXCR4 as HIV coreceptors a little over a decade ago, there has been hope that coreceptor inhibitors will be able to make an impact on the HIV epidemic, both as novel therapeutic drugs and as agents used in prevention. Significant progress has been made in the understanding of how coreceptor choice might impact HIV pathology and how coreceptor blockade may affect health. In this review, we focus on some of the key issues that are emerging now that CCR5 has been validated as a promising target for HIV prevention strategies and at a time when a CCR5 inhibitor has been approved in the United States as the first in a new class of anti-HIV therapeutic drugs.

Since the identification of CCR5 and CXCR4 as HIV coreceptors a little over a decade ago, there has been hope that coreceptor inhibitors will be able to make an impact on the HIV epidemic, both as novel therapeutic drugs and as agents used in prevention. Significant progress has been made in the understanding of how coreceptor choice might impact HIV pathology and how coreceptor blockade may affect health. In this review, we focus on some of the key issues that are emerging now that CCR5 has been validated as a promising target for HIV prevention strategies and at a time when a CCR5 inhibitor has been approved in the United States as the first in a new class of anti-HIV therapeutic drugs.

Several CCR5 ligands, including small molecules and monoclonal antibodies (MAbs), are being developed as therapies for infection with strains of human immunodeficiency virus type 1 (HIV-1) that use CCR5 for entry (R5 viruses). The efficacy of such therapies could be influenced by inter-individual differences in host factors, such as CCR5 expression levels. To study this, we used peripheral blood mononuclear cells (PBMCs) from humans and rhesus macaques. The half-maximal inhibitory concentrations (IC50) of the small-molecule CCR5 ligands CMPD167, UK427,857 and SCH-D, and of the PRO 140 MAb, differ by > 2 logs in a donor-dependent manner. We studied this variation by using flow cytometry to measure CCR5 expression on PBMCs from six of the human donors: the IC50 values of both SCH-D and PRO 140 correlated with CCR5 expression (R2 = 0.64 and 0.99, respectively). We also determined the efficacy of the CCR5 ligands against HIV-1 infection of HeLa-derived cell lines that express CD4 at the same level but vary 2-fold in CCR5 expression (JC.48 and JC.53 cells). The moderately greater CCR5 expression on the JC.53 than the JC.48 cells was associated with proportionately higher median IC50 values for all four CCR5 ligands but not for a soluble CD4-based inhibitor or a non-nucleoside reverse transcriptase inhibitor. We conclude that differences in CCR5 expression on human PBMCs, which can be affected by CCL3L1 gene dose, may influence the antiviral potency of CCR5 ligands in vitro, but other host factors are also likely to be involved. These host factors may affect the clinical activity of CCR5 inhibitors, including their use as topical microbicides to prevent HIV-1 transmission.

Human immunodeficiency virus type 1 (HIV-1) infection can be inhibited by small molecules that target the CCR5 coreceptor. Here, we describe some properties of clonal viruses resistant to one such inhibitor, SCH-D, using both chimeric, infectious molecular clones and Env-pseudotypes. Studies using combinations of CCR5 ligands, including small molecule inhibitors, monoclonal antibodies (MAbs) and chemokine derivatives such as PSC-RANTES, show that the fully SCH-D-resistant viruses enter target cells by using the SCH-D-bound form of CCR5. However, the way resistance to SCH-D and other small molecule CCR5 inhibitors is manifested depends on the target cell and the nature of the assay (single- vs. multi-cycle). In multi-cycle assays using primary lymphocytes, SCH-D does not inhibit resistant molecular clones, and it can even enhance their infectivity modestly. In contrast, the same viruses (as Env-pseudotypes) are significantly inhibited by SCH-D in single-cycle entry assays using U87-CD4/CCR5 cells, resistance being manifested by incomplete inhibition at high SCH-D concentrations. When a single-cycle, Env-pseudotype entry assay was performed using either U87-CD4/CCR5 cells or PBMC under comparable conditions, entry was inhibited by up to 88% in the former cells but by only 28% in the PBMC. Hence, there are both cell- and assay-dependent influences on how resistance is manifested. We also take this opportunity to correct our previous report that SCH-D-resistant isolates are also substantially cross-resistant to PSC-RANTES [Marozsan, A.J., Kuhmann, S.E., Morgan, T., Herrera, C., Rivera-Troche, E., Xu, S., Baroudy, B.M., Strizki, J., Moore, J.P., 2005. Generation and properties of a human immunodeficiency virus type 1 isolate resistant to the small molecule CCR5 inhibitor, SCH-417690 (SCH-D). Virology 338 (1), 182-199]. A substantial element of this resistance was attributable to the unappreciated carry-over of SCH-D from the selection cultures into analytical assays.

Fitness is a parameter used to quantify how well an organism adapts to its environment; in the present study, fitness is a measure of how well strains of human immunodeficiency virus type 1 (HIV-1) replicate in tissue culture. When HIV-1 develops resistance in vitro or in vivo to antiretroviral drugs such as reverse transcriptase or protease inhibitors, its fitness is often impaired. Here, we have investigated whether the development of resistance in vitro to a small molecule CCR5 inhibitor, AD101, has an associated fitness cost. To do this, we developed a growth-competition assay involving dual infections with molecularly cloned viruses that are essentially isogenic outside the env genes under study. Real-time TaqMan quantitative PCR (QPCR) was used to quantify each competing virus individually via probes specific to different, phenotypically silent target sequences engineered within their vif genes. Head-to-head competition assays of env clones derived from the AD101 escape mutant isolate, the inhibitor-sensitive parental virus, and a passage control virus showed that AD101 resistance was not associated with a fitness loss. This observation is consistent with the retention of the resistant phenotype when the escape mutant was cultured for a total of 20 passages in the absence of the selecting compound. Amino acid substitutions in the V3 region of gp120 that confer complete AD101 resistance cause a fitness loss when introduced into an AD101-sensitive, parental clone; however, in the resistant isolate, changes elsewhere in env that occurred prior to the substitutions within V3 appear to compensate for the adverse effect of the V3 changes on replicative capacity. These in vitro studies may have implications for the development and management of resistance to other CCR5 inhibitors that are being evaluated clinically for the treatment of HIV-1 infection.

The CC-chemokine receptor 5 (CCR5) is the major coreceptor for macrophage-tropic (R5) HIV-1 strains. Several small molecule inhibitors of CCR5 that block chemokine binding and HIV-1 entry are being evaluated as drug candidates. Here we define how CCR5 antagonists TAK-779, AD101 (SCH-350581) and SCH-C (SCH-351125), which inhibit HIV-1 entry, interact with CCR5. Using a mutagenesis approach in combination with a viral entry assay to provide a direct functional read out, we tested predictions based on a homology model of CCR5 and analyzed the functions of more than 30 amino acid residues. We find that a key set of aromatic and aliphatic residues serves as a hydrophobic core for the ligand binding pocket, while E283 is critical for high affinity interaction, most likely by acting as the counterion for a positively charged nitrogen atom common to all three inhibitors. These results provide a structural basis for understanding how specific antagonists interact with CCR5, and may be useful for the rational design of new, improved CCR5 ligands.

We describe the generation of two genetically related human immunodeficiency virus type 1 (HIV-1) isolates highly (>20,000-fold) resistant to the small molecule CCR5 inhibitor, SCH-417690 (formerly SCH-D). Both viruses were cross-resistant to other small molecules targeting entry via CCR5, but they were inhibited by some MAbs against the same coreceptor on primary CD4+ T-cells. The resistant isolates remained sensitive to inhibitors of other stages of virus entry, and to replication inhibitors acting post-entry. Neither escape mutant could replicate detectably in peripheral blood mononuclear cells (PBMC) from two donors homozygous for the CCR5-Δ32 allele and both were insensitive to the CXCR4-specific inhibitor, AMD3100. Hence, the SCH-D escape mutants retained the R5 phenotype. One of the resistant isolates was, however, capable of replication in U87.CD4.CXCR4 cells and, after expansion in those cells, was sensitive to AMD3100 in primary CD4+ T-cells. Hence, some X4 variants may be present in this escape mutant swarm. A notable observation was that the SCH-D escape mutants were also cross-resistant to PSC-RANTES and AOP-RANTES, chemokine derivatives that are reported to down-regulate cell surface CCR5 almost completely. However, the extent to which CCR5 is down-regulated was dependent upon the detection MAb. Hence, the escape mutants may be using a CCR5 configuration that is only detected by some anti-CCR5 MAbs. Finally, two SCH-D-resistant clonal viruses revealed no amino acid changes in the gp120 V3 region relative to the parental viruses, in marked contrast to clones resistant to the AD101 small molecule CCR5 inhibitor that possess 4 such sequence changes. Several sequence changes elsewhere in gp120 (V2, C3 and V4) were present in the SCH-D-resistant clones. Their influence on the resistant phenotype remains to be determined.

Replicative fitness of an HIV-1 isolate generally tracks to the region of the genome that is under the most selective pressure. During HIV-1 infection, significant selection pressure is applied by the immune system causing sequence changes in the env gene. The clinical use of entry inhibitors will add to this pressure. Drug selection pressure can often force the virus to compromise its fitness in favor of acquiring resistance. This loss of fitness may lead to lower viral loads in vivo despite high levels of drug resistance. If the virus has already undergone compromises to survive in the face of the immune response, the additional changes in env may cause a significant reduction in replicative capacity. This presentation will focus on in vitro evaluation of the replicative fitness of HIV-1 isolates resistant to entry inhibitors and possible implications of such findings. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

We have generated HIV-1 isolates that are resistant to several small molecule CCR5 inhibitors by serial passage in vitro. We have investigated the molecular basis of this resistance through genetic and phenotypic studies. We find that different genetic pathways leading to small molecule inhibitor resistance can be taken by the same starting virus. Additionally, these viruses can exhibit different levels of cross resistance to RANTES derivatives. These studies provide insight into potential mechanisms of CCR5 inhibitor resistance. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

BMS-378806 is a newly described small-molecule inhibitor of human immunodeficiency virus type 1 (HIV-1) replication. It acts by binding to the gp120 surface glycoprotein of the virus within, or very close to, the pocket normally occupied by the CD4 antigen, the primary cell-surface receptor protein of HIV-1. Consequently, BMS-378806 inhibits gp120–CD4 binding and the subsequent events of virus–cell attachment and fusion. Thus, BMS-378806 or a derivative could be highly suitable to treat HIV-1 infection, and perhaps also to prevent infection when used in combination with other inhibitors of HIV-1 entry.

Primary strains of human immunodeficiency virus type 1 (HIV-1) are known to adapt to replication in cell lines in vitro by becoming sensitive to soluble CD4 (sCD4) and neutralizing antibodies (NAb). T-cell lines favor isolation of variants that use CXCR4 as a co-receptor, while primary isolates predominantly use CCR5. We have now studied how a primary R5 isolate, CC1/85, adapts to prolonged replication in primary human peripheral blood mononuclear cells (PBMC). After 19 passages, a variant virus, CCcon.19, had increased sensitivity to both sCD4 and NAb b12 that binds to a CD4-binding site (CD4BS)-associated epitope, but decreased sensitivity to anti-CD4 antibodies. CCcon.19 retains the R5 phenotype, its resistance to other NAbs was unaltered, its sensitivity to various entry inhibitors was unchanged, and its ability to replicate in macrophages was modestly increased. We define CCcon.19 as a primary T-cell adapted (PTCA) variant. Genetic sequence analysis combined with mutagenesis studies on clonal, chimeric viruses derived from CC1/85 and the PTCA variant showed that the most important changes were in the V1/V2 loop structure, one of them involving the loss of an N-linked glycosylation site. Monomeric gp120 proteins expressed from CC1/85 and the PTCA variant did not differ in their affinities for sCD4, suggesting that the structural consequences of the sequence changes were manifested at the level of the native, trimeric Env complex. Overall, the adaptation process probably involves selection for variants with higher CD4 affinity and hence greater fusion efficiency, but this also involves the loss of some resistance to neutralization by agents directed at or near to the CD4BS. The loss of neutralization resistance is of no relevance under in vitro conditions, but NAbs would presumably be a counter-selection pressure against such adaptive changes in vivo, at least when the humoral immune response is intact.

Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the consecutive interaction of the envelope glycoprotein gp120 with CD4 and a coreceptor such as CCR5 or CXCR4. The CCR5 coreceptor is used by the most commonly transmitted HIV-1 strains that often persist throughout the course of infection. Compounds targeting CCR5-mediated entry are a novel class of drugs being developed to treat HIV-1 infection. In this study, we have identified the mechanism of action of two inhibitors of CCR5 function, SCH-350581 (AD101) and SCH-351125 (SCH-C). AD101 is more potent than SCH-C at inhibiting HIV-1 replication in primary lymphocytes, as well as viral entry and gp120 binding to cell lines. Both molecules also block the binding of several anti-CCR5 monoclonal antibodies that recognize epitopes in the second extracellular loop of CCR5. Alanine mutagenesis of the transmembrane domain of CCR5 suggests that AD101 and SCH-C bind to overlapping but nonidentical sites within a putative ligand-binding cavity formed by transmembrane helices 1, 2, 3, and 7. We propose that the binding of small molecules to the transmembrane domain of CCR5 may disrupt the conformation of its extracellular domain, thereby inhibiting ligand binding to CCR5.

ABSTRACT In virus-infected cells, the envelope glycoprotein (Env) precursor, gp160, of human immunodeficiency virus type 1 is cleaved by cellular proteases into a fusion-competent gp120-gp41 heterodimer in which the two subunits are noncovalently associated. However, cleavage can be inefficient when recombinant Env is expressed at high levels, either as a full-length gp160 or as a soluble gp140 truncated immediately N-terminal to the transmembrane domain. We have explored several methods for obtaining fully cleaved Env for use as a vaccine antigen. We tested whether purified Env could be enzymatically digested with purified protease in vitro. Plasmin efficiently cleaved the Env precursor but also cut at a second site in gp120, most probably the V3 loop. In contrast, a soluble form of furin was specific for the gp120-gp41 cleavage site but cleaved inefficiently. Coexpression of Env with the full-length or soluble form of furin enhanced Env cleavage but also reduced Env expression. When the Env cleavage site (REKR) was mutated in order to see if its use by cellular proteases could be enhanced, several mutants were found to be processed more efficiently than the wild-type protein. The optimal cleavage site sequences were RRRRRR, RRRRKR, and RRRKKR. These mutations did not significantly alter the capacity of the Env protein to mediate fusion, so they have not radically perturbed Env structure. Furthermore, unlike that of wild-type Env, expression of the cleavage site mutants was not significantly reduced by furin coexpression. Coexpression of Env cleavage site mutants and furin is therefore a useful method for obtaining high-level expression of processed Env.