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Anti-HIV Therapeutic Research

Lab Head

Robert Redfield, MD
Associate Director and Professor

Associated Faculty

Olga Latinovic, PhD
Assistant Professor

Alonso Heredia, PhD
Assistant Professor

Research Summary

Focus of our work is the CCR5 chemokine receptor that plays a crucial role in HIV-1 infection and as such offers an important potential therapeutic target [Fig.1]. We have established methods to quantify CCR5 density and to evaluate its impact on virus infectivity in spreading/single cycle infection and direct virus-cell fusion assays. Using low doses of the drug Rapamycin, a CCR5 suppressor, we demonstrate decreased R5 HIV-1 infectivity and enhanced potency of entry inhibitors. Our data show that Rapamycin reduction of CCR5 density restores sensitivity of drug-resistant R5 HIV-1 to fusion inhibitor Enfuvirtide,T-20, (Antimicrobial Reagents and Chemotherapy, 2007) and to the new generation of entry inhibitors-CCR5 antagonists (Antiviral Research, 2009; Clinical Medicine: Therapeutics, 2009).

We found that Rapamycin induced reduction of CCR5 density in lymphocytes increased sensitivity to Vicriviroc (VCV) in VCV-resistant strains, inhibiting production by ~ 90% (PNAS, 2008). Novel Beta-lactamase (BlaM) entry assay revealed the differences in the activity between CCR5 antagonist sensitive and CCR5 antagonist resistant virus. In the case of CCR5 antagonist sensitive virus, we observed complete inhibition in cell lines with high and physiological CCR5 levels. In the case of the resistant virus, there is no inhibition in higher CCR5 expressive cells, but in cells with physiological expression of CCR5, we do observe susceptibility to CCR5 antagonist resistant virus.

As an alternative anti-viral therapy approach, we currently employ the CCR5 antibodies in order to determine the affinity of resistant virus Envelope in cases when it is free versus occupied CCR5 site by the CCR5 antagonists. Looking for the mechanism of resistant to CCR5 antagonists viruses [Fig. 2], we found out that interaction between the antagonists occupied CCR5 and the CCR5 antibody is more potent than inhibition provided by CCR5 antibody per se. HIV-1 strains resistant to the only clinically approved CCR5 antagonist Maraviroc (MVC, Fig. 3) generally remain CCR5 tropic, but gain the ability to use drug-bound CCR5. We demonstrate that MVCresistant HIV-1 loses the ability to use MVC-bound CCR5 at low surface CCR5 densities, suggesting a lower affinity for antagonist-bound CCR5 compared to free CCR5. In accordance, antibodies directed against the second extracellular loop (ECL2) of CCR5 had greater antiviral activity in MVC-bound than in MVC-free CCR5 infection of cell lines. However, in primary peripheral blood lymphocytes (PBLs), a dichotomy in antibody efficacy became evident. ECL2 CCR5 mAbs HGS004 and HGS101, inhibited PBL infection by MVC resistant HIV-1 more potently with MVC-bound than with free CCR5. In addition, HGS004 and HGS101, but not the other mAbs, restored the antiviral activity of MVC against resistant virus in PBLs. Both, HGS004 and HGS101, currently in clinical development, could help overcome MVC Resistance (Virology, 2011).

In addition, we demonstrate that the CCR5 antibody HGS004 and the CCR5 antagonist Maraviroc have potent antiviral synergy against R5 HIV-1, translating into dose reductions of >10-fold for Maraviroc and >150-fold for HGS004 (AIDS, 2011). These data, together with the high barrier of resistance to HGS004, suggest that combinations of Maraviroc and HGS004 could provide effective preventive and therapeutic strategies against R5 HIV-1.

Figure 1

Therapeutic opportunities for inhibition of HIV-1 entry

HIV-1 entry is mediated by the viral Env protein, which comprises the glycoproteins gp120 and gp41 arranged in trimeric spikes on the viral surface. Entry encompasses three steps: CD4 binding, coreceptor binding and fusion. The viral gp120 first binds to CD4, causing a repositioning of the variable loops V1/V2 and V3 and thereby exposing the bridging sheet and forming a coreceptor binding site. Upon coreceptor binding, conformational changes in gp120 and gp41 lead to the insertion of gp41 fusion peptide into the cell membrane. Subsequent conformational changes result in the formation of a six-helix bundle, with the HR2 domains folding back and packing into grooves on the outside of the triple-stranded HR1 domains. This brings the fusion peptide and transmembrane region of gp41 in close proximity, forming a fusion pore that allows transfer of the viral core into the cell. Each step on HIV-1 entry can be targeted by inhibitors currently approved or in clinical development. CD4 binding is targeted by ibalizumab (formerly TNX-355), a humanized monoclonal antibody that binds to CD4. Coreceptor binding is blocked by small-molecule CCR5 antagonists (Maraviroc and Vicriviroc) and by CCR5 antibodies (PRO-140 and HGS-004). Finally, the formation of a six-helix bundle, and thereby fusion, is prevented by enfuvirtide. Figure 1. adapted from Melikyan GB, Retrovirology, 2008.

Figure 2

Model for Maraviroc mechanism of resistance

Maraviroc binds to the transmembrane region of CCR5, thereby inducing confomational changes that cannot be recognized by R5 HIV-1 gp120. One mechanism of resistance involves changes in HIV-1 Env that permit recognition of Maraviroc-bound CCR5. As such, resistant viruses are not blocked by increasing Maraviroc doses. Figure 2. adapted from M. Westby, Curr Opin HIV AIDS, 2007.

Figure 3

Model for maraviroc mechanism of action

Binding of HIV-1 gp120 to CD4 exposes the bridging sheet and creates a coreceptor binding site. In the absence of Maraviroc, the bridging sheet and the base of V3 interact with the N-terminus of CCR5, while more distal regions of V3 interact with extracellular loops (mainly ECL2). Binding of Maraviroc to the transmembrane region of CCR5 locks CCR5 in a conformation that does not recognize the distal regions of V3. Figure 3. adapted from Soriano V. et al, AIDS, 2008.