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Lab Head

Robert Redfield, MD

IHV> Research: Anti-HIV Therapeutic Research

ANTI-HIV THERAPEUTIC RESEARCH

Associated Faculty

Assistant Pr ofessor Olga L atinov ic, PhD

Assistant Pr ofessor Alonso Heredia, PhD

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

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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 andgp41 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.

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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.

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