Entry of HIV-1 into target cells involves the interaction of viral envelope proteins with specific cell surface receptors, leading ultimately to fusion of viral and host cell membranes. This multi-step process offers a number of potential targets for drug development. During the current funding period, significant progress was made towards understanding the viral dynamics, fitness consequences and clinical significance of ENF resistance. We now propose to extend these studies to HIV-1 isolates from patients treated with CCR5 inhibitors. Because of their critical role in virus entry, the chemokine co-receptors are attractive targets for drug development. Several small-molecule CCR5 inhibitors have demonstrated potent anti-HIV-1 activity in vitro and in HIV-1-infected subjects, two of which (maraviroc and vicriviroc) are in advanced stages of clinical development. Studies of maraviroc- and vicriviroc-resistant HIV-1 isolates selected by in vitro passage show that these resistant viruses retain their R5 phenotype and continue to rely on CCR5 for entry [Pugach]. However, little is known about the properties of HIV-1 isolates selected by exposure to these inhibitors in vivo. In addition, scant information is available on shifts in the viral quasispecies in persons infected with dual-tropic or mixed R5 and X4 viruses under drug pressure. We therefore propose to analyze viral adaptation to replication in the presence of the CCR5 inhibitor vicriviroc using samples obtained from subjects with virologic failure in the phase 2 study of vicriviroc, AIDS Clinical Trials Group protocol A5211. The following four specific aims are proposed: 1) To identify HIV-1 envelopes from vicriviroc-treated subjects with reduced CCR5 inhibitor susceptibility; 2) to identify genotypic changes in HIV-1 env associated with vicriviroc resistance in vivo; 3) to determine the effect of vicriviroc resistance on viral fitness; 4) to examine the entry kinetics of vicriviroc-resistant HIV-1. For this latter aim we will use the novel technology of massively parallel sequencing using microfabricated picoliter reactors. Results of these experiments will be highly relevant to understanding the mechanisms and consequences of resistance to this novel class of HIV- 1 therapeutics and will provide data that are essential to their proper use in the treatment of HIV-1-infected individuals. ? ? ? ?
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