In an effort to provide additional therapeutic options to patients with drug resistant virus or untoward toxicities on existing therapy, new drugs with potent antiretroviral activity are being developed that target and block HIV-1 entry into cells. Vicriviroc (VCV) is an investigational second-generation CCR5 (R5) antagonist that demonstrated potent and selective inhibition of HIV-1 in vitro and demonstrated potent virologic suppression through 24 weeks in HIV-1-infected treatment-experienced subjects enrolled in A5211, a phase IIb clinical trial. Subjects enrolled in clinical trials of vicriviroc and maraviroc have been identified with changes in viral co-receptor usage that emerge during therapy. The composition of the viral quasispecies is especially relevant during treatment with R5 inhibitors because there is legitimate concern that antagonism of CCR5 will result in viral adaptation, emergence of a dominant CXCR4 (X4)-using virus, and worsening of clinical disease. The characterization of the specific nature of CXCR4-using variants that develop in vivo and an accurate description of the viral quasispecies that emerge during therapy are hampered by current technological limitations. The purpose of this proposal is to assess the feasibility of using a novel ultra-deep sequencing technology to track dynamic shifts in the viral quasispecies in response to treatment with vicriviroc. Using subject samples from ACTG 5211, we will apply this technology to the sequencing of the V3 loop of gp120, a major determinant of co-receptor usage from subjects who have experienced changes in coreceptor usage on vicriviroc. We will investigate the emergence of CXCR4-using variants on R5 antagonist therapy and more accurately determine the origin of these viruses - either de novo emergence or expansion of a pre-existing minority X4 population. We hypothesize that the use of this highly parallel sequencing system will dramatically improve the speed and depth of sequencing and, for the first time, permit a comprehensive identification of minority viral populations that would not be possible using currently available techniques. More specifically, we propose to: sequence the V3 loop of gp120 from subjects experiencing tropism shifts using the highly parallel 454 sequencing system, investigate shifts in the proportion of CXCR4-using quasispecies variants in response to vicriviroc treatment, and determine the evolutionary origins and population dynamics of V3 loop sequences during vicriviroc treatment. Changes in HIV-1 envelope and the transition from R5 to X4 viruses even in the absence of drug therapy are important factors in pathogenesis and disease progression. The proposed studies will provide an unprecedented wealth of sequence data to illuminate the effects of CCR5 antagonist therapy on viral coreceptor usage and V3 loop sequence evolution across the entire quasispecies within an infected host. These studies will contribute to a better understanding of the role these changes may play in disease pathogenesis.
