579 Analysis of Latent HIV Infection and Sequence Evolution Post Therapy in Semen and Blood Compartments J.K. Craigo*1, B. Patterson2, M. Ding1, R.C. Montelaro1, J. Mellors1, P. Gupta1 1Univ of Pittsburgh, PA and 2Northwestern Univ, Evanston, IL
Background: HIV-1 RNA levels in semen and blood compartments decrease below detection limits during PI and DMP (an NNRTI) therapy. Despite these favorable therapeutic effects, it is clear that persistent reservoirs of HIV-1 capable of rebounding in the absence of drug treatment or by the evolution of escape mutants remain. To date there is little comparative quantitative and qualitative information on the nature of persistent HIV-1 populations harbored in blood and tissues during drug therapy. Thus, the current study was designed to provide detailed post therapy analysis of semen and blood compartment latent virus populations and evolution of their PR, RT and Env.
Methods: PBMC and semen samples were collected from HIV-1 infected patients pre and post DMP/PI treatment. Semen and blood mononuclear cells were co-cultured with anti-CD3-stimulated CD8-depleted donor PBMC. Supernatants were monitored for p24 production. Cultured cells were analyzed for CD45RO+ cells expressing HIV-1 RNA by a flow cytometric, in situ hybridization assay. Viral RNA was isolated from culture supernatants for amplification, cloning and sequencing. RT, PR, and C2-V5 of Env pre and post therapy were analyzed for polymorphisms, resistance mutations (RT/PR), and evolution.
Results: While p24 was only detected in PBMC culture supernatants, HIV RNA was detected in cells from semen and blood in all subjects out to 900 days after therapy. HIV RNA levels remained consistently higher in seminal cells than in PBMC. RT and PR sequences displayed polymorphisms, but lacked evidence of developing drug resistance in all samples. Env sequences displayed reduced levels of divergence post therapy, presumably reflecting the more stringent control of viral replication. Interestingly, pre and post therapy Env populations were distinct, indicating possible selection of HIV-1 quasispecies. Observed Env sequence alterations, however, did not indicate co-receptor usage modifications, perhaps suggesting immune selection accompanying drug treatment.
Conclusions: These observations indicate that genital and blood compartments may serve as distinct reservoirs of HIV-1 infection during drug therapy, harboring different virus populations and viral replication dynamics. Moreover, low levels of ongoing viral replication sustained low levels of Env variation, but persistence during drug treatment was not associated with defined resistance mutations in PR or RT genes of the seminal or blood viral reservoirs.
