Background:  HIV-1 RNA levels in semen and blood compartments decrease below detection limits during Indinavir (PI) and DMP-266 (NNRTI) therapy. Despite these favorable therapeutic effects persistent reservoirs capable of rebounding in the absence of drug treatment or by 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. The current study was designed to provide detailed post-therapy analysis of semen and blood latent virus populations.

Methods:  PBMC and semen samples were collected from HIV-1 patients pre and post DMP/PI treatment. Semen and blood mononuclear cells were co-cultured with anti-CD3-stimulated CD8-depleted donor PBMC and monitored for p24 production. Cultured cells were analyzed for CD45RO⁺ cells expressing HIV-1 mRNA. Viral RNA was isolated from culture supernatants for cloning and sequencing. RT, PR, and C2-V5 of Env pre and post-therapy were analyzed for polymorphisms, resistance mutations (RT/PR), population progression and evolution as determined by MRCA divergence.

Results:  CD4⁺, CD45R0⁺, HIV-1 gag-pol mRNA  was detected in semen and PBMC in all subjects up to 1800 days post-therapy, remaining consistently higher in seminal cells than in PBMC. RT and PR sequences displayed polymorphisms with several identical drug resistance residues in pre/post-therapy samples, lacking evidence of therapy-related drug resistance development. Env sequences displayed reduced levels of divergence post-therapy, presumably reflecting the stringent control of viral replication. Pre- and post-therapy Env populations were distinct, indicating possible selection of quasispecies. Divergence from the MRCA indicated the emergence of species from viral reservoirs during therapy.

Conclusions:  These observations indicate that genital and blood compartments may serve as distinct reservoirs of HIV-1 during drug therapy, harboring different populations and viral replication dynamics. Drug resistance profiles indicate that latent viral reservoirs of resistance mutations exist before therapy is initiated and could become problematic over longer periods of therapy. Persistence during treatment was not associated with evolution of the polymerase genes, however, low levels of ongoing viral replication sustained envelope variation and thus viral evolution persevered as reflected in the envelope sequences.

Keywords: Latent HIV Reservoirs; Therapy; Evolution