The Cell Associated Reservoir of HIV-1 is Highly Diverse and Harbors Multi-Drug Resistance in Fully Suppressed Patients

T Liegler1, J Harris2, M Warmerdam1, J Barbour1, LJ Montaner3,JM McCune1 and RM Grant1; 1JD Gladstone Inst of Virology and Immunology, San Francisco, CA; 2Univ of California San Francisco, 3Wistar Inst, Philadelphia, PA (USA).

Background: The genetic and cellular factors that determine the source, composition and dynamics of rebound plasma viremia are unknown. Viral variants that emerge in blood plasma during treatment interruption are not necessarily representative of viruses in the cellular reservoir. We hypothesize that the complexity of replication competent virus in the PBMC reservoir is high, and may therefore require extensive sampling to adequately assess the contribution to plasma rebound viremia. Material and Methods: We analyzed the  HIV-1 RT/PRO sequences of cellular and blood plasma viruses from chronically infected drug-suppressed patients undergoing STI. Thirteen (13) adults underwent up to 3 cycles of STI, typically 8 wks off and >12 wks on therapy. CD4 T-cell counts, plasma VL and genotypic drug resistance were assayed throughout the study. During periods of viral suppression, viral isolates were generated from patient  CD8 T-cell-depleted co-cultures at limiting dilution. Phylogenetic trees were inferred from nucleotide sequences using PAUP v4.0. Results: All patients had rebound viremia during all cycles off therapy. 5/13 cases showed genotypic drug resistance during the first or subsequent cycles. 3/3 cases showed resistance in biological clones isolated from blood cells at multiple cycles. MDR viruses were common despite long-term virologic suppression. During a single 8 week off cycle, shifts were seen in the predominant viral species. Early rebound plasma viremia was not necessarily represented in viral quasispecies from cultures at proximal off cycles, but emergence did occur at distant cycles of interruption. Viruses independently isolated from blood cells were typically diverse, with some but not all lineages clustering with viral variants appearing in the plasma. Conclusion: Replication-competent virus populations in peripheral blood cells are diverse, indicating that single isolates, or multiple clones from single isolates, are insufficient to characterize this reservoir of HIV-1.  Even with up to 8 biological clones of HIV-1 per time point, the rebounding virus in plasma could not always be identified.  MDR viruses persist in the cellular reservoir, and emerge readily during treatment interruption, despite long-term suppression. These data suggest that HIV-1 infection is compartmentalized, leading to diversity in reservoirs that contribute to viral genetic shifts over time.