BACKGROUND: Even after extended periods of effective, suppressive immune control, HIV-1 continues to dynamically change and evolve in body compartments, suggesting persisting and continuously active viral reservoirs.
METHODS: A longitudinal analysis of proviral Env sequences was performed by next-generation sequencing(NGS) in HIV-infected individuals from the Swiss-HIV-Cohort-Study right after diagnosis. HIV-1 proviral load, intracellular viral poly-A transcripts (pA) and Torque Teno Virus loads as independent marker were quantified by qPCR. PBMCs, sorted and cultured for 3 weeks for viral outgrowth, were monitored for viral reactivation by Tat-induced LTR-activation and HIV-1 protein expression by FACS. Single-genome sequencing (SGA) analysis of the 3' half of the HIV-genomes was carried out to assess re-activated viral RNA.
RESULTS: Specimens from nine viremic SHCS-HIV individuals with detectable HIV-1 provirus in PBMCs (median: 231/10^6 cells) and detectable HIV-1 polyA-RNA (median: 1'129/10^6 cells) were included. In 5/9 samples a high reservoir diversity persisted even for extended periods after therapy initiation, while 4/9 presented unique virus variants (241-1454 days). Lymphocyte counts (p=0.0068), CD4 (p<0.0001), and CD8 cells (p=0.0487) were lower in the population with high-diversity reservoirs, and as independent marker of immune reconstitution: Torque-Teno-Virus loads were higher.
In circulating T-cell subsets, predominantly TN(naïve) and TCM(central memory) T-cells harbored replication-competent HIV-1. In TTM(transitional memory) and TEM(effector memory) T-cells, most archived proviral sequences contained inactivating mutations and, at the same time, displayed a higher genetic diversity.
CONCLUSIONS: Our study demonstrates that less immune control, evidenced by low lymphocyte counts, makes it easier for the virus to replicate to high levels and further increase its genetic diversity. The main reservoirs for replication-competent, infectious virus include TN and TCM, with single or limited numbers of intact viral variants. A distinct, smaller contribution of archived HIV sequences stems from TTM and TEM, characterized by a higher viral variability. The latter potentially form the basis for an ongoing evolution of new viral variants during therapy.
These findings provide insights into the dynamics of the viral reservoir in individuals early during infection near the start of therapy and may have important implications for a better understanding of HIV-1 transmission and in vivo evolution.

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