The fate of HIV-infected cells after reversal of proviral latency is not well characterized. Simonetti, et al. recently showed that CD4 + T-cells containing intact proviruses can clonally expand in vivo and produce low-level infectious viremia. We hypothesized that reversal of HIV latency by activation of CD4 + T-cells can lead to the expansion of a subset of virus-producing cells rather than their elimination. We established an ex vivo cell culture system involving stimulation of CD4 + T-cells from donors on suppressive antiretroviral therapy (ART) with PMA/ionomycin (day 1–7), followed by rest (day 7–21), and then repeat stimulation (day 21–28), always in the presence of high concentrations of raltegravir and efavirenz to effectively block new cycles of viral replication. HIV DNA and virion RNA in the supernatant were quantified by qPCR. Single genome sequencing (SGS) of p6-PR-RT was performed to genetically characterize proviruses and virion-associated genomic RNA. The replication-competence of the virions produced was determined by the viral outgrowth assay (VOA) and SGS of co-culture supernatants from multiple time points. Experiments were performed with purified CD4 + T-cells from five consecutively recruited donors who had been on suppressive ART for > 2 years. In all experiments, HIV RNA levels in supernatant increased following initial stimulation, decreased or remained stable during the rest period, and increased again with repeat stimulation. HIV DNA levels did not show a consistent pattern of change. SGS of proviruses revealed diverse outcomes of infected cell populations, ranging from their apparent elimination to persistence and expansion. Importantly, a subset of infected cells expanded and produced infectious virus continuously after stimulation. These findings underscore the complexity of eliminating reservoirs of HIV-infected cells and highlight the need for new strategies to kill HIV-infected cells before they can proliferate.
Stable latent reservoirs of HIV persist despite suppressive antiretroviral therapy (ART) and cause rebound viremia following interruption of ART. New HIV cure strategies aim to deplete the latent reservoir by reversing HIV latency and promoting the death of cells containing inducible proviruses. Multiple latency reversing agents have been discovered, with the most effective compounds also causing T-cell activation. We investigated the ex vivo effects of cell activation on HIV-infected cells from individuals on stable suppressive ART. Latency reversal through robust cell activation led to diverse outcomes of infected cells. Although many infected cells appeared to be eliminated following T-cell activation, a subset of HIV-infected cells persisted and could expand, including those that can produce infectious virus. These findings highlight the need for new therapies that kill HIV-infected cells before they can proliferate.