HIV-1 persists in a latent reservoir in resting CD4⁺ T cells despite antiretroviral therapy (ART). The reservoir decays slowly over the first 7 years of ART (t_1/2 = 44 months). However, whether decay continues with long-term ART is unclear. Recent integration site studies indicate gradual selection against inducible, intact proviruses, raising speculation that decades of ART might allow treatment interruption without viral rebound. Therefore, we measured the reservoir in 42 people on long-term ART (mean 22 years) using a quantitative viral outgrowth assay. After 7 years of ART, there was no long-term decrease in the frequency of inducible, replication-competent proviruses but rather an increase with an estimated doubling time of 23 years. Another reservoir assay, the intact proviral DNA assay, confirmed that reservoir decay with t_1/2 of 44 months did not continue with long-term ART. The lack of decay reflected proliferation of infected cells. Most inducible, replication-competent viruses (79.8%) had env sequences identical to those of other isolates from the same sample. Thus, although integration site analysis indicates changes in reservoir composition, the proliferation of CD4⁺ T cells counteracts decay, maintaining the frequency of inducible, replication-competent proviruses at roughly constant levels over the long term. These results reinforce the need for lifelong ART.

Significance: The latent reservoir for HIV-1 is a major barrier to curing the infection. Current cure strategies are focused on eliminating the reservoir through a two-step “shock and kill” approach involving the use of small-molecule latency-reversing agents (LRAs) followed by immune interventions to kill infected cells. Numerous candidate LRAs have been identified in studies using in vitro models of latency, but none have been shown to reduce the reservoir in vivo. We show here that many candidate LRAs work through an unexpected pathway involving the transcription factor HSF1, a result with implications for developing effective combinations of LRAs for use in HIV cure strategies.

The eradication of the viral reservoir represents the major obstacle to the development of a clinical cure for established HIV-1 infection. Here, we demonstrate that the administration of N-803 (brand name Anktiva) and broadly neutralizing antibodies (bNAbs) results in sustained viral control after discontinuation of antiretroviral therapy (ART) in simian-human AD8 (SHIV-AD8)–infected, ART-suppressed rhesus macaques. N-803+bNAbs treatment induced immune activation and transient viremia but only limited reductions in the SHIV reservoir. Upon ART discontinuation, viral rebound occurred in all animals, which was followed by durable control in approximately 70% of all N-803+bNAb–treated macaques. Viral control was correlated with the reprogramming of CD8 ⁺ T cells by N-803+bNAb synergy. Thus, complete eradication of the replication-competent viral reservoir is likely not a prerequisite for the induction of sustained remission after discontinuation of ART.

The decay kinetics of HIV-1-infected cells are critical to understand virus persistence. We evaluated the frequency of simian immunodeficiency virus (SIV)-infected cells for 4 years of antiretroviral therapy (ART). The intact proviral DNA assay (IPDA) and an assay for hypermutated proviruses revealed short- and long-term infected cell dynamics in macaques starting ART ∼1 year after infection. Intact SIV genomes in circulating CD4⁺T cells showed triphasic decay with an initial phase slower than the decay of the plasma virus, a second phase faster than the second phase decay of intact HIV-1, and a stable third phase reached after 1.6-2.9 years. Hypermutated proviruses showed bi- or mono-phasic decay, reflecting different selective pressures. Viruses replicating at ART initiation had mutations conferring antibody escape. With time on ART, viruses with fewer mutations became more prominent, reflecting decay of variants replicating at ART initiation. Collectively, these findings confirm ART efficacy and indicate that cells enter the reservoir throughout untreated infection.

Significance: Novel approaches to promote killing of HIV-1–infected cells are necessary for elimination of the latent reservoir, the main barrier to a cure. Here, we utilized a diverse phage-display library to construct T cell receptor (TCR)-mimic antibodies to HIV-1 peptide-major histocompatibility complexes (pMHC). We show that single-chain diabody forms of these antibodies recognize distinct epitopes in Gag and reverse transcriptase in a specific manner and induce T cell-mediated killing of HIV-1–infected CD4 ⁺ T cells. This study lays the groundwork for future exploration of pMHC-based immunotherapeutic approaches toward elimination of the latent reservoir once effective latency-reversing strategies are developed.

Persistence of the human immunodeficiency virus type-1 (HIV-1) latent reservoir in infected individuals remains a problem despite fully suppressive antiretroviral therapy (ART). While reservoir formation begins during acute infection, the mechanisms responsible for its establishment remain unclear. CD8⁺ T cells are important during the initial control of viral replication. Here we examined the effect of CD8⁺ T cells on formation of the latent reservoir in simian immunodeficiency virus (SIV)-infected macaques by performing experimental CD8⁺ depletion either before infection or before early (that is, day 14 post-infection) ART initiation. We found that CD8⁺ depletion resulted in slower decline of viremia, indicating that CD8⁺ lymphocytes reduce the average lifespan of productively infected cells during acute infection and early ART, presumably through SIV-specific cytotoxic T lymphocyte (CTL) activity. However, CD8⁺ depletion did not change the frequency of infected CD4⁺ T cells in the blood or lymph node as measured by the total cell-associated viral DNA or intact provirus DNA assay. In addition, the size of the persistent reservoir remained the same when measuring the kinetics of virus rebound after ART interruption. These data indicate that during early SIV infection, the viral reservoir that persists under ART is established largely independent of CTL control.