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HIV persists in latently infected CD4 + T cells during antiretroviral therapy (ART). Immune checkpoint molecules, including PD-1, are preferentially expressed at the surface of persistently infected cells. However, whether PD-1 plays a functional role in HIV latency and reservoir persistence remains unknown. Using CD4 + T cells from HIV-infected individuals, we show that the engagement of PD-1 inhibits viral production at the transcriptional level and abrogates T-cell receptor (TCR)-induced HIV reactivation in latently infected cells. Conversely, PD-1 blockade with the monoclonal antibody pembrolizumab enhances HIV production in combination with the latency reversing agent bryostatin without increasing T cell activation. Our results suggest that the administration of immune checkpoint blockers to HIV-infected individuals on ART may facilitate latency disruption. The immune checkpoint molecule PD-1 is expressed on a fraction of CD4 + T cells latently infected with HIV, but whether PD-1 plays a functional role in reservoir persistence remains unknown. Here, Fromentin et al. show that PD-1 blockade potentiates latency reversal ex vivo in CD4 + T cells from ART suppressed individuals.

HIV persists in a small pool of latently infected cells despite antiretroviral therapy (ART). Identifying cellular markers expressed at the surface of these cells may lead to novel therapeutic strategies to reduce the size of the HIV reservoir. We hypothesized that CD4+ T cells expressing immune checkpoint molecules would be enriched in HIV-infected cells in individuals receiving suppressive ART. Expression levels of 7 immune checkpoint molecules (PD-1, CTLA-4, LAG-3, TIGIT, TIM-3, CD160 and 2B4) as well as 4 markers of HIV persistence (integrated and total HIV DNA, 2-LTR circles and cell-associated unspliced HIV RNA) were measured in PBMCs from 48 virally suppressed individuals. Using negative binomial regression models, we identified PD-1, TIGIT and LAG-3 as immune checkpoint molecules positively associated with the frequency of CD4+ T cells harboring integrated HIV DNA. The frequency of CD4+ T cells co-expressing PD-1, TIGIT and LAG-3 independently predicted the frequency of cells harboring integrated HIV DNA. Quantification of HIV genomes in highly purified cell subsets from blood further revealed that expressions of PD-1, TIGIT and LAG-3 were associated with HIV-infected cells in distinct memory CD4+ T cell subsets. CD4+ T cells co-expressing the three markers were highly enriched for integrated viral genomes (median of 8.2 fold compared to total CD4+ T cells). Importantly, most cells carrying inducible HIV genomes expressed at least one of these markers (median contribution of cells expressing LAG-3, PD-1 or TIGIT to the inducible reservoir = 76%). Our data provide evidence that CD4+ T cells expressing PD-1, TIGIT and LAG-3 alone or in combination are enriched for persistent HIV during ART and suggest that immune checkpoint blockers directed against these receptors may represent valuable tools to target latently infected cells in virally suppressed individuals.

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 (t1/2 = 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 t1/2 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.

Despite long-term antiretroviral therapy (ART), HIV-1 persists within a reservoir of CD4+ T cells that contribute to viral rebound if treatment is interrupted. Identifying the cellular populations that contribute to the HIV-1 reservoir and understanding the mechanisms of viral persistence are necessary to achieve an effective cure. In this regard, through Full-Length Individual Proviral Sequencing, we observed that the HIV-1 proviral landscape was different and changed with time on ART across naive and memory CD4+ T cell subsets isolated from 24 participants. We found that the proportion of genetically intact HIV-1 proviruses was higher and persisted over time in effector memory CD4+ T cells when compared with naive, central, and transitional memory CD4+ T cells. Interestingly, we found that escape mutations remained stable over time within effector memory T cells during therapy. Finally, we provided evidence that Nef plays a role in the persistence of genetically intact HIV-1. These findings posit effector memory T cells as a key component of the HIV-1 reservoir and suggest Nef as an attractive therapeutic target.

To investigate the possibility that HIV-1 replication in lymph nodes sustains the reservoir during ART, we looked for evidence of viral replication in 5 donors after up to 13 years of viral suppression. We characterized proviral populations in lymph nodes and peripheral blood before and during ART, evaluated the levels of viral RNA expression in single lymph node and blood cells, and characterized the proviral integration sites in paired lymph node and blood samples. Proviruses with identical sequences, identical integration sites, and similar levels of RNA expression were found in lymph nodes and blood samples collected during ART, and no single sequence with significant divergence from the pretherapy population was present in either blood or lymph nodes. These findings show that all detectable persistent HIV-1 infection is consistent with maintenance in lymph nodes by clonal proliferation of cells infected before ART and not by ongoing viral replication during ART.

The establishment of HIV-1 latency gives rise to persistent chronic infection that requires life-long treatment. To reverse latency for viral eradiation, the HIV-1 Tat protein and its associated ELL2-containing Super Elongation Complexes (ELL2-SECs) are essential to activate HIV-1 transcription. Despite efforts to identify effective latency-reversing agents (LRA), avenues for exposing latent HIV-1 remain inadequate, prompting the need to identify novel LRA targets. Here, by conducting a CRISPR interference-based screen to reiteratively enrich loss-of-function genotypes that increase HIV-1 transcription in latently infected CD4+ T cells, we have discovered a key role of the proteasome in maintaining viral latency. Downregulating or inhibiting the proteasome promotes Tat-transactivation in cell line models. Furthermore, the FDA-approved proteasome inhibitors bortezomib and carfilzomib strongly synergize with existing LRAs to reactivate HIV-1 in CD4+ T cells from antiretroviral therapy-suppressed individuals without inducing cell activation or proliferation. Mechanistically, downregulating/inhibiting the proteasome elevates the levels of ELL2 and ELL2-SECs to enable Tat-transactivation, indicating the proteasome-ELL2 axis as a key regulator of HIV-1 latency and promising target for therapeutic intervention.

The source and dynamics of persistent HIV-1 during long-term combinational antiretroviral therapy (cART) are critical to understanding the barriers to curing HIV-1 infection. To address this issue, we isolated and genetically characterized HIV-1 DNA from naïve and memory T cells from peripheral blood and gut-associated lymphoid tissue (GALT) from eight patients after 4–12 y of suppressive cART. Our detailed analysis of these eight patients indicates that persistent HIV-1 in peripheral blood and GALT is found primarily in memory CD4 ⁺ T cells [CD45RO ⁺/CD27(⁺/⁻)]. The HIV-1 infection frequency of CD4 ⁺ T cells from peripheral blood and GALT was higher in patients who initiated treatment during chronic compared with acute/early infection, indicating that early initiation of therapy results in lower HIV-1 reservoir size in blood and gut. Phylogenetic analysis revealed an HIV-1 genetic change between RNA sequences isolated before initiation of cART and intracellular HIV-1 sequences from the T-cell subsets after 4–12 y of suppressive cART in four of the eight patients. However, evolutionary rate analyses estimated no greater than three nucleotide substitutions per gene region analyzed during all of the 4–12 y of suppressive therapy. We also identified a clearly replication-incompetent viral sequence in multiple memory T cells in one patient, strongly supporting asynchronous cell replication of a cell containing integrated HIV-1 DNA as the source. This study indicates that persistence of a remarkably stable population of infected memory cells will be the primary barrier to a cure, and, with little evidence of viral replication, this population could be maintained by homeostatic cell proliferation or other processes.

BACKGROUNDEarly antiretroviral therapy initiation (ARTi) in HIV-1 restricts reservoir size and diversity while preserving immune function, potentially improving opportunities for immunotherapeutic cure strategies. For antibody-based cure approaches, the development of autologous neutralizing antibodies (anAbs) after acute/early ARTi is relevant but is poorly understood.METHODSWe characterized antibody responses in a cohort of 23 participants following ARTi in acute HIV (<60 days after acquisition) and early HIV (60-128 days after acquisition).RESULTSPlasma virus sequences at the time of ARTi revealed evidence of escape from anAbs after early, but not acute, ARTi. HIV-1 envelopes representing the transmitted/founder virus(es) (acute ARTi) or escape variants (early ARTi) were tested for sensitivity to longitudinal plasma IgG. After acute ARTi, no anAb responses developed over months to years of suppressive ART. In 2 of the 3 acute ARTi participants who experienced viremia after ARTi, however, anAbs arose shortly thereafter. After early ARTi, anAbs targeting those early variants developed between 12 and 42 weeks of ART and continued to increase in breadth and potency thereafter.CONCLUSIONResults indicate a threshold of virus replication (~60 days) required to induce anAbs, after which they continue to expand on suppressive ART to better target the range of reservoir variants.TRIAL REGISTRATIONClinicalTrials.gov NCT02656511.FUNDINGNIH grants U01AI169767, R01AI162646, UM1AI164570, UM1AI164560, U19AI096109, K23GM112526, T32AI118684, P30AI045008, P30AI027763, R24AI067039; Gilead Sciences grant INUS2361354; Viiv Healthcare grant A126326.

Persistence of HIV in people living with HIV (PWH) on suppressive antiretroviral therapy (ART) has been linked to physiological mechanisms of CD4+ T cells. Here, in the same 37 male PWH on ART we measure longitudinal kinetics of HIV DNA and cell turnover rates in five CD4 cell subsets: naïve (T N ), stem-cell- (T SCM ), central- (T CM ), transitional- (T TM ), and effector-memory (T EM ). HIV decreases in T TM and T EM but not in less-differentiated subsets. Cell turnover is ~10 times faster than HIV clearance in memory subsets, implying that cellular proliferation consistently creates HIV DNA. The optimal mathematical model for these integrated data sets posits HIV DNA also passages between CD4 cell subsets via cellular differentiation. Estimates are heterogeneous, but in an average participant’s year ~10 (in T N and T SCM ) and ~10 4 (in T CM , T TM , T EM ) proviruses are generated by proliferation while ~10 3 proviruses passage via cell differentiation (per million CD4). In simulations, therapies blocking proliferation and/or enhancing differentiation could reduce HIV DNA by 1-2 logs over 3 years. In summary, HIV exploits cellular proliferation and differentiation to persist during ART but clears faster in more proliferative/differentiated CD4 cell subsets and the same physiological mechanisms sustaining HIV might be temporarily modified to reduce it. The authors used mathematical modeling of human data to study how HIV persists despite suppressive antiretroviral therapy. They found that when latently infected CD4+ T cells proliferate or differentiate, they can create HIV DNA and passage it into other subsets. More mature CD4 cell subsets then clear HIV DNA faster.

Despite antiretroviral therapy (ART), HIV persists in latently-infected cells (the HIV reservoir) which decay slowly over time. Here, leveraging >500 longitudinal samples from 67 people living with HIV (PLWH) treated during acute infection, we developed a mathematical model to predict reservoir decay from peripheral CD4 + T cells. Nonlinear generalized additive models demonstrated rapid biphasic decay of intact DNA (week 0-5: t 1/2  ~ 2.83 weeks; week 5-24: t 1/2  ~ 15.4 weeks) that extended out to 1 year. These estimates were ~5-fold faster than prior decay estimates among chronic treated PLWH. Defective DNA had a similar biphasic pattern, but data were more variable. Predicted intact and defective decay rates were faster for PLWH with earlier timing of ART initiation, higher initial CD4 + T cell count, and lower pre-ART viral load. In this study, we advanced our limited understanding of HIV reservoir decay at the time of ART initiation, informing future curative strategies targeting this critical time. HIV reservoir decay is less well studied in acute infection. Here, the authors show that reservoir decay rates are biphasic and 5x faster in people initiating antiretroviral therapy during acute HIV than prior estimates for chronic HIV. Higher initial CD4+ counts and lower viral loads predicted faster decay.