ClinicalTrials.gov NCT02463227FUNDING. Funding was provided by the NIH.

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Type 1 interferons (IFN-I) are potent innate antiviral effectors that constrain HIV-1 transmission. However, harnessing these cytokines for HIV-1 cure strategies has been hampered by an incomplete understanding of their antiviral activities at later stages of infection. Here, we characterized the IFN-I sensitivity of 500 clonally derived HIV-1 isolates from the plasma and CD4+ T cells of 26 individuals sampled longitudinally after transmission or after antiretroviral therapy (ART) and analytical treatment interruption. We determined the concentration of IFNα2 and IFNβ that reduced viral replication in vitro by 50% (IC50) and found consistent changes in the sensitivity of HIV-1 to IFN-I inhibition both across individuals and over time. Resistance of HIV-1 isolates to IFN-I was uniformly high during acute infection, decreased in all individuals in the first year after infection, was reacquired concomitant with CD4+ T cell loss, and remained elevated in individuals with accelerated disease. HIV-1 isolates obtained by viral outgrowth during suppressive ART were relatively IFN-I sensitive, resembling viruses circulating just before ART initiation. However, viruses that rebounded after treatment interruption displayed the highest degree of IFNα2 and IFNβ resistance observed at any time during the infection course. These findings indicate a dynamic interplay between host innate responses and the evolving HIV-1 quasispecies, with the relative contribution of IFN-I to HIV-1 control affected by both ART and analytical treatment interruption. Although elevated at transmission, host innate pressures are the highest during viral rebound, limiting the viruses that successfully become reactivated from latency to those that are IFN-I resistant.

BACKGROUND. We performed a Phase I clinical trial that infused CCR5 gene edited CD4 T cells to determine how these T cells can better enable HIV cure strategies. METHODS. The trial addressed the method of zinc finger nuclease (ZFN) ex vivo delivery, whether CCR5 Δ32 heterozygotes preferentially benefit, the effect of CCR5 gene edited CD4 T cells on the HIV-specific T cell response, and the ability of infused CCR5 gene edited T cells to delay viral rebound during analytical treatment interruption. We enrolled 14 people living with HIV whose viral load was well controlled by antiretroviral therapy (ART). We measured time to viral rebound after ART withdrawal, persistence of CCR5-edited CD4 T cells, and whether infusion of 10 billion CCR5-edited CD4 T cells augmented the HIV-specific immune response. RESULTS. Infusion of the CD4 T cells was well tolerated with no serious adverse events. Modest delay to the time of viral rebound was observed relative to historical controls; however, three of 14 individuals of which two were CCR5 Δ32 heterozygotes appeared to regain control of viremia before ultimately rebounding. Interestingly, only these individuals had substantial restoration of HIV-specific CD8 T cell responses. Immune escape to one of these re-invigorated responses was observed at viral recrudescence, illustrating a direct link between viral control and enhanced CD8 T cell responses. CONCLUSION. These findings demonstrate how CCR5 gene edited CD4 T cell infusion could aid HIV cure strategies by augmenting pre-existing HIV-specific immune responses.

Understanding the complexity of the long-lived HIV reservoir during antiretroviral therapy (ART) remains a considerable impediment in research towards a cure for HIV. To address this, we developed a single-cell strategy to precisely define the unperturbed peripheral blood HIV-infected memory CD4+ T cell reservoir from ART-treated people living with HIV (ART-PLWH) via the presence of integrated accessible proviral DNA in concert with epigenetic and cell surface protein profiling. We identified profound reservoir heterogeneity within and between ART-PLWH, characterized by new and known surface markers within total and individual memory CD4+ T cell subsets. We further uncovered new epigenetic profiles and transcription factor motifs enriched in HIV-infected cells that suggest infected cells with accessible provirus, irrespective of reservoir distribution, are poised for reactivation during ART treatment. Together, our findings reveal the extensive inter- and intrapersonal cellular heterogeneity of the HIV reservoir, and establish an initial multiomic atlas to develop targeted reservoir elimination strategies.

Type 1 interferons (IFN-I) are potent innate antiviral effectors that constrain HIV-1 transmission. However, harnessing these cytokines for HIV-1 cure strategies has been hampered by an incomplete understanding of their anti-viral activities at later stages of infection. Here, we characterized the IFN-I sensitivity of 500 clonally-derived HIV-1 isolates from plasma and CD4+ T cells of 26 individuals sampled longitudinally following transmission and/or after antiretroviral therapy (ART) and analytical treatment interruption (ATI). Determining the concentration of IFNα2 and IFNβ that reduced HIV-1 replication by 50% (IC50), we found remarkably consistent changes in the sensitivity of viruses to IFN-I inhibition, both across individuals and over time. IFN-I resistance was uniformly high during acute infection, decreased in all subjects in the first year post-infection, was reacquired concomitant with CD4+ T cell loss, and remained elevated in subjects with accelerated disease. Isolates obtained by viral outgrowth during suppressive ART were relatively IFN-I sensitive, resembling viruses circulating just prior to ART initiation. However, viruses that rebounded following treatment interruption displayed the highest levels of IFNα2 and IFNβ resistance observed at any time during the infection course. These findings indicate a dynamic interplay between host innate immune responses and the evolving HIV-1 quasispecies, with the relative contribution of IFN-I to HIV-1 control impacted by both ART and ATI. Although elevated at transmission, IFN-mediated pressures are the highest during viral rebound, limiting the viruses that successfully reactivate from latency.

Defining the long-lived HIV reservoir during antiretroviral therapy remains a challenge for HIV cure research. To determine if unifying cell surface or genomic features can be identified in unperturbed HIV-1 infected CD4+ T-cells, we developed single-cell viral ASAPseq, which leverages scATACseq and surface marker profiling to define HIV-infected cells via the presence of integrated proviral DNA. Here, we employed this strategy to define infected CD4+ T-cells both in vitro and ex vivo from blood and lymph nodes of, respectively, treated and untreated people living with HIV. In each setting, we identified HIV-infected cells with both novel and known surface markers associated with provirus. We also uncovered epigenetic profiles and transcription factor motifs enriched in HIV-infected cells that more deeply define the reservoir. Our data highlight the broad heterogeneity of the HIV reservoir and create a foundation for a multiomic atlas to develop targeted elimination strategies.

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