Single-cell Proteogenomic profiling of HIV-1 reservoir cells

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Abstract HIV-1 reservoir cells are a small subset of CD4 T cells that harbor chromosomally integrated HIV-1 DNA, persist life-long and represent the major barrier to a cure of HIV-1 infection. For a long time, proviruses in these cells have been regarded as transcriptionally silent, which permits to escape from host immune recognition and to resist antiviral host immunity. However, recent studies enabled by single-genome and single-cell analytic technologies have shown that these cells can frequently be transcriptionally active and be vulnerable to host immune responses. In line with this observation, our recent work suggested that viral reservoir cells may be subject to immune-mediated host selection forces that promote elimination of proviruses integrated in permissive chromatin location supporting active viral transcription, while proviruses in repressive heterochromatin locations appear to have a selection advantage. In selected cases, such selection forces may, over extended periods of time, lead to a highly distinct reservoir profile dominated by intact proviruses integrated in heterochromatin locations; such a highly atypical proviral reservoir landscape may contribute to drug-free control of HIV-1 in elite and post-treatment-controllers. To better characterize the vulnerabilities and susceptibilities of viral reservoir cells to host immune cells, we here propose to use a novel single-cell proteogenomic profiling approach designed to evaluate the surface phenotype and intracellular markers of patient-derived HIV-1-infected cells from blood and tissues. We hypothesize that viral reservoir cells that persist during antiretroviral treatment are enriched and selected for phenotypic features conferring repression of proviral transcriptional activity and resistance to immune-mediated killing, resulting in reduced exposure to and protection from antiviral host effector cells. To test this hypothesis, we will conduct a detailed phenotypic analysis of HIV-1 reservoirs cells harboring intact and defective proviruses, focusing on more than 150 individual surface markers and evaluating cells collected ex vivo from the peripheral blood and from lymphoid tissues of ART-treated persons (specific aim 1). In addition, we will longitudinally track the evolution of the phenotypic viral reservoir profile, starting in early stages of ART initiation; simultaneously, the evolution of host immune responses will be analyzed to test the hypothesis that only a small subset of viral reservoir cells with optimal adaptation to host immune responses can survive long- term (specific aim 2). Finally, we will evaluate the susceptibility or resistance of HIV-1 reservoir cells to immune- mediated killing in functional assays and identify surface markers that can be pharmacologically targeted to reduce resistance of viral reservoir cells to immune-mediated killing (specific aim 3). By providing a detailed phenotypic characterization of HIV-1 reservoirs cells in direct ex vivo assessments, our studies will be highly informative for efforts to limit HIV-1 long-term persistence through clinical interventions.