Viral ASAPseq definition of CD4+ T cell viral reservoirs

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While combination antiretroviral therapy (ART) effectively controls HIV viremia in most people living with HIV (PLWH), broadly applicable strategies to induce viral remission have remained elusive due to HIV reservoir persistence. Substantial progress has been made in defining the virological characteristics of the proviral reservoir of latently infected cells during ART. However, significant gaps remain in our understanding of the infected CD4+ T cells that constitute the cellular HIV reservoir that limit our ability to develop effective strategies for HIV cure. We have developed a novel high throughput 10X Genomics single cell assay to directly identify HIV infected cells via the presence of integrated proviral DNA. Termed "viral ASAPseq" [Assay for Transposase Accessible Chomatin (ATAC) Surface Antigen Profile sequencing, ASAPseq, with viral alignment, viral ASAPseq], this assay allows detection of HIV proviral DNA within euchromatin in combination with 154-marker cell surface antigen labeling, yielding multimodal single cell resolution of HIV infected cells. This assay does not require ex vivo activation to detect the presence of integrated viral DNA, thereby preserving the native state of the infected cell. We have successfully piloted this procedure in several conditions, including in vitro infected primary CD4+ T cells, primary lymph node CD4+ T cells from viremic PLWH, and peripheral blood CD4+ T cells from ART treated PLWH. We have further developed bioinformatic strategies to identify proviral DNA using autologous viral sequence alignment, define the surface marker composition of HIV infected cells, and characterize the epigenetic structure of infected cells. Thus, we are poised for the first time to gain a direct understanding of HIV reservoir composition directly ex vivo. Here we will apply this new technique to test the hypothesis that HIV infected CD4+ T cells have a cell surface or epigenetic signature distinct from uninfected CD4+ T cells in ART treated PLWH. We will further pilot a new strategy, scGETseq, capable of simultaneously and separately sequencing euchromatin and heterochromatin to distinguish and profile integrated HIV provirus within the active and latent reservoir of ART treated PLWH. Together our studies have the potential to provide the first full understanding of the unmanipulated CD4+ T cell HIV reservoir directly ex vivo.