Project Summary HIV remains a global epidemic and, in the areas of greatest prevalence, disproportionately infects young women. However, most studies of hyperacute and acute HIV infection of the female genital tract (FGT) are conducted in in vitro, ex vivo, or nonhuman primate (NHP) models due to practical and ethical concerns. Because of this, the immune response to HIV in the human FGT during the earliest stages of infection is largely undefined and the degree to which current model systems recapitulate the events of natural infection is unclear. Critically, the first cells to be infected following natural HIV infection of the FGT remain unidentified, creating a major hurdle toward the development of interventions effect at preventing infection. The FRESH (Females Rising through Education, Support, and Health) cohort in KwaZulu-Natal, South Africa, is a long-running program that is designed to identify natural, hyperacute HIV infection and collect a variety of samples in a longitudinal manner, presenting an unprecedented opportunity to examine the immune and viral dynamics of the FGT during hyperacute and acute HIV infection. By utilizing cutting-edge transcriptomic techniques that have been validated by our group, we will be able to elucidate the events within the FGT during this critical time period of infection. Our overall hypothesis is that the FGT will have a strong inflammatory response that peaks earlier than the systemic response in peripheral blood and that early inflammation will primarily be driven by antigen-presenting cells (APCs), particularly plasmacytoid dendritic cells (pDCs), while CD4+ T cell subsets will bear the greatest burden of genital infection. In the first Aim of this proposal, we will investigate the immune response to genital HIV infection via population-level RNA sequencing using longitudinally collected bulk cellular population samples from hyperacutely and acutely infected FRESH participants. We will also conduct a single cell transcriptomic analysis of cells from the genital tract at the earliest time point following the detection of infection to investigate which specific cellular subpopulations are responsible for inflammation and immune modulation. These analyses will be supplemented by Luminex cytokine studies and immunofluorescence (IF) imaging of cervicovaginal biopsy tissues from FRESH participants. In the second Aim, we will investigate the viral dynamics of hyperacute genital HIV infection by identifying and visualizing infected cells using single cell RNA sequencing and tandem in situ hybridization (ISH) and IF imaging. Additionally, we will examine the diversity of viral quasispecies present in the FGT and in plasma at this time point to better ascertain the frequency with which local foci of infection in the FGT successfully develop to establish systemic infection. The proposed studies will for the first time enable a clear outline of key early immunological and viral events that occur during hyper...