Viral determinants of Bacterial Vaginosis and HIV acquisition risk in the female genital tract - The female genital tract (FGT) is colonized by millions of microbial taxa that have far reaching effects on reproductive health. Resident bacteria in the FGT affect mucosal immune responses, alter the risk of STI acquisition, and predict maternal health and birth outcomes, with lactobacilli typically associated with reduced overall bacterial diversity, inflammation, and risk of STI acquisition. Conversely, communities with little representation of Lactobacillus and an increased abundance of diverse anaerobes are characterized as Bacterial Vaginonsis (BV) and are associated with elevated levels of inflammatory cytokines, heightened risk of STI acquisition and adverse birth outcomes, vaginal discharge, and other sequelae. However, the mechanisms underlying transitions to BV and its high recurrence rates after antibiotic treatment (~50%) remain unknown. The vaginal virome remains largely uncharacterized outside of a few families of DNA viruses associated with adverse conditions (Herpesviridae, Papillomaviridae), with the RNA virome even less studied. Bacteriophages, including integrated prophages, are powerful mediators of bacterial community structure and dynamics across environments, though we know little of bacterial-phage interactions in the the FGT. This study proposes that the vaginal virome is one of the factors influencing transitions to BV, either by eliciting mucosal immune responses directly, or via bacterial-phage dynamics, including prophage induction via abiotic stressors. This proposal will utilize samples already collected from the Evidence for Contraceptive Options and HIV Outcomes (ECHO) Trial to longitudinally characterize (via shotgun metagenomics) interactions between viral and bacterial taxa in participants who do not experience BV (20), participants who experience and clear incident BV (20), and participants who experience persistent BV (20) (Aim 1). We will use strain-resolved analyses to identify alterations in prophage abundance, as well as non-integrated viruses, and we will integrate these data with measurements of 27 cytokines measured previously. In Aim 2, we will characterize the vaginal viral and bacterial metagenomes in our full cohort of participants that seroconverted (28) during our substudy and compare those with the same number of matched controls, identifying alterations in the abundance and functionality of bacterial and viral taxa between groups. In Aim 3, we will follow up the results from our previous work demonstrating that a copper intrauterine device (Cu-IUD) shifted vaginal bacteria toward BV states. We will sequence the bacterial and viral metagenomes across the full subset of women randomized to the Cu-IUD (60) prior to initiation and after six months of use to identify if Cu2+ released from the IUD induced prophages into lytic replication using strain resolved analyses similar to Aim 1, with special attention to lactobacilli prophages. We will assess the results from our metagenomic analyses in vitro via culture experiments using Cu2+ for prophage induction. Together, these aims will provide a characterization of the vaginal virome and its contributions to BV and associated sequelae, including HIV acquisition.
