Project Summary/Abstract BV is driver of the HIV epidemic, increasing HIV risk up to 60%. Bacterial Vaginosis (BV) is the most common vaginal condition among women of reproductive age, with a prevalence of 20-60% globally. Mediated by a non- optimal vaginal microbiome and characterized by vaginal inflammation, symptoms can cause extreme discomfort, including burning, vaginal malodor, and vaginal itching. A poorly understood component of BV are its effects on infiltrating immune cells. Tissue resident macrophages may play a role in increased HIV risk, but have not been investigated in the context of BV. The objective of this proposal is to determine the effects of a non-optimal vaginal microbiome on inflammatory macrophage activation and training towards an HIV permissive phenotype by establishing an ex-vivo macrophage stimulation model. Here we propose to assess changes in macrophage activation when exposed to vaginal fluid from a global cohort of women (BV and healthy). Macrophage activation will be characterized using both targeted analyses (HIV associated inflammatory markers) and multi-level proteomics (epigenetic, total proteome). Vaginal fluid will be characterized using metagenomics and metabolomics. Computational analyses and machine learning will allow for determination of the role of unique microbial combinations on macrophage activation. We expect to identify specific microbial compositions that correlate with an HIV promoting macrophage phenotype. This approach is highly innovative because it couples machine learning with an ex vivo macrophage stimulation model to study microbiome- macrophage interactions. Furthermore, vaginal fluid obtained from a global cohort of women in the US, South Africa, and Ghana will be obtained using a citizen scientist model of recruitment which empowers and educates participants. BV frequently recurs, and the vaginal microbiome is complexly variable among women. Completion of the proposed project will advance the field of HIV prevention science, by providing a tool to directly examine macrophage mediated-HIV risk. Additionally, it will provide a platform to test personalized, microbiome-informed, probiotic interventions. Further, these finding will be paradigm-shifting as there is no precedent data to determine if immune cells within the tissue micro-environment can be trained by a non-optimal vaginal microbiome, nor studies to determine how this impacts HIV risk. Trained immunity has been linked to increased risk of HIV infection, but has not been previously studied in the context of bacterial vaginosis. Completion of the proposed study will reveal the effects of the vaginal microbiome on macrophage activation, identifying previously unassessed novel mechanisms of inflammation. This multi-variate approach allows for deep proteomic and metabolic characterization of the macrophage, which will provide data to determine the effects of vaginal fluid on the macrophage training, differentiation and activation. ...