Project Summary Vaginal washing is a common practice that many women perceive as hygienic. However, vaginal washing has been linked to adverse reproductive health outcomes including increased HIV acquisition risk. The mechanism linking vaginal washing to HIV risk remains unknown. Although it has been hypothesized that disruption of vaginal microbiota may mediate the relationship between vaginal washing and HIV acquisition, results of studies evaluating this relationship are mixed. In preliminary studies, we found associations between vaginal washing and higher concentrations of IL-1 in cervicovaginal fluid and CD4+ T cells from cervical biopsy specimens. These associations were independent of the presence of bacterial vaginosis (BV), leading us to hypothesize that vaginal washing may increase HIV susceptibility by causing persistent activation of the IL-1 pathway, recruitment of HIV-susceptible target cells, and disruption of the mucosal barrier. The primary objective of this proposal is to test the hypothesis that a vaginal washing cessation intervention will lower concentrations of soluble inflammatory mediators in cervicovaginal fluid, lower total immune cells in mucosal tissue, reduce cervical epithelial disruption, and increase concentrations of protective vaginal Lactobacillus spp compared to control. These biologic changes have the potential to reduce HIV susceptibility based on substantial evidence that cervicovaginal inflammation, tissue breakdown, the absence of a Lactobacillus-dominated vaginal microbiota, and the presence of suboptimal vaginal bacteria increase HIV susceptibility. To achieve this objective, we will conduct a randomized controlled trial of vaginal washing cessation in a cohort of women who engage in sex work in Mombasa, Kenya, and compare the following outcomes between participants randomized to the intervention versus control arms of the study: i) concentrations of cervicovaginal cytokines, measured using a multiplex bead- based immunoassay (Luminex platform); ii) concentrations of activated CD4+ T cells and antigen presenting cells, measured from cervical biopsy specimens using flow cytometry; iii) expression of mucin and tight junction proteins detected by immunofluorescent staining of fixed cervical biopsy tissue sections and quantified using HALO image analysis software; iv) presence of cultivable Lactobacillus spp; and, v) concentrations of key Lactobacillus spp measured using quantitative polymerase chain reaction. Together, these data will provide mechanistic evidence for a causal link between vaginal washing and HIV acquisition risk and could be used to support expansion of public health programs to reduce vaginal washing and lower HIV susceptibility among populations at increased risk for HIV acquisition, such as women who engage in sex work. Furthermore, the detailed evaluation of the cervicovaginal immune system and mucosa may provide valuable insight into the mechanisms underlying other adverse reproducti...