Topical microbicides formulated for vaginal use may help stem further spread of HIV-1 by reducing the number of new infections in women. One microbicide strategy under consideration is to strengthen the natural defenses in the vaginal mucosa where HIV-1 transmission takes place. Lactobacillus species are the predominant commensal bacteria found in genital tract secretions of healthy women. Under anaerobic conditions, these bacteria produce lactic acid and certain strains also produce hydrogen peroxide, a potent antimicrobial that has been shown to inactivate HIV-1 in vitro. Lactic acid bacteria also produce an array of antimicrobial molecules, termed bacteriocins, that are effective against competing organisms in the local milieu. Whether these natural antimicrobial factors can inhibit transmission and replication of HIV-1 in vaginal tissues is unknown. In preliminary studies, we have shown that conditioned media (CM) from cultures of Lactobacillus rhamnosus GG (LGG) inhibits replication of HIV-1 by 2 to 4 logs10 in primary CD4+ T lymphocytes, and this antiviral activity is distinct from both lactic acid and hydrogen peroxide. We hypothesize that natural bacteriocin-like molecule(s) produced by commensal lactic acid bacteria can enhance innate immuity in the vaginal mucosa and can inhibit infection and/or replication of HIV-1 in these target tissues. The goal of studies proposed in the exploratory (R21) phase is to purify and identify the low molecular weight active factor(s) produced by LGG bacteria, and determine the extent to which the LGG-purified factors (LGG-PF): 1) inhibit replication of HIV-1 in vitro, 2) modulate cell activation, proliferation and transcriptional regulation, and 3) affect the secretion of innate immune factors from primary epithelial cells from the human female reproductive tract. In the developmental (R33) phase, we will evaluate the effect of LGG-PF on HIV-1 infection and secretion of innate immune factors in primary explant cultures of human cervical and vaginal tissues, and will further evaluate cervicovaginal toxicity and inflammation in a mouse model developed for preclinical evaluation of topical vaginal microbicides. If the purified factor(s) secreted by LGG bacteria are shown to inhibit HIV-1 replication in relevant target cells in vitro, and modulate expression of innate immune factors in female genital tract tissue explants, this would provide evidence of a novel and beneficial effect that extends well beyond the established antimicrobial function of these bacteria. Moreover these results would lay the foundation for application of lactobacilli-derived products as HIV microbicides either alone or in combination with targeted compounds that block HIV-1 infection and replication.