Immune activation is increasingly recognized as a critical element of HIV infection and pathogenesis, causing expansion of virus founder populations at the mucosal port of entry and eventual exhaustion of cellular immune effectors. A cohort of HIV-resistant (HIV-R) commercial sex workers (CSW) in Nairobi, Kenya, have increased levels of anti- inflammatory factors in vaginal secretions and reduced peripheral immune activation ("immune quiescence"). The mucosal immune micro-environment underlying HIV susceptibility is well-known to be influenced by concurrent sexually transmitted infections, however the role of commensal microbiota is poorly characterized. Bacterial vaginosis (BV), characterized by a shift from Lactobacillus to Gardnerella and Prevotella as dominant members of vaginal microbiota, is a risk factor for HIV acquisition in studies worldwide. However, the etiology and ecological dynamics of BV remain enigmatic, and the mechanisms by which BV increases HIV susceptibility are not fully defined. Protective functional characteristics of Lactobacillus microbiota, including acid and hydrogen peroxide (H2O2) production, may reinforce physicochemical defences of vaginal mucus, stimulate innate epithelial defences and/or modulate activation status of HIV target cells. Therefore, the goal of this study was to determine if reduced BV and increased Lactobacillus colonization are the basis for resistance to HIV in this cohort. Vaginal specimens from a group of 242 CSW were examined, including microscopic diagnosis of BV, culture-based functional analyses and phylogenetic profiling by ultra-deep sequencing. HIV-R individuals were just as likely to have BV compared to other HIV- negative (HIV-N) individuals, and no more likely to be colonized with acid- or H2O2- ii producing bacteria, however two BV-related phylotypes identified by deep sequencing were significantly more likely to be observed in HIV-N individuals (p=0.0002 and p=0.006). HIV+ individuals were significantly more likely than HIV– individuals to have E. coli detected by deep sequencing (p<0.0001) and less likely to have Lactobacillus crispatus (p=0.0006). A coherent set of differences in culture-based and culture- independent characteristics were observed in individuals with BV diagnoses compared to BV– individuals. This study has generated an unprecedented amount of information regarding the composition, structure and function of the vaginal microbiota in African CSW, fundamentally defining many aspects of BV microbiology. Elucidation of the relationship between complex microbial communities and protective mucosal responses against HIV infection should be a priority for future research.

Immune activation is increasingly recognized as a critical element of HIV infection and pathogenesis, causing expansion of virus founder populations at the mucosal port of entry and eventual exhaustion of cellular immune effectors. A cohort of HIV-resistant (HIV-R) commercial sex workers (CSW) in Nairobi, Kenya, have increased levels of anti- inflammatory factors in vaginal secretions and reduced peripheral immune activation ("immune quiescence"). The mucosal immune micro-environment underlying HIV susceptibility is well-known to be influenced by concurrent sexually transmitted infections, however the role of commensal microbiota is poorly characterized. Bacterial vaginosis (BV), characterized by a shift from Lactobacillus to Gardnerella and Prevotella as dominant members of vaginal microbiota, is a risk factor for HIV acquisition in studies worldwide. However, the etiology and ecological dynamics of BV remain enigmatic, and the mechanisms by which BV increases HIV susceptibility are not fully defined. Protective functional characteristics of Lactobacillus microbiota, including acid and hydrogen peroxide (H2O2) production, may reinforce physicochemical defences of vaginal mucus, stimulate innate epithelial defences and/or modulate activation status of HIV target cells. Therefore, the goal of this study was to determine if reduced BV and increased Lactobacillus colonization are the basis for resistance to HIV in this cohort. Vaginal specimens from a group of 242 CSW were examined, including microscopic diagnosis of BV, culture-based functional analyses and phylogenetic profiling by ultra-deep sequencing. HIV-R individuals were just as likely to have BV compared to other HIV- negative (HIV-N) individuals, and no more likely to be colonized with acid- or H2O2- ii producing bacteria, however two BV-related phylotypes identified by deep sequencing were significantly more likely to be observed in HIV-N individuals (p=0.0002 and p=0.006). HIV+ individuals were significantly more likely than HIV– individuals to have E. coli detected by deep sequencing (p<0.0001) and less likely to have Lactobacillus crispatus (p=0.0006). A coherent set of differences in culture-based and culture- independent characteristics were observed in individuals with BV diagnoses compared to BV– individuals. This study has generated an unprecedented amount of information regarding the composition, structure and function of the vaginal microbiota in African CSW, fundamentally defining many aspects of BV microbiology. Elucidation of the relationship between complex microbial communities and protective mucosal responses against HIV infection should be a priority for future research.