Vaginal bacteria associated with increased risk of HIV acquisition in African women McClelland RS, Lingappa J, John-Stewart G, Kinuthia, Yuhas K, Jaoko W, Srinivasan S, Mandaliya K, Fiedler T, Munch M, Richardson BA, Overbaugh J, Fredricks DN Disclosure: RSM has a research grant from Hologic Corp, paid to UW

Vaginal bacteria associated with increased risk of HIV acquisition in African women Background: Disruption of the vaginal microbiota associated with risk of HIV. Baseline Characteristics Median (IQR) or N (%)% Controls (N=262) Cases (N=87) Age 29 (23-36) 26 (22-30) Married 199 (76%) 66 (76%) DMPA 37 (14%) 18 (21%) Pregnant 57 (22%) 20 (23%) BV 67 (29%) 32 (42%) Methods Nested case-control Microbiota at pre-SC (N=72) or acute infection (N=15) sample vs. negative controls Characterized microbiota by deep sequencing and qPCR Nested case control in 5 cohorts of women from E and S Africa, and including FSW, PP, and HIV-negative women in DC. Vaginal microbiota were characterized using a combination of broad range PCR with deep sequencing and quantitative pcr. Characterized the microbiota by deep seq, then used the deep sequencing data to select bacterial taxa (some at genus and some at species level) to further investigate using highly sensitive qPCR probes.

Overall vaginal bacterial community diversity in 57 cases versus their 57 matched controls Because of the cost of deep seq, this was performed in a subset of 57 cases 1:1 matched with controls. Stacked colored bars represent taxa >1% relative abundance in controls on left and cases on right. Highest relative abundances of L. iners (orange), G. vag (purple), Prevotella (blue) in both cases and controls. Lower relative abundances of lacto species associated with vaginal health. Diversity (SDI) significantly higher in cases compared to controls. To identify potentially important species for quantitative analysis, Unadjusted logistic regression was applied to the relative abundance data, with case status as the outcome and relative abundance percentage for each taxon separately as the exposure. to identify 15 taxa that were most strongly associated with HIV acquisition for further analysis using qPCR. We also performed backward stepwise regression using the likelihood ratio method to identify taxa whose relative abundance was independently associated with HIV acquisition. Only Gemella genus remained significantly associated with HIV. Shannon Diversity Index higher in cases (median 0.9, IQR 0.4-2.3) vs. controls (median 0.7, IQR 0.1-1.4), p=0.03

Adj. ORs for association between bacterial quantity & HIV acquisition 5 species associated with HIV acquisition Moving to qPCR data, in analyses stratified by cohort, and after adjustment for PCF including age, pregnancy, HC, & sexual risk behaviors numpart, sexfreq, and UPS, 5 species showed significant, concentration-dependent associations with HIV acquisition when reference category (undetectable) was compared to tertiles of increasing bacterial concentration. The other 10 bacterial taxa did not show a statistically significant overall association between concentration and HIV acquisition, and included G. vaginalis, Prevotella genus, 3 species of Porphyromonas, 2 species of Dialister, BVAB2, Aerococcus christinsenii, and A. vaginae. We did create a qPCR probe for Prevotella, based on its observed association in the relative abundance analysis, but it did not show a significant concentration-dependent association with HIV in this dataset. While not possible to prove causality in observational study, these associations could plausibly be related to the effects of these species on vaginal mucosal immune activation, HIV inducing factors, effects on integrity of physical and chemical barriers like mucus and pH. May act together as high risk communities; was not possible to perform multivariable analysis of quant with >1 species because of collinearity. Two important messages: Key bacteria that are often present in the complex vaginal microbiota more commonly seen in African women may place them at greater risk for HIV acquisition. Hypothesis that interventions that reduce concentration or eliminate these species could lower women’s risk of HIV acquisition, though it is difficult to see how this hypothesis could be directly tested, given the current range of effective HIV prevention interventions (prep tasp) that would need to be offered to women in any new HIV prevention trial addressing vaginal microbiota. N=87 cases + 262 controls; undetectable compared to 1st, 2nd, 3rd tertile

Acknowledgements We gratefully acknowledge the contributions of the study participants, our clinical, laboratory and administrative staff. Funding: NIH P01 HD64915