The vaginal microbiota: the first line of defense against HIV Jacques Ravel Institute for Genome Sciences Dept. of Microbiology and Immunology University of Maryland School of Medicine Chaire Blaise Pascal Institut Pasteur Fondation de l’Ecole Normale Supérieure Composition of the vaginal microbiota in health but also some more recent unpublished data on the interaction between the vaginal microbiota, the host and Chlamydia. How descriptive data led us to investigate the role of the vaginal microbiota in disease susceptibility.

Conflict of Interest No conflicts of interest to declare

Vaginal Microbiota Lactobacillus spp. often (but not always) dominate the vaginal microbiota in reproductive age women. Lactobacillus spp. are lactic acid producers and acidify the vagina to pH < 4 An acidic environment is thought to restrict the growth of non- indigenous microbes • Self-collected mid-vaginal swabs • Vaginal pH measurements (Inverness VpH glove) • Nugent’s Gram stain scores - estimate of vaginal community disturbance • Questionnaire (health and sexual history, hygiene...) • Determine bacterial community composition by 454 pyrosequencing of barcoded V1-V2 16S rRNA gene Changing environment throughout a women’s lifespan - microbiota changes accordingly

Vaginal community composition Well-powered study of 400 asymptomatic women, equally representing 4 ethnic groups V Five major community state types that differ in their microbial composition and abundance Community state type IV lack significant number of Lactobacillus - higher diversity • Self-collected mid-vaginal swabs • Vaginal pH measurements (Inverness VpH glove) • Nugent’s Gram stain scores - estimate of vaginal community disturbance • Questionnaire (health and sexual history, hygiene...) • Determine bacterial community composition by 454 pyrosequencing of barcoded V1-V2 16S rRNA gene Ravel et al. The vaginal microbiome of reproductive age women. PNAS. 2011. 108 Suppl 1, 4680–4687.

Frequency of community types in ethnic groups We are very much interested in CST IV and we define it as…. Community abundance differ among ethnic groups (χ2 = 36.8 on 10 df P < .0001) when comparing community proportions on the five most common communities Ravel et al. The vaginal microbiome of reproductive age women. PNAS. 2011. 108 Suppl 1, 4680–4687.

Vaginal Microbiota Longitudinal profiles 160 women Daily sampling 10-week study

CST IV: an normal state that carry risks? At any given time, >25% of women are in a non-Lactobacillus dominated state. This state is associated with higher Nugent scores and higher pH High Nugent scores are strongly associated with increased risk of HIV and other sexually transmitted infections Women can be asymptomatic and apparently normal (may be not optimal), but potentially at increased risk of STIs Wiesenfeld HC, et al. : Bacterial vaginosis is a strong predictor of Neisseria gonorrhoeae and Chlamydia trachomatis infection. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 2003, 36(5):663-668. Taha TE, et al. : Bacterial vaginosis and disturbances of vaginal flora: association with increased acquisition of HIV. AIDS (London, England) 1998, 12(13):1699-1706. Cohen, C. R. et al. (2012). Bacterial Vaginosis Associated with Increased Risk of Female-to-Male HIV-1 Transmission: A Prospective Cohort Analysis among African Couples. (H. Watts, Ed.)PLoS Medicine, 9(6), e1001251.

Cervicovaginal mucus, the vaginal microbiota and HIV-1 HIV must penetrate CVM to reach target cells in the vaginal epithelium CVM that retards or immobilizes HIV virions can directly reduce or even prevent initial infection

n=14 n=17 Cervicovaginal mucus (CVM) and HIV-1 Collect CVM from 31 women using Instead Cups Measure the mobility of HIV-1 virus-like particles in fresh CVM Effective diffusion coefficient (Deff) n=14 n=17 Understand the contribution of the vaginal microbiota to these variations in the diffusional barrier properties of CVM samples against HIV Nunn KL, et al. Enhanced Trapping of HIV-1 by Human Cervicovaginal Mucus Is Associated with Lactobacillus crispatus-Dominant Microbiota. MBio 2015, 6 e01084-15

pH, Nugent score and total lactic acid do not explain differences Effect of pH, Nugent score, and lactic acid pH, Nugent score and total lactic acid do not explain differences Nunn KL, et al. Enhanced Trapping of HIV-1 by Human Cervicovaginal Mucus Is Associated with Lactobacillus crispatus-Dominant Microbiota. MBio 2015, 6 e01084-15

D-Lactic acid is associated with increased HIV-1 trapping Effect of isomers of lactic acid D-Lactic acid is associated with increased HIV-1 trapping Nunn KL, et al. Enhanced Trapping of HIV-1 by Human Cervicovaginal Mucus Is Associated with Lactobacillus crispatus-Dominant Microbiota. MBio 2015, 6 e01084-15

D/L lactic acid production - Lactobacillus spp. Witkin, S. S., Mendes-Soares, H., Linhares, I. M., Jayaram, A., Ledger, W. J., & Forney, L. J. (2013). Influence of vaginal bacteria and D- and L-lactic acid isomers on vaginal extracellular matrix metalloproteinase inducer: implications for protection against upper genital tract infections. mBio, 4(4).

Composition of the vaginal microbiota and HIV-1 mobility CVM associated with L. crispatus appears protective, while CVM associated with L. iners is somewhat deficient in trapping HIV-1 Nunn KL, et al. Enhanced Trapping of HIV-1 by Human Cervicovaginal Mucus Is Associated with Lactobacillus crispatus-Dominant Microbiota. MBio 2015, 6 e01084-15

Perspectives Need to redefine dysbiosis of the vaginal microbiota Bacterial vaginosis is a state of the vaginal microbiota that carries risk, but it is not the only one – treating BV appears insufficient Expand our understanding of the mechanisms by which vaginal microbiota provide the first line of defense and leverage the information into better preventive strategies Develop and implement strategies to manipulate (restore) the vaginal microbiota that aims at maintaining optimal protection against STIs

Acknowledgments ravel-lab.org National Institutes of Health Sam Lai Bing Ma Pawel Gajer Kenetta Nunn Larry Forney Vonetta Edwards Rebecca Brotman Ying-Ying Wang Steve Smith Mike Humphrys Li Fu Hongqiu Yang Elias McComb Mishka Terplan Katrina Mark Esther Colinetti National Institutes of Health NIAID U01 - AI070921- R03-AI061131 NIAID UH2 - AI083264 - U19 AI084044