Lactobacillus-Deficient Cervicovaginal Bacterial Communities Are Associated with Increased HIV Acquisition in Young South African Women

Christina Gosmann, Melis N Anahtar, Scott A Handley, Mara Farcasanu, Galeb Abu-Ali, Brittany A Bowman, Nikita Padavattan, Chandni Desai, Lindsay Droit, Amber Moodley, Mary Dong, Yuezhou Chen, Nasreen Ismail, Thumbi Ndung'u, Musie S Ghebremichael, Duane R Wesemann, Caroline Mitchell, Krista L Dong, Curtis Huttenhower, Bruce D Walker, Herbert W Virgin, Douglas S Kwon, Christina Gosmann, Melis N Anahtar, Scott A Handley, Mara Farcasanu, Galeb Abu-Ali, Brittany A Bowman, Nikita Padavattan, Chandni Desai, Lindsay Droit, Amber Moodley, Mary Dong, Yuezhou Chen, Nasreen Ismail, Thumbi Ndung'u, Musie S Ghebremichael, Duane R Wesemann, Caroline Mitchell, Krista L Dong, Curtis Huttenhower, Bruce D Walker, Herbert W Virgin, Douglas S Kwon

Abstract

Elevated inflammation in the female genital tract is associated with increased HIV risk. Cervicovaginal bacteria modulate genital inflammation; however, their role in HIV susceptibility has not been elucidated. In a prospective cohort of young, healthy South African women, we found that individuals with diverse genital bacterial communities dominated by anaerobes other than Gardnerella were at over 4-fold higher risk of acquiring HIV and had increased numbers of activated mucosal CD4+ T cells compared to those with Lactobacillus crispatus-dominant communities. We identified specific bacterial taxa linked with reduced (L. crispatus) or elevated (Prevotella, Sneathia, and other anaerobes) inflammation and HIV infection and found that high-risk bacteria increased numbers of activated genital CD4+ T cells in a murine model. Our results suggest that highly prevalent genital bacteria increase HIV risk by inducing mucosal HIV target cells. These findings might be leveraged to reduce HIV acquisition in women living in sub-Saharan Africa.

Keywords: HIV acquisition; HIV susceptibility; female genital tract (FGT); mucosal immunology; sub-Saharan Africa; vaginal microbiome.

Conflict of interest statement

The authors declare no competing interests.

Copyright © 2017 Elsevier Inc. All rights reserved.

Figures

Figure 1. Bacterial, but not viral, cervicovaginal…
Figure 1. Bacterial, but not viral, cervicovaginal community structures are distinct in HIV-uninfected women
(A) Stacked bar plot indicating the bacterial composition of the cervicovaginal microbiome of 236 HIV-uninfected women. Samples were grouped according to 4 cervicotypes (CTs), and the alpha diversity is shown below for each sample. (B) Principal coordinates analysis (PCoA) plot of the 236 individuals, distinguished by CT. (C) Normalized reads of alphapapillomaviruses and Anelloviridae detected in CVL fluid, grouped by CT (n=180 individuals). Groups were compared using Kruskal-Wallis test. The percentage of samples in which the indicated virus was detected is shown below. (D) Average linkage clustering of the Bray-Curtis dissimilarity of relative abundances of Caudovirales sequences detected in CVL fluid (n=180 individuals). Lines in the plots indicate the median and the interquartile range (IQR) of the dataset.
Figure 2. Cervicovaginal bacteria impact HIV acquisition
Figure 2. Cervicovaginal bacteria impact HIV acquisition
(A) Kaplan-Meier curve showing HIV infections in each CT group over time, including or excluding individuals with Chlamydia infection. Acquisition curves for CT2 (n=74/65 individuals), CT3 (n=68/57) and CT4 (n=68/54) were compared with the acquisition curve for CT1 (n=23/23). Log-rank (Mantel-Cox) test-based p values and Mantel-Haenszel hazard ratios (HR) are displayed. (B) Distribution of CT groups in participants who remained HIV-uninfected (n=205) versus those who acquired HIV (n=31), shown as % of total, and the difference in distribution between these two groups. (C) Stacked bar plot indicating the bacterial composition of the cervicovaginal microbiome of 31 women who subsequently acquired HIV. Samples were grouped by CT. (D) Bacterial PCoA plot indicating study participants who acquired HIV (in red, n=31) and CT groups for those who remained HIV-uninfected (faded coloring, n=205). See also Figure S1 and Table S1.
Figure 3. High-risk genital bacterial communities are…
Figure 3. High-risk genital bacterial communities are associated with increased HIV target cell numbers and pro-inflammatory cytokines
(A) Representative flow cytometry plots to identify HIV target cells isolated from cervical cytobrushes. Live T cells (CD45+CD3+) were gated on cells expressing CD4, HLA-DR, CD38 and CCR5. (B) Flow cytometry analysis of HIV target cell numbers in cytobrushes from 169 individuals, grouped by CT. (C,D,E) Chemokine and cytokine concentrations in CVL fluid from 219 individuals, grouped by CT. The bottom figure panel in (D) further distinguishes between samples of women who became infected with HIV and those who remained HIV-uninfected. Lines indicate the median and the interquartile range (IQR) of each dataset. Groups were compared using Kruskal-Wallis test with Dunn’s post-hoc analyses. See also Figures S2 and S3.
Figure 4. Specific bacterial taxa are associated…
Figure 4. Specific bacterial taxa are associated with genital inflammation and HIV infection
(A) Bacterial taxa in 219 individuals positively or negatively correlated with cytokine principal component 1 (PC1), an indicator of the presence of genital inflammation. Q-values reflect corrected p-values after multiple testing. (B) Cytokine and chemokine concentrations measured in the supernatant of cervical epithelial cells co-cultured with the indicated bacteria for 24 h (pooled results from 2–3 independent experiments; Kruskal-Wallis test). (C) Bacterial taxa significantly differentially abundant in women who became infected with HIV (n=31) and those who remained uninfected (n=205), including two strains of P. bivia. Error bars indicate the standard error estimate for the log2 fold change in abundance. (D) Germ-free mice were intravaginally inoculated with L. crispatus or P. bivia on day 0 and day 2 and sacrificed on day 6. (E) Numbers of activated (CD44+) CD4+ T cells detected in the female genital tract (FGT) and in 100 μl blood of the gnotobiotic mice on day 6 of the experiment (pooled results from 3 independent experiments; Mann-Whitney test). Lines in the plots indicate the median and the interquartile range (IQR) of the dataset. See also Figure S4.

References

    1. Anahtar MN, Bowman BA, Kwon DS. Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization. Journal of visualized experiments : JoVE 2016
    1. Anahtar MN, Byrne EH, Doherty KE, Bowman BA, Yamamoto HS, Soumillon M, Padavattan N, Ismail N, Moodley A, Sabatini ME, et al. Cervicovaginal bacteria are a major modulator of host inflammatory responses in the female genital tract. Immunity. 2015;42:965–976.
    1. Bradshaw CS, Pirotta M, De Guingand D, Hocking JS, Morton AN, Garland SM, Fehler G, Morrow A, Walker S, Vodstrcil LA, Fairley CK. Efficacy of oral metronidazole with vaginal clindamycin or vaginal probiotic for bacterial vaginosis: randomised placebo-controlled double-blind trial. PLoS One. 2012;7:e34540.
    1. Byrne EH, Anahtar MN, Cohen KE, Moodley A, Padavattan N, Ismail N, Bowman BA, Olson GS, Mabhula A, Leslie A, et al. Association between injectable progestin-only contraceptives and HIV acquisition and HIV target cell frequency in the female genital tract in South African women: a prospective cohort study. The Lancet Infectious Diseases. 2016;16:441–448.
    1. Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJ, Holmes SP. DADA2: High-resolution sample inference from Illumina amplicon data. Nature methods. 2016;13:581–583.
    1. Christensen-Quick A, Lafferty M, Sun L, Marchionni L, DeVico A, Garzino-Demo A. Human Th17 cells lack HIV-inhibitory RNases and are highly permissive to productive HIV infection. J Virol 2016
    1. Faith JJ, Ahern PP, Ridaura VK, Cheng J, Gordon JI. Identifying gut microbe-host phenotype relationships using combinatorial communities in gnotobiotic mice. Sci Transl Med. 2014:6.
    1. Gorgos LM, Sycuro LK, Srinivasan S, Fiedler TL, Morgan MT, Balkus JE, McClelland SR, Fredricks DN, Marrazzo JM. Relationship of Specific Bacteria in the Cervical and Vaginal Microbiotas With Cervicitis. Sex Transm Dis. 2015;42:475–481.
    1. Gosmann C, Mattarollo SR, Bridge JA, Frazer IH, Blumenthal A. IL-17 suppresses immune effector functions in human papillomavirus-associated epithelial hyperplasia. J Immunol. 2014;193:2248–2257.
    1. Haase AT. Early events in sexual transmission of HIV and SIV and opportunities for interventions. Annual review of medicine. 2011;62:127–139.
    1. Handley SA, Desai C, Zhao G, Droit L, Monaco CL, Schroeder AC, Nkolola JP, Norman ME, Miller AD, Wang D, et al. SIV Infection-Mediated Changes in Gastrointestinal Bacterial Microbiome and Virome Are Associated with Immunodeficiency and Prevented by Vaccination. Cell Host Microbe. 2016;19:323–335.
    1. Hay PE, Lamont RF, Taylor-Robinson D, Morgan DJ, Ison C, Pearson J. Abnormal bacterial colonisation of the genital tract and subsequent preterm delivery and late miscarriage. BMJ (Clinical research ed) 1994;308:295–298.
    1. Ivanov II, Atarashi K, Manel N, Brodie EL, Shima T, Karaoz U, Wei D, Goldfarb KC, Santee CA, Lynch SV, et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell. 2009;139:485–498.
    1. Koning FA, Otto SA, Hazenberg MD, Dekker L, Prins M, Miedema F, Schuitemaker H. Low-level CD4+ T cell activation is associated with low susceptibility to HIV-1 infection. J Immunol. 2005;175:6117–6122.
    1. Lamont RF, Nhan-Chang CL, Sobel JD, Workowski K, Conde-Agudelo A, Romero R. Treatment of abnormal vaginal flora in early pregnancy with clindamycin for the prevention of spontaneous preterm birth: a systematic review and metaanalysis. Am J Obstet Gynecol. 2011;205:177–190.
    1. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology. 2014;15:1–21.
    1. Low N, Chersich MF, Schmidlin K, Egger M, Francis SC, van de Wijgert JH, Hayes RJ, Baeten JM, Brown J, Delany-Moretlwe S, et al. Intravaginal practices, bacterial vaginosis, and HIV infection in women: individual participant data meta-analysis. PLoS Med. 2011;8:e1000416.
    1. Marchesi JR, Adams DH, Fava F, Hermes GD, Hirschfield GM, Hold G, Quraishi MN, Kinross J, Smidt H, Tuohy KM, et al. The gut microbiota and host health: a new clinical frontier. Gut. 2016;65:330–339.
    1. Martin HL, Richardson BA, Nyange PM, Lavreys L, Hillier SL, Chohan B, Mandaliya K, Ndinya-Achola JO, Bwayo J, Kreiss J. Vaginal lactobacilli, microbial flora, and risk of human immunodeficiency virus type 1 and sexually transmitted disease acquisition. J Infect Dis. 1999;180:1863–1868.
    1. Masson L, Passmore JA, Liebenberg LJ, Werner L, Baxter C, Arnold KB, Williamson C, Little F, Mansoor LE, Naranbhai V, et al. Genital inflammation and the risk of HIV acquisition in women. Clin Infect Dis. 2015;61:260–269.
    1. McKinnon LR, Karim QA. Factors Driving the HIV Epidemic in Southern Africa. Current HIV/AIDS reports. 2016;13:158–169.
    1. Meditz AL, Haas MK, Folkvord JM, Melander K, Young R, McCarter M, Mawhinney S, Campbell TB, Lie Y, Coakley E, et al. HLA-DR+ CD38+ CD4+ T lymphocytes have elevated CCR5 expression and produce the majority of R5-tropic HIV-1 RNA in vivo. J Virol. 2011;85:10189–10200.
    1. Monaco CL, Gootenberg DB, Zhao G, Handley SA, Ghebremichael MS, Lim ES, Lankowski A, Baldridge MT, Wilen CB, Flagg M, et al. Altered Virome and Bacterial Microbiome in Human Immunodeficiency Virus-Associated Acquired Immunodeficiency Syndrome. Cell Host Microbe. 2016;19:311–322.
    1. Morgan XC, Tickle TL, Sokol H, Gevers D, Devaney KL, Ward DV, Reyes JA, Shah SA, LeLeiko N, Snapper SB, et al. Dysfunction of the intestinal microbiome in inflammatory bowel disease and treatment. Genome Biol. 2012;13:R79.
    1. Myer L, Kuhn L, Stein ZA, Wright TC, Denny L. Intravaginal practices, bacterial vaginosis, and women's susceptibility to HIV infection: epidemiological evidence and biological mechanisms. The Lancet Infectious Diseases. 2005;5:786–794.
    1. Nunn KL, Wang YY, Harit D, Humphrys MS, Ma B, Cone R, Ravel J, Lai SK. Enhanced Trapping of HIV-1 by Human Cervicovaginal Mucus Is Associated with Lactobacillus crispatus-Dominant Microbiota. MBio. 2015;6:e01084–01015.
    1. Ravel J, Gajer P, Abdo Z, Schneider GM, Koenig SS, McCulle SL, Karlebach S, Gorle R, Russell J, Tacket CO, et al. Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci U S A. 2011;108(Suppl 1):4680–4687.
    1. Rodriguez-Garcia M, Barr FD, Crist SG, Fahey JV, Wira CR. Phenotype and susceptibility to HIV infection of CD4+ Th17 cells in the human female reproductive tract. Mucosal Immunol. 2014;7:1375–1385.
    1. Spurbeck RR, Arvidson CG. Lactobacilli at the front line of defense against vaginally acquired infections. Future microbiology. 2011;6:567–582.
    1. Stevenson M, Stanwick TL, Dempsey MP, Lamonica CA. HIV-1 replication is controlled at the level of T cell activation and proviral integration. The EMBO journal. 1990;9:1551–1560.
    1. Stieh DJ, Matias E, Xu H, Fought AJ, Blanchard JL, Marx PA, Veazey RS, Hope TJ. Th17 Cells Are Preferentially Infected Very Early after Vaginal Transmission of SIV in Macaques. Cell Host Microbe. 2016;19:529–540.
    1. UNAIDS, U.J.P.o.H.A. The gap report 2014: Adolescent girls and young women. 2014.
    1. Ward H, Ronn M. Contribution of sexually transmitted infections to the sexual transmission of HIV. Current opinion in HIV and AIDS. 2010;5:305–310.
    1. Woodman Z. Can one size fit all? Approach to bacterial vaginosis in sub-Saharan Africa. Annals of clinical microbiology and antimicrobials. 2016;15:16.

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