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Lactobacillus-Deficient Cervicovaginal Bacterial Communities Are Associated with Increased HIV Acquisition in Young South African Women
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Immunity Jan 2017 - Christina Gosmann,1,2 Melis N. Anahtar,1,2 Scott A. Handley,3 Mara Farcasanu,1 Galeb Abu-Ali,4 Brittany A. Bowman,1 Nikita Padavattan,5 Chandni Desai,3 Lindsay Droit,3 Amber Moodley,6 Mary Dong,1,6 Yuezhou Chen,7 Nasreen Ismail,5 Thumbi Ndung'u,1,5 Musie S. Ghebremichael,1,2 Duane R. Wesemann,7 Caroline Mitchell,8 Krista L. Dong,1,6 Curtis Huttenhower,4 Bruce D. Walker,1,9,10,11 Herbert W. Virgin,3 and Douglas S. Kwon1,2,10,12,*
Highlights
⋅ Women with high-diversity genital bacterial communities acquire HIV at 4× higher rates
⋅ Activated mucosal CD4+ T cell numbers are elevated in women with high-risk bacteria
⋅ Specific genital bacterial taxa are linked with reduced or elevated HIV acquisition
⋅ I.vag administered Prevotella increases activated genital CD4+ T cell numbers in mice
"Women with high-diversity genital bacterial communities acquire HIV at 4× higher rates....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.
......Clinical studies assessing the efficacy of regimens such as antibiotics and probiotic Lactobacillus vaginal suppositories in modifying the genital microbiome of women with bacterial vaginosis showed significant recurrence rates (Bradshaw et al., 2012; Woodman, 2016). Our findings underscore the importance of developing more effective regimens that achieve a sustained alteration of the genital microbiome since this could potentially reduce HIV acquisition. In this context, strategies targeting specific bacterial taxa associated with reduced or increased HIV acquisition might be useful, although the host mechanisms that maintain specific microbial communities in the genital tract need to be better understood. Our results advance our understanding of the cervicovaginal microbiome as an HIV risk factor and demonstrate the importance of considering the microbiome in the development of new treatments and preventive strategies to reduce HIV acquisition in young women living in sub-Saharan Africa."
Summary
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.
Introduction
24 million people in sub-Saharan Africa are infected with HIV, with the majority of new transmission events occurring following heterosexual sex. Young African women have an up to 8-fold increased HIV prevalence compared to young men (UNAIDS, 2014), emphasizing the need for a better understanding of the factors in the female genital tract (FGT) that influence HIV acquisition.
Elevated inflammation is associated with an increased risk of HIV infection (Masson et al., 2015), and cervicovaginal bacteria have been shown to impact baseline inflammation in the FGT (Anahtar et al., 2015), suggesting a potential role for FGT bacteria in modulating acquisition risk. A small number of prospective studies indicate that women with cervicovaginal bacteria deficient in lactobacilli, a bacterial genus considered beneficial for vaginal health (Spurbeck and Arvidson, 2011), are at higher risk of acquiring HIV (Low et al., 2011; Martin et al., 1999; Myer et al., 2005). However, the depth of these studies has been limited by the use of Gram-stained vaginal smears to assess the genital bacterial composition and by investigation of sex-worker and clinical cohorts not representative of the average healthy female population.
The recent development of high-throughput sequencing technologies to characterize the human microbiome has significantly improved our understanding of the role of the microbiome in diseases such as obesity, inflammatory bowel disease, colorectal cancer (Marchesi et al., 2016), and HIV-induced AIDS (Handley et al., 2016; Monaco et al., 2016). While these studies have focused on the enteric microbiome, less is understood regarding the role of the cervicovaginal microbiome in disease susceptibility, particularly in the developing world. Additionally, the majority of microbiome studies have focused on bacteria while viruses have remained incompletely characterized, particularly in the FGT.
Here we performed bacterial 16S ribosomal (r)RNA gene and viral shotgun sequencing of cervicovaginal microbiota and assessed their role in HIV acquisition in participants of the FRESH (Females Rising through Education, Support and Health) study, a prospective cohort study of healthy HIV-uninfected black South African women aged 18–23 years, monitored with high frequency testing for incident HIV infection. We demonstrated a differential association of four distinct bacterial community structures with genital inflammation, cervical HIV target cell numbers, and HIV acquisition. We additionally identified specific bacterial taxa that were closely associated with genital inflammation and HIV acquisition and showed that these same bacteria induced inflammation in in vitro and in vivo models, thus establishing their mechanistic potential for directly increasing HIV acquisition.
Women with Anaerobic FGT Bacteria Have Increased Genital HIV Target Cell Numbers and Th17-Associated Cytokines
In most women, HIV infection is established following viral replication in CD4+ T cells present in the genital mucosa (Haase, 2011). Activated CD4+ T cells expressing the HIV co-receptor CCR5 ("HIV target cells") are not only more prone to infection but also support higher degrees of viral replication than resting cells (Meditz et al., 2011; Stevenson et al., 1990), thus increasing HIV susceptibility (Haase, 2011; Koning et al., 2005). We examined the relationship between the bacterial microbiome and HIV target cell numbers in the genital tract and observed a 17-fold increase in HIV target cells in the FGT of women with CT4 versus L. crispatus-dominant communities (Figures 3A and 3B; Figure S2). Consistent with this finding, we demonstrated elevated levels of the chemokines MIP-1α and MIP-1β, which attract CCR5-expressing cells, in women with high-diversity bacterial communities (Figure 3C). We also observed higher concentrations of cytokines and chemokines associated with increased HIV acquisition (Masson et al., 2015), such as interleukin-1α (IL-1α), tumor necrosis factor-α (TNF-α), IL-8 (Figure 3D; upper row), IL-12p70, interferon-γ (IFN-γ), and IL-10 (Figure S3A), which confirmed our previous findings in a smaller cohort (Anahtar et al., 2015). Women who went on to become infected with HIV had similar pro-inflammatory cytokine levels as those who remained HIV-uninfected within the same CT group. This suggests comparable HIV susceptibility among women with similar genital communities (Figure 3D; lower row).
Discussion
Here we presented a comprehensive characterization of the genital bacterial and viral microbiome in healthy young women in sub-Saharan Africa, and identified a significant association of distinct cervicovaginal bacterial communities and specific bacterial taxa with HIV acquisition. The sequencing-based identification of cervicovaginal bacteria and viruses provides a substantial advancement over previous prospective studies that used Gram-stained vaginal smears (Low et al., 2011; Martin et al., 1999; Myer et al., 2005).
Our results indicate that high-diversity, low Lactobacillus abundance bacterial communities in the FGT negatively impact vaginal health by increasing the risk of HIV acquisition. In addition, prior studies have reported associations between Lactobacillus-deficient genital microbiota and other poor reproductive outcomes such as preterm delivery, late miscarriage (Hay et al., 1994; Lamont et al., 2011), and cervicitis (Gorgos et al., 2015). The increased rate of HIV acquisition in women with high-diversity, low Lactobacillus abundance bacterial communities observed in our study could be explained by the sensing of specific bacterial antigens or components such as lipopolysaccharide (LPS); we previously demonstrated that CT4 communities were enriched in LPS relative to CT1, and that LPS signaling pathways were significantly upregulated in cervical antigen-presenting cells of women with high-diversity communities (Anahtar et al., 2015).
This likely results in activation and recruitment of HIV target cells to the female genital mucosa. In support of this hypothesis, we show a 17-fold increase in HIV target cell numbers in the FGT mucosa of women with CT4 versus CT1 communities. This finding is further corroborated by the presence of increased numbers of activated CD4+ T cells in the genital mucosa of germ-free mice intravaginally inoculated with P. bivia compared to mice inoculated with L. crispatus, providing direct evidence of the impact of these bacteria on the recruitment of activated CD4+ T cells to the FGT. Increased concentrations of Th17-inducing cytokines IL-23 and IL-1β, and of IL-17 in the presence of CT3 and CT4 bacteria further indicated the presence of elevated numbers of Th17 cells, which are particularly susceptible to HIV infection and have been described as the first cells to be infected by lentivirus in the FGT (Christensen-Quick et al., 2016; Stieh et al., 2016). While the development of the Th17 subset in human epithelial tissues is not fully understood, in murine models, Th17 cells are specifically induced by bacteria in the gut (Ivanov et al., 2009). Thus, the bacterial composition of the FGT microbiome might influence HIV susceptibility not only by increasing local HIV target cell numbers, but also by influencing T helper cell differentiation within the genital mucosa. Whether FGT bacteria contribute to shaping mucosal T helper cell subsets will need to be addressed in future studies.
Reduced HIV acquisition in women with L. crispatus-dominant communities might be based on their ability to impair the growth of inflammatory bacteria and pathogens through the production of lactic acid, hydrogen peroxide, and bactericidal compounds (Spurbeck and Arvidson, 2011), thus diminishing inflammation and HIV target cell numbers. Consistent with this hypothesis, L. crispatus did not co-exist with the more diverse anaerobic communities associated with HIV acquisition. In contrast, L. iners was frequently found in CT3 and CT4 communities, and 29% of the women who became infected with HIV had L. iners-dominant communities while none had L. crispatus dominance. This observation might further be explained by reduced HIV virion diffusion rates in the mucus from women with L. crispatus dominated vaginal microbiota compared to women with L. iners dominance (Nunn et al., 2015). These findings identify L. crispatus as a Lactobacillus species associated with decreased HIV infection and highlight the importance of distinguishing between different lactobacilli in the context of HIV susceptibility. Our findings further indicate that individuals within a particular CT group are at similar risk of acquiring HIV. This suggests that more than half the population of young black South African women is at increased HIV infection risk based on the composition of their cervicovaginal microbiome.
The absence of distinct viral signatures in women with different bacterial communities suggests a primary role for bacteria in modulating HIV acquisition risk. However, it is possible that a greater depth of sequencing, or expansion of viral databases will reveal associations that were not observed here. The stability in bacteriophage community structure across CT groups further suggests that the observed bacterial communities were not being modified by bacteriophage predation, a mechanism that could explain the formation of distinct bacterial communities in different individuals.
Clinical studies assessing the efficacy of regimens such as antibiotics and probiotic Lactobacillus vaginal suppositories in modifying the genital microbiome of women with bacterial vaginosis showed significant recurrence rates (Bradshaw et al., 2012; Woodman, 2016). Our findings underscore the importance of developing more effective regimens that achieve a sustained alteration of the genital microbiome since this could potentially reduce HIV acquisition. In this context, strategies targeting specific bacterial taxa associated with reduced or increased HIV acquisition might be useful, although the host mechanisms that maintain specific microbial communities in the genital tract need to be better understood. Our results advance our understanding of the cervicovaginal microbiome as an HIV risk factor and demonstrate the importance of considering the microbiome in the development of new treatments and preventive strategies to reduce HIV acquisition in young women living in sub-Saharan Africa.
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