The mechanism(s) by which bacterial communities impact susceptibility to infectious diseases, such as HIV, and maintain female genital tract (FGT) health are poorly understood. Evaluation of FGT ...bacteria has predominantly been limited to studies of species abundance, but not bacterial function. We therefore sought to examine the relationship of bacterial community composition and function with mucosal epithelial barrier health in the context of bacterial vaginosis (BV) using metaproteomic, metagenomic, and in vitro approaches. We found highly diverse bacterial communities dominated by Gardnerella vaginalis associated with host epithelial barrier disruption and enhanced immune activation, and low diversity communities dominated by Lactobacillus species that associated with lower Nugent scores, reduced pH, and expression of host mucosal proteins important for maintaining epithelial integrity. Importantly, proteomic signatures of disrupted epithelial integrity associated with G. vaginalis-dominated communities in the absence of clinical BV diagnosis. Because traditional clinical assessments did not capture this, it likely represents a larger underrepresented phenomenon in populations with high prevalence of G. vaginalis. We finally demonstrated that soluble products derived from G. vaginalis inhibited wound healing, while those derived from L. iners did not, providing insight into functional mechanisms by which FGT bacterial communities affect epithelial barrier integrity.
Alterations to the mucosal environment of the female genital tract, such as genital inflammation, have been associated with increased HIV acquisition in women. As the microbiome and hormonal ...contraceptives can affect vaginal mucosal immunity, we hypothesized these components may interact in the context of HIV susceptibility. Using previously published microbiome data from 685 women in the CAPRISA-004 trial, we compared relative risk of HIV acquisition in this cohort who were using injectable depot medroxyprogesterone acetate (DMPA), norethisterone enanthate (NET-EN), and combined oral contraceptives (COC). In women who were Lactobacillus-dominant, HIV acquisition was 3-fold higher in women using DMPA relative to women using NET-EN or COC (OR: 3.27; 95% CI: 1.24-11.24, P = 0.0305). This was not observed in non-Lactobacillus-dominant women (OR: 0.95, 95% CI: 0.44-2.15, P = 0.895) (interaction P = 0.0686). Higher serum MPA levels associated with increased molecular pathways of inflammation in the vaginal mucosal fluid of Lactobacillus-dominant women, but no differences were seen in non-Lactobacillus dominant women. This study provides data suggesting an interaction between the microbiome, hormonal contraceptives, and HIV susceptibility.
Antiretroviral-based strategies for HIV prevention have shown inconsistent results in women. We investigated whether vaginal microbiota modulated tenofovir gel microbicide efficacy in the CAPRISA ...(Centre for the AIDS Program of Research in South Africa) 004 trial. Two major vaginal bacterial community types—one dominated by Lactobacillus (59.2%) and the other where Gardnerella vaginalis predominated with other anaerobic bacteria (40.8%)—were identified in 688 women profiled. Tenofovir reduced HIV incidence by 61%(P = 0.013) in Lactobacillus-dominant women but only 18% (P = 0.644) in women with non-Lactobacillus bacteria, a threefold difference in efficacy. Detectible mucosal tenofovir was lower in non-Lactobacillus women, negatively correlating with G. vaginalis and other anaerobic bacteria, which depleted tenofovir by metabolism more rapidly than target cells convert to pharmacologically active drug. This study provides evidence linking vaginal bacteria to microbicide efficacy through tenofovir depletion via bacterial metabolism.
Gastrointestinal (GI) mucosal dysfunction predicts and likely contributes to non-infectious comorbidities and mortality in HIV infection and persists despite antiretroviral therapy. However, the ...mechanisms underlying this dysfunction remain incompletely understood. Neutrophils are important for containment of pathogens but can also contribute to tissue damage due to their release of reactive oxygen species and other potentially harmful effector molecules. Here we used a flow cytometry approach to investigate increased neutrophil lifespan as a mechanism for GI neutrophil accumulation in chronic, treated HIV infection and a potential role for gastrointestinal dysbiosis. We report that increased neutrophil survival contributes to neutrophil accumulation in colorectal biopsy tissue, thus implicating neutrophil lifespan as a new therapeutic target for mucosal inflammation in HIV infection. Additionally, we characterized the intestinal microbiome of colorectal biopsies using 16S rRNA sequencing. We found that a reduced Lactobacillus: Prevotella ratio associated with neutrophil survival, suggesting that intestinal bacteria may contribute to GI neutrophil accumulation in treated HIV infection. Finally, we provide evidence that Lactobacillus species uniquely decrease neutrophil survival and neutrophil frequency in vitro, which could have important therapeutic implications for reducing neutrophil-driven inflammation in HIV and other chronic inflammatory conditions.
Abstract
Background
Adolescent girls and young women aged 15‒24 years in sub-Saharan Africa are at disproportionate risk of human immunodeficiency virus (HIV) infection. Given the known association ...between vaginal microbial dysbiosis and HIV susceptibility, we performed an age-stratified analysis of the vaginal microbiome in South African women and compared this to their risk of HIV acquisition.
Methods
Vaginal microbiome data were generated by mass spectrometry–based proteomic analysis of cervicovaginal lavages collected from participants (n = 688) in the Centre for the AIDS Programme of Research in South Africa (CAPRISA) 004 trial. Participants were grouped by age (18–19 years, n = 93; 20–24 years, n = 326; 25–41 years, n = 269).
Results
Four microbiome types were identified based on predominant taxa, including Lactobacillus crispatus (CST-LC, 12.2%), Lactobacillus iners (CST-LI, 43.6%), Gardnerella vaginalis (CST-GV, 26.6%), or polymicrobial (CST-PM, 15.1%). Women aged 18–19 and 20–24 years had increased CST-PM and a non-Lactobacillus-dominant microbiome compared to those 25–41 years (odds ratio OR, 3.14 95% confidence interval {CI}, 1.12–7.87, P = .017; OR, 2.81 95% CI, 1.07–7.09, P = .038, respectively; and OR, 1.65 95% CI, 1.02–2.65, P = .028; OR, 1.40 95% CI, 1.01–1.95, P = .030, respectively). The HIV incidence rate of women with CST-PM microbiome was 7.19-fold higher compared to women with CST-LC (hazard ratio HR, 7.19 95% CI, 2.11–24.5, P = .00162), which was also consistent in women aged 20–24 years (HR, 4.90 95% CI, 1.10–21.9, P = .0375).
Conclusions
Younger women were more likely to have a higher-risk polymicrobial microbiome suggesting that vaginal microbiota are contributing to increased HIV-1 susceptibility in this group.
Clinical Trials Registration
NCT00441298.
Young women are at disproportionate risk of HIV infection. In this study, polymicrobial vaginal microbiomes that predict increased risk of HIV acquisition are more common in younger women, which may be a contributing factor to health disparities in HIV.
Despite the efficacy of antiretroviral-based pre-exposure prophylactics (PrEP) in men who have sex with men, studies in women have produced widely varying outcomes. Recent evidence demonstrates that ...vaginal microbial communities are associated with increased HIV acquisition risk and may impact PrEP efficacy. Here, we investigate the mechanisms underlying how vaginal bacteria alter PrEP drug levels and impact HIV infection rates ex vivo. Using cervicovaginal lavages (CVLs) from women with or without bacterial vaginosis (BV), we identified microbial metabolism of PrEP drugs in BV samples through LC-MS/MS analysis of soluble drug levels and metabolite formation in dual T-cell cultures. CVL samples were assessed for microbiome analysis using sequencing of bacterial 16S rRNA genes. We also observed non-Lactobacillus bacteria that are associated with BV may potentially impact PrEP efficacy through increased HIV infection rates in co-cultures containing Lactobacillus or BV bacteria, PrEP drugs, CEM-GFP cells, and HIV-1LAI virus. Finally, we used these data to develop a novel predictive mathematical simulation modeling system to predict these drug interactions for future trials. These studies demonstrate how dysbiotic vaginal microbiota may impact PrEP drugs and provides evidence linking vaginal bacteria to PrEP efficacy in women.
Background. Increasing evidence suggests depot medroxyprogesterone acetate (DMPA) and intravaginal practices may be associated with human immunodeficiency virus (HIV-1) infection risk; however, the ...mechanisms are not fully understood. This study evaluated the effect of DMPA and intravaginal practices on the genital proteome and microbiome to gain mechanistic insights. Methods. Cervicovaginal secretions from 86 Kenyan women, including self-reported DMPA users (n = 23), nonhormonal contraceptive users (n = 63), and women who practice vaginal drying (n = 46), were analyzed using tandem-mass spectrometry. Results. We identified 473 human and 486 bacterial proteins from 18 different genera. Depot medroxyprogesterone acetate use associated with increased hemoglobin and immune activation (HBD, HBB, IL36G), and decreased epithelial repair proteins (TFF3, F11R). Vaginal drying associated with increased hemoglobin and decreased phagocytosis factors (AZU1, MYH9, PLAUR). Injury signatures were exacerbated in DMPA users who also practiced vaginal drying. More diverse (H index: 0.71 vs 0.45; P = .009) bacterial communities containing Gardnerella vaginalis associated with vaginal drying, whereas DMPA showed no significant association with community composition or diversity. Conclusions. These findings provide new insights into the impact of DMPA and vaginal drying on mucosal barriers. Future investigations are needed to confirm their relationship with HIV risk in women.
HIV infection damages the gut mucosa leading to chronic immune activation, increased morbidities and mortality, and antiretroviral therapies, do not completely ameliorate mucosal dysfunction. ...Understanding early molecular changes in acute infection may identify new biomarkers underlying gut dysfunction. Here we utilized a proteomics approach, coupled with flow cytometry, to characterize early molecular and immunological alterations during acute SIV infection in gut tissue of rhesus macaques. Gut tissue biopsies were obtained at 2 times pre-infection and 4 times post-infection from 6 macaques. The tissue proteome was analyzed by mass spectrometry, and immune cell populations in tissue and blood by flow cytometry. Significant proteome changes (p < 0.05) occurred at 3 days post-infection (dpi) (13.0%), 14 dpi (13.7%), 28 dpi (16.9%) and 63 dpi (14.8%). At 3 dpi, proteome changes included cellular structural activity, barrier integrity, and activation of epithelial to mesenchymal transition (EMT) (FDR < 0.0001) prior to the antiviral response at 14 dpi (IFNa/g pathways, p < 0.001). Novel EMT proteomic biomarkers (keratins 2, 6A and 20, collagen 12A1, desmoplakin) and inflammatory biomarkers (PSMB9, FGL2) were associated with early infection and barrier dysfunction. These findings identify new biomarkers preceding inflammation in SIV infection involved with EMT activation. This warrants further investigation of the role of these biomarkers in chronic infection, mucosal inflammation, and disease pathogenesis of HIV.
Sexual transmission of HIV occurs across a mucosal surface, which contains many soluble immune factors important for HIV immunity. Although the composition of mucosal fluids in the vaginal and oral ...compartments has been studied extensively, the knowledge of the expression of these factors in the rectal mucosa has been understudied and is very limited. This has particular relevance given that the highest rates of HIV acquisition occur via the rectal tract. To further our understanding of rectal mucosa, this study uses a proteomics approach to characterize immune factor components of rectal fluid, using saliva as a comparison, and evaluates its antiviral activity against HIV.
Paired salivary fluid (n = 10) and rectal lavage fluid (n = 10) samples were collected from healthy, HIV seronegative individuals. Samples were analyzed by label-free tandem mass spectrometry to comprehensively identify and quantify mucosal immune protein abundance differences between saliva and rectal fluids. The HIV inhibitory capacity of these fluids was further assessed using a TZM-bl reporter cell line.
Of the 315 proteins identified in rectal lavage fluid, 72 had known immune functions, many of which have described anti-HIV activity, including cathelicidin, serpins, cystatins and antileukoproteinase. The majority of immune factors were similarly expressed between fluids, with only 21 differentially abundant (p<0.05, multiple comparison corrected). Notably, rectal mucosa had a high abundance of mucosal immunoglobulins and antiproteases relative to saliva, Rectal lavage limited HIV infection by 40-50% in vitro (p<0.05), which is lower than the potent anti-HIV effect of oral mucosal fluid (70-80% inhibition, p<0.005).
This study reveals that rectal mucosa contains many innate immune factors important for host immunity to HIV and can limit viral replication in vitro. This indicates an important role for this fluid as the first line of defense against HIV.
Problem
Susceptibility to HIV is associated with the menstrual cycle and vaginal microbiome, but their collective impact on vaginal inflammation remains unclear. Here, we characterized the ...cervicovaginal proteome, inflammation, and microbiome community structure and function during the menstrual cycle.
Method of study
Cervicovaginal secretions were collected from regularly cycling women (n = 16) at median day 10, 16, and 24 of each menstrual cycle and analyzed by mass spectrometry, 16S rRNA gene sequencing, and a multiplex bead array immunoassay. Follicular, ovulatory, and luteal phases were defined by serum sex hormone levels.
Results
Ovulation showed the largest mucosal proteome changes, where 30% and 19% of the 406 human proteins identified differed compared to the luteal and follicular phases, respectively. Neutrophil/leukocyte migration pathways were lowest during ovulation and peaked in the luteal phase, while antimicrobial and epithelial barrier promoting proteins were highest during ovulation. Vaginal microbial community structure and function did not vary significantly during the menstrual cycle, with the majority consistently Lactobacillus‐dominant (63%) or non‐Lactobacillus‐dominant (25%). Fluctuations in the epithelial barrier protein RPTN between the ovulatory and luteal phase were amplified in women with Gardnerella vaginalis and anaerobic bacteria and reduced when Lactobacillus was dominant.
Conclusion
This small study demonstrates that sex hormones modulate neutrophil/leukocyte inflammation, barrier function, and antimicrobial pathways in the female genital tract with the strongest changes occurring during ovulation. The data further suggest a microbiome context for hormone‐driven changes in vaginal immunity which may have implications for HIV susceptibility.
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