Increased genital mucosal cytokines in Canadian women associate with higher antigen-presenting cells, inflammatory metabolites, epithelial barrier disruption, and the depletion of L. crispatus.

BACKGROUND: Cervicovaginal inflammation has been linked to negative reproductive health outcomes including the acquisition of HIV, other sexually transmitted infections, and cervical carcinogenesis. While changes to the vaginal microbiome have been linked to genital inflammation, the molecular relationships between the functional components of the microbiome with cervical immunology in the reproductive tract are understudied, limiting our understanding of mucosal biology that may be important for reproductive health. RESULTS: In this study, we used a multi'-omics approach to profile cervicovaginal samples collected from 43 Canadian women to characterize host, immune, functional microbiome, and metabolome features of cervicovaginal inflammation. We demonstrate that inflammation is associated with lower amounts of L. crispatus and higher levels of cervical antigen-presenting cells (APCs). Proteomic analysis showed an upregulation of pathways related to neutrophil degranulation, complement, and leukocyte migration, with lower levels of cornified envelope and cell-cell adherens junctions. Functional microbiome analysis showed reductions in carbohydrate metabolism and lactic acid, with increases in xanthine and other metabolites. Bayesian network analysis linked L. crispatus with glycolytic and nucleotide metabolism, succinate and xanthine, and epithelial proteins SCEL and IVL as major molecular features associated with pro-inflammatory cytokines and increased APCs. CONCLUSIONS: This study identified key molecular and immunological relationships with cervicovaginal inflammation, including higher APCs, bacterial metabolism, and proteome alterations that underlie inflammation. As APCs are involved in HIV transmission, parturition, and cervical cancer progression, further studies are needed to explore the interactions between these cells, bacterial metabolism, mucosal immunity, and their relationship to reproductive health. Video Abstract.

Vaginal epithelial dysfunction is mediated by the microbiome, metabolome, and mTOR signaling.

Bacterial vaginosis (BV) is characterized by depletion of Lactobacillus and overgrowth of anaerobic and facultative bacteria, leading to increased mucosal inflammation, epithelial disruption, and poor reproductive health outcomes. However, the molecular mediators contributing to vaginal epithelial dysfunction are poorly understood. Here we utilize proteomic, transcriptomic, and metabolomic analyses to characterize biological features underlying BV in 405 African women and explore functional mechanisms in vitro. We identify five major vaginal microbiome groups: L. crispatus (21%), L. iners (18%), Lactobacillus (9%), Gardnerella (30%), and polymicrobial (22%). Using multi-omics we show that BV-associated epithelial disruption and mucosal inflammation link to the mammalian target of rapamycin (mTOR) pathway and associate with Gardnerella, M. mulieris, and specific metabolites including imidazole propionate. Experiments in vitro confirm that type strain G. vaginalis and M. mulieris supernatants and imidazole propionate directly affect epithelial barrier function and activation of mTOR pathways. These results find that the microbiome-mTOR axis is a central feature of epithelial dysfunction in BV.

Nonoptimal bacteria species induce neutrophil-driven inflammation and barrier disruption in the female genital tract.

Neutrophil recruitment and activation within the female genital tract are often associated with tissue inflammation, loss of vaginal epithelial barrier integrity, and increased risk for sexually transmitted infections, such as HIV-1. However, the direct role of neutrophils on vaginal epithelial barrier function during genital inflammation in vivo remains unclear. Using complementary proteome and immunological analyses, we show high neutrophil influx into the lower female genital tract in response to physiological surges in progesterone, stimulating distinct stromal, immunological, and metabolic signaling pathways. However, despite the release of extracellular matrix-modifying proteases and inflammatory mediators, neutrophils contributed little to physiological mucosal remodeling events such as epithelial shedding or re-epithelialization during transition from diestrus to estrus phase. In contrast, the presence of bacterial vaginosis-associated bacteria resulted in a rapid and sustained neutrophil recruitment, resulting in vaginal epithelial barrier leakage and decreased cell-cell junction protein expression in vivo. Thus, neutrophils are important mucosal sentinels that rapidly respond to various biological cues within the female genital tract, dictating the magnitude and duration of the ensuing inflammatory response at steady state and during disease processes.

Non-Lactobacillus-Dominant and Polymicrobial Vaginal Microbiomes Are More Common in Younger South African Women and Predictive of Increased Risk of Human Immunodeficiency Virus Acquisition.

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.

Gut microbiome signatures linked to HIV-1 reservoir size and viremia control.

BACKGROUND: The potential role of the gut microbiome as a predictor of immune-mediated HIV-1 control in the absence of antiretroviral therapy (ART) is still unknown. In the BCN02 clinical trial, which combined the MVA.HIVconsv immunogen with the latency-reversing agent romidepsin in early-ART treated HIV-1 infected individuals, 23% (3/13) of participants showed sustained low-levels of plasma viremia during 32 weeks of a monitored ART pause (MAP). Here, we present a multi-omics analysis to identify compositional and functional gut microbiome patterns associated with HIV-1 control in the BCN02 trial. RESULTS: Viremic controllers during the MAP (controllers) exhibited higher Bacteroidales/Clostridiales ratio and lower microbial gene richness before vaccination and throughout the study intervention when compared to non-controllers. Longitudinal assessment indicated that the gut microbiome of controllers was enriched in pro-inflammatory bacteria and depleted in butyrate-producing bacteria and methanogenic archaea. Functional profiling also showed that metabolic pathways related to fatty acid and lipid biosynthesis were significantly increased in controllers. Fecal metaproteome analyses confirmed that baseline functional differences were mainly driven by Clostridiales. Participants with high baseline Bacteroidales/Clostridiales ratio had increased pre-existing immune activation-related transcripts. The Bacteroidales/Clostridiales ratio as well as host immune-activation signatures inversely correlated with HIV-1 reservoir size. CONCLUSIONS: The present proof-of-concept study suggests the Bacteroidales/Clostridiales ratio as a novel gut microbiome signature associated with HIV-1 reservoir size and immune-mediated viral control after ART interruption. Video abstract.

Corrigendum: Complement-Opsonized HIV Modulates Pathways Involved in Infection of Cervical Mucosal Tissues: A Transcriptomic and Proteomic Study.

[This corrects the article DOI: 10.3389/fimmu.2021.625649.].

Complement-Opsonized HIV Modulates Pathways Involved in Infection of Cervical Mucosal Tissues: A Transcriptomic and Proteomic Study.

Genital mucosal transmission is the most common route of HIV spread. The initial responses triggered at the site of viral entry are reportedly affected by host factors, especially complement components present at the site, and this will have profound consequences on the outcome and pathogenesis of HIV infection. We studied the initial events associated with host-pathogen interactions by exposing cervical biopsies to free or complement-opsonized HIV. Opsonization resulted in higher rates of HIV acquisition/infection in mucosal tissues and emigrating dendritic cells. Transcriptomic and proteomic data showed a significantly more pathways and higher expression of genes and proteins associated with viral replication and pathways involved in different aspects of viral infection including interferon signaling, cytokine profile and dendritic cell maturation for the opsonized HIV. Moreover, the proteomics data indicate a general suppression by the HIV exposure. This clearly suggests that HIV opsonization alters the initial signaling pathways in the cervical mucosa in a manner that promotes viral establishment and infection. Our findings provide a foundation for further studies of the role these early HIV induced events play in HIV pathogenesis.

An updated review on the effects of depot medroxyprogesterone acetate on the mucosal biology of the female genital tract.

BACKGROUND: Access to safe, effective, and affordable contraception is important for women's health and essential to mitigate maternal and fetal mortality rates. The progestin-based contraceptive depot medroxyprogesterone acetate (DMPA) is a popular contraceptive choice with a low failure rate and convenient administration schedule. AIM: In this review, we compiled observational data from human cohorts that examine how DMPA influences the mucosal biology of the female genital tract (FGT) that are essential in maintaining vaginal health, including resident immune cells, pro-inflammatory cytokines, epithelial barrier function, and the vaginal microbiome MATERIALS AND METHODS: This review focused on the recent published literature published in 2019 and 2020. RESULTS: Recent longitudinal studies show that DMPA use associates with an immunosuppressive phenotype, increase in CD4+CCR5+ T cells, and alterations to growth factors. In agreement with previous meta-analyses, DMPA use is associated with minimal effects of the composition of the vaginal microbiome. Cross-sectional studies associate a more pro-inflammatory relationship with DMPA, but these studies are confounded by inherent weaknesses of cross-sectional studies, including differences in study group sizes, behaviors, and other variables that may affect genital inflammation. DISCUSSION & CONCLUSION: These recent results indicate that the interactions between DMPA and the vaginal mucosa are complex emphasizing the need for comprehensive longitudinal studies that take into consideration the measurement of multiple biological parameters.

Vaginal microbiome-hormonal contraceptive interactions associate with the mucosal proteome and HIV acquisition.

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.

Treatment with Commonly Used Antiretroviral Drugs Induces a Type I/III Interferon Signature in the Gut in the Absence of HIV Infection.

Tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) are used for HIV treatment and prevention. Previously, we found that topical rectal tenofovir gel caused immunological changes in the mucosa. Here, we assess the effect of oral TDF/FTC in three HIV pre-exposure prophylaxis trials, two with gastrointestinal and one with cervicovaginal biopsies. TDF/FTC induces type I/III interferon-related (IFN I/III) genes in the gastrointestinal tract, but not blood, with strong correlations between the two independent rectal biopsy groups (Spearman r = 0.91) and between the rectum and duodenum (r = 0.81). Gene set testing also indicates stimulation of the type I/III pathways in the ectocervix and of cellular proliferation in the duodenum. mRNA sequencing, digital droplet PCR, proteomics, and immunofluorescence confirm IFN I/III pathway stimulation in the gastrointestinal tract. Thus, oral TDF/FTC stimulates an IFN I/III signature throughout the gut, which could increase antiviral efficacy but also cause chronic immune activation in HIV prevention and treatment settings.

The neovaginal microbiome of transgender women post-gender reassignment surgery.

BACKGROUND: Gender reassignment surgery is a procedure some transgender women (TW) undergo for gender-affirming purposes. This often includes the construction of a neovagina using existing penile and scrotal tissue and/or a sigmoid colon graft. There are limited data regarding the composition and function of the neovaginal microbiome representing a major gap in knowledge in neovaginal health. RESULTS: Metaproteomics was performed on secretions collected from the neovaginas (n = 5) and rectums (n = 7) of TW surgically reassigned via penile inversion/scrotal graft with (n = 1) or without (n = 4) a sigmoid colon graft extension and compared with secretions from cis vaginas (n = 32). We identified 541 unique bacterial proteins from 38 taxa. The most abundant taxa in the neovaginas were Porphyromonas (30.2%), Peptostreptococcus (9.2%), Prevotella (9.0%), Mobiluncus (8.0%), and Jonquetella (7.2%), while cis vaginas were primarily Lactobacillus and Gardnerella. Rectal samples were mainly composed of Prevotella and Roseburia. Neovaginas (median Shannon's H index = 1.33) had higher alpha diversity compared to cis vaginas (Shannon's H = 0.35) (p = 7.2E-3, Mann-Whitney U test) and were more similar to the non-Lactobacillus dominant/polymicrobial cis vaginas based on beta diversity (perMANOVA, p = 0.001, r2 = 0.342). In comparison to cis vaginas, toll-like receptor response, amino acid, and short-chain fatty acid metabolic pathways were increased (p < 0.01), while keratinization and cornification proteins were decreased (p < 0.001) in the neovaginal proteome. CONCLUSIONS: Penile skin-lined neovaginas have diverse, polymicrobial communities that show similarities in composition to uncircumcised penises and host responses to cis vaginas with bacterial vaginosis (BV) including increased immune activation pathways and decreased epithelial barrier function. Developing a better understanding of microbiome-associated inflammation in the neovaginal environment will be important for improving our knowledge of neovaginal health. Video Abstract.

Pregnancy associates with alterations to the host and microbial proteome in vaginal mucosa.

PROBLEM: Pregnant women are at increased risk of HIV acquisition, but the biological mechanisms contributing to this observation are not well understood. METHOD OF STUDY: Here, we assessed host immune and microbiome differences in the vaginal mucosa of healthy pregnant and non-pregnant women using a metaproteomics approach. Cervicovaginal lavage (CVL) samples were collected from 23 pregnant and 25 non-pregnant women. RESULTS: Mass spectrometry analysis of CVL identified 550 human proteins and 376 bacterial proteins from 11 genera. Host proteome analysis indicated 56 human proteins (10%) were differentially abundant (P < .05) between pregnant and non-pregnant women, including proteins involved in angiogenesis (P = 3.36E-3), cell movement of phagocytes (P = 1.34E-6), and permeability of blood vessels (P = 1.27E-4). The major bacterial genera identified were Lactobacillus, Gardnerella, Prevotella, Megasphaera, and Atopobium. Pregnant women had higher levels of Lactobacillus species (P = .017) compared with non-pregnant women. Functional pathway analysis indicated that pregnancy associated with changes to bacterial metabolic pathway involved in energy metabolism, which were increased in pregnant women (P = .035). CONCLUSION: Overall, pregnant women showed differences in the cervicovaginal proteome and microbiome that may be important for HIV infection risk.

Plasma concentration of injectable contraceptive correlates with reduced cervicovaginal growth factor expression in South African women.

Long-acting injectable contraceptives have been associated with mucosal immune changes and increased HIV acquisition, but studies have often been hampered by the inaccuracy of self-reported data, unknown timing of injection, and interactions with mucosal transmission co-factors. We used mass spectrometry to quantify the plasma concentrations of injectable contraceptives in women from the CAPRISA004 study (n = 664), with parallel quantification of 48 cytokines and >500 host proteins in cervicovaginal lavage. Higher DMPA levels were associated with reduced CVL concentrations of GCSF, MCSF, IL-16, CTACK, LIF, IL-1α, and SCGF-ß in adjusted linear mixed models. Dose-dependent relationships between DMPA concentration and genital cytokines were frequently observed. Unsupervised clustering of host proteins by DMPA concentration suggest that women with low DMPA had increases in proteins associated with mucosal fluid function, growth factors, and keratinization. Although DMPA was not broadly pro-inflammatory, DMPA was associated with increased IP-10 in HSV-2 seropositive and older women. DMPA-cytokine associations frequently differed by vaginal microbiome; in non-Lactobacillus-dominant women, DMPA was associated with elevated IL-8, MCP-1, and IP-10 concentrations. These data confirm a direct, concentration-dependant effect of DMPA on functionally important immune factors within the vaginal compartment. The biological effects of DMPA may vary depending on age, HSV-2 status, and vaginal microbiome composition.

A High-throughput Bead-based Affinity Assay Enables Analysis of Genital Protein Signatures in Women At Risk of HIV Infection.

Women at high risk of HIV infection, including sex workers and those with active genital inflammation, have molecular signatures of immune activation and epithelial barrier remodeling in samples of their genital mucosa. These alterations in the local immunological milieu are likely to impact HIV susceptibility. We here analyze host genital protein signatures in HIV uninfected women, with high frequency of condom use, living in HIV-serodiscordant relationships. Cervicovaginal secretions from women living in HIV-serodiscordant relationships (n = 62) were collected at three time points over 12 months. Women living in HIV-negative seroconcordant relationships (controls, n = 25) were sampled at one time point. All study subjects were examined for demographic parameters associated with susceptibility to HIV infection. The cervicovaginal samples were analyzed using a high-throughput bead-based affinity assay. Proteins involved in epithelial barrier function and inflammation were increased in HIV-serodiscordant women. By combining several methods of analysis, a total of five proteins (CAPG, KLK10, SPRR3, elafin/PI3, CSTB) were consistently associated with this study group. Proteins analyzed using the affinity set-up were further validated by label-free tandem mass spectrometry in a partially overlapping cohort with concordant results. Women living in HIV-serodiscordant relationships thus had elevated levels of proteins involved in epithelial barrier function and inflammation despite low prevalence of sexually transmitted infections and a high frequency of safe sex practices. The identified proteins are important markers to follow during assessment of mucosal HIV susceptibility factors and a high-throughput bead-based affinity set-up could be a suitable method for such evaluation.

Amniotic fluid proteomic signatures of cervical insufficiency and their association with length of latency.

PROBLEM: Cervical insufficiency is a precursor of preterm birth. Treatment with emergency cervical cerclage is contraindicated in the presence of intra-amniotic infection. Detecting infection with Gram stain and culture of amniotic fluid lacks sensitivity. Proteomic profiling of amniotic fluid in cervical insufficiency may help identify pregnancies best suited for emergency cerclage. METHOD OF STUDY: Thirty-two pregnant women underwent amniocentesis for routine genetic testing (n = 22) or after diagnosis of cervical insufficiency (n = 10). The proteomic profiles of the amniotic fluid samples were compared in a cross-sectional fashion, including sub-analyses of women with cervical insufficiency and latency periods of <1 week and >1 week post-diagnosis. RESULTS: Mean gestational age at diagnosis of cervical insufficiency was 21.4 weeks (95% CI 20.6-22.1). Proteomic analysis yielded 40 (7.2%, P < 0.05) differentially expressed proteins between women with delivery <1 week (n = 6) vs. >1 week (n = 4). Women who delivered <1 week had activated inflammatory response (z = 2.3, P = 6.71E-09), chemotaxis of immune cells (z = 2.9, P = 2.01E-08), and inhibited bacterial growth (z = -2.2, P = 5.82E-05). A multivariate model of eight biomarkers positively associated with cases of <1 week latency and distinguished cases from controls (97.8%, cross-validation accuracy 92.7%, P = 0.0009). CONCLUSION: In this pilot study, significant differences in the amniotic fluid proteomic profiles in cases of cervical insufficiency compared to genetic amniocentesis were observed. Proteomic signatures were predictive of achieving latency > 1 week after diagnosis of cervical insufficiency. These preliminary findings suggest that proteomic analysis may be of value in predicting outcome following cervical insufficiency and warrants further validation in larger studies.

Using safe, affordable and accessible non-steroidal anti-inflammatory drugs to reduce the number of HIV target cells in the blood and at the female genital tract.

INTRODUCTION: At its basic level, HIV infection requires a replication-competent virus and a susceptible target cell. Elevated levels of vaginal inflammation has been associated with the increased risk of HIV infection as it brings highly activated HIV target cells (CCR5+CD4+ T cells; CCR5+CD4+CD161+ Th17 T cells) to the female genital tract (FGT) where they interact with HIV. Decreased HIV risk has been associated with a phenotype of decreased immune activation, called immune quiescence, described among Kenyan female sex workers who were intensely exposed to HIV yet remain uninfected. Current prevention approaches focus on limiting viral access. We took the novel HIV prevention approach of trying to limit the number of HIV target cells in the genital tract by reducing inflammation using safe, affordable and globally accessible anti-inflammatory drugs. METHODS: We hypothesized that the daily administration of low doses of acetylsalicylic acid (ASA 81 mg) or hydroxychloroquine (HCQ 200 mg) would reduce inflammation thereby decreasing HIV target cells at the FGT. Low-risk HIV seronegative women from Nairobi, Kenya were randomized for six weeks therapy of ASA (n = 37) or HCQ (n = 39) and tested to determine the impact on their systemic and mucosal immune environment. RESULTS: The results showed that HCQ use was associated with a significant reduction in the proportion of systemic T cells that were CCR5+CD4+ (p = 0.01) and Th17 (p = 0.01). In the ASA arm, there was a 35% and 28% decrease in the proportion of genital T cells that were CD4+CCR5+ (p = 0.017) and Th17 (p = 0.04) respectively. Proteomic analyses of the cervical lavage showed ASA use was associated with significantly reduced amount of proteins involved in the inflammatory response and cell recruitment at the mucosa, although none of the individual proteins passed multiple comparison correction. These changes were more apparent in women with Lactobacillus dominant microbiomes. CONCLUSION: Together, these data indicate that taking low-dose ASA daily was associated with significant reduction in HIV target cells at the FGT. This study provides proof-of-concept for a novel HIV-prevention approach that reducing inflammation using safe, affordable and globally accessible non-steroidal anti-inflammatory agents is associated with significant reduction in the proportion of HIV-target cells at the FGT.

The vaginal microbiome amplifies sex hormone-associated cyclic changes in cervicovaginal inflammation and epithelial barrier disruption.

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.

Impact of the griffithsin anti-HIV microbicide and placebo gels on the rectal mucosal proteome and microbiome in non-human primates.

Topical microbicides are being explored as an HIV prevention method for individuals who practice receptive anal intercourse. In vivo studies of these microbicides are critical to confirm safety. Here, we evaluated the impact of a rectal microbicide containing the antiviral lectin, Griffithsin (GRFT), on the rectal mucosal proteome and microbiome. Using a randomized, crossover placebo-controlled design, six rhesus macaques received applications of hydroxyethylcellulose (HEC)- or carbopol-formulated 0.1% GRFT gels. Rectal mucosal samples were then evaluated by label-free tandem MS/MS and 16 S rRNA gene amplicon sequencing, for proteomics and microbiome analyses, respectively. Compared to placebo, GRFT gels were not associated with any significant changes to protein levels at any time point (FDR < 5%), but increased abundances of two common and beneficial microbial taxa after 24 hours were observed in HEC-GRFT gel (p < 2E-09). Compared to baseline, both placebo formulations were associated with alterations to proteins involved in proteolysis, activation of the immune response and inflammation after 2 hours (p < 0.0001), and increases in beneficial Faecalibacterium spp. after 24 hours in HEC placebo gel (p = 4.21E-15). This study supports the safety profile of 0.1% GRFT gel as an anti-HIV microbicide and demonstrates that current placebo formulations may associate with changes to rectal proteome and microbiota.

Vaginal bacteria modify HIV tenofovir microbicide efficacy in African women.

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.

Genital Injury Signatures and Microbiome Alterations Associated With Depot Medroxyprogesterone Acetate Usage and Intravaginal Drying Practices.

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.