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Abstract Background HIV-1 is usually transmitted in the presence of semen. We have shown that semen boosts HIV-1 infection and contains fragments of prostatic acid phosphatase (PAP) forming amyloid aggregates termed SEVI (semen-derived enhancer of viral infection) that promote virion attachment to target cells. Despite its importance for the global spread of HIV-1, however, the effect of semen on virus infection is controversial. Results Here, we established methods allowing the meaningful analysis of semen by minimizing its cytotoxic effects and partly recapitulating the conditions encountered during sexual HIV-1 transmission. We show that semen rapidly and effectively enhances the infectivity of HIV-1, HIV-2, and SIV. This enhancement occurs independently of the viral genotype and coreceptor tropism as well as the virus producer and target cell type. Semen-mediated enhancement of HIV-1 infection was also observed under acidic pH conditions and in the presence of vaginal fluid. We further show that the potency of semen in boosting HIV-1 infection is donor dependent and correlates with the levels of SEVI. Conclusions Our results show that semen strongly enhances the infectivity of HIV-1 and other primate lentiviruses and that SEVI contributes to this effect. Thus, SEVI may play an important role in the sexual transmission of HIV-1 and addition of SEVI inhibitors to microbicides may improve their efficacy.

Lichen sclerosus is a chronic inflammatory condition of unknown etiology that affects the genital and extragenital skin, which can lead to sexual dysfunction and has been associated with vulvar cancer. The vaginal microbiome has a critical role in gynecologic health, but little is known about the microbiome in lichen sclerosus. This study investigated the vaginal and vulvar microbiomes of 27 post-menopausal women with lichen sclerosus. The most abundant genera detected in the vaginal microbiome were Lactobacillus, Gardnerella, and Anaerococcus, while Lactobacillus, Anaerococcus, and Staphylococcus were the most abundant in the vulvar microbiome. The vaginal samples clustered into two main groups, Lactobacillus dominant (n = 6, > 50% microbiome Lactobacillus) and polymicrobial (n = 21) with no dominant genus. The vulvar samples were mainly polymicrobial (n = 25). Actinomyces, Anaerococcus, and Ezakiella in the vaginal microbiome and Actinomyces and Ezakiella in the vulvar microbiome were significantly associated with lichen sclerosus symptoms (adjusted p < 0.05). In this population of post-menopausal women with lichen sclerosus the majority have diverse, non-Lactobacillus dominant microbiomes, which is considered less optimal for gynecologic health based on studies of pre-menopausal women. Actinomyces, Ezakiella, and Anaerococcus were associated with lichen sclerosus symptoms. Understanding the role of these bacteria in lichen sclerosus pathogenesis will be an essential future investigation., (© 2024. The Author(s).)

The vaginal microbiome's role in risk, progression, and treatment of female cancers has been widely explored. Yet, there remains a need to develop methods to understand the interaction of microbiome factors with host cells and to characterize their potential therapeutic functions. To address this challenge, we developed a systems biology framework we term the Pharmacobiome for microbiome pharmacology analysis. The Pharmacobiome framework evaluates similarities between microbes and microbial byproducts and known drugs based on their impact on host transcriptomic cellular signatures. Here, we apply our framework to characterization of the Anti-Gynecologic Cancer Vaginal Pharmacobiome. Using published vaginal microbiome multi-omics data from the Partners PrEP clinical trial, we constructed vaginal epithelial gene signatures associated with each profiled vaginal microbe and metabolite. We compared these microbiome-associated host gene signatures to post-drug perturbation host gene signatures associated with 35 FDA-approved anti-cancer drugs from the Library of Integrated Network-based Cellular Signatures database to identify vaginal microbes and metabolites with high statistical and functional similarity to these drugs. We found that Lactobacilli and their metabolites can regulate host gene expression in ways similar to many anti-cancer drugs. Additionally, we experimentally tested our model prediction that taurine, a metabolite produced by L. crispatus, kills cancerous breast and endometrial cancer cells. Our study shows that the Pharmacobiome is a powerful framework for characterizing the anti-cancer therapeutic potential of vaginal microbiome factors with generalizability to other cancers, microbiomes, and diseases.

Background: The cervicovaginal epithelial barrier is crucial for defending the female reproductive tract against sexually transmitted infections. Hormones, specifically estradiol and progesterone, along with their respective receptor expressions, play an important role in modulating this barrier. However, the influence of estradiol and progesterone on gene and protein expression in the ectocervical mucosa of naturally cycling women is not well understood., Methods: Mucosal and blood samples were collected from Kenyan female sex workers at high risk of sexually transmitted infections. All samples were obtained at two time points, separated by two weeks, aiming for the follicular and luteal phases of the menstrual cycle. Ectocervical tissue biopsies were analyzed by RNA-sequencing and in situ immunofluorescence staining, cervicovaginal lavage samples (CVL) were evaluated using protein profiling, and plasma samples were analyzed for hormone levels., Results: Unsupervised clustering of RNA-sequencing data was performed using Weighted gene co-expression network analysis (WGCNA). In the follicular phase, estradiol levels positively correlated with a gene module representing epithelial structure and function, and negatively correlated with a gene module representing cell cycle regulation. These correlations were confirmed using regression analysis including adjustment for bacterial vaginosis status. Using WGCNA, no gene module correlated with progesterone levels in the follicular phase. In the luteal phase, no gene module correlated with either estradiol or progesterone levels. Protein profiling on CVL revealed that higher levels of estradiol during the follicular phase correlated with increased expression of epithelial barrier integrity markers, including DSG1. This contrasted to the limited correlations of protein expression with estradiol levels in the luteal phase. In situ imaging analysis confirmed that higher estradiol levels during the follicular phase correlated with increased DSG1 expression., Conclusion: We demonstrate that estradiol levels positively correlate with specific markers of ectocervical epithelial structure and function, particularly DSG1, during the follicular phase of the menstrual cycle. Neither progesterone levels during the follicular phase nor estradiol and progesterone levels during the luteal phase correlated with any specific sets of gene markers. These findings align with the expression of estradiol and progesterone receptors in the ectocervical epithelium during these menstrual phases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Bradley, Stern, Franzén Boger, Mousavian, Dethlefsen, Kaldhusdal, Lajoie, Omollo, Bergström, Månberg, Nilsson, Kimani, Burgener, Tjernlund, Sundling, Fowke and Broliden.)

Objectives: Bacterial vaginosis (BV) contributes to poor reproductive health and is characterized by a displacement of Lactobacillus in the vaginal microbiome. However, treatment for BV is limited to antibiotics and half of the women treated experience recurrence within a year. THRIVE (The Study of Host-Bacterial Relationships and Immune Function in Different Vaginal Environments) is a prospective study in Winnipeg, Manitoba, Canada, which is designed to capture the daily variation of the microbiome and host mucosal immunity during treatment. The objective of this study is to identify host and bacterial factors that associate with vaginal microbiome stability to better inform therapeutic interventions., Methods: Women treated for BV, and controls, are followed for 6 months collecting daily vaginal swabs and monthly questionnaires. Comprehensive mucosal sampling, including swabs, cytobrushes, biopsies, and blood are collected at baseline, months 1 and 6 post-enrolment., Results: We performed analysis on the first 52 participants, (19 BV+, 33 BV-). Molecular profiling by 16s RNA sequencing showed 20 women with non-Lactobacillus-dominant microbiomes and 32 with Lactobacillus-dominant microbiomes, with increased microbial diversity in non-Lactobacillus-dominant microbiomes (P = 3.1E-05). A pilot analysis in 2 participants demonstrates that multi-omics profiling of self-collected daily swabs provides high-quality data identifying 73 bacterial species, 1773 mucosal proteins and 117 metabolites. Initial flow cytometry analysis showed an increased cluster of differentiation (CD)4+ T cells and neutrophil activation (CD11b+CD62L neg/dim ) in the positive participant at baseline, while after treatment these shifted and resembled the control participant., Conclusions: This study provides a framework to comprehensively investigate the kinetics of vaginal mucosal microbiome alterations, providing further insight into host and molecular features predicting BV recurrence., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

A substantial percentage of the population remains at risk for cervical cancer due to pre-existing human papillomavirus (HPV) infections, despite prophylactic vaccines. Early diagnosis and treatment are crucial for better disease outcomes. The development of new treatments heavily relies on suitable preclinical model systems. Recently, we established a mouse papillomavirus (MmuPV1) model that is relevant to HPV genital pathogenesis. In the current study, we validated the use of Papanicolaou (Pap) smears, a valuable early diagnostic tool for detecting HPV cervical cancer, to monitor disease progression in the MmuPV1 mouse model. Biweekly cervicovaginal swabs were collected from the MmuPV1-infected mice for viral DNA quantitation and cytology assessment. The Pap smear slides were evaluated for signs of epithelial cell abnormalities using the 2014 Bethesda system criteria. Tissues from the infected mice were harvested at various times post-viral infection for additional histological and virological assays. Over time, increased viral replication was consistent with higher levels of viral DNA, and it coincided with an uptick in epithelial cell abnormalities with higher severity scores noted as early as 10 weeks after viral infection. The cytological results also correlated with the histological evaluation of tissues harvested simultaneously. Both immunocompromised and immunocompetent mice with squamous cell carcinoma (SCC) cytology also developed vaginal SCCs. Notably, samples from the MmuPV1-infected mice exhibited similar cellular abnormalities compared to the corresponding human samples at similar disease stages. Hence, Pap smear screening proves to be an effective tool for the longitudinal monitoring of disease progression in the MmuPV1 mouse model., Importance: Papanicolaou (Pap) smear has saved millions of women's lives as a valuable early screening tool for detecting human papillomavirus (HPV) cervical precancers and cancer. However, more than 200,000 women in the United States alone remain at risk for cervical cancer due to pre-existing HPV infection-induced precancers, as there are currently no effective treatments for HPV-associated precancers and cancers other than invasive procedures including a loop electrosurgical excision procedure (LEEP) to remove abnormal tissues. In the current study, we validated the use of Pap smears to monitor disease progression in our recently established mouse papillomavirus model. To the best of our knowledge, this is the first study that provides compelling evidence of applying Pap smears from cervicovaginal swabs to monitor disease progression in mice. This HPV-relevant cytology assay will enable us to develop and test novel antiviral and anti-tumor therapies using this model to eliminate HPV-associated diseases and cancers., Competing Interests: The authors declare no conflict of interest.

Although effective contraceptives are crucial for preventing unintended pregnancies, evidence suggests that their use may perturb the female genital tract (FGT). A comparative analysis of the effects of the most common contraceptives on the FGT have not been evaluated in a randomized clinical trial setting. Here, we evaluated the effect of three long-acting contraceptive methods: depot medroxyprogesterone acetate(DMPA-IM), levonorgestrel(LNG) implant, and a copper intrauterine device (Cu-IUD), on the endocervical host transcriptome in 188 women from the Evidence for Contraceptive Options and HIV Outcomes Trial (ECHO) trial. Cu-IUD usage showed the most extensive transcriptomic changes, and was associated with inflammatory and anti-viral host responses. DMPA-IM usage was enriched for pathways associated with T cell responses. LNG implant had the mildest effect on endocervical gene expression, and was associated with growth factor signaling. These data provide a mechanistic basis for the diverse influence that varying contraceptives have on the FGT., (Copyright © 2023. Published by Elsevier Inc.)

Purpose of Review: The long-acting reversible intramuscularly-injected contraceptive depot medroxyprogesterone acetate (DMPA-IM) is widely used by cisgender women in Africa. Although DMPA-IM provides reliable contraception, potential effects on the female genital tract (FGT) mucosa have raised concern, including risk of HIV infection. This review summarises and compares evidence from observational cohort studies and the randomised Evidence for Contraceptive Options in HIV Outcomes (ECHO) Trial., Recent Findings: Although previous observational studies found women using DMPA-IM had higher abundance of bacterial vaginosis (BV)-associated bacteria, increased inflammation, increased cervicovaginal HIV target cell density, and epithelial barrier damage, sub-studies of the ECHO Trial found no adverse changes in vaginal microbiome, inflammation, proteome, transcriptome, and risk of viral and bacterial STIs, other than an increase in Th17-like cells. Randomised data suggest that DMPA-IM use does not adversely change mucosal endpoints associated with acquisition of infections. These findings support the safe use of DMPA-IM in women at high risk of acquiring STIs, including HIV., (© 2023. The Author(s).)

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., (© 2023. The Author(s).)

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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests J.M.B. is an employee of Gilead Sciences, outside of the submitted work., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Potential conflicts of interest. M. P. reports participation as an editorial advisory board member for Global Graduate and Postgraduate Research Publishing (unpaid, started October 2022); and a leadership role as a board member of the not-for-profit charity Bridging Villages Inc (unpaid). All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

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., (© 2023. The Author(s).)

Background: Women with a cervicovaginal microbiota dominated by Lactobacillus spp. are at reduced risk of acquiring sexually transmitted infections including HIV, but the biological mechanisms involved remain poorly defined. Here, we performed metaproteomics on vaginal swab samples from young South African women (n = 113) and transcriptomics analysis of cervicovaginal epithelial cell cultures to examine the ability of lactic acid, a metabolite produced by cervicovaginal lactobacilli, to modulate genital epithelial barrier function., Results: Compared to women with Lactobacillus-depleted microbiota, women dominated by vaginal lactobacilli exhibit higher abundance of bacterial lactate dehydrogenase, a key enzyme responsible for lactic acid production, which is independently associated with an increased abundance of epithelial barrier proteins. Physiological concentrations of lactic acid enhance epithelial cell culture barrier integrity and increase intercellular junctional molecule expression., Conclusions: These findings reveal a novel ability of vaginal lactic acid to enhance genital epithelial barrier integrity that may help prevent invasion by sexually transmitted pathogens. Video abstract., (© 2022. The Author(s).)

People living with HIV (PLWH) who are immune nonresponders (INRs) are at greater risk of comorbidity and mortality than are immune responders (IRs) who restore their CD4+ T cell count after antiretroviral therapy (ART). INRs have low CD4+ T cell counts (<350 c/μL), heightened systemic inflammation, and increased CD4+ T cell cycling (Ki67+). Here, we report the findings that memory CD4+ T cells and plasma samples of INRs from several cohorts are enriched in gut-derived bacterial solutes p-cresol sulfate (PCS) and indoxyl sulfate (IS) that both negatively correlated with CD4+ T cell counts. In vitro PCS or IS blocked CD4+ T cell proliferation, induced apoptosis, and diminished the expression of mitochondrial proteins. Electron microscopy imaging revealed perturbations of mitochondrial networks similar to those found in INRs following incubation of healthy memory CD4+ T cells with PCS. Using bacterial 16S rDNA, INR stool samples were found enriched in proteolytic bacterial genera that metabolize tyrosine and phenylalanine to produce PCS. We propose that toxic solutes from the gut bacterial flora may impair CD4+ T cell recovery during ART and may contribute to CD4+ T cell lymphopenia characteristic of INRs.

In Sub-Saharan Africa, young women 15-24 years of age account for nearly 30% of all new HIV infections, however, biological and epidemiological factors underlying this disproportionate infection rate are unclear. In this study, we assessed biological contributors of SIV/HIV susceptibility in the female genital tract (FGT) using adolescent ( n  = 9) and adult ( n  = 10) pigtail macaques (PTMs) with weekly low-dose intravaginal challenges of SIV. Immunological variables were captured in vaginal tissue of PTMs by flow cytometry and cytokine assays. Vaginal biopsies were profiled by proteomic analysis. The vaginal microbiome was assessed by 16S rRNA sequencing. We were powered to detect a 2.2-fold increase in infection rates between age groups, however, we identified no significant differences in susceptibility. This model cannot capture epidemiological factors or may not best represent biological differences of HIV susceptibility. No immune cell subsets measured were significantly different between groups. Inflammatory marker MCP-1 was significantly higher (adj p  = .02), and sCD40L trended higher (adj p  = .06) in vaginal cytobrushes of adults. Proteomic analysis of vaginal biopsies showed no significant (adj p  < .05) protein or pathway differences between groups. Vaginal microbiomes were not significantly different between groups. No differences were observed between age groups in this PTM model, however, these animals may not reflect biological factors contributing to HIV risk such as those found in their human counterparts. This model is therefore not appropriate to explore human adolescent differences in HIV risk. Young women remain a key population at risk for HIV infection, and there is still a need for comprehensive assessment and intervention strategies for epidemic control of this uniquely vulnerable population.

The microbial colonization of the lower female reproductive tract has been extensively studied over the past few decades. In contrast, the upper female reproductive tract including the uterine cavity and peritoneum where the ovaries and fallopian tubes reside were traditionally assumed to be sterile under non-pathologic conditions. However, recent studies applying next-generation sequencing of the bacterial 16S ribosomal RNA gene have provided convincing evidence for the existence of an upper female reproductive tract microbiome. While the vaginal microbiome and its importance for reproductive health outcomes has been extensively studied, the microbiome of the upper female reproductive tract and its relevance for gynecologic cancers has been less studied and will be the focus of this article. This targeted review summarizes the pertinent literature on the female reproductive tract microbiome in gynecologic malignancies and its anticipated role in future research and clinical applications in personalized medicine.

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: The authors have declared that no competing interests exist.

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., (© 2020 The Authors. American Journal of Reproductive Immunology published by John Wiley & Sons Ltd.)

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-β 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.

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., Competing Interests: The authors have declared that no competing interests exist.

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., (© 2019 Månberg et al.)

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., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HIV and pathogenic SIV infection are characterized by mucosal dysfunction including epithelial barrier damage, loss of Th17 cells, neutrophil infiltration, and microbial translocation with accompanying inflammation. However, it is unclear how and when these contributing factors occur relative to one another. In order to determine whether any of these features initiates the cycle of damage, we longitudinally evaluated the kinetics of mucosal and systemic T-cell activation, microbial translocation, and Th17 cell and neutrophil frequencies following intrarectal SIV infection of rhesus macaques. We additionally assessed the colon proteome to elucidate molecular pathways altered early after infection. We demonstrate increased T-cell activation (HLA-DR+) beginning 3-14 days post-SIV challenge, reduced peripheral zonulin 3-14 days post-SIV, and evidence of microbial translocation 14 days post-SIV. The onset of mucosal dysfunction preceded peripheral and mucosal Th17 depletion, which occurred 14-28 days post-SIV, and gut neutrophil accumulation was not observed. Proteins involved in epithelial structure were downregulated 3 days post-SIV followed by an upregulation of immune proteins 14 days post-SIV. These data demonstrate that immune perturbations such as Th17 loss and neutrophil infiltration occur after alterations to epithelial structural protein pathways, suggesting that epithelial damage occurs prior to widespread immune dysfunction.

The mucosal surface of the female genital tract contains physiological, immunological, and microbial components that collectively comprise a functioning "mucosal system" that is critical for reproductive health. Alterations or imbalances to any of these components can have significant consequences for susceptibility to sexually transmitted infections, such as HIV. In recent years the advent of advanced systems biology technologies, such as metaproteomics, has provided new toolsets to studying mucosal systems. Studies have linked an altered mucosal proteome to many HIV risk factors including mucosal inflammation, bacterial vaginosis, hormonal contraceptives, and reduced efficacy of antiretroviral drugs for HIV prevention. Herein we will discuss how metaproteomics has been used to study mucosal system components, including epithelial barriers, inflammation, and the microbiome, with a focus on what alterations may contribute to increased HIV transmission risk in women., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

Risk of HIV acquisition varies, and some individuals are highly HIV-1-exposed, yet, persistently seronegative (HESN). The immunologic mechanisms contributing to this phenomenon are an area of intense interest. As immune activation and inflammation facilitate disease progression in HIV-1-infected persons and gastrointestinal-associated lymphoid tissue is a highly susceptible site for transmission, we hypothesized that reduced gut mucosal immune reactivity may contribute to reduced HIV-1 susceptibility in HESN men with a history of numerous rectal sexual exposures. To test this, we used ex vivo mucosal explants from freshly acquired colorectal biopsies from healthy control and HESN subjects who were stimulated with specific innate immune ligands and inactivated whole pathogens. Immune reactivity was then assessed via cytokine arrays and proteomic analysis. Mucosal immune cell compositions were quantified via immunohistochemistry. We found that explants from HESN subjects produced less proinflammatory cytokines compared with controls following innate immune stimulation; while noninflammatory cytokines were similar between groups. Proteomic analysis identified several immune response proteins to be differentially expressed between HIV-1-stimulated HESN and control explants. Immunohistochemical examination of colorectal mucosa showed similar amounts of T cells, macrophages, and dendritic cells between groups. The results of this pilot study suggest that mucosal innate immune reactivity is dampened in HESN versus control groups, despite presence of similar densities of immune cells in the colorectal mucosa. This observed modulation of the rectal mucosal immune response may contribute to lower risk of mucosal HIV-1 transmission in these individuals.

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., (Copyright © 2017, American Association for the Advancement of Science.)

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., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.)

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., Competing Interests: The authors have declared that no competing interests exist.

Objective: 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., Methods: 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., Results: 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)., Conclusions: 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.