ObjectiveTo compare the vaginal microbiota of women engaged in high-risk sexual behaviour (sex work) with women who are not engaged in high-risk sexual behaviour. Diverse vaginal microbiota, low in Lactobacillus species, like those in bacterial vaginosis (BV), are associated with increased prevalence of sexually transmitted infections (STIs) and human immunodeficiency virus (HIV) acquisition. Although high-risk sexual behaviour increases risk for STIs, the vaginal microbiota of sex workers is understudied.MethodsA retrospective cross-sectional study was conducted comparing vaginal microbiota of women who are not engaged in sex work (non-sex worker controls, NSW, N = 19) and women engaged in sex work (female sex workers, FSW, N = 48), using Illumina sequencing (16S rRNA, V3 region).ResultsBacterial richness and diversity were significantly less in controls, than FSW. Controls were more likely to have Lactobacillus as the most abundant genus (58% vs. 17%; P = 0.002) and composition of their vaginal microbiota differed from FSW (PERMANOVA, P = 0.001). Six microbiota clusters were detected, including a high diversity cluster with three sub-clusters, and 55% of women with low Nugent Scores fell within this cluster. High diversity was observed by 16S sequencing in FSW, regardless of Nugent Scores, suggesting that Nugent Score may not be capable of capturing the diversity present in the FSW vaginal microbiota.ConclusionsHigh-risk sexual behaviour is associated with diversity of the vaginal microbiota and lack of Lactobacillus. These factors could contribute to increased risk of STIs and HIV in women engaged in high-risk sexual behaviour.

The hormonal contraceptive medroxyprogesterone acetate (MPA) is associated with increased risk of human immunodeficiency virus (HIV), via incompletely understood mechanisms. Increased diversity in the vaginal microbiota modulates genital inflammation and is associated with increased HIV-1 acquisition. However, the effect of MPA on diversity of the vaginal microbiota is relatively unknown. In a cohort of female Kenyan sex workers, negative for sexually transmitted infections (STIs), with Nugent scores <7 (N=58 of 370 screened), MPA correlated with significantly increased diversity of the vaginal microbiota as assessed by 16S rRNA gene sequencing. MPA was also significantly associated with decreased levels of estrogen in the plasma, and low vaginal glycogen and α-amylase, factors implicated in vaginal colonization by lactobacilli, bacteria that are believed to protect against STIs. In a humanized mouse model, MPA treatment was associated with low serum estrogen, low glycogen and enhanced HIV-1 susceptibility. The mechanism by which the MPA-mediated changes in the vaginal microbiota may contribute to HIV-1 susceptibility in humans appears to be independent of inflammatory cytokines and/or activated T cells. Altogether, these results suggest MPA-induced hypo-estrogenism may alter key metabolic components that are necessary for vaginal colonization by certain bacterial species including lactobacilli, and allow for greater bacterial diversity in the vaginal microbiota..

It is well established that interferon gamma (IFN-γ) production by CD4 T cells is critical for antiviral immunity against herpes simplex virus 2 (HSV-2) genital infection. However, the role of interleukin-17A (IL-17A) production by CD4 T cells in HSV-2 antiviral immunity is yet to be elucidated. Here we demonstrate that IL-17A plays an important role in enhancing antiviral T helper type 1 (T1) responses in the female genital tract (FGT) and is essential for effective protection conferred by HSV-2 vaccination. While IL-17A did not play a critical role during primary genital HSV-2 infection, seen by lack of differences in susceptibility between IL-17A-deficient () and wild-type (WT) C57BL/6 mice, it was critical for mediating antiviral responses after challenge/reexposure. Compared to WT mice, mice (i) infected intravaginally and reexposed or (ii) vaccinated intranasally and challenged intravaginally demonstrated poor outcomes. Following intravaginal HSV-2 reexposure or challenge, vaccinated mice had significantly higher mortality, greater disease severity, higher viral shedding, and higher levels of proinflammatory cytokines and chemokines in vaginal secretions. Furthermore, mice had impaired T1 cell responses after challenge/reexposure, with significantly lower proportions of vaginal IFN-γ CD4 T cells. The impaired T1 cell responses in mice coincided with smaller populations of IFN-γ CD4 tissue resident memory T (T) cells in the genital tract postimmunization. Taken together, these findings describe a novel role for IL-17A in regulating antiviral IFN-γ T1 cell immunity in the vaginal tract. This strategy could be exploited to enhance antiviral immunity following HSV-2 vaccination. T helper type 1 (T1) immunity, specifically interferon gamma (IFN-γ) production by CD4 T cells, is critical for protection against genital herpesvirus (HSV-2) infection, and enhancing this response can potentially help improve disease outcomes. Our study demonstrated that interleukin-17A (IL-17A) plays an essential role in enhancing antiviral T1 responses in the female genital tract (FGT). We found that in the absence of IL-17A, preexposed and vaccinated mice showed poor disease outcomes and were unable to overcome HSV-2 reexposure/challenge. IL-17A-deficient mice () had smaller populations of IFN-γ CD4 tissue resident memory T (T) cells in the genital tract postimmunization than did wild-type (WT) mice, which coincided with attenuated T1 responses postchallenge. This has important implications for developing effective vaccines against HSV-2, as we propose that strategies inducing IL-17A in the genital tract may promote more effective T1 cell immunity and better overall protection.

While the prevalence of Human immunodeficiency virus-1 (HIV-1) infection has stabilized globally, it continues to be the leading cause of death among women of reproductive age. The majority of new infections are transmitted heterosexually, and women have consistently been found to be more susceptible to HIV-1 infection during heterosexual intercourse compared to men. This emphasizes the need for a deeper understanding of how the microenvironment in the female genital tract (FGT) could influence HIV-1 acquisition. This short review focuses on our current understanding of the interplay between estrogen, progesterone, and the cervicovaginal microbiome and their immunomodulatory effects on the FGT. The role of hormonal contraceptives and bacterial vaginosis on tissue inflammation, T cell immunity and HIV-1 susceptibility is discussed. Taken together, this review provides valuable information for the future development of multi-purpose interventions to prevent HIV-1 infection in women.

Herpes Simplex Virus Type 2 (HSV-2) is one of the most prevalent sexually transmitted viruses and is a known risk factor for HIV acquisition in the Female Genital Tract (FGT). Previously, we found that curcumin can block HSV-2 infection and abrogate the production of inflammatory cytokines and chemokines by genital epithelial cells in vitro. In this study, we investigated whether curcumin, encapsulated in nanoparticles and delivered by various in vivo routes, could minimize inflammation and prevent or reduce HSV-2 infection in the FGT. Female mice were pre-treated with curcumin nanoparticles through oral, intraperitoneal and intravaginal routes, and then exposed intravaginally to the tissue inflammation stimulant CpG-oligodeoxynucleotide (ODN). Local intravaginal delivery of curcumin nanoparticles, but not intraperitoneal or oral delivery, reduced CpG-mediated inflammatory histopathology and decreased production of pro-inflammatory cytokines Interleukin (IL)-6, Tumor Necrosis Factor Alpha (TNF-α) and Monocyte Chemoattractant Protein-1 (MCP-1) in the FGT. However, curcumin nanoparticles did not demonstrate anti-viral activity nor reduce tissue pathology when administered prior to intravaginal HSV-2 infection. In an alternative approach, intravaginal pre-treatment with crude curcumin or solid dispersion formulations of curcumin demonstrated increased survival and delayed pathology following HSV-2 infection. Our results suggest that curcumin nanoparticle delivery in the vaginal tract could reduce local tissue inflammation. The anti-inflammatory properties of curcumin delivered to the vaginal tract could potentially reduce the severity of HSV-2 infection and decrease the risk of HIV acquisition in the FGT of women.

The progestin-based hormonal contraceptive Depot Medroxyprogesterone Acetate (DMPA) is widely used in sub-Saharan Africa, where HIV-1 is endemic. Meta-analyses have shown that women using DMPA are 40% more likely than women not using hormonal contraceptives to acquire Human Immunodeficiency Virus (HIV-1). Therefore understanding how DMPA increases susceptibility to HIV-1 is an important public health issue. Using C57BL/6 mice and our previously optimized humanized mouse model (NOD-Rag1tm1Mom Il2rgtm1Wjl transplanted with hCD34-enriched hematopoietic stem cells; Hu-mice) where peripheral blood and tissues are reconstituted by human immune cells, we assessed how DMPA affected mucosal barrier function, HIV-1 susceptibility, viral titres, and target cells compared to mice in the diestrus phase of the estrous cycle, when endogenous progesterone is highest. We found that DMPA enhanced FITC-dextran dye leakage from the vaginal tract into the systemic circulation, enhanced target cells (hCD68+ macrophages, hCD4+ T cells) in the vaginal tract and peripheral blood (hCD45+hCD3+hCD4+hCCR5+ T cells), increased the rate of intravaginal HIV-1 infection, extended the window of vulnerability, and lowered vaginal viral titres following infection. These findings suggest DMPA may enhance susceptibility to HIV-1 in Hu-mice by impairing the vaginal epithelial barrier, increasing vaginal target cells (including macrophages), and extending the period of time during which Hu-mice are susceptible to infection; mechanisms that might also affect HIV-1 susceptibility in women.

38The hormonal contraceptive Medroxyprogesterone Acetate (MPA) is associated with increased 39 risk of Human Immunodeficiency Virus (HIV), via incompletely understood mechanisms. 40 Increased diversity in the vaginal microbiota modulates genital inflammation and is associated 41 with increased HIV-1 acquisition. However, the effect of MPA on diversity of the vaginal 42 microbiota is relatively unknown. In a cohort of female Kenyan sex workers, negative for sexually 43 transmitted infections (STIs), with Nugent Scores <7 (N=58 of 370 screened), MPA correlated 44 with significantly increased diversity of the vaginal microbiota as assessed by 16S rRNA gene 45 sequencing. MPA was also significantly associated with low vaginal glycogen and α-amylase, 46 factors implicated in vaginal colonization by lactobacilli, bacteria believed to protect against STIs. 47Introduction: 56 Meta-analyses suggest the injectable progestin-based contraceptive Depot-Medroxyprogesterone 57 Acetate (DMPA) increases heterosexual acquisition of Human Immunodeficiency Virus (HIV-1) 58 1.4-fold (1), while a prospective study reported women using injectable progestins were at 3.5 59 times increased risk of HIV acquisition compared to women not using long-term hormonal 60 contraceptives (2). These statistics are particularly troubling because DMPA is a popular 61 contraceptive in Africa (3), where HIV-1 prevalence is greatest. Studies have suggested several 62 biological mechanisms, including hypo-estrogenism, by which DMPA might enhance 63 susceptibility to HIV-1 and other sexually transmitted infections (STIs) (4). However, few (5-10) 64 examine the relationship between hormonal contraceptives and the vaginal microbiota (VMB), 65 even though diverse VMB, low in Lactobacillus species, confers a 4-fold increased risk of HIV-1 66 (11). Herein, we determined the effect of MPA (active component of DMPA) on several factors 67 within the vaginal microenvironment of Kenyan sex workers, including diversity of the VMB. 68 69 The VMB is a bacterial community lining vaginal epithelial cells (12). Unlike the diverse gut 70 microbiota, the VMB is generally low in diversity, and five community state types, including four 71 dominated by a Lactobacillus species have been described (12). Two factors thought to maintain 72 vaginal lactobacilli are glycogen, stored in epithelial cells and available as free glycogen in vaginal 73 fluid (13-15), and α-amylase, an enzyme that catabolizes glycogen for use by lactobacilli and other 74 bacteria (16) as energy. Similar to the gut, the VMB modifies immunity. The VMB alters 75 inflammation in the female genital tract (17), and is implicated in reproductive health and disease. 76 Several factors including ethnicity (12), STIs (18), bacterial vaginosis (BV) (19, 20), and sex work 77 (21) have been reported to impact VMB composition. This study was designed to examine the 78 4 effect of hormonal contraceptives on the VMB of healthy, asymptomatic Kenyan sex workers to 79 determine if DMPA is associated with changes...