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Rectal use of a 1% tenofovir (TFV) gel is currently being evaluated for HIV prevention. While careful assessment of mucosal safety of candidate microbicides is a primary concern, tools to assess mucosal toxicity are limited. Mass spectrometry-based proteomics is a sensitive and high-throughput technique that can provide in-depth information on inflammation processes in biological systems. In this study, we utilized a proteomics approach to characterize mucosal responses in study participants involved in a phase 1 clinical trial of a rectal TFV-based gel. Project Gel was a phase 1 randomized (1:1), double-blind, multisite, placebo-controlled trial in which 24 participants received rectal TFV or a universal placebo [hydroxyethyl cellulose (HEC)] over a course of 8 daily doses. Rectal mucosal swabs were collected after 0, 1, and 8 doses and were analyzed by label-free tandem mass spectrometry. Differential protein expression was evaluated using a combination of paired (time-effects) and unpaired (across study arm) t-tests, and multivariate [least absolute shrinkage and selection operator (LASSO)] modeling. Within the TFV arm, 7% (17/249, p

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.

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., Author Summary The female genital tract (FGT) is a key mucosal surface in the context of HIV transmission. Lactobacillus species are beneficial to the FGT, while Garderella vaginalis and other anaerobic bacteria are detrimental. Bacterial vaginosis (BV) is an inflammatory condition characterized by an outgrowth of G. vaginalis and other anaerobes, which is linked to increased HIV acquisition rates. However, the mechanism behind this remains unknown. Here, we used a novel proteomic approach to simultaneously evaluate host and bacterial functions in the FGT. We found that women with G. vaginalis-dominated FGT bacterial communities always displayed markers of decreased epithelial barrier integrity, and decreased wound healing capacity. We also demonstrated that the abundance of proteins from G. vaginalis associated with these signatures of disrupted epithelial integrity. Finally, we showed that products derived from G. vaginalis prevented healing of wounded cell monolayers while products derived from L. iners maintained the ability of the cell monolayers to close wounds. This study provides novel mechanistic insight into the link between BV and increased HIV acquisition rates.

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.

Elevated inflammatory cytokines (EMCs) at mucosal surfaces have been associated with HIV susceptibility, but the underlying mechanisms remain unclear. We characterized the soluble mucosal proteome associated with elevated cytokine expression in the female reproductive tract. A scoring system was devised based on the elevation (upper quartile) of at least three of seven inflammatory cytokines in cervicovaginal lavage. Using this score, HIV-uninfected Kenyan women were classified as either having EMC (n=28) or not (n=68). Of 455 proteins quantified in proteomic analyses, 53 were associated with EMC (5% false discovery rate threshold). EMCs were associated with proteases, cell motility, and actin cytoskeletal pathways, whereas protease inhibitor, epidermal cell differentiation, and cornified envelope pathways were decreased. Multivariate analysis identified an optimal signature of 16 proteins that distinguished the EMC group with 88% accuracy. Three proteins in this signature were neutrophil-associated proteases that correlated with many cytokines, especially GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-1β (interleukin-1β), MIP-3α (macrophage inflammatory protein-3α), IL-17, and IL-8. Gene set enrichment analyses implicated activated immune cells; we verified experimentally that EMC women had an increased frequency of endocervical CD4(+) T cells. These data reveal strong linkages between mucosal cytokines, barrier function, proteases, and immune cell movement, and propose these as potential mechanisms that increase risk of HIV acquisition.

BACKGROUND: Increasing evidence suggests that depot medroxyprogesterone acetate (DMPA) and intravaginal practices may be associated with an increased risk of HIV infection; however the mechanisms are not fully understood. This study evaluated the effect of DMPA and intravaginal practices on the genital mucosal proteome and microbiome to gain mechanistic insights. METHODS: Cervicovaginal secretions from 86 Kenyan women including self-reported DMPA (n=23) and non-hormonal contraceptive users (n=63) and women who practice vaginal drying (n=46) were analyzed using tandem-mass spectrometry in a cross sectional manner. RESULTS: We identified 473 human and 486 bacterial proteins from 18 different genera. DMPA use associated with increased hemoglobin and immune activation (HBD HBB ALAD IL36G) and decreased epithelial repair proteins (TFF3 F11R). Vaginal drying associated with increased hemoglobin and decreased phagocytosis factors (AZU1 MYH9 PLAUR PADI4). Injury signatures were exacerbated in DMPA users who also practiced vaginal drying. Vaginal drying associated with more diverse (H-index: 0.71 vs 0.45 p=0.0089) bacterial communities containing Gardnerella vaginalis whereas DMPA showed no significant changes in community composition or diversity. CONCLUSIONS: These findings provide new insights into the impact of DMPA and vaginal drying on mucosal barriers and future investigations are needed to confirm their relationship with HIV risk in women. (c) The Author 2016. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.