Vaginal microbicides are a female-controlled HIV prevention option whose early development faced challenges that necessitated the search for safety biomarkers in clinical trials. We started out with a group of 30 healthy Caucasian women in Antwerp, Belgium and characterized cellular and soluble immunity and the vaginal microbiome in the lower female genital tract. We also conducted both a cross-sectional and longitudinal characterization of the female genital tract in 430 sexually active women from Sub-Saharan Africa (Kenya, Rwanda and South Africa).

As generally expected, bacteria that are generally linked with good vaginal health, L. crispatus and L. vaginalis, were associated with lower levels of pro-inflammatory cytokines and higher levels of protective antimicrobial proteins. In contrast, BV-associated A. vaginae, G. vaginalis and P. bivia were associated with increased pro-inflammatory cytokines. Vaginal dysbiosis (specifically BV) was characterized by a clear pro-inflammatory signature and reduced levels of IP-10, SLPI and total protein.

Based on our findings, we propose pro-inflammatory IL-1 (α and β), IL-6, IL-8, IL-12(p70); anti-inflammatory IL-1RA; the chemokine IP-10; and the antimicrobial protein SLPI as a smaller panel of soluble biomarkers for consideration for safety measurement of candidate vaginal microbicides. Additionally, the assessment of microbicide safety should include clinical examination for visible signs of vaginal epithelia irritation and the effect of candidate microbicides on cellular markers of inflammation as well as the vaginal microbiome. An ideal outcome would be point-of-care tools that can be easily used in clinical setting in resource-limited settings to identify women with increased susceptibility to HIV infection.

Ryan K. Cheu1,2,3, Avid Mohammadi4, Tiffany Hensley-Mcbain1,2, Jennifer Manuzak1,2,3, Alexander S. Zevin1,2, Charlene Miller3, Mark Yudin5, Rupert Kaul4, Nichole R. Klatt1,2,3

1Department of Pharmaceutics, University of Washington, Seattle, WA, USA

2Washington National Primate Research Center, University of Washington, Seattle, WA, USA

3Department of Pediatrics, University of Miami Miller School of Medicine, Miami, FL, USA

4Department of Immunology, University of Toronto, Toronto, Canada

5Core Obstetrics & Gynecology, University of Toronto, Toronto, Canada

Background: Bacterial vaginosis (BV) is associated with an increased HIV transmission rate. Inflammatory cells such as neutrophils are critical for innate immune responses but can also contribute to barrier damage and inflammation. Currently, the role of neutrophils in HIV transmission and BV status is unknown. Here, we hypothesize that BV-associated bacteria increase HIV transmission rates by inducing activation and accumulation of neutrophils within the female reproductive tract (FRT), resulting in epithelial barrier damage.

Methods: In order to elucidate the mechanisms for the negative outcomes of BV, we collected cervicovaginal cytobrushes from 11 women with BV and 10 women without BV. We used flow cytometry to assess phenotype and functionality of neutrophils and performed in vitro whole blood co-cultures with bacteria associated with BV (G. vaginalis), healthy commensals such as L. iners and L. crispatus, media alone (negative control) and lipopolysaccharide and peptidoglycan (positive controls). To determine the impact neutrophils have on epithelial integrity, we isolated neutrophils from healthy human whole blood to co-culture with FRT bacteria across a vaginal epithelial cellular monolayer and used a transepithelial electrical resistance (TEER) set-up to assess barrier damage.

Results: We demonstrated increased neutrophil activation (p=0.0022), prolonged lifespan (p=0.0022), and more total neutrophils (p=0.0022) in the cytobrushes from women with non-Lactobacillus dominant (nLD) communities. Similarly, our co-culture experiments showed more neutrophil activation (p<0.0001), prolonged lifespan (p=0.008), and more total neutrophils (p<0.0001) in cultures with G. vaginalis compared with our negative controls and Lactobacillus cultures. Our 16S rRNA analysis identified two distinct community groups one dominated by Lactobacillus and the other a more diverse community consisting of G. vaginalis. Our TEER assays demonstrated significant barrier damage in cultures with neutrophils and G. vaginalis when compared with neutrophils alone (p<0.0001) or with neutrophils and Lactobacillus spp. (p<0.0001).

Conclusions: Here, we demonstrate that BVAB induce neutrophil activation, delay apoptosis leading to accumulation within the FRT, and jeopardize epithelial integrity. This study provides potential mechanistic insights into how BV may lead to FRT inflammation and increased HIV transmission.