Evaluation of immunological markers of ovine vaginal irritation: Implications for preclinical assessment of non-vaccine HIV preventive agents

Abstract

The presence of genital inflammatory responses and a compromised vaginal epithelial barrier have been linked to an increased risk of HIV acquisition. It is important to assure that application of candidate microbicides designed to limit HIV transmission will not cause these adverse events. We previously developed high resolution in vivo imaging methodologies in sheep to assess epithelial integrity following vaginal application of a model microbicide, however characterization of genital inflammation in sheep has not been previously possible. In this study, we significantly advanced the sheep model by developing approaches to detect and quantify inflammatory responses resulting from application of a nonoxynol-9-containing gel known to elicit vaginal irritation. Vaginal application of this model microbicide resulted in foci of disrupted epithelium detectable by confocal endomicroscopy. Leukocytes also infiltrated the treated mucosa and the number and composition of leukocytes obtained by cervicovaginal lavage (CVL) were determined by differential staining and flow cytometry. By 18 h post-treatment, a population comprised predominantly of granulocytes and monocytes infiltrated the vagina and persisted through 44 h post-treatment. The concentration of proinflammatory cytokines and chemokines in CVL was determined by quantitative ELISA. Concentrations of IL-8 and IL-1β were consistently significantly increased after microbicide application suggesting these cytokines are useful biomarkers for epithelial injury in the sheep model. Together, the results of these immunological assessments mirror those obtained in previous animal models and human trials with the same compound and greatly extend the utility of the sheep vaginal model in assessing the vaginal barrier and immune microenvironment.

Development of Gram Stain Scoring System Based on Pro-Inflammatory Cytokines in the Sheep Model for Testing Toxicity of Vaginal Products

“…This study also provided guidance for designation of vaginal inflammation based on the presence of three of four cytokines (TNF-α, IL-8, IL1-β, and CXCL10) with values above the mean value leading to the sample categorized as inflamed. There are few data on sheep VMB, no previous reports on quantitative evaluation of Gram-stained slides and only one study in sheep reporting on the vaginal inflammatory markers ( 17 ), therefore this study provides important advancements for determining dysbiotic states after vaginal product use and enables translation from the preclinical animal safety studies to clinical studies in women.…”

“…The Methods for specific cytokine analysis in the sheep vagina were described by our group and employed ( 17 ) with adaptation for automated processing on the archived fluids from vaginal swabs to establish useful standards for signs of inflammation in the ovine vaginal tract. The details are reported in a separate manuscript ( 15 ).…”

“…Periodically during the Main and Recovery Periods (Main: Days 1, 2, 3, 5, 7, and 9; Recovery: Days 11,13,15,17,and 21), the sterile calcium alginate swab samples of the vaginal wall were collected from each ewe. Vaginal swab fluid (10 µl) from each time point was used to create a smear on a glass microscope slide, heat fixed, and then, subjected to standard Gram staining (BD BBL Gram Stain kit with Stabilized Iodine, Thermo Fisher Scientific, MA, USA) completed in batches.…”

“…Kinetic vaginal sampling produced a dataset (n > 230 vaginal swab fluids) of natural and induced VMB dysbiosis and eubiosis to power the analyses. The ovine vaginal samples were evaluated for IL-1β, IL-6, IL-8, IL-17α, CXCL10, and TNF-α cytokine levels using the previously published methods (17) and are reported in a separate manuscript (15). These cytokines are shown previously to be the markers of vaginal inflammation and have been detected in sheep (17)(18)(19).…”

“…The ovine vaginal samples were evaluated for IL-1β, IL-6, IL-8, IL-17α, CXCL10, and TNF-α cytokine levels using the previously published methods (17) and are reported in a separate manuscript (15). These cytokines are shown previously to be the markers of vaginal inflammation and have been detected in sheep (17)(18)(19). Similarly, the established VMB CTs are reported elsewhere (15).…”