Characterization of the Ovine Vaginal Microbiome and Inflammation Patterns as an Improved Testing Model of Human Vaginal Irritation

Abstract

The development of therapies targeted to improve the health of women has utilized direct vaginal delivery as a more effective and less toxic method of protection from HIV and other pathogens. Vaginal applicants and delivery devices that provide sustained effects have been met with increasing acceptability, but the efficacy and toxicity outcomes have not been successfully predicted by preclinical in vitro studies and animal modeling. We have explored the utilization of sheep as a model for testing the safety of vaginal applicants and devices based on spatial and structural similarities to the human vagina. As recently noted by the FDA, an additional safety measure is an impact on the vaginal microbiome (VMB) that is known to contribute to vaginal health and influence pathogen susceptibility and drug metabolism. To advance the utility of the sheep vaginal model, we completed a thorough molecular characterization of the ovine VMB utilizing both next-generation sequencing (NGS) and PCR methods. The process also created a custom PCR array to quantify ovine VMB community profiles in an affordable, higher throughput fashion. The results from vaginal swabs (>475 samples) collected from non-pregnant crossbred Dorset and Merino ewes treated with selected vaginal applicants or collected as sham samples established 16 VMB community types (VMB CTs). To associate VMB CTs with eubiosis or dysbiosis, we also completed custom ELISAs for six cytokines identifying IL1B, IL8, TNFa, and CXCL10 as useful markers to support the characterization of ovine vaginal inflammation. The results indicated that Pasteurella, Actinobacillus, Pseudomonas, Bacteroides, Leptotrichia, and E. coli were common markers of eubiosis (low inflammatory marker expression), and that Haemophilus, Ureaplasma, and Corynebacterium were associated with dysbiosis (high cytokine levels). Utilizing the optimized workflow, we also confirmed the utility of three commonly used vaginal applicants for impact on the VMB and inflammatory state, producing a dataset that supports the recommendation for the use of sheep for testing of vaginal applicants and devices as part of preclinical pipelines.

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

“…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 ). Specifically, the enzyme-linked immunosorbent assays (ELISAs) were performed to quantify the concentrations of the following cytokines: (interleukin) IL-1β, IL-6, IL-8, IL-17α, CXCL10 (aka IP-10), and tumor necrosis factor-α (TNF-α) in solutions extracted from the sheep vaginal swab samples.…”

“…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).…”

“…A detailed study on the normal and dysbiotic VMB as well as VMB changes and proinflammatory cytokines after intravaginal placebo and irritant applicants was performed in sheep and reported elsewhere ( 15 ). Characterization of the sheep VMB was completed utilizing both the next generation sequencing (NGS) and quantitative PCR (qPCR) methods to establish common community types (CT) under the laboratory animal medicine conditions and has been reported separately ( 15 ).…”

“…A detailed study on the normal and dysbiotic VMB as well as VMB changes and proinflammatory cytokines after intravaginal placebo and irritant applicants was performed in sheep and reported elsewhere ( 15 ). Characterization of the sheep VMB was completed utilizing both the next generation sequencing (NGS) and quantitative PCR (qPCR) methods to establish common community types (CT) under the laboratory animal medicine conditions and has been reported separately ( 15 ). The goal of this work was to relate ovine VMBs to vaginal inflammatory markers through iterative development of a simple scoring system using the Gram staining of vaginal secretions similar to Nugent scoring in the clinical samples ( 16 ); such a system could be utilized in any laboratory without the need for complex assays.…”