Abstract
The human vaginal microbiome (VMB) is a complex bacterial community that interacts closely with vaginal epithelial cells (VECs) impacting the mucosal phenotype and its responses to pathogenic insults. The VMB and VEC relationship includes nutrient exchange and regulation of signaling molecules that controls numerous host functions and defends against invading pathogens. To better understand infection and replication of sexually transmitted viral pathogens in the human vaginal mucosa we used our ex vivo VEC multilayer culture system. We tested the hypothesis that selected VMB communities could be identified that alter the replication of sexually transmitted viruses consistent with reported clinical associations. Sterile VEC multilayer cultures or those colonized with VMB dominated by specific Lactobacillus spp., or VMB lacking lactobacilli, were infected with Zika virus, (ZIKV) a single stranded RNA virus, or Herpes Simplex Virus type 2 (HSV-2), a double stranded DNA virus. The virus was added to the apical surface of the cultured VEC multilayer to model transmission during vaginal intercourse. Viral replication was measured 48 h later by qPCR. The results indicated that VEC cultures colonized by VMB containing Staphylococcus spp., previously reported as inflammatory, significantly reduced the quantity of viral genomes produced by ZIKV. HSV-2 titers were decreased by nearly every VMB tested relative to the sterile control, although Lactobacillus spp.-dominated VMBs caused the greatest reduction in HSV-2 titer consistent with clinical observations. To explore the mechanism for reduced ZIKV titers, we investigated inflammation created by ZIKV infection, VMB colonization or pre-exposure to selected TLR agonists. Finally, expression levels of human beta defensins 1–3 were quantified in cultures infected by ZIKV and those colonized by VMBs that impacted ZIKV titers. Human beta defensins 1–3 produced by the VEC showed no association with ZIKV titers. The data presented expands the utility of this ex vivo model system providing controlled and reproducible methods to study the VMB impact on STIs and indicated an association between viral replication and specific bacterial species within the VMB.
Highlights
The vaginal mucosa supports an intricate ecosystem regulated by interactions between microbial communities and vaginal epithelial cells (VECs)
Microbiomes that are deficient in Lactobacillus spp. and have a predominance of Gram negative organisms are associated with bacterial vaginosis (BV) and inflammation of the vaginal mucosa (Ma et al, 2012)
After VEC fully matured in the transwell cultures, selected concentrations of each agonist in 10 μL of Keratinocyte Serum Free Medium (KSFM) were added to the apical surface of triplicate wells: CpG (0.1 μg/mL or 0.01 μg/mL), LPS (1 μg/mL or 0.1 μg/mL), FLAG (5 μg/mL or 0.5 μg/mL) as described previously (Rose et al, 2012)
Summary
The vaginal mucosa supports an intricate ecosystem regulated by interactions between microbial communities and vaginal epithelial cells (VECs). Microbiomes that are deficient in Lactobacillus spp. and have a predominance of Gram negative organisms are associated with bacterial vaginosis (BV) and inflammation of the vaginal mucosa (Ma et al, 2012) Such microbial communities, including those associated with symptomatic BV, are correlated clinically and in model systems with increased viral replication for sexually transmitted viruses including HIV and HSV-2 (Boris and Barbés, 2000; Brotman, 2011; Pyles et al, 2014). These studies have not included analyses of sexually transmitted Zika virus (ZIKV)
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