Homeostatic properties of Lactobacillus jensenii engineered as a live vaginal anti-HIV microbicide

Hidemi S Yamamoto, Qiang Xu, Raina N Fichorova

Vaginal probiotics are investigated as a binary strategy for prevention of bacterial vaginosis and HIV. We applied an innovative experimental model using primary and immortalized human cervical and vaginal epithelial cells to assess the functional properties of Lactobacillus jensenii, a predominant constituent of the healthy vaginal microbiome, engineered to express the HIV-1 entry inhibitor modified cyanovirin-N (mCV-N). In this model bacteria colonize the epithelial cells over a period of 24-72 h. Staurosporine and the Toll-like receptor 2/6 ligand macrophage-activating lipopeptide-2 (MALP-2) serve as positive controls for apoptosis and proinflammatory activation, respectively. In 24-hour intervals, the colonized epithelium is assessed microscopically, supernatants are collected for measurement of soluble immunoinflammatory mediators and production of CV-N, and cells are lysed for assessment of: 1) apoptosis by cleaved versus total caspase-3 assay; 2) NF-κB activation by a luciferase reporter assay; or 3) epithelia-associated colony forming units (CFU) in Brucella agar.