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

Abstract

BackgroundVaginal 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.ResultsWild type (WT) L. jensenii 1153 consistently colonized cervical and vaginal cells in the absence of epithelial damage and apoptosis. The bioengineered derivatives expressing mCV-N or control plasmids showed the same stable colonization pattern, which was reproducible between technologists and bacterial batches (CFU coefficient of variation <10% within and between experiments and epithelial cell types). MALP-2 activated NF-κB and caused fold-increased levels of proinflammatory mediators with clinically established significance in the cervicovaginal environment (IL-1α, IL-1β, IL-6, TNF-α, IL-8, RANTES, MIP-3α, and ICAM-1), measured by a multiplex electrochemiluminescence assay. At the same time levels of protective anti-inflammatory mediators interleukin 1 receptor antagonist (IL-1RA) and secretory leukocyte protease inhibitor (SLPI), both measured by ELISA, remained constant (IL-1RA) or moderately increased (SLPI). Similarly to MALP-2, colonization by L. jensenii WT activated NF-κB; however, unlike the synthetic TLR2/6 ligand, the live microorganisms did not induce significant changes in the secreted levels across all inflammation-associated proteins. The mCV-N production and function were confirmed by western blot and a HIV-1 gp120 binding assay, respectively. The bioengineered lactobacilli expressed mCV-N with anti-HIV activity preserved in the epithelial cell context and caused no significant immunoinflammatory changes as compared to the WT L. jensenii.ConclusionsThese results highlight the translational value of the colonization model and justify further clinical investigation of the homeostatic and anti-HIV effectiveness of the L. jensenii derivates.

The Villain Team-Up or howTrichomonas vaginalisand bacterial vaginosis alter innate immunity in concert

“…A vaginae did not grow under planktonic conditions but showed high numbers in the presence of colonised and TV-infected cells. Electron microscopy confirmed lack of cytotoxicity in our vaginal colonisation model, as previously shown,16 17 and also confirmed abundance of intracellular A vaginae (figure 1B) and P bivia (not shown). The ability of vaginal bacteria to reside inside the cervicovaginal epithelial cells is a phenomenon that warrants further investigation.…”

“…TV (1.25×10 5 parasites/cm 2 ), LPG (240 µg/ml) or CPI-GC (240 µg/ml) were then added, and epithelia-associated bacteria as well as immune responses were assessed after another 24 h. Synthetic TLR2/TLR6 ligand MALP-2 (25 nM; Alexis Biologicals) analogue of Mycoplasma fermentans lipopeptide macrophage activating lipopeptide was used as a pro-inflammatory control. At the end of each stimulation period, supernatants were unless specified collected for soluble mediators and epithelial cells were lysed for colony-forming units (CFU) counts, used for viability assessment or used for microscopy 16 17. For CFU counts, epithelial cells were washed twice with PBS and hypotonically lysed in HyPure water (Fisher) for 15 min.…”

Cross-Sectional Analysis of Selected Genital Tract Immunological Markers and Molecular Vaginal Microbiota in Sub-Saharan African Women, with Relevance to HIV Risk and Prevention

“…The presence of lactobacilli (specifically L. crispatus and L. vaginalis) was associated with lower levels of proinflammatory cytokines in our study. This is consistent with previous observations about the noninflammatory nature of specific Lactobacillus species (50,51). These species were also associated with higher levels of the protective antimicrobial proteins SLPI and elafin in our study.…”

Probiotic Properties of Lactobacillus crispatus 2,029: Homeostatic Interaction with Cervicovaginal Epithelial Cells and Antagonistic Activity to Genitourinary Pathogens

“…A nonvaginal Lactobacillus species induced production of proinflammatory protein MIP-3a [64]. Vaginal pathogens, associated with BV such as Atopobium vaginae and Prevotella bivia induced simultaneous NF-jB activation and upregulation of proinflammatory proteins in contrast to vaginal strains L. crispatus, L. acidophilus and L. jensenii [28,62]. BV is the most common vaginal infection among women of reproductive age.…”

“…The net effect of NF-jB activation depends on the cell and tissue properties, the interaction of intra-and extra-cellular factors, and the nature of the activating signal [60]. It has been previously shown that vaginal Lactobacillus species (L. crispatus, L. acidophilus and L. jensenii) can cause NF-jB activation and yet maintain low levels of IL-8 and RANTES [28,62]. L. jensenii can suppress IL-8 induced by TLR ligands [63].…”