title: Homeostatic properties of Lactobacillus jensenii engineered as a live vaginal anti-HIV microbicide.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  Wild 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.
  
  
  CONCLUSIONS
  These 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.
authors.affiliation: Laboratory of Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
authors.forename: Hidemi S
authors.initials: HS
authors.lastname: Yamamoto
authors.forename: Qiang
authors.initials: Q
authors.lastname: Xu
authors.forename: Raina N
authors.initials: RN
authors.lastname: Fichorova
mesh_terms: D000890:Anti-Infective Agents / Q000378:metabolism*; D017209:Apoptosis; D058491:Bacterial Load; D001426:Bacterial Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D004847:Epithelial Cells / Q000382:microbiology*; D005260:Female; D023581:HIV Fusion Inhibitors / Q000378:metabolism*; D006801:Humans; D007778:Lactobacillus / Q000235:genetics / Q000502:physiology*; D016328:NF-kappa B / Q000378:metabolism
chemical_list: D000890:Anti-Infective Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D023581:HIV Fusion Inhibitors; D016328:NF-kappa B; C107733:cyanovirin N