Establishment of an in vitro model to study co-infection of vaginal epithelium by Candida albicans and Herpes Simplex Virus 2

BACKGROUND AND AIM Most pathogens enter the human body by breaching the mucosal barriers, thus establishing a successful infection. Candida albicans (Ca) and Herpes Simplex Virus 2 (HSV-2) are among the most common pathogens of the genital tract. The present study aims to develop an in vitro model that, by closely mimicking human genital mucosa, will allow to dissect the fine host-pathogens interactions during mono- and polymicrobial infections. MATERIALS AND METHODS The A-431 vaginal cell line was used to set up cultures as: i) monolayers, produced on 96 or 24 multi-well plates, after 24 h of incubation in DMEM with 10% FBS; ii) reconstituted vaginal epithelium (RVE), established after 5-6 days of incubation under that same culture conditions, with medium renewal at day 3; iii) vaginal cells cultured on inert scaffolds. The cells, exposed or not to an artificial vaginal fluid (AVF), were infected with Ca and/or HSV-2, at the multiplicity of infection (MOI) of 10:5:1. Epithelial viability and cytokine production were assessed by LDH release and ELISA assays, respectively; Ca and viral load were also evaluated, at different time points and conditions, by CFU assay and PCR-based DNA quantification, respectively. RESULTS The epithelial cells, no matter whether assessed as monolayer or as RVE, were partially affected by the addition of the AVF. Differently, Ca exposure to AVF consistently enhanced its survival and growth ability. The epithelial cell viability was significantly impaired upon infection with Ca, while HSV-2 caused little or no effects; the combination of the two pathogens resulted in slightly additive effects in terms of cell damage. To date, the monolayer proved to be the best condition to assess cytokine production in response to a single or double infection. In particular, epithelial cells produced IL-8 following either HSV-2 or Ca infection, while the double infection did not further enhance such chemokine production. Differently, IL-1beta was released by Ca infected cells only; HSV-2 had no effects, either when added alone or in combination with Ca. Initial experiments performed by culturing the A-431 cells onto inert scaffolds indicated the ability of the cells to colonize such tridimensional matrix. Histological analysis of such cultures and viral load quantification are in progress. DISCUSSION AND CONCLUSIONS The A-431 epithelial cells are susceptible to both HSV-2 and Ca infection; they respond to the pathogens to a different extent, also depending upon the presence of the artificial vaginal fluid. These results lay the foundation for the development of an in vitro prototype that, by closely resembling the in vivo mucosal environment, will allow to highlight both single and cumulative microbial impact on host vaginal mucosa.