RECONSTRUCTED VAGINAL-ECTOCERVICAL AND ENDOCERVICAL TISSUE MODELS FOR HSV-2, CHLAMYDIA, AND GONORRHEA INFECTIONS.

Despite extensive efforts, limited success has been achieved in developing tissue models for sexually transmitted infections (STIs) such as herpes simplex virus 2 (HSV-2), Chlamydia trachomatis (Ct), and Neisseria gonorrhea (Ng). The purpose of this study was to utilize highly differentiated, normal human 3-dimensional (NHu-3D) models of human vaginal ectocervical (VEC) and endocervical (VEN) tissues to develop STI infection models.  We developed highly differentiated, normal human 3-dimensional (NHu-3D) vaginal-ectocervical (EpiVaginal™) and endocervical tissues models for STI infections. Immunohistochemistry and quantitative real time PCR were used to characterize the ectocervical tissues to monitor HSV-2 infection. Infection of the endocervical tissue model by the infectious elementary body (EB) of Ct was assessed by quantitative cultures. ELISA assays were used to quantify TNF-α release in response to Ng and Toll-Like Receptor (TLR) ligands. Results showed that the EpiVaginal tissue expresses nectin-1, a receptor for HSV-2, and infection experiments showed that the tissue model was infectable with HSV-2. Similar to the in vivo situation, infection of the vaginal-ectocervical tissue model with HSV-2 caused a separation of the epithelium from the lamina propria layer (“blister formation”). The results were confirmed by DNA PCR. The data from Ct infected endocervical tissues showed a cyclic level of EBs which were. 1) present at 12 hrs, representing bacteria that failed to be taken up by the cells, 2) not detectable at 24 hrs, all intracellular EBs might have been converted to the replicative reticular bodies (RB) and are therefore are not cultivatable, and 3) present at 40 hr, RBs have been converted back to EBs completing the first cycle. Furthermore, the reconstructed endocervical tissues respond to Ng infection and TLR ligands by secreting TNF-α into the culture medium. In conclusion, new in vitro reconstructed ectocervical and endocervical tissue models have been developed for HSV-2, Ct, and Ng infections. The models can be used to study the safety and efficacy of candidate therapeutics aimed at preventing or neutralizing HSV-2, Chlamydia, or gonorrheal infections.

Chlamydia, EndoCervical tissue (VEN-100), EpiVaginal (VEC-100-FT), Gonorrhea, herpes simplex virus 2 (HSV-2), infectious elementary body, nectin-1, sexually transmitted infections, TNF-α, Toll-Like Receptor (TLR)

Chlamydia trachomatis, herpes simplex virus 2 (HSV-2), LPS (TLR4 ligand), Neisseria gonorrhea, Pam2CSK4 (TLR2/6 ligand), Pam3CSK4 (TLR2/1 ligand), , poly I:C (TLR3 ligand)