Targeting EphA2 with a novel EFNA3-mimetic peptide enhances vaginal epithelial barrier function: Running title: EFNA3-MAP enhancing the vaginal epithelial barrier.

Barrier protection provided by vaginal stratified squamous epithelium (SSE) is supported by estrogen (E)-mediated stimulation of ephrin-Eph signaling that promote desmosome expression and function. Conversely, loss of E induced by use of the contraceptive depot-medroxyprogesterone acetate (DMPA) or menopause reduces desmosome expression and weakens this epithelial barrier. We previously reported treating mice with DMPA lowers expression of the desmosomal cadherin desmoglein-1 (DSG1) in vaginal SSE by dampening EFNA3-mediated signaling whereas these effects are partially reversed by intravaginal administration of recombinant EFNA3 (rEFNA3) protein. Herein we extended these results, developing an EFNA3-mimetic agonist peptide (EFNA3-MAP) that engages the EphA2 receptor in vitro. In vivo, EFNA3-MAP penetrated vaginal SSE more effectively than rEFNA3 and restored DSG1 levels and vaginal epithelial thickness in DMPA-treated mice. It also reduced susceptibility of these mice to genital infection with bacterial and viral pathogens. In separate studies, we showed that EFNA3-MAP bioactivity depended on G-H loop aromatic residues and EphA2 engagement and that topical EFNA3-MAP restored vaginal epithelial thickness and DSG1 expression in DMPA-treated rhesus macaques. Together, current studies establish EFNA3-EphA2 signaling as a key regulator of vaginal SSE health and offer strong rationale to explore if EFNA3-MAP similarly enhances cutaneous SSE barrier function.