Abstract This proposal is to produce IND-enabling data for supporting clinical trials of an attenuated clone of the mouse-adapted Chlamydia muridarum (CM) as a novel oral vaccine known as intrOv for protecting humans from sexually transmitted infection with Chlamydia trachomatis (CT). Extensive searching for a subunit vaccine against CT has not produced a licensed vaccine. One of the challenges is that all 8 STI-causing CT serovars (D to K) are significant human pathogens globally. We are proposing to develop intrOv into an oral vaccine against 8 CT serovars. Following a single oral inoculation, CM can establish long-lasting colonization in the mouse gastrointestinal (GI) tract but without causing any significant pathologies; Oral CM induces Th1-dominant transmucosal immunity against subsequent infection in the genital tract and airway but without altering CM colonization in the GI tract; CM colonization in the GI tract for 1 week is sufficient for inducing protective immunity in the genital tract and antibiotics clearance of GI tract CM does not compromise the genital protective immunity; To improve the safety of CM as an oral vaccine, genital tract pathogenicity-attenuated CM clones have been generated by various labs but the clone designated as intracellular Oral vaccine vector or intrOv is particularly attractive since it is still able to induce transmucosal protection in the genital tract but no longer maintains long-lasting colonization in the colon; IntrOv induces IFN+ILC3s and is also inhibited by IFN+ILC3s; Finally intrOv-induced transmucosal immunity conferred cross-species protection against CT in the genital tract, validating previously observed CM induction of anti-CT immunity. Thus, we hypothesize that oral intrOv protects human genital tract from CT infection and the intrOv-induced IFN+ILC3s in the gut may present cross-species epitopes to lymphocytes for mediating Th1- dorminant transmucosal immunity. We will test these hypotheses by validating the protection efficacy of intrOv against genital CT in mice and large animals, identifying protective correlates and CT antigens for improving clinical assays & intrOv efficacy and improving the quality and safety of intrOv as a human oral vaccine. Our hypotheses are consistent with these successful examples of using animal-adapted microbes to protect against human pathogens: cowpox as a vaccine against smallpox, BCG (M. bovis) against M. tuberculosis and meningococcal vaccine against gonorrhoea.