Neisseria gonorrhoeae (Ng) is a human-specific pathogen and the etiological agent of gonorrhea, a sexually transmitted infection with a significant global health burden of ~78 million new cases annually. While often asymptomatic, untreated gonorrhea can lead to pelvic inflammatory disease, ectopic pregnancy, infertility, and increased transmission/acquisition of HIV. Because of the inexorable increase in antibiotic resistance, a protective gonorrhea vaccine may be the only way to control disease transmission in the future. The recent successes of the MenZB and 4CMenB outer membrane vesicle vaccines for Group B N. meningitidis (Nm) provides a strong premise for development of an effective gonorrhea vaccine. Retrospective data suggested that the MenZB vaccine was 31% effective against gonorrhea in the immunized cohort. In support of this finding, we have shown that immunization with the similar 4CMenB vaccine markedly increased Ng clearance in the mouse model of gonorrhea, and sera from 4CMenB-vaccinated mice cross-reacted with MtrE, BamA, and PilQ from Ng outer membranes. Based on these data and the surface exposure, omnipresence, sequence conservation, and importance in vital cellular functions of MtrE, BamA, and PilQ, we hypothesize that antibodies directed at the extracellular regions of these antigens will provide protection against gonorrhea. Accordingly, the overarching goal of this collaborative translational project is to develop a gonorrhea vaccine(s) by targeting MtrE, BamA, and PilQ. Subunit antigens are proven candidates for vaccine development due to their safety, cost-effectiveness, and rapid preparation. To develop effective gonorrhea vaccine(s), we propose an innovative approach of incorporating the aforementioned antigens into nanoparticle platforms called nanodiscs (NDs) and combining with different adjuvants in our vaccine formulations. NDs will enable antigen multivalency and native shape that are important determinants of vaccine potency and efficacy, while adjuvants will be used to amplify robust antigen-specific responses. For Project 3 of the Gonorrhea Vaccine Cooperative Research Center (GV CRC), we will: i) purify full-length and/or the b-barrel regions of MtrE, BamA, and PilQ and natively display the proteins in NDs (Specific Aim 1); ii) protein-NDs will be combined with different adjuvant compositions to induce robust and balanced Th1/Th2 responses, and the resulting sera will be assessed for immunoglobulin subtypes, serum bactericidal and opsonophagocytolytic activity, and binding to intact Ng (Specific Aim 2); and iii) test the most promising antigen-ND/adjuvant combinations in the lower and upper reproductive tract mouse models of Ng infection, as well as in a mixed Ng/Chlamydia muridarum infection model, for their capacity to decrease the time of infection (Specific Aim 3). The success of the GV CRC will be greatly enhanced by the Outer Membrane Vesicles and Proteomics Core (Core B), Host Response Monitoring Core (Core C), the Functional Antibody Study Core (Core D), and the Mouse Immunization/Challenge Core (Core E), which are fully integrated into Aims 2 and 3, for testing the efficacy of the antigen-ND vaccines.