AEJ) 22 23 24 † These authors contributed equally to the work. 25 26 27 2 28 Abstract 29 30There is a pressing need for a gonorrhea vaccine due to the high disease burden 31 associated with gonococcal infections globally and the rapid evolution of antibiotic resistance in 32 Neisseria gonorrhoeae (Ng). Current gonorrhea vaccine research is in the stages of antigen 33 discovery and the identification of protective immune responses, and no vaccine has been tested 34 in clinical trials in over 30 years. Recently, however, it was reported in a retrospective case-control 35 study that vaccination of humans with a serogroup B Neisseria meningitidis (Nm) outer membrane 36 vesicle (OMV) vaccine (MeNZB) was associated with reduced rates of gonorrhea. Here we 37 directly tested the hypothesis that Nm OMVs induce cross-protection against gonorrhea in a well-38 characterized female mouse model of Ng genital tract infection. We found that immunization with 39 the licensed Nm OMV-based vaccine 4CMenB (Bexsero®) significantly accelerated clearance 40 and reduced the Ng bacterial burden compared to administration of alum or PBS. High titers of 41 serum IgG1 and IgG2a and vaginal IgG1 that cross-reacted with Ng OMVs were induced by 42 vaccination via either the subcutaneous or intraperitoneal routes, and a 4-fold increase in the 43 serum bactericidal 50 titers was detected against the challenge strain. Antibodies from vaccinated 44 mice recognized several surface proteins in a diverse collection of Ng strains, including PilQ, 45 BamA, MtrE, PorB, and Opa, and 4CMenB-induced antibodies bound PilQ and MtrE in native 46 form on the surface of viable bacteria. In contrast, the antibodies were only cross-reactive against 47 lipooligosaccharide species from a few Ng strains. Our findings directly support epidemiological 48 evidence that Nm OMVs confer cross-species protection against Ng and implicate several Ng 49 surface antigens as potentially protective targets. This work also validates the murine infection 50 model as a relevant experimental system for investigating mechanisms of vaccine-mediated 51 protection against gonorrhea. 52 3 53 Author summary 54 55Over 78 million Neisseria gonorrhoeae (Ng) infections occur globally each year and control 56 of gonorrhea through vaccination is challenged by a lack of strong evidence that immunity to 57 gonorrhea is possible. This contention was recently challenged by epidemiological evidence 58 suggesting that an outer membrane vesicle (OMV) vaccine from the related species Neisseria 59 meningitidis (Nm) protected humans against gonorrhea. Here we provide experimental evidence 60 in support of this hypothesis by demonstrating that a licensed, modified version of this Nm OMV-61 based vaccine accelerates clearance of Ng in a mouse infection model. These results confirm the 62 possibility cross-species protection and are important in that they support the biological feasibility 63 of vaccine-induced immunity against gonorrhea. We also showed that several Ng outer 64 membrane proteins are recogn...