Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection (STI) gonorrhea, a disease with high morbidity worldwide, estimated at 87 million cases annually, and severe reproductive tract complications in women. Current treatment approaches against gonorrhea are compromised by recent onset of antibiotic resistance. There is a pressing need for an effective vaccine against N. gonorrhoeae, but protective antigens have been limited, so far. Our previous work on the gonococcal transcriptome during human natural infection has described differences in mRNA expression of gonococcal genes in urethral and vaginal lavage samples fromnaturally-infected subjects as compared to mRNA expression when the correspondinggonococcal strains were cultured in vitro. We termed those genes “in vivo-expressed factors” (IVEFs). We also reported that ~30% of the gonococcal genes expressed during infection are hypothetical proteins. We hypothesized that gonococcal hypothetical proteins expressed and regulated during infection include new candidate antigens for a vaccine against N. gonorrhoeae. Through a bioinformatics-based candidate antigen selection strategy (CASS) that examined predicted immunogenicity, cellular localization, conservation in N. gonorrhoeae and structure features of the gonococcal hypothetical proteins, we identified 36 new potential targets (ongoing R21AI131004). We investigated an initial group of 6 antigens, confirming 3 as surface-exposed proteins (NGO0690, NGO0948 and NGO1701) that induced cross-reactive antibodies with complement-mediated serum bactericidal activity (SBA) against diverse N. gonorrhoeae strains. In preliminary studies, a combination of these antigens showed promise as protective vaccine candidates in a mouse model of gonococcal infection. Correlates of protection against gonorrhea in humans are not known but SBA and reduced vaginal colonization in mice are stepping- stones in preclinical gonococcal vaccine evaluation. An example is the (currently most advanced) N. gonorrhoeae vaccine candidate, the 2C7 LOS epitope (and its mimotope TCMP2). Other cited mechanisms of protection include antibody-dependent inhibition of bacterial adhesion or invasion of host cells at the colonization site, but these have yet to be confirmed in human studies. The goal of this project is to validate and improve the efficacy of our three candidates as gonococcal vaccine antigens. In Aim 1, we propose antigen dosing and adjuvant studies to achieve optimal immune responses to our candidates and SBA; in Aim 2 we will validate protection in the available female mouse model of gonococcal vaginal colonization, broaden strain coverage by including TMCP2, and correlate immune effector mechanisms and protection; in Aim 3, we will define the function of the hypothetical protein candidates and their impact on N. gonorrhoeae virulence, fitness and Immunity, a necessary complement for their selection as vaccine candidates. Our studies will provide...