Preclinical Evaluation of Novel Conserved Neisseria gonorrhoeae Vaccine Candidates Identified Through Infection-Informed Antigen Discovery

Background: The continued emergence of antimicrobial-resistant Neisseria gonorrhoeae highlights the urgent need for an effective vaccine. We evaluated the immunogenicity and vaccine potential of six new gonococcal conserved hypothetical proteins identified through our immunobioinformatics-based pipeline (Candidate Antigen Selection Strategy, CASS). Methods: NGO0588, NGO0694, NGO0757, NGO0861, NGO1438 and NGO1802 proteins were expressed in E. coli, purified, and used to immunize mice, adjuvanted with Alum and MPLA. Immunological analyses included quantitative and qualitative evaluation of antigen-specific antibody responses, cytokines, serum bactericidal activity (SBA), and protective efficacy in the mouse gonococcal vaginal colonization model. Results: The six antigens elicited robust antigen-specific IgG and IgM antibody responses against N. gonorrhoeae. Antisera to NGO0588, NGO0861, NGO1430 and NGO1802 were bactericidal against different gonococcal strains. IgG antibodies recognizing the antigens were detected in sera obtained from women with disseminated gonococcal infection, particularly against NGO1802, supporting immunogenicity in humans. Taking immunological and biochemical properties into consideration, NGO0861, NGO1438 and NGO1802 were combined in a trivalent vaccine formulation. The trivalent vaccine elicited robust antigen-specific and N. gonorrhoeae-reactive serum IgG and IgM, mucosal IgG, and higher SBA titers than each individual antigen. However, in the mouse gonococcal vaginal colonization model, vaccinated mice and adjuvant control mice did not show statistically significant differences in time to infection clearance or bacterial burdens. Conclusions: A multiantigen vaccine composed of NGO0861, NGO1438 and NGO1802 elicited antibodies that were functional in vitro but did not lead to protection in vivo. Immunological analyses in vitro and in vivo emphasize the importance of evaluating antigen and antibody quality and functional activity for vaccine target prioritization of next-generation gonococcal multicomponent vaccines.