Gonorrhea, a sexually transmitted disease caused by Neisseria gonorrhoeae, poses a significant global public health threat. Infection in women can be asymptomatic and may result in severe reproductive complications. Escalating antibiotic resistance underscores the need for an effective vaccine. Approaches being explored include subunit vaccines and outer membrane vesicles (OMVs), but an ideal candidate remains elusive. Meningococcal OMV-based vaccines have been associated with reduced rates of gonorrhea in retrospective epidemiologic studies, and with accelerated gonococcal clearance in mouse vaginal colonization models. Cross-protection is attributed to shared antigens and possibly cross-reactive, bactericidal antibodies. Using a Candidate Antigen Selection Strategy (CASS) based on the gonococcal transcriptome during human mucosal infection, we identified new potential vaccine targets that, when used to immunize mice, induced the production of antibodies with bactericidal activity against N. gonorrhoeae strains. The current study determined antigen recognition by human sera from N. gonorrhoeae-infected subjects, evaluated their potential as a multi-antigen (combination) vaccine in mice and examined the impact of different adjuvants (Alum or Alum+MPLA) on functional antibody responses to N. gonorrhoeae. Our results indicated that a stronger Th1 immune response component induced by Alum+MPLA led to antibodies with improved bactericidal activity. In conclusion, a combination of CASS-derived antigens may be promising for developing effective gonococcal vaccines.

Chlamydia trachomatis (Ct) is the most common cause of bacterial sexually transmitted infections (STIs) worldwide. Ct infections are often asymptomatic in women, leading to severe reproductive tract sequelae. Development of a vaccine against Chlamydia is crucial. The Chlamydia major outer membrane protein (MOMP) is a prime vaccine antigen candidate, and it can elicit both neutralizing antibodies and protective CD4+ T cell responses. We have previously designed chimeric antigens composed of immunogenic variable regions (VDs) and conserved regions (CDs) of MOMP from Chlamydia muridarum (Cm) expressed into a carrier protein (PorB), and we have shown that these were protective in a mouse model of Cm respiratory infection. Here, we generated corresponding constructs based on MOMP from Ct serovar F. Preliminary structure analysis of the three antigens, PorB/VD1-3, PorB/VD1-4 and PorB/VD1-2-4, showed that they retained structure features consistent with those of PorB. The antigens induced robust humoral and cellular responses in mice with different genetic backgrounds. The antibodies were cross-reactive against Ct, but only anti-PorB/VD1-4 and anti-PorB/VD1-2-4 IgG antibodies were neutralizing, likely due to the antigen specificity. The cellular responses included proliferation in vitro and production of IFN-γ by splenocytes following Ct re-stimulation. Our results support further investigation of the PorB/VD antigens as potential protective candidates for a Chlamydia subunit vaccine.

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. All the antigens reacted with sera obtained from women with disseminated gonococcal infection, which supports their 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. In the mouse gonococcal vaginal colonization model, a trend toward accelerated bacterial clearance and lower bacterial burden was observed in vaccinated mice compared to adjuvant control mice. Conclusions: NGO0861, NGO1438 and NGO1802 represent promising targets for a multicomponent gonococcal vaccine, supporting our infection-informed antigen discovery strategy. 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.

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.