Background: Epitope-level prioritization of N. gonorrhoeae outer membrane antigens integrating conservation, structural accessibility, and HLA-stratified population coverage has not been performed. Comprehensive in silico epitope mining enables prioritization of vaccine antigens more likely to elicit targeted immune responses while compressing experimental screening. Building on our proteomics-driven antigen discovery approach, we applied an epitope mining and structural mapping pipeline to 58 cell envelope and secreted proteins.
Methods: Candidate proteins were evaluated for antigenicity, toxicity, linear and conformational B-cell epitope density (ABCpred, BepiPred-3.0, ElliPro), MHC class I and II binding across representative HLA alleles (NetMHCpan, NetMHCIIpan), structural surface accessibility (PyMOL mapping onto experimental structures and AlphaFold models), sequence conservation (BLASTP), and HLA-stratified population coverage (IEDB). Top epitope–MHC interactions were validated by dual-platform peptide docking (ClusPro, HADDOCK). Computational predictions were experimentally validated by peptide ELISA using polyclonal antisera from rabbits and mice immunized with full-length recombinant proteins (rBamA, rSliC, rACP), and by IFN-γ ELISpot using splenocytes from rACP-immunized mice.
Results: Combined HLA class I and class II epitope coverage exceeded 95% across all IEDB geographic regions. Twelve top-tier candidates were prioritized by epitope density, conservation, and surface accessibility, spanning iron acquisition (LbpA, TbpA, TbpB), outer membrane biogenesis (BamA, BamE, LptD, NGO1985), adhesion and colonization (PilN, MafA, MetQ), and immune evasion (AniA, ACP, and SliC). ELISA confirmed that IgG antisera against full-length rBamA, rSliC, and rACP specifically recognized computationally predicted peptide epitopes in both species, with peptides B4, S3, and A3 consistently immunodominant. Dual-platform peptide–MHC docking confirmed stable binding of BamA epitopes AVAEQPEYV and TADGVSLGY to HLA-A*02:01 and HLA-A*01:01, respectively, with anchor residues occupying canonical B/C/F pockets. IFN-γ ELISpot detected specific T-cell responses against predicted MHC class I– and class II–restricted ACP peptides; pooled-peptide stimulation elicited 301 SFUs per 2.5×105 splenocytes, substantially exceeding the sum of individual peptide responses.
Conclusions: This integrated epitope-mining reverse vaccinology framework identifies computationally prioritized, immunologically validated vaccine candidates and provides a rational basis for multi-component gonococcal vaccines. In vivo evaluation of lead candidates in the female mouse lower genital tract infection model is the next step to determine whether the prioritized antigens confer cross-strain protection.