A protective vaccine is the only viable way to stop the spread of gonorrhea in the face of rising antibiotic resistance. However, the notorious phase and antigenic variation of Neisseria gonorrhoeae surface proteins remains one of the challenges in vaccine development. To facilitate vaccine advancement efforts, we carried out comprehensive bioinformatic analyses of sequence variation by comparing 34 gonorrhea antigen candidates among >5,000 clinical N. gonorrhoeae isolates deposited in the Neisseria PubMLST database. Eight protein antigens showed exceptional conservation by having a single allele variant distributed in >80% of isolates. An additional 18 vaccine candidates were represented by ≤3 alleles in >50% of N. gonorrhoeae isolates globally. Phylogenetic analyses highlighted closely related antigen variants and additionally showed that AniA and FetB were the closest between N. gonorrhoeae and N. meningitidis Up to 44% of N. meningitidis alleles for both antigens have premature stop codons, suggesting differential expression. Mapping polymorphisms to the available three-dimensional structures of 12 antigens revealed low-frequency surface polymorphisms. PorB and TbpB possessed numerous high-prevalence polymorphic sites. While TbpA was also highly variable, conserved loops were nonetheless identified. A high degree of sequence conservation, the distribution of a single antigen variant among N. gonorrhoeae strains globally, or low-frequency sequence polymorphisms in surface loops make ACP, AniA, BamA, BamE, MtrE, NspA, NGO0778, NGO1251, NGO1985, OpcA, PldA, Slam2, and ZnuD promising candidates for a gonorrhea vaccine. Finally, the commonly used N. gonorrhoeae FA1090 strain emerges as a vaccine prototype, as it carries antigen sequence types identical to the most broadly distributed antigen variants.IMPORTANCENeisseria gonorrhoeae, the Gram-negative bacterium responsible for the sexually transmitted infection gonorrhea, is categorized as a high-priority pathogen for research and development efforts. N. gonorrhoeae's "superbug" status, its high morbidity, and the serious health impact associated with gonorrhea highlight the importance of vaccine development. One of the longstanding barriers to developing an effective vaccine against N. gonorrhoeae is the remarkable variability of surface-exposed antigens. In this report, we addressed this roadblock by applying extensive bioinformatic analyses to 34 gonorrhea antigen candidates among >5,000 clinical N. gonorrhoeae isolates. Our studies are important, as they reveal promising, conserved gonorrhea vaccine candidates and aid structural vaccinology. Moreover, these approaches are broadly applicable to other infectious diseases where surface antigen variability impedes successful vaccine design.

Alaina K. Holm

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