Abolishing PorB-induced mitophagy enhances gonococcal outer membrane vesicle vaccine efficacy

<b>Abstract</b>The emergence and global spread of ceftriaxone-resistant <i>Neisseria gonorrhoeae</i> underscores the urgent need for an effective vaccine. Gonococcal outer membrane vesicles (OMVs) are promising as vaccine platform, but their efficacy is potentially compromised by the immunomodulatory properties of gonococcal surface-expressed proteins, particularly the essential outer membrane porin PorB. Our previous work identified that gonococcal OMVs induce epithelial cell mitophagy via PorB, dependent on lysine residues 117 and 171. Given the critical role of dendritic cells (DCs) in initiating adaptive immunity, this study investigated whether PorB-mediated mitophagy in DCs impacts OMV vaccine efficacy. Here, we demonstrated that gonococcal OMVs induce DC mitophagy in a PorB-dependent manner, a process abolished in OMVs expressing the mitophagy-deficient PorB mutant PorB_K117Q/K171Q. OMVs expressing PorB_K117Q/K171Q demonstrated significantly enhanced DC activation, as shown by increased CD86 and MHC-II expression, and promoted a Th1-skewed T cell response with elevated IFN-γ and TNF-α secretion. In immunized mice, OMVs containing PorB_K117Q/K171Q elicited significantly higher total IgG and IgG2a antibody titers against PorB compared with OMVs expressing wild-type PorB, with antibodies displaying enhanced bactericidal activity, including against a strain associated with the high-level ceftriaxone-resistant FC428 clone. Importantly, the OMV PorB_K117Q/K171Q vaccine provided enhanced protection in a mouse vaginal colonization model, accelerating bacterial clearance and reducing overall bacterial burden. Therefore, our results identify PorB-induced mitophagy in DCs as a potential immune evasion mechanism that may dampen adaptive immunity. Engineering OMV vaccines to circumvent this process represents a rational strategy to explore for enhancing gonococcal vaccine efficacy.