| Summary: | GMMA, outer membrane vesicles resulting from hyperblebbing mutated bacterial strains, are a versatile vaccine platform for displaying both homologous and heterologous antigens. Periplasmic expression is a popular technique for protein expression in the lumen of the blebs. However, the ability of internalized antigens to induce antibody responses has not been extensively investigated. Herein, the <i>Neisseria meningitidis</i> factor H binding protein (fHbp) was heterologously expressed in the lumen of O-antigen positive (OAg+) and O-antigen negative (OAg−) <i>Salmonella</i> Typhimurium GMMA. Only the OAg− GMMA induced an anti-fHbp IgG response in mice if formulated on Alum, although it was weak and much lower compared to the recombinant fHbp. The OAg− GMMA on Alum showed partial instability, with possible exposure of fHbp to the immune system. When we chemically conjugated fHbp to the surface of both OAg+ and OAg− GMMA, these constructs induced a stronger functional response compared to the fHbp immunization alone. Moreover, the OAg+ GMMA construct elicited a strong response against both the target antigens (fHbp and OAg), with no immune interference observed. This result suggests that antigen localization on GMMA surface can play a critical role in the induction of an effective immune response and can encourage the development of GMMA based vaccines delivering key protective antigens on their surface.
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