Modular design of a self-amplifying mRNA vaccine for multivalent immunization against Neisseria meningitidis B

Neisseria meningitidis group B (MenB) continues to pose challenges to vaccine development due to its antigenic diversity and immune escape mechanisms. Self-amplifying mRNA (SAM) vaccine platforms offer advantages such as long-lasting expression and flexible antigen combinations, which provide new strategies for tackling complex bacterial pathogens. In this study, we constructed and evaluated two multivalent SAM vaccine designs. The first design involved linking four antigens-factor H binding protein (fHbp), Neisseria Heparin-Binding Antigen (NHBA), Neisserial adhesin A (NadA), and Porin A (PorA)-in tandem within a single fusion SAM construct. The second strategy employed four individual SAM constructs, each encoding a single antigen, and these were delivered either by mixing equal masses of the four SAMs before lipid nanoparticle (LNP) encapsulation (PreMix) or by mixing the individually encapsulated mRNA-LNPs (PostMix). Systematic evaluation revealed that the tandem configuration exhibited limited in vitro expression but still induced immune responses in mice. In contrast, the single-antigen mixed-delivery approach demonstrated superior antigen expression and immunogenicity. The PreMix formulation (SAM-PreMix) elicited a serum bactericidal assay (SBA) titer of 1:256 at a 5 μg dose, which was markedly higher than the 1:32 titer observed with the tandem construct (SAM-SP2). Moreover, strong humoral and cellular immune responses were induced even at a low dose of 1 μg, underscoring the dose-sparing potential of the SAM platform. While the immunization effects of PreMix and PostMix are comparable, the former has more process advantages regarding manufacturability and standardization. Our technology demonstrates significant advantages in both immunization efficacy and process feasibility, offering a universal pathway for the design and platform development of multivalent bacterial mRNA vaccines with potential applications in research related to other complex bacterial pathogens.