An mRNA Vaccine with Tandem Mutated HA-NA Confers Protection Against Multiple Strains of H1N1 Influenza

Background/Objectives: Recurrent influenza epidemics impose a severe global burden, with conventional vaccines constrained by production time lags and rapid viral mutation. This study aims to explore a novel influenza mRNA vaccine design that balances conserved and mutable antigen regions. By combining hemagglutinin (HA) and neuraminidase (NA) into a dual-target approach, the objective is to simultaneously block viral entry and inhibit progeny release, potentially establishing a proposed “front-blockade, rear-containment” dual protective barrier against multiple H1N1 strains. Methods: We engineered a dual-target tandem mRNA vaccine linking mutated HA with conserved NA, with strategic amino acid mutations introduced into key antigenic sites within the HA head domain. Vaccine efficacy was evaluated in a mouse model. Humoral immunity was assessed by measuring antigen-specific antibody titers, and cellular immunity was evaluated via ELISpot assay. Protective capacity was determined through lethal challenge experiments using diverse H1N1 viral strains. Results: The vaccine successfully expressed the HA-NA tandem antigen at 130 kDa, and the in vitro-expressed antigen exhibited normal neuraminidase activity. Preliminary evidence supported the dual-target concept in model mice: hemagglutination-inhibiting and micro-neutralizing antibodies targeting HA were detected, and serum neuraminidase-inhibiting activity was also observed. In addition to triggering potent cellular immune responses, the vaccine offered total protection against lethal doses of various H1N1 variants. Conclusions: This study suggests a promising dual-target strategy that harmonizes antigen conservation and mutation while potentially establishing a synergistic front-blockade and rear-containment defense. The approach offers a viable pathway for developing improved H1N1 influenza vaccines.