Improved cross-protection and immunity against influenza A virus in mice using a novel mRNA vaccine with optimized design of RNA sequence

Conventional influenza vaccines provide strain-specific immunity, and their production can be affected by egg-culture-adapted mutations. A universal influenza vaccine with conserved antigens and novel vaccine platforms is urgently required. To induce the immune responses toward more conserved epitopes, we generated a multi-antigen influenza mRNA vaccine, lipopolyplex (LPP)-HNH mRNA, coding headless hemagglutinin stem and neuraminidase with three different sequences (HNH-ORI, HNH-E1, or HNH-E2) and delivered by LPP. The immunogenicity and protective efficacy of these single-chain mRNA vaccines against influenza A viruses were evaluated in mice models. Mice exhibited sustained and robust antibody and cellular immune responses against all three LPP-HNH mRNA vaccines, indicating that these vaccines provided mice with broad protection against H1N1, H3N2, or H5N1 influenza viruses. HNH-E1 and HNH-E2 with optimized sequence showed higher protein expression, stronger specific CD4⁺ and CD8⁺ T cell responses, and more effective protection than those of HNH-ORI, owing to their lower minimum free energy and higher codon adaptation index. These results reveal that the LPP-HNH mRNA vaccine encoding conserved antigens with optimized sequences is a promising strategy for the development of universal influenza vaccines.