Dynamic regulation-based stabilizing mutations are highly effective for designing RSV pre-fusion F mRNA vaccines.

Respiratory syncytial virus (RSV) is a leading cause of respiratory tract infections and poses a serious threat to young children, older adults, and immunocompromised individuals. We previously designed a pre-fusion stabilized F protein (pre-F), TriM-5, using a dynamic regulation mechanism, and produced a recombinant protein-based vaccine candidate that exhibited potent protection against RSV. However, several studies have revealed that the stabilization of pre-F achieved in protein form may not be sufficient for application in mRNA vaccine development. Here, based on TriM-5, we designed an mRNA vaccine candidate to assess whether the dynamic regulation-based stabilizing mutations of TriM-5 are also effective in an mRNA format. The TriM-5 mRNA was codon-optimized using in-house software named NVSI_GAmRNAopt, which employed a genetic algorithm incorporating multiple empirical parameters for coding sequence (CDS) optimization, and encapsulated using lipid nanoparticles (LNPs). Mouse immunization showed that after CDS optimization, the IgG antibody titers increased approximately threefold, indicating the effectiveness of our codon-optimization algorithm. Compared with the Moderna RSV mRNA sequence encapsulated with our proprietary BM028 LNPs, the TriM-5 mRNA-LNP induced 3.03-fold higher neutralizing antibody titers against RSV A2 virus, along with significantly higher IFN-γ, IL-5 and IL-17A secretion levels, indicating its superior immunogenicity. Virus challenge experiments demonstrated that both TriM-5 mRNA-LNP and Moderna mRNA-LNP reduced lung viral loads effectively, with no obvious lung pathology. The designed TriM-5 mRNA-LNP exhibits good immunogenicity and protective efficacy, and thereby may serve as a promising candidate for further vaccine development.