The continuous evolution and co-circulation of H5 and H7 subtype highly pathogenic avian influenza viruses (HPAIVs) have caused substantial economic losses to the global poultry industry and pose a persistent threat to public health. This study aimed to develop a bivalent nucleoside-modified messenger (mRNA) vaccine encoding the hemagglutinin (HA) antigens of circulating H5N1 and H7N9 strains and to evaluate its immunogenicity and protective efficacy in an SPF chicken model. The bivalent vaccine elicited robust humoral immunity in SPF chickens in a dose-dependent manner following immunization. The immune sera exhibited potent neutralizing activity against the homologous viruses and cross-reactivity with heterologous strains. Challenge experiments demonstrated that chickens immunized with high doses (50 μg and 80 μg) of the bivalent mRNA vaccine were 100% protected against lethal challenge with homologous and heterologous H5N1, as well as heterologous H7N9 viruses. Furthermore, the vaccine effectively suppressed viral replication in the lungs and significantly reduced or blocked viral shedding. Importantly, splenic transcriptome sequencing revealed that vaccination elicited extensive immune reprogramming, with marked upregulation of key genes associated with Th1-type immune responses, antigen presentation, and cytokine production. The bivalent mRNA-lipid nanoparticle (LNP) vaccine developed in this study exhibited excellent immunogenicity and broad-spectrum protective potential in SPF chickens. Transcriptomic analysis further elucidated the molecular mechanisms underlying the vaccine-induced protective immunity. This study provides a promising vaccine candidate for the control of the co-circulation of H5 and H7 subtype HPAIVs.
