Computationally designed multisubtype epigraph vaccine confers broad-spectrum protection against H1 and H3 swine influenza A viruses

H1 and H3 subtype swine influenza A viruses (IAV-S) threaten global agricultural economies and pose zoonotic risks, yet conventional vaccines provide limited protection against their vast antigenic diversity. Here, we developed a multisubtype vaccine utilizing adenoviral vectors expressing computationally designed H1 and H3 epigraph hemagglutinin (HA) immunogens. In mice, this All-in-One formulation elicited balanced humoral and cellular immunity and demonstrated neutralizing breadth against diverse IAV-S isolates compared to a commercially available vaccine. The multisubtype delivery system elicited concurrent immunity and protection against both H1 and H3 subtypes. The All-in-One vaccine conferred protection against heterologous H1 and H3 challenges and did not exacerbate clinical morbidity characteristic of mismatched vaccination. These findings demonstrate the feasibility of computationally optimized and multiplexed vaccines providing broad-spectrum protection against evolving influenza viruses without compromising safety.