M2e nanovaccines supplemented with recombinant hemagglutinin protect chickens against heterologous HPAI H5N1 challenge

Current poultry influenza A virus vaccines target the variable globular head of hemagglutinin (HA1) and provide limited cross-protection. Subunit vaccines using the conserved M2 ectodomain (M2e) generate cross-reactive antibodies but do not fully prevent virus shedding after LPAI challenge and are ineffective against HPAI viruses. This study evaluated combining nanoparticles bearing three tandem M2e repeats (NR-3M2e nanorings or NF-3M2e nanofilaments) with an HA1 subunit vaccine to protect chickens against heterologous HPAI H5N1 challenge. Chickens receiving combined formulations developed M2e- and HA1-specific antibodies, were fully protected from clinical disease and mortality, and displayed no histopathological lesions or virus shedding, in contrast to groups given HA1, NR-3M2e, or NF-3M2e alone. Thus, the combined formulations provided complete cross-protection against heterologous HPAI H5N1 and prevented environmental virus shedding, a key objective for controlling avian influenza outbreaks.