Incorporation of a tetrameric M2e antigen enhances cross-protective efficacy of an inactivated H5N1 avian influenza vaccine

Avian influenza (AI) viruses continuously undergo genetic mutations that reduce vaccine efficacy and allow outbreaks to persist. The ectodomain of the M2 protein (M2e) is highly conserved among AI viruses, making it a promising universal vaccine candidate, although its small size limits immunogenicity. This study evaluated a tetrameric M2e antigen (4×M2e51) to enhance vaccine performance. Initial attempts to fuse 4×M2e51 with Salmonella pullorum flagellin were successful at the DNA and expression levels, but the protein was unstable, and research continued with 4×M2e51 alone. Three inactivated H5N1 oil-emulsion vaccines containing mineral oil as adjuvant were prepared: one containing the M101 isolate, one with the M166 isolate, and one combining M101 with 4×M2e51. M101 (A/Ck/West_Java/M101/2016) and M166 (A/Quail/ West_Java/M166/2019) belong to clade 2.3.2.1g and share 95.72% hemagglutinin-1 protein similarity, with evidence of immune evasion. Separate groups of SPF chickens were vaccinated at 28 days of age and challenged with M166 three weeks later. Results showed that hemagglutination inhibition assays detected significant antibody titers only in chickens vaccinated with the homologous M166 strain before challenge. Challenge outcomes demonstrated that supplementation of the M101 vaccine with 4×M2e51 conferred complete protection (100% survival) against heterologous M166 challenge at both full and one-fifth antigen doses, comparable to the homologous vaccine, whereas the M101-only vaccine provided partial (90%) or poor (20%) protection. All unvaccinated controls died (100% mortality). These findings demonstrate, for the first time in field-relevant inactivated vaccines, that incorporating M2e enhances cross-protection, even when the vaccine antigen was not homologous to the challenge virus.