Development of an M2e-specific enzyme-linked immunosorbent assay for differentiating infected from vaccinated animals

Vaccination programs for the control of avian influenza (AI) in birds have restrictions because of some limited efficacy and the difficulty of discriminating between vaccinated and virus-infected poultry. We studied M2e, the highly conserved external domain of the influenza A M2 protein, as a potential differential diagnostic marker for influenza virus infection. The M2 protein is an integral membrane protein, scarcely present on virus particles, but abundantly expressed on virus-infected cells. M2e-specific enzyme-linked immunosorbent assays (ELISAs) for different avian influenza strains were developed by coating the peptides corresponding to the first 18 amino acids, without the first methionine, of the universal human consensus M2e sequence and the specific M2e sequence of two highly pathogenic AI (HPAI) strains, H7N7 and H5N1. Using the M2e ELISAs, M2e-specific antibodies were observed in chickens and ducks experimentally infected with H7 or H5 HPAI, respectively, that correlated well with hemagglutination inhibition (HI) antibodies. Conversely, sera from chicken and ducks inoculated with inactivated AI vaccines were positive for HI test but negative for the M2e ELISAs. Moreover, ducks inoculated with inactivated vaccine and challenged with a HPAI H5N1 seroconverted for antibodies to the M2e peptide, with significantly different levels from those measured between the vaccinated and infected groups. These results indicate the potential benefit of a simple and specific M2e ELISA in the assessment of the efficacy of vaccination as well as for diagnostic and survey applications.