Development of an M2e-Specific Enzyme-Linked Immunosorbent Assay for Differentiating Infected from Vaccinated Animals

Abstract

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.

Field Application of the H9M2e Enzyme-Linked Immunosorbent Assay for Differentiation of H9N2 Avian Influenza Virus-Infected Chickens from Vaccinated Chickens

“…However, in our study, anti-NS1 antibodies were not sufficient to differentiate infected from vaccinated sera in experimentally infected and vaccinated chickens using insect cell-expressed recombinant NS1 protein and NS1 peptides as the antigen (data not shown). Recently, the extracellular domain of the M2 protein (M2e) has been chosen as another putative differential diagnostic marker for H5 and H7 subtypes (14). Lambrecht et al (14) reported that M2e-specific antibodies were positive in chickens and ducks experimentally infected with H7 or H5 HPAI viruses, respectively, by M2e ELISAs but not in chicken and ducks inoculated with inactivated-AIV vaccines (14).…”

“…Recently, the extracellular domain of the M2 protein (M2e) has been chosen as another putative differential diagnostic marker for H5 and H7 subtypes (14). Lambrecht et al (14) reported that M2e-specific antibodies were positive in chickens and ducks experimentally infected with H7 or H5 HPAI viruses, respectively, by M2e ELISAs but not in chicken and ducks inoculated with inactivated-AIV vaccines (14). In addition, ducks inoculated with inactivated vaccine and challenged with an HPAI H5N1 virus were differentiated from ducks inoculated with inactivated vaccine (14).…”

“…Lambrecht et al (14) reported that M2e-specific antibodies were positive in chickens and ducks experimentally infected with H7 or H5 HPAI viruses, respectively, by M2e ELISAs but not in chicken and ducks inoculated with inactivated-AIV vaccines (14). In addition, ducks inoculated with inactivated vaccine and challenged with an HPAI H5N1 virus were differentiated from ducks inoculated with inactivated vaccine (14). A few changes were observed in the M2e segment of different influenza viruses, although the M2 protein is remarkably conserved among type A influenza viruses (14,18,19).…”

“…In addition, ducks inoculated with inactivated vaccine and challenged with an HPAI H5N1 virus were differentiated from ducks inoculated with inactivated vaccine (14). A few changes were observed in the M2e segment of different influenza viruses, although the M2 protein is remarkably conserved among type A influenza viruses (14,18,19). The reactivity of M2e ELISA was a little different for M2e sequences as an antigen peptide; the reactivity was better for the same peptide sequence than with different peptides (14).…”

“…Recently, an alternative to DIVA strategy was reported using the measurement of a differential immune response to the extracellular domain of the third integral membrane protein (M2 protein) of the influenza virus (14). The M2e protein (extracellular domain of M2), like NS1, is also associated with actively replicating virus which is exuberantly synthesized in infected cells but is not incorporated into the infectious viral particle, and M2 protein shares with NS1 protein the potential for elaborated DIVA testing, due to differential epitope density on the surfaces of infected cells and on infectious viral particles (10,18,22,35).…”