Abstract
Matrix 2 ectodomain (M2e) protein is a potential antigen for detection of influenza A virus infection in vaccinated poultry (DIVA test). However the M2e antigenicity and immune response it induces in either humans or animals are poorly understood. Seventeen M2e peptides and sixteen recombinant M2e (rM2e) proteins with amino acid (aa) changes introduced at position 10, 11, 12, 13 14, 16, 18 and 20 were compared by western blot (WB) and enzyme-linked immunosorbent assay (ELISA) using mouse anti-M2 monoclonal antibody (mAb) 14C2, and anti-M2e peptide chicken and rabbit polyclonal antibody (pAb). The mAb 14C had the best discriminating power and indicated that all six positions contributed to the M2e antigenicity. Position 11 was the important immunodominant and affected Mab14C binding to a greatest degree. Changes in the adjacent position 14, 16 and 18 also influenced the binding, and it detected regardless of the method (WB or ELISA), or the antigen used (M2e peptide or rM2e). For chicken pAb and rabbit pAb, the immunodominant aa was position 10 and the antibody reaction was not affected by aa change at 11. The binding of rabbit pAb was also affected by changes at 14 and 16, which confirm the contribution of these positions to the M2e antigenicity. Position 10 was the only important position for the binding of chicken pAb to M2e. Overall, the study showed that the M2e antigenic sites are located between residues 10 – 18 and that aa changes at position 10, 11, 12, 14, 16 and 18 may all affect the antibody binding within the M2e protein.
Highlights
Pathogenic avian influenza subtype H5N1, has continued to be a significant concern for more than a decade globally, and especially in several South EastAsia countries where the virus has become endemic in commercial poultry
The Matrix 2 (M2) lacking the ectodomain (M2e) based differentiation of infected from vaccinated animals (DIVA) test has been evaluated for possible use in commercial poultry (Lambrecht et al 2007; Hemmatzadeh et al 2013; Kim et al 2010, Tarigan et al 2015), very little is known about the antigenicity of the M2e and antibody response it induces in infected poultry
The M2e based DIVA test has been evaluated for possible use in commercial poultry, very little is known about the antigenicity of M2e and its specificity to detect infections caused by avian influenza virus (AIV)
Summary
Pathogenic avian influenza subtype H5N1, has continued to be a significant concern for more than a decade globally, and especially in several South EastAsia countries where the virus has become endemic in commercial poultry. Ectodomain matrix 2 (M2e) protein is a 24 amino acid long protein located in the Matrix 2 (M2) protein N terminal It is abundantly express on the cell surface infected by Influenza A virus (IAV), but very few are found in the virion of AIV (Lamb et al 1985). Limited studies have been carried on M2e antigenicity and immunogenicity and these studies were mostly focused on the M2e responses in humans, mice, pigs or ferrets (Schotsaert et al 2009) Those studies concluded that M2e was a poor immunogen and induced antibodies only in a fraction of infected individuals, and antibody titres were low and of short duration (Feng et al 2006); (Kitikoon et al 2008); (Bianchi et al 2001). Antigenic variations in the M2e protein have only been studied using monoclonal antibodies (Zharikova et al 2005; Zebedee & Lamb 1988)
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