Characterization of the M2e antibody response following highly pathogenic H5N1 avian influenza virus infection and reliability of M2e ELISA for identifying infected among vaccinated chickens

Abstract

A surveillance method able to differentiate between vaccinated and infected poultry is required for those countries that practice vaccination against highly pathogenic avian influenza H5N1. The external domain of the M2 protein (M2e) of influenza virus is a potentially useful differentiating-infected from vaccinated animals (DIVA) antigen but little is known about the M2e antibody response and factors influencing its detection. In this study, the M2e antibody response was characterized in layer birds vaccinated and challenged with an Indonesian H5N1 virus isolate, using a single M2e peptide or four-branched multiple antigenic peptide form of M2e (MAP-M2e) as antigens in two separate ELISAs. Anti-M2e antibodies were absent in chicks with high level of maternal haemagglutination inhibition antibodies and also in all layers vaccinated once, twice or three times with an inactivated commercial H5N1 vaccine. In contrast, anti-M2e antibodies were detected in vaccinated layers challenged with H5N1 virus and their presence was associated with virus isolation and an increase in haemagglutination inhibition titres. The number of birds that developed M2e antibodies, as well as the strength and duration of the M2e antibody response were strongly influenced by the length of the interval between vaccination and challenge. Birds challenged at six weeks after vaccination all developed M2e antibodies by 14 days that lasted until at least 56 days after infection. In birds challenged at two weeks after vaccination, only a proportion of birds developed M2e antibodies by 14 days that lasted only until 28 days post-infection. Both single M2e peptide and MAP-M2e ELISAs had high diagnostic specificity but the diagnostic sensitivity of MAP-M2e ELISA was significantly higher and more effective in detecting M2e antibody in immune and infected birds. The results show that MAP-M2e ELISA would be useful for surveillance in countries using vaccination to control highly pathogenic avian influenza H5N1.

Circulating H5N1 virus among native chicken living around commercial layer farms

“…A possible reasons is that HI, MM2e ELISA and NP ELISA all measure different type of antibodies and it is likely that the duration of these antibodies in serum following infection also differ. The HI antibodies last long in vaccinated chickens (Meulemans et al 1987) whereas the M2e antibodies last only about 8 weeks (Tarigan et al 2015), whereas the duration of NP antibodies is unknown.…”

“…Since not all birds infected with the influenza virus seroconvert to M2e, the percentage of native chicken that had been infected by influenza virus must therefore been higher than the M2e seroprevalence of 3.3% (Lambrecht et al 2007;Kim et al 2010;Hemmatzadeh et al 2013;Tarigan et al 2015).…”

“…(3) A MAP-M2e ELISA was used to detect antibody to external domain of M2 protein (M2e) of AI (H5N1) virus. The protocols for this test has been described previously (Tarigan et al 2015). Briefly, diluted sera were added to the 96-well microtitre plate that previously had been coated with with 4-symmetry-branched-M2e peptide.…”

Characterisation of M2e Antigenicity using anti-M2 Monoclonal Antibody and anti-M2e Polyclonal Antibodies

“…Previous study, using mouse mAb 14C2 reported that M2e was the most important part for antigenicity of M2 protein as mAb 14C2 could not recognise the M2 lacking the ectodomain (M2e) protein (Zebedee & Lamb 1988). M2e protein is considered to be a potential antigen for DIVA test and high specificity of enzyme-linked immunosorbent assay (ELISA) using synthetic M2epeptide or recombinant M2e protein has been reported by several studies (Lambrecht et al 2007;Hemmatzadeh et al 2013;Tarigan et al 2015). Although the M2e based 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.…”

“…M2e protein is considered to be a potential antigen for DIVA test and high specificity of enzyme-linked immunosorbent assay (ELISA) using synthetic M2epeptide or recombinant M2e protein has been reported by several studies (Lambrecht et al 2007;Hemmatzadeh et al 2013;Tarigan et al 2015). Although the M2e based 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. Limited studies, however, 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).…”