Recombinant M2e Protein-Based ELISA: A Novel and Inexpensive Approach for Differentiating Avian Influenza Infected Chickens from Vaccinated Ones

Abstract

Available avian influenza (AIV) serological diagnostic tests cannot distinguish vaccinated from naturally infected birds. Differentiation of vaccinated from infected animals (DIVA) is currently advocated as a means of achieving the full control of H5N1. In this study, for the first time, recombinant ectodomain of M2 protein (M2e) of avian influenza virus (H5N1 strain) was used for the DIVA serology test. M2e was cloned into pMAL-P4X vector and expressed in E. coli cells. We used Western blot to recognize the expressed M2e-MBP protein by chicken antisera produced against live H5N1 virus. Also, the specificity of M2e-MBP protein was compared to the M2e synthetic peptide via ELISA. In M2e-MBP ELISA, all sera raised against the live avian influenza viruses were positive for M2e antibodies, whereas sera from killed virus vaccination were negative. Furthermore, M2e-MBP ELISA of the field sera obtained from vaccinated and non-vaccinated chickens showed negative results, while challenged vaccinated chickens demonstrated strong positive reactions. H5N1-originated recombinant M2e protein induced broad-spectrum response and successfully reacted with antibodies against other AIV strains such as H5N2, H9N2, H7N7, and H11N6. The application of the recombinant protein instead of synthetic peptide has the advantages of continues access to an inexpensive reagent for performing a large scale screening. Moreover, recombinant proteins provide the possibility of testing the DIVA results with an additional technique such a Western blotting which is not possible in the case of synthetic proteins. All together, the results of the present investigation show that recombinant M2e-MBP can be used as a robust and inexpensive solution for DIVA test.

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

“…A peptide with a sequence identical to the 23-amino acid sequence of the N-terminal part of the M2 protein of the Indonesian H5N1 isolate, A/Indonesia/CDC540/2006 (GenBank accession no: EU014132.1) was used as the antigen in the ELISA (Hemmatzadeh et al, 2013). The sp-M2e peptide, synthetized by VCPBIO Ltd. (Shenzhen City, China), to a purity of >95%, was diluted in carbonate buffer pH 9.6 at concentration of 5 µg/mL, and added to a 96-well microtitration plate (Maxisorp, Nunc, Roskilde, Denmark), with 100 µL/well.…”

“…Antibodies to the external domain of the M2 protein (M2e) have been reported to be present only in chickens infected with live AIV but not in experimentally vaccinated chickens, thereby enabling the use of M2e protein as DIVA antigen (Lambrecht et al, 2007;Kim et al, 2010;Hemmatzadeh et al, 2013;Hadifar et al, 2014). The M2 protein is packed into the virion in a low copy number in comparison to the other influenza virus surface proteins: HA and neuraminidase (Zebedee & Lamb, 1988).…”

“…Despite its potential as a candidate for a DIVA test, there have been only a few studies on the characterization of the M2e antibody response in vaccinated and infected chickens (Lambrecht et al, 2007;Kim et al, 2010;Marché & van den Berg, 2010;Hemmatzadeh et al, 2013). In studies conducted in pigs and humans, the M2e antibodies were either absent or induced only at low titres (Feng et al, 2006;Kitikoon et al, 2008;Zhong et al, 2014b).…”

An impedance immunosensor based on low-cost microelectrodes and specific monoclonal antibodies for rapid detection of avian influenza virus H5N1 in chicken swabs

“…Currently available methods for detection of H5N1 virus mainly include viral isolation culture (gold standard method) (Woolcock, 2008;Bui et al, 2013), real-time Reverse Transverse Polymerase Chain Reaction (RT-PCR, WHO recommendation method) (WHO, 2007;Xie et al, 2006), and Enzyme Linked Immunosorbent Assay (ELISA) (Hemmatzadeh et al, 2013;Buehler et al, in press). However, these methods are not suitable for in-field screening of AI H5N1 now due to either time-consuming procedures, or complex sample pretreatment, or requirement of well-trained technicians and specialized facilities.…”

Circulating H5N1 virus among native chicken living around commercial layer farms

“…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).…”