Abstract
Killed avian influenza virus (AIV) vaccines have been used to control H5N1 infections in countries where the virus is endemic. Distinguishing vaccinated from naturally infected birds (DIVA) in such situations however, has become a major challenge. Recently, we introduced the recombinant ectodomain of the M2 protein (M2e) of H5N1 subtype as a novel tool for an ELISA based DIVA test. Despite being antigenic in natural infection the monomer form of the M2e used in ELISA had limited antigenicity and consequently poor diagnostic capability. To address this shortcoming, we evaluated the use of four tandem copies of M2e (tM2e) for increased efficiency of M2e antibody detection. The tM2e gene of H5N1 strain from Indonesia (A/Indonesia/CDC540/2006) was cloned into a pMAL- p4x expression vector and expressed in E.coli as a recombinant tM2e-MBP or M2e-MBP proteins. Both of these, M2e and tM2e antigens reacted with sera obtained from chickens following live H5N1 infection but not with sera from vaccinated birds. A significantly stronger M2e antibody reaction was observed with the tM2e compared to M2e antigen. Western blotting also supported the superiority of tM2e over M2e in detection of specific M2e antibodies against live H5N1 infection. Results from this study demonstrate that M2e tetramer is a better antigen than single M2e and could be more suitable for an ELISA based DIVA test.
Highlights
Outbreaks of highly pathogenic avian influenza (HPAI) subtype H5N1 and its possible transmission to humans are of worldwide concern [1,2]
Characterization of Recombinant tandem copies of M2e (tM2e)-maltose binding protein (MBP) by SDS-PAGE and Western Blotting tM2e-MBP protein was expressed in DH5a strain of E.coli and subsequently purified
Western blotting showed that tM2e-MBP reacted with positive sera in the same manner as Matrix protein 2 (M2) protein (M2e)-MBP, which was included as a control
Summary
Outbreaks of highly pathogenic avian influenza (HPAI) subtype H5N1 and its possible transmission to humans are of worldwide concern [1,2]. Transmission of H5N1 virus from infected birds to humans has been frequently reported resulting in severe disease and mortality [1,2,5,6]. Control of H5N1 infections in bird populations is widely considered as an important factor for limiting human exposure to this virus [7]. Utilization of the killed avian influenza virus (AIV) as a vaccine has been widely practiced in H5N1 endemic countries. Differentiation between vaccinated and infected birds (DIVA) is vital to achieve effective control leading to eventual eradication of H5N1 [14,15]
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