Multimeric Recombinant M2e Protein-Based ELISA: A Significant Improvement in Differentiating Avian Influenza Infected Chickens from Vaccinated Ones

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.

灭活禽流感病毒（AIV）疫苗已被用于在病毒流行国家防控H5N1病毒感染。然而在此类情形下，区分疫苗接种禽与自然感染禽（DIVA）已成为一项重大挑战。近期，我们将H5N1亚型M2蛋白的重组胞外域（M2e）开发为基于酶联免疫吸附试验（ELISA）的DIVA检测的新型工具。尽管M2e在自然感染中具有抗原性，但ELISA中使用的单体M2e抗原性有限，因此诊断能力欠佳。为弥补这一不足，我们评估了采用4个串联拷贝的M2e（tM2e）以提升M2e抗体检测效率的可行性。我们将来自印度尼西亚的H5N1毒株（A/Indonesia/CDC540/2006）的tM2e基因克隆至pMAL-p4x表达载体中，并在大肠杆菌（E.coli）中表达为重组tM2e-MBP或M2e-MBP蛋白。M2e与tM2e两种抗原均可与活H5N1感染鸡的血清产生免疫反应，但无法与疫苗接种禽的血清结合。与单体M2e抗原相比，tM2e与M2e抗体的反应强度显著更高。蛋白质印迹（Western blotting）实验结果也证实，在检测针对活H5N1感染的特异性M2e抗体时，tM2e的性能优于M2e。本研究结果表明，M2e四聚体相较于单体M2e是更优异的抗原，更适用于基于ELISA的DIVA检测。