Vaccination with Different M2e Epitope Densities Confers Partial Protection against H5N1 Influenza A Virus Challenge in Chickens

<i>Objective:</i> Currently, research is focused on universal influenza vaccines based on various ectodomains of the influenza matrix protein 2 (M2e). Such vaccines are tested mostly using mouse-adapted influenza viruses and in mouse or ferret models. The aim of this study was to investigate in a chicken model the protective efficacy of vaccines based on avian-type M2e at different epitope densities. <i>Methods:</i> On the basis of the optimized avian-type M2e gene, recombinant plasmids that contained tandem copies of different M2e were constructed and expressed in <i>Escherichia coli</i>. The expression and immunogenicity of the proteins were confirmed by SDS-PAGE and Western blot, as well as immunofluorescence assay and enzyme-linked immunosorbent assay. Animals were immunized with fusion proteins emulsified with an appropriate adjuvant and then infected with highly pathogenic influenza virus of A/chicken/Guangdong/04 (H5N1). Antibody levels, survival rate and weight loss were investigated. <i>Results:</i> Multiple copies of M2e were highly expressed; higher epitope density engendered better protection but there was not a linear increase. Among the fusion proteins, the MBP-3·M2e fusion protein showed the best protective efficacy. <i>Conclusions:</i> This study has provided evidence that the immune response to avian-type M2e-based subunit vaccines was greater in chickens than that in mice. In addition, higher M2e epitope density can yield better protection, but not in a linear fashion.