Influence of the Linking Order of Fragments of HA2 and М2е of the influenza A Virus to Flagellin on the Properties of Recombinant Proteins

The ectodomain of the M2 protein (M2e) and the conserved fragment of the second subunit of hemag-glutinin (HA2) are promising candidates for broadly protective vaccines. In this paper, we report on the design of chimeric constructs with differing orders of linkage of four tandem copies of M2e and the conserved fragment of НА2 (76–130) from phylogenetic group II influenza A viruses to the C-terminus of flagellin. The 3D-structure of two chimeric proteins showed that interior location of the M2е tandem copies (Flg-4M2e-HA2) provides partial α-helix formation nontypical of native М2е on the virion surface. The C-terminal position of the М2е tandem copies (Flg-HA2-4M2e) largely retained its native M2e conformation. These conformational differences in the structure of the two chimeric proteins were shown to affect their immunogenic properties. Different antibody levels induced by the chimeric proteins were detected. The protein Flg-HA2-4M2e was more immunogenic as compared to Flg-4M2e-HA2, with the former offering full protection to mice against a lethal challenge. We obtained evidence suggesting that the order of linkage of target antigens in a fusion protein may influence the 3D conformation of the chimeric construct, which leads to changes in immunogenicity and protective potency.