Influence of the Linking Order of Fragments of HA2 and M2e of the influenza A Virus to Flagellin on the Properties of Recombinant Proteins

Abstract

The ectodomain of the M2 protein (M2e) and the conserved fragment of the second subunit of hemagglutinin (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 HA2 (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 M2e tandem copies (Flg-4M2e-HA2) provides partial -helix formation nontypical of native M2e on the virion surface. The C-terminal position of the M2e 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.

Plant-Produced Recombinant Influenza A Virus Candidate Vaccine Based on Flagellin Linked to Conservative Fragments of M2 Protein and Hemagglutintin

“…However, immunization with Flg4M2eHA2-1 induced only a low level of antibodies capable of binding with HA2 synthetic peptides, and most likely anti-HA2 IgG in this vaccine design does not have a major role in protection. A similar result involving a strong anti-M2e immune response and a weak response to HA2 was reported for a similar candidate vaccine based on four copies of M2e and HA2 peptide from phylogenetic group 2 of influenza viruses linked to flagellin [13].…”

“…Five mice from each group were sacrificed (CO 2 -chamber, as described above) on day 6 postinfection, and the lungs were removed. The viral titer in the lungs was determined as described previously [13] and expressed as the log 50% tissue culture infectious dose (TCID 50 ).…”

“…A number of candidate vaccines based on M2e were able to induce a strong M2e-specific humoral immune response to protect animals against influenza A virus challenge [9,10]. A combination of M2e and HA2 in a single vaccine protein is a promising approach for the development of influenza vaccines with a broad spectrum of protection [11][12][13].…”

Comparative Immunological Study in Mice of Inactivated Influenza Vaccines Used in the Russian Immunization Program

“…The mice were routinely euthanized by placing them in a CO 2 euthanasia chamber (Vet Tech Solutions). Mouse sera were received as described above [ 17 ].…”

“…Lungs were harvested from mice as described above [ 18 ]. The titers of viruses reproducing in mice lungs were determined as set forth in [ 17 ]. Briefly, the virus titers were determined by reproduction on the MDCK cell line using the hemagglutination assay with 1% of chicken RBC.…”