Abstract
Matrix 2 protein ectodomain (M2e) is considered a promising candidate for a broadly protective influenza vaccine. M2e-based vaccines against human influenza A provide only partial protection against avian influenza viruses because of differences in the M2e sequences. In this work, we evaluated the possibility of obtaining equal protection and immune response by using recombinant protein on the basis of flagellin as a carrier of the M2e peptides of human and avian influenza A viruses. Recombinant protein was generated by the fusion of two tandem copies of consensus M2e sequence from human influenza A and two copies of M2e from avian A/H5N1 viruses to flagellin (Flg-2M2eh2M2ek). Intranasal immunisation of Balb/c mice with recombinant protein significantly elicited anti-M2e IgG in serum, IgG and sIgA in BAL. Antibodies induced by the fusion protein Flg-2M2eh2M2ek bound efficiently to synthetic peptides corresponding to the human consensus M2e sequence as well as to the M2e sequence of A/Chicken/Kurgan/05/05 RG (H5N1) and recognised native M2e epitopes exposed on the surface of the MDCK cells infected with A/PR/8/34 (H1N1) and A/Chicken/Kurgan/05/05 RG (H5N1) to an equal degree. Immunisation led to both anti-M2e IgG1 and IgG2a response with IgG1 prevalence. We observed a significant intracellular production of IL-4, but not IFN-γ, by CD4+ T-cells in spleen of mice following immunisation with Flg-2M2eh2M2ek. Immunisation with the Flg-2M2eh2M2ek fusion protein provided similar protection from lethal challenge with human influenza A viruses (H1N1, H3N2) and avian influenza virus (H5N1). Immunised mice experienced significantly less weight loss and decreased lung viral titres compared to control mice. The data obtained show the potential for the development of an M2e-flagellin candidate influenza vaccine with broad spectrum protection against influenza A viruses of various origins.
Highlights
Conserved proteins of influenza A virus (M2, HA2, M1, NP) are the target antigens for development of “universal” vaccines against human influenza A viruses of a particular subtype as well as against influenza viruses of distinct origin including potentially pandemic avian ones.Influenza М2 is a small, 97 amino acid protein that forms ion channels in the viral membrane
We demonstrated that intranasal immunisation of BALB/c mice with flagellin of Salmonella typhimurium (Flg)-2M2eh2M2ek resulted in equal antibody response to M2e of human influenza viruses (M2eh) and M2ek in serum and broncho-alveolar lavage (BAL)
The difference in survival rates and weight loss between immunised groups and control animals was significant (P = 0.0002, Montel-Cox test; P = 0.0156, Wilcoxon test). These results show that i.n. immunisation with the Flg-2M2eh2M2ek fusion protein protects mice against challenge with both human influenza A viruses (H1N1, H3N2) and avian influenza virus (H5N1)
Summary
Conserved proteins of influenza A virus (M2, HA2, M1, NP) are the target antigens for development of “universal” vaccines against human influenza A viruses of a particular subtype as well as against influenza viruses of distinct origin including potentially pandemic avian ones. Influenza М2 is a small, 97 amino acid (aa) protein that forms ion channels in the viral membrane. The tetramer М2 protein is expressed on viral surface in low quantities, but is abundantly presented on the plasma membrane of infected cells [2]. The М2 protein ectodomain (М2е)—is a short 24 aa length peptide that is highly conserved and presents an appropriate target for “universal” influenza vaccine development [3, 4]. Previous studies have shown that the immunogenicity of native М2е is poor, but it can be increased by using multimeric forms of M2e, the fusion of M2e to highly immunogenic carriers or application with adjuvants [5,6,7,8,9,10,11,12,13,14]
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