Flagellin-Fused Protein Targeting M2e and HA2 Induces Innate and T-Cell Responses in Mice of Different Genetic Lines.

Efficient control of influenza A infection can potentially be achieved through the development of broad-spectrum vaccines. Recombinant proteins incorporating conserved influenza A virus peptides are one of the platforms for the development of cross-protective influenza vaccines. We constructed a recombinant protein Flg-HA2-2-4M2ehs, in which the extracellular domain of the M2 protein (M2e) and the sequence (aa76-130) of the second subunit of HA (HA2) were used as target antigens. In this study, we investigated the ability of the Flg-HA2-2-4M2ehs protein to activate innate immunity and stimulate the formation of T-cell response in mice of different genetic lines after intranasal immunization. Our studies showed that the Flg-HA2-2-4M2ehs protein was manifested in an increase in the relative content of neutrophils, monocytes, and interstitial macrophages, against the backdrop of a decrease in the level of dendritic cells and increased expression in the CD86 marker. In the lungs of BALB/c mice, immunization with the Flg-HA2-2-4M2ehs protein induced the formation of antigen-specific CD4+ and CD8+ effector memory T cells, producing TNF-α. In mice C57Bl/6, the formation of antigen-specific effector CD8+ T cells, predominantly producing IFN-γ+, was demonstrated. The data obtained showed the formation of CD8+ and CD4+ effector memory T cells expressing the CD107a.

Flagellin-fused protein targeting M2e and HA2 induces potent humoral and T-cell responses and protects mice against various influenza viruses a subtypes.

BACKGROUND: Current influenza vaccines are mainly strain-specific and have limited efficacy in preventing new, potentially pandemic, influenza strains. Efficient control of influenza A infection can potentially be achieved through the development of broad-spectrum vaccines based on conserved antigens. A current trend in the design of universal flu vaccines is the construction of recombinant proteins based on combinations of various conserved epitopes of viral proteins (M1, M2, HA2, NP). In this study, we compared the immunogenicity and protective action of two recombinant proteins which feature different designs and which target different antigens. RESULTS: Balb/c mice were immunized subcutaneously with Flg-HA2-2-4M2ehs or FlgSh-HA2-2-4M2ehs; these constructs differ in the location of hemagglutinin's HA2-2(76-130) insertion into flagellin (FliC). The humoral and T-cell immune responses to these constructs were evaluated. The simultaneous expression of different M2e and HA2-2(76-130) in recombinant protein form induces a strong M2e-specific IgG response and CD4+/ CD8+ T-cell response. The insertion of HA2-2(76-130) into the hypervariable domain of flagellin greatly increases antigen-specific T-cell response, as evidenced by the formation of multi-cytokine-secreting CD4+, CD8+ T-cells, Tem, and Tcm. Both proteins provide full protection from lethal challenge with A/H3N2 and A/H7N9. CONCLUSION: Our results show that highly conserved M2e and HA2-2(76-130) can be used as important targets for the development of universal flu vaccines. The location of the HA2-2(76-130) peptide's insertion into the hypervariable domain of flagellin had a significant effect on the T-cell response to influenza antigens, as seen by forming of multi-cytokine-secreting CD4+ and CD8+ T-cells.

Protection against multiple influenza A virus strains induced by candidate recombinant vaccine based on heterologous M2e peptides linked to flagellin.

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.

Immunogenicity and protective efficacy of candidate universal influenza A nanovaccines produced in plants by Tobacco mosaic virus-based vectors.

A new approach for super-expression of the influenza virus epitope M2e in plants has been developed on the basis of a recombinant Tobacco mosaic virus (TMV, strain U1) genome designed for Agrobacterium-mediated delivery into the plant cell nucleus. The TMV coat protein (CP) served as a carrier and three versions of the M2e sequence were inserted into the surface loop between amino acid residues 155 and 156. Cysteine residues in the heterologous peptide were thought likely to impede efficient assembly of chimeric particles. Therefore, viral vectors TMV-M2e-ala and TMV-M2e-ser were constructed in which cysteine codons 17 and 19 of the M2e epitope were substituted by codons for serine or alanine. Agroinfiltration experiments proved that the chimeric viruses were capable of systemically infecting Nicotiana benthamiana plants. Antisera raised against TMV-M2e-ala virions appear to contain far more antibodies specific to influenza virus M2e than those specific to TMV carrier particle (ratio 5:1). Immunogold electron microscopy showed that the 2-epitopes were uniformly distributed and tightly packed on the surface of the chimeric TMV virions. Apparently, the majority of the TMV CP-specific epitopes in the chimeric TMV-M2e particles are hidden from the immune system by the M2e epitopes exposed on the particle surface. The profile of IgG subclasses after immunization of mice with TMV-M2e-ser and TMV-M2e-ala was evaluated. Immunization with TMV-M2e-ala induced a significant difference between the levels of IgG1 and IgG2a (IgG1/IgG2a=3.2). Mice immunized with the chimeric viruses were resistant to five lethal doses (LD50) of the homologous influenza virus strain, A/PR/8/34 (H1N1) and TMV-M2e-ala also gave partial protection (5LD50, 70% of survival rate) against a heterologous strain influenza A/California/04/2009 (H1N1) (4 amino acid changes in M2e). These results indicate that a new generation candidate universal nanovaccine against influenza based on a recombinant TMV construct has been obtained.