Original Article
Nanoparticles based on artificial self-assembling peptide and displaying M2e peptide and stalk HA epitopes of influenza A virus induce potent humoral and T-cell responses and protect against the viral infection

https://doi.org/10.1016/j.nano.2021.102463Get rights and content

Highlights

  • Proteins with influenza A virus M2e peptide and conserved region of HA were obtained.

  • Inclusion of artificial self-aggregating peptide enabled nanoparticles assembly.

  • Immunization of mice with nanoparticles induced humoral and T cellular response.

  • Immunized mice were protected against the lethal challenge with influenza A virus.

Abstract

The extracellular domain of the M2 protein (M2e) and conserved region of the second subunit of the hemagglutinin (HA2) could be used for the development of broad-spectrum vaccines against influenza A. Here we obtained and characterized recombinant mosaic proteins containing tandem copies of M2e and HA2 fused to an artificial self-assembling peptide (SAP). The inclusion of SAP peptides in the fusion proteins enabled their self-assembly in vitro into spherical particles with a size of 30-50 nm. Intranasal immunization of mice with these particles without additional adjuvants induced strong humoral immune response against M2e and the whole virus. Particles carrying both M2e and HA2 induced antigen-specific multifunctional CD4+ effector memory T cells. Immunization provided high protection of mice against the lethal challenge with different subtypes of influenza A virus. The obtained self-assembling nanoparticles can be used to develop a universal influenza vaccine.

Graphical Abstract

Fusion proteins comprising two conserved antigens of influenza A virus, the M2e peptide and conserved region of the second subunit of hemagglutinin (HA2), fused with artificial self-assembling peptide (SAP) were designed and expressed in Escherichia coli. Purified proteins comprising SAP self-assembled into nanosized particles. Immunization of mice revealed that the particles induced strong antigen-specific humoral and T cellular response. Immunized mice were protected against the lethal challenge with influenza A virus.

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Section snippets

Influenza A virus antigens and other parts of fusion proteins

Synthetic nucleotide sequences encoding the following antigens were used to construct fusion genes:

M2eh (SLLTEVETPIRNEWGSRSNDSSD) — consensus sequence of M2e of the human influenza A virus strains with the replacement of cysteines by serines.15

HA2 (RIQDLEKYVEDTKIDL WSYNAELLVALENQHTIDL TDSEMNKLFEKTRRQLR ENA) — conserved region of HA2 (a.a. 76-130) of influenza A viruses A/H3N2 and A/H7N9 (phylogenetic group 2).16

SAP (DMELRELQETLA ALQDVRELLRQQVKQ ITFLKCLLMGGRLLC RLEELERRLEE LERRLEELER) —

Ethic statement

The study was carried out according to the Russian Guidelines for the Care and Use of Laboratory Animals. The protocol was approved by the Committee for Ethics of Animal Experimentation at the Research Institute of Influenza (Permit ID 13/a, approved 21.10.2019). All possible efforts were made to minimize the suffering of the animals.

Design of recombinant proteins, expression and purification

Two conservative influenza antigens were used as the basis for recombinant vaccine proteins — the M2e peptide and the conservative region (a.a. 76-130) of the second (HA2) subunit of the hemagglutinin of the influenza A virus of the second phylogenetic group. The first set of recombinant proteins contained only M2e peptide, while the second pair comprised both M2e and HA2 antigens.

In the M2e sequence, cysteines at positions 17 and 19 were replaced by serines to prevent the formation of

Discussion

The development of a universal recombinant influenza vaccine is an urgent task. Seasonal influenza vaccines fail to protect against influenza for several seasons due to the high variability of the main antigenic determinants of the virus, hemagglutinin and neuraminidase.22 This problem could be overcome by inducing protective antibodies against conserved antigenic determinant such as M2e peptide and the second subunit (HA2) of the hemagglutinin that forms the “stem” of the protein molecule.

Author contributions

Conceptualization, N.V.R. and V.V.K.; investigation, A.A.Z., E.A.B., L.A.S., L.M.T., M.A.S and V.V.K.; resources, L.A.S. and L.M.T.; data curation, L.M.T. and N.V.R.; writing – original draft preparation, A.A.Z., and N.V.R.; writing – review & editing, N.V.R.; supervision, N.V.R.; funding acquisition, E.A.B. and N.V.R. All authors have read and agreed to the published version of the manuscript.

Acknowledgments

This work was supported by the Russian Foundation for Basic Research and Moscow City Government project 21-34-70038 and the Ministry of Science and Higher Education of the Russian Federation.

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    Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results.

    Funding: This work was supported by the Russian Foundation for Basic Research and Moscow city Government according to the project 21-34-70038 and the Ministry of Science and Higher Education of the Russian Federation.

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