Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A universal influenza A vaccine based on the extracellular domain of the M2 protein

Abstract

The antigenic variation of influenza virus represents a major health problem. However, the extracellular domain of the minor, virus-coded M2 protein is nearly invariant in all influenza A strains. We genetically fused this M2 domain to the hepatitis B virus core (HBc) protein to create fusion gene coding for M2HBc; this gene was efficiently expressed in Escherichia coli. Intraperitoneal or intranasal administration of purified M2HBc particles to mice provided 90–100% protection against a lethal virus challenge. The protection was mediated by antibodies, as it was transferable by serum. The enhanced immunogenicity of the M2 extracellular domain exposed on HBc particles allows broad-spectrum, long-lasting protection against influenza A infections.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: M2HBc fusion proteins.
Figure 2: Expression of M2e on the surface of HBc particles.
Figure 3: Vaccination with IPM2HBc particles administered intraperitoneally.
Figure 4: Titer of antibody against M2e.
Figure 5: Virus clearance from the lungs.
Figure 6: Titer of antibody against M2e in serum samples after intraperitoneal or intranasal immunization with IM2HBc.

Similar content being viewed by others

References

  1. Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M. & Kawaoka, Y. Evolution and ecology of influenza A viruses. Microbiol. Rev. 56, 152–179 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Verhoeyen, M. et al. Antigenic drift between the haemagglutinin of the Hong Kong influenza strains A/Aichi/2/68 and A/Victoria/3/75. Nature 286, 771–776 (1980).

    Article  CAS  PubMed  Google Scholar 

  3. Fang, R., Min Jou, W., Huylebroeck, D., Devos, R. & Fiers, W. Complete structure of A/duck/Ukraine/63 influenza hemagglutinin gene: animal virus as progenitor of human H3 Hong Kong 1968 influenza hemagglutinin. Cell 25, 315–323 (1981).

    Article  CAS  PubMed  Google Scholar 

  4. Lamb, R.A., Zebedee, S.L. & Richardson, C.D. Influenza virus M2 protein is an integral membrane protein expressed on the infected-cell surface. Cell 40, 627–633 (1985).

    Article  CAS  PubMed  Google Scholar 

  5. Zebedee, S.L. & Lamb, R.A. Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions. J. Virol. 62, 2762–2772 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Zebedee, S.L. & Lamb, R.A. Nucleotide sequences of influenza A virus RNA segment 7: a comparison of five isolates. Nucleic Acids Res. 17, 2870 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Ito, T., Gorman, O.T., Kawaoka, Y., Bean, W.J. & Webster, R.G. Evolutionary analysis of the influenza A virus M gene with comparison of the M1 and M2 proteins. J. Virol. 65, 5491–5498 ( 1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Sugrue, R.J. & Hay, A.J. Structural characteristics of the M2 protein of influenza A viruses: evidence that it forms a tetrameric channel. Virology 180, 617–624 (1991).

    Article  CAS  PubMed  Google Scholar 

  9. Holsinger, L.J. & Lamb, R.A. Influenza virus M2 integral membrane protein is a homotetramer stabilized by formation of disulfide bonds. Virology 183, 32– 43 (1991).

    Article  CAS  PubMed  Google Scholar 

  10. Davies, W.L. et al. Antiviral activity of 1-adamantanamine. Science 144, 862–863 ( 1964).

    Article  CAS  PubMed  Google Scholar 

  11. Appleyard G. Amantadine-resistance as a genetic marker for influenza viruses. J. Gen. Virol. 36, 249–255 (1977).

    Article  CAS  PubMed  Google Scholar 

  12. Lubeck, M.D., Schulman, J.L. &z Palese, P. Susceptibility of influenza A viruses to amantadine is influenced by the gene coding for M protein. J. Virol. 28, 710–716 (1978).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Hay, A.J., Kennedy, N.C.T., Skehel, J.J. & Appleyard, G. The matrix protein gene determines amantadine-sensitivity of influenza viruses. J. Gen. Virol. 42, 189– 191 (1979).

    Article  CAS  PubMed  Google Scholar 

  14. Hay, A.J., Wolstenholme, A.J., Skehel, J.J. & Smith, M.H. The molecular basis of the specific anti-influenza action of amantadine. EMBO J. 4, 3021–3024 ( 1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Belshe, R.B., Smith, M.H., Hall, C.B., Betts, R. & Hay, A.J. Genetic basis of resistance to rimantadine emerging during treatment of influenza virus infection. J. Virol. 62 , 1508–1512 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Treanor, J.J., Tierney, E.L., Zebedee, S.L., Lamb, R.A. & Murphy, B.R. Passively transferred monoclonal antibody to the M2 protein inhibits influenza A virus replication in mice. J. Virol. 64, 1375–1377 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Black, R.A., Rota, P.A., Gorodkova, N., Klenk, H.-D. & Kendal, A.P. Antibody response to the M2 protein of influenza A virus expressed in insect cells. J. Gen. Virol. 74, 143–146 ( 1993).

    Article  CAS  PubMed  Google Scholar 

  18. Slepushkin, V.A. et al. Protection of mice against influenza A virus challenge by vaccination with baculovirus-expressed M2 protein. Vaccine 13, 1399–1402 (1995).

    Article  CAS  PubMed  Google Scholar 

  19. Clarke, B.E. et al. Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein. Nature 330, 381–384 (1987).

    Article  CAS  PubMed  Google Scholar 

  20. Borisova, G.P. et al. Recombinant core particles of hepatitis B virus exposing foreign antigenic determinants on their surface. FEBS Lett. 259, 121–124 (1989).

    Article  CAS  PubMed  Google Scholar 

  21. Schödel, F. et al. The position of heterologous epitopes inserted in hepatitis B virus core particles determines their immunogenicity. J. Virol. 66, 106–114 ( 1992).

    PubMed  PubMed Central  Google Scholar 

  22. Ulrich, R., Nassal, M., Meisel, H. & Krüger, D.H. Core particles of hepatitis B virus as carrier for foreign epitopes. Adv. Virus Res. 50, 141–182 ( 1998).

    Article  CAS  PubMed  Google Scholar 

  23. Remaut, E., Stanssens, P. & Fiers, W. Plasmid vectors for high-efficiency expression controlled by the pL promoter of coliphage lambda. Gene 15, 81–93 ( 1981).

    Article  CAS  PubMed  Google Scholar 

  24. Cohen, B.J. & Richmond, J.E. Electron microscopy of hepatitis B core antigen synthesized in E. coli. Nature 296, 677–678 (1982).

    Article  CAS  PubMed  Google Scholar 

  25. Baez, M., Palese, P. & Kilbourne, E.D. Gene composition of high-yielding influenza vaccine strains obtained by recombination. J. Infect. Dis. 141, 362–365 (1980).

    Article  CAS  PubMed  Google Scholar 

  26. Nicholson, K.G., Webster, R.G. & Hay, A.J. (eds.). Textbook of Influenza (Blackwell Science, Oxford, 1998).

    Google Scholar 

  27. Cox, J.C. & Coulter, A.R. Adjuvants—a classification and review of their modes of action. Vaccine 15, 248–256 (1997).

    Article  CAS  PubMed  Google Scholar 

  28. Johansson, B.E., Bucher, D.J. & Kilbourne, E.D. Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce contrasting types of immunity to infection. J. Virol. 63, 1239–1246 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Deroo, T., Min Jou, W. & Fiers, W. Recombinant neuraminidase vaccine protects against lethal influenza. Vaccine 14, 561– 569 (1996).

    Article  CAS  PubMed  Google Scholar 

  30. Johansson, B.E., Matthews, J.T. & Kilbourne, E.D. Supplementation of conventional influenza A vaccine with purified viral neuraminidase results in a balanced and broadened immune response. Vaccine 16, 1009– 1015 (1998).

    Article  CAS  PubMed  Google Scholar 

  31. Ulmer, J.B. et al. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 259, 1745–1749 (1993).

    Article  CAS  PubMed  Google Scholar 

  32. Ulmer, J.B. et al. Protective CD4+ and CD8+ T cells against influenza virus induced by vaccination with nucleoprotein DNA. J. Virol. 72, 5648–5653 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Zhou, X. et al. Generation of cytotoxic and humoral immune responses by nonreplicative recombinant Semliki Forest virus. Proc. Natl. Acad. Sci. USA 92, 3009–3013 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Tsuji, M. et al. Recombinant Sindbis viruses expressing a cytotoxic T-lymphocyte epitope of a malaria parasite or of influenza virus elicit protection against the corresponding pathogen in mice. J. Virol. 72, 6907–6910 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Chen, Z. et al. Comparison of the ability of viral protein-expressing plasmid DNAs to protect against influenza. Vaccine 16, 1544–1549 (1998).

    Article  CAS  PubMed  Google Scholar 

  36. Ahmed, R. & Gray, D. Immunological memory and protective immunity: understanding their relation. Science 272 , 54–60 (1996).

    Article  CAS  PubMed  Google Scholar 

  37. US Department of Health and Human Services in Concepts and Procedures for Laboratory-Based Influenza Surveillance. U.S. Department of Health and Human Services, (Centers for Disease Control, Atlanta, p. B7–B12 and B17– B19 1982).

  38. Remaut, E., Stanssens, P. & Fiers, W. Inducible high level synthesis of mature human fibroblast interferon in Escherichia coli. Nucleic Acids Res. 11, 4677–4688 (1983).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Min Jou, W. et al. Complete structure of the hemagglutinin gene from the human influenza A/Victoria/3/75 (H3N2) strain as determined from cloned DNA. Cell 19, 683–696 ( 1980).

    Article  Google Scholar 

  40. Lamb, R.A., Lai, C.-J. & Choppin, P.W. Sequences of mRNAs derived from genome RNA segment 7 of influenza virus: colinear and interrupted mRNAs code for overlapping proteins. Proc. Natl. Acad. Sci. USA 78, 4170–4174 (1981).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Stanssens, P. et al. Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers. Nucleic Acids Res. 17, 4441– 4454 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Nassal, M. Total chemical synthesis of a gene for hepatitis B virus core protein and its functional characterization. Gene 66, 279–294 (1988).

    Article  CAS  PubMed  Google Scholar 

  43. Deng, W.P. & Nickolov, J.A. Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal. Biochem. 200, 81–88 ( 1992).

    Article  CAS  PubMed  Google Scholar 

  44. Köhler, G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497 ( 1975).

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank I. Bruggeman for technical assistance, G. Engler for electron microscopy analysis, F. Molemans for DNA sequencing and M. Goethals for determination of the N terminus of IPM2HBc.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Walter Fiers.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Neirynck, S., Deroo, T., Saelens, X. et al. A universal influenza A vaccine based on the extracellular domain of the M2 protein. Nat Med 5, 1157–1163 (1999). https://doi.org/10.1038/13484

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/13484

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing