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:

Antigenic drift originating from changes to the lateral surface of the neuraminidase head of influenza A virus

Nature Microbiology volume 4, pages 1024–1034 (2019)Cite this article

Subjects

Abstract

Influenza viruses possess two surface glycoproteins, haemagglutinin and neuraminidase (NA). Although haemagglutinin plays a major role as a protective antigen, immunity to NA also contributes to protection. The NA protein consists of a stalk and a head portion, the latter of which possesses enzymatic NA (or sialidase) activity. Like haemagglutinin, NA is under immune pressure, which leads to amino acid alterations and antigenic drift. Amino acid changes accumulate around the enzymatic active site, which is located at the top of the NA head. However, amino acid alterations also accumulate at the lateral surface of the NA head. The reason for this accumulation remains unknown. Here, we isolated seven anti-NA monoclonal antibodies (mAbs) from individuals infected with A(H1N1)pdm09 virus. We found that amino acid mutations on the lateral surface of the NA head abolished the binding of all of these mAbs. All seven mAbs activated Fcγ receptor (FcγR)-mediated signalling pathways in effector cells and five mAbs possessed NA inhibition activity, but the other two did not; however, all seven protected mice from lethal challenge infection through their NA inhibition activity and/or FcγR-mediated antiviral activity. Serological analysis of individuals infected with A(H1N1)pdm09 virus revealed that some possessed or acquired the anti-NA-lateral-surface antibodies following infection. We also found antigenic drift on the lateral surface of the NA head of isolates from 2009 and 2015. Our results demonstrate that anti-lateral-surface mAbs without NA inhibition activity can provide protection by activating FcγR-mediated antiviral activity and can drive antigenic drift at the lateral surface of the NA head. These findings have implications for NA antigenic characterization in that they demonstrate that traditional NA inhibition assays are inadequate to fully characterize NA antigenicity.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Fig. 1: Breadth of reactivity of seven isolated mAbs.
Fig. 2: Epitope analysis of the isolated anti-NA mAbs.
Fig. 3: In vitro characterization of the isolated anti-NA mAbs.
Fig. 4: In vivo protective efficacy of anti-NA mAbs.
Fig. 5: Competitive binding between antibodies in the sera of infected individuals and mAbs targeting the lateral surface of the NA head.
Fig. 6: Antigenic differences between CA/04/09-NA and YO/94/15-NA.

Similar content being viewed by others

Data availability

All data analysed during this study are included in this article. The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.

References

  1. Wilson, I. A., Skehel, J. J. & Wiley, D. C. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 Å resolution. Nature 289, 366–373 (1981).

    Article  CAS  Google Scholar 

  2. Weis, W. et al. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature 333, 426–431 (1988).

    Article  CAS  Google Scholar 

  3. Job, E. R. et al. Serum amyloid P is a sialylated glycoprotein inhibitor of influenza A viruses. PLoS ONE 8, e59623 (2013).

    Article  Google Scholar 

  4. Palese, P., Tobita, K., Ueda, M. & Compans, R. W. Characterization of temperature sensitive influenza virus mutants defective in neuraminidase. Virology 61, 397–410 (1974).

    Article  CAS  Google Scholar 

  5. Matrosovich, M. N., Matrosovich, T. Y., Gray, T., Roberts, N. A. & Klenk, H. D. Neuraminidase is important for the initiation of influenza virus infection in human airway epithelium. J. Virol. 78, 12665–12667 (2004).

    Article  CAS  Google Scholar 

  6. Potter, C. W. & Oxford, J. S. Determinants of immunity to influenza infection in man. Br. Med. Bull. 35, 69–75 (1979).

    Article  CAS  Google Scholar 

  7. Hobson, D., Curry, R. L., Beare, A. S. & Ward-Gardner, A. The role of serum haemagglutination-inhibiting antibody in protection against challenge infection with influenza A2 and B viruses. J. Hyg. 70, 767–777 (1972).

    Article  CAS  Google Scholar 

  8. Wrammert, J. et al. Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection. J. Exp. Med. 208, 181–193 (2011).

    Article  CAS  Google Scholar 

  9. Chen, Y. Q. et al. Influenza infection in humans induces broadly cross-reactive and protective neuraminidase-reactive antibodies. Cell 173, 417–429 (2018).

    Article  CAS  Google Scholar 

  10. Knossow, M. & Skehel, J. J. Variation and infectivity neutralization in influenza. Immunology 119, 1–7 (2006).

    Article  CAS  Google Scholar 

  11. Yamayoshi, S. et al. A broadly reactive human anti-hemagglutinin stem monoclonal antibody that inhibits influenza A virus particle release. EBioMedicine 17, 182–191 (2017).

    Article  Google Scholar 

  12. Brandenburg, B. et al. Mechanisms of hemagglutinin targeted influenza virus neutralization. PLoS ONE 8, e80034 (2013).

    Article  Google Scholar 

  13. Skehel, J. J. & Wiley, D. C. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu. Rev. Biochem. 69, 531–569 (2000).

    Article  CAS  Google Scholar 

  14. Schulman, J. L., Khakpour, M. & Kilbourne, E. D. Protective effects of specific immunity to viral neuraminidase on influenza virus infection of mice. J. Virol. 2, 778–786 (1968).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Webster, R. G. & Laver, W. G. Preparation and properties of antibody directed specifically against the neuraminidase of influenza virus. J. Immunol. 99, 49–55 (1967).

    CAS  PubMed  Google Scholar 

  16. Wilson, J. R. et al. An influenza A virus (H7N9) anti-neuraminidase monoclonal antibody with prophylactic and therapeutic activity in vivo. Antiviral Res. 135, 48–55 (2016).

    Article  CAS  Google Scholar 

  17. Wohlbold, T. J. et al. Broadly protective murine monoclonal antibodies against influenza B virus target highly conserved neuraminidase epitopes. Nat. Microbiol. 2, 1415–1424 (2017).

    Article  CAS  Google Scholar 

  18. Bosch, B. J. et al. Recombinant soluble, multimeric HA and NA exhibit distinctive types of protection against pandemic swine-origin 2009 A(H1N1) influenza virus infection in ferrets. J. Virol. 84, 10366–10374 (2010).

    Article  CAS  Google Scholar 

  19. Seto, J. T. & Chang, F. S. Functional significance of sialidase during influenza virus multiplication: an electron microscope study. J. Virol. 4, 58–66 (1969).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Monto, A. S. et al. Antibody to influenza virus neuraminidase: an independent correlate of protection. J. Infect. Dis. 212, 1191–1199 (2015).

    Article  CAS  Google Scholar 

  21. Monto, A. S. & Kendal, A. P. Effect of neuraminidase antibody on Hong Kong influenza. Lancet 1, 623–625 (1973).

    Article  CAS  Google Scholar 

  22. DiLillo, D. J., Palese, P., Wilson, P. C. & Ravetch, J. V. Broadly neutralizing anti-influenza antibodies require Fc receptor engagement for in vivo protection. J. Clin. Invest. 126, 605–610 (2016).

    Article  Google Scholar 

  23. Perussia, B., Starr, S., Abraham, S., Fanning, V. & Trinchieri, G. Human natural killer cells analyzed by B73.1, a monoclonal antibody blocking Fc receptor functions. I. Characterization of the lymphocyte subset reactive with B73.1. J. Immunol. 130, 2133–2141 (1983).

    CAS  PubMed  Google Scholar 

  24. Stuart, S. G. et al. Human IgG Fc receptor (hFcRII; CD32) exists as multiple isoforms in macrophages, lymphocytes and IgG-transporting placental epithelium. EMBO J. 8, 3657–3666 (1989).

    Article  CAS  Google Scholar 

  25. Futosi, K., Fodor, S. & Mócsai, A. Neutrophil cell surface receptors and their intracellular signal transduction pathways. Int. Immunopharmacol. 17, 638–650 (2013).

    Article  CAS  Google Scholar 

  26. Bournazos, S., DiLillo, D. J. & Ravetch, J. V. The role of Fc-FcγR interactions in IgG-mediated microbial neutralization. J. Exp. Med. 212, 1361–1369 (2015).

    Article  CAS  Google Scholar 

  27. Pincetic, A. et al. Type I and type II Fc receptors regulate innate and adaptive immunity. Nat. Immunol. 15, 707–716 (2014).

    Article  CAS  Google Scholar 

  28. Laver, W. G., Air, G. M., Webster, R. G. & Markoff, L. J. Amino acid sequence changes in antigenic variants of type A influenza virus N2 neuraminidase. Virology 122, 450–460 (1982).

    Article  CAS  Google Scholar 

  29. Luther, P., Bergmann, K. C. & Oxford, J. S. An investigation of antigenic drift of neuraminidases of influenza A (H1N1) viruses. J. Hyg. 92, 223–229 (1984).

    Article  CAS  Google Scholar 

  30. Webster, R. G., Laver, W. G., Air, G. M. & Schild, G. C. Molecular mechanisms of variation in influenza viruses. Nature 296, 115–121 (1982).

    Article  CAS  Google Scholar 

  31. Air, G. M., Els, M. C., Brown, L. E., Laver, W. G. & Webster, R. G. Location of antigenic sites on the three-dimensional structure of the influenza N2 virus neuraminidase. Virology 145, 237–248 (1985).

    Article  CAS  Google Scholar 

  32. Colman, P. M., Varghese, J. N. & Laver, W. G. Structure of the catalytic and antigenic sites in influenza virus neuraminidase. Nature 303, 41–44 (1983).

    Article  CAS  Google Scholar 

  33. Fanning, T. G., Reid, A. H. & Taubenberger, J. K. Influenza A virus neuraminidase: regions of the protein potentially involved in virus-host interactions. Virology 276, 417–423 (2000).

    Article  CAS  Google Scholar 

  34. Dreyfus, C. et al. Highly conserved protective epitopes on influenza B viruses. Science 337, 1343–1348 (2012).

    Article  CAS  Google Scholar 

  35. Simonelli, L. et al. Rational engineering of a human anti-dengue antibody through experimentally validated computational docking. PLoS ONE 8, e55561 (2013).

    Article  CAS  Google Scholar 

  36. Wright P., Neumann G. & Kawaoka Y. in Field Virology 5th edn, vol. 2 (eds Knipe, D. M. & Howley, P. M.) 1691–1740 (Lippincott Williams & Wilkins, Philadelphia, 2007).

  37. Iwatsuki-Horimoto, K. et al. Seroprevalence of pandemic 2009 (H1N1) influenza A virus among schoolchildren and their parents in Tokyo, Japan. Clin. Vaccine Immunol. 18, 860–866 (2011).

    Article  CAS  Google Scholar 

  38. Murphy, B. R., Kasel, J. A. & Chanock, R. M. Association of serum anti-neuraminidase antibody with resistance to influenza in man. N. Engl. J. Med. 286, 1329–1332 (1972).

    Article  CAS  Google Scholar 

  39. Bragstad, K., Nielsen, L. P. & Fomsgaard, A. The evolution of human influenza A viruses from 1999 to 2006: a complete genome study. Virol. J. 5, 40 (2008).

    Article  Google Scholar 

  40. Memoli, M. J. et al. Evaluation of antihemagglutinin and antineuraminidase antibodies as correlates of protection in an influenza A/H1N1 virus healthy human challenge model. mBio 7, e00417-16 (2016).

    Article  Google Scholar 

  41. Wan, H. et al. Structural characterization of a protective epitope spanning A(H1N1)pdm09 influenza virus neuraminidase monomers. Nat. Commun. 6, 6114 (2015).

    Article  CAS  Google Scholar 

  42. Jiang, L. et al. Comparative efficacy of monoclonal antibodies that bind to different epitopes of the 2009 pandemic H1N1 influenza virus neuraminidase. J. Virol. 90, 117–128 (2015).

    Article  Google Scholar 

  43. Kubota-Koketsu, R. et al. Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors. Biochem. Biophys. Res. Commun. 387, 180–185 (2009).

    Article  CAS  Google Scholar 

  44. Yasuhara, A. et al. Diversity of antigenic mutants of influenza A(H1N1)pdm09 virus escaped from human monoclonal antibodies. Sci. Rep. 7, 17735 (2017).

    Article  Google Scholar 

  45. Neumann, G. et al. Generation of influenza A viruses entirely from cloned cDNAs. Proc. Natl Acad. Sci. USA 96, 9345–9350 (1999).

    Article  CAS  Google Scholar 

  46. Sakabe, S., Ozawa, M., Takano, R., Iwastuki-Horimoto, K. & Kawaoka, Y. Mutations in PA, NP, and HA of a pandemic (H1N1) 2009 influenza virus contribute to its adaptation to mice. Virus Res. 158, 124–129 (2011).

    Article  CAS  Google Scholar 

  47. Yamayoshi S. et al. Human protective monoclonal antibodies against the HA stem of group 2 HAs derived from an H3N2 virus-infected human. J. Infect. 76, 177–185 (2017).

  48. Couzens, L. et al. An optimized enzyme-linked lectin assay to measure influenza A virus neuraminidase inhibition antibody titers in human sera. J. Virol. Methods 210, 7–14 (2014).

    Article  CAS  Google Scholar 

  49. Chen, G. W. et al. Genomic signatures of human versus avian influenza A viruses. Emerg. Infect. Dis. 12, 1353–1360 (2006).

    Article  CAS  Google Scholar 

  50. Shannon, C. E. A mathematical theory of communication. Bell Syst. Tech. J. 27, 379–423 (1948).

    Article  Google Scholar 

  51. Ottaviano, Y. & Gerace, L. Phosphorylation of the nuclear lamins during interphase and mitosis. J. Biol. Chem. 260, 624–632 (1985).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank P. C. Wilson for providing us with anti-NA mAbs; K. Iwatsuki-Horimoto and T. Koibuchi for assistance with experiments; C. Kawakami, E. Takashita and S. Nakajima for providing us with influenza viruses and S. Watson for editing the manuscript. This work was supported by the Japan Initiative for Global Research Network on Infectious Diseases from the Japan Agency for Medical Research and Development (AMED; grant no. JP18fm0108006), Leading Advanced Projects for medical innovation from the AMED (grant no. JP18am001007), Grants-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Science, Sports and Technology (MEXT) of Japan (grant nos. 16H06429, 16K21723 and 16H06434) and the Center for Research on Influenza Pathogenesis funded by the NIAID contract no. HHSN272201400008C.

Author information

Authors and Affiliations

  1. Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan

    Atsuhiro Yasuhara, Seiya Yamayoshi, Maki Kiso, Yuko Sakai-Tagawa, I-Hsuan Wang, Shinya Yamada & Yoshihiro Kawaoka

  2. Division of Infectious Diseases, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan

    Michiko Koga

  3. Department of Infectious Diseases and Applied Immunology, IMSUT Hospital of the Institute of Medical Science, University of Tokyo, Tokyo, Japan

    Eisuke Adachi

  4. AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan

    Tadashi Kikuchi

  5. Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin–Madison, Madison, WI, USA

    Yoshihiro Kawaoka

  6. Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo, Japan

    Yoshihiro Kawaoka

  7. ERATO Infection-Induced Host Responses Project, Japan Science and Technology Agency, Tokyo, Japan

    Yoshihiro Kawaoka

Authors
  1. Atsuhiro Yasuhara
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Seiya Yamayoshi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maki Kiso
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuko Sakai-Tagawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michiko Koga
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Eisuke Adachi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tadashi Kikuchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. I-Hsuan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Shinya Yamada
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yoshihiro Kawaoka
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.Y., S.Yamayoshi and Y.K. designed the study. A.Y. performed the experiments. A.Y., S.Yamayoshi and Y.K. analysed the data. M.K., Y.S.-T., M.K., E.A., T.K., I.-H.W. and S.Yamada assisted with the experiments. A.Y., S.Yamayoshi and Y.K. wrote the manuscript. All authors reviewed and approved the manuscript.

Corresponding authors

Correspondence to Seiya Yamayoshi or Yoshihiro Kawaoka.

Ethics declarations

Conflicts of interest

Y.K. has received speaker’s honoraria from Toyama Chemical and Astellas Inc., grant support from Chugai Pharmaceuticals, Daiichi Sankyo Pharmaceutical, Toyama Chemical, Tauns Laboratories, Inc., Otsuka Pharmaceutical Co., Ltd. and Denka Seiken Co., Ltd., and is a co-founder of FluGen.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–3 and Supplementary Table 1.

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yasuhara, A., Yamayoshi, S., Kiso, M. et al. Antigenic drift originating from changes to the lateral surface of the neuraminidase head of influenza A virus. Nat Microbiol 4, 1024–1034 (2019). https://doi.org/10.1038/s41564-019-0401-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41564-019-0401-1

This article is cited by

Search

Advanced 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