Abstract

Influenza viruses mutate frequently, necessitating constant updates of vaccine viruses. To establish experimental approaches that may complement the current vaccine strain selection process, we selected antigenic variants from human H1N1 and H3N2 influenza virus libraries possessing random mutations in the globular head of the haemagglutinin protein (which includes the antigenic sites) by incubating them with human and/or ferret convalescent sera to human H1N1 and H3N2 viruses. We also selected antigenic escape variants from human viruses treated with convalescent sera and from mice that had been previously immunized against human influenza viruses. Our pilot studies with past influenza viruses identified escape mutants that were antigenically similar to variants that emerged in nature, establishing the feasibility of our approach. Our studies with contemporary human influenza viruses identified escape mutants before they caused an epidemic in 2014–2015. This approach may aid in the prediction of potential antigenic escape variants and the selection of future vaccine candidates before they become widespread in nature.

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Acknowledgements

The authors thank S. Watson for scientific editing and P. Jester for technical support. This work was supported by the Bill & Melinda Gates Foundation Global Health Grant OPPGH5383; National Institute of Allergy and Infectious Diseases (NIAID) Public Health Service research grants (USA); ERATO (Japan Science and Technology Agency); the Center for Research on Influenza Pathogenesis (CRIP) funded by the NIAID contracts HHSN266200700010C and HHSN272201400008C; the Japan Initiative for Global Research Network on Infectious Diseases; Grants-in-Aid for Specially Promoted Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; Grants-in-Aid from the Ministry of Health, Labour and Welfare, Japan; grants from the Strategic Basic Research Program of the Japan Science and Technology Agency and by the Advanced Research & Development Programs for Medical Innovation from the Japan Agency for Medical Research and Development (AMED). C.A.R. was supported by a University Research Fellowship from the Royal Society. The authors acknowledge a Netherlands Organisation for Scientific Research (NWO) VICI grant, European Union (EU) FP7 programmes EMPERIE (223498) and ANTIGONE (278976); Human Frontier Science Program (HFSP) programme grant P0050/2008; Wellcome 087982AIA and NIH Director's Pioneer Award DP1-OD000490-01. D.F.B. and D.J.S. acknowledge support from CamGrid, the University of Cambridge distributed computer system. The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health. The origins of the HA and NA gene sequences used in this study are recognized in Supplementary Table 25. The content of this report is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Note: The methods used in this manuscript could potentially select for variants of A(H1N1)pdm09 viruses that could escape vaccine-based immunity; therefore, our manuscript was reviewed by the CDC's Institutional Biosecurity Board, which concluded this study does not constitute Dual Use Research of Concern (DURC). The selection of antigenic escape variants was completed before the US Government issued a Research Funding Pause on 17 October 2014, on selected gain-of-function research on influenza, MERS and SARS viruses.

Author information

Author notes

    • Chengjun Li
    • , Masato Hatta
    • , David F. Burke
    • , Jihui Ping
    •  & Ying Zhang

    These authors contributed equally to this work

    • Alexander I. Klimov

    Deceased.

Affiliations

  1. Department of Pathobiological Sciences, Influenza Research Institute, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53711, USA

    • Chengjun Li
    • , Masato Hatta
    • , Jihui Ping
    • , Ying Zhang
    • , Makoto Ozawa
    • , Andrew S. Taft
    • , Subash C. Das
    • , Anthony P. Hanson
    • , Jiasheng Song
    • , Masaki Imai
    • , Peter R. Wilker
    • , Eileen A. Maher
    • , Gabriele Neumann
    •  & Yoshihiro Kawaoka
  2. Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK

    • David F. Burke
    • , Sarah L. James
    • , Eugene Skepner
    •  & Derek J. Smith
  3. World Health Organization Collaborating Centre for Modelling, Evolution, and Control of Emerging Infectious Diseases, Cambridge CB2 3EJ, UK

    • David F. Burke
    • , Colin A. Russell
    • , Sarah L. James
    • , Eugene Skepner
    •  & Derek J. Smith
  4. Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan

    • Makoto Ozawa
    •  & Yoshihiro Kawaoka
  5. Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, Iwate 020-8550, Japan

    • Masaki Imai
  6. ERATO Infection-Induced Host Responses Project, Saitama 332-0012, Japan

    • Tokiko Watanabe
    • , Shinji Watanabe
    •  & Yoshihiro Kawaoka
  7. Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan

    • Mutsumi Ito
    • , Kiyoko Iwatsuki-Horimoto
    •  & Yoshihiro Kawaoka
  8. Fogarty International Center, National Institutes of Health, Bethesda, Maryland 20892, USA

    • Colin A. Russell
  9. Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, UK

    • Colin A. Russell
  10. Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30033, USA

    • Alexander I. Klimov
    • , Xiyan Xu
    • , David E. Wentworth
    • , Jacqueline M. Katz
    •  & Nancy J. Cox
  11. WHO Collaborating Centre for Reference and Research on Influenza (VIDRL) at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Australia

    • Anne Kelso
  12. Division of Virology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK

    • John McCauley
  13. Chinese National Influenza Center, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China

    • Dayan Wang
    •  & Yuelong Shu
  14. Influenza Virus Research Center, National Institute of Infectious Diseases, Musashi-Murayama, Tokyo 208-0011, Japan

    • Takato Odagiri
    •  & Masato Tashiro
  15. Department of Virology, Erasmus Medical Center, Rotterdam 3000 CA, Netherlands

    • Derek J. Smith

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Contributions

C.L., M.H., D.F.B., J.P., Y.Z., M.O., A.S.T., S.C.D., A.P.H., J.S., M.I., P.R.W., T.W., S.W., M.I., K.I.-H., C.A.R., S.L.J., E.S., E.A.M., G.N., A.I.K., A.K., J.M., D.W., Y.S., M.T., J.K., D.E.W., N.J.C., D.J.S. and Y.K. designed the experiments. C.L., M.H., D.F.B., J.P., Y.Z., M.O., A.S.T., S.C.D., A.P.H., J.S., M.I., P.R.W., T.W., S.W., M.I., K.I.-H., C.A.R., S.L.J., T.O., X.X. and E.S. performed the experiments. C.L., M.H., D.F.B., J.P., Y.Z., M.O., A.S.T., S.C.D., A.P.H., J.S., M.I., P.R.W., T.W., S.W., M.I., K.I.-H., C.A.R., S.L.J., E.S., E.A.M., G.N., A.I.K., A.K., J.M., D.W., Y.S., M.T., N.J.C., D.J.S. and Y.K. analysed the data. C.L., M.H., D.F.B., M.O., A.S.T., S.C.D., E.A.M., G.N., J.M.K., N.J.C., D.J.S. and Y.K. wrote the manuscript. C.L., M.H., D.F.B., J.P. and Y.Z. contributed equally to this work.

Competing interests

Y. Kawaoka has received speaker's honoraria from Toyama Chemical and Astellas, has received grant support from Chugai Pharmaceuticals, Daiichi Sankyo Pharmaceutical, Toyama Chemical, Tauns Laboratories, Otsuka Pharmaceutical Co., Tsumura and Co. and Denka Seiken Co. and is a founder of FluGen. G. Neumann is a founder of FluGen.

Corresponding authors

Correspondence to Derek J. Smith or Yoshihiro Kawaoka.

Supplementary information

PDF files

  1. 1.

    Supplementary information

    Supplementary Figures 1-11, Tables 1-4, 6, 7, 12–14, 16–19 and 22; and References.

Excel files

  1. 1.

    Supplementary Table 5

    HA mutations in antigenic escape mutants selected with human convalescent sera from a randomly mutagenized HA library of A/Norway/3858/2009.

  2. 2.

    Supplementary Table 8

    HA mutations identified in antigenic escape mutants selected from A/Norway/3858/2009 HA libraries randomly mutagenized at a single amino acid position.

  3. 3.

    Supplementary Table 9

    HA mutations in antigenic escape mutants selected from an A/Norway/3858/2009 HA library possessing random mutations at two amino acid positions.

  4. 4.

    Supplementary Table 10

    HA mutations in antigenic escape mutants selected from an A/Norway/3858/2009 HA library with random mutations at four amino acid positions.

  5. 5.

    Supplementary Table 11

    HA mutations in antigenic escape mutants selected from an A/Norway/3858/2009 HA library with the D127E mutation and random mutations at positions 153–156.

  6. 6.

    Supplementary Table 15

    HA mutations and HI titres of viruses selected from immunized mice that were subsequently infected with an A/Norway/3858/2009 HA library possessing the D127E mutation and random mutations at amino acid positions 153–156.

  7. 7.

    Supplementary Table 20

    HA mutations in antigenic escape mutants selected with human convalescent sera from a randomly mutagenized HA library of A/Hong Kong/CUHK5250/2002 (H3N2).

  8. 8.

    Supplementary Table 21

    HA mutations in antigenic escape mutants selected from a randomly mutagenized HA library of A/Kwangju/219/2002 (H3N2).

  9. 9.

    Supplementary Table 23

    HA mutations (ordered by frequency) in antigenic escape mutants selected with human sera from a randomly mutagenized HA library of A/Texas/50/2012.

  10. 10.

    Supplementary Table 24

    HA mutations (ordered by serum) in antigenic escape mutants selected with human sera from a randomly mutagenized HA library of A/Texas/50/2012.

  11. 11.

    Supplementary Table 25

    Database information and acknowledgments for the pandemic H1N1 and H3N2 virus HA and NA sequences used in this study.

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DOI

https://doi.org/10.1038/nmicrobiol.2016.58

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