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.
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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.
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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.
Supplementary information
Supplementary information
Supplementary Figures 1-11, Tables 1-4, 6, 7, 12–14, 16–19 and 22; and References. (PDF 1753 kb)
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. (XLSX 14 kb)
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. (XLSX 19 kb)
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. (XLSX 30 kb)
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. (XLSX 27 kb)
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. (XLSX 15 kb)
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. (XLSX 15 kb)
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). (XLSX 12 kb)
Supplementary Table 21
HA mutations in antigenic escape mutants selected from a randomly mutagenized HA library of A/Kwangju/219/2002 (H3N2). (XLSX 12 kb)
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. (XLSX 16 kb)
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. (XLSX 18 kb)
Supplementary Table 25
Database information and acknowledgments for the pandemic H1N1 and H3N2 virus HA and NA sequences used in this study. (XLSX 15 kb)
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Li, C., Hatta, M., Burke, D. et al. Selection of antigenically advanced variants of seasonal influenza viruses. Nat Microbiol 1, 16058 (2016). https://doi.org/10.1038/nmicrobiol.2016.58
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DOI: https://doi.org/10.1038/nmicrobiol.2016.58
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