Development of pre-pandemic influenza candidate vaccine viruses for use in vaccine manufacturing

development-of-pre-pandemic-influenza-candidate-vaccine-viruses-for-use-in-vaccine-manufacturing
Development of pre-pandemic influenza candidate vaccine viruses for use in vaccine manufacturing

Data availability

All example data used to illustrate the protocol steps are provided in the manuscript. This study did not generate new datasets beyond the influenza HA and NA gene sequences included in Table 1. These sequences have been deposited in GenBank under the following accession numbers: IDCDC-RG76A (HA: OQ871527; NA: OQ871528), IDCDC-RG79A (HA: PX070140; NA: PX070141), IDCDC-RG71A (HA: OM403993; NA: OM403994), IDCDC-RG64A (HA: MW403506; NA: MW403507), and IDCDC-RG61 (HA: MW403504; NA: MW403505). No additional datasets requiring deposition were generated.

References

  1. World Health Organization. WHO biosafety risk assessment and guidelines for the production and quality control of human influenza pandemic vaccines. https://cdn.who.int/media/docs/default-source/biologicals/vaccine-quality/who-biosafety-risk-assessment-and-guidelines-for-the-production-and-quality-control-of-human-influenza-pandemic-vaccinesa2e4b869-92e6-4927-9332-3416e7de7c46.pdf (2007).

  2. World Health Organization. Guidelines for the safe development and production of vaccines to human pandemic influenza viruses and influenza viruses with pandemic potential. https://cdn.who.int/media/docs/default-source/biologicals/vaccine-standardization/influenza/annex_3_who_trs_1016_web3.pdf (2019).

  3. World Health Organization. WHO guidance on development of influenza vaccine reference viruses by reverse genetics. https://iris.who.int/server/api/core/bitstreams/8381f662-1288-41c0-901b-0fcf6e0af674/content (2005).

  4. Kilbourne, E. D. Influenza pandemics of the 20th century. Emerg. Infect. Dis. 12, 9–14 (2006).

    Google Scholar 

  5. Iuliano, A. D. et al. Estimates of global seasonal influenza-associated respiratory mortality: a modelling study. Lancet 391, 1285–1300 (2018).

    Google Scholar 

  6. Harrington, W. N., Kackos, C. M. & Webby, R. J. The evolution and future of influenza pandemic preparedness. Exp. Mol. Med. 53, 737–749 (2021).

  7. World Health Organization. Up to 650 000 people die of respiratory diseases linked to seasonal flu each year. http://www.who.int/mediacentre/news/releases/2017/seasonal-flu/en/ (2017).

  8. Cruz-Aponte, M., McKiernan, E. C. & Herrera-Valdez, M. A. Mitigating effects of vaccination on influenza outbreaks given constraints in stockpile size and daily administration capacity. BMC Infect. Dis. 11, 207 (2011).

    Google Scholar 

  9. Uyeki, T. M. & Cox, N. J. Global concerns regarding novel influenza A (H7N9) virus infections. N. Engl. J. Med. 368, 1862–1864 (2013).

    Google Scholar 

  10. World Health Organization. Pandemic influenza preparedness and response: a WHO guidance document. https://iris.who.int/server/api/core/bitstreams/e41f13ed-9ca8-4689-b183-58bfc8944603/content (2009).

  11. Yen, H. L. & Webster, R. G. Pandemic influenza as a current threat. Curr. Top. Microbiol. Immunol. 333, 3–24, (2009).

    Google Scholar 

  12. O’Neill, E. & Donis, R. O. Generation and characterization of candidate vaccine viruses for prepandemic influenza vaccines. Curr. Top. Microbiol. Immunol. 333, 83–108 (2009).

    Google Scholar 

  13. Centers for Disease Control and Prevention. HHS Pandemic Influenza Plan 2017 UPDATE. https://www.cdc.gov/pandemic-flu/media/pan-flu-report-2017v2.pdf (2017).

  14. Biomedical Advanced Research and Development Authority. Influenza & emerging infectious diseases pandemic vaccines and adjuvants program. https://medicalcountermeasures.gov/barda/influenza-and-emerging-infectious-diseases/pandemic-vaccines-adjuvants/.

  15. Centers for Disease Control and Prevention. Allocating and targeting pandemic influenza vaccine during an influenza pandemic. https://www.cdc.gov/pandemic-flu/media/2018-Influenza-Guidance.pdf (2018).

  16. World Health Organization. Global Influenza Surveillance and Response System (GISRS). https://www.who.int/initiatives/global-influenza-surveillance-and-response-system.

  17. U.S. Food and Drug Administration. 21 CFR Part 58–Good laboratory Practice for Nonclinical Laboratory Studies. https://www.ecfr.gov/current/title-21/chapter-I/subchapter-A/part-58.

  18. World Health Organization. Zoonotic influenza candidate vaccine viruses. https://www.who.int/teams/global-influenza-programme/vaccines/who-recommendations/zoonotic-influenza-viruses-and-candidate-vaccine-viruses.

  19. Webby, R. J. et al. Responsiveness to a pandemic alert: use of reverse genetics for rapid development of influenza vaccines. Lancet 363, 1099–1103 (2004).

    Google Scholar 

  20. Subbarao, K. et al. Evaluation of a genetically modified reassortant H5N1 influenza A virus vaccine candidate generated by plasmid-based reverse genetics. Virology 305, 192–200 (2003).

    Google Scholar 

  21. Neumann, G., Ozawa, M. & Kawaoka, Y. Reverse genetics of influenza viruses. Methods Mol. Biol. 865, 193–206 (2012).

    Google Scholar 

  22. Zhou, B. et al. Single-reaction genomic amplification accelerates sequencing and vaccine production for classical and swine-origin human influenza A viruses. J. Virol. 83, 10309–10313 (2009).

  23. U.S. Department of Agriculture. Guidelines for Avian Influenza Viruses. https://www.selectagents.gov/compliance/guidance/avian/docs/aiv-guidelines-exemption-2024-august_508.pdf (2024).

  24. National Institutes of Health. NIH guidelines for research involving recombinant DNA molecules. https://osp.od.nih.gov/wp-content/uploads/NIH_Guidelines.pdf (2024).

  25. World Health Organization. The WHO Laboratory Biosafety Manual 4th edn. https://www.who.int/publications/i/item/9789240011311 (2020).

  26. National Institutes of Health. PHS Policy on Humane Care and Use of Laboratory Animals. https://olaw.nih.gov/policies-laws/phs-policy.htm (2015).

  27. World Health Organization. WHO Guidelines on Transmissible Spongiform Encephalopathies in Relation to Biological and Pharmaceutical Products https://www.who.int/publications/m/item/who-guidelines-on-transmissible-spongiform-encephalopathies (2003).

  28. Ozaki, H. et al. Generation of high-yielding influenza A viruses in African green monkey kidney (Vero) cells by reverse genetics. J Virol 78, 1851–1857 (2004).

    Google Scholar 

  29. World Health Organization. Manual for the laboratory diagnosis and virological surveillance of influenza. https://www.who.int/publications/i/item/manual-for-the-laboratory-diagnosis-and-virological-surveillance-of-influenza (2011).

  30. Pappas, C. et al. Identification of clinical and virological correlates associated with influenza A candidate vaccine virus (CVV) attenuation in a ferret model. J. Virol. 99, e0102325 (2025).

    Google Scholar 

  31. Federal Select Agent Program. Policy Statement Removal of Live Bird Lethality Testing Requirement. https://www.selectagents.gov/regulations/policy/birdlethality.htm (2018).

  32. Federal Select Agent Program. Exclusion Guidance Document. https://www.selectagents.gov/compliance/guidance/exclusions/docs/Exclusion_Guidance.pdf (2025).

  33. Federal Select Agent Program. Select agents and toxins exemption: H5 avian influenza virus. https://www.selectagents.gov/sat/exemptions/avian-influenza.htm (2024).

  34. Godeaux, O. et al. Immunogenicity and safety of AS03A-adjuvanted H5N1 influenza vaccine prepared from bulk antigen after stockpiling for 4 years. Vaccine 33, 2189–2195 (2015).

    Google Scholar 

  35. ClinicalTrials.gov. Safety and immunogenicity of different formulations of monovalent influenza A/Astrakhan/3212/2020 Like (H5N8) virus vaccine with AS03 adjuvant system in medically stable adults. https://clinicaltrials.gov/study/NCT05975840 (2025).

  36. U.S. Food and Drug Administration. Influenza Virus Vaccine, H5N1 (for National Stockpile). https://www.fda.gov/vaccines-blood-biologics/safety-availability-biologics/influenza-virus-vaccine-h5n1-national-stockpile (2024).

  37. Schild, G. C., Oxford, J. S., de Jong, J. C. & Webster, R. G. Evidence for host-cell selection of influenza virus antigenic variants. Nature 303, 706–709 (1983).

    Google Scholar 

  38. Steel, J. A paradigm shift in vaccine production for pandemic influenza. Ann. Transl. Med. 3, 165 (2015).

    Google Scholar 

  39. Dormitzer, P. R. et al. Synthetic generation of influenza vaccine viruses for rapid response to pandemics. Sci. Transl. Med. 5, 185ra168 (2013).

    Google Scholar 

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Acknowledgements

We extend our heartfelt gratitude to both current and former members of the CDC Influenza Division, particularly the Vaccine Preparedness Team, for their substantial contributions to the CVV development initiative at CDC. The high-confidence production and distribution of these CVVs would not have been possible without the immense support of our internal and external partners, who handled extensive QC testing and undertook QA and regulatory reviews. This work was funded by the Centers for Disease Control and Prevention. Use of trade names is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention or the U.S. Department of Health and Human Services. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention, the Department of Health and Human Services, or the U.S. Government. This manuscript reflects the policies, practices, and coordination mechanisms in place at the time of its finalization; subsequent changes may affect the applicability of certain information presented herein.

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Author notes

  1. David E. Wentworth

    Present address: GSK, Cambridge, MA, USA

Authors and Affiliations

  1. Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA

    Li Wang, Cindy Adolphus, Jieru Wang, Jaber Hossain, Michael Currier, C. Todd Davis, Vivien G. Dugan, David E. Wentworth & Bin Zhou

Authors

  1. Li Wang
  2. Cindy Adolphus
  3. Jieru Wang
  4. Jaber Hossain
  5. Michael Currier
  6. C. Todd Davis
  7. Vivien G. Dugan
  8. David E. Wentworth
  9. Bin Zhou

Contributions

Supervision: V.G.D., C.T.D., D.E.W., and B.Z. Performed the experiments: L.W., C.A., J.W., J.H., and M.C. Wrote the manuscript: L.W. and B.Z. Edited the manuscript: L.W., C.A., J.W., V.G.D., C.T.D., D.E.W., and B.Z. All authors reviewed and approved the manuscript.

Corresponding author

Correspondence to Bin Zhou.

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Competing interests

The authors declare no competing interests.

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Wang, L., Adolphus, C., Wang, J. et al. Development of pre-pandemic influenza candidate vaccine viruses for use in vaccine manufacturing. npj Vaccines (2026). https://doi.org/10.1038/s41541-026-01384-1

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