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Contemporary H3N2 influenza viruses have a glycosylation site that alters binding of antibodies elicited by egg-adapted vaccine strains

  1. Scott E. Hensleya,1
  1. aDepartment of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;
  2. bDepartment of Ecology and Evolution, University of Chicago, Chicago, IL 60637;
  3. cDepartment of Medicine, University of Chicago, Chicago, IL 60637;
  4. dDepartment of Medicine, University of Rochester Medical Center, Rochester, NY 14642;
  5. eDepartment of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, NY 14642
  1. Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved October 12, 2017 (received for review July 11, 2017)


The majority of influenza vaccine antigens are prepared in chicken eggs. Human vaccine strains grown in eggs often possess adaptive mutations that increase viral attachment to chicken cells. Most of these adaptive mutations are in the hemagglutinin protein, which functions as a viral attachment factor. Here, we identify a hemagglutinin mutation in the current egg-adapted H3N2 vaccine strain that alters antigenicity. We show that ferrets and humans exposed to the current egg-adapted H3N2 vaccine strain produce antibodies that poorly neutralize H3N2 viruses that circulated during the 2016–2017 influenza season. These studies highlight the challenges associated with producing influenza vaccine antigens in eggs, while offering a potential explanation of why there was only moderate vaccine effectiveness during the 2016–2017 influenza season.


H3N2 viruses continuously acquire mutations in the hemagglutinin (HA) glycoprotein that abrogate binding of human antibodies. During the 2014–2015 influenza season, clade 3C.2a H3N2 viruses possessing a new predicted glycosylation site in antigenic site B of HA emerged, and these viruses remain prevalent today. The 2016–2017 seasonal influenza vaccine was updated to include a clade 3C.2a H3N2 strain; however, the egg-adapted version of this viral strain lacks the new putative glycosylation site. Here, we biochemically demonstrate that the HA antigenic site B of circulating clade 3C.2a viruses is glycosylated. We show that antibodies elicited in ferrets and humans exposed to the egg-adapted 2016–2017 H3N2 vaccine strain poorly neutralize a glycosylated clade 3C.2a H3N2 virus. Importantly, antibodies elicited in ferrets infected with the current circulating H3N2 viral strain (that possesses the glycosylation site) and humans vaccinated with baculovirus-expressed H3 antigens (that possess the glycosylation site motif) were able to efficiently recognize a glycosylated clade 3C.2a H3N2 virus. We propose that differences in glycosylation between H3N2 egg-adapted vaccines and circulating strains likely contributed to reduced vaccine effectiveness during the 2016–2017 influenza season. Furthermore, our data suggest that influenza virus antigens prepared via systems not reliant on egg adaptations are more likely to elicit protective antibody responses that are not affected by glycosylation of antigenic site B of H3N2 HA.


  • ?1To whom correspondence should be addressed. Email: hensley{at}pennmedicine.upenn.edu.
  • Author contributions: S.J.Z., J.J.T., A.J.S., and S.E.H. designed research; S.J.Z., K.P., M.E.G., and S.D.P. performed research; P.C.W. contributed new reagents/analytic tools; S.J.Z., K.K., S.C., and S.E.H. analyzed data; and S.J.Z., S.C., and S.E.H. wrote the paper.

  • Conflict of interest statement: S.J.Z., K.P., M.E.G., K.K., S.D.P., P.C.W., A.J.S., S.C., and S.E.H. have no conflicts of interest. J.J.T. is an advisor (nonpaid) for Protein Sciences and is on the scientific advisory board or received consulting payments for Sequiris, Medicago, Takeda, and Flugen. J.J.T.’s laboratory has also received support from Sanofi.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.danielhellerman.com/lookup/suppl/doi:10.1073/pnas.1712377114/-/DCSupplemental.

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