The neuraminidase of A(H3N2) influenza viruses circulating since 2016 is antigenically distinct from the A/Hong Kong/4801/2014 vaccine strain

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A(H3N2) virus predominated recent influenza seasons, which has resulted in the rigorous investigation of haemagglutinin, but whether neuraminidase (NA) has undergone antigenic change and contributed to the predominance of A(H3N2) virus is unknown. Here, we show that the NA of the circulating A(H3N2) viruses has experienced significant antigenic drift since 2016 compared with the A/Hong Kong/4801/2014 vaccine strain. This antigenic drift was mainly caused by amino acid mutations at NA residues 245, 247 (S245N/S247T; introducing an N-linked glycosylation site at residue 245) and 468. As a result, the binding of the NA of A(H3N2) virus by some human monoclonal antibodies, including those that have broad reactivity to the NA of the 1957 A(H2N2) and 1968 A(H3N2) reference pandemic viruses as well as contemporary A(H3N2) strains, was reduced or abolished. This antigenic drift also reduced NA-antibody-based protection against in vivo virus challenge. X-ray crystallography showed that the glycosylation site at residue 245 is within a conserved epitope that overlaps the NA active site, explaining why it impacts antibody binding. Our findings suggest that NA antigenic drift impacts protection against influenza virus infection, thus highlighting the importance of including NA antigenicity for consideration in the optimization of influenza vaccines.

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Fig. 1: An N-linked glycosylation site acquired at NA residue 245 of circulating A(H3N2) viruses.
Fig. 2: Antigenic drift of the NA of circulating A(H3N2) viruses demonstrated by ferret and human antisera.
Fig. 3: Glycosylation at residue 245 impacts the binding of NA by human and mouse monoclonal antibodies.
Fig. 4: Crystal structure of the B10 Fab in complex with A(H3N2) virus NA.
Fig. 5: The NA245 glycosylation site is in an NA epitope that overlaps the NA active site.
Fig. 6: NA antigenic drift reduces in vivo protection against influenza virus challenge.

Data availability

The data that support the findings of this study are available from the corresponding author on request. The atomic coordinates and structure factors for the B10 Fab–MN/10 NA complex have been deposited to the PDB ( under the accession code 6N6B.


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This work was supported by intramural funds from the Food and Drug Administration and the Centers for Disease Control and Prevention. L.J. was supported by training funds administered by the Oak Ridge Institute for Science and Education. The work was also supported by the National Institute of Allergy and Infectious Disease (NIAID), National Institutes of Health (grant nos U19AI082724, U19AI109946 and U19AI057266 to P.C.W., and R01AI116744 to X.-F.W.), and the NIAID Centers of Excellence for Influenza Research and Surveillance (grant nos HSN272201400008C to F.K. and HHSN272201400005C to P.C.W.). We thank the St. Jude Children’s Research Hospital for providing the plasmids that were used to rescue viruses. We thank V. V. Lugovtsev for providing the ferret antisera, L. Li for help with the sequence analysis and L. Qi for help with the statistical analyses. We thank Y. An and A. Cheung for their helpful discussions in this study. We are indebted to the staff of the Division of Veterinary Services, CBER, FDA for the excellent animal care and the Facility for Biotechnology Resources, CBER, FDA for the sequencing work. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Food and Drug Administration and the Centers for Disease Control and Prevention.

Author information

H.W. conceived the project, designed and supervised the work, analysed data and wrote the manuscript. J.G. rescued reassortant viruses, performed the serological assays and assisted with the animal studies. H.Y., J.C. and P.J.C. expressed NA and defined the crystal structure. S.Y., W.W.W., R.-F.S. and J.F.C. analysed the glycans through mass spectrometry. R.H. contributed to the serological assays. Y.-Q.C., N.-Y.Z. and P.C.W. prepared the human monoclonal antibodies. X.L. and E.P. performed the western blots and virus counting. L.C., L.J and C.W. characterized the mouse monoclonal antibodies. S.S. and F.K. helped with the animal experiments. J.S. supervised the crystal structure work. X.-F.W. analysed the NA sequences and generated antigenic maps. M.C.E. and Z.Y. provided suggestions for the work. All authors helped improve the manuscript.

Correspondence to Hongquan Wan.

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