Structural characterization of influenza group 1 chimeric hemagglutinins as broad vaccine immunogens
Contributed by Ian A. Wilson; received September 2, 2024; accepted January 8, 2025; reviewed by Che Ma and Sriram Subramaniam
Significance
Influenza virus group 1 chimeric hemagglutinin (cHA) vaccines are in human clinical trials as universal influenza vaccine candidates. Here, we determined crystal and electron microscopy structures of group 1 cHAs cH5/1, cH8/1, and cH11/1 with broadly protective antibodies to elucidate their structural features. The conserved protective antibody epitopes were preserved in the cHA structures, although their chimeric head domains were sometimes rotated relative to the stem to accommodate transplantation of the chimeric heads onto an H1 stem, which adopted closed to more open base configurations. Binding of stem and head trimer interface antibodies to these cHAs also confirmed the integrity of the highly conserved epitopes in the cHAs. This structural information can aid in further cHA vaccine design.
Abstract
Chimeric hemagglutinins (cHA) appear to be promising for the design and development of universal influenza vaccines. Influenza A group 1 cHAs, cH5/1, cH8/1, and cH11/1, comprising an H1 stem attached to either an H5, H8, or H11 globular head, have been used sequentially as vaccine immunogens in human clinical trials and induced high levels of broadly protective antibodies. Using X-ray crystallography and negative-stain electron microscopy, we determined structures of cH5/1, cH8/1, and cH11/1 HAs in their apo (unliganded) and antibody Fab-bound states. Stem-reactive antibodies 3E1 and 31.b.09 recognize their cognate epitopes in cH5/1, cH8/1, and cH11/1 HAs. However, with cH5/1, the head domains are rotated by 35 to 45° around the threefold axis of the HA trimer compared to native HA with a more splayed-open conformation at the stem base. cH11/1 with 3E1 is structurally more native-like but resembles cH5/1 with 31.b.09, whereas cH8/1 with 31.b.09 exhibited a range of closed-to-open stem configurations with some separation of head and stem domains. Furthermore, all of these group 1 cHAs effectively bound a broad head trimer interface antibody and other broad stem antibodies. Thus, the cHAs exhibit structural plasticity without compromising the stem and head trimer interface epitopes for elicitation of influenza A group 1 cross-reactive antibodies.
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Data, Materials, and Software Availability
X-ray coordinates and structure factors have been deposited in the Research Collaboratory for Structural Bioinformatics Protein Data Bank under accession codes 9C0V for cH5/1 HA with 3E1, 9C0U for cH5/1 HA with 31.b.09, 9C22 for cH11/1 HA with 3E1, and 9C0X for cH11/1 HA with 31.b.09 (64–67). nsEM 3D maps have been deposited in the Electron Microscopy Databank (EMDB) under accession codes: EMD-46833, EMD-46834, EMD-46835, EMD-46836, EMD-46837, EMD-46838, EMD-46839, EMD-46840, EMD-46841, and EMD-46842. All other studies are included in the main text and/or SI Appendix.
Acknowledgments
We thank Henry Tien for help with automated robotic crystal screening at The Scripps Research Institute. The research was supported by the National Institutes of Health (NIH) National Institute of Allergy and Infectious Diseases (NIAID) Collaborative Influenza Vaccine Innovation Centers (CIVICS) contract grant 75N93019C00051 (to W.S., P.P., F.K., A.B.W., and I.A.W.). X-ray diffraction datasets were collected at the Advanced Photon Source (APS) beamline 23ID-D (GM/CA CAT) and Stanford Synchrotron Radiation Lightsource (SSRL) beamlines 12-1 and 12-2. The General Medical Sciences and Cancer Institute Structural Biology Facility at APS (GM/CA CAT) is funded in whole or in part with federal funds from the National Cancer Institute (Y1-CO-1020) and National Institute of General Medical Sciences (NIGMS) (Y1-GM-1104). Use of the APS was supported by US Department of Energy (DOE), Basic Energy Sciences, Office of Science, under Contract DE-AC02-06CH11357. SSRL is a Directorate of SLAC National Accelerator Laboratory, and an Office of Science User Facility operated for the US DOE of Science by Stanford University. SSRL Structural Molecular Biology Program is supported by DOE Office of Biological and Environmental Research, and by NIH, NIGMS (including P41GM103393), and National Center for Research Resources (P41RR001209).
Author contributions
Y.T.K.N., X.Z., A.B.W., and I.A.W. designed research; Y.T.K.N., J.H., A.J.R., and W.Y. performed research; W.S., P.P., F.K., and I.A.W. contributed new reagents/analytic tools; Y.T.K.N., X.Z., J.H., A.B.W., and I.A.W. analyzed data; and Y.T.K.N., X.Z., J.H., A.B.W., and I.A.W. wrote the paper.
Competing interests
W.S. and F.K. are co-founders and scientific advisory board members of CastleVax. F.K. has consulted for Merck, Curevac, Seqirus, GlaxoSmithKline (GSK) and Pfizer and is currently consulting for 3rd Rock Ventures, Gritstone and Avimex. The Krammer laboratory is collaborating with Dynavax on influenza vaccine development. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2 and another company, CastleVax, to develop SARS-CoV-2 vaccines. The Icahn School of Medicine at Mount Sinai has filed patent applications relating to influenza virus vaccines, which list P.P. and F.K. as co-inventors. The Icahn School of Medicine at Mount Sinai has also filed patent applications relating to SARS-CoV-2 serological assays, NDV-based SARS-CoV-2 vaccines, and influenza virus therapeutics which list F.K. as co-inventor. W.S. is listed as co-inventor of NDV-based SARS-CoV-2 vaccines.
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Copyright © 2025 the Author(s). Published by PNAS. This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).
Data, Materials, and Software Availability
X-ray coordinates and structure factors have been deposited in the Research Collaboratory for Structural Bioinformatics Protein Data Bank under accession codes 9C0V for cH5/1 HA with 3E1, 9C0U for cH5/1 HA with 31.b.09, 9C22 for cH11/1 HA with 3E1, and 9C0X for cH11/1 HA with 31.b.09 (64–67). nsEM 3D maps have been deposited in the Electron Microscopy Databank (EMDB) under accession codes: EMD-46833, EMD-46834, EMD-46835, EMD-46836, EMD-46837, EMD-46838, EMD-46839, EMD-46840, EMD-46841, and EMD-46842. All other studies are included in the main text and/or SI Appendix.
Submission history
Received: September 2, 2024
Accepted: January 8, 2025
Published online: February 12, 2025
Published in issue: February 18, 2025
Keywords
Acknowledgments
We thank Henry Tien for help with automated robotic crystal screening at The Scripps Research Institute. The research was supported by the National Institutes of Health (NIH) National Institute of Allergy and Infectious Diseases (NIAID) Collaborative Influenza Vaccine Innovation Centers (CIVICS) contract grant 75N93019C00051 (to W.S., P.P., F.K., A.B.W., and I.A.W.). X-ray diffraction datasets were collected at the Advanced Photon Source (APS) beamline 23ID-D (GM/CA CAT) and Stanford Synchrotron Radiation Lightsource (SSRL) beamlines 12-1 and 12-2. The General Medical Sciences and Cancer Institute Structural Biology Facility at APS (GM/CA CAT) is funded in whole or in part with federal funds from the National Cancer Institute (Y1-CO-1020) and National Institute of General Medical Sciences (NIGMS) (Y1-GM-1104). Use of the APS was supported by US Department of Energy (DOE), Basic Energy Sciences, Office of Science, under Contract DE-AC02-06CH11357. SSRL is a Directorate of SLAC National Accelerator Laboratory, and an Office of Science User Facility operated for the US DOE of Science by Stanford University. SSRL Structural Molecular Biology Program is supported by DOE Office of Biological and Environmental Research, and by NIH, NIGMS (including P41GM103393), and National Center for Research Resources (P41RR001209).
Author contributions
Y.T.K.N., X.Z., A.B.W., and I.A.W. designed research; Y.T.K.N., J.H., A.J.R., and W.Y. performed research; W.S., P.P., F.K., and I.A.W. contributed new reagents/analytic tools; Y.T.K.N., X.Z., J.H., A.B.W., and I.A.W. analyzed data; and Y.T.K.N., X.Z., J.H., A.B.W., and I.A.W. wrote the paper.
Competing interests
W.S. and F.K. are co-founders and scientific advisory board members of CastleVax. F.K. has consulted for Merck, Curevac, Seqirus, GlaxoSmithKline (GSK) and Pfizer and is currently consulting for 3rd Rock Ventures, Gritstone and Avimex. The Krammer laboratory is collaborating with Dynavax on influenza vaccine development. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2 and another company, CastleVax, to develop SARS-CoV-2 vaccines. The Icahn School of Medicine at Mount Sinai has filed patent applications relating to influenza virus vaccines, which list P.P. and F.K. as co-inventors. The Icahn School of Medicine at Mount Sinai has also filed patent applications relating to SARS-CoV-2 serological assays, NDV-based SARS-CoV-2 vaccines, and influenza virus therapeutics which list F.K. as co-inventor. W.S. is listed as co-inventor of NDV-based SARS-CoV-2 vaccines.
Notes
Reviewers: C.M., Academia Sinica; and S.S., University of British Columbia.
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Structural characterization of influenza group 1 chimeric hemagglutinins as broad vaccine immunogens, Proc. Natl. Acad. Sci. U.S.A.
122 (7) e2416628122,
https://doi.org/10.1073/pnas.2416628122
(2025).
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