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Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses

Nature Structural & Molecular Biology volume 16, pages 265–273 (2009)Cite this article

Abstract

Influenza virus remains a serious health threat, owing to its ability to evade immune surveillance through rapid genetic drift and reassortment. Here we used a human non-immune antibody phage-display library and the H5 hemagglutinin ectodomain to select ten neutralizing antibodies (nAbs) that were effective against all group 1 influenza viruses tested, including H5N1 'bird flu' and the H1N1 'Spanish flu'. The crystal structure of one such nAb bound to H5 shows that it blocks infection by inserting its heavy chain into a conserved pocket in the stem region, thus preventing membrane fusion. Nine of the nAbs employ the germline gene VH1-69, and all seem to use the same neutralizing mechanism. Our data further suggest that this region is recalcitrant to neutralization escape and that nAb-based immunotherapy is a promising strategy for broad-spectrum protection against seasonal and pandemic influenza viruses.

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Figure 1: In vitro binding and neutralization of anti-H5 antibodies.
Figure 2: Prophylactic and therapeutic efficacy of anti-H5 nAbs in mice.
Figure 3: Neutralization mechanism.
Figure 4: Structure of the H5–F10 complex.
Figure 5: Sequence conservation in hemagglutinin groups, clusters and subtypes at the F10 epitope.
Figure 6: Cross-subtype neutralization by nAbs.
Figure 7: Three-dimensional comparison of the F10 epitope in group 1 and group 2 hemagglutinins.

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Acknowledgements

We thank J. Appleton (Cornell University) for the gift of mouse mAbs against H5N1, 17A2.1.2 and 22F; A. Klimov (CDC) and A. Balish (CDC) for providing ferret antiserum and virus sequences; R. Webster (St. Jude Children's Research Hospital) for H11N9, H13N6 and H16N3; L. Quynh Mai (National Institute of Hygiene and Epidemiology, Vietnam Ministry of Health) for H5N1; W. Lim (Hong Kong Department of Health) for H5N1 and H9N2, as well as E. Sedyaningsih, T. Soendoro (National Institute of Health Research and Development, Indonesian Ministry of Health) for H5N1 specimens; P. Palese (Mount Sinai School of Medicine) for pCAGGS-H1(SC) plasmid encoding the full-length hemagglutinin protein of H1-SC1918; M. Farzan (New England Primate Research Center, Harvard Medical School) for pCAGGS-H1 (PR) plasmid encoding the hemagglutinin protein of H1-PR34 and X. Yang (Beth Israel Deaconess Medical Center, Harvard Medical School) for pCAGGS-H7 (FPV) encoding H7-FP34 hemagglutinin; and R. Fuller (University of Michigan) for furin cDNA. We thank W. Yuan and W. Li for helpful discussions and Y. Lin for assistance in crystallization and critical discussion. We thank the US National Institutes of Health (NIH) and the Department of Energy (DOE) for access to the Stanford Synchrotron Radiation Facility and the facility staff for assistance in X-ray data collection. Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081). This work was supported by NIH (U01-AI074518-01) to W.A.M. and in part by NIH (P01-AI055789) to R.C.L.

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  1. Jianhua Sui and William C Hwang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute,

    Jianhua Sui, Daniel Aird, Maryam Ali, Akikazu Murakami, Anuradha Yammanuru, Thomas Han & Wayne A Marasco

  2. Department of Medicine, Harvard Medical School, 44 Binney Street JFB 826, Boston, 02115, Massachusetts, USA

    Jianhua Sui, Daniel Aird, Maryam Ali, Akikazu Murakami, Anuradha Yammanuru, Thomas Han & Wayne A Marasco

  3. Infectious and Inflammatory Disease Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, 92037, California, USA

    William C Hwang, Ge Wei, Eugenio Santelli, Boguslaw Stec, Greg Cadwell, Laurie A Bankston & Robert C Liddington

  4. Influenza Division, Centers for Disease Control and Prevention, National Center for Immunization and Respiratory Diseases, 1600 Clifton Road, Mail Stop G-16, Atlanta, 30333, Georgia, USA

    Sandra Perez, Li-mei Chen, Hongquan Wan, Nancy J Cox & Ruben O Donis

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Contributions

In the DFCI team, J.S. and A.M. constructed H5-TH04 hemagglutinin; J.S. and D.A. performed phage-display antibody library selections and screening for antibodies by ELISA, FACS and pseudovirus-neutralization assays; J.S., M.A. and T.H. carried out epitope mapping using mutagenesis and FACS analysis; J.S., D.A. and M.A. purified antibodies; J.S. and A.Y. analyzed kinetics of antibody binding with hemagglutinin protein; J.S. performed hemagglutinin subtype cross-binding and neutralization assays, pseudovirus binding and fusion inhibition assay; J.S. and W.A.M. designed the study, analyzed data and wrote the sections about these studies. In the BIMR team, R.C.L. supervised all of the work; G.W. and G.C. cloned and expressed recombinant H5 for antibody panning and crystallization. W.C.H. expressed F10 scFv and crystallized the F10–HA0 complex; W.C.H., E.S. and B.S. collected diffraction data and solved and refined the structure; L.A.B. supervised cloning and expression; W.C.H., L.A.B. and R.C.L. wrote sections about these studies. In the CDC team, S.P., L.C., H.W., N.J.C. and R.O.D. designed the study and performed animal studies as well as virology studies with wild-type viruses; R.O.D wrote sections of these studies; J.S., R.O.D., R.C.L. and W.A.M. finalized the paper. All authors commented on the manuscript.

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention or the Agency for Toxic Substances and Disease Registry.

Corresponding authors

Correspondence to Jianhua Sui, Ruben O Donis, Robert C Liddington or Wayne A Marasco.

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Sui, J., Hwang, W., Perez, S. et al. Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat Struct Mol Biol 16, 265–273 (2009). https://doi.org/10.1038/nsmb.1566

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