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Cross-neutralization of influenza A viruses mediated by a single antibody loop

Nature volume 489, pages 526–532 (2012)Cite this article

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Abstract

Immune recognition of protein antigens relies on the combined interaction of multiple antibody loops, which provide a fairly large footprint and constrain the size and shape of protein surfaces that can be targeted. Single protein loops can mediate extremely high-affinity binding, but it is unclear whether such a mechanism is available to antibodies. Here we report the isolation and characterization of an antibody called C05, which neutralizes strains from multiple subtypes of influenza A virus, including H1, H2 and H3. X-ray and electron microscopy structures show that C05 recognizes conserved elements of the receptor-binding site on the haemagglutinin surface glycoprotein. Recognition of the haemagglutinin receptor-binding site is dominated by a single heavy-chain complementarity-determining region 3 loop, with minor contacts from heavy-chain complementarity-determining region 1, and is sufficient to achieve nanomolar binding with a minimal footprint. Thus, binding predominantly with a single loop can allow antibodies to target small, conserved functional sites on otherwise hypervariable antigens.

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Figure 1: C05 neutralizes multiple influenza virus subtypes from groups 1 and 2.
Figure 2: C05 protects mice from lethal virus challenge.
Figure 3: C05 binds the receptor-binding site on the HA1 head.
Figure 4: C05 epitope conservation across influenza A viruses.

Accession codes

Primary accessions

GenBank/EMBL/DDBJ

Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited in the Protein Data Bank under PDB codes 4FNK, 4FNL, 4FP8 and 4FQR. Electron microscopy maps have been deposited in the Electron Microscopy Data Bank (accession numbers 2138, 2139 and 2140). Nucleotide sequences for the C05 variable regions have been deposited in GenBank under accession numbers JX206996 and JX206997.

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Acknowledgements

We thank H. Tien and D. Marciano for automated crystal screening, the staff of the Advanced Photon Source (APS) GM/CA-CAT and Stanford Synchrotron Radiation Lightsource (SSRL) BL11-1 for beamline support, X. Dai and R. Stanfield for assistance with data collection and processing, M. Hothorn for assistance with ITC experiments, J. Paulson and D. Burton for comments and discussion, A. Estelles and R. Briante, L. Xu, S. Wang and D. Corn for technical support and P. Foreman for assistance with animal study design. This project was funded in part by National Institutes of Health (NIH) grant P01AI058113 (I.A.W.), a predoctoral fellowship from the Achievement Rewards for College Scientists Foundation (D.C.E.), grant GM080209 from the NIH Molecular Evolution Training Program (D.C.E.), the Skaggs Institute (I.A.W.), a career development fellowship from the Northeast Biodefense Center (U54-AI057158-Lipkin) (J.S.), National Institute of Allergy and Infectious Diseases (NIAID) grant U01AI070373 (R.W.), and Center for Research on Influenza Pathogenesis NIAID contract HHSN266200700010C (P.P.). Portions of this research were carried out at the SSRL, a national user facility operated by Stanford University on behalf of the US Department of Energy (DOE), Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the NIH, National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences (NIGMS). The GM/CA-CAT 23-ID-B beamline has been funded in whole or in part with federal funds from the National Cancer Institute (Y1-CO-1020) and NIGMS (Y1-GM-1104). Use of the APS was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract no. DE-AC02-06CH11357. The electron microscopy data presented here was collected at the National Resource for Automated Molecular Microscopy, which is supported by the NIH though the P41 program (RR017573) at the National Center for Research Resources. The JCSG is supported by NIH grant U54 GM094586. The content is solely the responsibility of the authors and does not necessarily represent the official views of NIGMS or the NIH. This is publication 21421 from The Scripps Research Institute.

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Author notes

  1. Damian C. Ekiert, John Steel, Michael A. Dillon & Ryann E. O’Neil

    Present address: Present addresses: Department of Microbiology and Immunology, University of California-San Francisco, 600 16th Street, San Francisco, California 94143, USA (D.C.E.); Genentech Inc., 1 DNA Way, South San Francisco, California 94080, USA (M.A.D.); Novartis Institutes for Biomedical Research, 4560 Horton Street, Emeryville, California 94608, USA (R.E.O.); Department of Microbiology and Immunology, Emory University School of Medicine, 3103 Rollins Research Center, 1510 Clifton Road, 194-001-1AD, Atlanta, Georgia 30322, USA (J.S.).,

  2. Damian C. Ekiert and Arun K. Kashyap: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA,

    Damian C. Ekiert, Gira Bhabha, Reza Khayat, Jeong Hyun Lee, Andrew B. Ward & Ian A. Wilson

  2. Sea Lane Biotechnologies, 2450 Bayshore Parkway, Mountain View, 94043, California, USA

    Arun K. Kashyap, Michael A. Dillon, Ryann E. O’Neil, Aleksandr M. Faynboym, Michael Horowitz, Lawrence Horowitz & Ramesh R. Bhatt

  3. Department of Microbiology, Mount Sinai School of Medicine, 1 Gustave Levy Place, New York, New York 10029-6574, USA,

    John Steel & Peter Palese

  4. Department of Infectious Diseases, St Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, Tennessee 38105, USA,

    Adam Rubrum & Richard Webby

  5. Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA,

    Richard A. Lerner

  6. The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA ,

    Richard A. Lerner & Ian A. Wilson

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Contributions

A.K.K., M.A.D., R.E.O., A.M.F., M.H., L.H., R.A.L. and R.R.B. isolated and performed the initial characterization of C05; A.K.K., J.S., A.R., P.P. and R.W. designed and performed virus neutralization and in vivo experiments; D.C.E., M.A.D., R.E.O., A.M.F and A.K.K. expressed and purified proteins; D.C.E. determined and analysed the crystal structures with guidance from I.A.W.; G.B., D.C.E., M.A.D., R.E.O., A.M.F. and A.K.K. performed binding experiments; R.K., J.H.L. and A.B.W. carried out electron microscopy studies; D.C.E. and A.K.K. analysed the sequence data sets and D.C.E., I.A.W., R.R.B. and A.K.K. wrote the manuscript. All authors commented on the paper.

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Correspondence to Ramesh R. Bhatt or Ian A. Wilson.

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A.K.K., M.A.D., R.E.O., A.M.F., M.H., L.H. and R.R.B. are employees or former employees of Sea Lane Biotechnologies.

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Ekiert, D., Kashyap, A., Steel, J. et al. Cross-neutralization of influenza A viruses mediated by a single antibody loop. Nature 489, 526–532 (2012). https://doi.org/10.1038/nature11414

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