Journal article

Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice

Phillip Pymm, Amy Adair, Li-Jin Chan, James P Cooney, Francesca L Mordant, Cody C Allison, Ester Lopez, Ebene R Haycroft, Matthew T O'Neill, Li Lynn Tan, Melanie H Dietrich, Damien Drew, Marcel Doerflinger, Michael A Dengler, Nichollas E Scott, Adam K Wheatley, Nicholas A Gherardin, Hariprasad Venugopal, Deborah Cromer, Miles P Davenport Show all

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | NATL ACAD SCIENCES | Published : 2021

Abstract

Neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19. Here, we identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2. Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD v..

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Grants

Awarded by Medical Research Future Fund


Awarded by Australian Research Council Future fellowship


Funding Acknowledgements

We thank Janet Newman from the CSIRO Collaborative Crystallization Centre for assistance with setting up the crystallization screens and the MX beamline staff and Rachel Williamson and Alan Riboldi-Tunnicliffe at the Australian Synchrotron for their assistance during data collection. This research was undertaken in part using the MX2 beamline at the Australian Synchrotron, part of Australian Nuclear Science and Technology Organisation (ANSTO), and made use of the Australian Cancer Research Foundation detector. We acknowledge use of Monash Ramaciotti Cryo-EM platform facilities and the Bio21 Advanced Microscopy Facility. This work was supported by the Multimodal Australian ScienceS Imaging and Visualization Environment (MASSIVE) high performance computing facility (https://massive.org.au/). We are grateful to Leonie Gibson for the excellent technical assistance with cloning of the constructs for retroviral overexpression of ACE2 and SARS-CoV-2 spike protein in mouse embryonic fibroblasts. This study was supported by the Victorian Government, Medical Research Future Fund Grant GNT2002073 (to W.-H.T., D.I.G., A.W.C., S.J.K., and A.K.W.), and a generous donation by Hengyi Pacific Pty Ltd. to support COVID-19 research. W-H.T. is a Howard Hughes Medical Institute-Wellcome Trust International Research Scholar (208693/Z/17/Z); D.I.G., W.-H.T., S.J.K., D.C., M.P.D., M.P., and A.K.W. are supported by National Health and Medical Research Council (NHMRC) fellowships; and N.E.S. is supported by an Australian Research Council Future fellowship (FT200100270). The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health. We acknowledge the Victorian State Government Operational Infrastructure Support and Australian Government NHMRC Independent Research Institutes Infrastructure Support Scheme.