Wheatley, Adam K.Pymm, PhillipEsterbauer, RobynDietrich, Melanie H.Lee, Wen ShiDrew, DamienKelly, Hannah G.Chan, Li JinMordant, Francesca L.Black, Katrina A.Adair, AmyTan, Hyon XhiJuno, Jennifer A.Wragg, Kathleen M.Amarasena, ThakshilaLopez, EsterSelva, Kevin J.Haycroft, Ebene R.Cooney, James P.Venugopal, HariprasadTan, Li LynnO Neill, Matthew T.Allison, Cody C.Cromer, DeborahDavenport, Miles P.Bowen, Richard A.Chung, Amy W.Pellegrini, MarcLiddament, Mark T.Glukhova, AlisaSubbarao, KantaKent, Stephen J.Tham, Wai Hong2025-06-302025-06-302211-1247PubMed:34610292ORCID:/0000-0002-4652-5609/work/182433353http://www.scopus.com/inward/record.url?scp=85116421941&partnerID=8YFLogxKhttps://hdl.handle.net/1885/733765975Potent neutralizing monoclonal antibodies are one of the few agents currently available to treat COVID-19. SARS-CoV-2 variants of concern (VOCs) that carry multiple mutations in the viral spike protein can exhibit neutralization resistance, potentially affecting the effectiveness of some antibody-based therapeutics. Here, the generation of a diverse panel of 91 human, neutralizing monoclonal antibodies provides an in-depth structural and phenotypic definition of receptor binding domain (RBD) antigenic sites on the viral spike. These RBD antibodies ameliorate SARS-CoV-2 infection in mice and hamster models in a dose-dependent manner and in proportion to in vitro, neutralizing potency. Assessing the effect of mutations in the spike protein on antibody recognition and neutralization highlights both potent single antibodies and stereotypic classes of antibodies that are unaffected by currently circulating VOCs, such as B.1.351 and P.1. These neutralizing monoclonal antibodies and others that bind analogous epitopes represent potentially useful future anti-SARS-CoV-2 therapeutics.We thank the generous participation of the trial subjects for providing samples. SARS-CoV-2 RBD expression plasmids were kindly provided by Florian Krammer, Mt Sinai School of Medicine, NY, USA. Human ACE2 expression plasmids were kindly provided by Merlin Thomas, Monash University, Australia. OC43 spike expression constructs were kindly provided by Dr. Barney Graham, NIAID. SARS1 RBD, and human ACE2 protein was kindly provided by Nicholas Gherardin and Dale Godfrey, University of Melbourne. We thank Damian Purcell for provision of the hCoV-19/Australia/VIC2089/2020 virus for mouse challenge studies. We thank the Melbourne Cytometry Platform (Melbourne Brain Centre node) for provision of flow cytometry services. We thank staff at CSL, including Matthew Hardy, Catherine Owczarek, James Munro, Lok Soon Law, Glenn Powers, and Ping Xu, for expression, purification, and analysis of antibodies scaled-up for use in animal models; Adam Quek for provision of the RBD protein; and Chao-Guang Chen for provision of IgG expression vectors. We thank Janet Newman from CSIRO Collaborative Crystallisation Centre for assistance with setting up the crystallization screens and the MX beamline staff 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 ANSTO and made use of the Australian Cancer Research Foundation (ACRF) detector. The authors acknowledge use of Monash Ramaciotti Cryo-EM platform facilities and the Bio21 Advanced Microscopy Facility. This work was supported by computational resources provided by the Australian Government through MASSIVE HPC facility (https://www.massive.org.au) under the National Computational Merit Allocation Scheme. This study was supported by the Victorian Government, the Medical Research Future Fund (MRFF) GNT2002073 (W.-H.T. A.W.C. S.J.K. and A.K.W.) and generous donations from the Paul Ramsay Foundation (A.K.W. A.W.C. and S.J.K.), and Hengyi Pacific to support COVID-19 research (W.-H.T.). W.-H.T. is a Howard Hughes Medical Institute-Wellcome Trust International research scholar (208693/Z/17/Z). A.G. is a CSL Centenary Fellow. W.-H.T. S.J.K. D.C. M.P.D. M.P. J.A.J. A.W.C. K.S. and A.K.W. are supported by NHMRC fellowships. The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health. The authors acknowledge the Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS. P.P. expressed and purified recombinant proteins and prepared samples for X-ray crystallography, crystallized RBD-WCSL Fab complexes, and collected diffraction data. M.H.D. with P.P. L.L.T. and L.-J.C. performed all the structural refinement for the crystal structures of RBD-Fab. D.D. performed the bio-panning using phage display and clonal analyses for WCSL antibodies and ELISA for specificity. A.A. performed the PRNT assays. L.-J.C. performed all bio-layer interferometry measurements for affinities and epitope competition, and established the FRET assay. J.P.C. performed the mouse experiments with the assistance of C.C.A. and the micro-neutralization assays for the WCSL antibodies. M.T.O. L.L.T. M.H.D. and D.D. assisted with expression and purification of recombinant proteins, WCSL antibodies, and the RBD variants. For cryo-EM, A.G. with the assistance of P.P. K.A.B. and H.V. prepared the samples, collected the data, and performed the data processing and model building. A.K.W. led human antibody recovery from convalescent individuals and drafted the manuscript. H.-X.T. performed single cell sorting of SARS-CoV-2-specific B cells. J.A.J. K.M.W. and S.J.K. recruited human subjects and processed blood samples. R.E. H.G.K. and T.A. cloned, expressed, and purified proteins and antibodies. W.S.L. F.L.M. and K.S. organized and performed live virus neutralization assays. E.L. K.J.S. E.R.H. and A.W.C. organized and performed multiplex RBD binding analysis. D.C. and M.P.D. provided statistical and high-level analytical support. R.A.B. led Syrian hamster studies. M.T.L. supervised the phage display, supplied RBD protein, provided high-level data analysis support, and led scaled-up antibody production to support animal studies. W.-H.T. and S.J.K. conceived the project, drafted the manuscript, and supervised the overall project. All authors assisted in manuscript preparation. The authors declare no competing interests. A provisional patent covering human mAbs isolated from convalescent donors has been submitted through the University of Melbourne. A provisional patent covering human mAbs isolated from phage display has been submitted through the Walter and Eliza Hall Institute. We thank the generous participation of the trial subjects for providing samples. SARS-CoV-2 RBD expression plasmids were kindly provided by Florian Krammer, Mt Sinai School of Medicine, NY, USA. Human ACE2 expression plasmids were kindly provided by Merlin Thomas, Monash University, Australia. OC43 spike expression constructs were kindly provided by Dr. Barney Graham, NIAID. SARS1 RBD, and human ACE2 protein was kindly provided by Nicholas Gherardin and Dale Godfrey, University of Melbourne. We thank Damian Purcell for provision of the hCoV-19/Australia/VIC2089/2020 virus for mouse challenge studies. We thank the Melbourne Cytometry Platform (Melbourne Brain Centre node) for provision of flow cytometry services. We thank staff at CSL, including Matthew Hardy, Catherine Owczarek, James Munro, Lok Soon Law, Glenn Powers, and Ping Xu, for expression, purification, and analysis of antibodies scaled-up for use in animal models; Adam Quek for provision of the RBD protein; and Chao-Guang Chen for provision of IgG expression vectors. We thank Janet Newman from CSIRO Collaborative Crystallisation Centre for assistance with setting up the crystallization screens and the MX beamline staff 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 ANSTO and made use of the Australian Cancer Research Foundation (ACRF) detector. The authors acknowledge use of Monash Ramaciotti Cryo-EM platform facilities and the Bio21 Advanced Microscopy Facility. This work was supported by computational resources provided by the Australian Government through MASSIVE HPC facility ( https://www.massive.org.au ) under the National Computational Merit Allocation Scheme. This study was supported by the Victorian Government , the Medical Research Future Fund (MRFF) GNT2002073 (W.-H.T., A.W.C., S.J.K., and A.K.W.) and generous donations from the Paul Ramsay Foundation (A.K.W., A.W.C., and S.J.K.), and Hengyi Pacific to support COVID-19 research (W.-H.T.). W.-H.T. is a Howard Hughes Medical Institute-Wellcome Trust International research scholar ( 208693/Z/17/Z ). A.G. is a CSL Centenary Fellow. W.-H.T., S.J.K., D.C., M.P.D., M.P., J.A.J., A.W.C., K.S., and A.K.W. are supported by NHMRC fellowships. The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health . The authors acknowledge the Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS.enPublisher Copyright: © 2021 The Authorsanti-viral therapeuticscryo-EMcrystallographyhuman antibodiesmAbSARS-CoV-22021-10-1210.1016/j.celrep.2021.10982285116421941