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; Raelene Pickering; Dale I. Godfrey; Damian F.J. Purcell; Stephen J. Kent; Amy W. Chung; Kanta Subbarao; Marc Pellegrini; Alisa Glukhova; Wai Hong Tham

doi:10.1073/pnas.2101918118

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. DavenportRaelene Pickering, Dale I. Godfrey, Damian F.J. Purcell, Stephen J. Kent, Amy W. Chung, Kanta Subbarao, Marc Pellegrini, Alisa Glukhova, Wai Hong Tham

Research output: Contribution to journal › Article › Research › peer-review

97 Citations (Scopus)

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 angiotensinconverting 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 variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody- Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.

Original language	English
Article number	e2101918118
Number of pages	12
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	118
Issue number	19
DOIs	https://doi.org/10.1073/pnas.2101918118
Publication status	Published - 11 May 2021

Keywords

Antiviral therapeutics
Cryo-EM
Crystallography
Nanobodies
SARS-CoV-2

Access to Document

10.1073/pnas.2101918118Licence: CC BY

Cite this

Pymm, P., Adair, A., Chan, L. J., Cooney, J. P., Mordant, F. L., Allison, C. C., Lopez, E., Haycroft, E. R., O'Neill, M. T., Tan, L. L., Dietrich, M. H., Drew, D., Doerflinger, M., Dengler, M. A., Scott, N. E., Wheatley, A. K., Gherardin, N. A., Venugopal, H., Cromer, D., ... Tham, W. H. (2021). Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice. Proceedings of the National Academy of Sciences of the United States of America, 118(19), Article e2101918118. https://doi.org/10.1073/pnas.2101918118

@article{a984636703644634bd9825be25517904,

title = "Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice",

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 angiotensinconverting 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 variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody- Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.",

keywords = "Antiviral therapeutics, Cryo-EM, Crystallography, Nanobodies, SARS-CoV-2",

author = "Phillip Pymm and Amy Adair and Chan, {Li Jin} and Cooney, {James P.} and Mordant, {Francesca L.} and Allison, {Cody C.} and Ester Lopez and Haycroft, {Ebene R.} and O'Neill, {Matthew T.} and Tan, {Li Lynn} and Dietrich, {Melanie H.} and Damien Drew and Marcel Doerflinger and Dengler, {Michael A.} and Scott, {Nichollas E.} and Wheatley, {Adam K.} and Gherardin, {Nicholas A.} and Hariprasad Venugopal and Deborah Cromer and Davenport, {Miles P.} and Raelene Pickering and Godfrey, {Dale I.} and Purcell, {Damian F.J.} and Kent, {Stephen J.} and Chung, {Amy W.} and Kanta Subbarao and Marc Pellegrini and Alisa Glukhova and Tham, {Wai Hong}",

note = "Funding Information: ACKNOWLEDGMENTS. 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. Publisher Copyright: {\textcopyright} 2021 National Academy of Sciences. All rights reserved. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.",

year = "2021",

month = may,

day = "11",

doi = "10.1073/pnas.2101918118",

language = "English",

volume = "118",

journal = "Proceedings of the National Academy of Sciences of the United States of America",

issn = "0027-8424",

publisher = "National Academy of Sciences",

number = "19",

}

Pymm, P, Adair, A, Chan, LJ, Cooney, JP, Mordant, FL, Allison, CC, Lopez, E, Haycroft, ER, O'Neill, MT, Tan, LL, Dietrich, MH, Drew, D, Doerflinger, M, Dengler, MA, Scott, NE, Wheatley, AK, Gherardin, NA, Venugopal, H, Cromer, D, Davenport, MP, Pickering, R, Godfrey, DI, Purcell, DFJ, Kent, SJ, Chung, AW, Subbarao, K, Pellegrini, M, Glukhova, A & Tham, WH 2021, 'Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice', Proceedings of the National Academy of Sciences of the United States of America, vol. 118, no. 19, e2101918118. https://doi.org/10.1073/pnas.2101918118

TY - JOUR

T1 - Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice

AU - Pymm, Phillip

AU - Adair, Amy

AU - Chan, Li Jin

AU - Cooney, James P.

AU - Mordant, Francesca L.

AU - Allison, Cody C.

AU - Lopez, Ester

AU - Haycroft, Ebene R.

AU - O'Neill, Matthew T.

AU - Tan, Li Lynn

AU - Dietrich, Melanie H.

AU - Drew, Damien

AU - Doerflinger, Marcel

AU - Dengler, Michael A.

AU - Scott, Nichollas E.

AU - Wheatley, Adam K.

AU - Gherardin, Nicholas A.

AU - Venugopal, Hariprasad

AU - Cromer, Deborah

AU - Davenport, Miles P.

AU - Pickering, Raelene

AU - Godfrey, Dale I.

AU - Purcell, Damian F.J.

AU - Kent, Stephen J.

AU - Chung, Amy W.

AU - Subbarao, Kanta

AU - Pellegrini, Marc

AU - Glukhova, Alisa

AU - Tham, Wai Hong

N1 - Funding Information: ACKNOWLEDGMENTS. 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. Publisher Copyright: © 2021 National Academy of Sciences. All rights reserved. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.

PY - 2021/5/11

Y1 - 2021/5/11

N2 - 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 angiotensinconverting 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 variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody- Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.

AB - 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 angiotensinconverting 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 variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody- Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.

KW - Antiviral therapeutics

KW - Cryo-EM

KW - Crystallography

KW - Nanobodies

KW - SARS-CoV-2

UR - http://www.scopus.com/inward/record.url?scp=85105252430&partnerID=8YFLogxK

U2 - 10.1073/pnas.2101918118

DO - 10.1073/pnas.2101918118

M3 - Article

C2 - 33893175

AN - SCOPUS:85105252430

SN - 0027-8424

VL - 118

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 19

M1 - e2101918118

ER -

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

Abstract

Keywords

Access to Document

Other files and links

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology

Cite this

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

Abstract

Keywords

Access to Document

Other files and links

Projects

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology

Cite this