Edited by Ian Wilson, The Scripps Research Institute, La Jolla, CA; received August 25, 2022; accepted February 10, 2023
March 24, 2023
120 (13) e2213690120
Significance
Structural features that govern neutralization potency differences between antiviral antibodies are not very well understood. In this report, we present cryo-EM reconstructions of Eastern equine encephalitis virus (EEEV) in complex with Fab fragments of three patient-derived anti-EEEV antibodies, that have greater than 20-fold differences in their neutralization potency (IC50) values. Our structural and biophysical analyses reveal constraints that correspond with the observed biological differences between these antibodies. EEEV is an alphavirus that causes severe encephalitis in humans and horses. Currently, there are no licensed vaccines or antiviral treatments against EEEV for human use. The research described here aids in the development of antibody therapeutics against alphaviruses and other icosahedral viral pathogens.
Abstract
Selection and development of monoclonal antibody (mAb) therapeutics against pathogenic viruses depends on certain functional characteristics. Neutralization potency, or the half-maximal inhibitory concentration (IC50) values, is an important characteristic of candidate therapeutic antibodies. Structural insights into the bases of neutralization potency differences between antiviral neutralizing mAbs are lacking. In this report, we present cryo-electron microscopy (EM) reconstructions of three anti-Eastern equine encephalitis virus (EEEV) neutralizing human mAbs targeting overlapping epitopes on the E2 protein, with greater than 20-fold differences in their respective IC50 values. From our structural and biophysical analyses, we identify several constraints that contribute to the observed differences in the neutralization potencies. Cryo-EM reconstructions of EEEV in complex with these Fab fragments reveal structural constraints that dictate intravirion or intervirion cross-linking of glycoprotein spikes by their IgG counterparts as a mechanism of neutralization. Additionally, we describe critical features for the recognition of EEEV by these mAbs including the epitope–paratope interaction surface, occupancy, and kinetic differences in on-rate for binding to the E2 protein. Each constraint contributes to the extent of EEEV inhibition for blockade of virus entry, fusion, and/or egress. These findings provide structural and biophysical insights into the differences in mechanism and neutralization potencies of these antibodies, which help inform rational design principles for candidate vaccines and therapeutic antibodies for all icosahedral viruses.
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Data, Materials, and Software Availability
All relevant data are included within the manuscript and are available upon request from the corresponding authors. Structural data have been deposited in Electron Microscopy Data Bank (EMDB) or Protein Data Bank (PDB) EMD-26947 or PDB ID: 7V0P (SINV/EEEV:EEEV-106 Fab), EMD-26946 or PDB ID: 7V0O (SINV/EEEV:EEEV-94 Fab), and EMD-26945 or PDB ID: 7V0N (SINV/EEEV:EEEV-21 Fab). Materials described in this paper are available for distribution for nonprofit use using templated documents from Association of University Technology Managers “Toolkit MTAs”, available at: https://autm.net/surveys-and-tools/agreements/material-transfer-agreements/mta-toolkit.
Acknowledgments
We thank C. Slaughter, R. Carnahan, and P. Gilchuk at Vanderbilt University for helpful discussions. We thank R. Nargi, R. Sutton, E. Armstrong, C. Gainza, J. Rodriguez, L. Handal, S. Monroig, S. Seeback, A. Trivette, J. Reidy, R. Irving, and R. Troseth for technical support. We thank M. Leksell, M. Mayo, G. DeBellis, K. Moton, T. Martin, T. Cooper, H. Darling, A. Bunnell, O. Battle, B. Haseltine, N. Lee, N. Suazo, and J. Mitchell for administrative assistance. We thank the Purdue Cryo-EM Facility for equipment access and support. L.E.W. was supported by the National Institutes of Health (NIH) grants T32 HL069765 and F31 AI145189. This project received support from the U.S. Defense Threat Reduction Agency-Joint Science and Technology Office for Chemical and Biological Defense (grant no. HDTRA1-13-1-0034), by Clinical Translational Science Award (CTSA) award National Center for Advancing Translational Sciences Clinical Translational Science Award (CTSA) Program, award number 5UL1TR002243, by the National Institute of Allergy and Infectious Diseases (NIAID) grant U19 AI142790 (R.J.K. and J.E.C.), by NIH grant R01 AI095366, and by NIH/NIAID contract HHSN272201700041I, Task A11. Its contents are solely the responsibility of the authors and do not necessarily represent official views of the National Center for Advancing Translational Sciences or the NIH.
Author contributions
L.E.W., A.B., R.J.K., and J.E.C. designed research; L.E.W., A.B., K.B., D.S., T.K., and J.G.J. performed research; L.E.W., A.B., K.B., D.S., T.K., J.G.J., R.J.K., and J.E.C. analyzed data; and L.E.W., A.B., R.J.K., and J.E.C. wrote the paper.
Competing interests
L.E.W. serves as a consultant for BigHat Biosciences. The content of this article is solely the responsibility of the authors and does not represent the official views of BigHat Biosciences. J.E.C. has served as a consultant for Luna Labs USA, Merck Sharp & Dohme Corporation, Emergent Biosolutions, GlaxoSmithKline, and BTG International Inc, is a member of the Scientific Advisory Board of Meissa Vaccines, a former member of the Scientific Advisory Board of Gigagen (Grifols), and is founder of IDBiologics. The laboratory of J.E.C. received unrelated sponsored research agreements from AstraZeneca, Takeda, and IDBiologics during the conduct of the study. The Crowe laboratory at Vanderbilt University Medical Center has received unrelated sponsored research agreements from IDBiologics, Takeda Pharmaceuticals, and AstraZeneca. All other authors report no conflicts. J.E.C. has owns stock in IDBiologics and has stock options in Meissa Vaccines. Vanderbilt University has applied for a patent related to antibodies described in this paper. The Crowe laboratory at Vanderbilt University Medical Center has received unrelated sponsored research agreements from IDBiologics, Takeda Pharmaceuticals, and AstraZeneca. J.E.C. is a member of the Scientific Advisory Board of Meissa Vaccines, a former member of the Gigagen Scientific Advisory Board, and is Founder of IDBiologics.
Supporting Information
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Information & Authors
Information
Published in
Proceedings of the National Academy of Sciences
Vol. 120 | No. 13
March 28, 2023
Classifications
Copyright
Data, Materials, and Software Availability
All relevant data are included within the manuscript and are available upon request from the corresponding authors. Structural data have been deposited in Electron Microscopy Data Bank (EMDB) or Protein Data Bank (PDB) EMD-26947 or PDB ID: 7V0P (SINV/EEEV:EEEV-106 Fab), EMD-26946 or PDB ID: 7V0O (SINV/EEEV:EEEV-94 Fab), and EMD-26945 or PDB ID: 7V0N (SINV/EEEV:EEEV-21 Fab). Materials described in this paper are available for distribution for nonprofit use using templated documents from Association of University Technology Managers “Toolkit MTAs”, available at: https://autm.net/surveys-and-tools/agreements/material-transfer-agreements/mta-toolkit.
Submission history
Received: August 25, 2022
Accepted: February 10, 2023
Published online: March 24, 2023
Published in issue: March 28, 2023
Keywords
- antibodies, human monoclonal
- alphavirus
- neutralization
- cryo-EM
- therapy
Acknowledgments
We thank C. Slaughter, R. Carnahan, and P. Gilchuk at Vanderbilt University for helpful discussions. We thank R. Nargi, R. Sutton, E. Armstrong, C. Gainza, J. Rodriguez, L. Handal, S. Monroig, S. Seeback, A. Trivette, J. Reidy, R. Irving, and R. Troseth for technical support. We thank M. Leksell, M. Mayo, G. DeBellis, K. Moton, T. Martin, T. Cooper, H. Darling, A. Bunnell, O. Battle, B. Haseltine, N. Lee, N. Suazo, and J. Mitchell for administrative assistance. We thank the Purdue Cryo-EM Facility for equipment access and support. L.E.W. was supported by the National Institutes of Health (NIH) grants T32 HL069765 and F31 AI145189. This project received support from the U.S. Defense Threat Reduction Agency-Joint Science and Technology Office for Chemical and Biological Defense (grant no. HDTRA1-13-1-0034), by Clinical Translational Science Award (CTSA) award National Center for Advancing Translational Sciences Clinical Translational Science Award (CTSA) Program, award number 5UL1TR002243, by the National Institute of Allergy and Infectious Diseases (NIAID) grant U19 AI142790 (R.J.K. and J.E.C.), by NIH grant R01 AI095366, and by NIH/NIAID contract HHSN272201700041I, Task A11. Its contents are solely the responsibility of the authors and do not necessarily represent official views of the National Center for Advancing Translational Sciences or the NIH.
Author Contributions
L.E.W., A.B., R.J.K., and J.E.C. designed research; L.E.W., A.B., K.B., D.S., T.K., and J.G.J. performed research; L.E.W., A.B., K.B., D.S., T.K., J.G.J., R.J.K., and J.E.C. analyzed data; and L.E.W., A.B., R.J.K., and J.E.C. wrote the paper.
Competing Interests
L.E.W. serves as a consultant for BigHat Biosciences. The content of this article is solely the responsibility of the authors and does not represent the official views of BigHat Biosciences. J.E.C. has served as a consultant for Luna Labs USA, Merck Sharp & Dohme Corporation, Emergent Biosolutions, GlaxoSmithKline, and BTG International Inc, is a member of the Scientific Advisory Board of Meissa Vaccines, a former member of the Scientific Advisory Board of Gigagen (Grifols), and is founder of IDBiologics. The laboratory of J.E.C. received unrelated sponsored research agreements from AstraZeneca, Takeda, and IDBiologics during the conduct of the study. The Crowe laboratory at Vanderbilt University Medical Center has received unrelated sponsored research agreements from IDBiologics, Takeda Pharmaceuticals, and AstraZeneca. All other authors report no conflicts. J.E.C. has owns stock in IDBiologics and has stock options in Meissa Vaccines. Vanderbilt University has applied for a patent related to antibodies described in this paper. The Crowe laboratory at Vanderbilt University Medical Center has received unrelated sponsored research agreements from IDBiologics, Takeda Pharmaceuticals, and AstraZeneca. J.E.C. is a member of the Scientific Advisory Board of Meissa Vaccines, a former member of the Gigagen Scientific Advisory Board, and is Founder of IDBiologics.
Notes
This article is a PNAS Direct Submission.
Authors
Affiliations
Lauren E. Williamson1
The Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville, TN 37232
Department of Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, TN 37232
Purdue Institute of Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, IN 47907
Institute for Antiviral Research, Utah State University, Logan, UT 84335
Purdue Institute of Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, IN 47907
Purdue Institute of Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, IN 47907
Institute for Antiviral Research, Utah State University, Logan, UT 84335
Purdue Institute of Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, IN 47907
The Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville, TN 37232
Department of Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, TN 37232
Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232
Notes
1
L.E.W. and A.B. contributed equally to this work.
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