Neutralizing monoclonal antibodies elicited by mosaic RBD nanoparticles bind conserved sarbecovirus epitopes

Creators: Fan, Chengcheng; Cohen, Alexander A.; Park, Miso; Hung, Alfur Fu-Hsin; Keeffe, Jennifer R.; Gnanapragasam, Priyanthi N. P.; Lee, Yu E.; Gao, Han; Kakutani, Leesa M.; Wu, Ziyan; Kleanthous, Harry; Malecek, Kathryn E.; Williams, John C.; Bjorkman, Pamela J.

Abstract

Increased immune evasion by SARS-CoV-2 variants of concern highlights the need for new therapeutic neutralizing antibodies. Immunization with nanoparticles co-displaying spike receptor-binding domains (RBDs) from eight sarbecoviruses (mosaic-8 RBD-nanoparticles) efficiently elicits cross-reactive polyclonal antibodies against conserved sarbecovirus RBD epitopes. Here, we identified monoclonal antibodies (mAbs) capable of cross-reactive binding and neutralization of animal sarbecoviruses and SARS-CoV-2 variants by screening single mouse B cells secreting IgGs that bind two or more sarbecovirus RBDs. Single-particle cryo-EM structures of antibody-spike complexes, including a Fab-Omicron complex, mapped neutralizing mAbs to conserved class 1/4 RBD epitopes. Structural analyses revealed neutralization mechanisms, potentials for intra-spike trimer cross-linking by IgGs, and induced changes in trimer upon Fab binding. In addition, we identified a mAb-resembling Bebtelovimab, an EUA-approved human class 3 anti-RBD mAb. These results support using mosaic RBD-nanoparticle vaccination to generate and identify therapeutic pan-sarbecovirus and pan-variant mAbs.

Additional Information

© 2022 The Author(s). This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). We thank J. Vielmetter, P. Hoffman, A. Rorick, K. Storm, and the Caltech Beckman Institute Protein Expression Center for protein production; D. Veesler for BtKY72 neutralization advice; A. Gonzales for isolation and sequencing of positive B cells; the Antibody Discovery Engine and the Drug Discovery and Structural Biology Shared Facility at City of Hope, Songye Chen, and the Caltech Cryo-EM facility for cryo-EM data collection; Jens Kaiser, staff at Stanford Synchrotron Radiation Lightsource, and the Caltech Molecular Observatory for X-ray data collection support; and the Bjorkman lab members for helpful discussions. Cryo-EM was performed in the Beckman Institute Resource Center for Transmission Electron Microscopy at Caltech. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (P30GM133894). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH. These studies were funded by the National Institutes of Health (NIH) P01-AI138938-S1 (P.J.B.) and City of Hope's Integrated Drug Development Venture supported by the National Cancer Institute of the National Institutes of Health P30 CA033572 (J.C.W.), Bill and Melinda Gates Foundation INV-034638, INV-004949 (P.J.B.), the Caltech Merkin Institute (P.J.B.), and a George Mason University Fast Grant (P.J.B.). Author contributions. C.F., A.A.C., J.R.K., H.K., J.C.W., and P.J.B. conceived the study and analyzed the data. C.F. performed single-particle cryo-EM, X-ray crystallography, interpreted structures, and analyzed antibody sequences. A.A.C. prepared nanoparticles and performed negative stain EM. M.P. and A.F.-H.H. isolated B cells and generated mAb sequences. C.F., Y.E.L., and H.G. prepared and purified proteins. A.A.C., J.R.K., and Z.W. performed ELISAs. P.N.P.G. and L.M.K. performed neutralization assays. J.R.K. and C.F. performed SPR assays. C.F., J.R.K., K.E.M., and P.J.B. wrote the paper with contributions from other authors. Data and code availability. Atomic models and cryo-EM maps generated from cryo-EM studies of the M8a-3–WA1 spike 6P, M8a-6–WA1 spike 6P, M8a-28–WA1 spike 6P, M8a-31–WA1 spike 6P, M8a-31–Omicron BA.1 spike 6P, M8a-34–WA1 spike 6P, HSW-1–WA1 spike 6P, and HSW-2–WA1 spike S1 domain complexes have been deposited at the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) under the following accession codes: PDB: 7UZ4, 7UZ5, 7UZ6, 7UZ7, 7UZ8, 7UZ9, 7UZA, and 7UZB; EMDB: EMD-26878, EMD-26879, EMD-26880, EMD-26881, EMD-26882, EMD-26883, EMD-26884, and EMD-26885. Atomic models generated from crystal structures of M8a-34–RBD and HSW-2–RBD complexes have been deposited at the PDB under accession codes PDB: 7UZC and 7UZD, respectively. Additional information required to analyze the data reported in this paper is available from the lead contact upon request. This paper does not report original code. P.J.B. serves on the scientific advisory boards for the Vir Biotechnology and Vaccine Company. P.J.B. and A.A.C. are inventors on a US patent application filed by the California Institute of Technology that covers the mosaic nanoparticles described in this work. P.J.B. and A.A.C. are inventors on a US patent application filed by the California Institute of Technology that covers the methodology to generate cross-reactive antibodies using mosaic nanoparticles. P.J.B., A.A.C., C.F., and J.C.W. are inventors on a US patent application filed by the California Institute of Technology that covers the mAbs elicited by vaccination with mosaic-8 RBD-mi3 nanoparticles described in this work. P.J.B., A.A.C., and J.R.K. are inventors on a US patent application filed by the California Institute of Technology that covers the methods of isolating cross-reactive antibodies by vaccination with mosaic nanoparticles. We support inclusive, diverse, and equitable conduct of research.

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