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Published July 8, 2022 | Submitted
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Neutralizing monoclonal antibodies elicited by mosaic RBD nanoparticles bind conserved sarbecovirus epitopes


Protection from SARS-related coronaviruses with spillover potential and SARS-CoV-2 variants could prevent and/or end pandemics. We show that mice immunized with nanoparticles co-displaying spike receptor-binding domains (RBDs) from eight sarbecoviruses (mosaic-8 RBD-nanoparticles) efficiently elicit cross-reactive anti-sarbecovirus antibodies against conserved class 1/4 and class 3 RBD epitopes. Monoclonal antibodies (mAbs) identified from initial screening of <10,000 single B-cells secreting IgGs binding two or more sarbecovirus RBDs showed cross-reactive binding and neutralization of SARS-CoV-2 variants and animal sarbecoviruses. Single-particle cryo-EM structures of antibody–spike complexes, including a Fab-Omicron complex, mapped neutralizing mAbs to conserved class 1/4 RBD epitopes and revealed neutralization mechanisms, potentials for intra-spike trimer crosslinking by single 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-nanoparticles to identify therapeutic pan-sarbecovirus and pan-variant mAbs and to elicit them by vaccination.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license. 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, and Jens Kaiser, staff at Stanford Synchrotron Radiation Lightsource, and the Caltech Molecular Observatory for X-ray data collection support. Cryo-Electron microscopy 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 (JCW), Bill and Melinda Gates Foundation INV-034638 INV-004949 (PJB), the Caltech Merkin Institute (PJB), and a George Mason University Fast Grant (PJB). Author contributions. C.F., A.A.C, J.R.K., J.C.W., and P.J.B. conceived the study and analyzed the data. C.F. performed single-particle cryo-electron microscopy, X-ray crystallography, interpreted structures and analyzed antibody sequences. A.A.C. prepared nanoparticles and performed negative stain electron microscopy. M.P. and A.F.H.H. isolated B cells and generated mAb sequences. C.F. and Y.E.L prepared and purified Fabs and IgGs. A.A.C., J.R.K., and Z.W. performed ELISAs. P.N.P.G., L.M.K., and A.A.C. performed neutralization assays. C.F., J.R.K., K.E.M. and P.J.B. wrote the paper with contributions from other authors. Data and materials 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 7UZC and 7UZD, respectively. Materials are available upon request to the corresponding author with a signed material transfer agreement. This work is licensed under a Creative Commons Attribution-NonCommercial 2.0 Generic (CC BY-NC 2.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited and not for commercial purposes. To view a copy of this license, visit https://creativecommons.org/licenses/by-nc/2.0/. This license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using such material. Competing Interest Statement. 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 monoclonal antibodies elicited by vaccination with mosaic-8 RBD-mi3 nanoparticles described in this work.

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