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Published May 22, 2025 | Published
Journal Article Open

Cross-reactive sarbecovirus antibodies induced by mosaic RBD nanoparticles

Fan, Chengcheng1 ORCID icon
Keeffe, Jennifer R.1 ORCID icon
Malecek, Kathryn E.1
Cohen, Alexander A.1 ORCID icon
West, Anthony P.1 ORCID icon
Baharani, Viren A.2
Rorick, Annie V.1 ORCID icon
Gao, Han1
Gnanapragasam, Priyanthi N. P.1
Rho, Semi1 ORCID icon
Alvarez, Jaasiel1
Segovia, Luisa N.1
Hatziioannou, Theodora2 ORCID icon
Bieniasz, Paul D.2 ORCID icon
Bjorkman, Pamela J.1 ORCID icon
  • 1. ROR icon California Institute of Technology
  • 2. ROR icon Rockefeller University

Abstract

Broad immune responses are needed to mitigate viral evolution and escape. To induce antibodies against conserved receptor-binding domain (RBD) regions of SARS-like betacoronavirus (sarbecovirus) spike proteins that recognize SARS-CoV-2 variants of concern and zoonotic sarbecoviruses, we developed mosaic-8b RBD nanoparticles presenting eight sarbecovirus RBDs arranged randomly on a 60-mer nanoparticle. Mosaic-8b immunizations protected animals from challenges from viruses whose RBDs were matched or mismatched to those on nanoparticles. Here, we describe neutralizing mAbs isolated from mosaic-8b-immunized rabbits, some on par with Pemgarda, the only currently FDA-approved therapeutic mAb. Deep mutational scanning, in vitro selection of spike resistance mutations, and single-particle cryo-electron microscopy structures of spike–antibody complexes demonstrated targeting of conserved RBD epitopes. Rabbit mAbs included critical D-gene segment RBD-recognizing features in common with human anti-RBD mAbs, despite rabbit genomes lacking an equivalent human D-gene segment, thus demonstrating that the immune systems of humans and other mammals can utilize different antibody gene segments to arrive at similar modes of antigen recognition. These results suggest that animal models can be used to elicit anti-RBD mAbs with similar properties to those raised in humans, which can then be humanized for therapeutic use, and that mosaic RBD nanoparticle immunization coupled with multiplexed screening represents an efficient way to generate and select broadly cross-reactive therapeutic pan-sarbecovirus and pan-SARS-CoV-2 variant mAbs.

Copyright and License

Copyright © 2025 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Acknowledgement

We thank Jost Vielmetter and the Caltech Beckman Institute Protein Expression Center for protein production, the Caltech Beckman Institute Beacon Center for Single Cell Biology for conducting Beacon experiments, 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. Cryoelectron 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 US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The Stanford Synchrotron Radiation Lightsource Structural Molecular Biology Program is supported by the Department of Energy Office of Biological and Environmental Research and by the NIH, National Institute of General Medical Sciences (P30GM133894). These studies were funded by the NIH P01-AI165075 (T.H., P.D.B., and P.J.B.), the Caltech Merkin Institute (P.J.B.), Wellcome Leap (P.J.B.), The Coalition for Epidemic Preparedness Innovations (P.J.B.), and the Boehringer Ingelheim Fonds PhD fellowship (V.A.B.). P.D.B. is a HHMI Investigator. This work was supported, in whole or in part, by the Bill & Melinda Gates Foundation grant INV-034638 (P.J.B.). Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission.

Funding

Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The Stanford Synchrotron Radiation Lightsource Structural Molecular Biology Program is supported by the Department of Energy Office of Biological and Environmental Research and by the NIH, National Institute of General Medical Sciences (P30GM133894). These studies were funded by the NIH P01-AI165075 (T.H., P.D.B., and P.J.B.), the Caltech Merkin Institute (P.J.B.), Wellcome Leap (P.J.B.), The Coalition for Epidemic Preparedness Innovations (P.J.B.), and the Boehringer Ingelheim Fonds PhD fellowship (V.A.B.). P.D.B. is a HHMI Investigator. This work was supported, in whole or in part, by the Bill & Melinda Gates Foundation grant INV-034638 (P.J.B.). Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission.

Contributions

C.F., J.R.K., K.E.M., A.A.C., V.A.B., and P.J.B. designed research; C.F., J.R.K., K.E.M., A.A.C., A.P.W., V.A.B., A.V.R., H.G., P.N.P.G., S.R., J.A., and L.N.S. performed research; J.R.K. and K.E.M. contributed new reagents/analytic tools; C.F., J.R.K., K.E.M., A.A.C., A.P.W., V.A.B., and P.J.B. analyzed data; and C.F., J.R.K., K.E.M., A.A.C., A.P.W., V.A.B., T.H., P.D.B., and P.J.B. wrote the paper.

Data Availability

Raw sequencing data from DMS experiments are deposited on NCBI SRA under BioProject PRJNA1067836 (48) and BioSample SAMN45169522 (49); processing folder is available upon request. Models and density maps for cryo-EM structures are deposited in the PDB [9ML4 (53), 9ML5 (56), 9ML6 (58), and 9ML7 (61)], and maps are available on EMDB [EMD-48347 (54), EMD-48348 (57), EMD-48349 (59), and EMD-48350 (62)]. Models and electron density maps for crystal structures were deposited in the PDB [9ML8 (63) and 9ML9 (60)]. All other data are included in the manuscript and/or SI Appendix.

Conflict of Interest

P.J.B. is a scientific advisor for Vaccine Company, Inc. and for ProtaBody, Inc., P.J.B. and A.A.C. are inventors on a US patent application (17/523,813) filed by the California Institute of Technology that covers mosaic RBD-NPs. P.J.B., C.F., J.R.K., K.E.M., and J.A. are inventors of US patent applications filed by the California Institute of Technology that cover the sequences of the Abs described in this publication.

Supplemental Material

Appendix 01 (PDF)

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