Mapping cross-variant neutralizing sites on the SARS-CoV-2 spike protein

ABSTRACT

The emergence of multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern threatens the efficacy of currently approved vaccines and authorized therapeutic monoclonal antibodies (MAbs). It is hence important to continue searching for SARS-CoV-2 broadly neutralizing MAbs and defining their epitopes. Here, we isolate 9 neutralizing mouse MAbs raised against the spike protein of a SARS-CoV-2 prototype strain and evaluate their neutralizing potency towards a panel of variants, including B.1.1.7, B.1.351, B.1.617.1, and B.1.617.2. By using a combination of biochemical, virological, and cryo-EM structural analyses, we identify three types of cross-variant neutralizing MAbs, represented by S5D2, S5G2, and S3H3, respectively, and further define their epitopes. S5D2 binds the top lateral edge of the receptor-binding motif within the receptor-binding domain (RBD) with a binding footprint centred around the loop^477–489, and efficiently neutralizes all variant pseudoviruses, but the potency against B.1.617.2 was observed to decrease significantly. S5G2 targets the highly conserved RBD core region and exhibits comparable neutralization towards the variant panel. S3H3 binds a previously unreported epitope located within the evolutionarily stable SD1 region and is able to near equally neutralize all of the variants tested. Our work thus defines three distinct cross-variant neutralizing sites on the SARS-CoV-2 spike protein, providing guidance for design and development of broadly effective vaccines and MAb-based therapies.

KEYWORDS:

• SARS-CoV-2
• antibody
• SD1
• epitope
• Cryo-EM

Acknowledgements

We thank the staffs of the NCPSS Electron Microscopy facility, Database and Computing facility, and Protein Expression and Purification facility for instrument support and technical assistance. We thank Jiang Shao and the other staffs of the Institutional Center for Shared Technologies and Facilities of Institut Pasteur of Shanghai for instrument support and technical assistance. The BSL-3 lab of Fudan University was supported by Shanghai Science and Technology Committee and Project of Novel Coronavirus Research from Fudan University. We thank the staffs of Chemical Biology Core Facility and Molecular Biology Core Facility in Center for Excellence in Molecular Cell Science for technical support.

Author contributions

Z.H., Y.C., Y.H.X., and G.W. designed the study; S.Q.X., Y.F.W., Y.X.W., C.Z., Q.H., C.J.G., R.X., T.F.W., Y.Y., J.K.Z., Y.Z., Z.Y.L., Q.X.L., B.J.Z., L.L.B., and Y.F.Z performed experiments; all authors analyzed data; S.Q.X., Y.F.W., Y.X.W., C.Z., Q.H., Y.C., and Z.H. created figures; Z.H., Y.C., S.Q.X., Y.F.W., Y.X.W., C.Z., and Q.H. wrote the manuscript with help from all authors; and Q.D., H.K.W., and D.L. advised the researcher and reviewed the article.

Disclosure statement

Z.H., S.Q.X., C.Z., and T.F.W. are listed as inventors on pending patent applications for MAbs S5D2, S5G2 and S3H3. The other authors declare that they have no competing interests.

Data availability statement

All data needed to evaluate the conclusions in the paper have been present in the paper and/or the Supplementary Materials. For the S-S5D2 Fab dataset, related cryo-EM maps have been deposited in the Protein Data Bank with accession codes 7WCZ, 7WD0, 7WD7, and 7WCR, and the associated models have been deposited in the Electron Microscopy Data Bank with accession codes EMD-32430, EMD-32431, EMD-32433, and EMD-32428 for S-S5D2-F1, S-S5D2-F2, S-S5D2-F3, and RBD-1-S5D2, respectively. For the S-S3H3 Fab dataset, related cryo-EM maps have been deposited in the Protein Data Bank Bank with accession codes 7WD9, 7WDF, and 7WD8, and the associated models have been deposited in the Electron Microscopy Data Bank with accession codes EMD-32435, EMD-32437 and EMD-32434 for S-S3H3-F3, S-S3H3-F2, and SD1-3H3, respectively.

Additional information

Funding

This work was supported by grants from the Chinese Academy of Sciences [grant number XDB29040300] and from the Ministry of Science and Technology of China [grant number 2020YFC0845900]. Y.C. was supported by grants from the CAS Pilot Strategic Science and Technology Projects B (XDB37040103), the National Basic Research Program of China [grant number 2017YFA0503503], the NSFC [grant numbers 31670754 and 31872714], the CAS Major Science and Technology Infrastructure Open Research Projects, and the CAS-Shanghai Science Research Center [grant number CAS-SSRC-YH-2015-01, DSS-WXJZ-2018-0002]. C.Z. was supported by the Youth Innovation Promotion Association of the Chinese Academy of Sciences [CAS].