Stabilization of the SARS-CoV-2 Spike Receptor-Binding Domain Using Deep Mutational Scanning and Structure-Based Design.

The unprecedented global demand for SARS-CoV-2 vaccines has demonstrated the need for highly effective vaccine candidates that are thermostable and amenable to large-scale manufacturing. Nanoparticle immunogens presenting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (S) in repetitive arrays are being advanced as second-generation vaccine candidates, as they feature robust manufacturing characteristics and have shown promising immunogenicity in preclinical models. Here, we used previously reported deep mutational scanning (DMS) data to guide the design of stabilized variants of the RBD. The selected mutations fill a cavity in the RBD that has been identified as a linoleic acid binding pocket. Screening of several designs led to the selection of two lead candidates that expressed at higher yields than the wild-type RBD. These stabilized RBDs possess enhanced thermal stability and resistance to aggregation, particularly when incorporated into an icosahedral nanoparticle immunogen that maintained its integrity and antigenicity for 28 days at 35-40°C, while corresponding immunogens displaying the wild-type RBD experienced aggregation and loss of antigenicity. The stabilized immunogens preserved the potent immunogenicity of the original nanoparticle immunogen, which is currently being evaluated in a Phase I/II clinical trial. Our findings may improve the scalability and stability of RBD-based coronavirus vaccines in any format and more generally highlight the utility of comprehensive DMS data in guiding vaccine design.
Competing Interests: DE, AW, TS, AG, JB, DV, and NK are named as inventors on patent applications filed by the University of Washington based on the studies presented in this paper. NK. is a co-founder, shareholder, paid consultant, and chair of the scientific advisory board of Icosavax, Inc. and has received an unrelated sponsored research agreement from Pfizer. DV is a consultant for and has received an unrelated sponsored research agreement from Vir Biotechnology Inc. JB consults for Moderna on viral evolution and epidemiology. JB and KC have the potential to receive a share of IP revenue as inventors on a Fred Hutch optioned technology/patent (application WO2020006494) related to deep mutational scanning of viral proteins. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
(Copyright © 2021 Ellis, Brunette, Crawford, Walls, Pham, Chen, Herpoldt, Fiala, Murphy, Pettie, Kraft, Malone, Navarro, Ogohara, Kepl, Ravichandran, Sydeman, Ahlrichs, Johnson, Blackstone, Carter, Starr, Greaney, Lee, Veesler, Bloom and King.)