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

Allison J Greaney ,John C Kraft,Daniel Ellis,Alexandra C Walls,Tyler N Starr ,Katharine H.d Crawford ,Deleah Pettie,David Veesler,Keara D Malone,Jesse D Bloom ,Michael Murphy,Karla-Luise Herpoldt,Claire Sydeman,Minh N Pham,Kelly K Lee,Neil P King,Chengbo Chen,Mary Jane Navarro,Brooke Fiala,Lauren Carter,Maggie Ahlrichs,Elizabeth Kepl,Alyssa Blackstone,Natalie Brunette,Rashmi Ravichandran,Max Johnson,Cassandra Ogohara

doi:10.3389/fimmu.2021.710263

Abstract

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.

Highlights

The rapid development of safe and effective vaccines in response to the SARS-CoV-2 pandemic is a significant achievement of modern vaccinology but has strained worldwide vaccine manufacturing and distribution capabilities
The other four mutations were observed to be near or within a recently identified linoleic acid-binding pocket formed between adjacent receptor-binding domain (RBD) in the closed Spike trimer [71, 72], with Y365 identified as a key gating residue for this interaction (Figures 1A, B)
The improved expression and stability observed by deep mutational scanning (DMS) for several mutations in the linoleic acid-binding pocket suggested that this region is structurally suboptimal in the isolated RBD

Summary

Introduction

The rapid development of safe and effective vaccines in response to the SARS-CoV-2 pandemic is a significant achievement of modern vaccinology but has strained worldwide vaccine manufacturing and distribution capabilities. The ability to reliably design, robustly produce, and widely distribute vaccines against SARS-CoV-2 variants and other pandemic threat coronaviruses is a public health priority, and there is room to improve existing vaccines against SARS-CoV-2 in these respects [5]. This is true for protein-based vaccines, which are yet to be widely deployed in response to the SARSCoV-2 pandemic but are likely to become a key part of the global portfolio of SARS-CoV-2 vaccines [6]. Even with stabilizing mutations, most the widely used “2P” mutations [2], protein-based immunogens containing the complete trimeric ectodomain can suffer from instability and low expression yields, which may limit their development and scalability [23, 24]

Methods

Results

Conclusion