Rapid characterization of spike variants via mammalian cell surface display.

The SARS-CoV-2 spike protein is a critical component of vaccines and a target for neutralizing monoclonal antibodies (nAbs). Spike is also undergoing immunogenic selection with variants that increase infectivity and partially escape convalescent plasma. Here, we describe Spike Display, a high-throughput platform to rapidly characterize glycosylated spike ectodomains across multiple coronavirus-family proteins. We assayed ∼200 variant SARS-CoV-2 spikes for their expression, ACE2 binding, and recognition by 13 nAbs. An alanine scan of all five N-terminal domain (NTD) loops highlights a public epitope in the N1, N3, and N5 loops recognized by most NTD-binding nAbs. NTD mutations in variants of concern B.1.1.7 (alpha), B.1.351 (beta), B.1.1.28 (gamma), B.1.427/B.1.429 (epsilon), and B.1.617.2 (delta) impact spike expression and escape most NTD-targeting nAbs. Finally, B.1.351 and B.1.1.28 completely escape a potent ACE2 mimic. We anticipate that Spike Display will accelerate antigen design, deep scanning mutagenesis, and antibody epitope mapping for SARS-CoV-2 and other emerging viral threats.

Mammalian Antigen Display for Pandemic Countermeasures.

Pandemic countermeasures require the rapid design of antigens for vaccines, profiling patient antibody responses, assessing antigen structure-function landscapes, and the surveillance of emerging viral lineages. Cell surface display of a viral antigen or its subdomains can facilitate these goals by coupling the phenotypes of protein variants to their DNA sequence. Screening surface-displayed proteins via flow cytometry also eliminates time-consuming protein purification steps. Prior approaches have primarily relied on yeast as a display chassis. However, yeast often cannot express large viral glycoproteins, requiring their truncation into subdomains. Here, we describe a method to design and express antigens on the surface of mammalian HEK293T cells. We discuss three use cases, including screening of stabilizing mutations, deep mutational scanning, and epitope mapping. The mammalian antigen display platform described herein will accelerate ongoing and future pandemic countermeasures.

Antibody escape and cryptic cross-domain stabilization in the SARS-CoV-2 Omicron spike protein.

The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the repeated emergence of variants of concern. For the Omicron variant, sub-lineages BA.1 and BA.2, respectively, contain 33 and 29 nonsynonymous and indel spike protein mutations. These amino acid substitutions and indels are implicated in increased transmissibility and enhanced immune evasion. By reverting individual spike mutations of BA.1 or BA.2, we characterize the molecular effects of the Omicron spike mutations on expression, ACE2 receptor affinity, and neutralizing antibody recognition. We identified key mutations enabling escape from neutralizing antibodies at a variety of epitopes. Stabilizing mutations in the N-terminal and S2 domains of the spike protein can compensate for destabilizing mutations in the receptor binding domain, enabling the record number of mutations in Omicron. Our results provide a comprehensive account of the mutational effects in the Omicron spike protein and illustrate previously uncharacterized mechanisms of host evasion.

Synthetic repertoires derived from convalescent COVID-19 patients enable discovery of SARS-CoV-2 neutralizing antibodies and a novel quaternary binding modality.

The ongoing evolution of SARS-CoV-2 into more easily transmissible and infectious variants has sparked concern over the continued effectiveness of existing therapeutic antibodies and vaccines. Hence, together with increased genomic surveillance, methods to rapidly develop and assess effective interventions are critically needed. Here we report the discovery of SARS-CoV-2 neutralizing antibodies isolated from COVID-19 patients using a high-throughput platform. Antibodies were identified from unpaired donor B-cell and serum repertoires using yeast surface display, proteomics, and public light chain screening. Cryo-EM and functional characterization of the antibodies identified N3-1, an antibody that binds avidly (Kd,app = 68 pM) to the receptor binding domain (RBD) of the spike protein and robustly neutralizes the virus in vitro. This antibody likely binds all three RBDs of the trimeric spike protein with a single IgG. Importantly, N3-1 equivalently binds spike proteins from emerging SARS-CoV-2 variants of concern, neutralizes UK variant B.1.1.7, and binds SARS-CoV spike with nanomolar affinity. Taken together, the strategies described herein will prove broadly applicable in interrogating adaptive immunity and developing rapid response biological countermeasures to emerging pathogens.

Discovery of Novel Gain-of-Function Mutations Guided by Structure-Based Deep Learning.

Despite the promise of deep learning accelerated protein engineering, examples of such improved proteins are scarce. Here we report that a 3D convolutional neural network trained to associate amino acids with neighboring chemical microenvironments can guide identification of novel gain-of-function mutations that are not predicted by energetics-based approaches. Amalgamation of these mutations improved protein function in vivo across three diverse proteins by at least 5-fold. Furthermore, this model provides a means to interrogate the chemical space within protein microenvironments and identify specific chemical interactions that contribute to the gain-of-function phenotypes resulting from individual mutations.