Molecular probes of spike ectodomain and its subdomains for SARS-CoV-2 variants, Alpha through Omicron.

Since the outbreak of the COVID-19 pandemic, widespread infections have allowed SARS-CoV-2 to evolve in human, leading to the emergence of multiple circulating variants. Some of these variants show increased resistance to vaccine-elicited immunity, convalescent plasma, or monoclonal antibodies. In particular, mutations in the SARS-CoV-2 spike have drawn attention. To facilitate the isolation of neutralizing antibodies and the monitoring of vaccine effectiveness against these variants, we designed and produced biotin-labeled molecular probes of variant SARS-CoV-2 spikes and their subdomains, using a structure-based construct design that incorporated an N-terminal purification tag, a specific amino acid sequence for protease cleavage, the variant spike-based region of interest, and a C-terminal sequence targeted by biotin ligase. These probes could be produced by a single step using in-process biotinylation and purification. We characterized the physical properties and antigenicity of these probes, comprising the N-terminal domain (NTD), the receptor-binding domain (RBD), the RBD and subdomain 1 (RBD-SD1), and the prefusion-stabilized spike ectodomain (S2P) with sequences from SARS-CoV-2 variants of concern or of interest, including variants Alpha, Beta, Gamma, Epsilon, Iota, Kappa, Delta, Lambda, Mu, and Omicron. We functionally validated probes by using yeast expressing a panel of nine SARS-CoV-2 spike-binding antibodies and confirmed sorting capabilities of variant probes using yeast displaying libraries of plasma antibodies from COVID-19 convalescent donors. We deposited these constructs to Addgene to enable their dissemination. Overall, this study describes a matrix of SARS-CoV-2 variant molecular probes that allow for assessment of immune responses, identification of serum antibody specificity, and isolation and characterization of neutralizing antibodies.

Ultrapotent antibodies against diverse and highly transmissible SARS-CoV-2 variants.

The emergence of highly transmissible SARS-CoV-2 variants of concern (VOCs) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identified four receptor binding domain-targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 23 variants, including the B.1.1.7, B.1.351, P.1, B.1.429, B.1.526, and B.1.617 VOCs. Two antibodies are ultrapotent, with subnanomolar neutralization titers [half-maximal inhibitory concentration (IC50) 0.3 to 11.1 nanograms per milliliter; IC80 1.5 to 34.5 nanograms per milliliter). We define the structural and functional determinants of binding for all four VOC-targeting antibodies and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting their potential in mitigating resistance development.

Paired heavy- and light-chain signatures contribute to potent SARS-CoV-2 neutralization in public antibody responses.

Understanding mechanisms of protective antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We report a monoclonal antibody, 910-30, targeting the SARS-CoV-2 receptor-binding site for ACE2 as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. Sequence and structural analyses of 910-30 and related antibodies explore how class recognition features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer reveal binding interactions and its ability to disassemble spike. Despite heavy-chain sequence similarity, biophysical analyses of IGHV3-53/3-66-encoded antibodies highlight the importance of native heavy:light pairings for ACE2-binding competition and SARS-CoV-2 neutralization. We develop paired heavy:light class sequence signatures and determine antibody precursor prevalence to be ∼1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These class signatures reveal genetic, structural, and functional immune features that are helpful in accelerating antibody-based medical interventions for SARS-CoV-2.

SARS-CoV-2 S2P spike ages through distinct states with altered immunogenicity.

The SARS-CoV-2 spike is the primary target of virus-neutralizing antibodies and critical to the development of effective vaccines against COVID-19. Here, we demonstrate that the prefusion-stabilized two-proline "S2P" spike-widely employed for laboratory work and clinical studies-unfolds when stored at 4 °C, physiological pH, as observed by electron microscopy (EM) and differential scanning calorimetry, but that its trimeric, native-like conformation can be reacquired by low pH treatment. When stored for approximately 1 week, this unfolding does not significantly alter antigenic characteristics; however, longer storage diminishes antibody binding, and month-old spike elicits virtually no neutralization in mice despite inducing high ELISA-binding titers. Cryo-EM structures reveal the folded fraction of spike to decrease with aging; however, its structure remains largely similar, although with varying mobility of the receptor-binding domain. Thus, the SARS-CoV-2 spike is susceptible to unfolding, which affects immunogenicity, highlighting the need to monitor its integrity.