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

Abstract

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.

XBB.1.5 monovalent booster improves antibody binding and neutralization against emerging SARS-CoV-2 Omicron variants

“…S2P and RBD antigens. Biotinylated probes of the stabilized SARS CoV-2 spike trimer S2P and the RBD subdomain were produced as previously described 39,40 . Briefly, the DNA sequence encoding either the S2P or RBD region from ancestral WA1 strain or XBB.1.5 variant and flanked by an HRV3C cleavable single-chain Fc tag and an AVI tag was cloned in a mammalian expression vector and transfected in FreeStyle 293 cells.…”

Adjuvanted SARS-CoV-2 spike protein vaccination elicits long-lived plasma cells in nonhuman primates

“…Biotinylated probes of the stabilized S-2P and the subdomains (NTD and RBD) were produced by an in-process biotinylation method as previously described ( 43 , 44 ). Briefly, the DNA sequences encoding either S-2P, NTD, or RBD from the WT, B.1.351, and B.1.617.2 SARS-CoV-2 strains were cloned in a mammalian expression vector with an HRV3C cleavable single-chain Fc before the coding sequence for purification and an Avi tag after the coding sequence for biotinylation.…”

“…Afterward, the probes were purified on a Superdex 200 16/600 gel filtration column equilibrated with PBS. Last, the biotinylation was confirmed using biolayer interferometry with streptavidin biosensors ( 43 ). Biotinylated proteins were incubated with fluorochrome-labeled streptavidin at a 1:4 molar ratio before being added to cells for staining.…”

Interaction dynamics between innate and adaptive immune cells responding to SARS-CoV-2 vaccination in non-human primates

“…Probes consisted of the SARS-CoV-2 proteins S-2P, S1, RBD, and NTD conjugated to APC, BV570, BV421 and BV711, respectively. Proteins were synthesized as described previously 80 , 81 . Additionally, each sample was labeled with 1 µl of TotalSeq-C Hashtag antibodies (Biolegend) that was incubated together with the staining cocktail.…”