Molecular probes of spike ectodomain and its subdomains for SARS-CoV-2 variants, Alpha through Omicron
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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 vaccines, 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 the 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.
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SciScore for 10.1101/2021.12.29.474491: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Expression and preparation of antibodies: DNA sequences of heavy and light chain variable regions of antibody CR3022 [32] and of donor S652 antibodies, S652-109, S652-118, and S652-112, and of SARS-CoV-2 antibodies A19-46.1, A23-58.1, A19-61.1, B1-182.1 [18], 5-7 [33], 2-51, and 5-24 [34] were synthesized and subcloned into the pVRC8400 vector, as described previously [43]. S652suggested: (Leinco Technologies Cat# S652, RRID:AB_2831816)S652-112suggested: NoneBriefly, VH and VL regions … SciScore for 10.1101/2021.12.29.474491: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Expression and preparation of antibodies: DNA sequences of heavy and light chain variable regions of antibody CR3022 [32] and of donor S652 antibodies, S652-109, S652-118, and S652-112, and of SARS-CoV-2 antibodies A19-46.1, A23-58.1, A19-61.1, B1-182.1 [18], 5-7 [33], 2-51, and 5-24 [34] were synthesized and subcloned into the pVRC8400 vector, as described previously [43]. S652suggested: (Leinco Technologies Cat# S652, RRID:AB_2831816)S652-112suggested: NoneBriefly, VH and VL regions of VRC01 (as a negative control) [46], S652-118, S652-112, S652-109 [19], and SARS-Cov2-specific antibodies LY-555 [40], CB6 [47], REGN10933, REGN10987 [48], A19-46.1, and A23-58.1 [18] were codon optimized for yeast expression using JCat [49], synthesized and cloned by Genscript into pCT-VHVL-K1 or pCT-VHVL-L1 yeast expression vectors [39]. VRC01suggested: (bNAber Cat# bNAberID_1, RRID:AB_2491019)SARS-Cov2-specificsuggested: NoneCB6suggested: NoneA19-46.1suggested: (Bethyl Cat# A303-978A, RRID:AB_2620327)Streptavidin, R-Phycoerythrin Conjugate, Thermo Fisher Scientific) or SA-APC (Streptavidin, Allophycocyanin Conjugate, Thermo Fisher Scientific), and an anti-FLAG fluorescein isothiocyanate (FITC) monoclonal antibody (clone M2-FITC, Sigma-Aldrich) was added as a marker for Fab expression. R-Phycoerythrinsuggested: Noneanti-FLAGsuggested: NoneExperimental Models: Cell Lines Sentences Resources Expression and preparation of wildtype and variant SARS-CoV-2 molecular probes: The wildtype and variant SARS-CoV-2 molecular probes were produced by transient transfection of FreeStyle 293-F cells [28] and in-process biotinylation [19] as previously described. 293-Fsuggested: RRID:CVCL_6642)Recombinant DNA Sentences Resources Construction of expression plasmids for wildtype and variant SARS-CoV-2 molecular probes: DNA sequences encoding the wildtype or variant SARS-CoV-2 spike or specific domains were cloned into a pVRC8400-based expression vector by GeneImmune. pVRC8400-basedsuggested: NoneSoftware and Algorithms Sentences Resources Micrographs were collected at a nominal magnification of 100,000x (pixel size: 0.22 nm) using SerialEM [44] on an FEI Tecnai T20 electron microscope operated at 200 kV and equipped with an Eagle CCD camera or at a nominal magnification of 57,000x (pixel size: 0.25 nm) on a Thermo Scientific Talos F200C electron microscope operated at 200 kV and equipped with a Ceta camera. SerialEMsuggested: (SerialEM, RRID:SCR_017293)Reference-free 2D classification was performed with Relion 1.4 [45] Relionsuggested: (RELION, RRID:SCR_016274)Obtained flow cytometry data was further analyzed using FlowJo 10.6.1 software (BD Biosciences). FlowJosuggested: (FlowJo, RRID:SCR_008520)Heat maps were created using GraphPad Prism 8 software (GraphPad Software Inc.) GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 49, 50, 51 and 52. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
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- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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