A Bispecific Antibody Targeting RBD and S2 Potently Neutralizes SARS-CoV-2 Omicron and Other Variants of Concern
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Abstract
The new, highly contagious SARS-CoV-2 Omicron variant caused substantial breakthrough infections and has become the dominant strain in countries across the world. Omicron variants usually bear high mutations in the spike protein and exhibit considerable escape of most potent neutralization monoclonal antibodies and reduced efficacy of current COVID-19 vaccines.
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SciScore for 10.1101/2022.05.11.491588: (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 Single-chain Fv format bispecific antibodies were designed from the sequences of the variable regions of monoclonal antibodies 35B5 and 47D10 (ScFv 35B5-47D10) or 47D10 and 35B5 (ScFv 47D10-35B5), utilizing tandem glycine-serine (G4S) 47D10suggested: NoneBispecific antibody isolation and purification: Briefly, bsAbs were efficiently purified by using Protein A Sepharose affinity chromatography medium (GenScript, L00210-10). L00210-10suggested: NoneFollowing washing 3 times, mouse … SciScore for 10.1101/2022.05.11.491588: (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 Single-chain Fv format bispecific antibodies were designed from the sequences of the variable regions of monoclonal antibodies 35B5 and 47D10 (ScFv 35B5-47D10) or 47D10 and 35B5 (ScFv 47D10-35B5), utilizing tandem glycine-serine (G4S) 47D10suggested: NoneBispecific antibody isolation and purification: Briefly, bsAbs were efficiently purified by using Protein A Sepharose affinity chromatography medium (GenScript, L00210-10). L00210-10suggested: NoneFollowing washing 3 times, mouse anti-human IgG Fc secondary antibody with HRP (Abcam) was added and incubated at 37°C for 1 h, followed by washing with PBST. mouse anti-human IgG Fc secondary antibodysuggested: NoneAfter further washing, bound SARS-CoV-2 RBD protein was detected with anti-mouse Fc HRP antibody (Abcam) diluted 1:10000 in blocking solution followed by washing with PBST. anti-mouse Fc HRPsuggested: NoneAfter washing, the cells were incubated with bsAbs, monoclonal human anti-SARS-CoV-2 RBD antibody 35B5 or monoclonal human anti-SARS-CoV-2 S2 antibody 47D10 (5 μg per well) for 1 hour on ice, followed by FITC-conjugated anti-human IgG (1:100) (ZSGB-Bio, ZF-0308) for 1 hour on ice away from light. anti-SARS-CoV-2 RBDsuggested: (Bio X Cell Cat# BE0357, RRID:AB_2894776)anti-SARS-CoV-2suggested: (Bio X Cell Cat# BE0358, RRID:AB_2894777)anti-human IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources Cells and plasmids: HEK293T cells producing pseudovirus and HEK293 cells overexpressing recombinant human ACE2 (293/hACE2) were preserved in our laboratory and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin–streptomycin and were incubated at 37ºC with 5% CO2 and 95% humidity. HEK293Tsuggested: NoneHEK293suggested: NoneIn brief, pseudoviruses were produced by using PEI to cotransfect 293T cells with psPAX2, pLenti-GFP and plasmids encoding SARS-CoV-2 S, SARS-CoV-2 VOCs S, SARS-CoV S, RaTG S or empty vector at a ratio of 1:1:1. 293Tsuggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)Recombinant DNA Sentences Resources Codon-optimized ScFvs DNA sequences were synthesized and cloned into the pUC57 cloning vector (GenScript, Piscataway, NJ) and subcloned into the eukaryotic cell expression vector AbVec-hIgG1 between the AgeI and Hind III sites. pUC57suggested: RRID:Addgene_40306)The pcDNA3.1-hACE2 plasmid with human codon optimization, plasmids encoding WT SARS-CoV-2 S glycoprotein, SARS-CoV-2 VBM spike protein, SARS-CoV-2 VOC S glycoprotein, RaTG S glycoprotein, lentiviral packaging plasmid psPAX2 and pLenti-GFP lentiviral reporter plasmid were generously gifted by Dr. Zhaohui Qian. pcDNA3.1-hACE2suggested: RRID:Addgene_145033)In brief, pseudoviruses were produced by using PEI to cotransfect 293T cells with psPAX2, pLenti-GFP and plasmids encoding SARS-CoV-2 S, SARS-CoV-2 VOCs S, SARS-CoV S, RaTG S or empty vector at a ratio of 1:1:1. psPAX2suggested: RRID:Addgene_12260)pLenti-GFPsuggested: RRID:Addgene_172394)For the pseudovirus neutralization assay, HEK293 (hACE2/293) cells 80-90% confluent in T75 cell culture flasks were transfected with 20 μg of plasmid encoding hACE2 for 36 hours and seeded into 24-well plates the day before transduction with pseudovirus. hACE2suggested: RRID:Addgene_1786)Software and Algorithms Sentences Resources Cells were acquired by flow cytometry (BD Biosciences) and analyzed using FlowJo. FlowJosuggested: (FlowJo, RRID:SCR_008520)Statistical analysis: All statistical analyses were performed using GraphPad Prism 9.0 software. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)PyMol was used to prepare all the structure figures (Schrodinger: https://www.schrodinger.com/pymol). PyMolsuggested: (PyMOL, RRID:SCR_000305)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: We did not find any issues relating to colormaps.
Results from rtransparent:- No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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