Humanized single domain antibodies neutralize SARS-CoV-2 by targeting the spike receptor binding domain
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Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads worldwide and leads to an unprecedented medical burden and lives lost. Neutralizing antibodies provide efficient blockade for viral infection and are a promising category of biological therapies. Here, using SARS-CoV-2 spike receptor-binding domain (RBD) as a bait, we generate a panel of humanized single domain antibodies (sdAbs) from a synthetic library. These sdAbs reveal binding kinetics with the equilibrium dissociation constant ( K D ) of 0.99–35.5 nM. The monomeric sdAbs show half maximal neutralization concentration (EC 50 ) of 0.0009–0.07 µg/mL and 0.13–0.51 µg/mL against SARS-CoV-2 pseudotypes, and authentic SARS-CoV-2, respectively. Competitive ligand-binding experiments suggest that the sdAbs either completely block or significantly inhibit the association between SARS-CoV-2 RBD and viral entry receptor ACE2. Fusion of the human IgG1 Fc to sdAbs improve their neutralization activity by up to ten times. These results support neutralizing sdAbs as a potential alternative for antiviral therapies.
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SciScore for 10.1101/2020.04.14.042010: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Antibodies were obtained from ThermoFisher for anti-His-HRP, anti-human IgG-HRP, anti-His-488 and anti-CM13. anti-His-HRPsuggested: Noneanti-human IgG-HRPsuggested: Noneanti-His-488suggested: NoneLibrary design and construction: A synthetic sdAb phage display library was used for the screening of SARS-CoV-2 neutralizing antibodies. SARS-CoV-2 neutralizing antibodies .suggested: NoneAfter 4 rounds of panning, phage ELISA identification was performed with 480 … SciScore for 10.1101/2020.04.14.042010: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Antibodies were obtained from ThermoFisher for anti-His-HRP, anti-human IgG-HRP, anti-His-488 and anti-CM13. anti-His-HRPsuggested: Noneanti-human IgG-HRPsuggested: Noneanti-His-488suggested: NoneLibrary design and construction: A synthetic sdAb phage display library was used for the screening of SARS-CoV-2 neutralizing antibodies. SARS-CoV-2 neutralizing antibodies .suggested: NoneAfter 4 rounds of panning, phage ELISA identification was performed with 480 individual colonies using Anti-CM13 antibody [B62-FE2] (HRP). Anti-CM13suggested: NoneMaxipreped plasmids were transiently transfected into 293-F cells (Thermofisher) and the cells were further cultured in suspension for 6 days before harvesting antibody-containing supernatant. antibody-containing supernatant .suggested: NoneThe primary antibody used for Western blot was a horseradish peroxidase conjugated goat anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA) anti-mouse IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources Cells and reagents: The Vero (African green monkey kidney), HEK293T (human kidney epithelial), 293F, Calu-3 (human lung adenocarcinoma) cells were obtained from China Infrastructure of Cell Line Resource (Beijing, China) and maintained in Dulbecco’s modified Eagle’s medium (DMEM, ThermoFisher, Waltham, MA, USA) supplemented with 2-10% fetal bovine serum (FBS, Calu-3suggested: NoneProduction of SARS-CoV-2 spike pseudotyped particle (SARS-CoV-2pp) and virus entry assay: To produce SARS-CoV-2pp, HEK293T cells were seeded 1 day prior to transfection at 2.5×106 cells in a 10-cm plate. HEK293Tsuggested: NoneTo conduct the virus entry assay, Vero E6 or other cells were seeded in a 96-well plate at 1 day prior to transduction. Vero E6suggested: NoneFor antibody neutralization assay, Vero cells were seeded in 96-well plates at 1 day prior to infection. Verosuggested: NoneImmunofluorescence microscopy and Western blot: Cultured 293T cells on coverslips were transfected with either SARS-CoV-2 S expression plasmid or empty vector for 24 h and then fixed using 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 10 min. 293Tsuggested: NoneSoftware and Algorithms Sentences Resources Statistical analysis: Data were analyzed using GraphPad Prism 6.01 (GraphPad Software, San Diego, CA, USA). 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: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- 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.
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