Neutralizing Antibodies Isolated by a site-directed Screening have Potent Protection on SARS-CoV-2 Infection
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Abstract
Neutralizing antibody is one of the most effective interventions for acute pathogenic infection. Currently, over three million people have been identified for SARS-CoV-2 infection but SARS-CoV-2-specific vaccines and neutralizing antibodies are still lacking. SARS-CoV-2 infects host cells by interacting with angiotensin converting enzyme-2 (ACE2) via the S1 receptor-binding domain (RBD) of its surface spike glycoprotein. Therefore, blocking the interaction of SARS-CoV-2-RBD and ACE2 by antibody would cause a directly neutralizing effect against virus. In the current study, we selected the ACE2 interface of SARS-CoV-2-RBD as the targeting epitope for neutralizing antibody screening. We performed site-directed screening by phage display and finally obtained one IgG antibody (4A3) and several domain antibodies. Among them, 4A3 and three domain antibodies (4A12, 4D5, and 4A10) were identified to act as neutralizing antibodies due to their capabilities to block the interaction between SARS-CoV-2-RBD and ACE2-positive cells. The domain antibody 4A12 was predicted to have the best accessibility to all three ACE2-interfaces on the spike homotrimer. Pseudovirus and authentic SARS-CoV-2 neutralization assays showed that all four antibodies could potently protect host cells from virus infection. Overall, we isolated multiple formats of SARS-CoV-2-neutralizing antibodies via site-directed antibody screening, which could be promising candidate drugs for the prevention and treatment of COVID-19.
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SciScore for 10.1101/2020.05.03.074914: (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 The phage library (Tomlison I library and Domain antibody library) and the coated wells were blocked with PBST with 5% milk at room temperature for 1 h. Domainsuggested: NoneAfter blocking, biotin-labeled ACE2, blocked phage or the indicated antibodies were added to the wells and incubated at 37 ° C for 0.5 h. ACE2suggested: NoneStreptavidin-HRP (Thermo, Pudong New Area, Shanghai), a rabbit anti-M13 HRP antibody (for phage) (GE Healthcare, Milwaukee, WI), … SciScore for 10.1101/2020.05.03.074914: (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 The phage library (Tomlison I library and Domain antibody library) and the coated wells were blocked with PBST with 5% milk at room temperature for 1 h. Domainsuggested: NoneAfter blocking, biotin-labeled ACE2, blocked phage or the indicated antibodies were added to the wells and incubated at 37 ° C for 0.5 h. ACE2suggested: NoneStreptavidin-HRP (Thermo, Pudong New Area, Shanghai), a rabbit anti-M13 HRP antibody (for phage) (GE Healthcare, Milwaukee, WI), or a goat anti-human Fcγ HRP antibody (Jackson ImmunoResearch, West Grove, PA) was added. anti-M13 HRPsuggested: NoneFor capture ELISA, an anti-his antibody (5 μg/ml) was coated on an ELISA plate at 4 °C overnight. anti-hissuggested: NoneAfter washing, a goat anti-human Fcγ HRP antibody (Jackson ImmunoResearch, West Grove, PA) was added and incubated at 37 °C for 0.5 h. anti-human Fcγ HRPsuggested: NoneAfter washing, the cell suspension was labeled with goat anti-human PE antibody (Thermo, Pudong New Area, Shanghai) and incubated for 1 h on ice. anti-human PEsuggested: NoneExperimental Models: Cell Lines Sentences Resources Cell binding and antibody blocking assays: For the cell binding assay, cell suspension of SARS-CoV-2-spike-CHO cells was incubated with the indicated antibody (5 μg/ml) for 1 h on ice and then incubated with goat anti-human PE antibody (Thermo, Pudong New Area, Shanghai) for 1 h on ice. SARS-CoV-2-spike-CHOsuggested: NoneFor the antibody blocking assay, antibodies were preincubated with 2.5 μg/ml SARS-CoV-1-RBD-hFc or SARS-CoV-2-RBD-hFc at different concentrations for 1 h on ice, and then, the mixture was incubated with ACE2-CHO cells for 1 h on ice. ACE2-CHOsuggested: NonePseudovirus neutralization assay: To generate SARS-CoV-2 pseudovirus, we replaced the coding sequence of VSV-G protein with the sequence of SARS-CoV-2 spike in lentiviral packaging system19,20 and then co-transfect HEK293T cells with the pLVX-EGFP-Luciferase reporter gene. HEK293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Viruses were amplified in Vero E6 cells and made as working stocks at 105 pfu/ml. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources The remodeled ectodomain trimers of SARS-CoV-2 spike (open state) and SARS-CoV-2 spike (closed state) were established by replacing the partially determined RBD of the ectodomain trimers of SARS-CoV-2 spike (open state) (PDB ID: 6VYB) and SARS-CoV-2 spike (closed state) (PDB ID: 6VXX) with the completely determined SARS-CoV-2-RBD (PDB ID: 6M0J) by using PyMOL, Discovery Studio and SWISS MODEL. PyMOLsuggested: (PyMOL, RRID:SCR_000305)All statistical analyses were conducted using GraphPad Prism 8.0. GraphPad Prismsuggested: (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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
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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