Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells
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Abstract
The global pandemic of Coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable in vitro neutralization assay is very important for the development of neutralizing antibodies, vaccines and other inhibitors. In this study, G protein-deficient vesicular stomatitis virus (VSVdG) bearing full-length and truncated spike (S) protein of SARS-CoV-2 were evaluated. The virus packaging efficiency of VSV-SARS-CoV-2-Sdel18 (S with C-terminal 18 amino acid truncation) is much higher than VSV-SARS-CoV-2-S. A neutralization assay for antibody screening and serum neutralizing titer quantification was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and human angiotensin-converting enzyme 2 (ACE2) overexpressed BHK21 cell (BHK21-hACE2). The experimental results can be obtained by automatically counting EGFP positive cell number at 12 hours after infection, making the assay convenient and high-throughput. The serum neutralizing titer of COVID-19 convalescent patients measured by VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with live SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting receptor binding domain (RBD) of SARS-CoV-2-S were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.
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SciScore for 10.1101/2020.04.08.026948: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consent: Written informed consents were obtained from all the volunteers. 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
Experimental Models: Cell Lines Sentences Resources Cells and Samples: Vero-E6 (American Type Culture Collection [ATCC], CRL-1586), Vero (ATCC, CCL-81), BHK21 (ATCC, CCL-10) and 293T (kindly gifted by Dr. Jiahuai Han) cells were maintained in high glucose DMEM (SIGMA-ALDRICH) supplemented with 10% FBS (GIBCO), penicillin (100 IU/mL), streptomycin (100 μg/mL) in a 5% CO2 environment at 37°C and passaged every 2 days. BHK21suggest…SciScore for 10.1101/2020.04.08.026948: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consent: Written informed consents were obtained from all the volunteers. 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
Experimental Models: Cell Lines Sentences Resources Cells and Samples: Vero-E6 (American Type Culture Collection [ATCC], CRL-1586), Vero (ATCC, CCL-81), BHK21 (ATCC, CCL-10) and 293T (kindly gifted by Dr. Jiahuai Han) cells were maintained in high glucose DMEM (SIGMA-ALDRICH) supplemented with 10% FBS (GIBCO), penicillin (100 IU/mL), streptomycin (100 μg/mL) in a 5% CO2 environment at 37°C and passaged every 2 days. BHK21suggested: None293Tsuggested: NoneBHK21-hACE2 cell was developed by stably transfection of hACE2-expressing plasmid following puromycin resistance selection. BHK21-hACE2suggested: NoneLive SARS-CoV-2-based neutralization assay: Seeded Vero cells in 96-well plates. Verosuggested: NoneSoftware and Algorithms Sentences Resources The IC50 (the half maximal inhibitory concentration) or ID 50 (plasma/serum dilutions causing a 50% reduction of GFP-positive cell number or RNA level) values were calculated with non-linear regression, i.e. log (inhibitor) vs. normalized response – Variable slope, using GraphPad Prism 7.00 (GraphPad Software, Inc., 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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