A Bifluorescent-Based Assay for the Identification of Neutralizing Antibodies against SARS-CoV-2 Variants of Concern In Vitro and In Vivo
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Abstract
SARS-CoV-2 is responsible of the COVID-19 pandemic that has warped daily routines and socioeconomics. There is still an urgent need for prophylactics and therapeutics to treat SARS-CoV-2 infections.
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SciScore for 10.1101/2021.06.28.450214: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: All experiments using SARS-CoV-2 were approved by the Institutional Biosafety Committee (IBC) at Texas Biomedical Research Institute. Cells: African green monkey kidney epithelial cells (Vero E6, CRL-1586) were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1X PSG (100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine), and incubated at 37°C in an 5% CO2 atmosphere. Sex as a biological variable Six-to- eight-week-old female K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice were purchased from The Jackson Laboratory and maintained in the Animal Biosafety Laboratory level 3 (ABSL-3) at … SciScore for 10.1101/2021.06.28.450214: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: All experiments using SARS-CoV-2 were approved by the Institutional Biosafety Committee (IBC) at Texas Biomedical Research Institute. Cells: African green monkey kidney epithelial cells (Vero E6, CRL-1586) were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1X PSG (100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine), and incubated at 37°C in an 5% CO2 atmosphere. Sex as a biological variable Six-to- eight-week-old female K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice were purchased from The Jackson Laboratory and maintained in the Animal Biosafety Laboratory level 3 (ABSL-3) at Texas Biomedical Research Institute. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources All experiments using SARS-CoV-2 were approved by the Institutional Biosafety Committee (IBC) at Texas Biomedical Research Institute. Cells: African green monkey kidney epithelial cells (Vero E6, CRL-1586) were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1X PSG (100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine), and incubated at 37°C in an 5% CO2 atmosphere. Vero E6suggested: NoneRecombinant DNA Sentences Resources Generation of pBeloBAC11-SARS-CoV-2 encoding fluorescent proteins (FP): The pBeloBAC11 plasmid (NEB) containing the entire viral genome of SARS-CoV-2 USA/WA1/2020 (WA-1) isolate (accession no. MN985325) has been described 20, 21. pBeloBAC11-SARS-CoV-2suggested: NonepBeloBAC11suggested: RRID:Addgene_60342)Briefly, confluent monolayers of Vero E6 cells (1.2 x 106 cells/well, 6-well plate format, triplicates) were transfected with 4 µg/well of pBeloBAC11-SARS-CoV-2 (WA-1), -2A/Venus, -2A/mCherry, or - 2A/mCherry-SA-RBD plasmids using Lipofectamine 2000 (Thermo Fisher). 2A/mCherry-SA-RBDsuggested: NoneSoftware and Algorithms Sentences Resources Cells were then fixed in 10% neutral buffered formalin overnight and washed with PBS, before fluorescence signal was measured and quantified using a Synergy LX microplate reader and Gen5 data analysis software (Bio- Tek). Gen5suggested: (Gen5, RRID:SCR_017317)The mean and SD of viral infections were calculated from individual wells of three independent experiments conducted in quadruplicates with Microsoft Excel software. Microsoft Excelsuggested: (Microsoft Excel, RRID:SCR_016137)Non-linear regression curves and NT50 values were determined using GraphPad Prism Software (San Diego, CA, USA, Version 8.2.1). GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Macroscopic pathological scoring was determined from the percent of total surface area affected by congestion, consolidation, and atelectasis of excised lungs, using NIH ImageJ software as previously described 21, 35. ImageJsuggested: (ImageJ, RRID:SCR_003070)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: We detected the following sentences addressing limitations in the study:Reporter-expressing recombinant viruses can circumvent limitations imposed by the need for secondary methods to detect the presence of viruses in infected cells. These reporter viruses have been used to evaluate viral infections, identify therapeutics, and to study viral virulence in vivo. Here, we have documented the generation of novel rSARS- CoV-2 to facilitate tracking infection of two different SARS-CoV-2 strains (WA-1 and SA) in vitro and in vivo based on the use of two different FPs (Venus and mCherry, respectively). The FP-expressing rSARS-CoV-2 encode the fluorescent Venus or mCherry proteins from the locus of the N protein, without the need for deletion of any viral protein 20. Notably, the use of this approach to generate FP-expressing rSARS- CoV-2 resulted in higher FP expression levels than those afforded by rSARS-CoV-2 expressing FPs from the locus of the viral ORF7a protein 20. Moreover, rSARS-CoV-2 expressing reporter genes from the N locus are more genetically stable than those expressing reporter genes from the ORF7a locus of the SARS-CoV-2 genome 20. We showed that rSARS-CoV-2 expressing Venus or mCherry from the N locus exhibited similar growth kinetics, peak titers, and plaque phenotype as the parental WT rSARS-CoV-2 WA-1 strain. Importantly, we were able to use these novel reporter rSARS-CoV-2 in bifluorescent-based assays to determine the neutralization efficacy of hMAbs based on FP expression levels. We also generated rSARS-CoV-2 mCherry SA, an mCherry...
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 40 and 38. 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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Results from scite Reference Check: We found no unreliable references.
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