Structural changes in the SARS-CoV-2 spike E406W mutant escaping a clinical monoclonal antibody cocktail
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SciScore for 10.1101/2022.01.21.477288: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: The study protocol was approved by the University of Washington Human Subjects Division Institutional Review Board (STUDY00010350) 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 Two hours after infection, the cells were washed 5 times with DMEM and grown in DMEM supplemented with 10% FBS and 1% PenStrep along with an anti-VSV-G antibody (I1-mouse hybridoma supernatant diluted 1:25, from CRL-2700, ATCC). anti-VSV-Gsuggested: NoneExperimental Models: Cell Lines Sentences Resources HEK-293T cells and HEK-293T cells stably expressing the human … SciScore for 10.1101/2022.01.21.477288: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: The study protocol was approved by the University of Washington Human Subjects Division Institutional Review Board (STUDY00010350) 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 Two hours after infection, the cells were washed 5 times with DMEM and grown in DMEM supplemented with 10% FBS and 1% PenStrep along with an anti-VSV-G antibody (I1-mouse hybridoma supernatant diluted 1:25, from CRL-2700, ATCC). anti-VSV-Gsuggested: NoneExperimental Models: Cell Lines Sentences Resources HEK-293T cells and HEK-293T cells stably expressing the human ACE2 receptor (HEK-ACE2)38 were grown in DMEM supplemented with 10% FBS and 1% PenStrep at 37°C and 5% CO2. HEK-293Tsuggested: NoneVero cells stably expressing the human protease TMPRSS2 (Vero-TMPRSS2) were grown in DMEM supplemented with 10% FBS, 1% PenStrep, and 8 µg/mL puromycin at 37°C and 5% CO2. Verosuggested: NoneRecombinant protein expression and purification: To produce the SARS-CoV-2 spike ectodomain containing the E406W mutation, 125 mL of Expi293 cells were grown to density of 2.5 × 106 cells per mL and transfected with 125 µg of DNA using PEI MAX diluted in Opti-MEM. Expi293suggested: RRID:CVCL_D615)Neutralization assays with vaccine-elicited sera and monoclonal antibodies: For neutralization assays using vaccine-elicited sera, HEK-ACE2 cells were seeded in 96-well poly-D-lysine coated plates at a density of 30,000 cells per well and grown overnight until they reached approximately 80% confluency. HEK-ACE2suggested: NoneNeutralizations were conducted as described above with one modification: prior to the addition of the virus-antibody mixture, Vero-TMPRSS2 cells were washed 3 times with DMEM. Vero-TMPRSS2suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)Recombinant DNA Sentences Resources The spike ectodomain was codon optimized, stabilized with the hexapro mutations39 and mutation of the furin cleavage site (682RRAR685 to 682GSAS685), and inserted into the pCDNA3.1 vector containing a C-terminal foldon followed by an avi tag and an octa-histidine tag. pCDNA3.1suggested: RRID:Addgene_79663)The construct encoding the E406W RBD was generated by performing around-the-horn mutagenesis using a pCMVR vector encoding the wildtype SARS-CoV-2 RBD containing an N-terminal mu-phosphatase signal peptide and a C-terminal avi tag and octa-histidine tag. pCMVRsuggested: NoneSoftware and Algorithms Sentences Resources Reference-free 2D classification was performed using cryoSPARC to select for well-defined particle images42. cryoSPARCsuggested: (cryoSPARC, RRID:SCR_016501)These selected particles were then used for 3D classification with 50 iterations (angular sampling 7.5° for 25 iterations followed by 1.8° with local search for 25 iterations) using Relion and a previously reported closed model for the SARS-CoV-2 spike ectodomain (PBD: 6VXX) as the initial model without imposing any symmetry. Relionsuggested: (RELION, RRID:SCR_016274)Luminescence readings from the neutralization assays were normalized and analyzed using GraphPad Prism 9. 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 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.
Results from scite Reference Check: We found no unreliable references.
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