Structural changes in the SARS-CoV-2 spike E406W mutant escaping a clinical monoclonal antibody cocktail

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1. Version published to 10.1016/j.celrep.2023.112621

   Jun 1, 2023
2. 

   ScreenIT

   Jan 29, 2022

   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-G suggested: None
   Experimental 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-G suggested: None
   Experimental 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-293T suggested: None
   Vero 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.	Vero suggested: None
   Recombinant 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.	Expi293 suggested: 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-ACE2 suggested: None
   Neutralizations 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-TMPRSS2 suggested: 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.	pCDNA3.1 suggested: 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.	pCMVR suggested: None
   Software and Algorithms
   Sentences	Resources
   Reference-free 2D classification was performed using cryoSPARC to select for well-defined particle images42.	cryoSPARC suggested: (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.	Relion suggested: (RELION, RRID:SCR_016274)
   Luminescence readings from the neutralization assays were normalized and analyzed using GraphPad Prism 9.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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   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).

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   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.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   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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   Results from scite Reference Check: We found no unreliable references.

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3. 

   Version published to 10.1101/2022.01.21.477288v1 on bioRxiv

   Jan 25, 2022