Structural basis and mode of action for two broadly neutralizing antibodies against SARS-CoV-2 emerging variants of concern
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SciScore for 10.1101/2021.08.02.454546: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources The conformational effects of 50 µg/ml CV3-1 and CV3-25 antibodies on SARS-CoV-2 spike were tested by pre-incubating fluorescently labeled viruses for 60 mins at 37°C before imaging in the continued presence of the antibodies. CV3-25suggested: NoneAt 48h post transfection, 293T cells were stained with anti-Spike monoclonal antibodies CV3-25, CV3-1 (5 µg/mL) or using the ACE2-Fc chimeric protein (20 µg/mL) for 45 min at 37°C. anti-Spikesuggested: None; mouse anti-S1 antibody (Sino Biological) and rabbit anti-S2 antibody (Sino Biological) were used as controls. anti-S1suggested: Noneanti-S2suggested: NoneTh… SciScore for 10.1101/2021.08.02.454546: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources The conformational effects of 50 µg/ml CV3-1 and CV3-25 antibodies on SARS-CoV-2 spike were tested by pre-incubating fluorescently labeled viruses for 60 mins at 37°C before imaging in the continued presence of the antibodies. CV3-25suggested: NoneAt 48h post transfection, 293T cells were stained with anti-Spike monoclonal antibodies CV3-25, CV3-1 (5 µg/mL) or using the ACE2-Fc chimeric protein (20 µg/mL) for 45 min at 37°C. anti-Spikesuggested: None; mouse anti-S1 antibody (Sino Biological) and rabbit anti-S2 antibody (Sino Biological) were used as controls. anti-S1suggested: Noneanti-S2suggested: NoneThe Western blots were developed with horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-human IgG, anti-mouse IgG or anti-rabbit IgG, correspondingly). anti-human IgGsuggested: Noneanti-mouse IgGsuggested: Noneanti-rabbit IgGsuggested: NoneTo evaluate antibody recognition of S glycoproteins lacking N-linked glycans, 293T-S cells expressing the wild-type SARS-CoV-2 S glycoprotein were lysed with lysis buffer, as described above. SARS-CoV-2 S glycoproteinsuggested: NoneExperimental Models: Cell Lines Sentences Resources 293T-ACE2 cells stably expressing human ACE2 are derived from 293T cells and were maintained in medium supplemented with 2 µg/mL of puromycin (Millipore Sigma 293T-ACE2suggested: None293Tsuggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)Briefly, the 4 x 105 Vero-E6 cells were seeded on 12-well plate. Vero-E6suggested: NonePseudoviruses decorated with SARS-CoV-2 Spike were prepared by transfecting HEK293T cells (70% confluent 10 cm dishes) with a plasmid mixture of 5 µg of psPAX2 (Gag-pol, Rev, and Tat expression vector; does not express Vpr), 5 µg of pCMV-d19 Spike (last 19 residues at C-terminal were deleted) from the B.1.1.7 variant or WH01 G614, and 2 µg of a pCAGGS-Cyclophilin A-HiBiT construct using polyetherimide (PEI). HEK293Tsuggested: NoneHEK293T-ACE2 target cells were transfected in a 24 well plate using PEI with 500ng/well of pMX Puro PH-LgBiT (LgBiT-tagged to pleckstrin homology domain of human phospholipase C the N terminus). HEK293T-ACE2suggested: None293T-S cells were seeded in 6-well plates at a density of 1×106 cells per well on day 0. 293T-Ssuggested: RRID:CVCL_LC70)Experimental Models: Organisms/Strains Sentences Resources Alternatively, for peptide epitope competition assay, CV3-25 (5µg/mL) was pre-incubated in presence of increasing concentrations of peptide #288 (1149-KEELDKYFKNHTSPD-1163), peptide #289 (1153-DKYFKNHTSPDVDLG-1167), a shorter version of peptide #289 (1153-DKYFKNHTSPD-1163) or a scramble version of the peptide #289 (DHDTKFLNYDPVGKS), which were synthesized by Genscript. 1153-DKYFKNHTSPD-1163suggested: NoneRecombinant DNA Sentences Resources The plasmids encoding the Spike from the B.1.617.1 (E154K, L452R, E484Q, D614G, P681R) and the B.1.617.2 (T19R, Δ156-158, L452R, T478K, D614G, P681R, D950N) lineages were generated by overlapping PCR using a codon-optimized wild-type SARS-CoV-2 Spike gene that was synthesized (Biobasic, Markham, ON, Canada) and cloned in pCAGGS as a template. pCAGGSsuggested: RRID:Addgene_18926)Pseudoviruses decorated with SARS-CoV-2 Spike were prepared by transfecting HEK293T cells (70% confluent 10 cm dishes) with a plasmid mixture of 5 µg of psPAX2 (Gag-pol, Rev, and Tat expression vector; does not express Vpr), 5 µg of pCMV-d19 Spike (last 19 residues at C-terminal were deleted) from the B.1.1.7 variant or WH01 G614, and 2 µg of a pCAGGS-Cyclophilin A-HiBiT construct using polyetherimide (PEI). psPAX2suggested: RRID:Addgene_12260)pCMV-d19suggested: NonepCAGGS-Cyclophilin A-HiBiTsuggested: NoneHEK293T-ACE2 target cells were transfected in a 24 well plate using PEI with 500ng/well of pMX Puro PH-LgBiT (LgBiT-tagged to pleckstrin homology domain of human phospholipase C the N terminus). pMXsuggested: RRID:Addgene_111810)Two short peptides labeling tags (Q3: GQQQLG; A4: DSLDMLEM) were introduced into designed positions in the S1 subunit on the plasmid encoding SB.1.1.7, pCMV-SB.1.1.7. pCMV-SB.1.1.7suggested: NonePlasmids pCMV-SB. pCMV-SBsuggested: None, dual-tagged pCMV-SB.1.1.7 Q3-1 A4-1, and pCMV delta R8.2 were transfected into 293T cells at a ratio of 20:1:21. pCMV-SB.1.1.7 Q3-1 A4-1suggested: NonepCMVsuggested: RRID:Addgene_20783)Flow Cytometry Analysis of Cell-Surface Staining: Using the standard calcium phosphate method, 10 μg of Spike expressor and 2 μg of a green fluorescent protein (GFP) expressor (pIRES2-eGFP; Clontech) was transfected into 2 × 106 293T cells. pIRES2-eGFPsuggested: RRID:Addgene_14998)Briefly, 293T cells were transfected by the calcium phosphate method with the lentiviral vector pNL4.3 R-E-Luc (NIH AIDS Reagent Program) and a plasmid encoding for SARS-CoV-2 Spike at a ratio of 5:4. pNL4.3suggested: NoneSoftware and Algorithms Sentences Resources Tomographic tilt series between −60° and +60° were collected by using SerialEM (Mastronarde, 2005)(Mastronarde, 2005) in a dose-symmetric scheme (Hagen et al., 2017; Mastronarde and Held, 2017)(Hagen et al., 2017) with increments of 3°. SerialEMsuggested: (SerialEM, RRID:SCR_017293)Tomograms were reconstructed by weighted back projection and tomographic slices were visualized with IMOD. IMODsuggested: (IMOD, RRID:SCR_003297)All the density maps were segmented in the UCSF Chimera (Pettersen et al., 2004), and ChimeraX (Goddard et al., 2018; Pettersen et al., 2021) was used for surface rendering and visualization of cryo-ET maps and models. “Fit in map” tool in Chimera and ChimeraX was used for rigid fitting. ChimeraXsuggested: (UCSF ChimeraX, RRID:SCR_015872)smFRET data analysis was performed using MATLAB ( MATLABsuggested: (MATLAB, RRID:SCR_001622)MathWorks)-based customized SPARTAN software package (Juette et al., 2016). SPARTANsuggested: (SPARTAN, RRID:SCR_014901), Model building and Analysis: Motion correction, CTF estimation, particle picking, curation and extraction, 2D classification, ab initio model reconstruction, volume refinements and local resolution estimation were carried out in cryoSPARC (Punjani et al., 2017; Rubinstein and Brubaker, 2015). cryoSPARCsuggested: (cryoSPARC, RRID:SCR_016501)Geometry validation and structure quality evaluation were performed by EM-Ringer (Barad et al., 2015) and Molprobity (Chen et al., 2010). Molprobitysuggested: (MolProbity, RRID:SCR_014226)Model-to-map fitting cross correlation and figures generation were carried out in USCF Chimera, Chimera X (Goddard et al., 2018; Pettersen et al., 2004; Pettersen et al., 2021) and PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.). PyMOLsuggested: (PyMOL, RRID:SCR_000305)Iterative cycles of model building and refinement were done in Coot(Emsley and Cowtan, 2004) and Phenix. Phenixsuggested: (Phenix, RRID:SCR_014224)Samples were acquired on a LSRII cytometer (BD Biosciences) and data analysis was performed using FlowJo v10.5.3 (Tree Star). FlowJosuggested: (FlowJo, RRID:SCR_008520)Hill coefficient analyses were done using GraphPad Prism version 9.1.0 (GraphPad). GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Quantification and Statistical Analysis: Data were analyzed and plotted using GraphPad Prism software (La Jolla, CA, USA). GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)P values were indicated as *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Schematics: Schematics for showing experimental design in figures were created with BioRender.com. BioRendersuggested: (Biorender, RRID:SCR_018361)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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 56 and 52. 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.
- 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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