Super-immunity by broadly protective nanobodies to sarbecoviruses

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   ScreenIT

   Jan 3, 2022

   SciScore for 10.1101/2021.12.26.474192: (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
   HRP-conjugated secondary antibodies against the T7-tag were diluted at 1:5,000 or 1:75,00 in the blocking buffer and incubated for 1 hour at room temperature.	T7-tag suggested: None
   A validated SARS-CoV-2 antibody-negative human serum control and a validated NIBSC SARS-CoV-2 plasma control were obtained from the National Institute for Biological Standards and Control, UK) and an uninfected cells control were also used to ensure that virus neutralization by antibodies was specific.	Control, UK suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   A 96-well ELISA plate (R&D system) was coated with …

   SciScore for 10.1101/2021.12.26.474192: (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
   HRP-conjugated secondary antibodies against the T7-tag were diluted at 1:5,000 or 1:75,00 in the blocking buffer and incubated for 1 hour at room temperature.	T7-tag suggested: None
   A validated SARS-CoV-2 antibody-negative human serum control and a validated NIBSC SARS-CoV-2 plasma control were obtained from the National Institute for Biological Standards and Control, UK) and an uninfected cells control were also used to ensure that virus neutralization by antibodies was specific.	Control, UK suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   A 96-well ELISA plate (R&D system) was coated with the RBD protein or the HEK-293T cell lysate at an amount of approximately 3-5 ng per well in a coating buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate, pH 9.6) overnight at 4°C, with subsequent blockage with a blocking buffer (DPBS, v/v 0.05% Tween 20, 5% milk) at room temperature for 2 hours.	HEK-293T suggested: None
   Pseudotyped SARS-CoV-2 neutralization assay: The 293T-hsACE2 stable cell line (Cat# C-HA101, Lot# TA060720C) and pseudotyped SARS-CoV-2 (Wuhan-Hu-1 strain, D614G, Alpha, Beta, Lambda, Delta and Omicron) particles with luciferase reporters were purchased from the Integral Molecular.	293T-hsACE2 suggested: None
   The Nb–virus mixes (220 μl total) were incubated at 37°C for 1 h, after which they were added dropwise onto confluent Vero E6 cell (ATCC® CRL-1586™, for Munich) or Vero E6-TMPRSS2-T2A-ACE2 cells (BEI cat# NR- 54970, for Delta) monolayers in the six-well plates.	Vero E6 suggested: None Vero E6-TMPRSS2-T2A-ACE2 suggested: None
   Recombinant DNA
   Sentences	Resources
   The monomeric Nbs 2-31 and 2-45 were also cloned into a pET-22b(+) vector at the BamHI and XhoI sites for periplasmic expression.	pET-22b(+) suggested: RRID:Addgene_12651)
   The PCR fragment was then inserted into the 2-45 pET-21b(+) vector at the same restriction sites to produce the heterodimer 2-45-(GGGGS)3-2-31.	pET-21b(+) suggested: RRID:Addgene_112204)
   Crystallographic analysis of psNbs with RBD: The receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (GenBank: QHD43416.1), used in the crystallographic study, was cloned into a customized pFastBac vector (69), and fused with an N-terminal gp67 signal peptide and C-terminal His6 tag (70).	pFastBac suggested: RRID:Addgene_1925)
   Software and Algorithms
   Sentences	Resources
   The MS data obtained from different RBD-specific VHH isolations were analyzed by AugurLlama to identify high-affinity Nbs for each RBD (15).	AugurLlama suggested: None
   Data were processed by Prism 9 (GraphPad) to fit into a 4PL curve and to calculate the logIC50 (half-maximal inhibitory concentration).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   Automated data collection was carried out using serialEM (46) at a nominal magnification of 64,000x with a physical pixel size of 1.329 Å/pixel (0.6645 Å/pixel at super-resolution).	serialEM suggested: (SerialEM, RRID:SCR_017293)
   Image processing was performed on-the-fly using CryoSPARC Live version 3.2 (47–49).	CryoSPARC suggested: (cryoSPARC, RRID:SCR_016501)
   Models were manually corrected in Coot (version 9.6.0) between rounds of read-space refinement in Phenix.	Coot suggested: (Coot, RRID:SCR_014222)
   Maps colored by local resolution were generated using RELION 3.1 (57).	RELION suggested: (RELION, RRID:SCR_016274)
   Iterative model building and refinement were carried out in COOT (74) and PHENIX (75), respectively.	PHENIX suggested: (Phenix, RRID:SCR_014224)
   Antigens were clustered at 70% sequence identity with a minimum length coverage of 15% using CD-HIT (78), the Bio.align pairwise sequence alignment module and CATH domain identifiers.	CD-HIT suggested: (CD-HIT, RRID:SCR_007105)
   Antibody hit rate calculation: We constructed a multiple sequence alignment for each antigen cluster using MAFFT (79) and selected a representative structure with highest structure coverage and resolution.	MAFFT suggested: (MAFFT, RRID:SCR_011811)
   Conservation scores range from 0 to ln(20) = 2.99, higher is more conserved.	Conservation suggested: (Conservation, RRID:SCR_016064)
   The simulation was run starting from the RBD structure (PDB 6lzg) using Gromacs 2020 version with the CHARMM36m force field (82).	Gromacs suggested: (GROMACS, RRID:SCR_014565)
   Sequences were aligned by MUSCLE (83) with default parameters.	MUSCLE suggested: (MUSCLE, RRID:SCR_011812)
   The phylogenetic tree was then constructed by the MEGA (84) using the maximized likelihood estimation method.	MEGA suggested: (Mega BLAST, RRID:SCR_011920)

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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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   Version published to 10.1101/2021.12.26.474192v1 on bioRxiv

   Dec 28, 2021