A panel of nanobodies recognizing conserved hidden clefts of all SARS-CoV-2 spike variants including Omicron

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1. Version published to 10.1038/s42003-022-03630-3

   Jul 6, 2022
2. 

   ScreenIT

   Nov 19, 2021

   SciScore for 10.1101/2021.10.25.465714: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IACUC: The KYODOKEN Institutional Animal Care and Use Committee approved the protocols for these studies (approval number 20200312) and monitored health conditions.
   Sex as a biological variable	half-siblings—a 19-month-old male named “Puta” and a 19-month-old female named “Christy”—were immunized.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Blotted membranes were incubated overnight at 4°C with the C9 antibody or the C-terminally 6×His-tagged homodimer of nanobodies—the dilution ratios of P158, P334, and P543 were 1:5000, 1:1000, and 1:2500, respectively—in Tris-buffered saline (TBS, …

   SciScore for 10.1101/2021.10.25.465714: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IACUC: The KYODOKEN Institutional Animal Care and Use Committee approved the protocols for these studies (approval number 20200312) and monitored health conditions.
   Sex as a biological variable	half-siblings—a 19-month-old male named “Puta” and a 19-month-old female named “Christy”—were immunized.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Blotted membranes were incubated overnight at 4°C with the C9 antibody or the C-terminally 6×His-tagged homodimer of nanobodies—the dilution ratios of P158, P334, and P543 were 1:5000, 1:1000, and 1:2500, respectively—in Tris-buffered saline (TBS, pH 7.4) containing 0.005% Tween 20 (TBST) and 5% skim milk.	C9 suggested: (LSBio (LifeSpan Cat# LS-C9-1000, RRID:AB_1276501) P334 suggested: (Leinco Technologies Cat# P334, RRID:AB_2831621)
   In the case of nanobody-based blotting, after 3 washes with TBST, the membranes were incubated with 1:5000-diluted anti-His-tag antibody (MBL) in TBST containing 5% skim milk at room temperature for 1 h.	anti-His-tag suggested: None
   The membranes were soaked with 1:5000-diluted HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibodies (GE Healthcare) in TBST containing 5% skim milk for 30 min at room temperature.	anti-rabbit suggested: None anti-mouse IgG suggested: None
   Anti-6×His-tagged antibody-positive fractions were gathered and concentrated via VIVAspin 6 size exclusion columns (30,000-MWCO) to reach a volume under 0.5 ml.	Anti-6×His-tagged suggested: None
   After 3 washes with PBST, HRP-conjugated anti-alpaca VHH antibody (Jackson) at a dilution of 1:5000 was reacted at room temperature for 30 min.	anti-alpaca VHH suggested: None
   After washing, the cells were incubated with an anti-His antibody (Abcam) on ice for 30 min and then Alexa 647-conjugated anti-rabbit IgG (Dako).	anti-His suggested: None anti-rabbit IgG suggested: None
   After washing with PBST, an appropriately diluted anti-His-tagged antibody and anti-C9-tagged antibody in blocking buffer were added and reacted at room temperature for 1 h.	anti-His-tagged suggested: (StressMarq Biosciences Cat# SPC-167, RRID:AB_2703750) anti-C9-tagged suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell culture and transfection: HEK and K562 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM: Invitrogen) supplemented with 10% foetal bovine serum (FBS) and antibiotics (1% penicillin and streptomycin).	K562 suggested: None
   Pseudotyped virus production: HIV-1-based SARS-CoV-2 spike pseudotyped virus was prepared as follows: LentiX-HEK293T cells were transfected using a polyethyleneimine transfection reagent (Cytiva) with plasmids encoding the C-terminally C9-tagged full-length SARS-CoV-2 spike variants (original, alpha, beta, and delta) and HIV-1 transfer vector encoding a luciferase reporter, according to the manufacturer’s protocol.	LentiX-HEK293T suggested: None
   Microscopy analyses for cell staining: HEK cells were transiently transfected with plasmids encoding the C-terminally C9-tagged full-length SARS-CoV-2 spike variants using Lipofectamine 3000 (Thermo) according to the manufacturer’s instructions.	HEK suggested: None
   Recombinant DNA
   Sentences	Resources
   The expression vector of the SARS-CoV-2 S2 domain (residues 744-1213) was constructed by removing an N-terminal part of the extracellular domain of the SARS-CoV-2 spike (residues 31-743) and subcloned into the pcDNA3.1(+) vector.	pcDNA3.1 ( + ) suggested: None
   The synthesized genes were subcloned in the pMES4 vector to express N-terminal PelB signal peptide-conjugated and C-terminal 6×His-tagged nanobodies into the bacterial periplasm.	pMES4 suggested: RRID:Addgene_98223)
   The lentiviral vector pWPI-IRES-Bla-Ak-ACE2-TMPRSS2 was acquired from AddGene (plasmid #154983).	pWPI-IRES-Bla-Ak-ACE2-TMPRSS2 suggested: RRID:Addgene_154983)
   Software and Algorithms
   Sentences	Resources
   coli TG1 cells (Agilent Technologies Japan, Ltd., Tokyo, Japan) were transformed with the ligated plasmids under chilled conditions (Bio-Rad Laboratories, Inc., Hercules, CA)	Bio-Rad Laboratories suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
   The raw data of reads were trimmed of the adaptor sequence using cutadapt v1.1859, and low-quality reads were subsequently removed using Trimmomatic v0.3960.	Trimmomatic suggested: (Trimmomatic, RRID:SCR_011848)
   The remaining paired reads were merged using fastq-join61 and then translated to the amino acid sequences using EMBOSS v6.6.0.062.	EMBOSS suggested: (EMBOSS, RRID:SCR_008493)
   Finally, unique amino acid sequences in each library were counted using a custom Python script combining seqkit v0.10.163 and usearch v.1164.	Python suggested: (IPython, RRID:SCR_001658)
   Cryo-EM image processing and refinement: The images were processed using RELION 3.169.	RELION suggested: (RELION, RRID:SCR_016274)
   Movies were motion corrected using MotionCor270, and the contrast transfer functions (CTFs) were estimated using CTFFIND 4.171.	CTFFIND suggested: (CTFFIND, RRID:SCR_016732)
   The data were imported and further processed with non-uniform refinement in cryoSPARC v3.2.072.	cryoSPARC suggested: (cryoSPARC, RRID:SCR_016501)
   After the models were manually fitted into the density using UCSF Chimera v1.1573 and modified in Coot v0.8.9.274, real space refinement was performed in PHENIX v1.19.175.	Coot suggested: (Coot, RRID:SCR_014222) PHENIX suggested: (Phenix, RRID:SCR_014224)
   Figures were prepared using UCSF Chimera73, ChimeraX77, and PyMOL v2.5.0 (Schrödinger, LLC, New York, NY).	PyMOL suggested: (PyMOL, RRID:SCR_000305)
   The program refmac582 in the ccp4 suite83 and the program Phenix-refine75 were used for structural refinement.	ccp4 suggested: (CCP4, RRID:SCR_007255)

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   Results from OddPub: Thank you for sharing your data.

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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 10.1101/2021.10.25.465714v1 on bioRxiv

   Oct 26, 2021