A potent alpaca-derived nanobody that neutralizes SARS-CoV-2 variants

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1. Version published to 10.1016/j.isci.2022.103960

   Mar 1, 2022
2. 

   ScreenIT

   Jan 22, 2022

   SciScore for 10.1101/2022.01.18.476801: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   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
   ELISAs were ran with bacterial supernatant containing secreted VHHs as primary antibody, then anti-nanobody biotin antibody (Jackson #128-065-232) and streptavidin-HRP antibody (Thermo #N100) were used as secondary.	streptavidin-HRP suggested: (Cell Signaling Technology Cat# 3999, RRID:AB_10830897)
   Anti-mouse IgG AF555 (Abcam ab150114) or anti-llama IgM AF488-conjugated secondary antibodies were added at 1:500 dilution for 1 hour at RT (Invitrogen).	Anti-mouse IgG suggested: (Abcam Cat# …

   SciScore for 10.1101/2022.01.18.476801: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   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
   ELISAs were ran with bacterial supernatant containing secreted VHHs as primary antibody, then anti-nanobody biotin antibody (Jackson #128-065-232) and streptavidin-HRP antibody (Thermo #N100) were used as secondary.	streptavidin-HRP suggested: (Cell Signaling Technology Cat# 3999, RRID:AB_10830897)
   Anti-mouse IgG AF555 (Abcam ab150114) or anti-llama IgM AF488-conjugated secondary antibodies were added at 1:500 dilution for 1 hour at RT (Invitrogen).	Anti-mouse IgG suggested: (Abcam Cat# ab150114, RRID:AB_2687594) anti-llama IgM suggested: None
   Cells were washed 2x with PBS then treated with anti-VHH biotin (Jackson 128-065-232) secondary antibody (Invitrogen #A16061) was added at 1:500 dilution for 1 hour at RT.	anti-VHH suggested: (GenScript Cat# A01860, RRID:AB_2734123)
   LzGreen SARS-COV-2 S pseudotyped lentiviruses were mixed with saRBD-1, or VHH52 control antibody.	VHH52 suggested: None
   RBD immunized alpaca sera was used as a primary antibody at 1:5,000 dilution in permeabilization buffer, and anti-Llama-HRP secondary was used at 1:20,000 dilution in permeabilization buffer.	anti-Llama-HRP suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Key resource table: Experimental model and subject details: HEK-293T stable cell lines expressing human ACE2 receptor (HEK-293T-ACE2) were a kind gift from Dr. Jesse D.	HEK-293T suggested: None
   Low-passage HEK-293T, HEK-293T-ACE2, and Vero E6 cells were cultured in D10, which consisted of Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% Penn-Strep, 1% non-essential amino acids (NEAA).	Vero E6 suggested: None
   SARS-CoV-2 immunofluorescence: 96-well TC plates were seeded to 50% confluency with VeroE6 or Caco2 cells.	Caco2 suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025)
   Spike-pseudotyped lentivirus production: 293T cells were seeded at 2 million cells/dish in 6cm TC-treated dishes.	293T suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
   Virus-antibody mixture was incubated at 37C for 1 hour after which polybrene was added up to 5 μg/ml and the mixture was added to 293T-ACE2 cells.	293T-ACE2 suggested: None
   In brief, Vero E6 cells were plated at 20,000 cells/well or Caco-2 cells were plated at 24,000 cells/well in 96-well plates and incubated overnight.	Caco-2 suggested: None
   Recombinant DNA
   Sentences	Resources
   Briefly, codon optimized his-tagged RBD or RBD containing the N501Y mutation in pLVX-IRES-puro plasmid (Takare Bio) was used to make lentivirus vectors in HEK-293T cells, which were then used to infect HEK-293F suspension cells.	pLVX-IRES-puro suggested: RRID:Addgene_140240)
   The amplified gene mixture was cloned into a phage-mid plasmid derived from pCANTA5BE, then transformed via electroporation into bacteriophage competent TG1 Escherichia coli for production of a VHH displaying bacteriophage library.	pCANTA5BE suggested: None
   Bivalent-saRBD-1 gene was synthesized containing two saRBD-1 genes separated by a flexible 20 a.a (GGGGS)4 linker, for cloning into pET24a bacterial expression plasmid.	pET24a suggested: RRID:Addgene_109417)
   For S transfection, the SARS-CoV2 structural protein plasmid pTwist-EF1alpha-nCoV-2019-S-2xStrep a kind gift from the Krogan lab at UCSF, was used as described previously (Gordon et al., 2020).	pTwist-EF1alpha-nCoV-2019-S-2xStrep suggested: None
   For pseudotyped lentivirus production, the following reporter plasmids and lentivirus packaging plasmids were used as described previously (Crawford et al., 2020): HDM_Hgpm2, HDM_tat1b, PRC_CMV_Rev1b packaging plasmids, SARS_CoV-2 S plasmid HDM_IDTSpike_fixK, and LzGreen GFP-reporter plasmid.	SARS_CoV-2 S suggested: None
   Software and Algorithms
   Sentences	Resources
   Maximum intensity z-projections were prepared in ImageJ.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   The normalized transduction data were fit to a logistic function to determine EC50 and IC50 values in python version 3.8.10.	python suggested: (IPython, RRID:SCR_001658)

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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 10.1101/2022.01.18.476801v1 on bioRxiv

   Jan 20, 2022