SARS-CoV-2 neutralizing camelid heavy-chain-only antibodies as powerful tools for diagnostic and therapeutic applications

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1. Version published to 10.3389/fimmu.2022.930975

   Sep 14, 2022
2. 

   ScreenIT

   Mar 26, 2022

   SciScore for 10.1101/2022.03.24.485614: (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
   Heavy chain Ab design, expression and purification: For the generation of a full length heavy-chain-only antibody, the different VHHs were amplified with SL13/14 and cloned into the vector pNEM_camhAb. pNEM_camAb was designed in our lab as vector for eukaryotic protein expression with a camelid Fc fragment (CH2-CH3 domain) resulting in hcAbs.	CH2-CH3 domain suggested: None
   Western Blot analyses and ELISA: To investigate the different hcAb candidates in Western Blot, 1 μg SARS-CoV-2 Spike …

   SciScore for 10.1101/2022.03.24.485614: (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
   Heavy chain Ab design, expression and purification: For the generation of a full length heavy-chain-only antibody, the different VHHs were amplified with SL13/14 and cloned into the vector pNEM_camhAb. pNEM_camAb was designed in our lab as vector for eukaryotic protein expression with a camelid Fc fragment (CH2-CH3 domain) resulting in hcAbs.	CH2-CH3 domain suggested: None
   Western Blot analyses and ELISA: To investigate the different hcAb candidates in Western Blot, 1 μg SARS-CoV-2 Spike protein (antibodies-online, ABIN6952734) per lane was applied onto a 4-12% SDS polyacrylamide gradient gel.	antibodies-online , ABIN6952734 suggested: None
   After washing the membrane, a horseradish peroxidase (HRP) conjugated secondary antibody (ABIN1981272, antibodies-online) was applied and incubated for 3 h at RT.	antibodies-online suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   In specific, VSV*ΔG either bearing the SARS-CoV-2 (D614G) or SARS-CoV-2 B.1.1.529 (Omicron) Spike protein was incubated with a two-fold dilution of hcAbs and subsequently used to inoculated VeroE6 cells.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Expi293 cells were grown at 37°C, 8% CO2 with 130-150 rpm on a Rotamax120 platform shaker (	Expi293 suggested: RRID:CVCL_D615)
   Recombinant DNA
   Sentences	Resources
   Amplified VHHs were cloned into phagemid vector pADL-22c (Antibody Design Labs).	pADL-22c suggested: None
   Positive clones were sequenced and become heavy chain only antibodies by cloning into the expression plasmid pNEM_camAb.	pNEM_camAb suggested: None
   The expression vector for the Omicron spike (based on isolate hCoV19/Botswana/R40B58_BHP_3321001245/2021; GISAID Accession ID: EPI_ISL_6640919) was generated by Gibson assembly using five overlapping DNA strings (Thermo Fisher Scientific, sequences available upon request), linearized (BamHI/XbaI digest) pCG1 plasmid and GeneArt™ Gibson Assembly HiFi Master Mix (Thermo Fisher Scientific).	pCG1 suggested: None
   VLPs): Human codon optimized sequences of genes encoding the S and E structural proteins of SARS-CoV-2 were synthesized by BioCat GmbH (Heidelberg, Germany) and subcloned into the pcDNA3.1 expression plasmid using the NheI 5’ and XhoI 3’ restriction site, respectively.	pcDNA3.1 suggested: RRID:Addgene_79663)
   Another plasmid (pEXP-M+N) for the dual expression of the human codon optimized sequences of the M and N protein was generated by Vectorbuilder Inc. (VB200528-1033wpt).	pEXP-M+N suggested: None
   The DNA mix was composed of pcDNA3.1-Spike (FKO), pcDNA3.1-E-Protein and pEXP-M+N-protein at a ratio of 6:2:3 diluted in Opti-MEM I Medium.	pcDNA3.1-Spike suggested: None pcDNA3.1-E-Protein suggested: None pEXP-M+N-protein suggested: None
   Software and Algorithms
   Sentences	Resources
   VLPs): Human codon optimized sequences of genes encoding the S and E structural proteins of SARS-CoV-2 were synthesized by BioCat GmbH (Heidelberg, Germany) and subcloned into the pcDNA3.1 expression plasmid using the NheI 5’ and XhoI 3’ restriction site, respectively.	BioCat suggested: (BioCAT, RRID:SCR_001440)

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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.
   • No funding statement was detected.
   • 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 10.1101/2022.03.24.485614v1 on bioRxiv

   Mar 24, 2022