Arsenal of Nanobodies for Broad-Spectrum Countermeasures against Current and Future SARS-CoV-2 Variants of Concerns

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   ScreenIT

   Dec 23, 2021

   SciScore for 10.1101/2021.12.20.473401: (What is this?)

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

   Table 1: Rigor

   Ethics

   Field Sample Permit: Experiments involving animals were conducted using protocols approved by the National Research Council Canada Animal Care Committee and in accordance with the guidelines set out in the OMAFRA Animals for Research Act, R.S.O. 1990, c. A.22. b) IACUC: Experiments involving animals were conducted using protocols approved by the National Research Council Canada Animal Care Committee and in accordance with the guidelines set out in the OMAFRA Animals for Research Act, R.S.O. 1990, c. A.22. b)

   Sex as a biological variable

   The denatured-induced unfolding of VHHs was considered to be reversible based on their small size as shown to be the case for small proteins and numerous times …

   SciScore for 10.1101/2021.12.20.473401: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: Experiments involving animals were conducted using protocols approved by the National Research Council Canada Animal Care Committee and in accordance with the guidelines set out in the OMAFRA Animals for Research Act, R.S.O. 1990, c. A.22. b) IACUC: Experiments involving animals were conducted using protocols approved by the National Research Council Canada Animal Care Committee and in accordance with the guidelines set out in the OMAFRA Animals for Research Act, R.S.O. 1990, c. A.22. b)
   Sex as a biological variable	The denatured-induced unfolding of VHHs was considered to be reversible based on their small size as shown to be the case for small proteins and numerous times for VHHs 56–59, 88, 89. d) Serum stability: Three female Syrian hamsters were injected intraperitoneally (IP) with 1 mg of 1d or VHH-72 VHH-Fc diluted in 200 µL PBS.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Reactions were stopped by adding 50 µL 1 M H2SO4 to wells, and absorbance were subsequently measured at 450 nm using a Multiskan™ FC photometer (Thermo Fisher). b) Binding to cognate anti-spike glycoprotein polyclonal antibody: The four spike glycoprotein antigens were passively adsorbed as described above.	anti-spike glycoprotein suggested: None
   After blocking with PBSC, wells were emptied, washed five times with PBST and incubated at room temperature for 1 h with 100 µL of 1 µg/mL anti-SARS-CoV-2 spike rabbit polyclonal antibody (Sino Biological, Beijing, China, Cat#40589-T62) in PBSCT.	anti-SARS-CoV-2 suggested: None
   Following 1 h incubation at room temperature, wells were washed 10 times with PBST and incubated with HRP-conjugated polyclonal goat anti-llama IgG heavy and light chain antibody (Bethyl Laboratories, Montgomery, TX, Cat#A160-100P) for 1 h at room temperature.	anti-llama IgG heavy and light chain suggested: None
   To determine the presence of antibodies that block the binding of S to ACE2 (surrogate for neutralization) in the immune sera of llamas, 400 ng of chemically biotinylated SARS-CoV-2 S was mixed with 1 × 105 Vero E6 cells in the presence of 2-fold dilutions of sera (pre immune, day 21 and day 28 sera) in a final volume of 150 µL.	ACE2 suggested: None
   Then plates were washed 10 times with PBST and binding of VHHs to S1-Fc was detected with rabbit anti-6xHis Tag antibody HRP Conjugate (Bethyl Laboratories, Cat#A190-114P), diluted at 10 ng/mL in PBST and added at 100 µL/well.	anti-6xHis suggested: None
   After 1 h incubation at room temperature, strips were washed 10 times with PBST and the binding of VHH-Fcs to denatured S was probed by incubating strips with 1 mL of 100 ng/mL anti-human Ig Fc antibody-peroxidase conjugate (Jackson ImmunoResearch, Cat#016-030-084) at room temperature for 1 h.	anti-human Ig Fc antibody-peroxidase conjugate suggested: None
   After 1 h incubation at room temperature, plates were washed 10 times with PBST and the ACE2-Fc binding was detected using 1 µg/mL goat anti-human IgG (Fc specific) HRP conjugate antibody (Sigma, Cat#A0170) in 100 µL PBSCT.	anti-human IgG suggested: (Sigma-Aldrich Cat# A0170, RRID:AB_257868)
   Immune cell infiltrate was detected using rabbit polyclonal antibodies against CD3 (1:500, Dako, Cat#A0452), and Iba-1 (ionized calcium binding adaptor protein, 1:5000, Dako, Cat#019-19741)	CD3 suggested: (Agilent Cat# A0452, RRID:AB_2335677) Iba-1 ( ionized calcium binding adaptor protein suggested: None
   Mouse anti-SARS-CoV-2 N monoclonal antibody (1:5000, R&D Systems, Cat#MAB10474) was used for the detection of SARS-CoV-2.	Mouse anti-SARS-CoV-2 N monoclonal antibody suggested: None anti-SARS-CoV-2 N suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Conversely, a binding response was seen during the second injection for VHHs that did not compete with ACE2. c) ACE2 competition assay by flow cytometry: Experiments were performed as described in 4.3c, except that biotinylated S/Vero E6 cells were mixed with VHHs or VHH-Fcs instead of sera.	S/Vero E6 suggested: RRID:CVCL_JX48)
   Pseudotyped and live virus neutralization assays: a) Pseudotyped virus neutralization assays: (i) Generation of SARS-CoV-2 spike pseudotyped lentiviral particles (LVP): HEK293T cells were plated in a 100-mm tissue culture dish and transfected the next day at about 75% confluency with a combination of a lentiviral transfer vector encoding eGFPLuc (addgene#119816), the packaging plasmid psPAX2 (addgene#12260), and a plasmid encoding the viral glycoprotein of interest SARS-CoV-2 Spike-ΔS1/S2-Δ20 expressed in pcDNA3.1+.	HEK293T suggested: None
   The pellet was then resuspended in PBS buffer at 1/10 of the original supernatant volume with gentle up-and-down pipetting, aliquoted and stored at −80°C. (ii) Viral neutralization assays: HEK293T-hACE2 cell line (BEI Resources, Manassas, VA, Cat#NR-52511) were seeded in poly-L-Lysine (PLL)-coated white, clear bottom 384-wells plate (NUNC, Thermo Fisher) at a density of 9,000 cells/well in 45 µL of media (DMEM without phenol red supplemented with 5% [v/v] FBS) and incubated for 24 h at 37°C, 5% CO2.	HEK293T-hACE2 suggested: RRID:CVCL_A7UK)
   Quantitative microneutralization assay was performed on Vero E6 cells with SARS-CoV-2 strains hCOV-19/Canada/ON-VIDO-01/2020, NR-53565; hCOV- 19/England/204820464/2020, NR-54000; or hCOV-19/South Africa/KRISP-EC-K005321 /2020, NR-54008.	Vero E6 suggested: None
   Virus titer was determined by plaque assay on Vero cells. b) Immunohistochemistry: Lungs were immersed in 10% neutral buffered formalin and fixed for 1 week at room temperature and then transferred into 70% ethanol.	Vero suggested: None
   Recombinant DNA
   Sentences	Resources
   VH/VHH genes were amplified using semi-nested PCR and cloned into the phagemid vector pMED1, followed by transformation of E. coli TG1 (Lucigen, Middleton, WI, Cat#60502-02) to construct two libraries with sizes of 1 × 107 and 2 × 107 independent transformants for Green and Red, respectively.	pMED1 suggested: None
   Expression and purification of VHHs and VHH-Fcs: a) Expression and validation of VHHs: Positive VHHs were cloned into a modified pET expression vector (pMRo.BAP.H6) for their production in BL21(DE3) E.coli as monomeric soluble protein 84.	pET suggested: None pMRo.BAP.H6 suggested: None
   For the VHH-72 benchmark 29, the sequence of the VHH was synthesized as a GeneBlock (Integrated DNA Technologies, Coralville, IA) flanked by SfiI sites for cloning into pMRo.	pMRo suggested: None
   Production of VHHs in mammalian cells in fusion with human IgG1 Fc (VHH-Fcs): Codon-optimized genes for bivalent VHH-Fcs were synthesized and cloned into pTT5 (GenScript; Piscataway, NJ.	pTT5 suggested: RRID:Addgene_52326)
   Pseudotyped and live virus neutralization assays: a) Pseudotyped virus neutralization assays: (i) Generation of SARS-CoV-2 spike pseudotyped lentiviral particles (LVP): HEK293T cells were plated in a 100-mm tissue culture dish and transfected the next day at about 75% confluency with a combination of a lentiviral transfer vector encoding eGFPLuc (addgene#119816), the packaging plasmid psPAX2 (addgene#12260), and a plasmid encoding the viral glycoprotein of interest SARS-CoV-2 Spike-ΔS1/S2-Δ20 expressed in pcDNA3.1+.	psPAX2 suggested: RRID:Addgene_12260) pcDNA3.1+ suggested: RRID:Addgene_117272)
   Software and Algorithms
   Sentences	Resources
   After a final wash, cells were resuspended in 100 µL PBSB and data were acquired on a CytoFLEX S flow cytometer (Beckman Coulter, Brea, CA) and analyzed by FlowJo software (FlowJo LLC, v10.6.2	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Proteins were buffer exchanged using Amicon® Ultra-15 Centrifugal Filter Units (Millipore-Sigma	Millipore-Sigma suggested: None
   IC50 was determined from non-linear regression [Inhibitor] vs. response, Variable slope (four parameters) using GraphPad Prism version 9 (La Jolla, CA). 4.11.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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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 10.1101/2021.12.20.473401v1 on bioRxiv

   Dec 21, 2021