Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies

SciScore for 10.1101/2020.03.26.010165: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	IACUC: Immunizations and handling of the llama were performed according to directive 2010/63/EU of the European parliament for the protection of animals used for scientific purposes and approved by the Ethical Committee for Animal Experiments of the Vrije Universiteit Brussel (permit No. 13-601-1).
Randomization	Periplasmic ELISA screen to select MERS- and SARS-CoV directed VHHs: After panning, 45 individual colonies of phage infected bacteria isolated after the first panning round on MERS-CoV S-2P or SARS-CoV-1 S-2P protein and 45 individual colonies isolated after the second panning round on MERS-CoV S-2P or SARS-CoV-1 S-2P protein were randomly selected for further analysis by ELISA for the presence of MERS-CoV and SARS-CoV-1 specific VHHs, respectively.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Llama immunization: A llama, negative for antibodies against MERS-CoV and SARS-CoV-1 S glycoprotein, was subcutaneously immunized with approximately 150 µg recombinant SARS-CoV-1 S-2P protein on days 0, 7, 28 and 150 µg recombinant MERS-CoV S-2P protein on days 14 and 28 and 150 µg of both MERS-CoV S-2P and SARS-CoV-1 S-2P protein on day 35 (Kirchdoerfer et al., 2018; Pallesen et al., 2017).	SARS-CoV-1 S glycoprotein suggested: None
In the PE-ELISA screen after panning on SARS-CoV-1 S protein wells of microtiter plates (type II, F96 Maxisorp, Nuc) were coated with 100 ng SARS-CoV-1 S-2P protein (with foldon), SARS-CoV-1 S-2P protein captured with an anti-foldon antibody (with foldon) or as negative controls coated with MERS-CoV S-2P (without foldon), HCoV-HKU1 S-2P (without foldon), DS-Cav1 (with foldon) or bovine serum albumin (BSA, Sigma-Aldrich).	anti-foldon suggested: None
Binding was detected by incubating the plates sequentially with either mouse anti-Histidine Tag antibody (MCA1396, Abd Serotec) followed horseradish peroxidase (HRP)-linked anti-mouse IgG (1/2000, NXA931, GE Healthcare) or Streptavidin-HRP (554066, BD Biosciences) or by an HRP-linked rabbit anti-camelid VHH monoclonal antibody (A01861-200, GenScript).	anti-Histidine Tag suggested: None MCA1396 suggested: None anti-mouse IgG ( 1/2000 , NXA931 , GE Healthcare ) suggested: None Streptavidin-HRP suggested: (BD Biosciences Cat# 554066, RRID:AB_2868972) anti-camelid VHH suggested: (GenScript Cat# A01860, RRID:AB_2734123)
Binding of the VHH-72-Fc and VHH-72-Fc (S) to cells was determined by an AF633 conjugated goat anti-human IgG antibody and binding to SARS-CoV-1 or SARS-CoV-2 S was calculated as the mean AF633 fluorescence intensity (MFI) of GFP expressing cells (GFP+) divided by the MFI of GFP negative cells (GFP-).	anti-human IgG suggested: (R and D Systems Cat# AF633, RRID:AB_355491) SARS-CoV-1 suggested: None GFP- suggested: None
Subsequently, the cells were washed 3 times with PBS containing 0.5% BSA and stained with an AF647 conjugated donkey anti-mouse IgG antibody (Invitrogen) for 1 hour.	anti-mouse IgG suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Briefly, pseudoviruses expressing spike genes for MERS-CoV England1 (GenBank ID: AFY13307) and SARS-CoV-1 Urbani (GenBank ID: AAP13441.1) were produced by co-transfection of plasmids encoding a luciferase reporter, lentivirus backbone, and spike genes in 293T cells (Wang et al., 2015).	293T suggested: None
Serial dilutions of VHHs were mixed with pseudoviruses, incubated for 30 min at room temperature, and then added to previously-plated Huh7.5 cells.	Huh7.5 suggested: RRID:CVCL_7927)
For the VSV pseudotype neutralization experiments, the pseudoviruses were incubated for 30 min at 37 °C with different dilutions of purified VHHs or with dilution series of culture supernatant of 293S cells that had been transfected with plasmids coding for SARS VHH-72 fused to human IgG1 Fc (VHH-72-Fc) or with GFP-binding protein (GBP: a VHH specific for GFP).	293S suggested: RRID:CVCL_A784)
The incubated pseudoviruses were subsequently added to confluent monolayers of Vero E6 cells.	Vero E6 suggested: RRID:CVCL_XD71)
Briefly, suspension-adapted and serum-free HEK 293S cells were seeded at 3 x 106 cells/mL in Freestyle-293 medium (ThermoFisher Scientific).	HEK 293S suggested: RRID:CVCL_A784)
Supernatant of non-transfected and VHH-72-Fc transfected HEK293-S cells was applied in a three-fold dilution series in kinetics buffer.	HEK293-S suggested: RRID:CVCL_A784)
Software and Algorithms
Sentences	Resources
Curve fitting was performed using nonlinear regression (Graphpad 7.0)	Graphpad suggested: (GraphPad, RRID:SCR_000306)
The resulting molecular replacement solutions were iteratively rebuilt and refined using Coot, ISOLDE and Phenix (Adams et al., 2002; Croll, 2018; Emsley and Cowtan, 2004).	Coot suggested: (Coot, RRID:SCR_014222)

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).

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.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

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Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies

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