An antibody targeting the N-terminal domain of SARS-CoV-2 disrupts the spike trimer

Jun 1, 2022

SciScore for 10.1101/2022.01.12.476120: (What is this?)

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

Table 1: Rigor

Ethics	IRB: The study was approved by the Institutional Review Board of Vanderbilt University Medical Center and specimens were obtained after written informed consent. Consent: The study was approved by the Institutional Review Board of Vanderbilt University Medical Center and specimens were obtained after written informed consent. IACUC: The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (assurance number A3381–01).
Sex as a biological variable	Eight to nine week-old mice of both sexes were inoculated with 103 PFU of SARS-CoV-2 by an intranasal route.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	Contamination: Mycoplasma testing of Expi293F and ExpiCHO cultures was performed monthly using a PCR- based mycoplasma detection kit (ATCC, 30-1012K).

Table 2: Resources

Antibodies
Sentences	Resources
In brief, synthesized antibody-encoding DNA (∼2 μg per transfection) was added to OptiPro serum free medium (OptiPro SFM), incubated with ExpiFectamine CHO Reagent and added to 800 µL of ExpiCHO cell cultures into 96-deep-well blocks using a ViaFlo 384 liquid handler (Integra Biosciences).	antibody-encoding DNA suggested: None
Antibody binding was detected with anti-IgG Alexa-Fluor-647-labelled secondary antibodies.	anti-IgG Alexa-Fluor-647-labelled secondary antibodies . suggested: None
The plates were incubated sequentially with 1 μg/mL of rCR3022 anti-S antibody or a murine anti-SARS-CoV-2 mAb, SARS2-16 (hybridoma supernatant diluted 1:6,000 to a final concentration of ∼20 ng/mL) and then HRP-conjugated goat anti-human IgG (Sigma-Aldrich, A6029) in PBS supplemented with 0.1% (w/v) saponin (Sigma) and 0.1% BSA.	anti-S suggested: None anti-SARS-CoV-2 suggested: None anti-human IgG suggested: (Sigma-Aldrich Cat# A6029, RRID:AB_258272)
Serum antibody competition binding ELISAs with biotinylated reference mAbs: mAb COV2-3434 was biotinylated using NHS-PEG4-biotin (Thermo Fisher Scientific, cat# A39259) according to manufacturer protocol.	NHS-PEG4-biotin suggested: None
Competition ELISA of mAbs, related to Figure 4 Competition ELISA of mAbs with previously mapped antibodies COV2-2130, COV2-2196, COV2-2676, COV2-2489, r4A8 or rCR3022.	COV2-2489 suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Cell lines: Vero (ATCC, CCL-81), HEK293 (ATCC, CRL-1573) and HEK293T (ATCC, CRL-3216) cells were maintained at 37°C in 5% CO2 in Dulbecco’s minimal essential medium (DMEM) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS), 10 mM HEPES pH 7.3, 1 mM sodium pyruvate, 1× non-essential amino acids and 100 U/mL of penicillin–streptomycin.	HEK293 suggested: None HEK293T suggested: None
Vero-furin cells were obtained from T. Pierson (NIAID, NIH) and have been described previously (45) Vero-hACE2-TMPRSS2 cells were a gift of A. Creanga and B. Graham (Vaccine Research Center, NIH)	Vero-hACE2-TMPRSS2 suggested: None
The antibody-virus complexes were added to Vero E6 cell-culture monolayers in 96-well plates for 1 h at 37°C.	Vero E6 suggested: None
A suspension of 18,000 Vero-E6 cells in 50 μL of cell culture medium was seeded in each well, and the plate was placed on the analyzer.	Vero-E6 suggested: None
Triplicate wells containing virus only (maximal CPE in the absence of mAb) and wells containing only Vero cells in medium (no-CPE wells) were included as controls.	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
Mice (8 to 9 weeks old) were inoculated with 1 × 104 focus forming units of SARS-CoV-2 (viral titer was determined on Vero-TMPRSS2-ACE2 cells) via the intranasal route.	Vero-TMPRSS2-ACE2 suggested: None
A plasmid encoding cDNA for each spike protein mutant was transfected into HEK-293T cells and allowed to express for 22 h.	HEK-293T suggested: None
Briefly, lung homogenates were serially diluted and added to Vero+TMPRSS2+hACE2 cell monolayers in 12-well plates.	Vero+TMPRSS2+hACE2 suggested: None
B. Neutralization of VSV-S by COV2-3434 was measured in the absence or presence of 25 μM biliverdin in Vero-CCL81 cells.	Vero-CCL81 suggested: None
Experimental Models: Organisms/Strains
Sentences	Resources
Heterozygous K18-hACE c57BL/6J mice (strain: 2B6.Cg-Tg(K18-ACE2)2Prlmn/J) were obtained from Jackson Laboratory (034860).	K18-hACE c57BL/6J suggested: None
Female heterozygous K18-hACE C57BL/6J mice were housed in groups of up to 5 mice per cage at 18 to 24°C ambient temperatures and 40 to 60% humidity.	C57BL/6J suggested: None
Eight-week-old female K18-hACE2 transgenic mice were inoculated by the intranasal route with 104 FFU of SARS-CoV-2 (WA1/2020 D614G).	K18-hACE2 suggested: RRID:IMSR_GPT:T037657)
Software and Algorithms
Sentences	Resources
) sequencing core laboratory at an appropriate target concentration for 10X Genomics library preparation and subsequent sequence analysis.	Genomics suggested: (UTHSCSA Genomics Core, RRID:SCR_012239)
Half maximal inhibitory concentration (IC50) values were determined by nonlinear regression analysis (with a variable slope) using Prism software.	Prism suggested: (PRISM, RRID:SCR_005375)
Briefly, 50 μL of cell culture medium (DMEM supplemented with 2% FBS) was added to each well of a 96-well E-plate using a ViaFlo384 liquid handler (Integra Biosciences) to obtain background reading.	Integra Biosciences suggested: None
Expressed protein was incubate with BioLock (IBA Lifesciences) and then isolated by Strep affinity chromatography on StrepTrap HP columns (GE Healthcare).	BioLock suggested: None
Image processing was performed using the cryoSPARC (Punjani et al., 2017) software package.	cryoSPARC suggested: (cryoSPARC, RRID:SCR_016501)
For each plate, background signal (signal from wells that were not coated with antigen) was subtracted and values were normalized to no-competition controls (signal from wells that had no competing serum or mAb) Four-parameter dose-response/inhibition curves were fit to the normalized data using Prism software (GraphPad) v8.1.1.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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 did not find any issues relating to the usage of bar graphs.

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

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.

Results from scite Reference Check: We found no unreliable references.

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An antibody targeting the N-terminal domain of SARS-CoV-2 disrupts the spike trimer

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