Potent neutralizing anti-SARS-CoV-2 human antibodies cure infection with SARS-CoV-2 variants in hamster model

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1. Version published to 10.1016/j.isci.2022.104705

   Aug 1, 2022
2. 

   ScreenIT

   Dec 1, 2021

   SciScore for 10.1101/2021.11.25.470011: (What is this?)

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

   Table 1: Rigor

   Ethics

   IRB: The study and corresponding experiments were approved by the local ethics committee (S64089) and all patients gave their written informed consent. Consent: The study and corresponding experiments were approved by the local ethics committee (S64089) and all patients gave their written informed consent. Euthanasia Agents: At day 4 post-infection, animals were euthanized by intraperitoneal injection of 500 μL Dolethal (200 mg/mL sodium pentobarbital, Vétoquinol SA).

   Sex as a biological variable

   Female Syrian hamsters (Mesocricetus auratus) were purchased from Janvier Laboratories and kept per two in individually ventilated isolator cages (IsoCage N Bio-containment System, Tecniplast) at 21°C, …

   SciScore for 10.1101/2021.11.25.470011: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: The study and corresponding experiments were approved by the local ethics committee (S64089) and all patients gave their written informed consent. Consent: The study and corresponding experiments were approved by the local ethics committee (S64089) and all patients gave their written informed consent. Euthanasia Agents: At day 4 post-infection, animals were euthanized by intraperitoneal injection of 500 μL Dolethal (200 mg/mL sodium pentobarbital, Vétoquinol SA).
   Sex as a biological variable	Female Syrian hamsters (Mesocricetus auratus) were purchased from Janvier Laboratories and kept per two in individually ventilated isolator cages (IsoCage N Bio-containment System, Tecniplast) at 21°C, 55% humidity and 12:12 day/night cycles.
   Randomization	No randomization methods were used and confounders were not controlled, though all caretakers and technicians were blinded to group allocation in the animal facility and to sample numbers for analysis (qPCR, titration, and histology).
   Blinding	No randomization methods were used and confounders were not controlled, though all caretakers and technicians were blinded to group allocation in the animal facility and to sample numbers for analysis (qPCR, titration, and histology).
   Power Analysis	Group size was calculated based on the independent t-test with an effect size of 2.0 and a power of 80% (effect size = delta mean/SD = 1 log10 decrease in viral RNA/0.5 log10), resulting in 5-6 animals/group.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   His-tag labeled SARS-CoV-2 RBD (The Native Antigen Company) was biotinylated with the EZ-Link Sulfo-NHS-LC-Biotin kit (Thermofisher Scientific) according to the manufacturer’s protocol, corresponding to 1-3 biotin groups per antibody molecule.	Sulfo-NHS-LC-Biotin suggested: None
   After the 60 minutes incubation, B cells were washed with FACS buffer and stained with PerCP-cy5.5 anti-human CD19 antibody (Biolegend, 363016)	anti-human CD19 suggested: (BioLegend Cat# 363016, RRID:AB_2564207)
   FITC anti-human CD3 antibody (Biolegend, 300306) and PE streptavidin (Biolegend, 405203) for 25 minutes on ice.	anti-human CD3 suggested: (BioLegend Cat# 300306, RRID:AB_314042)
   As a positive and negative control, an anti-SARS-CoV-2 RBD mAb (40150-D004, Sino Biological) and anti-SARS-CoV-2 nucleocapsid antibody (MBS2563841, MyBioSource) were used, respectively.	anti-SARS-CoV-2 suggested: None anti-SARS-CoV-2 nucleocapsid antibody ( MBS2563841 suggested: None
   First, mouse anti-human IgG (Fc) antibody (Human Antibody Capture Kit, Cytiva) was immobilized on a CM5 chip according to manufacturer instructions.	anti-human IgG suggested: None
   Monoclonal antibody neutralization assays: a. Production of S-pseudotyped virus and serum neutralization test (SARS2, SARS1, MERS, 229E): VSV S-pseudotypes were generated as described previously56.	SARS2 suggested: None
   Two hours later, the medium was replaced by medium containing anti-VSV-G antibody (I1-hybridoma, ATCC CRL-2700) to neutralize residual VSV-G input.	anti-VSV-G suggested: None
   In brief, serial dilutions of antibodies were mixed separately with live SARS-CoV-2 Wuhan, alpha, beta and gamma virus strains, incubated at 37 °C for 1h, and added to the monolayer of Vero E6 cells.	1h suggested: None
   Antibody protein treatments (anti-SARS-CoV-2 mAbs or human IgG1 isotype control Trastuzumab/Herceptin® (Roche)) were initiated 24 hours post infection by intraperitoneal injection.	human IgG1 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Briefly, HEK-293T cells (SARS-CoV, SARS-CoV-2 and MERS-S) or BHK-21J cells (229E) were transfected with the respective S protein expression plasmids, and one day later infected (MOI = 2) with GFP-encoding VSVΔG backbone virus (purchased from Kerafast).	HEK-293T suggested: None BHK-21J suggested: None
   To quantify nAbs, serial dilutions of serum samples were incubated for 1 hour at 37 °C with an equal volume of S pseudotyped VSV particles and inoculated on Vero E6 cells (SARS-CoV and SARS-CoV-2) or Huh-7 cells (229E and MERS-S) for 18 hours.	Huh-7 suggested: None
   Shortly, 104 VeroE6 cells/well were seeded in 96-well plates one day prior to the titration and inoculated with 10–fold serial dilutions of virus solutions and cultured for 3 days at 37°C.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Infectious virus was isolated by serial passaging on Huh7 and Vero E6 cells58; passage 6 virus was used for the study described here.	Huh7 suggested: None
   Recombinant DNA
   Sentences	Resources
   Intramuscular pDNA electroporation: 3B8 was delivered in vivo, encoded in the CMV-driven pcDNA3.4 vectors, as an equimolar mixture of the 3B8 heavy and light chain plasmids (jointly referred to as ‘p3B8’).	pcDNA3.4 suggested: RRID:Addgene_131198)
   Software and Algorithms
   Sentences	Resources
   Analysis was performed using Graphpad Prism 9.0 (Graphpad Software). b.	Graphpad suggested: (GraphPad, RRID:SCR_000306)
   Epitope binning graphs were made in Microsoft Excel and clustering was done with ClustVis web tool (	Microsoft Excel suggested: (Microsoft Excel, RRID:SCR_016137) ClustVis suggested: (ClustVis, RRID:SCR_017133)
   Neutralization IC50 values were determined by normalizing the serum neutralization dilution curve to a virus (100%) and cell control (0%) and fitting in Graphpad Prism (GraphPad Software, Inc.). b.	Graphpad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   Both strains were subjected to sequencing on a MinION platform (Oxford pore) directly from the nasopharyngeal swabs	MinION suggested: (MinION, RRID:SCR_017985)

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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: We found the following clinical trial numbers in your paper:
   

   Identifier	Status	Title
   NCT03831503	Active, not recruiting	A Study of INO-A002 in Healthy Dengue Virus-naive Adults

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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 10.1101/2021.11.25.470011v1 on bioRxiv

   Nov 29, 2021