A SARS-CoV-2 Spike Ferritin Nanoparticle Vaccine Is Protective and Promotes a Strong Immunological Response in the Cynomolgus Macaque Coronavirus Disease 2019 (COVID-19) Model

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1. Version published to 10.3390/vaccines10050717

   May 4, 2022
2. 

   ScreenIT

   Mar 30, 2022

   SciScore for 10.1101/2022.03.25.485832: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: Animals: Animal research was conducted at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). IACUC: Ethics statement: These experiments and procedures were reviewed and approved by the United States Army Medical Research Institute for Infectious Diseases Institutional Animal Care and Use Committee (IACUC).
   Sex as a biological variable	Genders were mixed male and female, and all animals were SARS-CoV-2 naïve at the outset of the study.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: R4719 was determined to have no detectable mycoplasma, endotoxin or adventitious agents based on …

   SciScore for 10.1101/2022.03.25.485832: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: Animals: Animal research was conducted at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). IACUC: Ethics statement: These experiments and procedures were reviewed and approved by the United States Army Medical Research Institute for Infectious Diseases Institutional Animal Care and Use Committee (IACUC).
   Sex as a biological variable	Genders were mixed male and female, and all animals were SARS-CoV-2 naïve at the outset of the study.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: R4719 was determined to have no detectable mycoplasma, endotoxin or adventitious agents based on the assays and techniques used.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Serum binding and ACE-2 inhibitory antibody assessment: SARS-CoV-2-specific binding IgG antibody responses were measured using MULTI-SPOT® 96-well plates, V-PLEX SARS-CoV-2 Panel 7 Kit (Meso Scale Discovery (MSD), Rockville, MD).	SARS-CoV-2-specific binding IgG suggested: None
   MSD SULFO-TAG™ conjugated anti-IgG antibody was added to each well.	anti-IgG suggested: None
   Stimulations consisted of two pools of peptides spanning the S protein of SARS-CoV-2 or SARS-CoV-1 (1 µg/mL, JPT, PM-WCPV-S and PM-CVHSA-S respectively) in the presence of Brefeldin A (0.65 µL/mL, GolgiPlug™, BD Cytofix/Cytoperm Kit, Cat. 555028), co-stimulatory antibodies anti-CD28 (BD Biosciences Cat.	SARS-CoV-1 (1 suggested: None
   Following stimulation, cells were stained serially with LIVE/DEAD Fixable Blue Dead Cell Stain (ThermoFisher #L23105) and a cocktail of fluorescent-labeled antibodies (BD Biosciences unless otherwise indicated) to cell surface markers CD4-PE-Cy5.5 (S3.5, ThermoFisher #MHCD0418, Lot 2118390 and 2247858), CD8-BV570 (RPA-T8, BioLegend #301038, Lot B281322), CD45RA BUV395 (5H9, #552888, Lot 154382 and 259854), CD28 BUV737 (CD28.2, #612815, Lot 0113886), CCR7-BV650 (GO43H7, # 353234, Lot B297645 and B316676) and HLA-DR-BV480 (G46-6, # 566113, Lot 0055314).	CD8-BV570 suggested: None RPA-T8 suggested: (BD Biosciences Cat# 563795, RRID:AB_2722501) CD45RA suggested: (BD Biosciences Cat# 552888, RRID:AB_394517) CD28 suggested: (BD Biosciences Cat# 612815, RRID:AB_2870140) CCR7-BV650 suggested: None B316676 suggested: None G46-6 suggested: (BD Biosciences Cat# 566113, RRID:AB_2739515)
   Experimental Models: Cell Lines
   Sentences	Resources
   Infectivity and neutralization titers were determined using ACE2-expressing HEK293 target cells (Integral Molecular, Philadelphia, PA) in a semi-automated assay format using robotic liquid handling (Biomek NXp Beckman Coulter, Brea, CA).	HEK293 suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
   Briefly, biotinylated SARS-CoV-2 Spike trimer (Hexapro) was incubated with red streptavidin-fluorescent beads (Molecular Probes, Eugene, OR) for 2h at 37°C. 10 μl of a 100-fold dilution of beads–protein was incubated 2h at 37°C with 100μl 900-fold diluted plasma samples before addition of THP-1 cells (25,000 cells per well; Millipore Sigma, Burlington, MA).	THP-1 suggested: CLS Cat# 300356/p804_THP-1, RRID:CVCL_0006)
   293F-Spike-S2-WT cells were incubated with 10-fold diluted heat-inactivated (56°C for 30 min) plasma samples for 30 min at 37°C.	293F-Spike-S2-WT suggested: None
   Recombinant DNA
   Sentences	Resources
   SARS-CoV-2 pseudovirions (PSV) were produced by co-transfection of HEK293T/17 cells with a SARS-CoV-2 S plasmid (pcDNA3.4), derived from the Wuhan-Hu-1 genome sequence (GenBank accession number: MN908947.3) and an HIV-1 (pNL4-3.Luc.R-E-, NIH HIV Reagent Program, Catalog number 3418).	pcDNA3.4 suggested: None pNL4-3.Luc.R-E- suggested: None
   Briefly, SARS-CoV-2 Spike-expressing 293 FreeStyle (293F) cells were generated by transfection with linearized plasmid (pcDNA3.1) encoding codon-optimized full-length SARS-CoV-2 Spike protein matching the amino acid sequence of the IL-CDC-IL1/2020 isolate (GenBank ACC# MN988713).	pcDNA3.1 suggested: RRID:Addgene_79663)
   Software and Algorithms
   Sentences	Resources
   Reduced data in the NSS files was extracted into Microsoft Excel workbooks using Notocord-derived formula add-ins, and the 30-minute (min) averages were calculated for each parameter for each subject.	Microsoft Excel suggested: (Microsoft Excel, RRID:SCR_016137)
   Assay equivalency was established by participation in the SARS-CoV-2 Neutralizing Assay Concordance Survey (SNACS) run by the Virology Quality Assurance Program and External Quality Assurance Program Oversite Laboratory (EQAPOL) at the Duke Human Vaccine Institute, sponsored through programs supported by the National Institute of Allergy and Infectious Diseases, Division of AIDS.	Quality Assurance Program suggested: None Quality Assurance Program Oversite Laboratory suggested: None
   Sample staining was measured on a FACSymphony(tm) A5 SORP (Becton Dickenson) and data was analyzed using FlowJo v.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Display of multicomponent distributions were performed with SPICE v6.0 (NIH, Bethesda, MD).	SPICE suggested: (SPICE, RRID:SCR_016603)

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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
   NCT04784767	Active, not recruiting	SARS-COV-2-Spike-Ferritin-Nanoparticle (SpFN) Vaccine With A…

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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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3. 

   Version published to 10.1101/2022.03.25.485832v1 on bioRxiv

   Mar 28, 2022