A live-attenuated SARS-CoV-2 vaccine candidate with accessory protein deletions

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1. Version published to 10.1038/s41467-022-31930-z

   Jul 27, 2022
2. 

   ScreenIT

   Feb 17, 2022

   SciScore for 10.1101/2022.02.14.480460: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at UTMB. IRB: The use of human COVID-19 sera was reviewed and approved by the UTMB Institutional Review Board (IRB#: 20-0070).
   Sex as a biological variable	Hamster immunization and challenge assay: Four- to six-week-old male golden Syrian hamsters, strain HsdHan:AURA (Envigo, Indianapolis, IN), were immunized intranasally with 100 µl WT virus (106 PFU, n=20) or Δ3678 mutant virus (106 PFU, n=20)
   Randomization	Statistics: Hamsters and mice were randomly allocated into different groups.
   Blinding	The blinded tissue sections were semi-quantitatively scored for pathological lesions.
   Power Analysis	No statistical …

   SciScore for 10.1101/2022.02.14.480460: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at UTMB. IRB: The use of human COVID-19 sera was reviewed and approved by the UTMB Institutional Review Board (IRB#: 20-0070).
   Sex as a biological variable	Hamster immunization and challenge assay: Four- to six-week-old male golden Syrian hamsters, strain HsdHan:AURA (Envigo, Indianapolis, IN), were immunized intranasally with 100 µl WT virus (106 PFU, n=20) or Δ3678 mutant virus (106 PFU, n=20)
   Randomization	Statistics: Hamsters and mice were randomly allocated into different groups.
   Blinding	The blinded tissue sections were semi-quantitatively scored for pathological lesions.
   Power Analysis	No statistical methods were used to predetermine sample size.
   Cell Line Authentication	Contamination: All cell lines were verified and tested negative for mycoplasma.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Anti-STAT1 (14994S, 1:1,000), anti-pSTAT1 (Y701) (7649S, 1:1,000), anti-STAT2 (72604S, 1:1,000), anti-pSTAT2 (Y690) (88410S, 1:1,000) antibodies were from Cell Signaling Technology (Danvers, MA); anti-GAPDH (G9545, 1:1,000) antibodies were from Sigma-Aldrich; SARS-CoV-2 (COVID-19	Anti-STAT1 suggested: (Cell Signaling Technology Cat# 14994, RRID:AB_2737027) anti-pSTAT1 ( Y701 suggested: None anti-STAT2 suggested: (Cell Signaling Technology Cat# 72604, RRID:AB_2799824) anti-pSTAT2 ( Y690 ) ( 88410S , 1:1,000 ) antibodies were from Cell Signaling Technology ( Danvers , MA) suggested: None anti-GAPDH suggested: (Sigma-Aldrich Cat# G9545, RRID:AB_796208) G9545 suggested: (Sigma-Aldrich Cat# G9545, RRID:AB_796208)
   ) nucleocapsid antibody (NB100-56576, 1:1000) were from Novus Biologicals (CO, USA)	NB100-56576 suggested: (Novus Cat# NB100-56576, RRID:AB_838838)
   Antiviral testing: A549-hACE2 cells were used to evaluate the efficacy of a monoclonal antibody IgG14 and antiviral drug remdesivir.	antiviral drug remdesivir. suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Vero-E6-TMPRSS2 cells were purchased from SEKISUI XenoTech, LLC (Kansas City, KS) and maintained in 10% fetal bovine serum (FBS; HyClone Laboratories, South Logan, UT) and 1% P/S and 1 mg/ml G418 (Gibco).	Vero-E6-TMPRSS2 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Human lung adenocarcinoma epithelial Calu-3 cells (ATCC, Manassas, VA, USA) were maintained in a high-glucose DMEM containing sodium pyruvate and GlutaMAX (Gibco) with 10% FBS and 1% penicillin/streptomycin at 37°C with 5% CO2.	Calu-3 suggested: None
   Suppression of STAT1 phosphorylation by ORF3a protein: A549-hACE2 cells were pre-treated with 1,000 units/ml IFN-α for 6 h.	A549-hACE2 suggested: RRID:CVCL_A5KB)
   The diluted serum was incubated with 100-150 fluorescent focus units (FFU) of mNG SARS-CoV-2 at 37°C for 1 h (final dilution range of 1:20 to 1:20,480), after which the serum-virus mixtures were inoculated onto the pre-seeded Vero-E6 cell monolayer in 96-well plates.	Vero-E6 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   K18-hACE2 mice were purchased from the Jackson Laboratory (Bar Harbor,	K18-hACE2 suggested: RRID:IMSR_GPT:T037657)
   BALB/c mice were purchased from Charles River Laboratories (Wilmington, MA).	BALB/c suggested: RRID:IMSR_ORNL:BALB/cRl)
   Software and Algorithms
   Sentences	Resources
   A nonlinear regression method with log (inhibitor) vs. response-variable slope (four parameters) model (bottom and top parameters were constrained to 0 and 100, respectively) was used to determine the dilution fold that neutralized 50% of mNG SARS-CoV-2 (defined as FFRNT50) in GraphPad Prism 9.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   The curves of the relative infection rates versus the concentration were plotted using Prism 9 (GraphPad).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   Analyses were performed in Prism version 9.0 (GraphPad, San Diego, CA).	Prism suggested: (PRISM, RRID:SCR_005375)

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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: We detected the following sentences addressing limitations in the study:

   One limitation of the current study is that we have not defined the attenuation mechanisms of the ORF 3b, 6, 7b, or 8 deletion, even though they reduced the lung viral loads in the K18- hACE2 mice (Fig. 5b). SARS-CoV-2 ORF8 protein was recently reported to contain a histone mimic that could disrupt chromatin regulation and enhance viral replication45. Truncations or deletions of ORF7b and ORF8 were reported in SARS-CoV-2 clinical isolates46, 47. Future studies are needed to understand the molecular functions of OFR 3b, 6, and 7b proteins. Nevertheless, our results indicate that Δ3678 virus could serve as a live-attenuated vaccine candidate and as an experimental system that can likely be performed at BSL-2 for COVID-19 research and countermeasure development.

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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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3. 

   Version published to 10.1101/2022.02.14.480460v1 on bioRxiv

   Feb 15, 2022