Prime-boost vaccinations with two serologically distinct chimpanzee adenovirus vectors expressing SARS-CoV-2 spike or nucleocapsid tested in a hamster COVID-19 model

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   ScreenIT

   Mar 21, 2022

   SciScore for 10.1101/2022.03.17.484786: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Antibody Assays: ELISA for anti-S1 and anti-S2 antibodies: Sera from individual hamsters were tested for S-specific antibodies by ELISA on plates coated overnight with 100 µl of a mixture of S1 and S2 (Native Antigen Company, Kidlington, UK) each diluted to 1 µg/ml in bicarbonate buffer.	anti-S1 suggested: None anti-S2 suggested: None
   An anti-hamster IgG (H+L)-Alkaline Phosphatase antibody produced in goat (Sigma-Aldrich, St Louis, MO) diluted to 1:1000 in 3% BAS-PBS was added at 60 …

   SciScore for 10.1101/2022.03.17.484786: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Antibody Assays: ELISA for anti-S1 and anti-S2 antibodies: Sera from individual hamsters were tested for S-specific antibodies by ELISA on plates coated overnight with 100 µl of a mixture of S1 and S2 (Native Antigen Company, Kidlington, UK) each diluted to 1 µg/ml in bicarbonate buffer.	anti-S1 suggested: None anti-S2 suggested: None
   An anti-hamster IgG (H+L)-Alkaline Phosphatase antibody produced in goat (Sigma-Aldrich, St Louis, MO) diluted to 1:1000 in 3% BAS-PBS was added at 60 µl/well for 1 hour at room temperature.	An anti-hamster IgG suggested: None anti-hamster IgG suggested: None H+L)-Alkaline Phosphatase suggested: None
   ELISA for RBD-binding antibodies: Sera were tested for inhibition of ACE binding to the RBD of the S protein by the Anti-SARS-CoV-2 Neutralizing Antibody Titer Serologic Assay Kit from Acro Biosystems (Newark, DE) following the manufacturer’s instructions.	Anti-SARS-CoV-2 suggested: None
   A positive standard with a known concentration provided by the kit was used to extrapolated anti-S antibody concentrations into µg per ml of serum.	anti-S suggested: None
   The VSV-S vector was diluted in medium containing 1 µg/ml of a mouse monoclonal antibody against VSV glycoprotein (sc-365019, Santa Crusz Biotechnology, Dallas, TX) and then incubated with the serum dilutions for 90 min at room temperature under gentle agitation starting with a serum dilution of 1 in 40.	antibody against VSV glycoprotein (sc-365019, Santa Crusz Biotechnology, Dallas, TX) suggested: None
   A monoclonal antibody to N protein (Abbexa, MCA6373, lot# 156962) served as a positive control (data not shown).	MCA6373 suggested: None
   Cells were then incubated with an anti-INF-γ-FITC antibody (clone XMG1.2 BioLegend, San Diego, CA) at 4°C for 30 min in the dark.	anti-INF-γ-FITC suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell lines: HEK 293 cells were grown in Dulbecco’s Modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics.	HEK 293 suggested: None
   Challenge virus: Challenge was conducted with the WT WAS stock, which was generated from seed stock (2019-nCoV/USA-WA1/2020), obtained from Biodefense and Emerging Infections Research (BEI) resources (Cat # NR-52281, Lot # 70036318) and expanded in Calu-3 cells at 37°C for 3 days.	Calu-3 suggested: None
   The stock (Lot # 12152020-1235) titers in Vero TMPRSS2 cells are 2.34×109 Median Tissue Culture Infectious Dose (TCID50)/mL and 3.68 × 107 plaque forming units (PFU)/mL, and 6 × 105 PFU/mL in Vero76 cells.	Vero TMPRSS2 suggested: None Vero76 suggested: JCRB Cat# IFO50410, RRID:CVCL_0603)
   Generation of VSV-S vectors: VSV vectors pseudotyped with S of SARS-CoV-2 were generated in BHK-21/WI-2 cells using a the ΔG-GFP (G*ΔG-GFP) rVSV kit (Kerafast, Boston, MA, USA) and S sequences cloned into an expression plasmid under the control of the CMV promoter as described previously [51].	BHK-21/WI-2 suggested: RRID:CVCL_HB78)
   A549A/T cells were plated into Terasaki plate wells as described above.	A549A/T suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   A549-hACE-TMPRSS2 (A549A/T) cells (InvivoGen, cat no. a549-hace2tpsa) were maintained in DMEM with 10%FBS, 300µg/ml of hygromycin, 0.5 µg/ml of puromycin and antibiotics.	A549-hACE-TMPRSS2 (A549A/T suggested: None
   Recombinant DNA
   Sentences	Resources
   For the neutralization assay a dose of VSV-S vector that infected ∼20-40 cells/well was selected.	VSV-S suggested: None
   Software and Algorithms
   Sentences	Resources
   Post-acquisition analyses were performed with FlowJo (TreeStar, Ashland, OR).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   The analyses were carried out by GraphPad Prism.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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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: 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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   Version published to 10.1101/2022.03.17.484786v1 on bioRxiv

   Mar 18, 2022