Immunogenicity and efficacy of the COVID-19 candidate vector vaccine MVA SARS 2 S in preclinical vaccination

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   ScreenIT

   Jan 13, 2021

   SciScore for 10.1101/2021.01.09.426032: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: For use of patient serum ethical approval was granted by the Ethics Committee at the Medical Faculty of LMU Munich (vote 20-225 KB) in accordance with the guidelines of the Declaration of Helsinki.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Vaccination experiments in mice: Female BALB/c mice (6 to 10 week-old) were purchased from Charles River Laboratories (Sulzfeld, Germany).
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The blots were blocked in a phosphate buffered saline (PBS) buffer containing 5% Bovine Serum Albumin (BSA) (Sigma-Aldrich, Taufkirchen, Germany) …

   SciScore for 10.1101/2021.01.09.426032: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: For use of patient serum ethical approval was granted by the Ethics Committee at the Medical Faculty of LMU Munich (vote 20-225 KB) in accordance with the guidelines of the Declaration of Helsinki.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Vaccination experiments in mice: Female BALB/c mice (6 to 10 week-old) were purchased from Charles River Laboratories (Sulzfeld, Germany).
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The blots were blocked in a phosphate buffered saline (PBS) buffer containing 5% Bovine Serum Albumin (BSA) (Sigma-Aldrich, Taufkirchen, Germany) and 0.1% Tween-20 (Sigma-Aldrich, Taufkirchen, Germany) and incubated for 60 min with primary antibody, monoclonal anti-HAtag antibody (1:8000; HA Tag mAb 2-2.2.14, Thermo Fisher Scientific, Planegg, Germany) or COVID-19 patient serum (1:200).	anti-HAtag suggested: (InvivoGen Cat# ab-hatag, RRID:AB_391833)
   Permeabilized cells were probed with a monoclonal antibody against the HA-tag epitope (1:1000; HA Tag mAb 2-2.2.14, Thermo Fisher Scientific, Planegg, Germany) to detect SARS-2-S protein.	HA-tag epitope suggested: None
   Non-permeabilized cells were stained with a mouse monoclonal antibody obtained against the S protein of SARS-CoV-1 (SARS-1-S; 1:200; GeneTex) before fixation with PFA.	SARS-1-S suggested: None
   Polyclonal goat anti-mouse secondary antibody (1:1000; Life Technologies, Darmstadt, Germany) was used to visualize S-specific staining by red fluorescence.	anti-mouse suggested: None
   Surrogate virus neutralization assay (sVNT): To test for the presence of neutralizing anti-SARS-CoV-2-S serum antibodies we used surrogate virus neutralization test as described before with slight modifications (25).	anti-SARS-CoV-2-S suggested: None
   Plates were extensively washed with phosphate-buffered saline/0.05% Tween-20 (PBST), followed by incubation for 1h at 37°C with an HRP-conjugated anti-His-tag antibody (1.2 μg/ml; clone HIS 3D5).	anti-His-tag suggested: (MBL International Cat# M136-3, RRID:AB_11125943)
   To remove background effects, the mean percentage of inhibition from non-specific mouse serum (Invitrogen) was deducted from sample values and neutralizing anti-SARS-CoV2-S antibodies titres were determined as serum dilution that still had binding reduction > mean + 2 SD of values from sera of vehicle-treated mice.	anti-SARS-CoV2-S suggested: None
   After incubation, we fixed the cells with 4% formaldehyde/phosphate-buffered saline (PBS) and stained the cells with polyclonal rabbit anti-SARS-CoV antibody (Sino Biological, https://www.sinobiological.com) and a secondary peroxidase-labeled goat anti-rabbit IgG (Dako, https://www.agilent.com).	anti-SARS-CoV suggested: None anti-rabbit IgG suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Human A549 cells (ATCC® CCL-185™) (LGC standards) were maintained in DMEM with high glucose and 10% FBS.	A549 suggested: None
   The replicative capacity of recombinant MVA was tested in multi-step-growth experiments on monolayers of DF-1, HaCat, HeLa or A549 cells grown in 6-well-tissue-culture plates.	HaCat suggested: None HeLa suggested: None
   Western Blot analysis of recombinant protein: To monitor production of the recombinant SARS-2-S protein, DF-1 cells were infected at MOI 10 with recombinant or non-recombinant MVA or remained uninfected (mock).	DF-1 suggested: None
   Immunostaining of recombinant SARS-2-S protein: Vero cells were infected with 0.05 MOI MVA-SARS-2-S or non-recombinant MVA and incubated at 37 °C.	Vero suggested: None
   We then added 50 μL of virus suspension (400 plaque-forming units) to each well and incubated at 37°C for 1 h before placing the mixtures on Vero-E6 cells.	Vero-E6 suggested: None
   Cytopathic effects (CPE) on VeroE6 cells (ATCC CRL1586) were analyzed 4 days after infection.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Vaccination experiments in mice: Female BALB/c mice (6 to 10 week-old) were purchased from Charles River Laboratories (Sulzfeld, Germany).	BALB/c suggested: None
   Software and Algorithms
   Sentences	Resources
   After incubation, we fixed the cells with 4% formaldehyde/phosphate-buffered saline (PBS) and stained the cells with polyclonal rabbit anti-SARS-CoV antibody (Sino Biological, https://www.sinobiological.com) and a secondary peroxidase-labeled goat anti-rabbit IgG (Dako, https://www.agilent.com).	https://www.sinobiological.com suggested: (Sino Biological, RRID:SCR_003697) https://www.agilent.com suggested: (Agilent Technologies, RRID:SCR_013575)
   Next, cells were permeabilized using Intracellular Staining Permeabilization Wash Buffer (Perm Wash buffer; Biolegend; dilution 1:10), and stained intracellularly in 100 μl/well of anti-mouse IFN-γ (clone XMG1.2, 1:200, Biolegend) plus anti-mouse TNF-α (clone MP6-XT22, 1:200, Biolegend) diluted in Perm Wash buffer for 30 min at room temperature.	Biolegend suggested: (BioLegend, RRID:SCR_001134)
   For each antibody, single colour controls were prepared using OneComp eBeads™ Compensation Beads (eBioscience, Thermo Fisher Scientific) and cells for the viability dye Zombie Aqua.	OneComp suggested: None
   Data was acquired by the MACSQuant VYB Flow Analyser (Miltenyi Biotec) and analyzed using FlowJo (FlowJo LLC, BD Life Sciences, Ashland, OR, USA).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Statistical analysis: Data were prepared using GraphPad Prism version 5 (GraphPad Software Inc., San Diego CA, USA) and expressed as mean ± standard error of the mean (SEM).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) GraphPad 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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2. 

   Version published to 10.1101/2021.01.09.426032 on bioRxiv

   Jan 11, 2021