Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response

Article activity feed

1. 

   ScreenIT

   Jul 2, 2020

   SciScore for 10.1101/2020.06.27.175166: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: All animal experiments were reviewed and approved by the Animal Care and Use Committee of the Emory University.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Briefly, 6–8-week-old female BALB/c mice (n=5) were immunized with 10^7 plaque-forming-units (pfu) of rMVA/S or rMVA/S1 by intramuscular (i.m.) route on weeks 0 and 3.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The expression of spike on the MVA-infected cells from MVA/S construct was determined using anti-SARS-CoV-2 spike antibody (Cat #135356, GeneTex).	anti-SARS-CoV-2 suggested: None
   For MVA/S1, anti …

   SciScore for 10.1101/2020.06.27.175166: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: All animal experiments were reviewed and approved by the Animal Care and Use Committee of the Emory University.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Briefly, 6–8-week-old female BALB/c mice (n=5) were immunized with 10^7 plaque-forming-units (pfu) of rMVA/S or rMVA/S1 by intramuscular (i.m.) route on weeks 0 and 3.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The expression of spike on the MVA-infected cells from MVA/S construct was determined using anti-SARS-CoV-2 spike antibody (Cat #135356, GeneTex).	anti-SARS-CoV-2 suggested: None
   For MVA/S1, anti SARS-CoV-2 RBD antibody (Cat# 40592-T62, Sino Biological) was used to stain intracellularly.	anti SARS-CoV-2 RBD suggested: None
   MVA/S infected cells were harvested, stained with live dead marker and anti SARS-CoV-2 spike antibody (#GTX135356, GeneTex) followed by donkey anti-rabbit IgG coupled to PE (# 406421, BioLegend) on the surface.	anti SARS-CoV-2 suggested: None anti-rabbit IgG suggested: (BioLegend Cat# 406421, RRID:AB_2563484)
   The membrane was washed in PBS containing Tween-20 (0.05%) and was incubated for 1 h with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (Southern Biotech) diluted 1:20,000 accordingly.	anti-mouse suggested: None anti-rabbit suggested: None
   Plates were then incubated with serial dilutions of mouse sera for 2h at RT, washed 6 times and then incubated with 1: 6000 dilution of horseradish peroxidase (HRP) conjugated anti-mouse IgG secondary antibody (Southern Biotech; Birmingham, AL, USA) for 1 h at RT.	anti-mouse IgG suggested: None
   For investigating the formation of iBALT B cell and T cells were stained in lungs using primary antibody cocktail containing rat anti-mouse CD45R/B220 (BioLegend, Cat#103202) and Hamster anti mouse CD3 (BD, Cat#550277) antibodies.	anti-mouse CD45R/B220 suggested: (Fluidigm Cat# 3144011B, RRID:AB_2811239) anti mouse CD3 suggested: None
   Secondary antibodies cocktail contained goat antirat IgG-Alexa488 (abcam, Cat#ab150157) and goat anti-hamster IgG-Alexa546 (Thermofisher, Cat#A21111).	antirat IgG-Alexa488 suggested: None anti-hamster IgG-Alexa546 suggested: None
   Systems serology by Luminex analysis: For relative quantification of antigen-specific antibody titers, a customized multiplexed approach was applied, as previously described (30).	antigen-specific suggested: None
   Anti-mouse IgG-, IgG2a-, IgG3-, IgA- and IgM-PE coupled (Southern Biotech) detection antibodies were diluted in Luminex assay buffer to 0.65ug/ml.	Anti-mouse IgG- suggested: None
   Following washing of stained immune complexes, a tertiary goat anti-mouse IgG-PE antibody (Southern Biotech) was added and incubated for 1h at RT on a shaker.	anti-mouse IgG-PE suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Similarly human ACE2 binding to surface expressed spike on MVA/S infected DF-1 cells was done using biotinylated human ACE2 protein (#10108-H08H-B, Sino Biological) followed by streptavidin-PE (BD Pharmingen) and intracellularly for MVA as described earlier.	DF-1 suggested: None
   Protein expression and purification: RBD-His and S1 proteins were produced in Amara laboratory by transfecting HEK293 cells using plasmids pCAGGS-RBD-His and pGA8-S1, respectively.	HEK293 suggested: None
   Briefly, HEK 293F cells were seeded at a density of 2×106 cells/ml in Expi293 expression medium and incubated in an orbital shaking incubator at 37°C and 127 rpm with 8% CO2 overnight.	HEK 293F suggested: RRID:CVCL_6642)
   The stock virus was passaged twice (P2) and viral titers were determined by plaque assay on Vero E6 cells (ATCC).	Vero E6 suggested: RRID:CVCL_XD71)
   Vero cells were cultured in complete DMEM medium consisting of 1x DMEM (Corning Cellgro), 10% FBS, 25mM HEPES Buffer (Corning Cellgro), 2mM L-glutamine, 1mM sodium pyruvate, 1x Non-essential Amino Acids, and 1x antibiotics.	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Briefly, 6–8-week-old female BALB/c mice (n=5) were immunized with 10^7 plaque-forming-units (pfu) of rMVA/S or rMVA/S1 by intramuscular (i.m.) route on weeks 0 and 3.	BALB/c suggested: None
   Software and Algorithms
   Sentences	Resources
   Briefly, Nunc high-binding ELISA plates (Thermo Fisher Scientific; Waltham, MA, USA) were coated with 2 μg/ml of recombinant SARS-CoV-2 proteins (S1 and RBD-His proteins were produced in the lab and S1 +S2 ECD spike protein was purchased from Sino Biologicals) in Dulbecco’s phosphate-buffered saline (DPBS) and incubated overnight at 4 °C.	Thermo Fisher Scientific suggested: (Thermo Fisher Scientific, RRID:SCR_008452)
   FRNT50 curves were generated by non-linear regression analysis using the 4PL sigmoidal dose curve equation on Prism 8 (Graphpad Software).	Graphpad suggested: (GraphPad, RRID:SCR_000306)
   The slides were imaged by Olympus FV1000 confocal imaging using 20X objective and images were analyzed using ImageJ.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Data analysis and statistical procedures: GraphPad Prism v7.0 was used to perform data analysis and statistics.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

   ---

   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).

   ---

   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.

   ---

   Results from TrialIdentifier: No clinical trial numbers were referenced.

   ---

   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

   ---

   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 17. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

   ---

   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.

   ---

   Read the original source
2. 

   Version published to 10.1101/2020.06.27.175166v1 on bioRxiv

   Jun 27, 2020