A Highly Immunogenic Measles Virus-based Th1-biased COVID-19 Vaccine

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   ScreenIT

   Jul 12, 2020

   SciScore for 10.1101/2020.07.11.198291: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	For the experiments, animals were randomized for age- and sex-matched groups and housed in IVC cages in groups of 3 to 5 animals with nist packs as environmental enrichment at room temperature with regular 12 h day and night intervalls.
   Blinding	not detected.
   Power Analysis	Group sizes were calculated based on statistical considerations to yield sufficient statistical power as authorized by the respective competent authority.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The cells were then probed for 1 h with a polyclonal rabbit anti-SARS-CoV-2-S protein antibody …

   SciScore for 10.1101/2020.07.11.198291: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	For the experiments, animals were randomized for age- and sex-matched groups and housed in IVC cages in groups of 3 to 5 animals with nist packs as environmental enrichment at room temperature with regular 12 h day and night intervalls.
   Blinding	not detected.
   Power Analysis	Group sizes were calculated based on statistical considerations to yield sufficient statistical power as authorized by the respective competent authority.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The cells were then probed for 1 h with a polyclonal rabbit anti-SARS-CoV-2-S protein antibody (1:2,250; ab252690; Abcam, Cambridge, UK) or a rabbit anti-MeV N protein antibody (1:1,000, ab23974, Abcam) in PBS with 2% BSA.	anti-SARS-CoV-2-S protein suggested: None anti-MeV N protein suggested: None
   The cells were washed 3 times with 1 ml PBS and subsequently incubated with the secondary HRP-coupled donkey anti-rabbit IgG(H+L) polyclonal antibody (1:1,000; 611-7202; Rockland, Gilbertsville, USA) for 1 h at 37°C.	anti-rabbit IgG(H+L suggested: (Rockland Cat# 611-7202, RRID:AB_219746)
   Rabbit anti-SARS-S protein antibody (1:3,000; ab252690; Abcam), rabbit anti-MeV-N protein polyclonal antibody (1:5,000; ab23974; Abcam), and a mouse anti-ß-actin antibody (1:5,000; ab6276; Abcam) were used.	anti-SARS-S protein suggested: None anti-MeV-N suggested: None anti-ß-actin suggested: (Abcam Cat# ab6276, RRID:AB_2223210)
   Donkey anti-rabbit IgG-HRP (H&L) polyclonal antibody (1:10,000; 611-7202; Rockland) and goat anti-mouse IgG-HRP (1:10,000; A2554-1ML; Merck, Darmstadt, Germany) served as secondary antibodies.	anti-rabbit IgG-HRP suggested: None anti-mouse IgG-HRP suggested: None
   IFN-γ ELISpot Analysis: Murine interferon gamma (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assays were performed using the Mouse IFN-γ ELISPOT Pair kit including capture and detection antibody (BD Bioscience, Franklin Lakes, NJ, USA) and HRP Streptavidin (BD Bioscience) for ELISpot detection in combination with multiscreen immunoprecipitation (IP) ELISpot polyvinylidene difluoride (PVDF) 96-well plates (Merck Millipore, Darmstadt, Germany) according to the manufacturer’s instructions.	IFN-γ suggested: None
   Stimulated cells were subsequently stained with CD3-PacBlue (1:50; clone 500A2; Invitrogen Life Technologies), CD8-APC (1:100; clone 53-6.7; ebioscience) and CD4-PE (1:2000; Cat. 553049; BD) antibodies and fixed with 1% PFA in PBS.	CD3-PacBlue suggested: None CD8-APC suggested: None CD4-PE suggested: (BD Biosciences Cat# 553049, RRID:AB_394585)
   Experimental Models: Cell Lines
   Sentences	Resources
   Cells: Vero (African green monkey kidney) (ATCC# CCL-81), Vero clone E6 (ATCC# CRL-1586), 293T (ATCC CRL-3216) and EL-4 (ATCC TIB-39) cell lines were purchased from ATCC (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Biowest, Nuaillé, France) supplemented with 10% fetal bovine serum (FBS; Biochrom, Berlin, Germany) and 2 mM L-glutamine (L-Gln; Biochrom).	Vero clone E6 suggested: None 293T suggested: None
   Subsequent purification by filtration and ultracentrifugation of supernatants yielded virus stocks were used to transduce murine DC cell lines, DC2.4 and JAWSII, as well as the murine T cell line EL-4, resulting in DC2.4-SARS2-S, JAWSII-SARS2-S, and EL-4green-SARS2-S, respectively, that express the SARS-CoV-2 S protein and GFP and present the respective peptides via MHC-I.	EL-4 suggested: None
   Single syncytia were picked and overlaid onto 50% confluent Vero cells cultured in 6-well plates and harvested as “passage 0” (P0) by scraping and freeze-thaw cycle of cells at the time of maximal infection.	Vero suggested: None
   It was propagated on Vero E6 cells and was titrated via TCID50 as described above for recombinant MeV.	Vero E6 suggested: None
   200 μL Medium containing 10 µg/ml Concanavalin A (Con A, Sigma-Aldrich), 10 μg/ml MeV bulk antigen (Virion Serion), or 5×103 DC2.4-SARS2-S cells were added to each well, and cultured for 6 days.	DC2.4-SARS2-S suggested: None
   CTL killing assay: For re-stimulation of T cells isolated 3 weeks after the second immunization, 5×106 splenocytes were co-cultured with 5×104 DC2.4-SARS2-S cells for 6 days in 12-wells in RPMI 1640 supplemented with 10% FBS, 2 nM L-Glutamin, 1 mM HEPES, 1% penicillin/streptomycin, 100 μM 2-mercaptoethanol, and 100 U/ml murine rIL-2 (Peprotech, Hamburg, Germany). 5×103 EL-4red cells were labeled with 0.5 µM CFSE and mixed with 5×103 EL-4green-SARS2-S cells per well.	EL-4red suggested: None EL-4green-SARS2-S suggested: None
   Software and Algorithms
   Sentences	Resources
   NGS library preparation and sequencing: Total RNA was isolated from Vero cells after 4 days post infection using the Direct-zol RNA isolation kit (Zymo Research).	NGS suggested: (PM4NGS, RRID:SCR_019164)
   Mapping was performed with BWA mem v 0.7.12-r1039 (43), using default parameters unless stated otherwise.	BWA suggested: (BWA, RRID:SCR_010910)
   Host-derived reads were removed by mapping quality controlled reads against the African green monkey genome (Chlorocebus sabeus, RefSeq assembly GCA_000409795.2), specifying the minimum seed length (-k 31).	RefSeq suggested: (RefSeq, RRID:SCR_003496)
   Unmapped reads were extracted using samtools v1.7 (44) and bamToFastq v2.17.0 (45), and subsequently mapped to the plasmid reference genomes of either MeVvac2-SARS2-S(H) or MeVvac2-SARS2-S(P), as appropriate.	samtools suggested: (SAMTOOLS, RRID:SCR_002105)
   Host-free alignments were deduplicated using picard-tools MarkDuplicates (http://broadinstitute.github.io/picard) and left-aligned using GATK LeftAlignIndels v4.0 (46).	GATK suggested: (GATK, RRID:SCR_001876)
   Variant calling was performed with LoFreq v2.1.3 (47) using default parameters.	LoFreq suggested: (LoFreq, RRID:SCR_013054)

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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: Please consider improving the rainbow (“jet”) colormap(s) used on page 39. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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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 10.1101/2020.07.11.198291v1 on bioRxiv

   Jul 11, 2020