A booster dose enhances immunogenicity of the COVID-19 vaccine candidate ChAdOx1 nCoV-19 in aged mice

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1. 

   ScreenIT

   Oct 30, 2020

   SciScore for 10.1101/2020.10.27.357426: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: All mouse experimentation was approved by the Babraham Institute Animal Welfare and Ethical Review Body.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Following three wash steps with PBS-T, the slides were stained with primary antibody mix, which included AF647-conjugated rat anti-mouse IgD (clone 11-26 c.2a, Biolegend; 1:200), FITC-conjugated rat anti-mouse Ki67 (clone SolA15, Invitrogen; 1:100), rat antimouse biotin-conjugated CD21/35 (clone 8D9, ThermoFisher Scientific; 1:400) and hamster antimouse CD3ε …

   SciScore for 10.1101/2020.10.27.357426: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: All mouse experimentation was approved by the Babraham Institute Animal Welfare and Ethical Review Body.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Following three wash steps with PBS-T, the slides were stained with primary antibody mix, which included AF647-conjugated rat anti-mouse IgD (clone 11-26 c.2a, Biolegend; 1:200), FITC-conjugated rat anti-mouse Ki67 (clone SolA15, Invitrogen; 1:100), rat antimouse biotin-conjugated CD21/35 (clone 8D9, ThermoFisher Scientific; 1:400) and hamster antimouse CD3ε (clone 500A2, ThermoFisher Scientific; 1:200), in PBS-T containing 1% BSA at 4°C overnight.	anti-mouse IgD suggested: None anti-mouse suggested: None antimouse biotin-conjugated CD21/35 suggested: None antimouse CD3ε suggested: None
   The next day, slides were washed three times with PBS-T and incubated with secondary antibody mix, which included AF568-conjugated goat anti-hamster IgG (Thermofisher, 1:1000) and BV421-conjugated Streptavidin (Biolegend, 1:1000), in PBS-T containing 2% goat serum for 2hr at room temperature.	anti-hamster IgG suggested: None
   Following washing, bound antibodies were detected by addition of AP-conjugated goat anti-mouse IgG (Sigma-Aldrich) for 1h at RT or addition of AP-conjugated goat anti-mouse IgM or IgA (Abcam and Sigma-Aldrich, respectively) and addition of pNPP substrate (Sigma-Aldrich).	anti-mouse IgG suggested: None anti-mouse IgM suggested: None IgA suggested: None
   In addition, all serum samples were diluted to 1 total IgG ELISA unit and then detected with anti-mouse IgG subclass-specific secondary antibodies (Southern Biotech or Abcam).	anti-mouse IgG subclass-specific secondary antibodies (Southern Biotech or Abcam). suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Target HEK293T cells, previously transfected with 500 ng of a human ACE2 expression plasmid (Addgene, Cambridge, MA, USA) were seeded at a density of 2 × 104 in 100 μL DMEM-10% in a white flat-bottomed 96-well plate one day prior to harvesting SARS-CoV-2 pps.	HEK293T suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Mouse housing and husbandry: C57BL/6Babr mice were bred, aged and maintained in the Babraham Institute Biological Support Unit (BSU).	C57BL/6Babr suggested: None
   Software and Algorithms
   Sentences	Resources
   Manual gating of flow cytometry data was done using FlowJo v10 software (Tree Star). tSNE, FlowSOM and heatmap analysis were performed on aLN samples from day 7 postvaccination using R (version 4.0.2) using code that has previously been described(63).	FlowJo suggested: (FlowJo, RRID:SCR_008520) FlowSOM suggested: (FlowSOM, RRID:SCR_016899)
   Following three wash steps with PBS-T, the slides were stained with primary antibody mix, which included AF647-conjugated rat anti-mouse IgD (clone 11-26 c.2a, Biolegend; 1:200), FITC-conjugated rat anti-mouse Ki67 (clone SolA15, Invitrogen; 1:100), rat antimouse biotin-conjugated CD21/35 (clone 8D9, ThermoFisher Scientific; 1:400) and hamster antimouse CD3ε (clone 500A2, ThermoFisher Scientific; 1:200), in PBS-T containing 1% BSA at 4°C overnight.	Biolegend suggested: (BioLegend, RRID:SCR_001134) ThermoFisher Scientific suggested: None
   Images were acquired using a Zeiss 780 confocal microscope with 10x and 20x objectives and analysed using ImageJ.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   RT-qPCR was performed using TaqMan Gene Expression Assays for Mx1 (Mm 00487796_m1), Gbp2 (Mm 00494576_g1) and Hprt (Mm 03024075_m1) directly on RNA using Thermo Fisher Scientific’s TaqMan RNA-to-CT 1-Step Kit (#4392656) following the manufacturer’s protocol.	Thermo Fisher Scientific’s suggested: None
   Analyses were performed within the Prism v8 software (GraphPad).	Prism suggested: (PRISM, RRID:SCR_005375) 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 10.1101/2020.10.27.357426 on bioRxiv

   Oct 27, 2020