Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants

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   ScreenIT

   Mar 24, 2021

   SciScore for 10.1101/2021.03.16.435594: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: The animal experiments were approved by Peking University Laboratory Animal Center (Beijing), and undertaken in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	For mouse vaccination, groups of 6-8 week-old female BLAB/c mice were intramuscularly immunized with LNP-circRNARBD (10 μg, N = 5; 50 μg, N = 5), or Placebo (empty LNP, N = 5) in 150 μL using a 1 mL sterile syringe, and 2 weeks later a second dose was immunized to boost the immune responses.
   Cell Line Authentication	not detected.

   Table 2: Resources

   …

   SciScore for 10.1101/2021.03.16.435594: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: The animal experiments were approved by Peking University Laboratory Animal Center (Beijing), and undertaken in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	For mouse vaccination, groups of 6-8 week-old female BLAB/c mice were intramuscularly immunized with LNP-circRNARBD (10 μg, N = 5; 50 μg, N = 5), or Placebo (empty LNP, N = 5) in 150 μL using a 1 mL sterile syringe, and 2 weeks later a second dose was immunized to boost the immune responses.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The SARS-CoV-2-specific IgG antibody titer was measured by ELISA.	SARS-CoV-2-specific IgG suggested: None
   Cells were then washed in stain buffer (PBS supplemented with 2.5% FBS) and suspended in Fc Block for 5 min at RT prior to staining with a surface stain of following antibodies: CD3 (Invitrogen, 45-0031-82)/CD4 (BD, 562285)/CD8 (BD, 553035)/CD69 (BD, 557392)/CD44 (BD, 563058)/CD62L (BD, 560507).	CD3 suggested: (Abcam Cat# ab52305, RRID:AB_955118)
   Experimental Models: Cell Lines
   Sentences	Resources
   circRNA transfection in vitro: For the circRNA transfection in HEK293T or NIH3T3 cells, 3×105 cells per well were seeded in 12-well plates.	NIH3T3 suggested: None
   Then the supernatant of HEK293T-hACE2 cells were removed and the mixer of serum and pseudovirus were added to each well. 36-48 hr later, the luciferase activity, which reflecting the degree of SARS-CoV-2 pseudovirus transfection, was measured using the Nano-Glo Luciferase Assay System (Promega).	HEK293T-hACE2 suggested: None
   Briefly, serum was diluted at 1:100 with 5 additional serial 5-fold dilution, and incubated with the same volume of pseudovirus with a TCID50 of 1.3×104 for 60 min at 37°C. 20,000 Huh-7 cells/well were cultured with mixture at 37°C for 24 h.	Huh-7 suggested: None
   Expression of neutralizing nanobodies or ACE2 decoys by circular RNAs: HEK293T cells were transfected with circular RNA in transfection reagent.	HEK293T suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Mouse vaccination and serum collection: The BALB/c mice were ordered from Beijing Vital River Laboratory Animal Technology Co., Ltd.	BALB/c suggested: RRID:IMSR_ORNL:BALB/cRl)
   For mouse vaccination, groups of 6-8 week-old female BLAB/c mice were intramuscularly immunized with LNP-circRNARBD (10 μg, N = 5; 50 μg, N = 5), or Placebo (empty LNP, N = 5) in 150 μL using a 1 mL sterile syringe, and 2 weeks later a second dose was immunized to boost the immune responses.	BLAB/c suggested: None
   Software and Algorithms
   Sentences	Resources
   The half-neutralization titer of serum (NT50) was determined using four-parameter nonlinear regression in Prism 8 (GraphPad).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   Relative luciferase units (RLU) were normalized to untreated groups, and analyzed by four-parameter nonlinear regression in Prism (GraphPad).	Prism suggested: (PRISM, RRID:SCR_005375)
   For the neutralization assay of circRNAnAB or circRNAACE2 decoys, the HEK293T-hACE2 cells were seeded in 96-well plates (50,000 cells/well) and incubated for approximate 24 hr until reaching over 90% confluent.	circRNAnAB suggested: None
   Analysis was performed using FlowJo software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)

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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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 19. 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 10.1101/2021.03.16.435594 on bioRxiv

   Mar 16, 2021