Vandetanib Reduces Inflammatory Cytokines and Ameliorates COVID-19 in Infected Mice

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   ScreenIT

   Dec 23, 2021

   SciScore for 10.1101/2021.12.16.472155: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: Mouse studies: Ethical approval: All the experimental procedures were performed in accordance with the guide for the use of laboratory animals of the University of Sao Paulo and approved by the institutional ethics committee under the protocol number 105/2021. SARS-CoV-2: SARS-CoV-2 was isolated from a COVID-19 positive-tested patient.
   Sex as a biological variable	SARS-CoV-2 experimental infection and treatments: Female K18-hACE2 mice, aged 8 weeks, were infected with 2×104 PFU of SARS-CoV-2 (in 40 μL) by intranasal route.
   Randomization	A total of 10 photomicrographs in 40X magnification per animal were randomly obtained using a microscope Novel (Novel L3000 LED, China) coupled to a HDI …

   SciScore for 10.1101/2021.12.16.472155: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: Mouse studies: Ethical approval: All the experimental procedures were performed in accordance with the guide for the use of laboratory animals of the University of Sao Paulo and approved by the institutional ethics committee under the protocol number 105/2021. SARS-CoV-2: SARS-CoV-2 was isolated from a COVID-19 positive-tested patient.
   Sex as a biological variable	SARS-CoV-2 experimental infection and treatments: Female K18-hACE2 mice, aged 8 weeks, were infected with 2×104 PFU of SARS-CoV-2 (in 40 μL) by intranasal route.
   Randomization	A total of 10 photomicrographs in 40X magnification per animal were randomly obtained using a microscope Novel (Novel L3000 LED, China) coupled to a HDI camera for images capture.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Expression and purification of Spike RBD of SARS-CoV-2: A codon-optimized gene encoding for SARS-CoV-2 (331 to 528 amino acids, QIS60558.1) was expressed in Expi293 cells (Thermo Fisher Scientific) with human serum albumin secretion signal sequence and fusion tags (6xHistidine tag, Halo tag, and TwinStrep tag) as described before 1.	Expi293 suggested: RRID:CVCL_D615)
   HUVEC single cell donor (Lonza, cat#C2517A) cells were transduced at room temperature with ACE2 using a BacMam viral vector at a concentration of 2e9 VG/ml (Montana Molecular #C1120G Pseudo SARS-CoV-2 D614G Green Reporter) followed by incubation at 36°C for 24 hours.	HUVEC suggested: None
   SARS-Cov-2 tested in A549-ACE2 cells: A549-ACE2 cells were plated in Corning black walled clear bottom 96 well plates 24 hours before infection for confluency.	A549-ACE2 suggested: None
   SARS-Cov-2 tested in Calu-3 cells: Calu-3 (ATCC, HTB-55) cells were pretreated with test compounds for 2 hours prior to continuous infection with SARS-CoV-2 (isolate USA WA1/2020) at a MOI=0.5.	Calu-3 suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
   Supernatant from the Caco-2 cells are collected on day 3 post-infection and titrated on Vero 76 cells for virus titer as before.	Caco-2 suggested: None Vero 76 suggested: KCLB Cat# 21587, RRID:CVCL_0603)
   HCoV 229E antiviral assay: HCoV 229E, (a gift from Ralph Baric, UNC, Chapel Hill) was propagated on Huh-7 cells and titers were determined by TCID50 assay on Huh-7 cells.	Huh-7 suggested: None
   The virus was propagated and titrated in Vero E6 cells in a biosafety level 3 laboratory (BSL3) at the Ribeirao Preto Medical school (Ribeirao Preto, Brazil).	Vero E6 suggested: RRID:CVCL_XD71)
   Penicillin 10,000 U/mL; Streptomycin10,000 μ Vero cells in DMEM (FBS 2%) incubated at 37 °C with 5% CO2 for 48 h.	Vero suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   K18-hACE2 mice: To evaluate the effects of vandetanib in vivo, we infected the K18-hACE2 humanized mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J)7, 8, 9.	B6.Cg-Tg(K18-ACE2)2Prlmn/J)7 suggested: None
   K18-hACE2 mice were obtained from The Jackson Laboratory and were breed in the Centro de Criação de Animais Especiais	K18-hACE2 suggested: RRID:IMSR_GPT:T037657)
   Recombinant DNA
   Sentences	Resources
   MHV-A59 with nano-Luciferase: The MHV-A59 G plasmid was engineered to replace most of the coding sequence for orf4a and 4b with nano-luciferase (nLuc).	The MHV-A59 G suggested: None
   A sequence verified G-nLuc plasmid was used with MHV-A59 wild type A, B, C, D, E and F plasmids to recover virus expressing nLuc, using our previously described molecule clone (Systematic assembly of a full-length infectious cDNA of mouse hepatitis virus strain A59 4.	G-nLuc suggested: None
   Software and Algorithms
   Sentences	Resources
   The total septal area and total area were analyzed with the aid of the Pro Plus 7 software (Media Cybernetics, Inc., MD, USA).	Pro Plus suggested: (Image-Pro Plus, RRID:SCR_016879)

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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: We found the following clinical trial numbers in your paper:
   

   Identifier	Status	Title
   NCT0427541464	Trial number did not resolve on clinicaltrials.gov. Is the number correct?	NA

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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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   Results from scite Reference Check: We found no unreliable references.

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   Version published to 10.1101/2021.12.16.472155v1 on bioRxiv

   Dec 20, 2021