Preclinical evaluation of RQ3013, a broad-spectrum mRNA vaccine against SARS-CoV-2 variants

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1. Version published to 10.1016/j.scib.2023.11.024

   Dec 1, 2023
2. 

   ScreenIT

   May 14, 2022

   SciScore for 10.1101/2022.05.10.491301: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: All experiments with mice, hamsters, and macaques were carried out in accordance with the Regulations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China. IACUC: All animal procedures were approved by the Institutional Animal Care and Use Committee of the Institute of Medical Biology, Chinese Academy of Medical Science.
   Sex as a biological variable	The 6- to 8-year-old male or female rhesus macaque experiments were performed in the animal biosafety level 4 (ABSL-4) facility at Wuhan Institute of Virology (WIV), Hubei, China.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.

   SciScore for 10.1101/2022.05.10.491301: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: All experiments with mice, hamsters, and macaques were carried out in accordance with the Regulations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China. IACUC: All animal procedures were approved by the Institutional Animal Care and Use Committee of the Institute of Medical Biology, Chinese Academy of Medical Science.
   Sex as a biological variable	The 6- to 8-year-old male or female rhesus macaque experiments were performed in the animal biosafety level 4 (ABSL-4) facility at Wuhan Institute of Virology (WIV), Hubei, China.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The following primary antibodies were used in this study: anti-SARS-CoV-2 (2019-nCoV) Spike Antibody (40589-T62, Sino Biological), and anti-GAPDH Antibody (60004, Proteintech).	anti-SARS-CoV-2 suggested: None anti-GAPDH suggested: (Proteintech Cat# 60004-1-Ig, RRID:AB_2107436)
   The secondary antibodies used were Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (111-035-003, Jackson ImmunoResearch), Peroxidase	Anti-Rabbit IgG suggested: (Jackson ImmunoResearch Labs Cat# 111-035-003, RRID:AB_2313567)
   For determination of S-specific antibody response, plates were incubated with goat anti-mouse IgG HRP (for mouse sera, Proteintech Cat: SA00001-1) or goat anti-Syrian hamster IgG HRP (for hamster sera, abcam Cat: ab6892) or goat anti-monkey IgG HRP (for NHP sera, Invitrogen Cat: PA1-84631) at 37°C for 1 hour and then substrate tetramethylbenzidine (TMB) solution (Invitrogen) was used to develop.	anti-mouse IgG suggested: (Proteintech Cat# SA00001-1, RRID:AB_2722565) anti-Syrian hamster IgG suggested: (Abcam Cat# ab6892, RRID:AB_955427) anti-monkey IgG suggested: (Thermo Fisher Scientific Cat# PA1-84631, RRID:AB_933605)
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell lines and antibodies: HEK 293F cells were grown in FreeStyle Media (Gibco-Thermo Fisher Scientific) and transiently transfected using polyethylenimine (PEI) (Polysciences, Inc.) in an 8% CO2 environment at 37°C.	HEK 293F suggested: RRID:CVCL_6642)
   HEK 293A and Vero E6 cells were maintained in high glucose DMEM(GIBCO) supplemented with 10% FBS(GIBCO) and 1% penicillin/streptomycin (P/S) (GIBCO) in a 5% CO2 environment at 37°C.	HEK 293A suggested: None
   Virus titration: Virus titrations were performed by endpoint titration in Vero E6 cells.	Vero E6 suggested: RRID:CVCL_XD71)
   After 1 hour of incubation, 100 μL mixtures were inoculated onto monolayer Vero cells in a 24- well plate for 1 hour with shaking every 15 minutes.	Vero suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Animal vaccination and serum collection: Mice: For mouse vaccination, groups of 6- to 8-week-old female BALB/c mice or female K18-hACE2 Transgenic Mice were intramuscularly immunized with LNP vaccine candidates or a placebo in 50 μL, and 3 weeks later, a second dose was administered to boost the immune responses.	BALB/c suggested: None
   Recombinant DNA
   Sentences	Resources
   Cloning, expression, and preparation of the RQ3013 encoded Spike proteins: The gene encoding the RQ3013 was fused with a C-terminal twin Strep-tag (LEVLFQGPSGS WSHPQFEK GGGSGGGSGGSA WSHPQFEK) and cloned into a mammalian cell expression vector pcDNA3.1.	pcDNA3.1 suggested: RRID:Addgene_79663)
   The resulting plasmid, pcDNA3.1-RQ3013-Twinstrep, was transformed into HEK 293F cells using polyethylenimine (PEI) in FreeStyle Media (Gibco-Thermo Fisher Scientific).	pcDNA3.1-RQ3013-Twinstrep suggested: None
   Software and Algorithms
   Sentences	Resources
   Other procedures of cryo-EM data processing were performed within RELION v3.1 or CryoSPARC v3 using the dose-weighted micrographs23, 24.	RELION suggested: (RELION, RRID:SCR_016274) CryoSPARC suggested: (cryoSPARC, RRID:SCR_016501)
   Briefly, the initial templates were fit into the map using Chimera and Coot28, followed by a ten-cycle rigid body refinement using Phenix.	Phenix suggested: (Phenix, RRID:SCR_014224)
   Then, a combined manual refinement and real-space refinement were carried out for both prefusion state and postfusion state S structures in Coot and Phenix29.	Coot suggested: (Coot, RRID:SCR_014222)
   The dose-response curves were plotted from viral RNA copies versus the drug concentrations using GraphPad Prism 8.0 software.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   Statistical Analysis: All statistics data were performed and graphed using GraphPad Prism8.0.	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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   Results from scite Reference Check: We found no unreliable references.

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3. 

   Version published to 10.1101/2022.05.10.491301v1 on bioRxiv

   May 10, 2022