A Novel CRISPR-Engineered, Stem Cell-Derived Cellular Vaccine

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   ScreenIT

   Jan 3, 2022

   SciScore for 10.1101/2021.12.28.474336: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: All animal studies were conducted in compliance with all relevant local, state, and federal regulations and were approved by the Bioqual Institutional Animal Care and Use Committee (IACUC) IACUC: All animal studies were conducted in compliance with all relevant local, state, and federal regulations and were approved by the Bioqual Institutional Animal Care and Use Committee (IACUC)
   Sex as a biological variable	Animals and study design: Outbred adult male and female rhesus macaques (M. mulatta) and cynomolgus macaques (M. fascicularis), 6–12 years old, were randomly allocated to groups.
   Randomization	Animals and study design: Outbred adult male and female rhesus macaques (M. …

   SciScore for 10.1101/2021.12.28.474336: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: All animal studies were conducted in compliance with all relevant local, state, and federal regulations and were approved by the Bioqual Institutional Animal Care and Use Committee (IACUC) IACUC: All animal studies were conducted in compliance with all relevant local, state, and federal regulations and were approved by the Bioqual Institutional Animal Care and Use Committee (IACUC)
   Sex as a biological variable	Animals and study design: Outbred adult male and female rhesus macaques (M. mulatta) and cynomolgus macaques (M. fascicularis), 6–12 years old, were randomly allocated to groups.
   Randomization	Animals and study design: Outbred adult male and female rhesus macaques (M. mulatta) and cynomolgus macaques (M. fascicularis), 6–12 years old, were randomly allocated to groups.
   Blinding	All immunological and virological assays were performed blinded.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: The UVC preparations for use in non-human primate studies were analyzed for endotoxin levels (Wickham Laboratories Ltd) and absence of mycoplasma (Mycoplasma Experience Ltd).

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Cells were then stained for intracellular spike protein using an Anti-SARS-CoV-2 Spike Glycoprotein S1 antibody (Abcam, ab275759, 1:50) followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) (Abcam, ab150077, 1:500).	ab275759 suggested: (Abcam Cat# ab275759, RRID:AB_2892127) Anti-Rabbit IgG suggested: (Abcam Cat# ab150077, RRID:AB_2630356)
   Flow cytometry analysis of cell surface antigen expression: For flow cytometric analysis of cell surface expression of MHC-I, MICA and SARS-CoV-2 spike protein, cells were harvested from culture plates and washed using PBS with 1% Bovine Serum Albumen (Thermo Scientific) and were then stained with PE anti-human MICA/MICB Antibody (6D4, Biolegend)	SARS-CoV-2 spike protein suggested: None anti-human MICA/MICB suggested: None 6D4 suggested: None
   Alexa Fluor 647 anti-human HLA-A,B,C (W6/32, Biolegend), and anti-SARS-CoV-2 Spike Glycoprotein S1 antibody (Abcam, ab275759, 1:50) followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) (Abcam, ab150077, 1:500).	anti-human HLA-A suggested: None W6/32 suggested: (Bio-Rad Cat# MCA81A488, RRID:AB_324712) anti-SARS-CoV-2 Spike Glycoprotein S1 suggested: (Abcam Cat# ab275759, RRID:AB_2892127) ab150077 suggested: (Abcam Cat# ab150077, RRID:AB_2630356)
   Membranes were blocked in blocking buffer (5% non-fat powdered milk in TBST), before incubation with primary antibodies in blocking buffer (Rabbit polyclonal anti-SARS-Cov2, Sino Biological 40591-T62, 1:6000 dilution or Mouse b-actin, Abcam 8226, 1 μg/ml), detected with HRP conjugated secondaries in blocking buffer (Goat anti-Rabbit HRP, Sino Biological SSA003, 0.5 μg/ml or Goat anti-Mouse HRP, Abcam ab205719, 1: 4000 dilution) and visualised using the SuperSignal West Femto kit (ThermoFisher) as per kit instructions.	anti-SARS-Cov2 suggested: None b-actin , Abcam 8226 , 1 suggested: None anti-Rabbit suggested: (Sino Biological Cat# SSA003, RRID:AB_2814815) anti-Mouse HRP suggested: (Abcam Cat# ab205719, RRID:AB_2755049)
   After 48 hours of culture, transfected cells were stained with aqua dye for live/dead discrimination and corresponding antibodies-MICA/MICB (Clone 6D4, PE, BioLegend) or ULBP-1 (clone 170818, PE, R & D Systems).	antibodies-MICA/MICB suggested: None
   Anti-CD107a antibody (clone H4A3, ECD conjugate, BD Biosciences)	Anti-CD107a suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   , luciferase reporter plasmid pLenti-CMV Puro-Luc (Addgene), and spike protein expressing pcDNA3.1-SARS-CoV-2 SΔCT were co-transfected into HEK293T cells with calcium phosphate.	HEK293T suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
   To determine the neutralization activity of the antisera from vaccinated macaques, HEK293T-hACE2 cells were seeded in 96-well tissue culture plates at a density of 1.75 × 104 cells per well overnight.	HEK293T-hACE2 suggested: RRID:CVCL_A7UK)
   Recombinant DNA
   Sentences	Resources
   In brief, to generate a standard curve, the SARS-CoV-2 E gene sgRNA was cloned into a pcDNA3.1 expression plasmid; this insert was transcribed using an AmpliCap-Max T7 High Yield Message Maker Kit (Cellscript) to obtain RNA for standards.	pcDNA3.1 suggested: RRID:Addgene_79663)
   In brief, the packaging construct psPAX2 (AIDS Resource and Reagent Program)	psPAX2 suggested: RRID:Addgene_12260)
   , luciferase reporter plasmid pLenti-CMV Puro-Luc (Addgene), and spike protein expressing pcDNA3.1-SARS-CoV-2 SΔCT were co-transfected into HEK293T cells with calcium phosphate.	pLenti-CMV suggested: None pcDNA3.1-SARS-CoV-2 suggested: None
   Software and Algorithms
   Sentences	Resources
   For analysis by TIDE, PCR amplicons were Sanger sequenced (Eurofins or Genewiz) and paired .	Genewiz suggested: (GENEWIZ, RRID:SCR_003177)
   Intracellular spike protein staining: Engineered UVC were harvested and then fixed and permeabilized using BD Cytofix/Cytoperm Fixation/Permeabilization Solution (ThermoFisher).	BD Cytofix/Cytoperm Fixation/Permeabilization Solution suggested: None
   Flow analysis was carried out on a Fortessa flow cytometer (BD Bioscience), and data analyzed, and flow cytometry figures generated using FlowJo 10 software (BD Biosciences)	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   GraphPad Prism was used to plot a standard curve and interpolate the sample values using a 4-parameter logistic fit.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   In brief, the packaging construct psPAX2 (AIDS Resource and Reagent Program)	AIDS Resource and Reagent Program suggested: None
   Statistical Analyses: Statistical differences between two sample groups, where appropriate, were analyzed by a standard Student’s two-tailed, non-paired, t-test and between three or more sample groups using two-way or three-way ANOVA using GraphPad Prism 9.	GraphPad Prism 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: We detected the following sentences addressing limitations in the study:

   A limitation of the WA1/2020 UVC in a Delta heterologous challenge was the absence of any meaningful protection in nasal swabs. This discordance between vaccine variant, versus virus variant, is the principal challenge facing current vaccinated populations and might suggest the reason for ongoing infectious spread but more limited morbidity and mortality against these emerging variants29, 31. As regards duration of protection, the theoretical hyper-immunity postulated by creating a self-adjuvanting, hyper-immune UVC established robust initial nAb titers. When these animals were rechallenged 6-month later, the nAb response stayed robust at the higher, and now established for clinical use, 1e8 UVC dose. Moreover, the persistent nAb response at 6-months remained robust for SARS-CoV-2 WA1/2020, Beta and Delta variants. As regards intrinsic safety, the UVC undergoes lethal irradiation during manufacture and rapid apoptosis in the immune microenvironment upon vaccination. This is the principal mechanism of efficacy of the UVC, and fortunately its most redeeming safety feature, by virtue of the impossibility of in vivo persistence and teratogenicity of the cellular antigen carrier. The irradiation-induced apoptosis is further enhanced by CRISPR genetic engineering to remove MHC-I expression and introduce cell surface expression of the NKG2D ligand MICA, making the UVC potent targets for host NK cells. Recruited NK cells will likely recognize the UVC as virally infected cell through ...

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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.
   • No funding statement was detected.
   • No protocol registration statement was detected.

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

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2. 

   Version published to 10.1101/2021.12.28.474336v1 on bioRxiv

   Dec 29, 2021