Nucleic acid delivery of immune-focused SARS-CoV-2 nanoparticles drive rapid and potent immunogenicity capable of single-dose protection

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   ScreenIT

   Apr 30, 2021

   SciScore for 10.1101/2021.04.28.441474: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: All studies were performed in accordance with Wistar Institutional Animal Care and Use Committees under approved animal protocols. Euthanasia Agents: For cellular responses, mice were euthanized under CO2 overdose. Field Sample Permit: Serum samples were collected at indicated timepoints via saphenous vein blood collection throughout the experiment.
   Sex as a biological variable	Female Hartley guinea pigs (8 weeks old, Elm Hill Labs, Chelmsford MA) were housed at Acculab (San Diego CA).
   Randomization	not detected.
   Blinding	For the lethal challenge study, Texas Biomed were blinded to identity of vaccination groups and weight loss cutoff for euthanasia was 20%.
   Power Analysis	not detected.
   Cell …

   SciScore for 10.1101/2021.04.28.441474: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: All studies were performed in accordance with Wistar Institutional Animal Care and Use Committees under approved animal protocols. Euthanasia Agents: For cellular responses, mice were euthanized under CO2 overdose. Field Sample Permit: Serum samples were collected at indicated timepoints via saphenous vein blood collection throughout the experiment.
   Sex as a biological variable	Female Hartley guinea pigs (8 weeks old, Elm Hill Labs, Chelmsford MA) were housed at Acculab (San Diego CA).
   Randomization	not detected.
   Blinding	For the lethal challenge study, Texas Biomed were blinded to identity of vaccination groups and weight loss cutoff for euthanasia was 20%.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Blocking Buffer (LI-COR) for >1 hour at ambient temperature then incubated with *** μg / protein gel of MonoRab anti-his tag C-term (Genscript) in Intercept T20 (PBS) Antibody Diluent (LI-COR) overnight at 4°C.	anti-his tag C-term suggested: None
   The membrane was then incubated in a 1:10000 IRDye 800CW goat anti-rabbit IgG (LI-COR Biosciences) in Intercept T20 (PBS) Antibody Diluent (LI-COR) at room temperature for 1 h.	anti-rabbit IgG suggested: None
   Goat anti-Human IgG-Fc fragment cross-adsorbed antibody HRP conjugated (Bethyl Laboratories) secondary at a 1:10,000 dilution for 1 hour at ambient temperature.	Goat anti-Human IgG-Fc fragment cross-adsorbed antibody HRP suggested: None anti-Human IgG-Fc suggested: None
   For BL6, BALB/c, and K18 ACE2 mouse studies, goat anti-mouse IgG h+l HRP-tagged antibody (Bethyl Laboratories) diluted 1:20000.	ACE2 suggested: None anti-mouse IgG suggested: None
   Fragment Goat Anti-Rat IgM, μ chain specific (Jackson ImmunoResearch) at 1:10000, Goat anti-Human Kappa Light Chain Antibody HRP Conjugated (Bethyl Laboratories) at 1:10000,	Anti-Rat IgM suggested: None anti-Human Kappa Light Chain suggested: None
   Goat anti-Human Lambda Light Chain Antibody HRP Conjugated (Bethyl Laboratories) at 1:10000, and Goat anti-Mouse IgG-heavy and light chain Antibody HRP Conjugated (Bethyl Laboratories) at 1:20000, and Goat anti-guinea pig IgG whole molecule (Sigma) at 1:10,000 were used.	anti-Human Lambda Light Chain suggested: None anti-Mouse IgG-heavy suggested: None anti-guinea pig IgG whole molecule ( Sigma ) suggested: None
   Anti-Hamster HRP antibody (Sigma) was diluted in diluent buffer 1:10,000 and were incubated for 1 hr at room temperature.	Anti-Hamster HRP antibody suggested: None Anti-Hamster HRP suggested: None
   Intracellular cytokine staining and Flow cytometry: Splenocytes were processed as described in the previous section and stimulated with RBD peptides for 5 hours at 37°C with protein transport inhibitor (Invitrogen) and anti-mouse CD107a-FITC antibody (BioLegend).	anti-mouse CD107a-FITC suggested: (SouthernBiotech Cat# 1920-02, RRID:AB_2795531)
   Anti-mouse CD4-BV510, CD8-APC-Cy7, CD44-A700, and CD62L-BV711 antibodies were used for surface staining and CD3e-PE-Cy5, IFN-γ-APC, and TNF-α-BV605 (all from BioLegend) were used for intracellular staining.	Anti-mouse CD4-BV510 suggested: None CD8-APC-Cy7 suggested: None CD44-A700 suggested: None CD62L-BV711 suggested: None CD3e-PE-Cy5 , IFN-γ-APC , suggested: None TNF-α-BV605 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   ExpiF293 cells were transfected with the pVAX plasmid vector either carrying the nanoparticles or the His-Tagged monomer transgene with PEI/Opti-MEM and harvested 6-7 days post transfection.	ExpiF293 suggested: None
   Pseudovirus Neutralization Assay: HEK293T (CRL-3216) and CHO cells (CRL-12023: double check) were obtained from ATCC (Manassas, VA, USA).	HEK293T suggested: None CHO suggested: None
   CHO-ACE2 cells were seeded at 10,000 cells/well in 96-well plates and incubated for 24 hours.	CHO-ACE2 suggested: None
   To grow a stock of virus, 3 million Vero cells were seeded in a T-75 flask for overnight incubation (37⍰C, 5% CO2).	Vero suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   For BL6, BALB/c, and K18 ACE2 mouse studies, goat anti-mouse IgG h+l HRP-tagged antibody (Bethyl Laboratories) diluted 1:20000.	BALB/c suggested: None
   Animal Studies: C57BL/6, BALBc, and K18-hACE2 mice were obtained from Charles River Laboratories (Malvern, PA) and The Jackson Laboratory (Bar Harbor, ME).	C57BL/6 suggested: None K18-hACE2 suggested: RRID:IMSR_GPT:T037657)
   All animals were housed in the Wistar animal facility in ventilated cages and given free access to food and water.	Wistar suggested: RRID:MGI:5657554)
   Recombinant DNA
   Sentences	Resources
   DNA encoding the variants were codon optimized for homo sapiens and cloned with a IgE secretion sequence into the pVAX vector.	pVAX suggested: None
   For antibody production, heavy and light chains were encoded in pFUSEss-CHIg-hG1, and pFUSE2ss-CLIg-hk or pFUSEss-CLIg-hL2 respectively and were co-transfected in equal parts using ExpiFectamine™ 293 Transfection Kit(Gibco) according to manufacturer’s protocol.	pFUSEss-CHIg-hG1 suggested: None pFUSE2ss-CLIg-hk suggested: None pFUSEss-CLIg-hL2 suggested: None
   6μg S_IgE_deltaCterm19_plasmid (Genscript), and 6μg pNL4-3.luc.R-E- backbone (Aldevron) and incubated for 48 hours.	pNL4-3.luc.R-E- suggested: None
   Software and Algorithms
   Sentences	Resources
   Curves were analyzed in GraphPad Prism 8 with Sigmoidal, 4PL, X is concentration and AUC.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   For variant pseudoviruses, cells were similarly treated with GeneJammer and backbone with 6μg of S_SA_IgE_deltaCterm19, S_UK_IgE_deltaCterm19, or S_Brazil_IgE_deltaCterm19 plasmid.	GeneJammer suggested: None
   The virus titer (TCID50/ml) was calculated using the Reed-Munch method and the Microsoft Excel based calculator published by Lei et al[73] For neutralization assays, Vero cells were seeded in DMEM with 1% FBS at 20,000 cells/well in 96 well flat bottom plates for overnight incubation (37C, 5% CO2)	Microsoft Excel suggested: (Microsoft Excel, RRID:SCR_016137)
   Data processing was performed in Relion v3.1.2[74]	Relion suggested: (RELION, RRID:SCR_016274)
   The samples were run on an 18-color LSRII flow cytometer (BD Biosciences) and analyzed by FlowJo software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   After being washed, the plates were further incubated at room temperature for 1 hour with goat-anti human IgG-Fc fragment cross-adsorbed Ab (A80-340P; Bethyl Laboratories) at a 1: 10,000 dilution, followed by addition of TMB substrates (ThermoFisher), and then quenched with 1M H2SO4.	ThermoFisher suggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)

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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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   Version published to 10.1101/2021.04.28.441474 on bioRxiv

   Apr 28, 2021