Systems immune profiling of variant-specific vaccination against SARS-CoV-2

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   ScreenIT

   Dec 7, 2021

   SciScore for 10.1101/2021.12.02.471028: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: Single cell profiling: Splenocytes were collected from mRNA-LNP vaccinated and control mice were collected as described above for mouse immunization and sample collection, and normalized to 1000 cells/μL.
   Sex as a biological variable	Animals: M. musculus (mice), 6-8 weeks old females of C57BL/6Ncr, were purchased from Charles River and used for immunogenicity study.
   Randomization	All experiments utilize randomized littermate controls.
   Blinding	Replication, randomization, blinding and reagent validations: Replicate experiments have been performed for all key data shown in this study.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: All cell lines tested negative …

   SciScore for 10.1101/2021.12.02.471028: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: Single cell profiling: Splenocytes were collected from mRNA-LNP vaccinated and control mice were collected as described above for mouse immunization and sample collection, and normalized to 1000 cells/μL.
   Sex as a biological variable	Animals: M. musculus (mice), 6-8 weeks old females of C57BL/6Ncr, were purchased from Charles River and used for immunogenicity study.
   Randomization	All experiments utilize randomized littermate controls.
   Blinding	Replication, randomization, blinding and reagent validations: Replicate experiments have been performed for all key data shown in this study.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: All cell lines tested negative for mycoplasma.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Thereafter, cells were washed twice in MACS buffer and incubated with PE–anti-human FC antibody (Biolegend, M1310G05) in MACS buffer for 30 min on ice.	PE–anti-human FC suggested: None
   anti-mouse CD28 antibody (Biolegend, Clone 37.51) and seed into 96-well plate for overnight.	anti-mouse CD28 suggested: None
   Anti-mouse secondary antibody was diluted to 1:2500 in blocking buffer and incubated at room temperature for one hour.	Anti-mouse suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   In vitro mRNA expression: HEK293T cells were electroporated with mRNA encoding B.1351 variant (6P) or B.1.617 variant (6P) proteins using Neon™ Transfection System 10 µL Kit following the standard protocol provided by manufacturer.	HEK293T suggested: None
   Briefly, 293T cells were seeded in 150 mm plates, and transfected with 21 µg pHIVNLGagPol, 21 µg pCCNanoLuc2AEGFP, and 7.5 µg of a SARS-CoV-2 SΔ19 or B.1.351 variant-Δ19 or SARS-CoV-2 SA SΔ19 plasmid, utilizing 198 µl PEI.	293T suggested: None
   Plates were incubated at 37°C supplied with 5% CO2. 48 hr later, 293T-hACE2 cells were collected and the GFP+ cells were analyzed with Attune NxT Acoustic Focusing Cytometer (Thermo Fisher).	293T-hACE2 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Animals: M. musculus (mice), 6-8 weeks old females of C57BL/6Ncr, were purchased from Charles River and used for immunogenicity study.	C57BL/6Ncr suggested: RRID:MGI:2160593)
   Recombinant DNA
   Sentences	Resources
   Plasmid construction: The DNA sequences of B.1.351 and B.1.617 SARS-CoV-2 spikes for the mRNA transcription and pseudovirus assay were synthesized as gBlocks (IDT) and cloned by Gibson Assembly (NEB) into pcDNA3.1 plasmids.	pcDNA3.1 suggested: RRID:Addgene_79663)
   Plasmid expressing a C-terminally truncated SARS-CoV-2 S protein (pSARS-CoV-2Δ19) was from Dr Bieniasz’ lab	pSARS-CoV-2Δ19 suggested: None
   Briefly, 293T cells were seeded in 150 mm plates, and transfected with 21 µg pHIVNLGagPol, 21 µg pCCNanoLuc2AEGFP, and 7.5 µg of a SARS-CoV-2 SΔ19 or B.1.351 variant-Δ19 or SARS-CoV-2 SA SΔ19 plasmid, utilizing 198 µl PEI.	pCCNanoLuc2AEGFP suggested: None
   Software and Algorithms
   Sentences	Resources
   , CD4 FITC (Biolegend, Clone GK1.5,1:200) in MACS buffer (D-PBS with 2 mM EDTA and 0.5% BSA) on ice for 20 min, cells were washed with MACS buffer then fixed and permeabilized using the BD Cytofix/Cytoperm fixation/permeabilization solution kit according to the manufacturer’s instructions.	BD Cytofix/Cytoperm suggested: None
   Analysis was performed using FlowJo software according to the gating strategy outlined in a Supplemental Figure.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   The 50% inhibitory concentration (IC50) was calculated with a four-parameter logistic regression using GraphPad Prism (GraphPad Software Inc.)	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   The pooled library was sequenced using MiSeq (Illumina) with 2*300 read length.	MiSeq suggested: (A5-miseq, RRID:SCR_012148)
   Bulk VDJ sequencing data analysis: Raw fastq files from bulk BCR and TCR sequencing were processed by MiXCR v2.1.5 to clonotypes.	MiXCR suggested: (MiXCR, RRID:SCR_018725)
   For T-cell specific analyses, cells associated with the following terms were taken as a subset and used for standard Seurat pipeline analyses as described above: “CD4 T cell”, “CD8 T / NKT cell”, “CD8 T cell”, “T cell-like.	Seurat suggested: (SEURAT, RRID:SCR_007322)
   ” For B-cell specific analyses, cells associated with the following terms were taken as a subset: “B cell”, “B cell-like”, “Progenitor B cell”, “Plasma cell.” For functional annotation, differentially upregulated and downregulated genes with cutoff of adjusted p-value 0.05 were used for DAVID analysis.	DAVID suggested: (DAVID, RRID:SCR_001881)
   The statistical significance was labeled as follows: n.s., not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Prism (GraphPad Software) and RStudio were used for these analyses.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: Thank you for sharing your code.

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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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 32 and 37. 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.
   • 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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   Version published to 10.1101/2021.12.02.471028 on bioRxiv

   Dec 3, 2021