DOI: 10.5061/dryad.g79cnp64d
title: N1-methylpseudouridine enhances immunogenicity of RNA/lipidnanoparticle vaccines targeting SARS-CoV-2 spike and the model antigen ovalbumin

description: The recent success of RNA/lipid nanoparticle (LNP)-based vaccine
 formulations in the clinic against SARS-CoV-2 and RSV has highlighted the
 potential of this platform.  While RNA-LNP vaccines share the
 fundamental principle of using lipid nanoparticles to deliver RNA
 molecules into host cells to induce antigen expression and
 antigen-specific immune responses, several factors including RNA type
 (i.e., messenger, self-amplifying), RNA chemistry and/or nanoparticle
 formulation can influence the vaccine’s stability, tolerability and
 overall activity.  Although these differences may influence
 clinical performance, the lack of head-to-head comparative studies limits
 the ability to identify which factors have contributed most to the success
 of the clinically approved vaccines. Herein, we evaluated the influence of
 N1-methylpseudouridine incorporation into the RNA molecule on the overall
 immunogenicity of RNA vaccines encoding either the pathogen derived
 antigen SARS-CoV-2 spike or the model antigen ovalbumin.  Across
 repeated in vivo mouse studies, we demonstrate that the replacement of
 canonical uridine with N1-methylpseudouridine in the RNA molecules leads
 to more immunogenic vaccine formulations, corresponding to higher
 antigen-specific antibody titers as well as neutralizing
 activity.  Both types of RNA vaccines induced antigen-specific T
 cells, with the chemically modified RNA eliciting significantly higher
 levels of spike-specific CD4+ T cells.  As such, the
 incorporation of N1-methylpseudouridine can enhance vaccine
 immunogenicity, providing insight into the activity of previously
 evaluated formulations and supporting its continued use in vaccines under
 development.
description: # N1-methylpseudouridine enhances immunogenicity of RNA/lipidnanoparticle
 vaccines targeting SARS-CoV-2 spike and the model antigen ovalbumin
 Dataset DOI:
 [10.5061/dryad.g79cnp64d](https://doi.org/10.5061/dryad.g79cnp64d) ##
 Description of the data and file structure \# Data from:
 N1-methylpseudouridine Enhances Immunogenicity of RNA/Lipid Nanoparticle
 Vaccines Targeting SARS-CoV-2 Spike and the Model Antigen Ovalbumin This
 README.md file was generated on 2025-12-18 by Bassel Akache. GENERAL
 INFORMATION 1.  Title of Dataset: N1-methylpseudouridine Enhances
 Immunogenicity of RNA/Lipid Nanoparticle Vaccines Targeting SARS-CoV-2
 Spike and the Model Antigen Ovalbumin     Related Article of the same
 title submitted to Scientific Reports. 2.  Author information:     Hiva
 Azizi^1a^, Tyler M. Renner^1a^, Gerard Agbayani^1^, Renu Dudani^1^, Blair
 A. Harrison^1^, Martin A. Rossotti^2^, Jamshid Tanha^2,3,4^, Michael J.
 McCluskie^1^, Felicity C. Stark^1^ and Bassel Akache^1*^   1Human Health
 Therapeutics Research Centre, National Research Council Canada, 1200
 Montreal Road, Ottawa, Ontario, Canada, K1A 0R6 2Human Health Therapeutics
 Research Centre, National Research Council Canada, 100 Sussex Drive,
 Ottawa, Ontario, Canada, K1A 0R6 3Department of Biochemistry, Microbiology
 and Immunology, University of Ottawa, 451 Smyth Road, Ottawa, Ontario,
 Canada, K1H 8M5 4uOttawa Center for Infection, Immunity, and Inflammation
 (CI3), 451 Smyth Road, Ottawa, Ontario, Canada, K1H 8L1 aBoth authors
 contributed equally to this work.   \* Corresponding author: Bassel
 Akache,
 [Bassel.Akache@nrc-cnrc.gc.ca](mailto:Bassel.Akache@nrc-cnrc.gc.ca) 3. 
 Date of data collection: 2024 - 2025 4.  Geographic location of data
 collection: Ottawa, Canada. 5.  Funding sources that supported the
 collection of data: National Research Council of Canada. 6.  Recommended
 citation for this data set: Azizi, Hiva et al. (2025), Data from:
 N1-methylpseudouridine Enhances Immunogenicity of RNA/Lipid Nanoparticle
 Vaccines Targeting SARS-CoV-2 Spike and the Model Antigen Ovalbumin,
 Dryad, Dataset, [https://doi.org/10.5061/dryad.g79cnp64d] 
 
 Material and Methods In vitro RNA transcription The plasmid DNA templates
 used to generate the synthetic RNAs were previously described.^14^  For
 SARS-CoV-2 spike, the design was based on the publicly available
 Pfizer-BioNTech mRNA/LNP vaccine sequence^15^ and synthesized by Genscript
 (Piscataway, NJ, USA) in a pUC57 backbone to include the same 5’ and 3’
 untranslated regions (UTRs). The coding sequence for OVA was derived from
 pcDNA3-OVA (Addgene plasmid # 64599; http://n2t.net/addgene:64599;
 RRID:Addgene_64599; a gift from Sandra Diebold & Martin Zenke) and
 included the native UTRs.  DNA templates were linearized with XbaI and
 purified with a PCR purification kit (Qiagen, Hilden, Germany). RNA was
 generated using Megascript T7 transcription kit (Thermo Fisher Scientific,
 Waltham, MA, USA) and co-transcriptionally capped using Cap analog
 [m7Gppp(2’-OMeA) pG] reagent from Northern RNA (Calgary, AB, Canada) to
 generate RNA with stabilized Cap 1 structure. In place of uridine, m1Ψ
 (Jena Bioscience, Jena, Germany) was included to generate the mRNAs with
 modified nucleotide chemistry. Following transcription of RNA, plasmid DNA
 was digested using Turbo DNase (Invitrogen, Waltham, MA, USA) as per
 manufacturer’s instructions. RNA was purified by lithium chloride
 precipitation, washed with 70% ethanol and resuspended in RNase-free
 water. To assess RNA size and integrity, mRNA samples were analyzed on the
 Agilent Tapestation 4150 using the RNA ScreenTape (Agilent Technologies,
 Santa Clara, CA, USA). The concentrations of RNA samples were measured
 using Invitrogen’s RiboGreen assay reagent (Thermo Fisher Scientific), as
 per manufacturer’s recommendations.   Preparation of mRNA/LNPs In
 vitro-transcribed RNA was encapsulated within SM-102 (Cayman Chemical, Ann
 Arbor, Michigan, USA) or Genvoy-ILM ionizable lipid mixture (Precision
 NanoSystems, Vancouver, BC, Canada) at an N:P (Amine:Phosphate) ratio of
 6. Formation of LNPs was achieved by microfluidic mixing of RNA (aqueous)
 and ionizable lipids (organic) using the NanoAssemblr Ignite system
 (Precision NanoSystems) according to manufacturer’s recommendations with a
 flow rate ratio of 3 (aqueous: organic) and total flow rate of 12 mL/min.
 Formulations were then diluted in Mg2+/Ca2+-free phosphate-buffered saline
 (PBS), before undergoing buffer exchange and concentration using 100k kDa
 molecular weight cut-off Amicon Ultra Centrifugal Filters (Millipore
 Sigma, St. Louis, MO, USA). Encapsulation efficiency of RNA was determined
 by ribogreen assay (Thermo Fisher Scientific) as per instructions
 accompanying the Genvoy-ILM ionizable lipid and found to be &gt;85%. In
 addition, particle size, polydispersity index (pdi) and zeta potential of
 the LNPs were verified using a Zetasizer NanoZS (Malvern Instruments,
 Malvern, UK) and found to be generally consistent within formulations.  
 In vitro expression in HEK-293T cells HEK-293T cells (American Type
 Culture Collection, Manassas, VA, USA) were seeded at a density of 50,000
 cells/well in a 96 well plate and cultured in DMEM supplemented with 10%
 fetal bovine serum (FBS) and 1% penicillin/streptomycin (Thermo Fisher
 Scientific) and incubated overnight in a humidified incubator at 37°C, 5%
 CO2. The following day, RNA/LNPs or RNA complexed with Lipofectamine 3000
 (Thermo Fisher Scientific) were added to the cells.  A total of 300 or
 1,000 ng of RNA was added per well for SARS-CoV-2 spike and OVA,
 respectively. At 24 hours post-transfection, the plates were centrifuged
 and cell pellets lysed with tissue protein extraction reagent (Thermo
 Fisher Scientific). As OVA is a secreted protein, supernatants were
 collected instead and used in the ELISA.  Samples were stored frozen until
 analysis for SARS-CoV-2 spike or OVA protein expression by enzyme-linked
 immunosorbent assay (ELISA).   Protein Quantification by Sandwich ELISA
 Measurement of expression of SARS-CoV-2 spike from the RNA formulations
 was conducted by ELISA using previously described nanobodies.^16^ In
 brief, the capture antibody (biotinylated VHH 11) was immobilized at 5
 µg/mL on plates previously coated with Neutravidin (2 µg/mL) which had
 been prepared overnight and subsequently blocked with 1% (w/v) bovine
 serum albumin (BSA). Then, test samples and kit standard curve samples
 were added and incubated for 1 hour at room temperature. Spike was
 detected using human IgG1 Fc-fused VHH 12 (1 µg/mL) followed by anti-human
 Fc IgG conjugated to horseradish peroxidase (HRP) (Thermo Fisher
 Scientific) at 0.1 µg/mL. Finally, plates were washed and incubated with
 100 µL of peroxidase substrate solution (TMB, Seracare Life Sciences,
 Gaithersburg, MD, USA) for 15 mins at room temperature.  Reaction was then
 stopped with 1M H2SO4 and absorbance was measured spectrophotometrically
 at 450 nm. Reference Material of SARS-CoV-2 spike glycoprotein (National
 Research Council Canada, Montreal, QC, Canada; Cat. no.: SMT1-1)^17^ was
 used to generate the calibration curve. Levels of OVA were measured using
 a commercial OVA ELISA kit (Abcam, Cambridge, UK) according to
 manufacturer’s instructions.   Institutional Review Board statement: Mice
 were maintained at the small animal facility of the National Research
 Council Canada (NRC) in accordance with the guidelines of the Canadian
 Council on Animal Care. All procedures performed on animals in this study
 were approved by our Institutional Review Board (NRC Human Health
 Therapeutics Animal Care Committee) and covered under animal use protocol
 2024.14. All experiments were carried out in accordance with the ARRIVE
 guidelines.   Vaccination studies Female C57BL/6 mice (6-8 weeks old;
 16-20 grams) were obtained from Charles River Laboratories
 (Saint-Constant, QC, Canada). Mice (n=10 per group) were immunized by
 intramuscular (i.m.) injection (50 µL) into the left tibialis anterior
 muscle on Days 0 and 21 with 1 µg of encapsulated RNA corresponding to the
 various vaccine formulations. On Day 20, mice were anesthetized with
 isoflurane and bled via the submandibular vein with recovered serum used
 for quantification of antigen-specific antibody levels and neutralization
 activity. On Days 28 or 35 (depending on the study design), mice were bled
 as above for serum collection and then euthanized by cervical dislocation
 prior to collection of spleens for measurement of cellular immune
 responses by IFN-γ ELISpot. Samples were also collected from naïve animals
 to establish background immune response levels. Each individual mouse
 sample was analyzed separately across the various readouts.   Anti-OVA or
 -spike IgG ELISA Anti-OVA or anti-spike IgG titers in serum were measured
 by indirect ELISA as previously described.^18^ Briefly, 96–well
 high-binding ELISA plates (Thermo Fisher Scientific) were coated with 0.3
 µg/mL recombinant spike or 10 µg/mL OVA protein diluted in PBS. Serum
 samples were serially diluted 3.162-fold and added to the plates to allow
 for binding of antibodies to the protein. Bound IgG was detected with goat
 anti-mouse IgG-, IgG1- or IgG2c-HRP conjugate (1:4,000, Southern Biotech,
 Birmingham, AL, USA) prior to the addition of the substrate
 o-phenylenediamine dihydrochloride (Sigma-Aldrich). Bound IgG Abs were
 detected spectrophotometrically at 450 nm. Titers for IgG in serum were
 defined as the dilution that resulted in an absorbance value (OD450) of
 0.2 and were calculated using XLfit software (ID Business Solutions,
 Guildford, UK). Samples that did not reach the target OD were assigned the
 value of the lowest tested dilution (i.e., 10) for analysis purposes. No
 detectable titers were measured in serum samples from naïve control
 animals.   Cell-based SARS-CoV-2 spike-ACE2 binding assay The ability of
 serum to neutralize the binding of labeled SARS-CoV-2 spike trimers to
 HEK-293T-hACE2 cells (BEI Resources, Manassas, VA, USA) was measured as
 previously described.^19^ Mouse serum was diluted to a final concentration
 of 1:75 and mixed with 250 ng of recombinant biotinylated spike and
 1 × 105 HEK-293T-hACE2 cells. The amount of bound spike was quantified
 using a streptavidin-phycoerythrin conjugate prior to acquisition on an
 LSRFortessa (Becton Dickinson, Franklin Lakes, NJ, USA). For data
 analysis, samples with calculated values ≤0 of % of neutralization were
 assigned a value of 0.   IFN-γ ELISpot IFN-γ ELISpot was also conducted as
 previously described.^20^ The levels of spike- or OVA-specific T cells
 were quantified by ELISpot using a mouse IFN-γ kit (either from Mabtech
 Inc., Cincinnati, OH, USA or BD Biosciences, Franklin Lakes, NJ, USA
 depending on the study). For OVA, a peptide corresponding to the
 well-recognized CD8+ T cell epitope OVA257-264: SIINFEKL (JPT Peptide
 Technologies GmbH, Berlin, Germany) was used. For SARS-CoV-2 spike,
 peptide libraries composed of either 181 peptides (17- or 13-mers
 overlapping by 10 amino acids; BEI Resources; NR-52402) or 315 peptides
 (15-mers overlapping by 11 amino acids, with the final peptide consisting
 of a 17-mer; JPT Peptide Technologies GmbH) were used for Figures 2 and 4,
 respectively. The libraries were divided into sub-pools used to separately
 stimulate 4x 10^5^ cells in duplicate at a concentration of 2 µg/mL per
 peptide. Cells were also incubated without any stimulants to measure
 background responses. Spots were counted using an automated ELISpot plate
 reader (Cellular Technology LTD, Beachwood, OH, USA). For each animal,
 values obtained with media alone were subtracted from those obtained with
 each of the peptides, and then converted to yield the number of
 antigen-specific IFN-γ+ spot forming cells (SFC)/10^6^ splenocytes per
 animal. For spike protein, the values with the subpools were combined to
 generate a single count per animal.   Intracellular cytokine staining
 (ICCS) The procedure for ICCS was repeated as described previously.^21^
 Briefly, the phenotype (CD4+ vs. CD8+) and polyfunctionality (expression
 of IFN-γ, TNF-α, and/or IL-2) of spike-specific T cells were determined by
 ICCS of splenocytes. Cells (2 × 10^6^ per sample) were stimulated
 separately with spike peptide subpools (JPT Peptide Technologies GmbH) at
 a concentration of 2 μg/mL in the presence of GolgiPlug™ (Becton
 Dickinson) for ~ 20 h at 37°C with 5% CO2. Cells were also incubated
 without any peptides to measure background responses. Following
 incubation, splenocytes were washed with PBS (Thermo Fisher Scientific)
 and stained with the LIVE/DEAD™ fixable blue dead cell stain (Thermo
 Fisher Scientific). Cells were then stained with an antibody cocktail to
 identify immune cell types through binding of cell surface markers:
 anti-CD14-BV510, anti-CD16-BV510, anti-CD19-BV510, anti-CD4-APC-Cy7, and
 anti-CD8-PerCp-Cy5.5 (all from Becton Dickinson) diluted in staining
 buffer (PBS + 2% FBS + 2 mM EDTA). Cells were then washed in staining
 buffer and permeabilized for intracellular staining using the BD
 Cytofix/Cytoperm™ kit (Becton Dickinson) according to the manufacturer’s
 instructions. Samples were then stained with an antibody cocktail to
 anti-CD3-AF700 (eBioscience, San Diego, CA, USA), anti-CD69-PE-CF594
 (Becton Dickinson), anti-IFN-γ-AF488 (Becton Dickinson), anti-TNF-α-BV421
 (Becton Dickinson) and anti-IL-2-APC (Becton Dickinson) diluted in
 permeabilization wash buffer (Becton Dickinson). All samples were washed
 and resuspended in staining buffer prior to acquisition with the
 LSRFortessa flow cytometer (Becton Dickinson). Cell populations were
 characterized as follows: cells were identified by forward scatter (FSC)
 and side scatter (SSC), doublets were excluded, non-T cells and dead cells
 were excluded based on staining for BV510 (dump channel) and the
 LIVE/DEAD™ fixable blue dead cell stain. Activated CD3+ CD4+ or
 CD3+ CD8+ T cells were identified through positive staining of the CD69
 activation marker prior to classifying them as IFN-γ-, TNF-α-, or
 IL-2-positive cells. As above with ELISpot, values were background
 subtracted and then combined for each animal.   Statistical analysis Data
 were analyzed using GraphPad Prism® version 10 (GraphPad Software, Boston,
 MA, USA). Statistical significance of the difference between groups was
 calculated by two-tailed Student’s t-test, as indicated in the figure
 legends. Data were log transformed for IgG ELISA and IFN-γ ELISpot prior
 to statistical analysis. For all analyses, differences were considered to
 be not significant with p &gt; 0.05. Significance was indicated in the
 graphs as follows: *p &lt; 0.05, **p &lt; 0.01, ***p&lt;0.001 and ****:
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N1-methylpseudouridylation of mRNA causes +1 ribosomal frameshifting. Nature 625, 189–194 (2024). DATA & FILE OVERVIEW 1.  These data were collected to investigate the impacts of the modified nucleotide N1-methylpseudouridine on the expression and immunogenicity of proteins encoded by mRNA vaccines. 2.  File list:     File Name: 251218_Azizi_et_al_Source_Data_File.xlsx         Sheet 1 Name: Figure 1d     Sheet 1 Description: HEK-293T cells were transfected in vitro with RNA encoding SARS-CoV-2 Spike. After 24 hours, cellular supernatant was collected and analyzed for Spike protein levels by sandwich ELISA.     Sheet 2 Name: Figure 2b     Sheet 2 Description: On Days 0 and 21, C57BL/6 mice were injected intramuscularly with 1 µg canonical or m1Ψ-modified SARS-CoV-2 spike mRNA encapsulated within Genvoy ILM-based LNPs. Relative levels of Spike antigen specific IgG titer in immunized mouse serum from Days 20 and 28 as determined by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2.     Sheet 3 Name: Figure 2c     Sheet 3 Description: On Days 0 and 21, C57BL/6 mice were injected intramuscularly with 1 µg canonical or m1Ψ-modified SARS-CoV-2 spike mRNA encapsulated within Genvoy ILM-based LNPs. Relative levels of Spike antigen specific IgG1 and IgG2c titers in immunized mouse serum from Day 28 were measured by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2, and then divided by each other to calculate the anti-spike IgG2c/IgG1 ratio.     Sheet 4 Name: Figure 2d     Sheet 4 Description: On Days 0 and 21, C57BL/6 mice were injected intramuscularly with 1 µg canonical or m1Ψ-modified SARS-CoV-2 spike mRNA encapsulated within Genvoy ILM-based LNPs. The neutralization activity of immunized mouse serum from Day 28 as measured by its ability to prevent binding of soluble spike protein to hACE2-expressing HEK-293T cells.     Sheet 5 Name: Figure 2e     Sheet 5 Description: On Days 0 and 21, C57BL/6 mice were injected intramuscularly with 1 µg canonical or m1Ψ-modified SARS-CoV-2 spike mRNA encapsulated within Genvoy ILM-based LNPs. Extrapolated levels of peptide reactive splenocytes for SARS-CoV-2 Spike on Day 28 splenocytes as determined by IFN-γ ELISpot, where the background of splenocytes in culture media is subtracted.       Sheet 6 Name: Figure 3b     Sheet 6 Description: On Days 0 and 21, C57BL/6 mice were injected intramuscularly with 1 µg canonical or m1Ψ-modified OVA spike mRNA encapsulated within SM102-based LNPs. Relative levels of OVA antigen specific IgG titer in immunized mouse serum from Days 20 and 35 as determined by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2.     Sheet 7 Name: Figure 3c     Sheet 7 Description: On Days 0 and 21, C57BL/6 mice were injected intramuscularly with 1 µg canonical or m1Ψ-modified OVA spike mRNA encapsulated within SM102-based LNPs. Relative levels of OVA antigen specific IgG1 and IgG2c titers in immunized mouse serum from Day 35 were measured by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2, and then divided by each other to calculate the anti-spike IgG2c/IgG1 ratio.     Sheet 8 Name: Figure 3d     Sheet 8 Description: On Days 0 and 21, C57BL/6 mice were injected intramuscularly with 1 µg canonical or m1Ψ-modified OVA spike mRNA encapsulated within SM102-based LNPs. Extrapolated levels of peptide reactive splenocytes for OVA on Day 35 splenocytes as determined by IFN-γ ELISpot, where the background of splenocytes in culture media is subtracted.     Sheet 9 Name: Figure 4b     Sheet 9 Description: On Days 0 and 21, C57BL/6 mice were injected intramuscularly with 1 µg canonical or m1Ψ-modified SARS-CoV-2 spike mRNA encapsulated within SM102-based LNPs. Relative levels of Spike antigen specific IgG titer in immunized mouse serum from Days 20 and 35 as determined by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2.     Sheet 10 Name: Figure 4c     Sheet 10 Description: On Days 0 and 21, C57BL/6 mice were injected intramuscularly with 1 µg canonical or m1Ψ-modified SARS-CoV-2 spike mRNA encapsulated within SM102-based LNPs. Relative levels of Spike antigen specific IgG1 and IgG2c titers in immunized mouse serum from Day 35 were measured by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2, and then divided by each other to calculate the anti-spike IgG2c/IgG1 ratio.     Sheet 11 Name: Figure 4d     Sheet 11 Description: On Days 0 and 21, C57BL/6 mice were injected intramuscularly with 1 µg canonical or m1Ψ-modified SARS-CoV-2 spike mRNA encapsulated within SM102-based LNPs. The neutralization activity of immunized mouse serum from Day 35 as measured by its ability to prevent binding of soluble spike protein to hACE2-expressing HEK-293T cells.     Sheet 12 Name: Figure 4e     Sheet 12 Description: On Days 0 and 21, C57BL/6 mice were injected intramuscularly with 1 µg canonical or m1Ψ-modified SARS-CoV-2 spike mRNA encapsulated within SM102-based LNPs. Extrapolated levels of peptide reactive splenocytes for SARS-CoV-2 Spike on Day 35 splenocytes as determined by IFN-γ ELISpot, where the background of splenocytes in culture media is subtracted.     Sheet 13 Name: Figure 4f     Sheet 13 Description: On Days 0 and 21, C57BL/6 mice were injected intramuscularly with 1 µg canonical or m1Ψ-modified SARS-CoV-2 spike mRNA encapsulated within SM102-based LNPs. Extrapolated levels of peptide reactive splenocytes for SARS-CoV-2 Spike on Day 35 splenocytes as determined by Intracellular cytokine staining for IFN-γ, IL-2, TNF-α within CD4 and CD8 T cell populations, where the background of splenocytes in culture media is subtracted.       Sheet 14 Name: Supp Figure 1a     Sheet 14 Description: On Days 0 and 21, C57BL/6 mice were injected intramuscularly with 1 µg canonical or m1Ψ-modified SARS-CoV-2 spike mRNA encapsulated within Genvoy ILM-based LNPs. Relative levels of Spike antigen specific IgG1 and IgG2c titers in immunized mouse serum from Day 28 as determined by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2.       Sheet 15 Name: Supp Figure 1b     Sheet 15 Description: On Days 0 and 21, C57BL/6 mice were injected intramuscularly with 1 µg canonical or m1Ψ-modified OVA mRNA encapsulated within SM102-based LNPs. Relative levels of Spike antigen specific IgG1 and IgG2c titers in immunized mouse serum from Day 35 as determined by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2.       Sheet 16 Name: Supp Figure 1c     Sheet 16 Description: On Days 0 and 21, C57BL/6 mice were injected intramuscularly with 1 µg canonical or m1Ψ-modified SARS-CoV-2 spike mRNA encapsulated within SM102-based LNPs. Relative levels of Spike antigen specific IgG1 and IgG2c titers in immunized mouse serum from Day 35 as determined by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2.       Sheet 17 Name: Supp Figure 2     Sheet 17 Description: HEK-293T cells were transfected in vitro with RNA encoding OVA. After 24 hours, cellular supernatant was collected and analyzed for OVA protein levels by sandwich ELISA.   DATA-SPECIFIC INFORMATION FOR: Figure 1d 1.  Number of variables: 3 2.  Number of rows: 14 3.  Variable list:     Replicate     Formulation     Spike protein levels 4.  Missing data codes:     None 5.  Abbreviations used:     m1Ψ = N1-Methylpseudouridine; Canon. = Canonical; Neg.= negative; LNP= Lipid Nanoparticle; ng/mL= nanogram/milliliter. 6.  Other relevant information:     Values obtained by sandwich ELISA on cellular supernatants collected 24 hours post transfection. DATA-SPECIFIC INFORMATION FOR: Figure 2b 1.  Number of variables: 4 2.  Number of rows: 41 3.  Variable list:     Replicate     Nucleotides     Day     Anti-Spike IgG Titers 4.  Missing data codes:     None 5.  Abbreviations used:     IgG = Immunoglobulin G; m1Ψ = N1-Methylpseudouridine; 6.  Other relevant information:     Relative levels of SARS-CoV-2 Spike antigen specific titer on serum as determined by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2. DATA-SPECIFIC INFORMATION FOR: Figure 2c 1.  Number of variables: 4 2.  Number of rows: 21 3.  Variable list:     Replicate     Nucleotides     Day     Anti-Spike IgG2c/IgG1 Ratios 4.  Missing data codes:     None 5.  Abbreviations used:     IgG = Immunoglobulin G; m1Ψ = N1-Methylpseudouridine; 6.  Other relevant information:     Relative levels of Spike antigen specific IgG1 and IgG2c titers in immunized mouse serum from Day 28 were measured by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2, and then divided by each other to calculate the anti-spike IgG2c/IgG1 ratio.     DATA-SPECIFIC INFORMATION FOR: Figure 2d 1.  Number of variables: 4 2.  Number of rows: 21 3.  Variable list:     Replicate     Nucleotides     Day     %neutralization of binding 4.  Missing data codes:     None 5.  Abbreviations used:     m1Ψ = N1-Methylpseudouridine; 6.  Other relevant information:     The neutralization activity of immunized mouse serum from Day 28 as measured by its ability to prevent binding of soluble spike protein to hACE2-expressing HEK-293T cells.         DATA-SPECIFIC INFORMATION FOR: Figure 2e 1.  Number of variables: 4 2.  Number of rows: 11 3.  Variable list:     Replicate     Nucleotides     Day     % IFN-γ+ spot forming cells/10^6^ Splenocytes 4.  Missing data codes:     None 5.  Abbreviations used:     m1Ψ = N1-Methylpseudouridine;IFN-γ+ = interferon gamma positive 6.  Other relevant information:     Spots were counted using an automated ELISpot plate reader (Cellular Technology LTD) and the background of splenocytes in culture media is subtracted. The value from the ELISpot was extrapolated from the experimental value obtained from 4x10^5 splenocytes.       DATA-SPECIFIC INFORMATION FOR: Figure 3b 1.  Number of variables: 4 2.  Number of rows: 41 3.  Variable list:     Replicate     Nucleotides     Day     Anti-OVA IgG Titers 4.  Missing data codes:     None 5.  Abbreviations used:     IgG = Immunoglobulin G; m1Ψ = N1-Methylpseudouridine; OVA= ovalbumin 6.  Other relevant information:     Relative levels of OVA antigen specific titer on serum as determined by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2. DATA-SPECIFIC INFORMATION FOR: Figure 3c 1.  Number of variables: 4 2.  Number of rows: 21 3.  Variable list:     Replicate     Nucleotides     Day     Anti-OVA IgG2c/IgG1 Ratios 4.  Missing data codes:     None 5.  Abbreviations used:     IgG = Immunoglobulin G; m1Ψ = N1-Methylpseudouridine; OVA= ovalbumin 6.  Other relevant information:     Relative levels of OVA antigen specific IgG1 and IgG2c titers in immunized mouse serum from Day 35 were measured by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2, and then divided by each other to calculate the anti-spike IgG2c/IgG1 ratio.       DATA-SPECIFIC INFORMATION FOR: Figure 3d 1.  Number of variables: 4 2.  Number of rows: 21 3.  Variable list:     Replicate     Nucleotides     Day     % IFN-γ+ spot forming cells/10^6^ Splenocytes 4.  Missing data codes:     None 5.  Abbreviations used:     m1Ψ = N1-Methylpseudouridine; IFN-γ+ = interferon gamma positive 6.  Other relevant information:     Spots were counted using an automated ELISpot plate reader (Cellular Technology LTD) and the background of splenocytes in culture media is subtracted. The value from the ELISpot was extrapolated from the experimental value obtained from 4x10^5 splenocytes.   DATA-SPECIFIC INFORMATION FOR: Figure 4b 1.  Number of variables: 4 2.  Number of rows: 41 3.  Variable list:     Replicate     Nucleotides     Day     Anti-Spike IgG Titers 4.  Missing data codes:     None 5.  Abbreviations used:     IgG = Immunoglobulin G; m1Ψ = N1-Methylpseudouridine; 6.  Other relevant information:     Relative levels of SARS-CoV-2 Spike antigen specific titer on serum as determined by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2. DATA-SPECIFIC INFORMATION FOR: Figure 4c 1.  Number of variables: 4 2.  Number of rows: 21 3.  Variable list:     Replicate     Nucleotides     Day     Anti-Spike IgG2c/IgG1 Ratios 4.  Missing data codes:     None 5.  Abbreviations used:     IgG = Immunoglobulin G; m1Ψ = N1-Methylpseudouridine; 6.  Other relevant information:     Relative levels of Spike antigen specific IgG1 and IgG2c titers in immunized mouse serum from Day 35 were measured by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2, and then divided by each other to calculate the anti-spike IgG2c/IgG1 ratio.     DATA-SPECIFIC INFORMATION FOR: Figure 4d 1.  Number of variables: 4 2.  Number of rows: 21 3.  Variable list:     Replicate     Nucleotides     Day     %neutralization of binding 4.  Missing data codes:     None 5.  Abbreviations used:     m1Ψ = N1-Methylpseudouridine; 6.  Other relevant information:     The neutralization activity of immunized mouse serum from Day 35 as measured by its ability to prevent binding of soluble spike protein to hACE2-expressing HEK-293T cells.         DATA-SPECIFIC INFORMATION FOR: Figure 4e 1.  Number of variables: 4 2.  Number of rows: 21 3.  Variable list:     Replicate     Nucleotides     Day     % IFN-γ+ spot forming cells/10^6^ Splenocytes 4.  Missing data codes:     None 5.  Abbreviations used:     m1Ψ = N1-Methylpseudouridine; IFN-γ+ = interferon gamma positive 6.  Other relevant information:     Spots were counted using an automated ELISpot plate reader (Cellular Technology LTD) and the background of splenocytes in culture media is subtracted. The value from the ELISpot was extrapolated from the experimental value obtained from 4x10^5 splenocytes.     DATA-SPECIFIC INFORMATION FOR: Figure 4f 1.  Number of variables: 6 2.  Number of rows: 121 3.  Variable list:     Replicate     Nucleotides     Cell Type     Cytokine     Day     Cytokine+/10^6 T cells 4.  Missing data codes:     None 5.  Abbreviations used:     m1Ψ = N1-Methylpseudouridine; IFN-γ= interferon gamma; IL-2= interleukin 2 ; TNF-α= Tumor necrosis factor alpha; CD: Cluster of differentiation 6.  Other relevant information:     Cells were acquired on a LSRFortessa flow cytometer (Becton Dickinson) and the background signal obtained with splenocytes in culture media is subtracted.   DATA-SPECIFIC INFORMATION FOR: Supp Figure 1a 1.  Number of variables: 5 2.  Number of rows: 41 3.  Variable list:     Replicate     Nucleotides     Day     Anti-Spike Titers     IgG Subtype 4.  Missing data codes:     None 5.  Abbreviations used:     IgG = Immunoglobulin G; m1Ψ = N1-Methylpseudouridine; 6.  Other relevant information:     Relative levels of SARS-CoV-2 Spike antigen specific IgG1 or IgG2c titers on serum as determined by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2.       DATA-SPECIFIC INFORMATION FOR: Supp Figure 1b 1.  Number of variables: 5 2.  Number of rows: 41 3.  Variable list:     Replicate     Nucleotides     Day     Anti-OVA Titers     IgG Subtype 4.  Missing data codes:     None 5.  Abbreviations used:     IgG = Immunoglobulin G; m1Ψ = N1-Methylpseudouridine; OVA= Ovalbumin 6.  Other relevant information:     Relative levels of OVA antigen specific IgG1 or IgG2c titers on serum as determined by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2. DATA-SPECIFIC INFORMATION FOR: Supp Figure 1c 1.  Number of variables: 5 2.  Number of rows: 41 3.  Variable list:     Replicate     Nucleotides     Day     Anti-Spike Titers     IgG Subtype 4.  Missing data codes:     None 5.  Abbreviations used:     IgG = Immunoglobulin G; m1Ψ = N1-Methylpseudouridine; 6.  Other relevant information:     Relative levels of SARS-CoV-2 Spike antigen specific IgG1 or IgG2c titers on serum as determined by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2.     DATA-SPECIFIC INFORMATION FOR: Supp Figure 2 1.  Number of variables: 3 2.  Number of rows: 7 3.  Variable list:     Replicate     Formulation     OVA protein levels (ng/mL) 4.  Missing data codes:     None 5.  Abbreviations used:     m1Ψ = N1-Methylpseudouridine; Canon. = Canonical; Neg.= negative; LNP= Lipid Nanoparticle; ng/mL= nanogram/milliliter; OVA= ovalbumin. 6.  Other relevant information:     Values obtained by sandwich ELISA on cellular supernatants collected 24 hours post transfection.