Dataset DOI: 10.5061/dryad.g79cnp64d

Description of the data and file structure

This README.md file was generated on 2026-08-11 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 Agbayani¹, Renu Dudani¹, Blair A. Harrison¹, Martin A. Rossotti², Jamshid Tanha^2,3,4, Michael J. McCluskie¹, Felicity C. Stark¹ 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

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 sequence¹⁵ 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. While not tested directly with the RNA samples generated for this study, previous studies by our group have shown that the in vitro transcription process used above generates RNA with <0.4% double-stranded vs. single-stranded RNA, regardless if all canonical or modified nucleotides are used.

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 >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 10⁵ 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)/10⁶ splenocytes per animal. For spike protein, the values with the subpools were combined to generate a single count per animal. In some of the studies, splenocytes from naïve animals were similarly analyzed to determine the background responses present in non-immunized animals. The number of IFN-γ+ SFC /106 splenocytes was measured to be ≤20 in samples from each of the naïve animals tested.

Intracellular cytokine staining (ICCS)

The procedure for ICCS was repeated as described previously.²¹ Briefly, the phenotype (CD4+ vs. CD8+) and expression profile of IFN-γ, TNF-α, and/or IL-2 from spike-specific T cells were determined by ICCS of splenocytes. Cells (2 × 10⁶ 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. The flow cytometry gating strategy for identifying cytokine-positive cells is outlined in Supplementary Figure 1. As above with ELISpot, values were background subtracted and then combined for each animal. In addition, splenocytes from naïve animals were similarly analyzed side-by-side to determine the background responses present in non-immunized animals.

Statistical analysis

Data were analyzed using GraphPad Prism® version 10 (GraphPad Software, Boston, MA, USA). Statistical significance of the difference between the animals immunized with the m1Ψ-modified or canonical RNA formulations 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 > 0.05. Significance was indicated in the graphs as follows: *p < 0.05, **p < 0.01, ***p<0.001 and ****: p<0.0001.

References

1.       Soheili, M. et al. The efficacy and effectiveness of COVID-19 vaccines around the world: a mini-review and meta-analysis. Ann. Clin. Microbiol. Antimicrob. 22, 42 (2023).

2.       Savinkina, A. et al. Estimating deaths averted and cost per life saved by scaling up mRNA COVID-19 vaccination in low-income and lower-middle-income countries in the COVID-19 Omicron variant era: a modelling study. BMJ Open 12, e061752 (2022).

3.       Zhang, Z. et al. Research progress of mRNA vaccines for infectious diseases. Eur. J. Med. Res. 30, 792 (2025).

4.       Morais, P., Adachi, H. & Yu, Y.-T. The Critical Contribution of Pseudouridine to mRNA COVID-19 Vaccines. Front. Cell Dev. Biol. 9, 789427 (2021).

5.       Kremsner, P. G. et al. Efficacy and safety of the CVnCoV SARS-CoV-2 mRNA vaccine candidate in ten countries in Europe and Latin America (HERALD): a randomised, observer-blinded, placebo-controlled, phase 2b/3 trial. Lancet Infect. Dis. 22, 329–340 (2022).

6.       Karikó, K. et al. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol. Ther. J. Am. Soc. Gene Ther. 16, 1833–1840 (2008).

7.       Karikó, K., Buckstein, M., Ni, H. & Weissman, D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23, 165–175 (2005).

8.       McComb, S., Thiriot, A., Akache, B., Krishnan, L. & Stark, F. C. Introduction to the Immune System. Methods Mol. Biol. 2980, 1–31 (2026).

9.       Wang, Z. et al. Reducing cell intrinsic immunity to mRNA vaccine alters adaptive immune responses in mice. Mol. Ther. Nucleic Acids 34, 102045 (2023).

10.     Mu, X., Greenwald, E., Ahmad, S. & Hur, S. An origin of the immunogenicity of in vitro transcribed RNA. Nucleic Acids Res. 46, 5239–5249 (2018).

11.     Svitkin, Y. V. et al. N1-methyl-pseudouridine in mRNA enhances translation through eIF2α-dependent and independent mechanisms by increasing ribosome density. Nucleic Acids Res. 45, 6023–6036 (2017).

12.     Pardi, N. et al. Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses. J. Exp. Med. 215, 1571–1588 (2018).

13.     Ramos da Silva, J. et al. Single immunizations of self-amplifying or non-replicating mRNA-LNP vaccines control HPV-associated tumors in mice. Sci. Transl. Med. 15, eabn3464 (2023).

14.     Kuzmin, I. V. et al. Comparison of uridine and N1-methylpseudouridine mRNA platforms in development of an Andes virus vaccine. Nat. Commun. 15, 6421 (2024).

15.     Engstrand, O. et al. Immunogenicity and innate immunity to high-dose and repeated vaccination of modified mRNA versus unmodified mRNA. Mol. Ther. Nucleic Acids 36, 102588 (2025).

16.     Renner, T. M. et al. Reduced cross-protective potential of Omicron compared to ancestral SARS-CoV-2 spike vaccines against potentially zoonotic coronaviruses. Npj Viruses 2, 58 (2024).

17.     World Health Organization Messenger RNA Encoding the Full-Length SARS-CoV-2 Spike Glycoprotein. Available from: https://web.archive.org/web/20210105162941/https://mednet-communities.net/inn/db/media/docs/11889.doc

18.     Azizi, H. et al. Self-amplifying RNAs generated with the modified nucleotides 5-methylcytidine and 5-methyluridine mediate strong expression and immunogenicity in vivo. NAR Mol. Med. 1, ugae004 (2024).

19.     Rossotti, M. A. et al. Arsenal of nanobodies shows broad-spectrum neutralization against SARS-CoV-2 variants of concern in vitro and in vivo in hamster models. Commun. Biol. 5, 933 (2022).

20.     Stuible, M. et al. Rapid, high-yield production of full-length SARS-CoV-2 spike ectodomain by transient gene expression in CHO cells. J. Biotechnol. 326, 21–27 (2021).

21.     Akache, B., Stark, F. C. & McCluskie, M. J. Measurement of Antigen-Specific IgG Titers by Direct ELISA. Methods Mol. Biol. Clifton NJ 2183, 537–547 (2021).

22.     Rossotti, M. A. et al. A Safe and Accessible Cell-Based Spike-ACE2 Binding Assay for Evaluating SARS-CoV-2 Neutralization Activity in Biological Samples Using Flow Cytometry. Methods Protoc. 8, 104 (2025).

23.     Akache, B. & McCluskie, M. J. The Quantification of Antigen-Specific T Cells by IFN-γ ELISpot. Methods Mol. Biol. Clifton NJ 2183, 525–536 (2021).

24.     Akache, B. et al. Immunogenic and efficacious SARS-CoV-2 vaccine based on resistin-trimerized spike antigen SmT1 and SLA archaeosome adjuvant. Sci. Rep. 11, 21849 (2021).

25.     Akache, B. et al. Immunogenicity of SARS-CoV-2 spike antigens derived from Beta & Delta variants of concern. NPJ Vaccines 7, 118 (2022).

26.     Renner, T. M. et al. Tuning the immune response: sulfated archaeal glycolipid archaeosomes as an effective vaccine adjuvant for induction of humoral and cell-mediated immunity towards the SARS-CoV-2 Omicron variant of concern. Front. Immunol. 14, 1182556 (2023).

27.     Zhang, L. et al. Effect of mRNA-LNP components of two globally-marketed COVID-19 vaccines on efficacy and stability. NPJ Vaccines 8, 156 (2023).

28.     Akache, B. et al. Sulfated archaeol glycolipids: Comparison with other immunological adjuvants in mice. PloS One 13, e0208067 (2018).

29.     McComb, S., Thiriot, A., Akache, B., Krishnan, L. & Stark, F. Introduction to the Immune System. Methods Mol. Biol. Clifton NJ 2024, 1–24 (2019).

30.     Akache, B., Stark, F. C., Agbayani, G., Renner, T. M. & McCluskie, M. J. Adjuvants: Engineering Protective Immune Responses in Human and Veterinary Vaccines. Methods Mol. Biol. Clifton NJ 2412, 179–231 (2022).

31.     Tregoning, J. S., Wang, Z., Sridhar, S., Shattock, R. J. & DeRosa, F. Immunology of RNA-based vaccines: The critical interplay between inflammation and expression. Mol. Ther. J. Am. Soc. Gene Ther. S1525-0016(25)00739–7 (2025) doi:10.1016/j.ymthe.2025.09.011.

32.     Tregoning, J. S. et al. Formulation, inflammation, and RNA sensing impact the immunogenicity of self-amplifying RNA vaccines. Mol. Ther. Nucleic Acids 31, 29–42 (2023).

33.     Ying, B. et al. Protective activity of mRNA vaccines against ancestral and variant SARS-CoV-2 strains. Sci. Transl. Med. 14, eabm3302 (2022).

34.     Rauch, S. et al. mRNA-based SARS-CoV-2 vaccine candidate CVnCoV induces high levels of virus-neutralising antibodies and mediates protection in rodents. NPJ Vaccines 6, 57 (2021).

35.     Mulroney, T. E. et al. 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: 260712_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 2a

    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 2b

    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 2c

    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 3

    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.

    Sheet 18 Name: Supp Figure 4

    Sheet 18 Description: Ratio of total number of CD4+ vs. CD8+ cells measured by ICCS in spleens of mice from Figure 4.

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/10⁶ 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: 26

3.  Variable list:

    Replicate

    Nucleotides

    Day

    % IFN-γ+ spot forming cells/10⁶ 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: 31

3.  Variable list:

    Replicate

    Nucleotides

    Day

    % IFN-γ+ spot forming cells/10⁶ 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: 181

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 2a

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 2b

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 2c

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 3

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.

DATA-SPECIFIC INFORMATION FOR: Supp Figure 4

1.  Number of variables: 4

2.  Number of rows: 31

3.  Variable list:

    Replicate

    Nucleotides

    CD4/CD8 Ratios

    Day

4.  Missing data codes:

    None

5.  Abbreviations used:

    m1Ψ = N1-Methylpseudouridine; CD: Cluster of differentiation.

6.  Other relevant information:

        Cells were acquired on a LSRFortessa flow cytometer (Becton Dickinson) and the ratio of CD4 to CD8 counts per sample was calculated.