RECOMMENDED README FILE FOR GREDOS_USAL This readme.txt file was generated on 20260415 by Javier Sánchez Montejo ---------------------------------- GENERAL INFORMATION ---------------------------------- 1. Title of Dataset: Raw data of Multiepitope mRNA vaccine against Fasciola hepatica confers T-cell-mediated protection in mice. 2. Authors: ✓ Name: Javier Sánchez-Montejo 1,2, Cristina Teodosio 2,3,4,5,6, Julio López-Abán 1,2, Raúl Manzano-Román 1,2, Julio Pozo 4,7, María Ángeles Solinís 8,9, Ana del Pozo-Rodríguez 8,9, Tania Strilets 10, Mariano A. García-Blanco 10,11, Belén Vicente 1,2*, and Antonio Muro 1,2* ✓ Institution: 1. Infectious and Tropical Diseases Research Group (e-INTRO), Research Center for Tropical Diseases at the University of Salamanca (IBSAL-CIETUS), 37007 Salamanca, Spain. 2. Biomedical Research Institute of Salamanca (IBSAL), Salamanca, Spain. 3. Cancer Research Center (IBMCC, USAL-CSIC), Salamanca, Spain. 4. Cytometry Service (NUCLEUS), University of Salamanca (USAL), Salamanca, Spain. 5. Department of Medicine, University of Salamanca (USAL), Salamanca, Spain. 6. Centro de Investigación Biomédica en Red Cáncer (CIBERONC; CB16/12/00400), Madrid, Spain. 7. Cytognos SL/Water Biosciences (formerly BD Biosciences), Salamanca, Spain. 8. PharmaNanoGene, Faculty of Pharmacy, Department of Pharmacy and Food Sciences, Centro de Investigación Lascaray Ikergunea, University of Basque Country, UPV/EHU, Vitoria-Gasteiz, Spain. 9. Bioaraba, Microbiology, Infectious Disease, Antimicrobial Agents and Gene Therapy, Vitoria-Gasteiz, Spain. 10. Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA 22908, USA. 11. Center for RNA Science and Medicine, University of Virginia, Charlottesville, VA 22908, USA. ✓ Email: s.montejo@usal.es, belvi25@usal.es, ama@usal.es ✓ ORCID: 0000-0002-4227-9834 ------------------- DESCRIPTION ------------------- 1. Dataset language: English 2. Abstract: This dataset contains raw spectral flow cytometry files (FCS format) generated in a mouse immunization and challenge study evaluating an mRNA–solid lipid nanoparticle (mRNA-SLN) vaccine candidate encoding three MHC class II T-cell epitopes from Fasciola hepatica fused to eGFP (eGFP-Fh3Tq). BALB/c mice (n = 33) were allocated into four experimental groups: Untreated (n = 9), Infection control (n = 6), eGFP (n = 9), and eGFP-Fh3Tq (n = 9). Groups eGFP and eGFP-Fh3Tq received a prime–boost immunization regimen (21 days apart) with the corresponding mRNA-SLN formulation. Peripheral blood was collected from three groups (Untreated, eGFP, and eGFP-Fh3Tq) at day 1 post-vaccination (innate panel) and day 42 post-vaccination (adaptive panel); the Infection control group was not differentiated from the Untreated group at these time points and was therefore not sampled separately. For blood panels, samples from two mice were pooled and barcoded with anti-CD45 conjugated to either PerCP or BUV496 before acquisition, yielding three FCS files per group (6 mice/group). At day 42, spleens from three mice per group (Untreated, eGFP, eGFP-Fh3Tq) were harvested for intracellular cytokine staining (ICS) after ex vivo restimulation with the synthetic peptides T14, T15, and T16, PMA/ionomycin (positive control), or left unstimulated (negative control). For ICS, individual splenocyte samples from different conditions were barcoded with anti-CD45 and combined in pairs within each acquisition tube. Where sufficient cells were available, stimulation conditions were performed in duplicate. Remaining mice were orally challenged with 7 F. hepatica metacercariae for survival and protection assessment. All samples were acquired on a Cytek Aurora 5L spectral flow cytometer equipped with five lasers (355, 405, 488, 561, 640 nm). 3. Keywords: Fasciola hepatica, parasite, trematode, mRNA vaccine, solid lipid nanoparticles, T-cell epitopes, cytometry, multiepitope 4. Date of data collection: 27/02/2025-11/03/2026 5. Date of data publication on repository: 6. Funding (Information about funding sources that supported the collection of the data): Financial support from Grant PID2022-136462NB-I00 funded by "Ministerio de Ciencia e Innovación" and cofinanced by "European Union". T.S. and M.A.G.-B. acknowledge funding from the University of Virginia. J.S.-M. acknowledges the predoctoral fellowship program of Junta de Castilla y León, co-funded by "Fondo Social Europeo" (Orden EDU875/2021). C.T. was supported by an Andrés Laguna fellowship (Junta de Castilla y León, co-financed by the Fondo Social Europeo Plus, FSE+; ORDEN EDU/300/2025). M.A.S. and A.dP.-R. acknowledge the funding from the Department of Education of the Basque Government (IT1587-22, GIC21/34). 7. Geographic location of data collection : Salamanca, Spain 8. Recommended citation for this dataset: --------------------------------------------------------- SHARING/ACCESS/CONTEXT INFORMATION --------------------------------------------------------- 1. Usage Licenses: Creative commons 4.0- BY-NC-ND 2. Related publications: Sánchez-Montejo et al., 2026 3. Dataset DOI: -------------------------------- DATA & FILE OVERVIEW -------------------------------- 1. File List: Group key: G1 = Untreated (PBS control), G2 = eGFP (eGFP mRNA-SLN), G3 = eGFP-Fh3Tq (eGFP-Fh3Tq mRNA-SLN). R = biological replicate (individual mouse). Blood files contain two barcoded mice per tube (e.g., R1_R2). ICS files contain two barcoded conditions per tube. Stimulation conditions (ICS): PHAIono = PMA/ionomycin positive control; CTRL = unstimulated negative control; Pep = T14+T15+T16 peptide pool. Suffixes _1 and _2 denote technical duplicates of the same condition. Folder D1 — Peripheral blood, day 1 post-vaccination (innate panel) 1. PROT_Blood_D1_G1-Untreated_R1_R2_Unmixed.fcs 2. PROT_Blood_D1_G1-Untreated_R3_R4_Unmixed.fcs 3. PROT_Blood_D1_G1-Untreated_R5_R6_Unmixed.fcs 4. PROT_Blood_D1_G2-eGFP_R1_R2_Unmixed.fcs 5. PROT_Blood_D1_G2-eGFP_R3_R4_Unmixed.fcs 6. PROT_Blood_D1_G2-eGFP_R5_R6_Unmixed.fcs 7. PROT_Blood_D1_G3-eGFP-Fh3Tq_R1_R2_Unmixed.fcs 8. PROT_Blood_D1_G3-eGFP-Fh3Tq_R3_R4_Unmixed.fcs 9. PROT_Blood_D1_G3-eGFP-Fh3Tq_R5_R6_Unmixed.fcs Folder D42 — Peripheral blood, day 42 post-vaccination (adaptive panel) 1. PROT_Blood_D42_G1-Untreated_R1_R2_Unmixed.fcs 2. PROT_Blood_D42_G1-Untreated_R3_R4_Unmixed.fcs 3. PROT_Blood_D42_G1-Untreated_R5_R6_Unmixed.fcs 4. PROT_Blood_D42_G2-eGFP_R1_R2_Unmixed.fcs 5. PROT_Blood_D42_G2-eGFP_R3_R4_Unmixed.fcs 6. PROT_Blood_D42_G2-eGFP_R5_R6_Unmixed.fcs 7. PROT_Blood_D42_G3-eGFP-Fh3Tq_R1_R2_Unmixed.fcs 8. PROT_Blood_D42_G3-eGFP-Fh3Tq_R3_R4_Unmixed.fcs 9. PROT_Blood_D42_G3-eGFP-Fh3Tq_R5_R6_Unmixed.fcs Folder ICS — Splenocytes, day 42 post-vaccination (intracellular cytokine staining panel) 1. PROT_Spleen_ICS_01_G1R1-PHAIono1_G1R1-CTRL1_Unmixed.fcs 2. PROT_Spleen_ICS_02_G1R1-Pep1_G1R1-Pep2_Unmixed.fcs 3. PROT_Spleen_ICS_03_G1R2-PHAIono1_G1R2-PHAIono2_Unmixed.fcs 4. PROT_Spleen_ICS_04_G1R2-CTRL1_G3R1-CTRL1_Unmixed.fcs 5. PROT_Spleen_ICS_05_G1R2-Pep1_G1R2-Pep2_Unmixed.fcs 6. PROT_Spleen_ICS_06_G1R3-PHAIono1_G1R3-PHAIono2_Unmixed.fcs 7. PROT_Spleen_ICS_07_G1R3-CTRL1_G1R3-CTRL2_Unmixed.fcs 8. PROT_Spleen_ICS_08_G1R3-Pep1_G1R3-Pep2_Unmixed.fcs 9. PROT_Spleen_ICS_09_G2R1-PHAIono1_G2R1-CTRL1_Unmixed.fcs 10. PROT_Spleen_ICS_10_G2R1-Pep1_G2R1-Pep2_Unmixed.fcs 11. PROT_Spleen_ICS_11_G2R2-PHAIono1_G2R2-PHAIono2_Unmixed.fcs 12. PROT_Spleen_ICS_12_G2R2-CTRL1_G2R2-CTRL2_Unmixed.fcs 13. PROT_Spleen_ICS_13_G2R2-Pep1_G2R2-Pep2_Unmixed.fcs 14. PROT_Spleen_ICS_14_G2R3-PHAIono1_G2R3-PHAIono2_Unmixed.fcs 15. PROT_Spleen_ICS_15_G2R3-CTRL1_G2R3-CTRL2_Unmixed.fcs 16. PROT_Spleen_ICS_16_G2R3-Pep1_G2R3-Pep2_Unmixed.fcs 17. PROT_Spleen_ICS_17_G3R1-PHAIono1_G3R1-PHAIono2_Unmixed.fcs 18. PROT_Spleen_ICS_18_G3R1-Pep1_G3R1-Pep2_Unmixed.fcs 19. PROT_Spleen_ICS_19_G3R2-PHAIono1_G3R2-Pep1_Unmixed.fcs 20. PROT_Spleen_ICS_20_G3R2-CTRL1_G3R3-CTRL1_Unmixed.fcs 21. PROT_Spleen_ICS_21_G3R3-PHAIono1_G3R3-PHAIono2_Unmixed.fcs 22. PROT_Spleen_ICS_22_G3R3-Pep1_G3R3-Pep2_Unmixed.fcs 2. Relationship between files, if important: For D1 and D42 folders, each FCS file contains two barcoded mice from the same group, identified by anti-CD45 conjugate (PerCP vs. BUV496). For the ICS folder, each FCS file contains two barcoded splenocyte samples from the same or different conditions, identified by anti-CD45 conjugate. Tubes 04 and 20 contain barcoded samples from different groups to accommodate unequal cell availability. 3. File format: fcs ----------------------------------------------- METHODOLOGICAL INFORMATION ---------------------------------------------- 1. Instrument- or software-specific information needed to interpret/reproduce the data, please indicate their location: Samples were acquired on a Cytek Aurora 5L spectral flow cytometer (Cytek Biosciences, Fremont, CA) equipped with five lasers (355 nm, 405 nm, 488 nm, 561 nm, 640 nm). Spectral unmixing was performed using SpectroFlo software (v3.3.0; Cytek). Flow cytometric data were analyzed using Infinicyt software version 2.1.0.a (BD Biosciences, San Jose, CA, USA). 2. Describe any quality-assurance procedures performed on the data: Raw data. Daily instrument quality control was performed using SpectroFlo QC beads (Cytek) before sample measurement. Single-stained reference controls and unstained controls were used for spectral unmixing validation. 3. Author contact information: Javier Sánchez-Montejo s.montejo@usal.es