Dataset for 'Identification of immunodominant T cell epitopes in SARS-CoV-2 spike protein in Syrian hamsters'
Description
This dataset contains all raw data underlying the main and supplementary figures of the manuscript “Identification of Immunodominant T Cell Epitopes in the SARS-CoV-2 Spike Protein in Syrian Hamsters” published in The Journal of Immunology. It includes measurements of T cell responses in Syrian hamsters immunized with a replication-defective ChAd-SARS-CoV-2-S vaccine encoding a pre-fusion–stabilized Wuhan-1 spike protein. The dataset also provides comparative T cell–response data following in vivo CD4 or CD8 depletion. In addition, it identifies key CD4 and CD8 epitopes within the most immunogenic regions of the spike protein.
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To generate the presented dataset, we conducted a systematic analysis of T cell responses in Syrian hamsters following intranasal immunization with a replication-defective ChAd-SARS-CoV-2-S vaccine encoding a pre-fusion stabilized Wuhan-1 S protein. All animal experiments complied with NIH guidelines and were approved by the Washington University IACUC (A3381–01). Five-week-old male hamsters from Charles River Laboratories and Envigo/Inotiv were acclimated prior to immunization. Animals received a single intranasal dose (10¹⁰ VP in 100 µL PBS) or, for comparison, two intramuscular doses of mRNA vaccine (BNT162b2, 5 µg). At 7 or 30 days post-immunization, animals were euthanized and lungs, cervical draining lymph nodes (DLNs), and spleens were collected. Tissues were digested enzymatically (Liberase + DNase I) and dissociated to single-cell suspensions. Cell viability was assessed by acridine orange/propidium iodide staining on an automated Nexcelom counter. Antigen-specific responses were measured using an ELISpot assay (MABTECH Hamster IFN-γ kit) on PVDF plates. Cells (5×10⁵/well) were stimulated with overlapping peptide pools (15-mers overlapping by 10 aa) spanning the SARS-CoV-2 S protein, sub-pools (10–11 peptides each), or individual CD4⁺ (13–15-mer) and CD8⁺ (8–10-mer) peptides. Peptides were synthesized by solid-phase peptide synthesis (SPPS), purified by reverse-phase HPLC, and validated by ESI-MS/MALDI-TOF before lyophilization and storage at −80°C. Positive controls included PMA/ionomycin, and negative controls contained 1% DMSO. Spot development used biotinylated anti-IFN-γ, streptavidin-ALP, and BCIP/NBT substrate, quantified by a CTL BioSpot analyzer. To define MHC restriction, peptide-stimulated splenocytes were pretreated with anti-MHC class II mAbs (I-Eᵏ clone 14-4-4S, I-A clone Y3JP, and HLA-DR clone L243) before ELISpot measurement. CD4⁺ and CD8⁺ T cell depletions were performed using Invitrogen magnetic bead kits conjugated to anti-CD4 (GK1.5) or anti-CD8β (341) antibodies, with depletion efficiency verified by flow cytometry (Cytek Aurora). For immunophenotyping, single-cell suspensions were stained with CD4-PE, CD8β-BB700, and B220-PE/Cy7, excluding dead cells via Zombie Aqua dye, and analyzed with FlowJo v10. Expression of MHC class I and II molecules was verified using in-house purified monoclonal antibodies (validated via ATCC hybridomas) on Bio-Rad ZE5 cytometer, with FITC-conjugated secondary antibodies and isotype controls for specificity. Protein domain mapping and cross-species epitope conservation were assessed using Clustal Omega v1.2.4 for multiple sequence alignment of S protein sequences from Wuhan-Hu-1 and Omicron sublineages. Domain annotations were based on PDB structural references, and motif conservation was analyzed across NCBI and GISAID datasets. All statistical analyses were performed in GraphPad Prism 10.1, using one-way ANOVA with post hoc tests or paired t-tests, considering p < 0.05 as significant.
Institutions
- Washington University in St. Louis
- La Jolla Institute for Allergy and Immunology
- Yeshiva University Albert Einstein College of Medicine
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Additional Metadata for Digital Commons Data@Becker
| Keywords | SARS-CoV-2, Syrian hamsters, T cells, T cell epitopes, Interferon-gamma |