The induction of antigen-specific T cells was determined using ICS. For ICS, splenocytes from vaccinated and control mice were isolated and single-cell suspensions were prepared in supplemented medium. Then, 2 × 106 splenocytes (200 µl) per well were stimulated for 5–6 h at 37 °C using an SARS-CoV-2 peptide library (JPT, PM-WCPV-S2) at 0.5 µg/ml. After 1 h, Golgi Plug (BD Biosciences, Cat: 555029) was added in a dilution of 1:200 (50 µl) to the splenocytes to inhibit the secretion of intracellular cytokines. After stimulation, splenocytes were centrifuged, resuspended in supplemented medium and stored at 4 °C overnight. Following this, splenocytes were washed twice in PBS and stained with AquaDye (Invitrogen, Cat: L34957) solution at 4 °C for 30 min. After an additional washing step in FACS buffer (PBS with 0.5% BSA), cells were surface stained for Thy1.2, CD4 and CD8 and incubated with FcƴR-block for 30 min at 4 °C in FACS buffer. After surface staining, splenocytes were washed in FACS buffer and fixed using Cytofix/Cytoperm (BD Biosciences) according to the manufacturer’s instructions. After fixation, splenocytes were washed in perm buffer and stained for IFN-ƴ and TNF for 30 min at 4 °C. After staining, the cells were washed with perm buffer, resuspended in FACS buffer supplemented with 2 mM EDTA and 0.01% Natriumacid and stored at 4 °C until analysis. Splenocytes were analysed on a Canto II flow cytometer (BD Biosciences). Flow cytometry data were analysed using FlowJo software (Tree Star, Inc, Ashland, USA.).

The following antibodies were used for flow cytometry analysis: anti-Thy1.2 FITC (clone 53-2.1; Biolegend, Cat.14304), anti-CD4 V450 (clone RM4-5; BD Biosciences, Cat. 560468), anti-CD8a APC-H7 (clone 53-6.7; BD Biosciences, Cat. 560182), anti-IFNƴ APC (clone XMG1.2, BD Biosciences, Cat. 554413) and anti-TNF PE (clone MP6-XT22, eBioscience, Cat. 25-7321-82).

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