*2.7. Detection of T-Cell Immune Response Using IFN*γ *ELISpot and Intracellular Cytokine Staining (ICS) Assay*

Enzyme-Linked ImmunoSpot (ELISpot) and intracellular cytokine staining (ICS) assays were used to characterize the immune response of mice after immunization with DNA vaccines. Stimulation of splenocytes was carried out using a mix of synthetic peptides (KFINKLDALH, NYNGLLSSI, PGPAKFSLL, YFTFDLTALK, EYLFEVDNL, LFLRATTEL, and LYDRLASTV) from the compound of the designed antigens. Peptides were synthesized by Synpeptide Co., Ltd. (Shanghai, China) with >80% purity. Analysis of IFNγ ELISpot was performed with Mouse IFN-γ ELISPOT Set (BD, cat 551083, San Diego, CA, USA) according to the manufacturer's instruction and as previously described [29]. To stimulate splenocytes, we used a mix of peptides at concentration 20 μg/mL of each peptide to 1 <sup>×</sup> 10<sup>6</sup> cells followed by co-cultivation for 24 h. IFNγ-producing cells were calculated using an ELISpot-analyzer (Zeiss, Germany). ICS was performed according to the standard protocol of BD Biosciences as previously described [30]. To stimulate splenocytes, we used a mix of peptides at concentration 20 <sup>μ</sup>g/mL of each peptide to 1 <sup>×</sup> 106 cells and incubated for 20 h at 37 ◦C and 5% CO2 and additionally for 5 h with Brefeldin A. Cells were washed with PBS and permeabilized with Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit (BD Biosciences, San Diego, CA, USA). When staining, the following monoclonal antibodies were used: PerCP Rat Anti-Mouse CD4, FITC Rat Anti-Mouse CD8a, PE Hamster Anti-Mouse CD3ε, and APC Rat Anti-Mouse IFN-γ (BD Pharmingen, San Diego, CA, USA). The samples were analyzed using flow cytometer FACSCalibur (Becton Dickinson, San Jose, CA, USA) and Cell Quest software.
