Fig. 5.

Induction of SARS-CoV-2-specific cellular immunity by AdCoV2 vaccines. Five mice per group of BALB/c were individually primed and boosted at 14-day intervals though i.n. or s.c. routes with 1 × 10⁷ pfu of AdCoV2 or Ad-LacZ. Ad-immunized mice were sacrificed 3 months after the vaccine boost, and splenocytes were collected and cultured in the presence or absence of 10 µg/mL S protein or 10 µg/ml Con A. A total of 5 x10⁵ splenocytes were seeded on (A) anti-IFN-ɣ or (B) anti-IL-4 capture antibody-coated ELISPOT plates for 24 or 48 h, respectively, for the ELISPOT assay. Cytokine-positive spots were developed, and the obtained number of spots from S-stimulated lymphocytes was then subtracted to the number of spots gained from the respective well with medium only. The results are expressed as the number of spots for each mice in the experimental group. (C) The ratio of IFN-γ-positive spot numbers/IL-4-positive spot numbers was calculated and shown. Two independent experiments were performed and one of the representative data was shown. In parallel, the results regarding the percentage of (D) IFN-γ⁺ or (E) IL-4⁺ populations in CD3⁺CD4⁺ splenocytes with or without S protein restimulation were shown. The method was described in the section of Materials and Methods. Briefly, S-stimulated splenocytes were double stained with anti-CD3 and anti-CD4 antibodies conjugated with APC and PE-Cyanine5- dye, respectively. After fixation and permeabilization, splenocytes were stained with (D) FITC-conjugated IFN-γ-specific antibody or (E) PE-conjugated IL-4-specific antibody. The 3-colouried splenocytes were individually analyzed by flow cytometry.