ELISPOT assay

ELISPOT assay cellular immune responses in vaccinated mice were assessed using IFN‐γ or IL‐4 precoated ELISPOT kits (Dakewe Biotech Co., Ltd.) according to the manufacturer's protocol. Briefly, single‐cell suspensions obtained from mouse spleens (1 × 10⁵ cells/well) were ground in cell strainers and seeded onto precoated ELISPOT plates. Subsequently, the cells were incubated with peptide pools (15‐mers overlapping by 13 aas, 2 µg/ml each) covering A/Victoria/2570/2019 (H1N1) HA stem proteins (GenScript) and cultured at 37°C with 5% CO₂ for 20 h. The same volume of 40% dimethyl sulfoxide (Sigma‐Aldrich), which was used to dissolve the peptide pool, was used as the negative control. Phorbol 12‐myristate 13‐acetate/ionomycin (Dakewe Biotech) was used as the positive control. The spots were counted and analyzed using CTL‐ImmunoSpot S5 (Cellular Technology Limited).