Enzyme-linked immunosorbent spot

Mice were vaccinated with different combinations of QIV ± adjuvant ± LNPs and spleens were harvested at 10 DPV from all vaccinated as well as unvaccinated animals. Spleens were collected in 5 mL of RPMI-1640 media supplemented with 2% FBS and 1× penicillin/streptomycin and kept on ice. A single-cell suspension of splenocytes was obtained by homogenizing the spleens against a 70-μm strainer. Interferon gamma (IFN-γ) or interleukin-4 (IL-4) ELIspot assays were performed using 10⁵ splenocytes per well in a 96-well polyvinylidene difluoride (PVDF) ELIspot plates provided in the ELIspot kits (Supplementary Table), precoated with IFN-γ or IL-4 capture antibodies, respectively, according to the manufacturer’s protocol. Splenocytes were left unstimulated or restimulated either with hemagglutination (HA-H1N1) overlapping 15-mer peptides or whole live IVR-180 (H1N1) virus and incubated overnight in 37°C incubator. The wells were washed thrice with water to get rid of cells and incubated with 100 μL of biotinylated polyclonal detection antibody against IFN-γ or IL-4 for 1.5 h at RT, followed by an incubation with streptavidin-HRP-conjugated antibody for 1 h at RT. The plates were finally incubated with 100 μL of the substrate for 1 h in dark, followed by thorough washing under tap water multiple times. The plates were air-dried in the dark and the number of spots in each well was manually counted using a dissection microscope. The results were represented as number of IFN-γ- or IL-4-producing splenocytes per million splenocytes.