Splenocyte proliferation assay

All mice were killed on day-42 to collect spleen. Spleen were washed with PBS (0.01 M, pH 7.4) and placed in cell culture dish containing complete media, i.e., Roswell Park Memorial Institute media (RPMI1640) supplemented with 10% Fetal Bovine Serum (FBS) and 100 U/mL penicillin–streptomycin. Spleen were further processed to prepare a single cell suspension of splenocytes. Using sterile blade, spleen was cut into small pieces in a culture dish containing RPMI1640 media followed by crushing the slices with the plunger end of needle. Above suspension was passed through a cell strainer (70 µm) and collected in a 50-mL falcon to prepare single cell suspension of splenocytes. Cell suspensions were centrifuged, supernatants were discarded and pellets resuspended in RBC lysis buffer followed by centrifugation to collect pellet. Pellet was washed with PBS three times followed by suspension in RPMI1640 media. Splenocytes were cultured in a 96-well plate and stimulated in the presence of HEV ORF2 protein (20 µg/mL), wells containing untreated cells served as positive control and wells containing RPMI1640 media alone was taken as blank. Cells treated with Concanavalin A served as positive control. Splenocytes isolated from mice immunized with all formulation with or without antigen (GNP26, GNP54, IFA26, IFA54, GNP only, IFA only) were stimulated with 26 kDa and 54 kDa HEV protein, respectively. To measure antigen-specific proliferation, MTS assay was performed. After 2 days, MTS was added to the microwells and incubated for 3 h and finally absorbance (OD) was taken at 490 nm. Stimulation index was calculated as: