Isolation of splenocyte and Interferon‐gamma (IFN-γ) ELISpot assay

The spleens of the mice were dissected and crushed using a Stomacher device (Seward, UK) and the splenocytes were filtered through a 40μm cell strainer (ThermoFisher, Waltham, MA, USA). For the lysis of red blood cells, the splenocytes were treated with Ammonium-Chloride-Potassium (ACK) lysis buffer (Quality Biologicals, MD, USA) for 5 minutes. Subsequently, Mouse IFN-γ ELISpot PLUS assay (Mabtech, Cincinnati, OH, USA) was carried out using the splenocytes resuspended in R10 as per the manufacturer’s protocol. Precisely, splenocytes from PCaA-SEV or pMV101 immunized mice were added at a density of 2x10⁵/well in plates and then incubated separately in the presence of only media (negative control), media along with cell activation cocktail (BioLegend, San Diego, CA, USA), pre-mixed phorbol 12-myristate-13-acetate (PMA) and ionomycin (positive control), and media with peptides with a final concentration of 1μg/ml, for 18 hours at 37°C in a 5% CO₂ regulated incubator. PCaA-SEV derived synthetic peptides were synthetized by Genscript, USA. The peptides were dissolved in DMSO and stored at -80°C. Bioinformatics approach using the SYFPEITHI website (www.syfpeithi.com) was utilized to define the dominant epitopes. Subsequently, upon addition of 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) color development substrate (R&D Systems, Minneapolis, MN, USA), formation of spots were observed and the spot forming units (SFU) were then quantified with the help of automated ELISpot reader (CTL Limited, Ohio, USA).