An ELISPOT assay was performed as previously described with minor modifications [2] . Briefly, a 96-well nitrocellulose plate (MultiScreen-HA, Millipore, Billerica, MA, USA) was coated with an anti-mouse IFN-c antibody (5 lg/mL, eBioscience) at 4°C for 20 h followed by blocking with an IMDM medium (Invitrogen) supplemented with 5% FBS (Invitrogen) at 37°C for 2 h. Splenocytes (1 · 10 6 ) were seeded into the four wells of a 96-well nitrocellulose plate, and the corresponding target peptides (10 lg/mL) were added. The plate was incubated at 37°C for 24 h. The mice immunized with N220HHDpBudCE4.1 were used as experiment controls. The splenocytes with N220 peptide added were used as positive controls while those added with N160 peptide served as negative controls [2] . Subsequently, the plate was washed with 1· PBS containing 0.01% Tween 20 and incubated with a biotinylated anti-mouse IFN-c antibody (0.5 lg/mL, eBioscience) at room temperature for 1 h. After washing, a streptavidin-AP solution (1 lg/mL, Sigma-Aldrich Corp) was added and the plate was incubated at room temperature for 30 min. After a final wash, spots were developed by adding a BCIP/NBT solution (Invitrogen) and the visible spots were counted by an ELISPOT reader (Thermo Labsystems, Franklin, MA, USA).