Female HHD mice (3 per group) were inoculated subcutaneously on each side of the base of tail (50 ml per dose) with VLPs (30 mg) either alone, emulsified in an equal volume of complete Freund's adjuvant (CFA) or with an equal amount of E 8 Pam 2 Cys on days 0 and 14. Spleens were removed 28 days after the second dose and splenocytes restimulated in vitro at a concentration of 3610 6 cells/ml in RF-10 medium consisting of RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal calf serum (CSL, Parkville, Australia) 7.5 mM HEPES, 2 mM L-glutamine, 76 mM 2-mercaptoethanol, 150 U/ml penicillin, 150 mg/ml streptomycin, 150 mM non-essential amino acids and 10 U/ml of recombinant IL-2 (Roche, Indianapolis, USA) at 37uC in an atmosphere of 5% CO 2 . Restimulation was carried out in the presence of 10 mg of VLPs or 10 mM of an irrelevant HCVderived HLA-A2-restricted epitope (NS5B2594-2602) that is not contained in the VLP construct.