Cells were harvested 5 days later, washed and serial dilutions commencing at 5610 5 cells/ml performed in polyvinylidene fluoride (PVDF) membrane-lined 96-well plates (Millipore, Ireland) previously coated with 5 mg/ml anti-IFN-c capture antibody (clone R4-6A2-BD Pharmingen, San Diego, USA). Cells were then cultured in the presence of 3.75610 5 irradiated autologous VLP-pulsed (10 mg) splenocytes for 40 hours at 37uC and 5% CO 2 . After washing with PBST (PBS containing 0.05% Tween 20), biotinylated anti-IFN-c detection antibody (clone XMG1.2; Becton Dickinson, USA) was added and incubated for 2 hours at room temperature in a humidified atmosphere. Plates were then washed and streptavidin-alkaline phosphatase (Becton Dickinson, USA) added and incubated for a further 2 hours. Spots representative of IFN-c-producing cells were developed by the addition of 100 ml of 1 mg/ml 5-bromo-4-chloro-3-indolyl phosphate in 2-amino-2-methyl-1-propanol buffer (Sigma-Aldrich, USA) for 30 minutes. Individual spots were counted using an AID iSpot EliSpot Reader (GmbH, Strassberg, Germany). Analysis of variance in all experiments and all p values in this study were conducted and obtained using one-way ANOVA nonparametric statistical analysis and Tukey's post-hoc range tests performed with Prism 5 (GraphPad Software, La Jolla, California USA). Flat bottom 96-well polyvinyl plates were coated with either purified HCV VLPs (10 mg/ml) or recombinant E2 protein (5 mg/ ml) in PBSN 3 overnight at 4uC. Prior to coating plates with HCV VLPs, wells were pre-incubated with Galanthus nivalis lectin (2 mg/ml; Sigma Aldrich Australia) in carbonate buffer (15 mM NaCO 3 , 35 mM NaHCO 3 , 0.21 mM NaCl) for 30 minutes at room temperature Following removal of antigen, 100 ml of BSA (10 mg/ml) in PBS was added and plates incubated for 1 hour at room temperature before washing four times with PBST (PBS containing v/v 0.05% Tween-20 [Sigma Aldrich, Milwaukee, USA]). Serial dilutions of sera obtained from immunised mice were added to wells and incubated in a humidified atmosphere overnight. After washing, bound antibody was detected using horseradish peroxidase-conjugated rabbit antimouse IgG antibodies (Dako, Glostrup, Denmark) in conjunction with enzyme substrate (0.2 mM 2,2_-azino-bis 3-ethylbenzthiazoline-sulfonic acid in 50 mM citric acid containing 0.004% hydrogen peroxide). The reaction was stopped by addition of 50 ml of 0.05 M NaF. Titers of antibody are expressed as the reciprocal of the highest dilution of serum required to achieve an optical density of 0.2.