Evaluation of in vitro detectable CTLs and tumor-inhibiting responses in C57BL/6J mice immunized with wtNS3/4A-pVAX1 or wtNS3/4AΔ5,9-pVAX1 plasmid DNA. (a) Groups of five C57BL/6J mice were immunized once with 2 μg plasmid DNA using transdermal gene gun (gg) delivery or were left untreated (non-immunized). Two weeks after the last immunization, the NS3-specific lytic activity was determined using peptide-loaded (GAVQNEVTL) RMA-S cells at E:T ratios of 60:1, 20:1 and 7:1 in a standard 51Cr-release assay. Specific lysis above 10% was considered positive. Each line indicates an individual mouse. In (b), the in vitro peptide stabilization of MHC–peptide complexes on transporter associated with antigen processing 2-deficient RMA-S cells transfected with H-2Db is shown. Binding affinities were determined by flow cytometry measuring the mean fluorescence intensity (MFI) for each peptide in descending concentrations. (c) Groups of 10 C57BL/6J mice were either left untreated or were given one gg immunizations of 2 μg wtNS3/4A or wtNS3/4AΔ5,9 plasmid DNA. At 2 weeks after immunization, mice were subcutaneously inoculated with 1 × 106 NS3/4A-expressing EL-4 cells. Tumor sizes were measured through the skin at days 6–18 after tumor inoculation. Values are given as the mean tumor size±s.e.m. Also given is the P-value obtained from the statistical comparison of wtNS3/4A and non-immunized or wtNS3/4AΔ5,9 and non-immunized or wtNS3/4A and wtNS3/4AΔ5,9 using the AUC and analysis of variance. NS, not significant.
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