Characterization of antibody responses to rBLS after immunization with pcDNA-BLS or rBLS. Mice (five per group) were immunized with pcDNA-BLS or rBLS and bled retroorbitally. The titers of antibodies were evaluated by ELISA. Data are representative of two separate experiments. (A) Kinetics of the anti-BLS humoral response in mice immunized with 100 μg of pcDNA-BLS (solid lines) or 10 μg of rBLS (dashed lines). The squares represent the mean titers± SD (error bars) of antibodies in five animals. The arrows indicate the days of inoculation. PI, postimmunization. (B) Antibody isotype profiles of mice immunized with pcDNA-BLS or rBLS. IgG1 (filled bars) and IgG2a (open bars) antibody titers of mice immunized with pcDNA-BLS or rBLS were studied by ELISA 60 days after the immunization. Each bar represents the mean titers ± SD of antibodies in five animals. ★, significantly different from titer of IgG2a in mice immunized with rBLS (P < 0.01). P values were calculated by the Kruskal-Wallis test.

(A) Concentrations of rBLS-induced IFN-γ in cells from mice immunized with pcDNA-BLS. Spleen cells (4 × 10⁶/ml) from pcDNA3- and pcDNA-BLS-immunized mice were stimulated with RPMI 1640, rBLS (5 μg/ml), or ConA (2.5 μg/ml) for 48 h. Levels of IFN-γ in the cell supernatants were quantified by antibody capture ELISA. Each bar represents the geometric mean ± SD (error bar) of the responses in spleen cells from five individual mice, with experiment run in duplicate. ★, significantly different from results for RPMI 1640-stimulated cells of mice immunized with pcDNA-BLS (P < 0.05). The data are representative of two separate experiments. (B) rBLS-induced IL-2 mRNA expression in cells from mice immunized with pcDNA-BLS. Spleen cells (4 × 10⁶/ml) from pcDNA3- and pcDNA-BLS-immunized mice were stimulated with RPMI 1640, rBLS (5 μg/ml), or ConA (2.5 μg/ml) for 24 h. The induced mRNA levels of IL-2 were determined by RT-PCR. The responses are shown in terms of the increases (n-fold) in the IL-2 levels in stimulate cells over that in unstimulated cells. All values were normalized with respect to β-actin mRNA levels. Each bar represents the geometric mean ± SD of the responses in spleen cells from five individual mice, with experiments run in duplicate. ★, significantly different from the rBLS-induced IL-2 levels in cells of mice immunized with pcDNA3 (P < 0.05). The data are representative of two separate experiments.