Figure 3.

Evaluation of humoral and cellular immune responses in mice

BalB/c mice were immunized via intramuscular and oral routes. The intramuscular injection consisted of a single dose of 1 × 10⁷ CFU. The oral route of administration consisted of two doses of 1 × 10⁸ CFU at a 2-week interval. The immune responses were evaluated 3 weeks after the final inoculation. (A) IgG, IgG1, and IgG2a response in mice sera determined by ELISA. Data denote the OD₄₉₂ values against the corresponding sera dilution, collected from five biologically independent mice per group. Dotted lines indicate the cutoff value determined by multiplying the mean OD₄₉₂ values of healthy sera by 2.1. (B) The ratio of IgG2a to IgG1, compared with a theoretical Th1 or Th2 immune response. The ratio was calculated from the data obtained at 1:100 sera dilution. Percentages of (C) CD4⁺, (D) CD8⁺, (E) CD4⁺IFNγ, and (F) CD8⁺IFNγ T cells. Data represent the total change in cell population in response to stimulation of splenocytes with respective immunogens. (G) Splenocyte proliferation index in immunized mice compared with the controls. (H) Representative photographs of IFN-γ ELISpot in splenocytes stimulated with respective proteins. Images were taken at a magnification of 50×. The total number of spots counted per 1 × 10⁵ splenocytes is presented in the right panel. IM, intramuscular; PO, per os; M, membrane glycoprotein, Data in (A) were analyzed by unpaired Student's t test. Data in (C)–(H) were analyzed by two-way ANOVA using Šídák's or Tukey's multiple comparisons test. Data in (C)–(H) represent six biologically independent mice per group. The data points represent the individual value from each animal and error bars denote the SEM at 95% confidence interval (CI). ns, p > 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.