Fig. 5: The NVT-formulated intranasal peptide vaccine did not show synergistic effects in the BCG prime and vaccine boost models.

a Experimental design. C57BL/6 mice (n = 23 per group) were divided into BCG-primed and non-primed groups, and mice in the BCG-primed group were immunized subcutaneously (s.c.) with BCG. At 13 weeks post-priming, all mice were immunized intranasally (i.n.) twice at 3-week intervals with NVT or Pep 5′ + NVT. Four weeks after the final immunization, immune response analysis (n = 5 per group) or aerosol challenge with HN878 (n = 20 per group) were performed. At 4 and 16 weeks post-infection (wpi), mice (n = 9 per group at each time point) were sacrificed for bacterial counts (n = 5 per group) and lung histopathology (n = 4 per group). b, c IFN-γ⁺CD4⁺ T cell responses to Pep 5′ in the lungs were analyzed by flow cytometry. Representative dot plots (b) and percentages (c) of IFN-γ⁺CD4⁺ T cells. Bacterial loads in the lungs (d) and spleen (e) at 4 wpi. f, g Bacterial loads in the lungs (f) and spleen (g) at 16 wpi. H&E staining (h) and inflamed area (i) in lung tissues at 4 wpi. j, k H&E staining (j) and inflamed area (k) in lung tissues at 16 wpi. The box-and-whisker plots display the median (center line), 25th and 75th percentiles (box), and the minimum and maximum values (whiskers). Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparisons test. This experiment was performed once. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.