Fig. 3: The intranasal peptide vaccine provides effective protection against TB in the lungs.

a Experimental design. C57BL/6 mice (n = 14 per group) were immunized intramuscularly (i.m.) or intranasally (i.n.) twice at 3-week intervals with NVT-formulated Pep 5′. Four weeks after the final immunization, immune response analysis (n = 5 per group) or aerosol challenge with H37Rv (n = 9 per group) were performed. b Body weight was monitored daily for 5 days after both the first and second immunizations. c–f IFN-γ⁺CD4⁺ T cell responses to Pep 5’ in the lungs and spleen were analyzed by flow cytometry. Representative dot plots (c) and percentages (d) of IFN-γ⁺CD4⁺ T cells in the lung. Representative dot plots (e) and percentages (f) of IFN-γ⁺CD4⁺ T cells in the spleen. CD4⁺ T_RM cells (CD4⁺CD44⁺CD62L^-CD69⁺) in the lungs were analyzed by flow cytometry. Representative dot plots (g), percentages (h), and absolute numbers (i) of CD4⁺ _RM cells in the lung. j Lung CD4⁺ T cell subsets based on CD44 and CD62L expression. CD4⁺ T cells were classified into naïve (T_N; CD44⁻CD62L⁺), central memory (T_CM; CD44⁺CD62L⁺), effector (T_E; CD44⁻CD62L⁻), and effector memory (T_EM; CD44⁺CD62L⁻) subsets. k–m The immunized mice (n = 9 per group) were infected with H37Rv via aerosol exposure. At 4 weeks post-infection, mice were sacrificed for bacterial counts (n = 5 per group) and lung histopathology (n = 4 per group). Bacterial loads in the lungs (k) and spleen (l). H&E staining (m) and inflamed area (n) in lung tissues. 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. Data are representative of two independent experiments. Unstim., unstimulated; *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant.