Recombinant BCG vaccine expressing multistage antigens of Mycobacterium tuberculosis provides long-term immunity against tuberculosis in BALB/c mice

Figures & data

Figure 1. Schematic diagram of T-cell epitope distribution in nDnak and DnaK. The scale unit is amino acid (AA).

Figure 2. Construction of rBCG-ECD003. (a) Schematic of the mycobacteria-E. coli shuttle vector pMV361-ECD003. (b) Agarose gel electrophoresis of the PCR products amplified from rBCG-ECD003, transformed with pMV361 shuttle vectors. Lane 1, DNA marker; lane 2, the PCR product amplified from rBCG-pMV361; lane 3, the PCR product amplified from rBCG-ECD003.

Figure 3. Superior cytokines production profile induction in the spleen lymphocytes of the BALB/c mice immunized with rBCG-ECD003. The cytokines production profile was analyzed in isolated spleen lymphocytes at the 4^th and 12^th week after immunization. 1 × 10⁵ cells were restimulated with the fusion protein ECD003 (a) or PPD (b) and the concentration of cytokines (IFN-γ, TNF-α, IL-2, IL-12, IL-4, IL-6, IL-10, IL-17 and GM-CSF) was analyzed with multiplex cytokine assay. Results were shown as the mean ± SEM (n = 3) per group. Statistical significance was determined by ANOVA with Tukey’s multiple comparisons tests (*p < .05, **p < .01).

Figure 4. Vaccination with rBCG-ECD003 enhanced long-term responses of polyfunctional CD4⁺T cells in the spleen lymphocytes. Intracellular cytokine staining was performed in isolated spleen lymphocytes at the 12^th week post-vaccination. The proportion of cytokine-producing CD4⁺T cells after stimulation with the ECD003 (a) and PPD (b) respectively were shown as the mean ± SEM (n = 3) per group. Pie charts reflected the average proportion of ECD003-responding (c) and PPD-responding (d) CD4⁺T cells with each combination of cytokine production. Statistical significance was determined by ANOVA with Tukey’s multiple comparisons tests (*p < .05, **p < .01).

Figure 5. rBCG-ECD003 conferred superior humoral immunity in BALB/c mice. Assay of IgG antibodies against ECD003 (a) and PPD (b) in the sera of the immunized BALB/c mice. Serum samples were collected at the 4^th, 8^th, and 12^th week after immunization, and the titers of IgG were detected by ELISA. The results were shown as the Log₂ of the mean ± SEM (n = 6) per group. Statistical significance was determined by Student’s t-test. (*p < .05, **p < .01).

Figure 6. The protective efficacy of rBCG-ECD003 in vitro. The BALB/c mice (n = 6) were s.C. immunized with PBS, BCG, and rBCG-ECD003 respectively. The spleen lymphocytes were isolated 4th and 12th week post-immunization and co-cultured with ~ 500 CFU of mtb H37Rv. The bacterial load results of the 4th week (a) and 12th week (b) were presented as mean ± SEM. Statistical significance was determined by ANOVA with Tukey’s multiple comparisons tests (*p < .05, **p < .01).

Figure 7. The safety analysis of the rBCG-ECD003 vaccine after immunization. The BALB/c mice were immunized with 1 × 10⁶ CFU of the parental BCG or rBCG-ECD003. At the 4th and 12th week, the mice were sacrificed, and the parental BCG and rBCG-ECD003 CFU recovered from lung (a), and spleens (b) The liver, spleen, and lung samples were collected and performed with H&E staining (c). The typical pathological changes in each group were shown.