Recombinant BCG-Prime and DNA-Boost Immunization Confers Mice with Enhanced Protection against Mycobacterium kansasii

Abstract

The incidence of infections with nontuberculous mycobacteria (NTM) has been increasing worldwide. The emergence of multidrug-resistant NTM is a serious clinical concern, and a vaccine for NTM has not yet been developed. We previously developed a new recombinant Bacillus Calmette-Guérin (rBCG) vaccine encoding the antigen 85B (Ag85B) protein of Mycobacterium kansasii-termed rBCG-Mkan85B-which was used together with a booster immunization with plasmid DNA expressing the same M. kansasii Ag85B gene (DNA-Mkan85B). We reported that rBCG-Mkan85B/DNA-Mkan85B prime-boost immunization elicited various NTM strain-specific CD4⁺ and CD8⁺ T cells and induced Mycobacterium tuberculosis-specific immunity. In this study, to investigate the protective effect against M. kansasii infection, we challenged mice vaccinated with a rBCG-Mkan85B or rBCG-Mkan85B/DNA-Mkan85B prime-boost strategy with virulent M. kansasii. Although BCG and rBCG-Mkan85B immunization each suppressed the growth of M. kansasii in the mouse lungs, the rBCG-Mkan85B/DNA-Mkan85B prime-boost vaccination reduced the bacterial burden more significantly. Moreover, the rBCG-Mkan85B/DNA-Mkan85B prime-boost vaccination induced antigen-specific CD4⁺ and CD8⁺ T cells. Our data suggest that rBCG-Mkan85B/DNA-Mkan85B prime-boost vaccination effectively enhances antigen-specific T cells. Our novel rBCG could be a potential alternative to clinical BCG for preventing various NTM infections.

Keywords: CD4+ T Cells; CD8+ T Cells; Mycobacterium kansasii; recombinant BCG.

Figure 1

BCG or rBCG-Mkan85B vaccination protects against M. kansasii infection. The CB6F1 (H2b/d) mice were either left unimmunized or immunized with BCG or rBCG-Mkan85B for 6 weeks, followed by intratracheal infection with a virulent M. kansasii strain. The mice were sacrificed at 6 weeks after M. kansasii infection. The immunization schedule is shown Figure S1A. The number of M. kansasii in individual mouse lungs was analyzed using the colony assay method. The data represent two independent experiments with three to four mice per group. The detection limit for bacilli in the tissue homogenate was 15 CFU. The error bars represent the SD. ** p < 0.01.

Figure 2

Histopathology of lungs from M. kansasii-infected mice. Representative pathological observations of the lungs of the unvaccinated (A,D,G,J), BCG-vaccinated (B,E,H), and rBCG-Mkan85B-vaccinated (C,F,I) mice 6 weeks after M. kansasii infection are shown. First-row panels, 100× of HE-stained specimens; second-row panels, 400× of HE-stained specimens; third-row panels, 400× of Ziehl-Neelsen-stained specimens; (panel J), 1000× of Ziehl-Neelsen-stained specimens. The yellow arrowheads indicate the acid-fast bacilli. Bars: 100 μm (first-row panels), 20 μm (second- and third-row panels), and 10 μm (panel J).

Figure 3

rBCG-Mkan85B/DNA-Mkan85B vaccination protects against M. kansasii infection. The CB6F1 mice were either left unimmunized or immunized with BCG or rBCG-Mkan85B/DNA-Mkan85B and then challenged with a virulent M. kansasii strain for another 6 weeks. The immunization schedule is shown in Figure S1B. The mice were sacrificed at 6 weeks after M. kansasii infection. The number of M. kansasii in individual mouse lungs was analyzed using the colony assay method. The data represent two independent experiments with five to six mice per group. The detection limit for bacilli in the tissue homogenate was 15 CFU. The error bars represent the SD. ** p < 0.01.

Figure 4

M.kansasii-specific polyfunctional CD4⁺ and CD8⁺ T cells in BCG- or rBCG-Mkan85B-vaccinated CB6F1 mice. (A) A representative fluorogram of epitope-specific polyfunctional CD4⁺ and CD8⁺ T cell inductions in splenocytes from unvaccinated and vaccinated mice infected with M. kansasii; TNF is shown on the x-axis and IFN-γ is shown on the y-axis. (B) Polyfunctional CD4⁺ and CD8⁺ T cells from unvaccinated and vaccinated CB6F1 mice infected with M. kansasii. Polyfunctional CD4⁺ T cell induction by stimulation with PPD (left panel), polyfunctional CD4⁺ T cell induction by stimulation with peptide 25 (middle panel), and polyfunctional CD8⁺ T cell induction by stimulation with Pep8 (right panel) in unvaccinated, BCG-, and rBCG-Mkan85B-vaccinated mice are shown. The data represent two independent experiments with three to four mice per group. The error bars represent the SD. * p < 0.05.

Figure 5

M.kansasii-specific polyfunctional CD4⁺ and CD8⁺ T cells in rBCG-Mkan85B/DNA-Mkan85B-prime–boost-vaccinated CB6F1 mice. (A) A representative fluorogram of epitope-specific polyfunctional CD4⁺ and CD8⁺ T cell inductions in splenocytes from unvaccinated and vaccinated CB6F1 mice infected with M. kansasii; TNF is shown on the x-axis and IFN-γ is shown on the y-axis. (B) Polyfunctional CD4⁺ and CD8⁺ T cells from the unvaccinated and vaccinated CB6F1 mice infected with M. kansasii. Polyfunctional CD4⁺ T cell induction by stimulation with PPD (left panel), polyfunctional CD4+ T cell induction by stimulation with peptide 25 (middle panel), and polyfunctional CD8⁺ T cell induction by stimulation with Pep8 (right panel) in unvaccinated, BCG-, and rBCG-Mkan85B/DNA-Mkan85B-vaccinated mice are shown. The data represent two independent experiments with five to six mice per group. The error bars represent the SD. * p < 0.05. ** p < 0.01.