Repeated Aerosolized-Boosting with Gamma-Irradiated Mycobacterium bovis BCG Confers Improved Pulmonary Protection against the Hypervirulent Mycobacterium tuberculosis Strain HN878 in Mice

Abstract

Mycobacterium bovis bacillus Calmette-Guerin (BCG), the only licensed vaccine, shows limited protection efficacy against pulmonary tuberculosis (TB), particularly hypervirulent Mycobacterium tuberculosis (Mtb) strains, suggesting that a logistical and practical vaccination strategy is urgently required. Boosting the BCG-induced immunity may offer a potentially advantageous strategy for advancing TB vaccine development, instead of replacing BCG completely. Despite the improved protection of the airway immunization by using live BCG, the use of live BCG as an airway boosting agent may evoke safety concerns. Here, we analyzed the protective efficacy of γ-irradiated BCG as a BCG-prime boosting agent for airway immunization against a hypervirulent clinical strain challenge with Mycobacterium tuberculosis HN878 in a mouse TB model. After the aerosol challenge with the HN878 strain, the mice vaccinated with BCG via the parenteral route exhibited only mild and transient protection, whereas BCG vaccination followed by multiple aerosolized boosting with γ-irradiated BCG efficiently maintained long-lasting control of Mtb in terms of bacterial reduction and pathological findings. Further immunological investigation revealed that this approach resulted in a significant increase in the cellular responses in terms of a robust expansion of antigen (PPD and Ag85A)-specific CD4+ T cells concomitantly producing IFN-γ, TNF-α, and IL-2, as well as a high level of IFN-γ-producing recall response via both the local and systemic immune systems upon further boosting. Collectively, aerosolized boosting of γ-irradiated BCG is able to elicit strong Th1-biased immune responses and confer enhanced protection against a hypervirulent Mycobacterium tuberculosis HN878 infection in a boosting number-dependent manner.

Fig 1. Immunogenicity in the lungs of immunized mice.

Three weeks after the final immunization, mice from each group (n = 5) were sacrificed and lung cells were prepared as described in the materials and methods section. The percentages of CD4⁺, CD8⁺ central memory (CD44^hiCD62L⁺CD127⁺), effector memory (CD44^hiCD62L^-CD127⁺), effector (CD44^hiCD62L^-CD127^-), and naïve (CD44^loCD62L⁺CD127⁺) T cells were analyzed by flow cytometry (A). A total of 2 × 10⁶ cells were added to each well of microtiter plates and incubated with PPD (2 μg/ml) or Ag85A-specific CD4 or CD8 T-cell peptides (2 μg/ml) for 24 h at 37°C. The IFN-γ concentrations in the suspension were detected using commercial ELISA kits (B). The induction of PPD-specific IgG2c antibodies in the serum from each group of mice (C). The data are presented as the means ± SD from five mice in each group. An unpaired t-test was used to determine the significance of differences. A value of p<0.05 was considered to be statistically significant. ^# p<0.05, ^## p<0.01, and ^### p<0.001 compared to G1. * p<0.05, ** p<0.01, and *** p<0.001 compared to G3. n.s.: not significant.

Fig 2. Induction of antigen-specific multifunctional T cells in the lungs of immunized mice.

Each group of mice was immunized and sacrificed as described in the materials and methods section. Three weeks after the final immunization, mice from each group (n = 5) were euthanized and their lung cells (2 × 10⁶ cells) were stimulated with PPD (2 μg/ml) or Ag85A-specific CD4 or CD8 T cell peptides (2 μg/ml) for 12 h at 37°C in the presence of GolgiStop. The percentages of antigen-specific CD4⁺CD62L^- and CD8⁺CD62L^- T cells producing IFN-γ, TNF-α, and/or IL-2 in the cells isolated from the lungs of each group of mice were analyzed by multicolor flow cytometry by gating for CD4⁺ and CD8⁺ lymphocytes (A). Pie charts (B) show the mean frequencies of cells coexpressing IFN-γ, TNF-α, and/or IL-2. The data are presented as the mean ± SD from five mice in each group. An unpaired t-test was used to determine the significance of differences. A value of p<0.05 was considered to be statistically significant. ^# p<0.05, ^## p<0.01, and ^### p<0.001 compared to G1. * p<0.05, ** p<0.01, and *** p<0.001 compared to G3.

Fig 3. Bacterial loads in the lungs and spleens after immunization and aerosol challenge with the M. tuberculosis HN878 strain.

Seven weeks after the final immunization, mice were challenged with 200 CFU of the M. tuberculosis HN878 strain via the aerosol route. Five and ten weeks after challenge, the CFU in the lungs (A) and spleens (B) of each group were analyzed by culturing lung and spleen homogenates and enumerating the bacteria. The data are presented as the median ± IQR log₁₀CFU/organ (n = 5), and the levels of significance for comparisons between samples were determined by a one-way ANOVA, followed by Dunnett’s test. A value of p<0.05 was considered to be statistically significant. ^# p<0.05, ^## p<0.01, and ^### p<0.001 compared to G2. * p<0.05, ** p<0.01, and *** p<0.001 compared to G3.

Fig 4. Histopathological lesions of the lungs.

Mice were infected with 200 CFU of the M. tuberculosis HN878 strain via the aerosol route, and the lungs were removed at 5 weeks (A) and 10 weeks (B) post-infection. Representative lung pathological changes from the different groups and the percentages of inflamed areas in lung section are also shown. The data regarding the percentages of the inflamed areas are presented as Whisker box plots (Whiskers represent minimum and maximum values) (n = 5), and a one-way ANOVA followed by Dunnett’s test was used to determine the significance of the findings. A value of p<0.05 was considered to be statistically significant. ^# p<0.05, ^## p<0.01 and ^### p<0.001 compared to G2. * p<0.05, ** p<0.01, and *** p<0.001 compared to G3.

Fig 5. Induction of multifunctional T cells in the lungs of mice after challenge with the M. tuberculosis HN878 strain.

Five and ten weeks post-infection, mice from each group (n = 5) were euthanized and their lung cells (2 × 10⁶ cells) were stimulated with PPD (2 μg/ml) for 12 h at 37°C in the presence of GolgiStop. The percentage of antigen-specific CD4⁺CD62L^- and CD8⁺CD62L^- T cells producing IFN-γ, TNF-α, and/or IL-2 in the cells isolated from the lungs of each group of mice were analyzed by multicolor flow cytometry by gating for CD4⁺ and CD8⁺ lymphocytes (A). Pie charts (B) show the mean frequencies of cells coexpressing IFN-γ, TNF-α, and/or IL-2. The data are presented as the mean ± SD from five mice in each group. An unpaired t-test was used to determine the significance of differences. A value of p<0.05 was considered to be statistically significant. ^# p<0.05, ^## p<0.01, and ^### p<0.001 compared to G2. * p<0.05, ** p<0.01, and *** p<0.001 compared to G3.