Differential Mycobacterium bovis BCG vaccine-derived efficacy in C3Heb/FeJ and C3H/HeOuJ mice exposed to a clinical strain of Mycobacterium tuberculosis

Abstract

The global epidemic caused by the bacterial pathogen Mycobacterium tuberculosis continues unabated. Moreover, the only available vaccine against tuberculosis, Mycobacterium bovis bacillus Calmette-Guérin (BCG), demonstrates variable efficacy. To respond to this global threat, new animal models that mimic the pathological disease process in humans are required for vaccine testing. One new model, susceptible C3Heb/FeJ mice, is similar to human tuberculosis in that these animals are capable of forming necrotic tubercle granulomas, in contrast to resistant C3H/HeOuJ mice. In this study, we evaluated the impact of prior BCG vaccination of C3Heb/FeJ and C3H/HeOuJ mice on exposure to a low-dose aerosol of Mycobacterium tuberculosis W-Beijing strain SA161. Both BCG-vaccinated murine strains demonstrated reduced bacterial loads 25 days after infection compared to controls, indicating vaccine efficacy. However, during chronic infection, vaccine efficacy waned in C3H/HeOuJ but not in C3Heb/FeJ mice. Protection in vaccinated C3Heb/FeJ mice was associated with reduced numbers of CD11b(+) Gr1(+) cells, increased numbers of effector and memory T cells, and an absence of necrotic granulomas. BCG vaccine efficacy waned in C3H/HeOuJ mice, as indicated by reduced expression of gamma interferon (IFN-γ) and increased expressions of interleukin-17 (IL-17), IL-10, and Foxp3 by T cells compared to C3Heb/FeJ mice. This is the first murine vaccine model system described to date that can be utilized to dissect differential vaccine-derived immune efficacy.

FIG 1

BCG-induced protection is lost in infected C3H/HeOuJ but not C3Heb/FeJ mice. Shown are bacterial counts in the lungs (A) and spleens (B) of control C3Heb/FeJ and C3H/HeOuJ mice as well as BCG-vaccinated immune C3Heb/FeJ and C3H/HeOuJ mice infected with a low-dose aerosol of M. tuberculosis W-Beijing strain SA161. CFU were determined on days 25 and 50 after infection by plating serial dilutions of organ homogenates onto nutrient 7H11 agar and counting CFU after 3 weeks of incubation at 37°C. For both murine strains, BCG vaccination resulted in a reduced bacterial burden at day 25 in comparison to the bacterial burden in nonvaccinated animals. However, protection in both the lungs and spleens of C3H/HeOuJ mice was lost at day 50 after infection. C3Heb/FeJ mice demonstrated strong BCG vaccine efficacy throughout infection. Results represent the average (n = 5) bacterial loads in each group and are expressed as log₁₀ CFU (±SEM). *, P < 0.050; **, P < 0.010; ***, P < 0.001 (determined by ANOVA and the Tukey test).

FIG 2

Changes in lung pathology in control and BCG-vaccinated C3H/HeOuJ and C3Heb/FeJ mice. Shown are representative photomicrographs of hematoxylin-eosin-stained slides (left) and acid-fast staining of slides (right) from the lungs of control or vaccinated mice. (A) As early as 25 days after infection, significant areas of necrosis were observed in C3Heb/FeJ mice. As disease progressed (day 50), areas of necrosis and bacterial burden (denoted by acid-fast staining) significantly increased in C3Heb/FeJ mice. Clusters of bacilli can be observed, which accumulated in areas of necrosis (arrows). (B) In contrast, BCG vaccination of C3Heb/FeJ mice significantly diminished necrosis and bacillary loads on both days 25 and 50 after infection. (D) BCG vaccination limited lesion size and the presence of acid-fast bacilli in C3H/HeOuJ mice temporarily 25 days after infection, and this protection was lost during chronic infection. (C) In control C3H/HeOuJ mice, bacillary numbers increased at day 50 albeit to a much lesser extent than in C3Heb/FeJ mice. Magnifications, ×4 (left) and ×100 (right).

FIG 3

Decreasing percentages of pulmonary CD4⁺ IFN-γ-producing cells with concomitant increasing percentages of Th17 and regulatory T cells during disease progression in C3H/HeOuJ mice. Shown are the percentages of pulmonary CD4⁺ T (A), Th1 (CD4⁺ IFN-γ⁺) (B), proinflammatory Th17 CD4⁺ IL-17⁺ (C), and regulatory CD4⁺ Foxp3⁺ LL-10⁺ (D to F) cells obtained from control C3Heb/FeJ and C3H/HeOuJ mice as well as BCG-vaccinated immune C3Heb/FeJ and C3H/HeOuJ mice infected with a low-dose aerosol of M. tuberculosis W-Beijing strain SA161, analyzed by flow cytometry. BCG-vaccinated C3Heb/FeJ mice demonstrated lower percentages of CD4⁺ T cells and cytokines (B to F), denoting bacterial control, than did control mice. As chronic disease progressed, the percentages of T cell cytokines in BCG-vaccinated C3Heb/FeJ mice significantly decreased as the bacterial burden declined (B to F). In contrast, percentages of T cell cytokines in control C3Heb/FeJ mice began to increase during chronic disease, indicative of attempted immune control of bacterial infection. Results represent the average (n = 5) percentages of cells (±SEM). *, P < 0.050 (determined by ANOVA and the Tukey posttest).

FIG 4

Increased percentages of effector and memory IFN-γ-and IL-17-producing cells confer BCG-induced protection in C3Heb/FeJ mice. Shown are the percentages of pulmonary effector (CD44^hi CD62L^lo) and memory (CD44^hi CD62L^hi) cells obtained from control C3Heb/FeJ and C3H/HeOuJ mice as well as BCG-vaccinated immune C3Heb/FeJ and C3H/HeOuJ mice infected with a low-dose aerosol of M. tuberculosis W-Beijing strain SA161, analyzed by flow cytometry. During chronic infection, BCG-vaccinated C3Heb/FeJ mice expressed increased numbers of effector and memory cells capable of producing IFN-γ and IL-17, which was associated with bacterial clearance (D to F). In contrast, BCG-vaccinated C3H/HeOuJ mice showed reduced levels of IFN-γ- and IL-17-producing memory T cells during chronic infection. Results represent the average (n = 5) percentages of cells (±SEM). *, P < 0.050; **, P < 0.010; ***, P < 0.001 (determined by ANOVA and the Tukey posttest).

FIG 5

BCG vaccination curtails the influx of Gr1^hi and Gr1^int cells present in areas of primary granuloma necrosis in C3Heb/FeJ mice. Shown are percentages of pulmonary CD11b⁺ Gr1^int (A) and CD11b⁺ Gr1^hi (B) cells obtained from control C3Heb/FeJ and C3H/HeOuJ mice as well as BCG-vaccinated immune C3Heb/FeJ and C3H/HeOuJ mice infected with a low-dose aerosol of M. tuberculosis W-Beijing strain SA161, analyzed by flow cytometry. Control C3Heb/FeJ and C3H/HeOuJ mice had higher percentages of both populations of GR-1⁺ cells. (A) As the disease progressed, the numbers of Gr1^int cells increased in control C3Heb/FeJ mice but not in C3H/HeOuJ mice (A). Interestingly, BCG-vaccinated C3Heb/FeJ mice showed a reduced influx of Gr1⁺ cells (A and B), which was also observed in the flow cytometric contour plots (C) and confirmed by immunohistochemistry (D). Results represent the average (n = 5) percentages of cells (±SEM). *, P < 0.050; **, P < 0.010; ***, P < 0.001 (determined by ANOVA and the Tukey posttest).