M72 Fusion Proteins in Nanocapsules Enhance BCG Efficacy Against Bovine Tuberculosis in a Mouse Model

Abstract

Mycobacterium bovis is the causative pathogen of bovine tuberculosis (bTB), a disease that affects cattle and other mammals, including humans. Currently, there is no efficient vaccine against bTB, underscoring the need for novel immunization strategies. The M72 fusion protein, composed of three polypeptides derived from Mycobacterium tuberculosis and M. bovis, has demonstrated protective efficacy against M. tuberculosis in clinical trials when combined with the AS01E adjuvant. Given the established efficacy of nanocapsule formulations as vaccine delivery systems, this study evaluated a novel immunization strategy combining BCG with either full-length M72 or a truncated M72 fused to a streptococcal albumin-binding domain (ABDsM72). Both antigens were encapsulated in chitosan/alginate nanocapsules and assessed in a murine M. bovis challenge model. Priming with BCG followed by an M72 boost significantly improved splenic protection compared to BCG alone, but it did not enhance pulmonary protection. Notably, boosting with ABDsM72 further increased the proportion of CD4+KLRG1-CXCR3+ T cells in the lungs of M. bovis-challenged mice, a key correlate of protective immunity. These findings demonstrate that chitosan/alginate-encapsulated antigens enhance BCG-induced immunity, supporting their potential as next-generation vaccine candidates for bTB control.

Keywords: M72; Mycobacterium bovis; bovine tuberculosis; chitosan nanocapsules; mice; vaccine.

Figure 1

Purification of recombinant M72 and ABDsM72. Recombinant M72 (A) and ABDsM72 (B) were expressed in E. coli and purified using Ni-affinity chromatography. The purified proteins were resolved using a 12% SDS-PAGE gel and visualized by Coomassie blue staining. Arrows indicate the positions of the recombinant proteins. (C) Schematic representation of M72 and ABDsM72 and sequence of ABD.

Figure 2

Protection of vaccinated mice against M. bovis challenge. (A) Schematic representation of the mouse vaccination trial for M. bovis infection. (B) Bacterial burden in lung tissue was quantified as colony-forming units (CFUs). Data are presented as mean ± standard error of the mean (SEM) and were analysed using one-way ANOVA followed by Bonferroni’s post hoc test for multiple pairwise comparisons (n = 7–8 animals/group) (*** p < 0.001). (C) Splenic bacterial loads were similarly determined and are shown as mean ± SEM. Statistical analysis was performed using the Shapiro–Wilk normality test and Kruskal–Wallis test followed by Dunn’s multiple comparison test (n = 7–8 animals/group) (* p < 0.05).

Figure 2

Protection of vaccinated mice against M. bovis challenge. (A) Schematic representation of the mouse vaccination trial for M. bovis infection. (B) Bacterial burden in lung tissue was quantified as colony-forming units (CFUs). Data are presented as mean ± standard error of the mean (SEM) and were analysed using one-way ANOVA followed by Bonferroni’s post hoc test for multiple pairwise comparisons (n = 7–8 animals/group) (*** p < 0.001). (C) Splenic bacterial loads were similarly determined and are shown as mean ± SEM. Statistical analysis was performed using the Shapiro–Wilk normality test and Kruskal–Wallis test followed by Dunn’s multiple comparison test (n = 7–8 animals/group) (* p < 0.05).

Figure 3

Lung CD4+ T cell immunophenotyping following vaccination and M. bovis challenge. Flow cytometric analysis of lymphocyte populations from lung homogenates showing percentages of (A) CXCR3+, (B) KLRG1+, and (C) CXCR3+KLRG1-subsets among CD4+ T cells. Bars represent mean values ± SEM for BCG-, M72-, and ABDsM72-vaccinated groups versus PBS controls (n = 8 animals/group). Significant differences were determined by one-way ANOVA with Bonferroni’s post hoc test (* p < 0.05; ** p < 0.01; *** p < 0.001). Complete gating strategy is presented in Supplementary Figure S2A.

Figure 4

Correlations between number of CFUs and CD4+ T cell populations. A linear regression model was used to assess the relationship between lung CFU counts in vaccinated versus control animals and the proportions of (A) KLRG1-CXCR3+, (B) KLRG1+, and (C) PD1+ CD4+ T cell subsets. The regression line is represented by a solid line, with the dashed lines marking the 95% confidence intervals. The coefficient of determination (r²) and p-value from the regression analysis are provided.