Vaccination with a BCG Strain Overexpressing Ag85B Protects Cattle against <em>Mycobacterium bovis</em> Challenge

<div><p><em>Mycobacterium bovis</em> is the causative agent of tuberculosis in cattle but also infects other animals, including humans. Previous studies in cattle have demonstrated that the protection induced by BCG is not complete. In order to improve the protection efficacy of BCG, in this study we overexpressed Ag85B in a BCG Pasteur strain, by using an expression system based on the use of an auxotrophic strain for the leucine amino acid, and complementation with <em>leuD</em>. We found that vaccination of cattle with BCG overexpressing Ag85B induced higher production of IL-17 and IL-4 mRNA upon purified protein derivative (PPDB) stimulation of peripheral blood mononuclear cells (PBMCs) than vaccination with BCG. Moreover, the IL-17 mRNA expression after vaccination negatively correlated with disease severity resulting from a subsequent challenge with <em>M. bovis</em>, suggesting that this cytokine is a potential biomarker of cattle protection against bovine tuberculosis. Importantly, vaccination with the recombinant BCG vaccine protected cattle better than the wild-type BCG Pasteur.</p> </div

Protective efficacy as measured by gross pathology.

<p>The calves were euthanized sixteen weeks after infection and thin slices of lungs and lymph nodes were analysed looking for granuloma formations. Pathology scores were established using the scoring system described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051396#s2" target="_blank">Material and Methods</a> (A) Mean pathology scores of lungs in vaccinated and nonvaccinated groups. (B) Mean pathology scores of total lesions (head lymph nodes, respiratory tract-associated lymph nodes and lungs) in vaccinated and nonvaccinated groups. Pathology scores for individual animals are plotted. Horizontal lines indicate median values. Significance was determined by Mann Whitney test: * Statistically significantly different, <i>P</i><0.05.</p

Correlation between IL-17 and IFN-γ mRNA expression in PPDB-stimulated PBMCs and disease severity.

<p> Results are expressed as mean increases in IL-17 (A) and IFN-γ (B) transcription at 15 days post-vaccination and IFN-γ transcription at 40 days post-challenge (C) of individual animals in relation to the corresponding total gross pathology scores (head lymph nodes, respiratory tract-associated lymph nodes and lungs). Animals vaccinated with either BCG (squares) or Δ<i>leuD</i> BCG-85B (triangles) and nonvaccinated (circles) were infected eight weeks after vaccination and sacrificed sixteen weeks post-challenge. Solid lines in panels A and B indicate linear regression; dashed lines indicate 95% confidence intervals. Values for <i>r²</i> and <i>p</i> of linear regression analysis are indicated.</p

Mean tuberculin skin test responses to PPDB.

<p>Animals were vaccinated with BCG (n = 5, squares), Δ<i>leuD</i> BCG-85B (n = 5, triangles) or inoculated with PBS (n = 6, circles) and challenged after eight weeks with <i>M. bovis</i>. Values indicate skin thickness at one month post-vaccination and prior to challenge (A), and three months post-challenge (B). Horizontal lines indicate mean values. Data were analysed using a Mann Whitney test,*<i>P</i><0.05, **<i>P</i><0.005.</p

Determination of lymphocyte subsets in PPDB-stimulated PBMCs.

<p>Percentages of lymphocyte cell subsets CD4+ (A) and CD8+ (B) expressing CD25 for PPDB stimulated-PBMCs from animals vaccinated with BCG (n = 5, triangles), Δ<i>leuD</i> BCG-85B (n = 6, circles) or nonvaccinated (n = 6, squares) at 15, 30 and 60 days after vaccination and 20, 40, 70 and 100 days after challenge. The arrow indicates the challenge time point. Data were analysed using Mann-Whitney test for comparison between groups. (Statistically significantly different to that for the nonvaccinated group *<i>P</i><0.05 and ** <i>P</i><0.01).</p

IFN-γ production in PPDB-stimulated PBMCs.

<p>(A) IFN-γ responses in vaccinated and nonvaccinated animals following vaccination and after challenge. IFN-γ production was measured in PPDB-stimulated blood from animals vaccinated with BCG (squares), Δ<i>leuD</i> BCG-85B (triangles) or nonvaccinated (circles) at different time points (0, 30 and 60, 120 and 150 days post-vaccination). Animals were infected eight weeks after vaccination and sacrificed sixteen weeks post-challenge. Arrow indicates the challenge time point. Significance was determined by ANOVA test. Statistically significantly different to that for the nonvaccinated group, *<i>P</i><0.05. (B) Correlation between IFN-γ production in PPDB-stimulated PBMCs and disease severity. Results are expressed as IFN-γ 40 days post-challenge of individual animals in relation to the corresponding total gross pathology scores. Solid line indicates linear regression; dashed lines indicate 95% confidence intervals. Values for <i>r²</i> and <i>p</i> of linear regression analysis are indicated.</p

Relative cytokine gene expression.

<p>Gene expression of IFN-γ (A), IL-2 (B), IL-4 (C) and IL-17 (D) was measured in PPDB-stimulated PBMCs from animals vaccinated with BCG (gray), Δ<i>leuD</i> BCG-85B (white) or nonvaccinated (black) at different time points (15 and 30 days post-vaccination and 20, 40, 70 and 100 days post-challenge). Transversal line indicates the challenge time point. Relative gene expression was calculated using the 2-ΔΔCt method with E correction, using <i>gadph</i> mRNA expression as reference gene and the pre-immune condition as the calibrator. Data were analysed using Mann-Whitney test, statistically significantly different, * <i>P</i><0.05 and ** <i>P</i><0.01. The bars indicate the median fold change.</p