Evaluation of a Mycobacterium avium subsp. paratuberculosis leuD mutant as a vaccine candidate against challenge in a caprine model

Abstract

Johne's disease (JD) is prevalent worldwide and has a significant impact on the global agricultural economy. In the present study, we evaluated the protective efficacy of a leuD (Δleud) mutant and gained insight into differential immune responses after challenge with virulent M. avium subsp. paratuberculosis in a caprine colonization model. The immune response and protective efficacy were compared with those of the killed vaccine Mycopar. In vitro stimulation of peripheral blood mononuclear cells with johnin purified protein derivative showed that Mycopar and ΔleuD generated similar levels of gamma interferon (IFN-γ) but significantly higher levels than unvaccinated and challenged phosphate-buffered saline controls. However, only with ΔleuD was the IFN-γ response maintained. Flow cytometric analysis showed that the increase in IFN-γ correlated with proliferation and activation (increased expression of CD25) of CD4, CD8, and γδT cells, but this response was significantly higher in ΔleuD-vaccinated animals at some time points after challenge. Both Mycopar and ΔleuD vaccines upregulated Th1/proinflammatory and Th17 cytokines and downregulated Th2/anti-inflammatory and regulatory cytokines at similar levels at almost all time points. However, significantly higher levels of IFN-γ (at weeks 26 and 30), interleukin-2 (IL-2; week 18), IL-1b (weeks 14 and 22), IL-17 (weeks 18 and 22), and IL-23 (week 18) and a significantly lower level of IL-10 (weeks 14 and 18) and transforming growth factor β (week 18) were detected in the ΔleuD-vaccinated group. Most importantly, ΔleuD elicited an immune response that significantly limited colonization of tissues compared to Mycopar upon challenge with wild-type M. avium subsp. paratuberculosis. In conclusion, the ΔleuD mutant is a promising vaccine candidate for development of a live attenuated vaccine for JD in ruminants.

Fig 1

Antibody response. Sera isolated from blood collected at different time points were diluted (1:200) and analyzed for PPDj-specific IgG antibodies by indirect ELISA, as described in Material and Methods. The response was measured in individual goats, and data are presented in the form of whisker-box plots. The white box indicates the lower quartile (25% of data greater than this value), the shaded box indicates the upper quartile (25% of data less than this value), the middle line is the median (50% of data greater than this value), and error bars indicate the minimum and maximum values. *, P < 0.05, ΔleuD versus Mycopar.

Fig 2

IFN-γ response. PBMCs isolated from animals bled at different time points were stimulated with 10 μg/ml PPDj in 200 μl RPMI for 72 h at 37°C in a humidified atmosphere supplemented with 5% CO₂. Cells were centrifuged, culture supernatant was recovered, and IFN-γ levels were determined by using a BOVIGAM kit following the manufacturer's protocol. Results are expressed as the OD, and error bars indicate standard deviations from the means. *, P < 0.05 at week 30 and P < 0.02 at week 26 (ΔleuD versus Mycopar); **, P < 0.05 (ΔleuD versus PBS and Mycopar versus PBS).

Fig 3

Lymphoproliferation. PBMCs isolated from animals bled at different time points were stimulated with 10 μg/ml PPDj in 200 μl RPMI for 72 h at 37°C in a humidified atmosphere supplemented with 5% CO₂. The proliferative response was measured with a cell proliferation ELISA and BrdU colorimetric kit (Roche Diagnostics, Indianapolis, IN) as per the manufacturer's protocol. The results are expressed as the SI, and the error bars indicate standard deviations from the means. *, P < 0.05 at week 26 and P < 0.03 at week 30 (ΔleuD versus Mycopar); **, P < 0.05 (ΔleuD versus PBS and Mycopar versus PBS).

Fig 4

Analysis of activation status of T cells, based on comparison of activation status of T cells in PBMCs isolated from animals bled at various time points after immunization and challenge, stimulated with PPDj for 6 days, and subjected to FC analysis. The data were analyzed with the BD FACS Diva software. The data are presented as a bar graph for simplicity. Activation statuses of CD4 T cells (A), CD8 T cells (B), and γδ T cells (C) at different time points are shown. V, vaccination; B, booster; C, challenge; *, P < 0.05 (ΔleuD versus Mycopar).

Fig 5

(A) Results of cytokine analysis by RT-PCR. Relative transcription of cytokine message was measured by RT-PCR in PBMCs isolated from animals bled at different time points after immunization and stimulation with PPDj for 3 days. The relative transcription level was calculated using the value of unstimulated cells as the calibrator, with the housekeeping gene GAPDH as an internal control. Data are presented as the relative mRNA expression level (mean fold change) of each group with error bars indicating the standard deviations. Panels are grouped together as follows: Th1 cytokines (A, B, and C); proinflammatory cytokines (D, E, F, and G); Th2/anti-inflammatory cytokines (H, I, and J); Th17/regulatory cytokines (K, L, M, N). *, P < 0.05 for ΔleuD versus Mycopar; **, P < 0.05 for Mycopar versus ΔleuD.