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

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 (Delta leud) 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 Delta leuD generated similar levels of gamma interferon (IFN-gamma) but significantly higher levels than unvaccinated and challenged phosphate-buffered saline controls. However, only with Delta leuD was the IFN-gamma response maintained. Flow cytometric analysis showed that the increase in IFN-gamma correlated with proliferation and activation (increased expression of CD25) of CD4, CD8, and gamma delta T cells, but this response was significantly higher in Delta leuD-vaccinated animals at some time points after challenge. Both Mycopar and Delta leuD 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-gamma (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 beta (week 18) were detected in the Delta leuD-vaccinated group. Most importantly, Delta leuD 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 Delta leuD mutant is a promising vaccine candidate for development of a live attenuated vaccine for JD in ruminants.