Evaluation of subsp. isocitrate lyase () and ABC transporter () knockout mutants as vaccine candidates.

There has been little success in controlling Johne's disease, caused by subsp. , due to suboptimal diagnostics and the ineffectiveness of available vaccines. By knocking out and , genes required for MAP survival in dairy calves, two live-attenuated vaccine candidates were created. This study evaluated the host-specific attenuation of MAP and mutants in mouse and calf models, as well as the elicited immune responses. Deletion mutants were generated in MAP strain A1-157 through specialized transduction and found viable . First, the mutants' attenuation and elicited cytokine secretion were assessed in a mouse model, 3 weeks after intraperitoneal inoculation with MAP strains. Later, vaccine strains were assessed in a natural host infection model where calves received 10CFU oral dose of MAP wild-type or mutant strains at 2 weeks old. Transcription levels of cytokines in PBMCs were evaluated at 12-, 14-, and 16-weeks post-inoculation (WPI) and MAP colonization in tissue was assessed at 4.5 months after inoculation. Whereas both vaccine candidates colonized mouse tissues similarly to wild-type strain, both failed to persist in calf tissues. In either mouse or calf models, gene deletion did not reduce immunogenicity. Instead, inoculation with Δ induced a greater upregulation of proinflammatory cytokines than Δ and wild-type in both models and a greater expansion of cytotoxic and memory T-cells than uninfected control in calves. Δ and wild-type strains significantly increased secretion of IP-10, MIG, TNFα, and RANTES in mice serum compared to uninfected control. This agreed with upregulation of IL-12, IL-17, and TNFα in calves inoculated with Δ at all time points. The Δ also gave rise to greater populations of CD4+CD45RO+, and CD8+ cells than uninfected control calves at 16 WPI. Low survival rate of MAP in macrophages co-incubated with PBMCs isolated from the Δ group indicated that these cell populations are capable of killing MAP. Overall, the immune response elicited by Δ is stronger compared to Δ and it is maintained over two different models and over time in calves. Further investigation is warranted to evaluate the mutant's protection against MAP infection as a live attenuated vaccine candidate.