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Efforts to develop live attenuated vaccines against  Mycobacterium avium  subspecies  paratuberculosis  ( Map ), using indirect methods to screen  Map  deletion mutants for potential efficacy, have not been successful. A reduction in the capacity to survive in macrophages has not predicted the ability of mutants to survive in vivo. Previous studies for screening of three deletion mutants in cattle and goats revealed one mutant, with a deletion in  relA  (Δ Map / relA ), could not establish a persistent infection. Further studies, using antigen presenting cells (APC), blood dendritic cells and monocyte derived DC, pulsed with Δ Map / relA  or a 35 kDa  Map  membrane protein (MMP) revealed a component of the response to Δ Map / relA  was directed towards MMP. As reported herein, we developed a bacterium viability assay and cell culture assays for analysis and evaluation of cytotoxic T cells generated against Δ Map / relA  or MMP. Analysis of the effector activity of responding cells revealed the reason Δ Map / relA  could not establish a persistent infection was that vaccination elicited development of cytotoxic CD8 T cells (CTL) with the capacity to kill intracellular bacteria. We demonstrated the same CTL response could be elicited with two rounds of antigenic stimulation of APC pulsed with Δ Map / relA  or MMP ex vivo. Cytotoxicity was mediated through the perforin granzyme B pathway. Finally, cognate recognition of peptides presented in context of MHC I and II molecules to CD4 and CD8 T cells is required for development of CTL.

veterinary research

A Mycobacterium avium subsp. paratuberculosis relA deletion mutant and a 35 kDa major membrane protein elicit development of cytotoxic T lymphocytes with ability to kill intracellular bacteria