A major T-cell-inducing cytosolic 23 kDa protein antigen of the vaccine candidate Mycobacterium habana is superoxide dismutase

This study describes the purification and immunochemical characterization of a major 23 kDa cytosolic protein antigen of the vaccine candidate Mycobacterium habana (TMC 5135). The 23 kDa protein alone was salted out from the cytosol at an ammonium sulfate saturation of 80-95%. It represented about 1.5% of the total cytosolic protein, appeared glycosylated by staining with periodic acid/Schiff's reagent, and showed a pI of approximately 5.3. Its native molecular mass was determined as approximately 48 kDa, suggesting a homodimeric configuration. Immunoblotting with the WHO-IMMLEP/IMMTUB mAbs mc5041 and IT61 and activity staining after native PAGE established its identity as a mycobacterial superoxide dismutase (SOD) of the Fe/Mn type. The sequence of the 18 N-terminal amino acids, which also contained the binding site for mc5041, showed a close resemblance, not only with the reported deduced sequences of Mycobacterium leprae and Mycobacterium tuberculosis Fe/MnSODs, but also with human MnSOD. In order to study its immunopathological relevance, the protein was subjected to in vivo and in vitro assays for T cell activation. It induced, in a dose-related manner, skin delayed hypersensitivity in guinea-pigs and lymphocyte proliferation in BALB/c mice primed with M. habana. Most significantly, it also induced lymphocyte proliferative responses, in a manner analogous to M. leprae, in human subjects comprising tuberculoid leprosy patients and healthy contacts.