Deglycosylation of the 45/47-kilodalton antigen complex of Mycobacterium tuberculosis decreases its capacity to elicit in vivo or in vitro cellular immune responses

A protection against a challenge with Mycobacterium tuberculosis is induced by previous immunization with living attenuated mycobacteria, usually baci llus Calmette-Guerin (BCG), The 45/47-kDa antigen complex (Apa) present in culture filtrates of BCG of M. tuberculosis has been identified and isolate d based on its ability to interact mainly with T lymphocytes and/or antibod ies induced by immunization with living bacteria. The protein is glycosylat ed, A large batch of Apa was purified from M. tuberculosis culture filtrate to determine the extent of glycosylation and its role on the expression of the immune responses. Mass spectrometry revealed a spectrum of glycosylate d molecules, with the majority of species bearing six, seven, or eight mann ose residues (22, 24, and 17%, respectively), while others three, four, or five mannoses (5, 9, and 14%, respectively). Molecules with one, two, or ni ne mannoses were rare (1.5, 3, and 3%, respectively), as were unglycosylate d species (in the range of 1%), To eliminate the mannose residues linked to the protein, the glycosylated Apa molecules were chemically or enzymatical ly treated. The deglycosylated antigen was 10-fold less active than native molecules in eliciting delayed-type hypersensitivity reactions in guinea pi gs immunized with BCG, It was 30-fold less active than native molecules whe n assayed in vitro for its capacity to stimulate T lymphocytes primed in vi vo. The presence of the mannose residues on the Apa protein was essential f or the antigenicity of the molecules in T-cell-dependent immune responses i n vitro and in vivo.