EXPRESSION OF MYCOBACTERIUM-TUBERCULOSIS MPT64 IN RECOMBINANT MYCOBACTERIUM-TSMEGMATIS - PURIFICATION, IMMUNOGENICITY AND APPLICATION TO SKIN-TESTS FOR TUBERCULOSIS

Proteins secreted across the cell wall of mycobacteria are important a ntigens recognized early in the host response to mycobacterial infecti on. MPT64 is a 23-kD secreted protein restricted to members of the Myc obacterium tuberculosis complex which elicits T cell responses and cut aneous DTH reactions in Myco. tuberculosis-infected animals. Patients with tuberculosis and their tuberculin-positive contacts respond to th e protein, but recipients of bacille Calmette-Guerin rin (BCG) vaccine strains lacking the mpt64 gene do not. In the present study, we descr ibe the development of a unique recombinant mycobacterial vector which secretes the encoded Myco. tuberculosis protein MPT64 at high levels into the culture filtrate, from which the protein is isolated by a sin gle-step affinity chromatographic step. The purified protein was recog nized by both polyclonal and monoclonal anti-MPT64 antibodies. The T c ell reactivity of the protein was confirmed by its ability to stimulat e human anti-rMPB64 T cell lines. The Myco. smegmatis recombinant MPT6 4 protein was superior to the Escherichia coli rMPB64 protein, which h as identical amino acid sequence, in eliciting cutaneous DTH reactions in guinea pigs sensitized with Myco. tuberculosis. Animals sensitized with BCG strains lacking the mpb64 gene failed to respond to MPT64. S imilarly, interferon-gamma (IFN-gamma) responses in tuberculosis patie nts and their contacts were higher to the Myco. smegmatis form of the protein. The potential of this form of the Myco. tuberculosis MPT64 pr otein as a skin test reagent for tuberculosis is discussed.