Serological distinction of integral plasma membrane proteins as a class of mycobacterial antigens and their relevance for human T cell activation.
This study pertains to classification and antigenic analysis of mycobacterial plasma membrane proteins in relation to human T cell proliferative responses, using a 'fast grower' Mycobacterium fortuitum as model. Membrane vesicles, prepared by sonication and differential centrifugation, were subjected to biphasic Triton X-114 extraction for isolation of integral peripheral (aqueous phase) proteins. Neither protein pool showed any appreciable overlap serologically. SDS-PAGE showed five prominent bands in peripheral and three in the integral protein pool, whereas immunoblotting with rabbit antisera identified only two major antigens (60 and 67 kD) in the former and five (24, 34, 42, 51 and 54 kD) in the latter. ELISA with a panel of anti-mycobacterial MoAbs revealed that nine out of 12 previously known antigens were present in the peripheral protein pool. Only two of them (33 and 40 kD) were additionally detected amongst integral proteins. The membrane-associated immunosuppressive moiety lipoarabinomannan was semiquantitatively located in aqueous phase. In bulk T cell proliferation assays, seven out of 10 subjects belonging to a 'responder' background (BT-BB leprosy patients and healthy contacts) showed high responses for Myco. fortuitum antigens. Proliferative response with integral proteins was comparable to that with whole membrane, but it was significantly higher (P < 0.0005) than the response with peripheral proteins. The distinction and relevance of integral membrane proteins as a class of mycobacterial antigens make them worthy of consideration in a subunit vaccine design.