IMMUNOGENICITY OF PLASMA MEMBRANE PROTEINS OF MYCOBACTERIAL SPECIES

Abstract

Successive failures of integral vaccines in the prevention of mycobacterial diseases - tuberculosis and leprosy have intensified the quest for development of subunit vaccines resulting in exquisite definition of an array of mycobacterial antigens. The focus has obviously been on protein antigens as they are capable of generating a "cell (T cell) mediated immunity' which is regarded as the principle host-protective mechanism against intracellular pathogens. Mycobacterial proteins well characterised to date are mainly the ones derived from cytosol, cell wall, or those released in the medium. Little knowledge is available regarding antigens localised within the mycobacterial plasma membrane. Yet, data from other intracellular pathogens, like Treponema pallidum, Leishmania spec.> and Plasmodium falciparum, suggest that such proteins specially the detergent soluble 'integral membrane proteins' are highly immunogenic and in some cases protective. This study is aimed at antigenic definition of plasma membrane proteins of BCG vaccine and identification of immunodominant T cell activating subunits. BCG despite all controversies is the only vaccine which has afforded considerable protection against tuberculosis and more so against leprosy. Apart from being clinically safe it is also bestowed with adjuvant properties. An understanding of immunodominant antigens of BCG is likely to provide clues to the development of more effective immunoprophylactic / immunotherapeutic agents based on cross-reactive mycobacterial antigens. Plasma membrane isolated from culture-grown BCG (Indian vaccine strain) was subjected to Triton X-114 based biphasic extraction procedure for isolation of 'peripheral' (water soluble) and 'integral' (detergent soluble) proteins (PMP and IMP) . Distinction between the two protein pools was evident from results of SDS-PAGE and immunoblotting using antisera raised in rabbits. ELISA with a panel of WHO- IMMYC monoclonal antibodies against various mycobacterial antigens revealed that three well known antigens - 19kDa, 33/36kDa ("proline rich") and 38kDa (Pst-S homolog) were a part of IMP pool; and another such antigen, 14/16kDa °c-crystalline homolg, partly constituted the PMP pool. Apparently, antigenically distinct species of the immunomodulatory moiety lipoarabinomannan partiotioned in both aqueous and detergent phases. Human T cell proliferation assay showed a significantly greater potency of IMP over PMP. IMP subunits of <56kDa were resolved into 15 fractions using a continuous elution SDS-PAGE based protocol and all fractions, after SDS removal, were subjected to T cell proliferation assays for the identification of immunodominant subunits. Proteins falling within three low molecular weight zones (all <35kDa) did better than the rest, particularly the = 22kDa protein which strongly stimulated T cells from all 5 donors. Partial overlap between IMP and secretory proteins, as noticed in this study, could proof clues to the immunodominance of the latter. The apparent uniquness of a high T cell activity ptJtency make IMPs attractive candidates for designing future vaccines or immunotherapeutic agents.