03083nas a2200361   4500000000100000008004100001260001300042653002700055653002400082653001600106653003100122653001100153653002800164653002500192653002600217653002200243653002100265653002400286653002500310653001800335653002800353100001500381700001300396700001600409700001600425700001700441700001200458245016400470300001000634490000700644520205600651022001402707       1999                            d  c1999 Oct10aAntibodies, Monoclonal10aAntigens, Bacterial10aBCG Vaccine10aCD4-Positive T-Lymphocytes10aHumans10aImmunodominant Epitopes10aLeprosy, Tuberculoid10aLymphocyte Activation10aMembrane Proteins10aMolecular Weight10aMycobacterium bovis10aT-Lymphocyte Subsets10aT-Lymphocytes10aTuberculosis, Pulmonary1 aMehrotra J1 aMittal A1 aRastogi A K1 aJaiswal A K1 aBhandari N K1 aSinha S00aAntigenic definition of plasma membrane proteins of Bacillus Calmette-Guérin: predominant activation of human T cells by low-molecular-mass integral proteins.  a411-90 v503 a<p>Mycobacterial plasma membrane proteins, in particular the detergent-soluble or 'integral' ones, comprise a class of mostly unexplored antigens capable of inducing potent activation of human T cells. Plasma membrane isolated from culture-grown Bacillus Calmette-Guérin (BCG; Indian vaccine; Danish strain) was subjected to a Triton X-114-based biphasic extraction procedure for isolation of peripheral (water-soluble) and integral proteins (PMP and IMP). A distinction between the two protein pools was evident from results of SDS-PAGE and immunoblotting using antisera raised in rabbits. An enzyme-linked immunosorbant assay with a panel of WHO-IMMYC monoclonal antibodies against various mycobacterial antigens revealed that three well-known antigens, 19 kDa, 33/36 kDa (proline rich) and 38 kDa (PstS homologue), were part of the IMP pool; and another such antigen, 14/16 kDa alpha-crystallin homologue, partly constituted the PMP pool. Apparently, antigenically distinct species of the immunomodulatory moiety lipoarabinomannan partitioned in aqueous and detergent phases. Human T-cell proliferation assays in donors comprising tuberculoid leprosy and pulmonary tuberculosis patients and healthy BCG vaccinees showed significantly greater potency of IMP over PMP and this immunodominance appeared to be directed towards CD4+ cells. IMP of < 56 kDa were resolved by 'continuous elution SDS-PAGE' into 15 fractions which, after extraction of SDS, were used in T-cell proliferation assays for the identification of immunodominant constituents. Proteins falling within three low-molecular-mass zones (all < 35 kDa) performed better than the rest, particularly a approximately 22 kDa fraction, which strongly stimulated T cells from all five donors. Partial overlap between IMP and secreted proteins, as noticed in this study, could provide clues to immunodominance of the latter. The apparent uniqueness and a high T-cell activating potency make mycobacterial IMP attractive candidates for designing future vaccines or immunotherapeutic agents.</p>  a0300-9475