Antigenic definition of plasma membrane proteins of Bacillus Calmette-Guérin: predominant activation of human T cells by low-molecular-mass integral proteins.

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.

Quality control tests for vaccines in leprosy vaccine trial, Avadi.

All the vaccines supplied for the large scale comparative leprosy vaccine trial of ICRC bacilli, M.w, BCG plus killed M. leprae (candidate vaccines), BCG and normal saline (control arms) at CJIL Field Unit, Chennai were tested for quality control by the suppliers following the procedures laid down in the WHO protocol for killed M. leprae. Quality control for BCG was carried out at BCG vaccine laboratory as per protocol. Toxicity and sterility tests were done on all the vaccine batches/lots received. As part of the quality control, bacterial count, and protein estimation were also done. Studies showed that the bacterial content and protein concentration were comparable with the original preparations. Vaccines were free from micro-organisms, toxic materials and safe for human use. Thus the quality of all vaccine preparations was satisfactory.