No abstract

The attributes of immunodominance, predominant expression during mycobacterial dormancy and restriction to the Mycobacterium tuberculosis complex make the 16 kDa protein an important candidate for the study of the immune response in humans. We therefore investigated the relationship between T‐ and B‐cell reactivity to the recombinant antigen and disease in a total of 127 subjects. The percentage of T‐cell responders towards both the intact antigen and its permissively recognised peptide 16p91‐110 was highest in healthy bacillus Calmette–Guérin (BCG)‐sensitized controls (96% and 68%, respectively) and lowest in those with extensive untreated tuberculosis (26% and 18%) (P < 0.001). By contrast, antibody levels (ABT50 > 100) were highest in patients with extensive disease (46–50%) (P < 0.005). There was significantly higher production of IFN‐γ in the BCG‐sensitized controls by comparison with untreated patients (P < 0.05), but complete antituberculous chemotherapy abolished this deficit in patients. The significance of these findings to immunodiagnosis and protective immunity is discussed.

The diagnosis of infection caused by Mycobacterium tuberculosis is of increased public health concern following increases in the number of cases in developed countries and major increases in developing countries associated with the spread of human immunodeficiency virus (HIV) infection. The specificity of purified protein derivative skin testing for the detection of infection is compromised by exposure to environmental mycobacteria. Examination of sputum detects the most infectious patients, but not those with extrapulmonary disease. The 38-kDa antigen of M. tuberculosis contains two M. tuberculosis-specific B-cell epitopes. We overexpressed the gene for this antigen in Escherichia coli and evaluated the recombinant product in in vitro assays of T-cell function and as a target for the antibody response in humans. The sensitivity and specificity of the antigen as a skin test reagent were also assessed in outbred guinea pigs. We found that 69% of healthy sensitized humans recognize the antigen in vitro, as manifested by both cell proliferation and the production of gamma interferon. Untreated patients initially have a lower frequency of response (38%); this recovers to 72% during therapy. A total of 292 patients (20 with HIV coinfection) and 58 controls were examined for production of antibody to the 38-kDa antigen by using a commercially available kit. The sensitivity of the test in comparison with that of culture was 72.6%, and the specificity was 94.9%. The antigen was also tested for its ability to induce skin reactions in outbred guinea pigs sensitized by various mycobacterial species. The antigen provoked significant skin reactions in M. tuberculosis-, M. bovis BCG-, and M. intracellulare-sensitized animals. The significance of these findings and the usefulness of this antigen in immunodiagnosis are discussed.

To study the immunological activity of proteins secreted by Mycobacterium tuberculosis, we carried out comparative studies in guinea pigs infected intravenously with 2.5 ؋ 10 3 CFU of this organism or with 2.5 ؋ 10 4 CFU of Mycobacterium bovis BCG. Groups of infected guinea pigs were skin tested with fractions of secreted proteins covering well-defined narrow-molecular-mass regions, or such fractions were used for lymphocyte stimulation experiments. The lymphocyte stimulation experiments showed that the fraction containing proteins with molecular masses below 10 kDa had a superior stimulating capacity in tuberculous guinea pigs whereas the 24-to 30-kDa fraction gave significantly higher skin reactions in this group compared with BCG-vaccinated guinea pigs. A precise mapping within the region from 23 to 35 kDa by using a combination of narrow overlapping fractions and purified proteins enabled the identification of the 24-kDa antigen MPT64 as a molecule specific for tuberculous infection. Thus, MPT64 is a promising candidate for a specific diagnostic skin test reagent for human tuberculosis.

We have developed monoclonal antibodies (MoAb) reactive with a protein from Mycobacterium tuberculosis of apparent molecular mass 24 kDa. This protein was shown to be identical with MPB 64 (Harboe et al.,) MoAb bound to four different epitopes of which two were restricted to the 'tuberculosis complex' and two were also found in mycobacteria not belonging to the 'tuberculosis complex'. The cross-reactive MoAb demonstrate that MPB 64 is present in more mycobacterial species than previously assumed. MPB 64 was shown to induce strong delayed type hypersensitivity (Dth) reactions in outbred guinea pigs immunized with M. tuberculosis and M. bovis bacille Calmette-Guérin (BCG). No reaction was observed in animals immunized with mycobacteria not belonging to the 'tuberculosis complex'. The Dth-inducing capacity of MPB64 was compared with that of another 24 kDa protein purified from M. tuberculosis and of the previously described 38 kDa protein. The Dth responses to these three antigens were further analysed in four inbred guinea pig strains. A genetic restriction of the ability of the animals to respond to MPB 64 as well as to the 38 kDa protein was observed.