Protective responses against experimental Mycobacterium leprae infection in mice induced by recombinant Bacillus Calmette-Guérin over-producing three putative protective antigen candidates.

The components of Ag85 (Ag85A, Ag85B, and Ag85C) are putative protective antigen candidates against mycobacterial infection. A recombinant Mycobacterium bovis Bacillus Calmette-Guérin (rBCG) over-producing Ag85A, Ag85B, and MPB51 (rBCG/BA51) was constructed. rBCG/BA51 could secrete these antigens at levels more than five times higher than parental BCG. Immunization of C57BL/6 and BALB/c mice with this rBCG reduced the multiplication of Mycobacterium leprae in the foot pads of both strains of mice. The inhibition by rBCG/BA51 was more evident than that by parental BCG.

[Study on recombinant BCG].

The progress of study on recombinant BCG was stated briefly. And then our studies on recombinant BCG were mentioned. Recombinant BCG secreting alpha antigen-fused merozoite surface protein 1 (MSP 1) was prepared and tested for its ability to control infections of Plasmodium yoelii. Result turned out it controlled the infection better than recombinant MSP 1 mixed with Freund incomplete adjuvant did. Recombinant BCG secreting excess amounts of antigen 85 complex A controlled infection of Mycobacterium leprae. Addition of recombinant BCG secreting alpha antigen-fused IL-2 to peritoneal exudate cells induced IFN-gamma resulting in killing bladder cancer cells more efficiently than parental BCG did.

Inhibition of multiplication of Mycobacterium leprae in mouse foot pads by recombinant Bacillus Catmette-Guérin (BCG).

Immunization of mice with recombinant Mycobacterium bovis Bacillus Calmette-Guérin (rBCG) which over-produces a putative protective antigen candidate, the A component of antigen 85 complex (Ag85A), reduced the multiplication of Mycobacterium leprae in the foot pads of mice. The inhibition by this rBCG (rBCG/85A) was more evident than that with parental BCG. Repeated rBCG/85A immunization significantly could reduce M. leplae multiplication in mice. This is first report of rBCG to control mycobacterial infection in animal model. Therefore, rBCG technique may be useful for the development of a more effective mycobacteria vaccine.

The gene encoding mycobacterial DNA-binding protein I (MDPI) transformed rapidly growing bacteria to slowly growing bacteria.

Pathogenic species of Mycobacterium are slowly growing intracellular bacteria. Slow growth is important for the parasitism of these organisms and chronicity of the disease, but its precise mechanism has not been elucidated. Recently, we found that a novel DNA-binding protein (MDPI) was expressed (7-10% in total protein) in mycobacteria, such as Mycobacterium bovis bacillus Calmette-Guérin, Mycobacterium tuberculosis, and Mycobacterium leprae. In this study, we observed that MDPI interfered with replication, transcription, and translation in the analysis in in vitro E. coli cell-free macromolecular biosynthesizing systems. Furthermore, MDPI inhibited the rapid growth of both Escherichia coli and Mycobacterium smegmatis, and NH(2)-terminal second amino acid, asparagine, was observed to be important in terms of this function. These data suggest an important role of MDPI for suppression of growth rates of mycobacteria.

The antigen 85 complex vaccine against experimental Mycobacterium leprae infection in mice.

The proteins in culture filtrate derived from Bacillus Calmette-Guérin (BCG) were examined for protection against infection by Mycobacterium leprae. Immunization with the major secreted proteins, antigen 85 complex (Ag 85) A, B and C, induced effective protective immunity against multiplication of M. leprae in the foot pads of mice. The most effective protection was observed when mice were immunized with Ag 85A. A single immunization with Ag 85 could induce antigen-specific interferon gamma (IFNgamma) synthesis and more effective protection than live BCG vaccine. This study demonstrates that Ag 85 is an important immunoprotective molecule against leprosy infection.

Ribosomal protein L7 included in tuberculin purified protein derivative (PPD) is a major heat-resistant protein inducing strong delayed-type hypersensitivity.

The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis. It consists of more than 100 denatured proteins in a culture filtrate of a heated culture of Mycobacterium tuberculosis. In two-dimensional electrophoretic analysis of PPDs from M. tuberculosis and M. bovis BCG, most proteins were diffusely separated and could not be seen as spots because of denaturation, whereas a few proteins showed relatively clear spots, indicating heat resistance. Two such proteins corresponded to ribosomal proteins L7 and L12. The mixture of these proteins L7/L2 induced a strong delayed-type hypersensitivity reaction. Another protein showing a clear spot was a GroES analogue, but this did not induce delayed-type hypersensitivity. There were a few other unidentified proteins. It is well known that L7 and L12 are encoded by the same gene and that they differ from each other only by an acetylic post-translational modification that occurs at the N-terminus of L12 converting it to L7 in Escherichia coli. L12, but not L7, was found in two-dimensional electrophoresis of BCG ribosomes, although we found two proteins corresponding to L7 and L12 in PPDs and a native culture filtrate of BCG. We compared the delayed-type hypersensitivity reaction elicited by L7/L12 derived from a culture filtrate of BCG and L12 derived from BCG ribosomes. L7/L12 from the culture filtrate could induce delayed-type hypersensitivity, but L12 from ribosomes could not, indicating that L7 was attributable to the induction of delayed-type hypersensitivity. The activity of L7/L12 was heat resistant. Neither glycosylation nor phosphorylation of L7/L12 from a culture filtrate could be detected. The acetylation at N-terminal of L12 was essential for the delayed-type hypersensitivity activity.

Two-dimensional electrophoretic analysis of humoral responses to culture filtrate of Mycobacterium bovis BCG in patients with leprosy and tuberculosis.

Sera from 3 lepromatous (LL), 3 borderline lepromatous (BL), 3 mid-borderline (BB), 3 borderline tuberculoid (BT), 2 tuberculoid (TT) and 4 tuberculosis (TB) patients and 3 healthy individuals were examined for their reactivities against the proteins in the culture filtrate of BCG separated by two-dimensional electrophoresis (2-DE). The sera were obtained from patients who were untreated. Sera from LL and BL patients reacted strongly with the antigen 85 (Ag85) complex and MPB51. Sera from LL and BL patients also weakly reacted with the newly identified 29-, 24- and 23-kDa spots. Sera from the other patients reacted similarly, but the levels of reaction were remarkably lower than those form LL and BL patients. Mycobacterium leprae antigens that are analogous to BCG AG85 and MPB51 are suggested as the main targets for the humoral immunity of untreated patients. The reactivities of sera with newly identified antigens may provide the potential for predicting the severity and prognosis of diseases.

Inhibition of multiplication of Mycobacterium leprae in mouse foot pads by immunization with ribosomal fraction and culture filtrate from Mycobacterium bovis BCG.

Immunization of mice with the ribosomal fraction from ruptured Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and the culture filtrate reduced remarkably the multiplication of Mycobacterium leprae in the foot pads of mice. This is the first reported case of the protective activity against M. leprae multiplication in mice of the BCG ribosomal fraction and culture filtrate. The inhibition was more evident with the culture filtrate than with the ribosomal fraction. When the ribosomal proteins separated from ribosomal RNA were injected into mice, only slight inhibition was observed. Ribosomal RNA alone did not inhibit at all, in contrast to the conclusion reported by Youmans and Youmans.

[BCG vaccination to Mycobacterium leprae infection in mice].

BCG vaccine (Tokyo strain) was given in BALB/cA mice intradermally 1 or 3 months before Mycobacterium leprae (M. leprae) challenge as modified Shepard's method. The vaccine dosage was 10(7-8) or 10(6). The BCG gave good protection in both dosages and both challenges against M. leprae infection. Lymphocytes proliferations of BCG-vaccinated splenocyte cultures in response to M. leprae lysate or BCG components (hsp65, 38 kD, 30 kD or 12 kD protein) were tested, and potent proliferative responses were seen in the cultures with M. leprae lysate and hsp65. Furthermore, gamma-IFN productions were positive in the cultures with M. leprae lysate or hsp65, but negative with other antigens. The production of gamma-IFN with hsp65 was never inhibited with polymyxin B, but inhibited with IL-10. These results show that BCG (Tokyo strain) is a useful vaccine for M. leprae infection in mice, and one of the components of BCG, hsp65, may be a effective antigen component for protection of M. leprae infection inducing Th1 type cytokine.

Cloning, sequencing and expression in Escherichia coli of the gene for alpha antigen from Mycobacterium scrofulaceum.

The gene for the extracellular alpha antigen of Mycobacterium scrofulaceum (S-alpha) was cloned by using the alpha antigen gene fragments of Mycobacterium bovis BCG as probes. The complete nucleotide sequence was determined. The gene was expressed in Escherichia coli. The gene encodes 330 amino acids, including 40 amino acids for the signal peptide, followed by 290 amino acids for the mature protein. The deduced amino acid sequences were highly homologous to the alpha antigen of the species of other mycobacteria. Interestingly, the alpha antigens of MAIS complex (Mycobacterium avium-Mycobacterium intracellulare-M. scrofulaceum) were closely homologous even at the C-terminal regions which were variable among those of M. bovis BCG, Mycobacterium leprae and Mycobacterium kansasii.

Cloning and sequencing of the gene for alpha antigen from Mycobacterium avium and mapping of B-cell epitopes.

The complete nucleotide sequence of alpha antigen secreted from Mycobacterium avium (A-alpha) was determined. The gene encodes 330 amino acids, including 40 amino acids for the signal peptide, followed by 290 amino acids for the mature protein with a molecular mass of 30,811 Da. This is the first sequence of A-alpha. Comparisons between A-alpha and alpha antigens of Mycobacterium leprae, Mycobacterium bovis BCG, and Mycobacterium kansasii showed highly homologous regions which suggested a conserved functional domain and two less-homologous regions. Serological analysis of recombinant A-alpha, expressed by a series of deletion constructs, indicated the possibility that A-alpha carries at least six B-cell epitopes. The three antigenic determinants were common to Mycobacterium tuberculosis, M. kansasii, and M. avium. The results also suggested the possibility that there are three species-specific epitopes.