Adoptive transfer of tolerance induced by ICRC bacilli against Mycobacterium leprae in mice.

ICRC bacilli, the cultivable leprosy-derived mycobacteria, isolated from lepromatous nodules of leprosy patients were found to be immunogenic in BALB/c mice at a dose of 2 X 10(7) acid-fast bacilli when injected by the intradermal (i.d.) route. The sensitization to lepromin and ICRC antigens was measured by the foot pad enlargement (FPE) method. The same dose of bacilli when injected by intravenous (i.v.), intraperitoneal, and subcutaneous routes induced immune tolerance in mice as indicated by reduction in the FPE to the test antigens. The spleen cells obtained after i.v. injection of ICRC bacilli/Mycobacterium leprae after adoptive transfer brought about suppression of delayed-type hypersensitivity in sensitized as well as nonsensitized recipients, indicating production of suppressor cells after i.v. injection. Similarly, the tolerance induced by i.v. injection of M. leprae in mice could be partially converted to immunity by i.d. sensitization with live BCG and two strains of ICRC bacilli (C-44 and C-75).

Immunological properties of M. leprae culture isolates ICRC bacilli: hypothesis on relationship between M. leprae and ML-culture isolates.

The M. leprae-culture isolate, being a culture, is identified by biochemical criteria. Majority belong to either M. avium-intracellulare-scrofulacieum complex, or corynobacterium group. The identification is also determined by serological methods using soluble sonicates. Skin tests with heat-killed suspension often give positive Mitsuda response and hence the isolates are discarded as non-M. leprae. The data on ICRC strains show that Dharmendra type antigen prepared from ICRC bacilli compares very well with lepromin in lepromatous patients. The CMI/DTH tests in mice by FPE and LMI technique have experimentally demonstrated that ICRC bacillus is the first cultivable mycobacterium that can sensitize mice against lepromin. These data also brought out antigenic differences between the ICRC strains. The clinical trial on ICRC vaccine has shown that the ICRC bacilli is immunogenic in Man. The biochemical data on ICRC strain show that they possess DOPA-Oxidase and can express biochemical character of 9/10 similarity index with M. leprae with appropriate substrates in the medium. Thus, the ICRC strains possess both M. leprae and M. avium characters. An hypothesis is proposed to explain these observations--The 'Janus face' of M. leprae--by proposing a relationship between M. leprae and its culture isolates, e.g. ICRC bacilli (1). The M. leprae-culture isolate may by a recombinent of M. leprae with M. avium background (2). The ML-isolate may express the CMI related antigenic determinants corresponding to that of M. leprae in the biopsy (3). The CMI related antigens of whole bacilli in the ML-culture isolate may be inducing type as against only eliciting antigen expressed by M. leprae (4). Recombination may provide immunogenicity as well as 'in vitro' growth potential to the ML-culture isolate (5). Absence or failure to express inducing antigen by M. leprae maybe alternate biological escape mechanism from immune surveillance for survival in vivo, with or without suppressor mechanisms.

Induction of delayed-type hypersensitivity by ICRC anti-leprosy vaccine and the adoptive transfer of cell-mediated immunity in mice.

The immunogenic potency of anti-leprosy ICRC vaccine preparations was determined by foot pad enlargement in mice. Female Balb/c mice were sensitized by I.D. route with irradiated, heat-killed or live antigens of 2 strains (C-44 and C-75) of ICRC bacilli and tested against lepromin and ICRC-antigens. ICRC antigens sensitized mice against lepromin; similarly, BCG and M. leprae sensitized mice, which showed good cross reactivity with ICRC antigens. A mixture of irradiated ICRC bacilli and live BCG was not more immunogenic to mice than its single constituents. The immunity induced by ICRC antigens could be adoptively transferred to syngeneic recipients by sensitized spleen cells.

Comparison of biochemical characterisation of ICRC bacilli with M. leprae: effect of substrate alteration in the medium.

ICRC-bacilli strain C-44 when grown in Dubos medium of its equivalent, express M. avium taxonomic biochemical characters. Assuming that difference in characters of M. leprae and ICRC bacilli, could be due to 'in vivo' and 'in vitro' milieu, we altered the substrates in the medium. The bacilli grow well in the new medium containing selenium, ferric nitrate, magnesium chloride and deleting Tween 80. The ICRC strain C-44 grown in new medium expressed characters: 9/10 similarity with M. leprae. The 10 day tween hydrolysis reaction in weak but positive. It is probable that 'M. leprae culture isolate', may have acquired 'in vitro' growth potential by recombination with M. avium, an ubiquitous mycobacterium. The M. leprae culture isolate thus may express some characters of both M. leprae and M. avium.

Antigeneic cross-reactivity between ICRC-bacilli and M. leprae--"in vitro" evaluation.

Leucocyte migration inhibition, in presence of specific particulate antigen, is a good correlate of CMI. It can detect small differences in related antigens. In the present study, LMI was used to study the cross-reactivity between ICRC bacilli and M. leprae and also to examine the antigenic relationship amongst different ICRC isolates. The study showed a close antigenic cross-reactivity in lepromin and ICRC-in. LMI has brought out strain differences in two ICRC culture isolates, C-44 and C-75. The data appear to elucidate the possible strain specific antigenic relationship of M. leprae with its culture isolate e.g. ICRC strains. This may have important bearing on the development effective antileprosy vaccine for human use.

Dopa-oxidase activity on ICRC bacilli.

Presence of O-phenoloxidase is regarded as M. leprae specific character. This enzyme activity was found to be present in ICRC bacilli, Strain C-44. Though this strain is cultivable 'in vitro', the expression of DOPA-Oxidase activity strongly suggests that it carries M. leprae genome. The ICRC bacilli, therefore, may thus from a group of M. leprae culture isolates, distinct from other known cultivable mycobacteria which do not possess this enzyme activity.

Reversal reaction in lepromatous patients induced by a vaccine containing killed ICRC bacilli--a report of five cases.

Clinico-pathological features of five cases of lepromatous leprosy exhibiting "reversal" reaction with upgrading of lesions following vaccination with ICRC vaccine have been described. Two patients also developed ENL. Associated with the "reversal" reaction, the patients exhibited lepromin conversion. No evidence of fresh nerve lesions was observed in any patient. Besides lepromin conversion, occurrence of "reversal" reaction is yet another evidence of immunogenicity of the ICRC vaccine.

Growth of the ICRC bacilli in the foot-pad of mice.

The ICRC bacilli are acid-fast bacilli cultivated from M. leprae isolates of lepromatous tissue. The ICRC bacilli from C-44 in the conditioned medium were subjected to foot-pad test, in both normal and T/900r mice. The bacilli exhibits a limited multiplication in normal mice while a continuous growth in T/900r mice. The maximum yield for normal and T/900r mice was 10(7) and 10(9)/foot-pad, respectively. The infiltration of voluntary muscle tissue as the main localization site was common for both normal and T/900r mice with evidence of dissemination in the latter. The spread of AFB to sciatic nerve, induction of liver granuloma and the foot-drop was observed only in T/900r mice. These experiments show that the growth of ICRC bacilli in mouse foot-pad is very similar to that of M. leprae confirming a test for identification.