Activity of phenazine analogs against Mycobacterium leprae infections in mice.

Twenty-five compounds structurally related to clofazimine were tested for their ability to inhibit the growth of Mycobacterium leprae using the kinetic method of drug evaluation in the mouse foot pad model of leprosy. Seven of the phenazine derivatives displayed anti-M. leprae activity comparable to that of clofazimine when administered at a concentration of 0.01% (w/w) in the diet. Three of the compounds, B746, B4087, and B4101, were active when administered at 0.001% in the diet. At a dietary concentration of 0.0001%, B4087 and B4101 were slightly more active than clofazimine, while B746 was less active. In the kinetic method of drug evaluation, greater anti-M. leprae activity of phenazine derivatives was generally associated with greater pigmentation of abdominal fat. Of the compounds which did not cause pigmentation when fed at a concentration of 0.01% in the diet B4090 was the most active. This compound also inhibits the growth of a clofazimine-resistant M. smegmatis strain.

Clarithromycin is bactericidal against strains of Mycobacterium leprae resistant and susceptible to dapsone and rifampin.

The anti-Mycobacterium leprae activity of clarithromycin when administered alone and in combination with rifampin and dapsone in the diet was determined using the kinetic method of drug evaluation in mice. Clarithromycin when administered at a concentration of 0.1% (w/w) in the diet completely prevented growth of 2 pan-susceptible, 3 dapsone-resistant, 2 rifampin-resistant, and 2 rifampin and dapsone double resistant strains of M. leprae. A 0.03% (w/w) concentration also completely prevented growth of M. leprae in all mice infected with 2 of 7 strains tested, but in only some of the mice infected with the remaining 5 strains. No antagonistic drug interactions were observed between clarithromycin and dapsone or rifampin. The addition of clarithromycin to the currently recommended multidrug regimen should improve the rate of killing of M. leprae and help to prevent the growth of dapsone-resistant and rifampin-resistant strains.

Activity of selected beta-lactam antibiotics against Mycobacterium leprae.

Twelve beta-lactam antibiotics were tested for activity against Mycobacterium leprae growing in the foot pads of mice. Two cephalosporins (7-aminocephalosporanic acid and cefuroxime) and one cephamycin (cefoxitin) showed significant activity against M. leprae, and one penicillin (mezlocillin) exerted possible growth-promoting activity. These results suggest that particular molecular structures may be required for activity against M. leprae.

Comparison of the immunogenicity of vaccines prepared from viable Mycobacterium bovis BCG, heat-killed Mycobacterium leprae, and a mixture of the two for normal and M. leprae-tolerant mice.

Intradermal vaccines consisting of viable Mycobacterium bovis BCG, heat-killed Mycobacterium leprae, or mixtures of the two were titrated in mice in doses of 10(5.2), 10(5.8), 10(6.4), 10(7.0), and 10(7.6) acid-fast bacilli. The immune response was measured by sensitization (48 to 72 h foot pad enlargement on challenge with 10(7.0) heat-killed M. leprae) and by protection against infection with a viable M. leprae challenge. There was increasing response with increasing dose of vaccine, and overall the responses to the three vaccines were similar. At the lowest dose, however, the combination of BCG and M. leprae gave superior protection. The local reaction to the vaccines in the lower dose range was less severe with the M. leprae vaccine. In another experiment, the three vaccines were compared in normal mice and in mice that had been rendered tolerant by intravenous injection of M. leprae. The tolerant mice developed no measurable sensitization on vaccination with M. leprae, but they developed partial but distinct sensitization on vaccination with BCG, alone or in combination with M. leprae. The tolerant mice developed little or no protection with any of the vaccines, however.

Sensitization or tolerance to Mycobacterium leprae antigen by route of injection.

Aqueous suspensions of heat-killed Mycobacterium leprae in a dose of 10(7) organisms were highly immunogenic when injected intradermally (i.d.). The same dose of bacteria did not sensitize when given intraperitoneally (i.p.) or intravenously (i.v.), and did so only minimally at best when given subcutaneously. The i.d. route was the most immunogenic for sheep erythrocytes also. M. leprae injected i.p. or i.v. stimulated immune tolerance to M. leprae challenge i.d. In older mice (greater than or equal to 8 weeks), the i.v. injections gave more complete tolerance. Mice that had been rendered tolerant by i.v. injections maintained their tolerance for at least 168 days. Prior UV irradiation of intact mice prevented sensitization by the i.d. route. In normal mice, living M. bovis BCG given i.d. produced good sensitization to M. leprae. Mice that had been made tolerant by i.v. injection of M. leprae could be partially sensitized to M. leprae by i.d. immunization with BCG; mixtures of living BCG and heat-killed M. leprae were no more effective than BCG alone. These findings appear to have relevance to the pathogenesis of lepromatous leprosy and its immunoprophylaxis.

Foot pad enlargement as a measure of induced immunity to Mycobacterium leprae.

Foot pad enlargement (FPE) has been used as a measure of induced immunity to M. leprae, FPE peaked at 2-3 days, but it sometimes persisted for 4 weeks or more. Both as the inducing and eliciting antigen, heat-killed M. leprae were effective, and the optimum dose was about 1 x 10(7) bacilli. Higher doses were associated with flattening of the dose-response curve. Disrupted bacilli were not effective in immunizing mice, but they elicited FPE responses in mice immunized with intact bacilli. Cord factor was not found to have adjuvant activity for M. leprae. In immunization, the intradermal route was confirmed to be more effective than the foot pad route; the subcutaneous route was effective in providing protection against infection. FPE tests were used to investigate the steps of standard purification procedures for M. leprae in armadillo livers. A trypsin-chymotrypsin digestion step was found to be harmful to immunogenicity in one of two experiments.

Searches among mycobacterial cultures for antileprosy vaccines.

All mycobacteria species share some antigens, so there may be cultivable mycobacterial cultures that can provide vaccine protection against leprosy. Vaccine protection against Mycobacterium leprae infections in mice has been demonstrated for M. leprae itself, as living or heat-killed suspensions, and for Mycobacterium bovis (BCG), as living suspensions. Results are reported here with 17 other cultures. The mycobacterial suspensions were injected intradermally, and the mice were challenged in the footpad with infectious suspensions of M. leprae. In two experiments the mice were also challenged by footpad injections of 10(7) heat-killed M. leprae so the footpad enlargment could be measured. That some mycobacterial suspensions were immunogenic for some of their own antigens was suggested by reactions at the vaccine site and enlargement of the regional lymph nodes. Some mycobacterial suspensions also stimulated footpad enlargement on challenge by homologous suspensions or by challenge with M. leprae suspensions. Consistent protection against infectious challenge with M. leprae was observed only with BCG and M. leprae, however.

Heat stability of Mycobacterium leprae immunogenicity.

The protection provided to mice by vaccines administered intradermally was measured after footpad challenge with Mycobacterium leprae. The protection offered by M. leprae suspensions was not decreased when the vaccines were killed by 60 degrees C heat or at the higher temperatures tested, which included 215 degrees C (autoclave). Even highly purified suspensions retained their immunogenicity. In contrast, the vaccine protection provided by intradermal M. bovis (strain BCG) was markedly reduced when heated to 60 degrees C. The enlargement of the lymph nodes regional to the intradermal vaccines was measured and found generally to parallel the vaccine protection provided by M. leprae and by BCG.

Immunity to Mycobacterium leprae infections induced in mice by BCG vaccination at different times before or after challenge.

Viable suspensions of BCG, an attenuated strain of Mycobacterium bovis, have been previously shown to immunize mice against infections with M. leprae. Usually, the mice have been vaccinated about 1 month before challenge. Experiments have now been carried out with single intradermal injections of BCG given before or after the M. leprae challenge. Approximately equal immunizing effect was seen in one experiment when the BCG was given at -168, -119, -70, and -28 days relative to challenge. Approximately equal protection was observed in another experiment when the vaccine was given at -28, +28, and +56 days. In the latter experiment, however, vaccine given at +91 days appeared to be somewhat less effective. Enlargement of the lymph nodes regional to the intradermal vaccine site persisted for at least the duration of the experiment, approximately 400 days. Thus, antigenic stimulation appears to have continued throughout the period of observation.

Effect of levamisole on Mycobacterium leprae in mice.

Levamisole, an antihelminthic drug that is capable of enhancing immune responses in mice and in humans, was tested in experimental Mycobacterium leprae infections in mice by a number of schedules. Intermittent schedules were used, and administration of the drug was started (i) around the time of inoculation with M. leprae, (ii) when the M. leprae population was approaching the plateau level, (iii) after the onset of the plateau phase, or (iv) after BCG vaccination 28 days following the inoculation with M. leprae. No effect of drug could be discerned with any of the schedules.

Effect of levamisole on Mycobacterium leprae in mice

Levamisole, an antihelminthic drug that is capable of enhancing immune responses in mice and in humans, was tested in experimental Mycobacterium leprae infections in mice by a number of schedules. Intermittent schedules were used, and administration of the drug was started (i) around the time of inoculation with M. leprae, (ii) when the M. leprae population was approaching the plateau level, (iii) after the onset of the plateau phase, or (iv) after BCG vaccination 28 days following the inoculation with M. leprae. No effect of drug could be discerned with any of the schedules.

Immunity to Mycobacterium leprae infections in mice stimulated by M. leprae, BCG, and graft-versus-host reactions.

Infections of mice with Mycobacterium leprae in one rear foot pad immunized them against a second infection in the other rear foot pad. Purified bacilli harvested from the first infection also produced immuniy when injection into the foot pads of previously uninfected mice. Injections of BCG afforded similar protection, but had no adjuvant effect on M. leprae. M. duvali, a cultivable mycobacterium that is reported to be more closely related antigenically to M. leprae than BCG is, provided much less protection against M. leprae challenge than BCG did. Moreover, when M. duvali was mixed with BCG, it was not any more effective than BCG alone. Graft-versus-host reactions, induced by injections of parental spleen cells into F1 hybrids, provided no protection against M. tuberculosis and M. marinum challenge. They gave moderate protection against M. leprae in one experiment but not in another with a different schedule. Allogenic spleen cells had a protective effect when injected locally into the infected foot pad. The effect produced by these injections of spleen cells was a delay in the appearance of bacterial growth; however, there was no decrease in the rate of logarithmic growth when it did appear and no reduction in the eventual plateau level.