Activity of combinations of dapsone, rifampin, minocycline, clarithromycin, and sparfloxacin against M. leprae-infected mice.

In these studies we evaluated the activity of low levels of five antimicrobials against Mycobacterium leprae-infected mice when administered singly and in all possible two- and three-drug combinations. Antibiotics studied were: dapsone (D) 0.0001% in the diet, rifampin (R) 20 mg/kg by gavage once monthly, minocycline (M) 0.004% in the diet, clarithromycin (C) 0.001% in the diet, and sparfloxacin (S) 5 mg/kg by gavage five times weekly. Singly each agent was found bacteriostatic (D + R) or partially bactericidal (M, C, and S) but not fully bactericidal. All 10 two-drug regimens were found at least bacteriostatic, 2 being "partially bactericidal" and 4 being "fully bactericidal." Of the 10 three-drug regimens, 9 were found "fully bactericidal" and the other "partially bactericidal." We conclude that combinations of antibiotics active against M. leprae are generally additive in combination.

Efficacy of minocycline in single dose and at 100 mg twice daily for lepromatous leprosy.

A clinical trial of minocycline in a total of 10 patients with previously untreated lepromatous leprosy was conducted in order to evaluate the efficacy of a single, initial, 200-mg dose and 100 mg twice daily of minocycline for a total duration of up to 3 months. Patients improved remarkably quickly. Although single-dose therapy did not result in a significant killing of Mycobacterium leprae, viable M. leprae were cleared from the dermis regularly by 3 months of twice-daily therapy, a rate similar to that achieved by minocycline 100 mg once daily. Because more side effects were noted herein than previously with 100 mg daily, we recommend that minocycline, when applied, be administered at 100 mg daily to leprosy patients.

Vaccination with pure Mycobacterium leprae proteins inhibits M. leprae multiplication in mouse footpads.

In this study, we evaluated vaccination with a number of purified, as well as recombinant, Mycobacterium leprae proteins for protective efficacy in mice. BALB/c mice were immunized intradermally with various native somatic (purified) or recombinant M. leprae proteins and their synthetic polypeptides emulsified in Freund's incomplete adjuvant. The protective efficacy of these preparations was assessed by enumeration of bacilli in the footpads of mice challenged with viable M. leprae 1 to 2 months following immunization. Protection was afforded by the purified and recombinant 10-kDa M. leprae cytoplasmic heat shock protein, the recombinant cell wall-associated 65-kDa M. leprae heat shock protein, and to a lesser extent, the purified 28-kDa M. leprae cytoplasmic protein (superoxide dismutase). Vaccination with either the purified or recombinant 35-kDa M. leprae cell membrane protein, the synthetic 27-amino-acid N-terminal peptide of the 10-kDa protein, the recombinant 18-kDa M. leprae protein, or the purified 22-kDa cell membrane protein was ineffective. When the interval between immunization and challenge was increased to 6 months, the purified 10-kDa M. leprae protein and the recombinant 65-kDa M. leprae protein lost vaccine efficacy, while a sodium dodecyl sulfate-soluble protein fraction of the M. leprae cell wall (soluble proteins), as had been found previously, continued to protect, suggesting that multiple M. leprae protein epitopes are critical for solid vaccine protection.

Effective vaccination of mice against Mycobacterium leprae with density-gradient subfractions of soluble M. leprae proteins: clues to effective protein epitopes.

It had previously been discovered that intradermal mouse vaccination with a protein fraction of Mycobacterium leprae (called soluble proteins) in Freund's incomplete adjuvant (FIA) resulted in consistent and long-lived protection against M. leprae multiplication from subsequent viable footpad challenges. In this study certain density-gradient subfractions of this soluble protein, but not others, in FIA afforded vaccine protection. The results of this study suggest which M. leprae proteins may be involved in protective immunity, particularly 1-3 kD, 10 kD, 65 kD, and those of higher molecular weight.

Activities of various quinolone antibiotics against Mycobacterium leprae in infected mice.

Previously, pefloxacin and ofloxacin were found to be active against Mycobacterium leprae in vitro, in experimental animals, and in clinical trials of lepromatous leprosy patients. In this study, we compared certain more recently developed fluoroquinolones (lomefloxacin, PD 124816, WIN 57273, temafloxacin, and sparfloxacin) with pefloxacin and ofloxacin in M. leprae-infected mice at doses of 50, 150, and 300 mg/kg given five times weekly. All seven of the fluoroquinolones studies were active against M. leprae; temafloxacin and sparfloxacin were the most active, being fully bactericidal at all three dosage schedules. Additionally, sparfloxacin was found to be fully bactericidal at 15 and 30 mg/kg given five times weekly.

Vaccination of mice with a soluble protein fraction of Mycobacterium leprae provides consistent and long-term protection against M. leprae infection.

Groups of BALB/c mice were vaccinated intradermally with either Freund's incomplete adjuvant (FIA) alone, 10(7) heat-killed Mycobacterium leprae organisms in FIA, or a number of fractions of M. leprae containing soluble and/or cell wall components. At 1, 3, 6, 9, and 12 months later, vaccinated mice were challenged in the right hind footpad with 5,000 live M. leprae organisms, and vaccine protection was assessed 6 to 8 months later, at the peak of M. leprae multiplication in the negative control (FIA alone), by the two-sample rank-sum test. In these studies, a cell wall fraction rich in peptidoglycan was consistently ineffective. Both heat-killed M. leprae and a fraction containing cell wall and fixed proteins generally protected when the interval between vaccination and challenge was 1 or 3 months but not subsequently. On the other hand, soluble proteins of M. leprae alone or in combination (with cell wall fractions) consistently (14 of 14 instances) afforded highly significant protection (P less than or equal to 0.01) at all challenge intervals up to 1 year after vaccination. These results suggest that the soluble protein fraction of M. leprae offers promise for a vaccine against leprosy.

Effect of low-level and intermittent minocycline therapy on the growth of Mycobacterium leprae in mice.

We evaluated the minimal concentrations of minocycline in the diet and in serum required to inhibit the growth of seven Mycobacterium leprae isolates in mice. Minocycline concentrations of 0.01 and 0.04% in the diet, which resulted in levels in serum of less than or equal to 0.17 and 0.51 microgram/ml, respectively, were consistently and completely inhibitory. Even 0.004% dietary minocycline (levels in serum, less than or equal to 0.08 microgram/ml) partially inhibited five of these strains, while 0.001% minocycline was consistently inactive. For five of these isolates, minocycline at a concentration of 0.04% in the diet given 3 days (Monday, Wednesday, Friday) and 1 day weekly completely inhibited the growth of M. leprae, and minocycline given even 1 day monthly was partially inhibitory for three of these five M. leprae isolates.

Activities of various macrolide antibiotics against Mycobacterium leprae infection in mice.

We evaluated the activities of several macrolide antibiotics against M. leprae infections in mouse footpads. Erythromycin and azithromycin were inactive, while both roxithromycin and clarithromycin were found to be consistently active and, in fact, bactericidal. By both methods, clarithromycin was found to be superior to roxithromycin, a finding which, at least in part, may be a consequence of the higher levels of clarithromycin at the site of infection.

Primary dapsone-resistant Hansen's disease in California. Experience with over 100 Mycobacterium leprae isolates.

We found that in the years 1978 through 1981 only one of 54 previously untreated patients with Hansen's disease was found to harbor dapsone-resistant Mycobacterium leprae. That single strain was only partially resistant, ie, it was resistant to 0.0001% dapsone in a mouse diet but not to higher concentrations. During the years 1983 through 1988, M leprae from 47 previously untreated patients presenting to clinics in San Francisco, Calif, and Los Angeles, Calif, grew in mice. None of these strains was found to be dapsone resistant. Thus, from 1978 through 1988 only one of 101 M leprae isolates obtained from skin biopsy specimens from patients with leprosy was found to be resistant to dapsone. We have concluded that primary dapsone resistance still does not appear to be a significant problem in California. Owing to the fact that our single resistant case and those reported from international sources are, in general, partially resistant, the potential importance of partial dapsone resistance is discussed.

Effective vaccination of mice against leprosy bacilli with subunits of Mycobacterium leprae.

Model vaccines against leprosy bacilli have consisted of nonvirulent, live, attenuated Mycobacterium bovis BCG and irradiated, heat-killed, or autoclaved intact M. leprae. We report that immunization with various cell wall fractions of M. leprae, progressively depleted of lipids, carbohydrates, and soluble proteins, as well as a partially purified protein(s) derived from a pellet fraction of sonicated M. leprae, conferred significant protection against subsequent infection with live leprosy bacilli. Moreover, lymphocytes from regional lymph nodes and spleens of mice immunized with these M. leprae-derived subunits responded by proliferation when stimulated with M. leprae in vitro. Our results provide the first evidence that vaccination with M. leprae-derived fractions protects mice against leprosy bacilli.