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A DNA vaccine composed of the gene for the common mycobacterial secreted protein antigen 85B was demonstrated to protect the mouse foot pad against infection with Mycobacterium leprae. The protective effect was demonstrated by a 61%-88% reduction in the bacterial number, a protective effect less than that of BCG. The same DNA vaccine has been shown to protect mice against M. tuberculosis infection, and the importance of testing other candidate tuberculosis vaccines for their potential to protect against leprosy is discussed.

A new rapid immuno-chromatographic test card for the detection of antibodies to the Mycobacterium leprae 35-kD protein is described. The new assay is compared in the same group of subjects with a direct enzyme ELISA method for 35-kD antibodies and with assays for anti-phenolic glycolipid-I (PGL-I) antibodies using a standard ELISA as well as the recently described "dipstick" method. Good concordance was found between the rapid methods and the corresponding ELISA methods. The detection of untreated paucibacillary leprosy by the 35-kD test card was 59% compared with 27% for the PGL-I dipstick; however, the specificity for the 35-kD test card was 90% compared with 100% for the PGL-I dipstick in an endemic population. The potential application of these new, rapid serologic methods for the diagnosis of leprosy under field conditions is discussed.

Although multidrug therapy (MDT) was introduced into Nepal in 1983, the MDT coverage only recently exceeded 67%. In view of the large number of patients who were still receiving dapsone monotherapy, it is relevant to investigate the current levels of dapsone and rifampin resistance. The study was undertaken at a leprosy referral hospital near Kathmandu. Over a 5 1/2-year period, 157 leprosy patients with a bacterial index (BI) > or = 2.0 were investigated for drug resistance according to the method of Rees. Among previously untreated cases, 6% of 88 isolates showed low-dose dapsone resistance; among previously treated patients with a presumed relapse, 47% of 34 isolates demonstrated dapsone resistance. In the remaining 35 cases there was no growth in control mice. Rifampin resistance was not confirmed in any case.

Sixty-five patients initially seropositive for IgM anti-phenolic glycolipid-I (PGL-I) antibodies were tested for antibody levels to PGL-I, lipoarabinomannan (LAM), and the 35-kDa protein of Mycobacterium leprae at regular intervals for up to 30 months following the commencement of multidrug therapy (MDT). There was a steady decline in IgM anti-PGL-I and anti-35-kDa antibody levels to a mean of 17% and 14%, respectively, of the starting level at 24 months. The development of type 1 and type 2 reactions or the presence of drug-resistant organisms in a small number of patients had no significant influence on the changes in antibody level. The rate of decline was similar in different disease categories, but a higher proportion of lepromatous patients remained seropositive at the end of 2 years of treatment than borderline tuberculoid patients. By contrast, the mean IgG anti-LAM antibody levels remained stable or increased. Again the occurrence of type 1 or type 2 reactions had no significant effect on antibody level over 2 years. Falls in the IgM anti-PGL-I antibody levels mirrored the falls in the bacterial index in individual patients and provide an additional parameter for monitoring the response to chemotherapy.

The 18-kDa protein of Mycobacterium leprae, as recognized by the monoclonal antibody L5, has a restricted species distribution, being confined to M. leprae and M. habana. We have developed a solid-phase ELISA using purified, recombinant M. leprae 18-kDa protein and compared the serological responses of Nepali leprosy and tuberculosis patients and endemic control subjects to the protein and the M. leprae phenolic glycolipid-I (PGL-I). Few control subjects had anti-18-kDa antibodies. A small proportion of paucibacillary (PB) leprosy and 42% of multibacillary (MB) leprosy patients had IgG anti-M. leprae antibodies. A similar proportion (47%) of Nepali tuberculosis (TB) patients were seropositive, and IgG anti-18-kDa antibody levels were significantly higher in MB and TB patients than in control subjects. By comparison, IgM anti-PGL-I antibodies were detected in 88% of MB leprosy patients and only 7% of TB patients. The possible reasons for the 18-kDa protein seroreactivity in TB patients are discussed, and the anti-18-kDa assay is compared with other antibody assays for protein and nonprotein antigens of M. leprae. It is concluded that the sensitivity and specificity of the anti-M. leprae 18-kDa ELISA are insufficient for the assay to be of clinical utility in leprosy patients.

Sixty-three leprosy patients, representing the entire leprosy spectrum from tuberculoid to lepromatous disease, and 17 healthy Nepali subjects were tested for their T-cell responses to the purified p65 and p70 protein antigens of Mycobacterium bovis BCG using a lymphocyte proliferative assay. There was strong correlation between the responses to BCG and M. leprae and the responses to the two antigens. Patients and controls lacking a response to either of the whole cell preparations failed to respond to the purified antigens, while BCG responders at the lepromatous pole of the disease did mount a response to both antigens. Significant differences in the magnitude of the responses to these antigens were obtained between controls and the disease groups. In individual subjects, responses to the two antigens were significantly correlated to each other.

The antibody responses of 100 previously untreated multibacillary (MB) leprosy patients to one protein and two carbohydrate antigens were examined: 94% of the patients had Mycobacterium leprae-specific antibodies; 89% directed to the species-specific epitope on phenolic glycolipid (PGL-I), 89% against the specific epitope on the 35-kDa protein, and 94% against one or both of the two. By contrast, 67% of the patients had anti-lipoarabinomannan (LAM) antibodies. There were trends for the seropositivity rate and the antibody level to rise with the increasing extent of the disease and as patients moved to the polar lepromatous end of the spectrum. The bacillary load, as measured by the bacterial index, was moderately correlated with the IgM anti-PGL-I and the anti-35-kDa antibody levels and, to a lesser extent, with the IgG antibodies directed at the common mycobacterial carbohydrate LAM. The sensitivity of the IgM anti-PGL-I antibodies for detecting smear-positive MB disease was 91%; that for the anti-35-kDa antibodies was 92%.

We have examined the serological responses of 154 untreated paucibacillary (PB) leprosy patients to two carbohydrate and one protein antigens of Mycobacterium leprae. There was a heterogeneous response with 20% of PB patients having IgM anti-PGL-I antibodies and a similar proportion with IgG anti-LAM antibodies, while 33% had antibodies to the M. leprae-specific epitope on the 35-kDa protein. There was overlap in the responses such that 43% of the patients were seropositive in one of the two M. leprae-specific assays, while 49% were positive in any assay. There was a gradation in seropositivity with increasing extent of disease for each of the clinical parameters measured. Those with established disability at the time of presentation were more likely to be seropositive in each assay.

Monoclonal antibodies (Mabs) raised against Mycobacterium leprae sonicate defined two different determinants on related, cell-wall-associated, carbohydrate antigens common to M. leprae, M. bovis (BCG), and M. tuberculosis. Antigen inhibition ELISA and antigen capture assays demonstrated that the two antigens were present in a cell-wall fraction, M. leprae resonicate. There was species variation in the distribution of the antigens; the 4.5-6 kD antigen was more abundant in M. tuberculosis and M. bovis, while the 30-40 kD antigen was more concentrated in M. leprae preparations. Although both were present in the cell wall, only determinants on the 30-40 kD antigen were accessible on intact bacilli. The results from capture assays and Mab affinity chromatography with both L9 and L4 indicated that the 4.5-6 kD antigen was probably a fragment of the larger molecule. Both antigens are significant immunogens in the human B-cell response to M. leprae.