Immune responses to recombinant proteins of Mycobacterium leprae

Identification of antigenic determinants of the polar immune response in leprosy may illuminate both protection and pathogenesis. Thirty subjects were studied (22 with polar disease and 8 healthy controls who were heavily exposed but disease-free) by assaying the proliferative, interferon (IFN)-gamma, and antibody responses to recombinant antigens of Mycobacterium leprae (10, 28, 36, and 65 kDa). The 10-kDa antigen elicited IFN-gamma production from all tuberculoid (TT) and borderline tuberculoid (BT) patients but little from controls, lepromatous (LL), or borderline lepromatous (BL) patients (P<.05). Production of 65-kDa-specific IFN-gamma was higher in TT/BT than in controls or LL/BL patients (P<.006). All subjects produced 65-kDa-specific antibody, but it was higher in LL/BL patients than in healthy controls, whose responses were higher than in TT/BT subjects (P=.035). The 36-kDa antibody responses were selectively increased in LL/BL subjects (P<.02). The intermediate phenotype of the controls suggests that M. leprae-specific production of IFN-gamma may contribute to pathology and to protection in leprosy.     

Serologic response to mycobacterial proteins in hansen's patients during multidrug treatment

Humoral immune responses were studied in 24 leprosy patients treated with multidrug therapy (MDT) and 16 contacts. The patients were monitored for 2 to 3 years with repeated determination of IgG antibody levels directed to different mycobacterial proteins (Mycobacterium tuberculosis, Mt70; M. bovis, Mb65; M. leprae, Ml36, 28, 18, 10 kDa, and the complete protein M. leprae extract, MLSA). All recombinant antigens were used at 5 micrograms/ml concentration and the complete soluble M. leprae extract at 2 micrograms/ml. The results shown in this study reveal a clear decline in IgG antibodies directed toward mycobacterial proteins in the 12 multibacillary (MB) patients when they were submitted to MDT. Initially we found strong reactivity toward complete cytosolic protein and M. leprae membrane protein. The most reactive recombinant proteins in MB patients were Ml10, Ml36, Mt70 kDa and, finally, Ml18 kDa when compared to the paucibacillary (PB) group. After treatment was completed all lepromatous and borderline lepromatous patients showed low or undetectable levels as compared with their initial values before starting treatment.     

Salivary immunoglobulins and antibody activities in leprosy

The technics of immunodiffusion and the fluorescent leprosy antibody absorption (FLA-ABS) test were used to determine the levels of immunoglobulins and their antibody activities against Mycobacterium leprae in the serum and the saliva collected from a total of 110 patients with leprosy (50 lepromatous, 24 borderline, and 36 tuberculoid). The average levels of serum IgG, IgM, and IgA were not significantly different among these patients. In saliva, however, IgM was detected in only two cases with lepromatous leprosy and three tuberculoid cases. Salivary IgG and IgA levels and their ratios to those in the sera were not significantly different according to the classification of leprosy. The percentages of positive FLA-ABS tests in the sera and saliva were compared by using fluorescent antibodies specific for IgG, IgM, and IgA, respectively. The results indicated that M. leprae-specific antibodies in the serum were mainly found in IgG and IgM and, less frequently, in IgA. IgG antibodies were found more frequently in lepromatous and borderline patients than in tuberculoid cases. On the other hand, salivary IgA antibodies against M. leprae were found in a significant number of specimens; whereas IgG and IgM antibodies were scarcely found. However, the percentage of positive FLA-ABS tests caused by salivary IgA antibodies was higher in the patients with tuberculoid or borderline leprosy than in those with lepromatous leprosy. A significant number of patients with tuberculoid or borderline leprosy secreted M. leprae-specific IgA antibodies into saliva without detection of circulating IgA antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of T-cell-activating recombinant antigens shared among three candidate antileprosy vaccines, killed M. leprae, M. bovis BCG, and mycobacterium w

Antigenic crossreactivity among three candidate antileprosy vaccines, killed Mycobacterium leprae, BCG, and Mycobacterium w, was studied using T-cell lines and clones raised from BCG- and killed-M. leprae-vaccinated subjects. To identify the crossreactive antigens, the T-cell lines and clones were tested against Escherichia coli lysates containing 65-, 36-, 28-, 18-, and 14-kilodalton (kDa) and 13B3 M. leprae antigens and 65-, 19-, and 12-kDa M. tuberculosis antigens. The short-term T-cell lines, which compared to T-cell clones are easy to raise and maintain, were equally effective in identifying the T-cell-activating recombinant antigens. The reactivity pattern of the T-cell lines and the clones suggested that 65-kDa M. leprae and M. tuberculosis antigens are present in M. leprae, BCG, and Mycobacterium w; 18-kDa M. leprae antigen is shared between M. leprae and Mycobacterium w, 13B3 M. leprae antigen is possessed by M. leprae and BCG. These and other unidentified T-cell-activating antigens shared among candidate leprosy vaccines may be the basis for induction of in vivo sensitization to M. leprae antigens after vaccination with BCG or Mycobacterium w.     

Antigenic protein from Mycobacterium leprae released in macrophages in vitro as indicator of viability of bacteria

Peritoneal macrophages from randombred, Swiss white mice, when cultured and infected with Mycobacterium leprae for 24 hours, are able to show the presence of antigen(s) with binding affinity to antibodies present in the sera of bacteriologically positive, lepromatous leprosy patients. Such antibodies are not seen in sera from normal and healthy persons, tuberculoid leprosy patients, or long-term-treated, bacteriologically negative, lepromatous leprosy patients. The production of the antigen(s) is blocked by the anti-M leprae drug rifampin. Other mycobacteria when incubated with macrophages from mice show very little antigens in the lysate but the antigens have an equal affinity for antibodies in sera from both normal individuals and lepromatous patients. Only the lysates from macrophages exposed to live M. leprae could discriminate and could exhibit differential binding to sera from leprosy patients compared to sera from normal individuals. This antigen(s) does not have any binding ability to the monoclonal antibodies available to the antigens of M. leprae identified at present and shown to be specific to M. leprae. This indicates a separate identity of this product which has potential for further exploitation in exploring host-pathogen interactions related specifically to the leprosy infection and the tolerance of M. leprae inside cells.     

Leprosy reactions: humoral and cellular immune responses to M. leprae, 65kDa, 28kDa, and 18 kDa antigens

This study examines the immune responses against some stress proteins of Mycobacterium leprae in leprosy patients with and without leprosy reactions. Leprosy patients showed a higher level of antibodies to all antigens compared to healthy controls. The antibody response to 18kDa antigen was significantly higher in patients with Type 1 reaction compared to those of TT or borderline patients without Type 1 reaction, or those with Type 2 reaction. Borderline (BT/BL), lepromatous (LL) and patients with reactions (Type 1 and Type 2) had higher levels of antibodies to M. leprae soluble extract (MLSE) and 65kDa than those of the tuberculoid (TT) group. LL, borderline patients, and patients with Type 1 reaction had a higher level of antibody to 28kDa than those of healthy controls. However, no significant differences could be observed in antibody response to these antigens (MLSE, 65kDa, and 28kDa) between patients with reaction and without reaction. A significant proportion of TT/BT patients showed positive lymphoproliferative response to MLSE compared to BL/LL patients. In addition, the lymphoproliferative response to MLSE was significantly greater in patients with Type 1 reaction compared to patients without reaction. No difference in proliferative response to 65kDa could be observed in any of these groups. The finding of high levels of antibodies against stress proteins in patients with Type 1 reactions, especially to 18 kDa antigen, along with a heightened lymphoproliferative response to MLSE is suggestive of a coexistence of cell mediated and humoral immunity in leprosy patients during Type 1 reactions. On the other hand, in Type 2 reactions no significant role of stress proteins could be demonstrated except a heightened lymphoproliferative response to the 28 kDa antigen.     

A peptidoglycan protein complex purified from M. leprae cell walls contains most or all immunodominant M. leprae T-cell antigens

The outcome of an infection with Mycobacterium leprae is correlated with the T-cell-mediated immune response developed against this pathogenic agent. The identification of M. leprae antigens that are recognized by T cells is therefore of great importance. In this paper we present the results of in vitro lymphoproliferation assays in which T-cell reactivity was measured against a peptidoglycan-protein complex (PPC) which was purified from the cell wall of M. leprae. Twelve M. leprae-reactive T-cell clones with different antigen specificities from a tuberculoid (TT) leprosy patient showed proliferative responses, but only when PPC was presented by HLA-DR-matched antigen-presenting cells (APCs). Four of these clones were known to react with the recombinant mycobacterial 65-kDa protein. A tetanus-toxoid-reactive T-cell line from a healthy control was not stimulated by this complex, supporting the idea that the stimulation by PPC was antigen specific. Both PPD-reactive and M. leprae-reactive T-cell lines from healthy individuals were stimulated by PPC. However, when this complex was presented to PPD-reactive T-cell lines derived from two lepromatous (LL) leprosy patients, we did not observe any proliferative responses. From these results we conclude that PPC contains most or all of the antigens which stimulate M. leprae-reactive T cells in association with relevant HLA class II molecules, including the 65-kDa protein or at least some immunogenic parts of it.     

Humans respond predominantly with IgM immunoglobulin to the species-specific glycolipid of Mycobacterium leprae

The immunoglobulin classes of the antibody response to the species-specific phenolic glycolipid antigen of Mycobacterium leprae have been characterized for serum specimens from 78 patients with leprosy. These patients included the entire clinical spectrum from paucibacillary to multibacillary disease, including polar tuberculoid (TT; 11 patients), borderline tuberculoid (BT; 15), borderline (BB; 17), borderline lepromatous (BL; 13), and lepromatous (LL; 22)--clinical classifications according to Ridley-Jopling criteria. In each patient group, the levels of IgM antibody to phenolic glycolipid were significantly higher than levels of IgG or IgA. Inhibition experiments with purified antigen showed that antibodies to the phenolic glycolipid dominated the human IgM antibody response to the surface of M. leprae.     

Lymphocyte transformation test in healthy contacts of patients with leprosy. I. Influence of exposure to leprosy within a household.

Fifty-three household contacts of lepromatous patients, 37 household contacts of tuberculoid patients, and 91 control persons were examined with the lymphocyte transformation test (LTT) for their responses to whole and sonicated antigen preparations from M. leprae, to BCG, M. avium, M. gordonae, and phytohemagglutinin (PHA). The study was carried out in the Gurage area of Ethiopia in 15 households with a leprosy patient and 15 matched control households. Household contacts of lepromatous patients showed significantly greater LTT responses to antigens from M. leprae than the controls, whereas household contacts of tuberculoid patients did not respond differently from controls. Household contacts of lepromatous patients had significantly greater responses to M. leprae antigens when the index patients were "active," i.e., highly bacilliferous, than when they were "inactive," i.e., having a low bacillary load. The degree of sensitization, as indicated by the LTT response, in different exposure groups paralleled the degree of probable infectivity of the index patient. A preparation of antigen from whole M. leprae proved to be more sensitive and more specific in the LTT than did a sonicated preparation. A significant degree of cross-reactivity was found among the various mycobacteria in their LTT responses.     

Enzyme linked immunosorbent assay of a 12-kDa protein of Mycobacterium leprae with sera from leprosy patients.

A low molecular weight protein was obtained from a sonicate of armadillo-derived Mycobacterium leprae cells and from a lambda gt11 phage lysate of Escherichia coli (specifying the M. leprae 12-kDa protein) by a single step of ultrafiltration. Both proteins had an approximate molecular weight of about 12,000 (by SDS-PAGE) and were recognized by the M. leprae 12-kDa-specific monoclonal antibody ML06 by immunoblotting. Sera from 79 leprosy patients across the clinical spectrum, 17 contacts, and 12 normal healthy individuals were screened in an enzyme-linked immunosorbent assay (ELISA) using the 12-kDa proteins as the antigens. Antibodies to the 12-kDa protein (from lysate as well as sonicate) were detected in patients' sera across the clinical spectrum (44%-100% positivity), while no detectable reactivity was observed with control or contact sera. Sera from patients who had undergone a year or more of chemotherapy exhibited no reactivity compared to those from patients with only 3-6 months of chemotherapy. The 12-kDa proteins were also recognized by rabbit hyper-immune M. leprae antiserum.     

Increased expression of regulatory T cells and down-regulatory molecules in lepromatous leprosy

T regulatory cells (Tregs) play an important role in the mechanism of host's failure to control pathogen dissemination in severe forms of different chronic granulomatous diseases, but their role in leprosy has not yet been elucidated; 28 newly diagnosed patients (16 patients with lepromatous leprosy and 12 patients with tuberculoid leprosy) and 6 healthy Mycobacterium leprae-exposed individuals (contacts) were studied. Tregs were quantified by flow cytometry (CD4+ CD25+ Foxp3+) in peripheral blood mononuclear cells stimulated in vitro with a M. leprae antigenic preparation and phytohemagglutinin as well as in skin lesions by immunohistochemistry. The lymphoproliferative (LPR), interleukin-10 (IL-10), and interferon-γ (IFN-γ) responses of the in vitro-stimulated peripheral blood mononuclear cells and the in situ expression of IL-10, transforming growth factor-β (TGF-β), and cytotoxic T-lymphocyte antigen 4 (CTLA-4) were also determined. We show that M. leprae antigens induced significantly lower LPR but significantly higher Treg numbers in lepromatous than tuberculoid patients and contacts. Mitogen-induced LPR and Treg frequencies were not significantly different among the three groups. Tregs were also more frequent in situ in lepromatous patients, and this finding was paralleled by increased expression of the antiinflammatory molecules IL-10 and CTLA-4 but not TGF-β. In lepromatous patients, Tregs were intermingled with vacuolized hystiocyte infiltrates all over the lesion, whereas in tuberculoid patients, Tregs were rare. Our results suggest that Tregs are present in increased numbers, and they may have a pathogenic role in leprosy patients harboring uncontrolled bacillary multiplication but not in those individuals capable of limiting M. leprae growth.     

Comparative study of skin reactions in leprosy patients to M. leprae-lepromin and to ICRC-in, an antigen from cultivable acid-fast bacilli from M. leprae isolated from lepromatous nodules.

Skin test antigens (Dharmendra type) were prepared from fresh M. leprae (lepromin) and from a culture of strain C-44 ICRC bacilli (ICRCin) grown 'in vitro' from M. leprae isolate from lepromatous nodules. Comparative study of skin reactivity to lepromin and ICRCin--both "early" and "late" reactions in 76 leprosy patients was conducted. In 29 lepromatous (LL) cases, 25 exhibited totally negative reaction at the end of third week. In tuberculoid (TT) 22 and 23 out of 31 were positive (greater than 4.5 mm) at 3 weeks to lepromin and ICRCin respectively. In the 16 BB group, the reactions were comparable in the same patient. The cellular reaction in tuberculoid cases consisted of lymphocytic infiltration, epitheloid giant cells and Langhan type cells and indistinguishable from each other. These data with characteristic total lack of reaction in 25/29 lepromatous leprosy cases and identical cellular reaction in TT patients, provide strong evidence that ICRC bacillus strain C-44 is antigenically identical with M. leprae.     

Variable lepromin response to Mycobacterium leprae in resistant armadillos

Eight armadillos resistant to the infection of Mycobacterium leprae were lepromin tested. The tissue response was tuberculoid in 5, borderline in 2, and lepromatous in 1, thus showing a wide variation. It is seen that although cell-mediated immunity as evidenced by a tuberculoid granulomatous response to killed M. leprae is associated with resistance to the disease, there may be other yet unknown factors which protect armadillos from the infection. Lepromin responses were recognized histologically even at a low dose of 10(3) organisms, and the response increased with the dose up to 10(7) organisms. The tissue reaction to live organisms was the same as that to killed ones, and autoclaving of M. leprae produced no change in the tissue response to the antigens of M. leprae.     

Detection of Mycobacterium leprae DNA for 36kDa protein in urine from leprosy patients: a preliminary report

We have searched for Mycobacterium leprae DNA for 36kDa protein in urine using a M. leprae specific PCR technique. A limited number of 16 patients (of which 11 belonged to lepromatous leprosy and five to tuberculoid leprosy) and eight healthy individuals were included for the present study. The number of urine samples positive by PCR were 36.4% (4/11) in lepromatous patients and 40% (2/5) in tuberculoid patients. None of the samples from healthy individuals was positive. To our knowledge, the results indicate, for the first time, the presence of M. leprae DNA in urine from leprosy patients. Another important finding obtained out of the study is that amongst treated patients 66.6% (4/6) were positive whereas amongst untreated only 20% (2/10) were positive. From the present indicative data it appears that treatment improves the PCR results with urine as a sample. Thus, the approach could prove to be useful for monitoring the treatment response of individual patients and needs to be further evaluated with a large number of patients.     

Lymphocyte transformation test in healthy contacts of patients with leprosy. II. Influence of consanguinity with the patient, sex, and age.

The study was carried out in the Gurage area of Ethiopia, where 53 household contacts of lepromatous patients, 37 household contacts of tuberculoid patients, and 91 control persons were examined with the lymphocyte transformation test (LTT) for their responses to whole and sonicated antigen preparation from M. leprae to BCG, M. avium, M. gordonae and phytohemagglutinin. The potential influence of host factors, namely the state of consanguinity with the leprosy patient, sex and age on the LTT responses was evaluated. In the 35 household contacts of "active," i.e., highly bacilliferous, lepromatous patients, consanguinity with a lepromatous patient was not associated with a significant depression of the LTT responses to M. leprae antigens. Male household contacts of active lepromatous patients showed significantly greater LTT responses to M. leprae antigens than female household contacts. Possible confounding factors for this finding are discussed. Sensitization of M. leprae antigens was present already in a high proportion of the 6 to 14 year old household contacts of active lepromatous patients, which was the youngest age group examined in our study. No significant results were found in any of the other patient contact groups with regard to the host factors examined.     

Learning from lesions: patterns of tissue inflammation in leprosy.

The clinical forms of leprosy constitute a spectrum that correlates closely with the degree of cell-mediated immunity. Patients with tuberculoid leprosy develop strong cell-mediated responses and have only a few, localized lesions, whereas patients with multibacillary lepromatous leprosy are specifically unresponsive to antigens of Myobacterium leprae. T cells of the CD4+ subset predominate in tuberculoid lesions, whereas CD8+ cells predominate in lepromatous lesions. Monoclonal antibodies that distinguish subpopulations of CD4+ and CD8+ cells were used to analyze the distribution of T cells infiltrating lesions across the disease spectrum. In lepromatous lesions, T cells of T-suppressor phenotype (9.3-) were the predominant CD8+ cells and suppressor/inducer cells (2H4+, Leu-8+) represented half of the CD4+ subset. In tuberculoid lesions, helper T cells (CD4+4B4+) outnumbered suppressor/inducer T cells by 14:1, compared with a ratio of 1.2:1 in peripheral blood. Analysis of the precursor frequency of antigen-reactive T cells permitted us to estimate that there was a 100-fold enrichment of T cells able to proliferate in response to M. leprae antigens in tuberculoid lesions (2/100), when compared with blood from the same patients. The methods used here to characterize the T-lymphocyte subsets and frequency of antigen-reactive T cells in leprosy may be useful in analyzing immunological reactions occurring in lesions of other inflammatory and autoimmune diseases.     

Crossreactivity between Mycobacterium leprae and various actinomycetes and related organisms

Serological crossreactivity was analyzed between M. leprae and strains of various species of Corynebacterium, Mycobacterium, Nocardia, Rhodococcus, Streptomyces, and related organisms. M. leprae shares antigens with most of these organisms, and sera from patients with lepromatous leprosy contain antibodies against them. The results demonstrate that M. leprae shares more antigens with the mycobacteria than with strains of the other tested genera, thus supporting the view that the leprosy organism belongs to the genus Mycobacterium. One precipitinogen (designated p beta) was found to be common to M. leprae and the streptomycetes, and sera from patients with lepromatous leprosy contain antibodies against this antigen.     

Analysis of circulating immune complexes from leprosy patients for Mycobacterium leprae antigens.

Circulating immune complexes (CICs) from 31 leprosy patients (16 tuberculoid, 15 lepromatous) and 12 healthy volunteers, precipitated by 3.5% polyethylene glycol, were individually subjected to SDS-PAGE and immunoblotting using a variety of monoclonal and polyclonal antibodies against Mycobacterium leprae. A common mycobacterial antigen of an apparent molecular size of 65 kDa was identified in CICs from about 40% of the patients. No correlation was observed between the positivity for this antigen and any of the following parameters: bacterial index, M. leprae-specific antibody titers, motor nerve involvement, duration of disease or treatment. Nevertheless, patients with a relatively recent and massive infection were more frequently positive for antigen than the others.     

Histologic responses in sixty multibacillary leprosy patients inoculated with autoclaved Mycobacterium leprae and live BCG

Sixty lepromatous or borderline lepromatous patients were submitted to immunotherapy with a mixture of autoclaved Mycobacterium leprae and BCG. The histopathologic findings in skin biopsy specimens taken before and after immunotherapy were evaluated independently by six histopathologists in a workshop setting. Their pooled observations on diagnosis and classification were analyzed to assess the histopathologic changes following various periods of immunotherapy. Expressing the results as the average value of five to six independent observations, there were changes in classification of reversal or upgrading toward the tuberculoid end of the leprosy spectrum in 90.5% of the patients initially classified as lepromatous (LL), and in 83.3% of those initially classified as borderline lepromatous (BL). The histopathologic findings amply support the clinical, bacteriologic and immunological changes following immunotherapy from LL or BL, to BL, mid-borderline (BB) or even borderline tuberculoid (BT) leprosy.     

Cross-reactivity of antigens from the cytoplasm and cell walls of some corynebacteria and mycobacteria

Leprosy-derived corynebacteria (LDC) are non-acid-fast organisms isolated from leprosy lesions in humans. In this study 20 antigens of native LDC cytoplasm were identified by immunoelectrophoresis, and autoclaving yielded the M1 component, which strongly cross-reacted with antigen 60 of Mycobacterium bovis BCG (bacille Calmette-Guérin) and antigen 7 of Mycobacterium leprae. The polysaccharide moiety of M1 was immunologically related to the LDC cell wall polysaccharide previously characterized as arabinogalactomannan. The latter polysaccharide competitively inhibited the formation of immune complexes by labeled M1 and antisera to the LDC cell wall; cytoplasm and wall polysaccharides from other bacteria produced lower-level inhibition. In a radioimmunoassay with 125I-labeled antigen 7 of M. leprae, sera from patients with leprosy and antisera to the LDC cell wall yielded overlapping curves. Sera from patients with tuberculoid leprosy and those from patients with lepromatous leprosy afforded different levels of inhibition in this radioimmunoassay; this result indicated a difference in antibody specificity in the two forms of leprosy. In conclusion, the cell wall polysaccharide of LDC corresponds to the main thermostable cytoplasmic antigen M1, which strongly crossreacts with sera from patients with leprosy and, more specifically, with antigen 7 of M. leprae.