Humoral responses against the 85A and 85B antigens of Mycobacterium bovis BCG in patients with leprosy and tuberculosis.

Immunoglobulin G antibodies against the 85A and 85B components of the Mycobacterium bovis BCG antigen 85 complex separated by isoelectric focusing were investigated in serum samples from 129 patients representing the major forms of leprosy, 111 tuberculous patients, and 153 healthy subjects. For both of the antigens, a higher degree of staining was observed for lepromatous leprosy patients and patients with active tuberculosis than for the other groups. Because sera from some healthy subjects recognized the 85A antigen, we suggest that antigen 85B is the most useful component of the antigen 85 complex for the serodiagnosis of the multibacillary forms of leprosy or of the active forms of tuberculosis.     

Antigens of Mycobacterium leprae identified by immunoprecipitation with sera from leprosy and tuberculosis patients.

Mycobacterial antigens which react with human B lymphocytes were investigated by immunoprecipitation of radiolabelled sonicates of Mycobacterium leprae and M. bovis (BCG) with sera from patients with leprosy and tuberculosis in the presence of Staphylococcus aureus. SDS-PAGE analysis of the immunoprecipitates demonstrated that dense bands of Mr 12,000 (12K), 15K, 27K, 32-33K, 36K and 48K were the major antigens of M. leprae recognized by antibodies in lepromatous leprosy sera. Of these, only the 15-16K band reacted significantly with sera from patients with tuberculoid leprosy and tuberculosis. Other antigens including the T cell immunogens of Mr 18K and 70K reacted with some of the BL/LL sera tested. There were differences in the pattern of antigens precipitated from BCG sonicate by leprosy sera with the 65K antigen and a high molecular weight band (greater than 94K) being readily detected. These results differ in part to these obtained by probing immunoblots of M. leprae sonicate with leprosy sera. Factors contributing to these differences are discussed.     

High antibody levels to the mycobacterial fibronectin-binding antigen of 30-31 kD in tuberculosis and lepromatous leprosy.

Immunoblot assays showed that mycobacterial fibronectin-binding antigens are important targets of the humoral immune response in tuberculosis and leprosy. Using culture filtrate antigens of Mycobacterium tuberculosis, strong reactivity with the fibronectin-binding of 30-31 kD (Fn 30-31) was demonstrated in 55.9% of tuberculosis sera and in 56.5% of lepromatous leprosy sera. Sera from patients with tuberculoid leprosy and control sera gave very weak binding. Reactivity of tuberculosis and lepromatous leprosy sera with the fibronectin-binding antigen of 58-60 kD (Fn 58-60) was less conspicuous. The ability to react with fibronectin of the antigens of 58-60 and 30-31 kD was demonstrated by parallel labelling with a fibronectin-biotin conjugate. Fn 30-31 was purified to homogeneity by a two-step procedure and used for ELISA. Positive titres were found in 63% out of 65 tuberculosis sera and in 60.5% out of 43 lepromatous leprosy sera. Antibody titres in lepromatous leprosy sera were higher than in tuberculosis sera. Our observations indicate indirectly that M. leprae possess a highly immunogenic molecule homologous to M. tuberculosis Fn 30-31, which elicits a high antibody response in lepromatous leprosy but not in tuberculoid leprosy. In this investigation, direct evidence for the presence of this antigen in M. leprae was obtained by immunochemistry of lepromatous leprosy lesions with a monospecific antibody raised against M. tuberculosis Fn 30-31.     

IgG Humoral Response Against the Antigen 85 Complex Homologues in Leprosy

Antigen 85 complex is the major protein component present in M. bovis BCG culture filtrate (CF). It consists of a family of three proteins: 85A, 85B and 85C. Combining isoelectric focusing and Western blot analysis, we have previously identified different antigenically related proteins present in the CF of other mycobacteria ( M. tuberculosis, M. kansasii, M. avium, M. gordonae, M. fortuitum and M. plilei ) using monoclonal antibodies (MoAbs) directed against the antigen 85 complex of M. bovis BCG.
Humoral immune response directed against these cross-reactive homologues was analysed in sera from 20 patients with multibacillary leprosy (BL/LL), from 20 patients with paucibacillary leprosy (BT/ TT) and from 15 healthy leprosy contacts.
All the antigen 85 homologues identified in the seven CFs by MoAbs were also recognized by IgG present in sera from multibacillary leprosy patients, but not or very faintly in sera from paucibacillary leprosy patients or from healthy subjects.
These results suggest that some of the M. leprae epitopes inducing a significant humoral response in multibacillary leprosy are common to the various 85 antigenically related proteins present in all mycobacterial species.     

The variable C-terminal region of the Mycobacterium leprae 70-kilodalton heat shock protein is the target for humoral immune responses.

The 70-kDa heat shock protein of Mycobacterium leprae has a high degree of homology with the human hsp70 protein, yet it still elicits T-lymphocyte responses in subjects infected with M. leprae or vaccinated with the related Mycobacterium bovis BCG. We examined the serological responses to this protein by using recombinant protein fragments expressed from mutants with deletions of the M. leprae p70 gene. Monoclonal antibodies raised against either M. bovis or M. leprae p70 reacted with the C-terminal fragments but not the N-terminal fragments in a solid-phase enzyme-linked immunosorbent assay and an immunoblot assay. Inhibition enzyme-linked immunosorbent assays confirmed that two separate epitopes were defined by these monoclonal antibodies. Murine polyclonal sera also showed stronger binding to the C-terminal fragments. Sera from 33 and 48% of lepromatous leprosy patients reacted with M. leprae and M. bovis p70. This reactivity was mycobacterium specific, since few sera from control subjects in the same leprosy-endemic region were seropositive. The levels of anti-mycobacterial hsp70 antibodies were higher in patients with lepromatous leprosy than in those with tuberculoid leprosy or tuberculosis. The reactivity of sera from patients with leprosy was maximal with the C-terminal fragments. Therefore the C-terminal portion of M. leprae hsp70, which includes the region of maximum divergence from human hsp70, is the major target for the humoral immune response to the protein.     

Serology and leprosy: immunoassays comparing immunoglobulin G antibody responses to 28- and 30-kilodalton proteins purified from Mycobacterium bovis BCG.

Two major proteins from Mycobacterium bovis BCG culture filtrates with molecular masses of 28 kDa (P28) and 30 kDa (P30), identified as components of the BCG 85 complex, were purified and used in enzyme-linked immunosorbent assays (ELISAs) for the determination of specific immunoglobulin G (IgG) levels in patients with leprosy or tuberculosis or with exposure to these diseases. High reactivity to both antigens was observed with sera from lepromatous leprosy patients, whereas antibody levels in sera from paucibacillary leprosy patients were not significantly different from those in sera from healthy individuals from an area in which leprosy is endemic. High IgG responses were also found in some contacts of lepromatous leprosy patients. A comparison of the levels of anti-P28 and anti-P30 within the multibacillary leprosy patient group showed much higher IgG reactivity to P28 than to P30, suggesting that the antibody response of lepromatous patients is directed predominantly against the 28-kDa protein. A high degree of correlation in values of ELISAs based on P28 and on the phenolic glycolipid of Mycobacterium leprae was observed in all groups analyzed. The potential use of an assay based on the 28-kDa protein to selectively distinguish individuals destined to develop multibacillary leprosy is discussed, as also is the likelihood that the 28-kDa-30-kDa complex, part of the fibronectin-binding family, is an important component of M. leprae.     

Identification of an immunostimulating protein from Mycobacterium leprae.

Despite the recent identification of a number of Mycobacterium leprae proteins, the major immunogenic determinants of this organism remain obscure. We isolated from M. leprae a potent immunostimulatory preparation, designated the MLP fraction, which contains a major protein of 35 kilodaltons (kDa). This protein was precipitated by monoclonal antibody ML03-A1, which recognizes a 35-kDa protein of M. leprae, and by sera obtained from patients with lepromatous leprosy. Neither sera from healthy controls nor sera from patients with pulmonary tuberculosis recognized the 35-kDa protein, and only one of four serum samples from patients with borderline tuberculoid leprosy reacted with this protein. The MLP fraction stimulated T-cell proliferation in patients with leprosy whose T cells proliferate in response to whole M. leprae cells. Apparently, the T-cell epitope associated with MLP is also expressed on M. tuberculosis and M. bovis BCG, since patients with pulmonary tuberculosis and BCG-vaccinated individuals demonstrated significant responses to the MLP fraction. The 35-kDa M. leprae protein, purified to homogeneity in the laboratory of P. J. Brennan, stimulated T-cell proliferative responses in all MLP-responsive subjects. These findings suggest that the 35-kDa protein present in MLP is an immunostimulatory component of M. leprae. In addition to serving as a useful probe for study of the T-cell anergy associated with lepromatous disease, this protein may ultimately be useful as a component of a vaccine designed to provide protection against infection with M. leprae.     

Amyloid-related serum component (protein ASC) IN LEPROSY PATIENTS.

The presence of amyloid-related serum component, protein ASC, in serum samples from 63 leprosy patients was investigated. Protein ASC was detected in 38% of the patients. A correlation to the disease spectrum of leprosy was apparent: polar lepromatous cases, 64% positive; borderline lepromatous, 50%; borderline tuberculoid, 36%; subpolar tuberculoid, 17%; and polar tuberculoid, negative. Antibody activity against the a antigen of Mycobacterium leprae was also determined, showing a similar correlation to the disease spectrum. Serum samples from 23 apparently healthy Ethiopians serving as controls showed a protein ASC incidence of 22%. This figure is significantly higher than the frequency found by others among healthy Norwegian blood donors. Immunoglobulin M levels among patients were elevated in the borderline lepromatous and poplar lepromatous groups. The three tuberculoid groups did not differ in this respect from the control group but were all elevated as compared to a normal Caucasian serum pool. Although raised immunoglobulin M levels seemed to parallel increased frequencies of protein ASC in the patient groups as well as in controls, this correlation might be only secondary to a primary derangement in T-cell function.     

Anti-peripheral nerve antibodies in leprosy patients recognize an epitope shared by the M. leprae 65 kDa heat shock protein.

Binding of leprosy sera to peripheral nerve from different species (mouse, guinea pig and rabbit) was evaluated by ELISA. A majority of sera, whatever the clinical form of leprosy, bind to these antigens. Absorption with Mycobacterium bovis BCG demonstrated that these antibodies recognize cross-reactive epitopes between peripheral nerve and mycobacteria. In immunoblot analysis, both leprosy patient sera and a monoclonal antibody directed at the 65 kDa heat shock protein of M. leprae were shown to react with a heat-shock 67-68 kDa sciatic nerve protein. Binding of the monoclonal antibody to this sciatic nerve antigen was prevented by incubation with lepromatous patient sera, showing that some peripheral nerve epitopes recognized by patient antibodies are shared by the 65 kDa heat shock protein of M. leprae.     

Humoral immune response of tuberculous patients against the three components of the Mycobacterium bovis BCG 85 complex separated by isoelectric focusing.

An isoelectric-focusing technique followed by Western blot (immunoblot) analysis was used to investigate the immunoglobulin G response of tuberculous patients against each of the three components of the Mycobacterium bovis BCG antigen 85 complex. The 85A component was stained by the tuberculous as well as the non-tuberculous sera. In contrast, the 85B and the 85C proteins of the complex were not stained by the control sera but were stained by 20 of 28 tuberculous serum samples.     

Major proteins of mycobacterial strain ICRC and Mycobacterium leprae, identified by antibodies in sera from leprosy patients and their contacts.

Sera from leprosy patients across the clinical spectrum, healthy contacts, tuberculosis patients, and healthy donors were tested for their reactivity with antigens of mycobacterial strain ICRC (a cultivable mycobacterium) and Mycobacterium leprae by immunoprecipitation technique. Using M. leprae antigens, it was not possible to distinguish between reactivities of sera from lepromatous, borderline lepromatous, borderline tuberculoid, and tuberculoid leprosy patients. All these sera identified M. antigens with molecular masses of 47, 36, 21, and 14 kDa. When the same sera were tested for their reactivities with antigens of mycobacterial strain ICRC, several differences were observed. The 21-kDa antigen of mycobacterial strain ICRC was exclusively precipitated by sera from all lepromatous leprosy patients and from those undergoing erythema nodosum leprosum reaction. Sera from all the other donors tested failed to identify the 21-kDa antigen of mycobacterial strain ICRC. The 14-kDa protein of mycobacterial strain ICRC was identified by sera from a few lepromatous leprosy patients (5 of 26) and all their contacts. Our studies indicate that antigens present on cultivable mycobacteria rather than species-specific antigens may prove to be useful in the serodiagnosis of leprosy.     

T cell responses to fractionated Mycobacterium leprae antigens in leprosy. The lepromatous nonresponder defect can be overcome in vitro by stimulation with fractionated M. leprae components

Protective immunity against Mycobacterium leprae is dependent on M. leprae-reactive T lymphocytes. M. leprae-directed T cell reactivity is high in the localized tuberculoid form of leprosy but specifically absent in the disseminated lepromatous type of the disease. Two important questions that are relevant for the understanding of the immune response in leprosy as well as for the design of rational immunoprophylaxis and -therapy strategies are: (a) what are the antigens that trigger T cell responses in tuberculoid patients and thus protect these individuals from developing lepromatous leprosy and (b) is it possible to restore T cell responsiveness to M. leprae in lepromatous patients by rechallenging the immune system with selected antigens that will trigger help but not suppression? We have addressed these question by directly probing the peripheral T cell repertoire of 10 tuberculoid and 18 lepromatous patients with large numbers of different M. leprae and BCG antigenic components that had been separated on the basis of their relative molecular mass (M_r) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose. This technique allows the identification of T cell-stimulating antigens independent of the expression of B cell epitopes by these antigens. So far T cell epitopes have only been mapped on M. leprae proteins that had previously been defined by antibodies. Our results show that: (a) tuberculoid patients' T cells responded preferentially to M. leprae and BCG antigens in the lower (i.e. < 70 kDa) M_r range with a peak in the 10–25 kDa range; (b) 6 out of 18 lepromatous patients that did not respond to whole M. leprae responded strongly to isolated M. leprae components; antigens in the lower M_r. range were recognized by five out of these six patients and thus commonly seen by both tuberculoid and lepromatous patients' T cells; however, antigens in the higher M_r range, in particular > 150 kDa, were only recognized by lepromatous patients' T lymphocytes; (c) furthermore, the T and B cell repertoires in leprosy patients are skewed towards different antigenic fractions.     

Expression of a common idiotype PR4 in the sera of patients with leprosy.

The sera of 187 patients from across the leprosy spectrum were screened for the expression of the PR4 idiotype, which was first identified on a human hybridoma-derived monoclonal antibody from a patient with leprosy and found to react with the Mycobacterium leprae phenolic glycolipid and a variety of polynucleotides. Sixty per cent (51 out of 85) of patients with lepromatous leprosy (LL), 66% (33 out of 49) with borderline lepromatous (BL) disease, 47% (14 out of 30) with borderline tuberculoid (BT) leprosy, and 56% (13 out of 23) of tuberculoid (TT) patients were found to have significantly elevated titres of the PR4 idiotype in their sera compared with endemic controls, irrespective of the presence or absence of endemic malaria. Sera from 52 patients with tuberculosis were also screened as a control for mycobacterial infection. The PR4 idiotype was significantly elevated in 37% (19 out of 52) of these patients. No correlation between idiotype and serum immunoglobulins IgG and IgM was found, indicating that the concentrations of idiotype levels in sera were not merely a reflection of changes in serum immunoglobulin levels. It is hypothesized that the expression of the PR4 idiotype is due to certain germline genes preferentially expressed rather than being the result of polyclonal B cell activation.     

Serological specificity of phenolic glycolipid I from Mycobacterium leprae and use in serodiagnosis of leprosy

The serological activities of the specific phenolic glycolipid I from Mycobacterium leprae, its dissected parts, and related glycolipids from other mycobacteria were examined by enzyme-linked immunosorbent assay against hyperimmune anti-M. leprae rabbit antiserum and sera from patients with leprosy and other mycobacterial diseases. High anti-phenolic glycolipid I immunoglobulin M antibodies were found in 23 of 24 (96%) of lepromatous leprosy patients on short term chemotherapy and in 8 of 13 tuberculoid leprosy patients (62%). Sera from patients with tuberculosis or atypical mycobacterial infections were devoid of anti-phenolic glycolipid I activity. The structurally related phenolic glycolipids from Mycobacterium kansasii and Mycobacterium bovis and the aglycone segments of the M. leprae product showed no significant activity. Thus, the trisaccharide determinant of phenolic glycolipid I is specific in its structure, serological activity, and, to a lesser extent, the antibody class it evokes.     

T-cell-epitope mapping of the major secreted mycobacterial antigen Ag85A in tuberculosis and leprosy.

Lymphoproliferation and gamma interferon (IFN-gamma) secretion in response to 28 overlapping 20-mer synthetic peptides covering the complete sequence of the mature (295-amino-acid) 85A component of the major secreted, fibronectin-binding antigen 85 complex from Mycobacterium tuberculosis and Mycobacterium bovis BCG (MTAg85A) was examined by using peripheral blood mononuclear cell (PBMC) cultures from healthy tuberculin- and lepromin-positive volunteers and from patients with tuberculosis and leprosy. Peptide recognition was largely promiscuous, with a variety of human leukocyte antigen haplotypes reacting to the same peptides. PBMC from all tuberculin-positive subjects reacted to Ag85, and the majority proliferated in response to peptide 6 (amino acids 51 to 70), peptides 13, 14, and 15 (amino acids 121 to 160), or peptides 20 and 21 (amino acids 191 to 220). PBMC from tuberculosis patients demonstrated a variable reactivity to Ag85 and its peptides, and the strongest proliferation was observed against peptide 7 (amino acids 61 to 80). MTAg85A peptides were also recognized by PBMC from healthy lepromin-positive volunteers and paucibacillary leprosy patients (again in a promiscuous manner), but despite a 90% homology between the 85A proteins of M. leprae and M. tuberculosis, the peptides recognized were different. PBMC from lepromin-positive healthy contacts reacted against peptide 2 (amino acids 11 to 30), peptide 5 (amino acids 41 to 60), and peptides 25 and 26 (amino acids 241 to 270). PBMC from paucibacillary patients reacted preferentially against peptide 1 (amino acids 1 to 20) and peptide 5. Multibacillary patients were not reactive to Ag85 or the MT85A peptides. IFN-gamma production was generally detected simultaneously with positive lymphoproliferative responses, although peptide 1 mostly stimulated proliferation and peptides 27 and 28 mostly elicited an IFN-gamma response. In conclusion, regions 41 to 80 and 241 to 295 demonstrated powerful and promiscuous T-cell-stimulatory properties, resulting in proliferative responses and IFN-gamma secretion, respectively, in the majority of reactive subjects tested in this study. These results could be of value in the development of a subunit vaccine for tuberculosis and leprosy.     

Serological response of patients with leprosy to a 28- to 30-kilodalton protein doublet from early cultures of Mycobacterium bovis BCG.

Analysis by Western immunoblotting of Mycobacterium bovis BCG short-term culture filtrates with a pool of serum samples from lepromatous leprosy patients revealed an immunodominant protein doublet with apparent molecular masses of 28 and 30 kilodaltons (kDa). The humoral response to these antigens was also investigated by using individual serum samples from patients representative of the whole spectrum of leprosy and from tuberculosis patients, as well as from contacts of leprosy patients and control groups. The protein doublet was recognized by 92% of the sera from patients with lepromatous leprosy (51 of 56), whereas essentially negative results were obtained with sera from the other groups. Similar immunodominant bands were also detected by Western blotting analysis of sonic extracts of seven other slow- and fast-growing mycobacterial species, suggesting a broad distribution of these antigens within the genus. Analysis of the purified doublet by Western blotting after two-dimensional gel electrophoresis fractionation showed that the 28- and 30-kDa doublet consisted of at least five different components with pls from 5.2 to 5.7 and molecular masses from 28 to 31 kDa. These results indicate that the protein doublet could be used as a potential marker in the diagnosis of lepromatous leprosy.     

Use of recombinant antigens expressed in Escherichia coli K-12 to map B-cell and T-cell epitopes on the immunodominant 65-kilodalton protein of Mycobacterium bovis BCG

In gene libraries of Mycobacterium bovis BCG, Mycobacterium tuberculosis, and Mycobacterium leprae, recombinants were frequently encountered that expressed an immunodominant 65-kilodalton (kDa) protein antigen that was shown to react with a high proportion of mycobacterium-reactive human and murine T cells and murine monoclonal antibodies. In this study, recombinant antigens were used to map T-cell and B-cell epitopes on the M. bovis BCG 65-kDa protein that was previously designated MbaA. Four different T-cell-epitope-containing regions (amino acid residues 1 through 16, 17 through 61, 85 through 108, and 235 through 279) were defined that were recognized by seven T-cell clones from patients with tuberculoid leprosy. These regions are distinct from two previously described T-cell epitopes recognized by T cells from a tuberculosis patient. As T-cell clones restricted by different class II determinants were shown to be specific for different regions on the 65-kDa protein, the presented data suggested that the products of different human leukocyte antigen class II loci and alleles present different parts of MbaA to the immune system. B-cell epitopes recognized by 20 monoclonal antibodies were assigned to eight different regions of MbaA. Using 15 of these antibodies, we previously showed that MbaA was antigenically related to a common antigen present in many bacterial species. The dispersed localization of the involved epitopes defined here shows that various different parts of MbaA are indeed conserved. These results show that well-defined recombinant antigens are useful tools for the localization of both B- and T-cell-epitope-containing regions of a protein. Peptides synthesized from the sequences of such regions may then exactly define the epitopes relevant for the development of specific diagnostic tests or of vaccines against mycobacteria.     

Members of the 30- to 32-kilodalton mycolyl transferase family (Ag85) from culture filtrate of Mycobacterium avium subsp. paratuberculosis are immunodominant Th1-type antigens recognized early upon infection in mice and cattle

The characterization of protective antigens is essential for the development of an effective, subunit-based vaccine against paratuberculosis. Surface-exposed and secreted antigens, present abundantly in mycobacterial culture filtrate (CF), are among the well-known protective antigens of Mycobacterium tuberculosis and Mycobacterium bovis. Culture filtrate, prepared from Mycobacterium avium subsp. paratuberculosis ATCC 19698 grown as a surface pellicle on synthetic Sauton medium, was strongly and early recognized in experimentally infected B6 bg/bg beige mice and cattle, as indicated by elevated spleen cell gamma interferon (IFN-gamma) secretion and lymphoproliferative responses of peripheral blood mononuclear cells, respectively. Strong proliferative and ex vivo IFN-gamma responses against antigen 85 (Ag85) complex (a major protein component from M. bovis BCG culture filtrate) could be detected in cattle as early as 10 weeks after oral M. avium subsp. paratuberculosis infection. Synthetic peptides from the Ag85A and Ag85B components of this complex were strongly recognized, whereas T-cell responses were weaker against peptides from the Ag85C protein. A promiscuous T-cell epitope spanning amino acids 145 to 162 of Ag85B (identical sequence in M. bovis and M. avium subsp. paratuberculosis) was identified in experimentally infected cattle. Finally, young calves, born from cows with confirmed paratuberculosis, demonstrated proliferative responses to purified, recombinant Ag85A and Ag85B from M. avium subsp. paratuberculosis. These results indicate that the M. avium subsp. paratuberculosis Ag85 homologues are immunodominant T-cell antigens that are recognized early in experimental and natural infection of cattle.     

Mycobacterium leprae produces extracellular homologs of the antigen 85 complex.

The antigen 85 complex is a set of at least three closely related secreted proteins (85A, 85B, and 85C) of 30 to 32 kDa produced by Mycobacterium tuberculosis and other mycobacteria. Their prominence in Mycobacterium leprae, the one obligate intracellular pathogen of the genus, had been assumed on the basis of immunological evidence and proof of the existence of the gene encoding the 85B protein of the complex. We have now observed the production of this family of proteins by M. leprae through analysis of various fractions by Western blotting (immunoblotting) with monospecific rabbit antisera raised against the individual Mycobacterium bovis BCG 85A, 85B, and 85C proteins. A predominant cross-reactive band with an apparent molecular mass of 30 kDa was detected in extracts of nondisrupted whole M. leprae and in soluble fractions prepared from the tissues of M. leprae-infected armadillos. Further studies of the subcellular distribution of this protein within the bacterium confirmed that it is secreted by the organism, an observation that explains past difficulties in detecting the antigen 85 complex in M. leprae. Confirmation that the M. leprae product is a member of the antigen 85 complex was obtained by comparison of peptide fingerprints with those from the BCG product. The pattern of reactivity of the M. leprae antigen 85 complex with anti-M. bovis BCG 85B serum, as well as two-dimensional electrophoresis, established that the 85B component was the predominant member of the complex in M. leprae. The fibronectin-binding capacity of the M. leprae and BCG 85 complexes was reinvestigated by new approaches and is questioned. Nevertheless, the results obtained with the native proteins reinforce previous reports, derived primarily from the use of homologous proteins, that the antigen 85 complex is one of the dominant protein immunogens of the leprosy bacillus.     

Characterization of the cellular immune defect in lepromatous leprosy: a specific lack of circulating mycobacterium leprae-reactive lymphocytes

The blastogenic response of leucocyte cultures from patients with tuberculoid and lepromatous leprosy has been studied. The leucocytes from the two groups were studied simultaneously and cultivated in the same pool of normal human serum. While the leucocytes from twenty-eight tuberculoid patients responded quite strongly to Mycobacterium leprae after 7 days of culture (average lymphocyte transformation 11·1%), there was a complete lack of response in similar cultures from twenty-seven lepromatous patients (average 0·1% transformed cells). These results were confirmed by studies on cellular incorporation of ³H-thymidine in the cultures from four tuberculoid and four lepromatous patients.

This lack of response was quite specific as leucocytes from several lepromatous patients responded to BCG. Furthermore, four patients with both lepromatous leprosy and tuberculosis responded as strongly to BCG and PPD as tuberculous patients without leprosy. In the mixed leucocyte reaction, between two lepromatous or two tuberculoid patients respectively, the lepromatous cells responded well (average 15·0%) and comparably to tuberculoid cells (average 12·1%).

The blastogenic response of purified lymphocytes to M. leprae revealed a similar pattern, i.e. the tuberculoid cells responded well, while again there was a lack of response in the lepromatous group.

It is concluded that the lepromatous patients lack circulating lymphocytes responding to M. leprae, indicating that their immunological defect as observed in the present study has features in common with immunological tolerance.