Detection of Mycobacterium leprae antigens in the sera of leprosy patients by sandwich immunoradiometric assay using monoclonal antibodies.

An immunological technique for demonstration of Mycobacterium leprae antigen in sera was developed by using specific as well as cross-reactive monoclonal antibodies. The sandwich immunoradiometric assay which we developed is a simple, robust assay that is sensitive to the nanogram level. Sera from 72 leprosy patients were screened for the presence of antigen by this assay. A total of 69% of untreated tuberculoid leprosy patients showed 35-kDa antigen positivity, and 45% of these patients showed anti-35-kDa antibody positivity. Consistently higher antigen positivity rates for the 35-, 12-, and 30- to 40-kDa components of M. leprae were observed in lepromatous leprosy patients than in tuberculoid leprosy patients. During the course of therapy the antigen positivity rate gradually declined, and the antigen could not be detected in any of the 15 patients with subsided cases of leprosy. As antigen is presumably in excess before the antibody response is evoked, our experimental approach for antigen detection is likely to be useful by itself or along with antibody detection for diagnosis of early leprosy.     

T cell subsets in leprosy lesions: in situ characterization using monoclonal antibodies

Cryostat sections of dermal lesions from 30 untreated leprosy patients were studied by indirect immunofluorescence using monoclonal antibodies defining T cell subsets and Ia like antigens. Most lymphocytes in leprosy lesions were positive for OKT3 and Ia like antigens indicating thereby the presence of activated T cells. Maximal numbers of these cells were seen in localized paucibacillary tuberculoid leprosy lesions in close association with epithelioid cells. A decline in their numbers was observed over the leprosy spectrum with a marked reduction in disseminated, multi-bacillary, lepromatous leprosy where only scattered OKT3+ cells were visualized. OKT4 and OKT8 positive cells defining T cell subsets, were frequently found within the OKT3+ lymphocytes throughout the leprosy spectrum. The ratio of OKT4+/OKT8+ cells ranged from 1.2 to 5.0 in tuberculoid and from 0.2 to 1.0 in lepromatous lesions. Macrophages in the granulomas stained intensely with anti-Ia antisera. Ia like antigens were expressed to the same degree on macrophages with or without intracellular acid fast bacilli.     

Human monoclonal antibodies against Mycobacterium leprae.

Human hybridomas were constructed which produce antibodies against three different extracts of Mycobacterium leprae. A thioguanine-resistant (Thgr), ouabain-resistant (Ouar), human lymphoblastoid cell line, KR-4, was hybridized with Epstein-Barr virus-transformed cell lines from lepromatous leprosy patients with fusion frequencies of greater than 10(-5). Non-Epstein-Barr virus-transformed donor cells fused at much lower rates (less than 2 X 10(-7]. Hybrids were selected in medium containing hypoxanthine aminopterin thymidine and 10(-5) M ouabain. An enzyme-linked immunosorbent assay was used to screen for antibodies against three crude extracts of armadillo-derived M. leprae, including (i) a soluble sonic extract preparation, (ii) sodium dodecyl sulfate extract of insoluble sonicated M. leprae, and (iii) a purified phenolic glycolipid antigen. Of a total of 2,200 final clones screened, 359 were found to secrete antibody which bound to soluble sonic extracts and the sodium dodecyl sulfate extract (6.7 and 9.6%, respectively), whereas 12.5% (21 out of 168) showed positivity to the glycolipid antigen. Four selected hybridomas also reacted with the deacylated derivative of M. leprae phenolic-glycolipid antigen. The specificity of these monoclonal antibodies was partially determined by screening on a panel of crude extracts from four other mycobacteria. Nine clones of 122 showed reactivity to M. leprae only. The predominant immunoglobulin was immunoglobulin M, and quantities up to 10 micrograms/ml were produced. Antibody production by hybrid clones was stable in more than 75% of the clones grown in continuous culture. By comparison, 10,000 Epstein-Barr virus-transformed lymphocyte clones from lepromatous leprosy patients were screened for anti-M. leprae antibody production, and all of the 42 clones that were initially positive in the enzyme-linked immunosorbent assay lost their antibody-producing capabilities within 6 weeks in culture. These results suggest that a combination of Epstein-Barr virus transformation and hybridization may be an optimal method in producing human monoclonal antibodies from leprosy patients.     

In vitro lymphocyte stimulation in leprosy; simultaneous stimulation with Mycobacterium leprae antigens and phytohaemagglutinin

Peripheral blood lymphocytes from 105 subjects with different forms of leprosy and healthy contacts of leprosy patients were stimulated in vitro with different preparations of mycobacterial antigens alone or in combination with a suboptimal dose of phytohaemagglutinin (PHA). In nearly all individuals sonicated leprosy bacilli and PHA together gave a lower ³H-thymidine incorporation than did the same dose of PHA alone. There was no difference in the degree of inhibition seen in the different patient groups or the healthy contacts. High doses of whole, washed Mycobacterium leprae, combined with PHA led to an increased thymidine incorporation in borderline tuberculoid leprosy patients who had experienced a reversal reaction, and in healthy contacts with more than 6 months of exposure, while most lepromatous patients and contacts with less than 6 months exposure did not show an augmentation of the PHA-induced thymidine incorporation. The inhibition exerted by sonicated M. leprae was dose-dependent, seen even with very low doses of antigen, and was not due to direct cytotoxicity. M. bovis, strain BCG, was weakly suppressive in combination with PHA, and sonicated M. duvalii had a very marked suppressive effect. There was no correlation between the suppressive effect of M. leprae antigens and the other mycobacteria neither was there any correlation with the responses to the mycobacterial antigens alone. Many lepromatous leprosy patients showed significant suppression of background incorporation with addition of M. leprae antigens. This paper discusses whether the apparent `non-responsiveness' in lepromatous leprosy could be due to active suppressor mechanisms operative in vivo.     

Serodiagnosis of leprosy: relationships between antibodies to Mycobacterium leprae phenolic glycolipid I and protein antigens.

Sera from leprosy patients and controls were assayed for immunoglobulin M (IgM) and IgG antibodies to the Mycobacterium leprae-specific phenolic glycolipid I antigen (PG) by enzyme-linked immunosorbent assay, for IgG antibodies to M. leprae protein antigens by Western immunoblot, and for antibodies to a 65-kilodalton (kDa) protein antigen of M. leprae by a competition antibody binding assay. Elevated levels of anti-PG IgM were seen in lepromatous and borderline lepromatous patients, and elevated levels of anti-PG IgG were seen in borderline lepromatous patients. There was a significant correlation between the bacillary index (BI) and anti-PG IgM whether all leprosy patients or only multibacillary patients were analyzed. A significant correlation was seen between anti-PG IgG and BI when all leprosy patients were used for analysis, but not when only multibacillary patients were used. IgG antibodies to protein antigens of M. leprae, as detected by Western immunoblot, were more prevalent in lepromatous and borderline lepromatous patients than in borderline tuberculoid patients, while one of eight controls showed one weak band. There were significant correlations between the number of M. leprae protein antigens detected by the sera of patients and both BI and the level of anti-PG IgM. The 65-kDa competition antibody binding assay detected active multibacillary leprosy. Patients positive for antibody to the 65-kDa antigen had a significantly higher BI and levels of anti-PG IgM and anti-PG IgG than did patients that were negative. In addition, the level of antibody to the 65-kDa antigen correlated with both the BI and anti-PG IgM. We conclude that testing for antibodies to protein antigens of M. leprae may provide a useful adjunct to testing for antibodies to PG.     

Cellular immune responses of leprosy contacts to fractionated Mycobacterium leprae antigens.

Antigens of armadillo-derived Mycobacterium leprae sonic extract were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane, and the unstained blot was converted into 20 fractions of antigen-bearing particles. These were tested in cellular proliferation assays, and reproducible results were obtained between batches of fractions. Peripheral blood mononuclear cells from healthy contacts of leprosy patients (presumed to have protective immunity) were tested with the fractions to investigate which antigens they recognized. A small group of tuberculoid leprosy patients were also tested. Both groups showed a wide range of responses. Almost every fraction stimulated proliferation with at least one donor, yet none was clearly immunodominant or inhibitory in either group. Thus, protective immunity did not appear to be associated with proliferation caused by any single fraction.     

Most Mycobacterium leprae carbohydrate-reactive monoclonal antibodies are directed to lipoarabinomannan

Each of more than 30 monoclonal antibodies that had been raised against Mycobacterium leprae and previously classified as reactive with carbohydrate was shown to be directed against lipoarabinomannan, a prominent, highly pervasive, myo-inositol-phosphate-containing, cross-reactive antigen within the leprosy bacillus. Some of the antibodies preferentially bound to the lipopolysaccharide of M. leprae rather than to that of Mycobacterium tuberculosis, suggesting the presence of distinguishing structural features. The presence of alkali-labile inositol 1-phosphate in the lipopolysaccharide from M. tuberculosis and its apparent absence from the M. leprae product may account for the difference.     

Protein antigens of Mycobacterium leprae

Protein antigens of Mycobacterium leprae have been identified by screening the lambda gt11, pYA626 and pHC79::M. leprae genomic libraries with pooled sera from leprosy patients and with antiserum to M. leprae cell wall protein (CWP) aggregate. Immunological screening of the lambda gt11 library with pooled sera from 21 lepromatous (LL) leprosy patients resulted in the identification of 19 antigens that are apparently different from previously identified M. leprae antigens. Five additional antigens were identified by screening the lambda gt11 library with pooled sera from 30 borderline tuberculoid or tuberculoid patients. Four other antigens were identified by screening the lambda gt11 library with anti-CWP. Two groups of recombinant cosmids were identified by screening the pHC79 library with LL patients' sera: one group specified proteins that reacted with monoclonal antibodies (mAb) against the 65-kDa protein and against the 18-kDa protein; the other group specified a 15-kDa protein that did not react with any of the mAb that were tested. One pYA626 clone also specified a 15-kDa protein that reacted with LL patients' sera, but did not react with any mAb. Genes specifying several of these antigens have been subcloned into the Asd+ plasmid vector pYA292 and have been introduced into a delta cya delta crp delta asd Salmonella typhimurium strain to evaluate the ability of individual M. leprae proteins to elicit immune responses against M. leprae infection.     

Definition of species specific and cross-reactive antigenic determinants of Mycobacterium leprae using monoclonal antibodies.

Four soluble antigens of Mycobacterium leprae have been identified using 12 murine monoclonal antibodies. Their specificity, taxonomic distribution and molecular nature were analysed by radioimmunoassays and by immunoblotting from polyacrylamide electrophoresis gels. A protein antigen MY1 (12K) reacted with one antibody (ML06) without demonstrable cross-reactivity for any of the other 20 tested species of mycobacteria. Another four antibodies which identified antigen MY2 revealed only a marginal degree of cross-reactivity with three other species of mycobacteria. Two antigens shared by several other mycobacteria species were: MY3 represented by five protein bands (35-70K) and a subtilisin resistant molecule MY4 (40-50K) with two distinct determinants and presumably of polysaccharide nature. The described monoclonal antibodies may represent novel valuable diagnostic reagents as well as tools for the purification of antigens which could be explored towards prophylactic or therapeutic immunization against leprosy.     

Production and partial characterization of monoclonal antibodies to Mycobacterium leprae.

Monoclonal antibodies to Mycobacterium leprae were produced by the fusion of BALB/c splenocytes and lymph node cells to BALB/c myeloma (NSI/1) cells. Eleven monoclonal antibodies were characterized as to their reactivity with M. leprae and 18 other mycobacterial species by enzyme-linked immunosorbent assay and immunofluorescence. Two monoclonal antibodies reacted only with M. leprae, and the other nine showed unique patterns of reactivity by enzyme-linked immunosorbent assay. One monoclonal antibody (IIH9) reacted with a 68,000-dalton protein present in extracts from M. leprae, M. tuberculosis H37Rv, M. gastri, and M. smegmatis. Potential uses for these antibodies in serological tests and immunochemical analyses are discussed.     

The immunohistochemical demonstration of major histocompatibility antigens in the human kidney using monoclonal antibodies

The distribution of HLA-ABC and DR antigens in human kidneys was studied using monoclonal antibodies to monomorphic determinants and a 4-layer immunoperoxidase technique applied to frozen and paraffin sections. Enzyme digested paraffin sections provided the best localization. HLA-ABC antigens were located on all renal endothelium, Bowman's capsule and in proximal tubules. HLA-DR antigen was restricted to capillary and venous endothelium plus, at low density, in proximal tubules. Minor differences in distribution were found with different monoclonal antibodies, but dendritic cells were not detected using monoclonal antibodies to HLA-ABC, HLA-DR or leukocyte-common antigens.     

In situ demonstration of tissue proliferative activity using anti-bromo-deoxyuridine monoclonal antibody.

Immunohistochemical staining with anti-bromo-deoxyuridine (BrdU) monoclonal antibody was performed on a variety of human tissues following in vitro incubation with BrdU. The effect of different fixatives and DNA denaturation techniques on the reactivity with anti-BrdU was investigated. Optimal preservation of the antigenicity of BrdU incorporated into the DNA of proliferating cells was seen in tissues fixed in Bouin's fluid, while samples which had been fixed with cross-linking reagents, such as formalin, were usually unreactive. Positivity for BrdU was restored in formalin fixed tissues after digestion with pepsin, but this was usually associated with loss of morphological details. Acid and thermal DNA denaturation techniques gave similar results. It is concluded that Bouin fixation followed by acid or thermal denaturation of DNA is the method of choice for the in situ detection of cells in S-phase using anti-BrdU monoclonal antibody.     

Novel monoclonal antibodies against major antigens of Mycobacterium bovis

MPB70 and MPB83 are among the most characteristically exported proteins defining a strongly expressed phenotype of Mycobacterium bovis. These proteins are known to be homologous to osteoblast-specific factor 2. By in vitro culture of mycobacteria they appear to have a limited species distribution and to be relatively specific for M. bovis. Virtually identical genes are however, present in Mycobacterium tuberculosis. In order to facilitate further research into the immunobiology of these proteins and their potential application for differential diagnosis of tuberculosis as a result of M. bovis, we describe the reactivities of 20 monoclonal antibodies (MoAbs) to these proteins. Immunizing with bovine PPD generated 10 MoAbs. These antibodies reacted preferentially with the soluble MPB70 antigen using reducing conditions in SDS-PAGE with western blotting. Ten MoAbs were generated by immunizing mice with fractions derived from a whole cell sonic extract of M. bovis. These antibodies reacted preferentially with the surface exposed MPB83 lipoglycoprotein.     

Dominant cell wall proteins of Mycobacterium leprae recognized by monoclonal antibodies.

A cell wall fraction of Mycobacterium leprae has enhanced potency in activating immune T cells. By using a panel of monoclonal antibodies (MoAb), the dominant immunogen in this preparation was shown to be a complex of proteins of apparent molecular weight (Mr) 65 to 50 kD with a major antigen of 65 kD. Antigen capture assays supported the results of immunoblots and ELISA that this protein was concentrated in the cell wall. By varying the MoAb used as capture or tracer antibody, one of the three MoAb-defined epitopes on the 65 kD protein proved to be unique to M. leprae while the other two were shared by M. bovis (BCG) and M. tuberculosis. The cross-reactive epitope defined by MoAb L22 was present on a protein of Mr 12 kD as well as the 65 kD protein. The 12 kD protein was strongly radiolabelled with 125I and was immunoprecipitated by L22 but not by two other MoAb, L12 or L14. By contrast the higher molecular weight forms were only weakly precipitated by the three MoAb. Competitive inhibition assays with lepromatous leprosy sera demonstrated that the MoAb-defined epitopes were recognized by human B cells. The proteins bearing one of the cross-reactive determinants was purified from M. bovis (BCG) sonicate by affinity chromatography with MoAb L22 coupled to Sepharose 4B. This antigen fraction stimulated proliferation in peripheral blood mononuclear cells from BCG vaccinated, mantoux positive individuals indicating that the cell wall protein has cellular as well as humoral reactivity. The three MoAb defined epitopes are encoded by the DNA clone Y3178 recently isolated from M. leprae.     

Study of idiotypes expressed by monoclonal antibodies to the 35 kD and 12 kD antigens of Mycobacterium leprae.

Rabbit antisera were raised against four monoclonal antibodies (MoAb) binding with the 35 kD protein and four MoAb binding with the 12 kD protein antigen of Mycobacterium leprae. Antisera showed idiotype (Id) specificity following cross-absorption with normal mouse globulin. One Id on a single MoAb and another Id shared between three MoAb were identified for each group. Functional studies were carried out with the Rb04 anti [anti-35 kD] specificity. The expression of this Id and paratope in antigen immunized mice was associated with Igh alleles. Inoculation of mice with anti-Id Rb04 induced an 'Ab3' serum response of corresponding Id specificity only when the anti-Id was given in emulsion with incomplete Freund's adjuvant (IFA). Conversely, prior injection of soluble anti-Id inhibited the subsequent Ab3 response to Rb04/IFA. Moreover, the suppressive effect of soluble anti-Id was abrogated by prior injection of 50 mg/kg cyclophosphamide. These results indicate that regulatory mechanisms similar to those involved in antigenic stimulation may explain the stimulatory or suppressive potency of anti-Id antibodies. Finally, the Ab3 responses to the two tested anti-Ids did not contain any antigen binding activity.     

Selection and characterization of recombinant clones that produce Mycobacterium leprae antigens recognized by antibodies in sera from household contacts of leprosy patients.

A Mycobacterium leprae expression library was constructed in the vectors EX1, pEX2, and pEX3 and screened with a pool of 19 well-absorbed sera from household contacts of leprosy patients. Twelve selected recombinants that were further characterized differed clearly from recombinants selected with murine monoclonal antibodies. Whereas the monoclonal antibodies recognized mainly six recombinant antigens, the human sera from contacts reacted with a range of different recombinant antigens. None of the contact recombinant antigens was identical or related to well-characterized antigens from M. leprae or other mycobacteria selected with monoclonal antibodies, including proteins of the heat shock families. Two groups of recombinant antigens could be distinguished: one that was recognized by all sera used in the pool and one that was recognized by only a limited number of sera. These antigens, selected with sera from household contacts of previously untreated lepromatous leprosy patients, may be relevant to the immune responses during the early phase of infection with M. leprae.     

ML0405 and ML2331 are antigens of Mycobacterium leprae with potential for diagnosis of leprosy

Despite the success of multidrug therapy in reducing the number of registered leprosy cases worldwide, evidence suggests that Mycobacterium leprae continues to be transmitted. A serological diagnostic test capable of identifying and allowing treatment of early-stage disease could reduce transmission and prevent the onset of the disability, a common complication of the disease in later stages. Serological diagnosis based on antibody recognition of phenolic glycolipid I (PGL-I) cannot reliably identify individuals with lower bacterial indices (BI). One strategy that might improve this situation is the provision of highly specific serological antigens that may be combined with PGL-I to improve the sensitivity of diagnosis. Using serological expression cloning with a serum pool of untreated lepromatous leprosy (LL) patients, we identified 14 strongly reactive M. leprae proteins, 5 of which were previously unstudied. We present results suggesting that two of these proteins, ML0405 and ML2331, demonstrate the ability to specifically identify LL/borderline lepromatous (BL) patients on the basis of immunoglobulin G (IgG) reactivity. In a household contact study, LL index cases were identified on the basis of this reactivity, while household contacts of these patients demonstrated undetectable reactivity. At a serum dilution of 1:800, suitable to reduce background PGL-I IgM reactivity, two BL patients with a BI of <4 showed anti-human polyvalent immunoglobulin G, A, and M reactivity measured with a combination of ML0405, ML2331, and natural disaccharide O-linked human serum albumin (NDOHSA) (synthetic PGL-I) that was markedly higher than IgM reactivity to NDOHSA alone. We suggest that ML0405 and ML2331 may have utility in serological leprosy diagnosis.     

Generation and characterization of monoclonal antibodies to the phenolic glycolipid of Mycobacterium leprae.

Nine cloned cell lines producing antibodies to the unique phenolic glycolipid of Mycobacterium leprae have been established as a result of fusions with spleens from mice immunized with the glycolipid complexed with methylated bovine serum albumin. One of the antibodies was relatively nonspecific, binding to a related glycolipid from Mycobacterium kansasii, but the remaining antibodies were specific for the M. leprae lipid. Some of the antibodies required the intact (trisaccharide) carbohydrate portion for recognition of the glycolipid antigen, whereas others recognized partially hydrolyzed forms lacking one or two sugar residues. Monoclonal antibodies directed at the terminal saccharide of the glycolipid showed the greatest specificity for M. leprae in enzyme-linked immunoassays. These antibodies brightly labeled whole mycobacteria in indirect immunofluorescence experiments, demonstrating the surface location of M. leprae-specific determinants of the glycolipid antigen. In addition to their use in providing information about the antigenic properties of the phenolic glycolipid, these antibodies have potential applications for elucidating the roles of glycolipid in the pathogenesis of leprosy.     

C-reactive protein and apoB containing lipoproteins are associated with Mycobacterium leprae in lesions of human leprosy.

Skin biopsies from patients with leprosy across the spectrum from tuberculoid (TT) to lepromatous (LL), including histoid lepromas and erythema nodosum leprosum (ENL) reactions, were stained immunohistochemically for the presence of C-reactive protein (CRP) and the apolipoprotein, apoB. Mycobacterium leprae bacillary material comprising cell walls, cytoplasmic and soluble components was present with increasing abundance towards the lepromatous end of the spectrum and always stained positively with anti-CRP. M. leprae from armadillos did not stain with anti-human CRP indicating that the staining of M. leprae in human tissues was not due to a cross-reaction between anti-CRP and the organism itself. When CRP was present in large amounts apoB was also demonstrated in the same distribution. CRP was detected on bacilli and their degradation products within the cytoplasm of macrophages even in the absence of a raised serum CRP level in some ENL patients and also in two cases of advanced resolving lepromas. These findings demonstrate remarkable persistence of CRP in association with M. leprae in vivo, and raise intriguing questions about the possible role of CRP in relation to the handling of leprosy bacilli.     

Studies of murine malaria antigens using monoclonal antibodies

A panel of ten monoclonal antibodies made againstPlasmodium chabaudi andPlasmodium yoelii infected mouse erythrocytes were used for characterization of antigens present in murine malaria. Screening of the antibodies in ELISA with different fractions of infected erythrocytes revealed both species-specific and fraction-specific monoclonal antibodies (MAbs), but also MAbs cross-reacting between the species. Two MAbs bound normal erythrocyte components. Subcellular localization of the target antigens was studied by immunofluorescence and their molecular identity by immunoblotting after SDS-PAGE. Of the MAbs toP. yoelii, one reacted with a cytoplasmic granule component of 137 k and two others reacted with vacuole-associated antigens of 26 k and 25/70/73 k, respectively. The latter antibodies cross-reacted withP. chabaudi antigens. Of the MAbs toP. chabaudi, all were species specific, one reacting with parasite surface antigens of 79 and 250 k and two with a vacuole-associated antigen of 70 k.