Development of a mouse food pad model for detection of sub clinical leprosy

Early diagnosis of leprosy and a multi-drug therapy (MDT) regimen will block the trajectory of nerve damage, disability and deformity that are the hallmarks of this chronic disease. However, the diagnosis of leprosy is made solely by recognition of clinical signs and symptoms, requiring special expertise. These limitations also result in the under reporting of worldwide prevalence and incidence rates for leprosy. Sorely needed is an objective laboratory test for detecting early leprosy. As the antigenic burden of M. leprae can be virtually undetectable in early clinical leprosy, cell mediated immunity and antibody responses will likely be weak. So the sensitivity of new diagnostic tests is as important as specificity. Major efforts are underway employing recombinant M. leprae antigens and synthetic peptides, to develop diagnostic assays for early leprosy infection, using in vitro T cell reactivity or serological tests. We have used the initial phase of the mouse foot pad model as an 'early' model of leprosy infection to screen T cell responses against M. leprae specific antigens and synthetic peptides. Unlike human disease in animal models we can control infection progress and monitor bacillary growth relative to time course of development of T cell response to specific M. leprae antigens. The study employed splenic T cells instead of draining lymph node T cells to model the systemic response as opposed to a local one. We found that 10(5) live M. leprae is the minimum dose required for any meaningful and consistent in vitro splenic IFN-gamma response against M. leprae antigens 3 months after foot pad inoculation. Using this model we found that several M. leprae recombinant proteins, ML0840, ML2028, ML2307, ML2346, ML2478, and ML2532, induced significant levels of IFN-gamma secretion. By controlling for variables that can be confounding factors in the sensitivity of human testing, this mouse model provides an interface between M. leprae diagnostic antigen development and the screening of these antigens in humans under field conditions.     

Evaluation of four semi-synthetic Mycobacterium leprae antigens with sera from healthy populations in endemic and non-endemic areas.

In order to determine the frequency of occurrence of antibodies to semisynthetic antigens of Mycobacterium leprae in clinically healthy nonpatient populations and to establish a 'baseline' for comparison with antibody frequencies in both patients with a history of leprosy and their contacts, ELISAs were conducted using representative sera from two areas: a leprosy endemic area, Cebu City, Philippines and a nonendemic area for leprosy Chicago, Illinois, USA. These sera were tested, by an indirect IgM ELISA, for the presence of antibodies reacting with four semisynthetic antigens based on the phenolic glycolipid I antigen of M. leprae: ND-O-BSA (natural disaccharide with octyl linkage to bovine serum albumin), NT-O-BSA (natural trisaccharide with octyl linkage to BSA), ND-P-BSA (natural disaccharide with phenolic ring linkage to BSA) and NT-P-BSA (natural trisaccharide with phenolic ring linkage to BSA). Using an OD reading > or = 0.16 as positive, the antigen with the lowest background seroreactivity was ND-O-BSA, which reacted with 5/398 (1.3%) sera from Cebu, and 3/426 (0.7%) sera from Chicago. A total of 10 (2.5%) of 398 sera from the endemic area reacted with at least one antigen and 5 (1.3%) sera reacted with all four semisynthetic antigens. Of the 426 sera from Chicago, 12 (2.8%) were reactive with at least one antigen and 3 (0.7%) were reactive with all four semisynthetic antigens. Mean ELISA values for the 22 positive sera for each antigen ranged from 0.17 to 0.3 OD units, while the mean values for all sera in each area ranged from 0.01 to 0.04 OD units for all four antigens. Reactivity of 14 of the positive sera to some antigens, but not all four semisynthetic antigens, indicated that the carrier and linker arms might be associated with this background reactivity. Investigation of alternative linker arms and carriers is warranted. We conclude that nonspecific background reactivity to the semisynthetic antigens representing the PG-I molecule of M. leprae is 0.7-1.3%, based on a > or = 0.16 OD cutoff value. From these data it was concluded that reactivity in individuals free of leprosy was low enough to warrant use of these antigens in a diagnostic setting, such as screening household contacts and highly endemic populations. When incidence and prevalence of leprosy are low, testing with these antigens would not be cost effective, unless applied to high risk individuals.(ABSTRACT TRUNCATED AT 400 WORDS)     

M. leprae recombinant antigens important for T-cell reactivity.

Identification of M. leprae antigens recognized by T-cell is important for specific diagnosis, vaccine development and understanding the basic mechanisms involved in protection against and pathogenesis of leprosy. Screening of an M. leprae recombinant DNA library with antibody probes led to the identification of half a dozen M. leprae antigens recognized by B-cells. When tested for T-cell reactivity, all the antigens recognized by antibodies were shown to have T-cell reactivity. However, among these antigens 18 kDa, 65 kDa and 70 kDa heat shock proteins (hsps) were most frequently recognized by T-cell lines and clones established from healthy donors vaccinated with killed M. leprae. A 24 kDa secreted antigen of M. leprae with T-cell epitope specific for M. leprae and M. tuberculosis complex was identified by direct screening of the recombinant DNA library with T-cell clones. The recombinant T-cell antigens of M. leprae were recognized by memory T-cells of Th1 type in association with multiple HLA-DR molecules. Epitope mapping with synthetic peptides identified M. leprae-specific as well as cross-reactive T-cell epitopes on the 18 kDa, 65 kDa and 70 kDa hsp antigens. In conclusion, our studies suggest that the recombinant antigens of M. leprae could be useful as reagents for specific diagnosis as well as in subunit and recombinant vaccine design against leprosy.     

Recent advances in immunodiagnosis of leprosy

Although prevalence of leprosy is considerably reduced, the unabated emergence of about 300,000 cases worldwide indicates that the source of infection and transmission are not being addressed. Early diagnosis and treatment still remain the cornerstone of leprosy control. Many diagnostic issues hinder the correct and timely diagnosis and classification of leprosy. Delayed and missed diagnosis of infectious leprosy patients and the lack of tests to measure asymptomatic M. leprae infection in contacts also hamper the assessment of transmission of M. leprae infection. An important goal would be the development of improved diagnostic tools to diagnose difficult cases and to detect M. leprae infection before clinical manifestation. The search for an ideal immunodiagnostic tool for leprosy had gone through various phases and development over the years, with inherent limitations in the sensitivity and specificity of the immunodiagnostic tests for leprosy. With improvement in technology many modifications of previously used PGL-1 assay in the form of rapid and less expensive techniques, such as dipstick, ELISA, ML flow test, have been introduced. Many new skin test antigens with potential for improving their efficiency, such as MLSA LAM, MLCwA and their fractionates, have been studied. After the completion of genome sequencing of M. leprae in 2000, many genes that were studied in M. tuberculosis and found potential for the immunodiagnosis of tuberculosis, such as CFP-10 and ESAT-6 proteins, have been investigated in M. leprae also. Genes that are unique to M. leprae with no homologous in M. tuberculosis have been explored for novel M. leprae-specific antigens. In order to overcome the problem of cross-reactivity, a number of workers have synthesized overlapping short peptides of different M. leprae recombinant proteins and studied their sequence divergence and attempted to identify M. leprae-specific B- and T-cell epitopes. This review makes an effort to present an overview of all these developments in the field of immunodiagnosis of leprosy.     

Use of non-conventional antigen: M. habana in detecting M. leprae antibodies from leprosy patients and contacts in FLA-ABS test

An indirect immunofluorescent (FLA-ABS) test has been developed to detect M. leprae specific antibodies in the active and subclinical cases of leprosy. An antigenically related mycobacterium, M. habana, was used as an antigen to detect M. leprae specific antibodies in the sera samples of leprosy patients. A comparison was made with M. leprae antigen using same set of sera samples. M. habana is capable of detecting anti-M. leprae antibodies in the serum samples of leprosy patients, previously absorbed with various mycobacterial antigens, cardiolipin and lecithin, almost to the same percentage as M. leprae. Possible use of M. habana antigen as an alternative to M. leprae, in the serodiagnosis of leprosy, has been discussed.     

Observations on the cultivation of M. leprae and M. tuberculosis in medium 'V' and 'V 1'.

Skin scrapings from five different active sites were collected from 14 leprosy patients and inoculated into medium V. Skin scrapings from three leprosy patients were inoculated into medium V 1. All the cultures were incubated at 8-10 degrees C. M. tuberculosis H37Rv, pretreatment isolates and streptomycin resistant strains were inoculated into medium V, with and without antibiotics, and incubated at 8-10 degrees C as well as 37 degrees C. Smears were made from the M. leprae and M. tuberculosis cultures at 0 hours and at different time points. The number of bacilli in the smears were counted. There was no increase in the number of M. leprae or M. tuberculosis in any of the cultures.     

High Incidence of IgG Antibodies to Phenolic Glycolipid in Non-Leprosy Patients in India

Purified phenolic glycolipid (PGL-1) from Mycobacterium leprae was used to detect IgG antibodies against PGL-1 in leprosy patients in an enzyme-linked immunosorbent assay (ELISA). A total of 698 sera were screened; they came from patients suffering from leprosy, autoimmune disease, myeloma, tuberculosis and sexually transmitted diseases (STDs). Cases with miscellaneous diseases and persons undergoing AIDS screening were also included. Sera from lepromatous and tuberculoid leprosy patients gave positivity rates of 60.5% and 41.7%, respectively. In non-leprosy cases, the PGL-1 ELISA showed an overall positivity rate of 6.9%; this was greatest in patients with tuberculosis (43.8%) followed by autoimmune diseases (40.9%) and miscellaneous cases including liver diseases (37.9%). This study emphasizes that PGL-1 ELISA has a low predictive value for diagnosis of active infection by Mycobacterium leprae. Positive reactions in a significant percentage of patients with autoimmune disease are intriguing and need indepth study.     

Immunogenicity of Mycobacterium leprae unique antigens in leprosy endemic populations in Asia and Africa

Ongoing transmission of leprosy is evident from the stable disease incidence in high burden areas. Tools for early detection of Mycobacterium leprae (M. leprae) infection, particularly in sub-clinically infected individuals, are urgently required to reduce transmission. Following the sequencing of the M. leprae genome, many M. leprae-unique candidate proteins have been identified, several of which have been tested for induction of M. leprae specific T cell responses in different leprosy endemic areas. In this study, 21 M. leprae-unique proteins and 10 peptide pools covering the complete sequence of five M. leprae-unique proteins (ML0576, ML1989, ML1990, ML2283, and ML2567) were evaluated in 160 individuals in Nepal and Ethiopia. These included: tuberculoid and borderline tuberculoid (TT/BT), borderline borderline and borderline lepromatous (BB/BL) leprosy patients; healthy household contacts (HHC); tuberculosis (TB) patients and endemic controls (EC). Immunogenicity of the proteins was determined by IFN-gamma secretion via stimulation of PBMC in 6 days lymphocyte stimulation tests (LST) or in whole blood assays (WBA). In LST, BB/BL patients (40%) responded to ML0573 and ML1601 whereas ML1604 was most immunogenic in TT/BT (35%) and HHC (36%). Additionally, significant numbers of EC displayed IFN-gamma production in response to ML0573 (54%), ML1601 (50%) and ML1604 (54%). TB patients on the other hand, hardly responded to any of the proteins except for ML1989. Comparison of IFN-gamma responses to ML0121, ML0141 and ML0188 for TT/BT patients showed specific increase in diluted 6 days WBA compared to the undiluted 24 hours WBA, whereas EC showed a reduced response in the diluted WBA, which may indicate detection of disease-specific responses in the 6 days WBA. In summary, identification of multiple M. leprae proteins inducing M. leprae-specific T cell responses in groups at high risk of developing leprosy may contribute to improve early detection for M. leprae infection.     

Experiences with Mycobacterium leprae soluble antigens in a leprosy endemic population

Rees and Convit antigens prepared from armadillo-derived Mycobacterium leprae were used for skin testing in two leprosy endemic villages to understand their use in the epidemiology of leprosy. In all, 2602 individuals comprising 202 patients with leprosy detected in a prevalence survey, 476 household contacts and 1924 persons residing in non-case households were tested with two antigens. There was a strong and positive correlation (r = 0.85) between reactions to the Rees and Convit antigens. The distribution of reactions was bimodal and considering reactions of 12 mm or more as 'positive', the positivity rate steeply increased with the increase in age. However, the distributions of reactions to these antigens in patients with leprosy, their household contacts and persons living in non-case households were very similar. These results indicate that Rees and Convit antigens are not useful in the identification of M. leprae infection or in the confirmation of leprosy diagnosis in a leprosy endemic population with a high prevalence of nonspecific sensitivity.     

Demonstration of Mycobacterium leprae specific antigens in leprosy lesions using monoclonal antibodies

Cryostat sections of dermal lesions from 13 untreated patients of leprosy were studied by indirect immunoperoxidase using monoclonal antibodies (MLO4 & MLO6), defining M. leprae specific antigens. The lymphocytes and macrophages in both the tuberculoid and lepromatous granulomas showed membranous staining with the above antibodies. M. leprae organisms in the lepromatous granulomas and the cells in the section of lymph nodes of patients with tuberculosis, or sections of normal skin or psoriatic lesions did not show any staining with these antibodies. These observations suggest that M. leprae specific antigens are present and expressed on the cells infiltrating the granulomas of leprosy lesions.     

Similarity between mycobacterial and human epidermal antigens

Eight out of 17 mouse anti-Mycobacterium leprae monoclonal antibodies (MAb) were previously observed to react with human nerve and skin antigenic determinants in cryostat sections, using an indirect immunoperoxidase technique. These observations suggested that antigenic mimicry may be involved in the development of the clinical manifestations of leprosy. In the present study we have extended our earlier findings by investigating sera from leprosy patients and MAb using Western blot technique. It was observed that 30 sera and their corresponding F(ab')2 fragments from isolated IgG fractions of both tuberculoid and lepromatous patients reacted with 40-50 epidermal proteins of molecular weights (MW) ranging from 10 to 130 kDa. Sera from 14 controls, however, showed similar reactivity patterns. Absorption of nine patient and control sera with M. tuberculosis, M. marinum and M. kansasii resulted in the removal of several components of different MW in nine, four and three cases, respectively. No consistent differences between sera from leprosy patients and controls were observed. Four out of eight MAb against M. leprae which reacted with determinants in human epidermis and/or dermis in skin cryostat sections reacted with epidermal proteins of MW higher than 39 kDa in Western blot. Four MAb which showed reactivity in cryostat sections did not react in Western blot. Another four MAb did react with human epidermal proteins in Western blot but did not react in cryostat sections, indicating that the MAb were reacting with different epitopes in the two systems. Five MAb did not react with human epidermal proteins either in cryostat sections or in Western blot.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anti-leprosy protective vaccination of rhesus monkeys with BCG or BCG plus heat-killed Mycobacterium leprae: lepromin skin test results

Groups of rhesus monkeys (RM) were vaccinated and boosted with living Mycobacterium bovis Bacillus Calmette-Guerin (BCG) or BCG + low dose (LD) heat-killed Mycobacterium leprae (HKML) or high dose (HD) HKML or were unvaccinated. Animals vaccinated with BCG + LD and HD HKML were lepromin skin tested 2 weeks after boosting. All groups were lepromin tested 37 and 46 months after challenge with live M. leprae. Fernandez (72 h) and Mitsuda (28 day) responses were recorded. Ten of 10 rhesus monkeys in each of the two BCG + HKML-vaccinated groups significantly converted to strong positive Fernandez status within 2 weeks of boosting, compared to one of six positives in the unvaccinated unchallenged normal control group. Both BCG + HKML groups were significantly protected from clinical leprosy. Six of 10 in each of the two BCG + HKML groups significantly converted to Mitsuda positivity within 2 weeks of boosting compared to zero of six in the normal control group. The sizes of the Mitsuda responses were larger in the LD group than the HD HKML vaccinated/boosted group, suggesting suppression by vaccination with higher doses of HKML in combination with BCG. Fernandez responses were negative in normal RM as well as in the unvaccinated, ML-challenged group and the BCG-vaccinated, ML-challenged group at 37 or 46 months after ML inoculation, although the BCG-vaccinated group was significantly protected from leprosy and the unvaccinated group was not. In contrast, at 37 months the Fernandez reaction was positive in the BCG plus LD and the BCG plus HD HKML-vaccinated groups, both of which were significantly protected from clinical leprosy. By 46 months, the Fernandez responses were below significance in all groups. Thus, Fernandez reactivity is not a reliable correlate to protection from experimental leprosy in RM. Mitsuda responses became strongly positive in all four ML-challenged groups by 37 months and remained strongly positive at 46 months after ML inoculation, suggesting that strong Mitsuda reactivity reflects responses to living ML. BCG or BCG + LD or HD HKML vaccination/boosting of RM produced significant clinical protection from leprosy and there was a good correlation between protection from LL forms of leprosy and positive Mitsuda skin test responses after challenge with live ML. Positive Mitsuda responses were generated in essentially all individuals after challenge with live ML, and this response was primed by prior vaccination/boosting with BCG + HKML as shown by conversion to positivity 2 weeks after boosting. The data show that resistance to clinical leprosy is reflected by Mitsuda responses in ML-exposed RM, similar to results from human studies, and confirm the suitability of RM as a model for leprosy vaccine studies.     

Anti-Mycobacterium leprae monoclonal antibodies cross-react with human skin: an alternative explanation for the immune responses in leprosy.

A panel of 17 mouse monoclonal antibodies (MoAb) raised against Mycobacterium leprae (M. leprae) antigens was used to detect antigenic determinants in normal human skin. An indirect immunoperoxidase technique was used. Eight of the MoAb detected epidermal antigens similar to patterns well known for human sera. Five of these MoAb detected determinants in the dermis, too. These observations may indicate a certain degree of similarity between the antigenic determinants occurring in M. leprae and in the human host. We propose that such a similarity on the one hand may facilitate the survival of M. leprae in the human host when the antigens are not recognized as "non-self," a situation which seems to occur in lepromatous leprosy, when the patients' tissues are loaded with bacteria virtually without any immune response. On the other hand, M. leprae antigens which mimic host antigens may induce an auto-immune reaction against the host's own antigens, which could explain the immune reaction in tuberculoid leprosy and during a "reversal reaction" when M. leprae is not observed in the host tissues, but extensive granuloma formation occurs.     

Detection of lepromatous leprosy patients shedding M. leprae in nasal droppings by enzyme immunoassay

Enzyme immunoassays (EIAs) for detection of lepromatous leprosy (LL) patients harbouring M. leprae in nasal mucosa are described. One EIA measures IgM antibodies against the synthetic disaccharide (ND-BSA) residue of phenolic glycolipid I of M. leprae, whereas the other titrates primarily IgG antibodies against sonicate supernatant antigens of Mycobacterium w. (M.w.). Fifty coded leprosy sera were analysed by EIAs under a double blind code. Amongst the 20 LL patients with positive nasal smear, 18 (90%) were positive in EIA based on ND-BSA, in comparison to 19 (95%) in EIA using M.w. antigens. The assays can be performed on fresh serum samples or on blood samples collected on filter paper discs. These assays can be useful for leprosy control programmes.     

Evaluation of Mycobacterium leprae antigens in the serological monitoring of a clofazimine-based chemotherapeutic study of dapsone resistant lepromatous leprosy patients in Cebu, Philippines

Thirty-one dapsone resistant lepromatous leprosy patients receiving clofazimine based therapy were serologically monitored throughout their 5-year period of treatment. Sequentially collected sera were used to examine 4 Mycobacterium leprae antigens to evaluate their usefulness in ELISA's for monitoring the progress of their therapy. The ELISA results were compared with decline in bacterial load over the treatment period and with duration of treatment. In addition the ELISA's were compared with each other. The ELISA's based on the measurement of IgM antibodies to the two neoglycoproteins (NDO and NTO) representing the phenolic glycolipid antigen of M. leprae were found to be the most effective with regard to monitoring treatment. A whole M. leprae based ELISA was less efficient in monitoring treatment because it failed to measure antibodies in 5 out of 31 patients. The ELISA-inhibition test based on the detection of antibodies to a species-specific epitope on the 36 K antigen of M. leprae was less suitable because of persistent reactivity during therapy.     

Comparison of PCR mediated amplification of DNA and the classical methods for detection of Mycobacterium leprae in different types of clinical samples in leprosy patients and contacts

Traditional staining and microscopic examination techniques for the detection of Mycobacterium leprae, DNA amplification by polymerase chain reaction (PCR) of a 531-bp fragment of the M. leprae specific gene encoding the 36-kDa antigen, and serodiagnosis with M. leprae specific antigens (PGL-1 and D-BSA) were compared on different clinical specimens (serum samples, slit-skin smears, biopsies and swabs) from 60 leprosy patients attending the Sanatorium of Fontilles. Patients were divided into groups; (i) 20 multibacillary patients (MB) with positive bacteriological index (BI) by conventional methods and on WHO multidrug therapy (MDT); (ii) 30 MB patients with negative BI and completed minimum 2 years treatment MDT; (iii) 10 paucibacillary (PB) patients who had completed 6 months MDT at least 8 years ago. Control groups included four non-leprosy patients for PCR methods and 40 health control patients and 10 tuberculosis patients for serological methods. In the multibacillary BI positive group, there was a good correlation between all methods. All tests were negative in the paucibacillary group, although only a few patients were tested and all had been treated many years ago. One must be cautious concerning the diagnostic potential of these techniques in this type of leprosy. We also studied different combinations of leprosy diagnosis methods to determine the potential risk in a leprosy contact individuals group. The prevalence of antibodies to M. leprae antigens in serum was measured, together with the presence of M. leprae DNA in the nose and lepromin status in a group of 43 contacts of leprosy patients (12 household and 31 occupational) to evaluate the maintenance of infection reservoirs and transmission of the disease. Only two individuals were found to form a potential high risk group.     

Relative cross reactivity of habanin, lepromin and tuberculin in guinea pigs sensitized with homologous and heterologous mycobacteria

An atypical strain Mycobacterium habana has been studied for its antigenic cross reactivity with delayed type of hypersensitivity responses in guinea pigs. Guinea pigs sensitized with M. habana, M. leprae and M. tuberculosis when challenged with habanin, lepromin and tuberculin in criss-cross fashion have demonstrated strong cross reactivity with each other. Possibilities of developing M. habana as a vaccine against tuberculosis and/or leprosy has been discussed.     

Delipidified cell components of Mycobacterium leprae and its applications.

Delipidified cell components (DCC) of Mycobacterium leprae obtained as an insoluble material consist of several proteins. This preparation, DCC, has ability to differentially bind to sera from lepromatous leprosy patients and antibodies to this complex get reduced as patients improve under chemotherapy. The antigenic complex has no ability to bind to proteins of sera from normal healthy individuals or tuberculoid leprosy patients. The DCC is antigenic and is recognised by immune deficient cells of lepromatous leprosy patients, leading to lymphocyte proliferation, production of Interleukin II and interferon gamma, and resulting in activation of the phagocytes to initiate killing of endocytosed M.leprae through reactive oxygen intermediates, primarily superoxide. The DCC has also immunomodulatory properties to protect mice against M.leprae infection. Experiments with mice and isolated peripheral blood cells from patients have indicated the probable molecular mechanism of immunomodulation by DCC.     

Studies on lepromin and soluble antigens of M.leprae: their classification standardization and use.

Before the discovery of armadillo as a susceptible animal the source of M.leprae was limited and hence the use of lepromin was not common in the field. In recent times, the soluble antigens of armadillo-derived M.leprae have been used extensively in the field. Although the results of the study show that these antigens do not differentiate always a susceptible form from the resistant form, they are able to segregate the polar forms of leprosy. In a given field situation the criteria for diagnosis is so stressed that leprosy is overdiagnosed and within one year of follow up nearly half the number of cases are noted as not leprosy. Hence, in such situations lepromin reaction would be definitely a poor correlate with the type of leprosy. However, in hospital based studies the lepromin reaction has always been and would remain useful in confirming the classification (Sengupta et al 1984). Lepromins and M.leprae soluble antigens have gone through extensive standardization procedures. As these antigens contain mostly common mycobacterial antigens along with the M.leprae-specific antigens, these antigens are unable to specifically diagnose M.leprae infection. After purification of M.leprae from infected armadillo tissue, it was expected that the soluble antigen of M.leprae would probably be as useful as tuberculin. However, this was not found to be true in case of lepromin. Specificity for M.leprae has been noted in the epitopes (antigenic sites) on cross reacting molecules (12 kd, 18 kd, 28 kd, 35 kd, 36 kd) of mycobacteria (Ivanyi et al 1983; Watson 1989). These specific epitopes, if synthesized, could be of use as skin test antigens for determining M.leprae infection.(ABSTRACT TRUNCATED AT 250 WORDS)