The role of Mycobacterium leprae phenolic glycolipid I (PGL-I) in serodiagnosis and in the pathogenesis of leprosy

PGL-I (phenolic glycolipid I) emerged in the early 1980s on the one hand as part of intensive efforts to define the typing antigens of a host of Mycobacterium spp. and also from characterisation of the lipids of skin biopsies from highly bacillary positive lepromatous leprosy patients. PGL-I, despite its extreme lipophilicity due to its inherent phthiocerol dimycocerosyl component, is highly antigenic evoking high titre IgM antibodies in lepromatous leprosy patients, attributable largely to the unique 3,6-di-O-methyl-beta-D-glucosyl entity at the non-reducing terminus of its trisaccharide. PGL-I itself or in the form of semisynthetic neoglycoproteins containing the synthetic terminal disaccharide or the whole trisaccharide chemically conjugated to such as bovine or human serum albumin, has found its greatest utility in the serological diagnosis, confirmation and management of lepromatous leprosy. PGL-I has also been implicated in the tropism of M. leprae for Schwann cells, through specific binding to laminin, and to play an important role in downregulation of the inflammatory immune response and inhibition of dendritic cell maturation and activation, thereby facilitating the persistence of M. leprae/leprosy.     

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.     

Challenges presented by nerve damage in leprosy

The basis of nerve damage in leprosy is the unique tendency of Mycobacterium leprae to invade Schwann cells. alphaBeta-Dystroglycan on the basement membrane of Schwann cells binds to laminin alpha2, in turn binding to receptors on the M. leprae surface, comprising a histone-like protein and phenoglycolipid-1. When nerve damage during reversal reactions was found to be associated with an abrupt increase in delayed type hypersensitivity against M. leprae antigenic determinants released from Schwann cells, it suggested that the nerve is damaged as an innocent bystander during the immune response. This strongly influenced the introduction of therapy based on immunosuppression combined with continued anti-mycobacterial medication. Lysis of Schwann cells presenting M. leprae antigenic determinants by activated CD4+ T cells and interaction of M. leprae with Toll-like receptors on Schwann cells are additional mechanisms implicated in nerve damage. Persistence of M. leprae antigen in local lesions after regular multiple drug therapy (MDT) is an important risk factor for late reactions. In spite of significant advances in the provision of MDT globally, early diagnosis, together with effective treatment of the disease and associated nerve damage at initial presentation remains a major challenge for the health services. Reduced prevalence as a result of MDT should not be taken to indicate that the challenges of leprosy control are diminished as long as nerve damage is not controlled and new case detection rates are not declining.     

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)     

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)     

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.     

Study of cytokine response against panel of purified Mycobacterium leprae antigens by using whole blood assay in subjects residing in a resettlement village of cured leprosy patients

Mycobacterium leprae being an intracellular pathogen, cell mediated immunity is very important in the clinical outcome of leprosy. Manifestation of the disease is correlated with the level and type of cell mediated immune response. The main objective of this study was to analyse TNF-alpha and IFN-gamma production by T-cells when challenged with different M. leprae purified antigens in subjects with known exposure. 50 subjects residing in resettlement village of cured leprosy patients were included in the study. Whole Blood assay studies were undertaken in which the blood was placed in culture and was challenged with 35-kDa antigen, whole M. leprae cells, M. leprae cell wall antigen and M. leprae soluble antigen minus LAM. T-cell derived cytokines TNF-alpha and IFN-gamma were measured by ELISA. It was observed that challenging the lymphocytes with 35-kDa antigen, the cell wall antigen and M. leprae soluble antigen minus LAM resulted in increased levels of IFN-gamma whereas challenge with 35-kDa antigen and M. leprae cell wall antigen resulted in increased levels of TNF-alpha.     

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.     

Leprosy immunopathogenesis and vaccine development.

The truly effective immunity against intracellular parasites, including mycobacteria, is mediated by monocyte/macrophages, and in the immunologically responding (resistant) host these phagocytes need minimal antigenic stimulus, specific or non-specific, to become activated and be microbicidal. T-cell mediated delayed hypersensitivity (DTH) causes tissue damage and destruction, which is particularly unwelcome in leprosy because of its nerve-damaging potential. Gamma interferon (INF-gamma), the terminal lymphokine of a DTH response, promotes mycobacterial survival and growth. There are T-cells (TH1 subtypes) that produce DH response either independent of, or, only partly dependent on INF-gamma; this type of DH peaking at 24 hours appears similar to the Jones-Mote type rather than to the tuberculin type of DTH peaking at 48-72 hours and is devoid of the necrotic component of tuberculin type of DTH. M. leprae antigens normally elicit this Jones-Mote type of DH. Suppressor T-cells are associated with a protective immune response, while helper T-cells mediating DTH are harmful. In view of this immunobiology, it would appear that pathogenic mycobacteria that generate a tuberculin type DTH response should not be used as immunogens in leprosy.     

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.     

Presence of M. leprae in tissues in slit skin smear negative multibacillary (MB) patients after WHO-MBR

This study looked for M. leprae in the lymph node, nerve and skin of multibacillary (MB) leprosy patients who become slit skin smear negative after the completion of WHO-MBR. Twenty-five WHO-MBR-treated multibacillary leprosy patients were studied; borderline lepromatous (BL) leprosy (n = 11) and lepromatous (LL) leprosy (n = 14)). Fifteen patients had reaction (erythema nodosum leprosum 11, upgrading reaction 4) either at presentation or during therapy. All patients attained slit skin smear negativity after WHO-MBR (range 24-39 months. Sixteen (64%) patients with multibacillary leprosy showed fragmented bacilli in skin and nerve biopsy or lymph node aspirates after WHO-MBR. Lymph node aspirates alone revealed M. leprae in seven patients, followed by nerve in two and skin in one patient. Four cases showed M. leprae at all sites followed by nerve and skin or lymph node in one case each. A pretreatment bacteriological index (BI) of 4+ or more was significantly associated with the presence of M. leprae at the end of treatment. Also, significantly more lymph node aspirates contained M. leprae in comparison with nerve or skin biopsies. All seven cases in whom treatment was extended beyond 24 months showed M. leprae in tissues even after attaining slit smear negativity. In conclusion, M. leprae persist in tissues after 2 years of WHO-MBR and patients with an initial BI of 4+ or more need to be closely followed up after stopping MDT.     

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.     

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.     

Immunohistochemical detection of PGL-1, LAM, 30 kD and 65 kD antigens in leprosy infected paraffin preserved skin and nerve sections

A panel of lipid, carbohydrate and protein antibodies were optimized for use in detecting M. leprae antigens in paraffin embedded material. Skin and nerve biopsies from 13 patients across the leprosy spectrum were studied. All antibodies detected antigen in tissues with a BI > 1. Phenolic-glycolipid was not detected in bacteriologically negative tissue but lipoarabinomanan (LAM) and protein antigens were detected. Staining with LAM was strongest and gave least background. The transfer of this immunohistochemical technique to paraffin embedded material will allow examination of tissue with better morphology and from clinics without access to tissue freezing facilities.     

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.     

Leprosy: review of the epidemiological,clinical, and etiopathogenic aspects - Part 1

Leprosy is caused by Mycobacterium leprae and has been known since biblical times. It is still endemic in many regions of the world and a public health problem in Brazil. The prevalence rate in 2011 reached 1.54 cases per 10,000 inhabitants in Brazil. The mechanism of transmission of leprosy consists of prolonged close contact between susceptible and genetically predisposed individuals and untreated multibacillary patients. Transmission occurs through inhalation of bacilli present in upper airway secretion. The nasal mucosa is the main entry or exit route of M. leprae. The deeper understanding of the structural and biological characteristics of M. leprae, the sequencing of its genome, along with the advances in understanding the mechanisms of host immune response against the bacilli, dependent on genetic susceptibility, have contributed to the understanding of the pathogenesis, variations in the clinical characteristics, and progression of the disease. This article aims to update dermatologist on epidemiological, clinical, and etiopathogenic leprosy aspects.     

Recent histopathological studies in leprosy, with particular reference to early diagnosis and leprous neuropathy

In histopathological studies in leprosy, two important areas were identified in recently published work. They are early diagnosis and neuropathy. In histopathological examination, finding of M. leprae in tissues and/or granulomatous destruction of nerves are the two important findings to confirm the diagnosis. Immunopathological staining of M. leprae, PCR amplification of M. leprae antigen and S100 staining of Schwaann cells have considerably enhanced the sensitivity of histopathological diagnosis. If the two clinical findings such as hypopigmented patches with impaired sensation and thickened nerves accompanied by loss of sensation are the only ones that are taken into account for diagnosis, then a significant number of early patients will be missed. It is pointed out that biopsy examination of skin and nerves, when necessary, and skin-smear studies are indispensable diagnostic procedures. In the study of leprous neuropathy, there are several studies trying to decipher the entry of M. leprae into Schwann cells. The sharing of antigens between M. leprae and surface membrane of Schwann cells may be an important factor. However, there is much more to be learned in this area. In the control and prevention of neuritis, although corticosteroids administered along with multi-drug therapy was helpful, the benefit was not sustained.     

Recombinant Mycobacterium leprae protein associated with entry into mammalian cells of respiratory and skin components

BACKGROUNDS: The transmission of Mycobacterium leprae, the causative pathogen of leprosy, has been postulated to occur mainly through upper respiratory route rather than skin-to-skin contact via minor injuries. The M. leprae genome contains mce1A gene, which encodes a putative mammalian cell entry protein. However, to date, there have been no functional analyses of the M. leprae mce1A gene product. OBJECTIVE: The aim of this study was to elucidate a possible relationship between this transmission mechanism and the mce1A gene product. METHODS: We analyzed the cell uptake activity in vitro of polystyrene latex beads coated with a purified recombinant (r-) protein expressed by a 849-bp locus within the mce1A gene. RESULTS: The r-protein promoted uptake of the beads into human nasal epithelial cells derived from nasal polyps, human bronchial epithelial cell line, normal human dermal fibroblasts, normal human microvascular endothelial cells and normal human keratinocytes cultured at 0.01 mM extracellular calcium concentration [Ca]; no uptake occurred with keratinocytes cultured at 1.2mM [Ca]. CONCLUSION: These results suggest that the mce1A gene product can mediate M. leprae entry into respiratory epithelial cells as their natural target cells, which may be the main mode of transmission. Endothelial cells, on the other hand, may serve as the reservoir of the bacilli for long-term infection. The M. leprae Mce1A protein has potential important implications for mode of transmission and pathogenesis of leprosy.     

Comparative leprosy vaccine trial in south India

This report provides results from a controlled, double blind, randomized, prophylactic leprosy vaccine trial conducted in South India. Four vaccines, viz BCG, BCG+ killed M. leprae, M.w and ICRC were studied in this trial in comparison with normal saline placebo. From about 3,00,000 people, 2,16,000 were found eligible for vaccination and among them, 1,71,400 volunteered to participate in the study. Intake for the study was completed in two and a half years from January 1991. There was no instance of serious toxicity or side effects subsequent to vaccination for which premature decoding was required. All the vaccine candidates were safe for human use. Decoding was done after the completion of the second resurvey in December 1998. Results for vaccine efficacy are based on examination of more than 70% of the original "vaccinated" cohort population, in both the first and the second resurveys. It was possible to assess the overall protective efficacy of the candidate vaccines against leprosy as such. Observed incidence rates were not sufficiently high to ascertain the protective efficacy of the candidate vaccines against progressive and serious forms of leprosy. BCG+ killed M. leprae provided 64% protection (CI 50.4-73.9), ICRC provided 65.5% protection (CI 48.0-77.0), M.w gave 25.7% protection (CI 1.9-43.8) and BCG gave 34.1% protection (CI 13.5-49.8). Protection observed with the ICRC vaccine and the combination vaccine (BCG+ killed M. leprae) meets the requirement of public health utility and these vaccines deserve further consideration for their ultimate applicability in leprosy prevention.