Presence of human T-cell responses to the Mycobacterium leprae 45-kilodalton antigen reflects infection with or exposure to M. leprae

The ability of the 45-kDa serine-rich Mycobacterium leprae antigen to stimulate peripheral blood mononuclear cell (PBMC) proliferation and gamma interferon (IFN-gamma) production was measured in leprosy patients, household contacts, and healthy controls from areas of endemicity in Mexico. Almost all the tuberculoid leprosy patients gave strong PBMC proliferation responses to the M. leprae 45-kDa antigen (92.8%; n = 14). Responses were lower in lepromatous leprosy patients (60.6%; n = 34), but some responses to the 45-kDa antigen were detected in patients unresponsive to M. leprae sonicate. The proportion of positive responses to the M. leprae 45-kDa antigen was much higher in leprosy contacts (88%; n = 17) than in controls from areas of endemicity (10%; n = 20). None of 15 patients with pulmonary tuberculosis gave a positive proliferation response to the 45-kDa antigen. The 45-kDa antigen induced IFN-gamma secretion similar to that induced by the native Mycobacterium tuberculosis 30/31-kDa antigen in tuberculoid leprosy patients and higher responses than those induced by the other recombinant antigens (M. leprae 10- and 65-kDa antigens, thioredoxin, and thioredoxin reductase); in patients with pulmonary tuberculosis it induced lower IFN-gamma secretion than the other recombinant antigens. These results suggest that the M. leprae 45-kDa antigen is a potent T-cell antigen which is M. leprae specific in these Mexican donors. This antigen may therefore have diagnostic potential as a new skin test reagent or as an antigen in a simple whole-blood cytokine test.     

Enhancing Sensitivity of Detection of Immune Responses to Mycobacterium leprae Peptides in Whole-Blood Assays

Although worldwide leprosy prevalence has been reduced considerably following multidrug therapy, new case detection rates remain relatively stable, suggesting that transmission of infection still continues. This calls for new efforts, among which is development of assays that can identify subclinical/early-stage Mycobacterium leprae-infected subjects, a likely source of transmission. Areas in which leprosy is endemic often lack sophisticated laboratories, necessitating development of field-friendly immunodiagnostic tests for leprosy, like short-term whole-blood assays (WBA). In classical, peripheral blood mononuclear cell (PBMC)-based gamma interferon (IFN-γ) release assays, M. leprae peptides have been shown to discriminate in a more specific fashion than M. leprae proteins between M. leprae-exposed contacts and patients as opposed to healthy controls from the same area of endemicity. However, peptides induced significantly lower levels of IFN-γ than did proteins, particularly when whole blood was used. Therefore, possibilities of specifically enhancing IFN-γ production in response to M. leprae peptides in 24-h WBA were sought by addition of various cytokines and antibodies or by mannosylation of peptides. In addition, other cytokines and chemokines were analyzed as potential biomarkers in WBA. We found that only interleukin 12 (IL-12), not other costimulants, increased IFN-γ production in WBA while maintaining M. leprae peptide specificity, as evidenced by lack of increase of IFN-γ in control samples stimulated with IL-12 alone. The IL-12-induced increase in IFN-γ was mainly mediated by CD4⁺ T cells that did not produce IL-2 or tumor necrosis factor (TNF). Mannosylation further allowed the use of 100-fold-less peptide. Although not statistically significantly, macrophage inflammatory protein 1β (MIP-1β) and macrophage c protein 1 (MCP-1) levels specific for M. leprae peptide tended to be increased by IL-12. IP-10 production was also found to be a useful marker of M. leprae peptide responses, but its production was enhanced by IL-12 nonspecifically. We conclude that IFN-γ-based WBA combined with IL-12 represents a more sensitive and robust assay for measuring reactivity to M. leprae peptides.Leprosy is a disabling and stigmatizing disease caused by infection with Mycobacterium leprae. The characteristic immunological and clinical leprosy spectrum, classified by Ridley and Jopling in 1966 (25), ranges from tuberculoid (TT) or paucibacillary (PB) leprosy to lepromatous (LL) or multibacillary (MB) leprosy. In between these poles the borderline states borderline lepromatous (BL), borderline borderline (BB), and borderline tuberculoid (BT) leprosy are positioned. TT/BT patients in general show high cellular responses to M. leprae antigens injected in the skin as well as in in vitro T-cell assays; have low antibody titers to M. leprae antigens, including phenolic glycolipid I (PGL-I); and develop localized granulomatous disease with few, if any, detectable bacilli in their lesions. At the opposite end of the spectrum are LL/BL patients with a characteristic inability to generate M. leprae-specific Th1-cell responses and with disseminating progressive infection and high antibody titers to M. leprae antigens, including the M. leprae-specific cell surface antigen PGL-I.Over the last 2 decades the WHO leprosy elimination program, partly in combination with wide coverage of Mycobacterium bovis BCG vaccination (28), has had a massive effect on the registered number of cases, which dropped from approximately 5.4 million in 1985 to 212,802 worldwide at the beginning of 2008. In addition, since 2003 the global number of new cases detected showed a drastic decrease at an average rate of nearly 20% per year, and a reported year-end prevalence below 1 per 10,000 was obtained in 2007 in all countries with a population of >1 million except for Brazil, Nepal, and East Timor (32). However, part of the decrease was achieved by changing leprosy control policies and does not necessarily reflect the reality of infection. Concomitantly, the elimination campaign has had a severe downside as it led to a discontinuation of leprosy control programs and a decrease in leprosy clinics, specialists, and research. Thus, leprosy patients have to be treated in integrated programs, where health workers lack the knowledge and time to diagnose and treat leprosy. This resulted in sustained transmission as evidenced by the hundreds of thousands of new cases of leprosy that keep being detected globally every year (254,525 in 2007) and a 3.1% increase between 2007 and 2008 of new case detection in children (32). In addition, countries that do not exceed this prevalence rate nationwide still harbor regions of high endemicity, where leprosy remains a public health problem (e.g., Angola, Central African Republic, India, and Tanzania). These figures demonstrate that M. leprae-infected contacts and persons with subclinical, undiagnosed leprosy, likely the major sources of unidentified transmission, are an incessant source of active transmission, making early detection of leprosy or M. leprae infection and prompt multidrug treatment (MDT) of utmost importance for control of the disabling effects of leprosy.Diagnosis of leprosy is at present based only on clinical features and the number of skin lesions. Due to the loss of diagnostic skills and the decrease in skin smear services, the detection of M. leprae infection occurs in many cases only after significant and irreversible nerve damage has occurred. Since M. leprae is not cultivable in vitro, bacterial enumeration by microscopic examination is required for leprosy classification, monitoring chemotherapy regimens, and diagnosis of relapse, yielding, in inexperienced hands, data of limited specificity and sensitivity. There is no test available that can detect asymptomatic M. leprae infection or predict progression of infection to clinical disease. Assays that demonstrate the presence of IgM antibodies against PGL-I are useful for most MB patients but have limited value in identifying or predicting PB patients who typically develop cellular rather than humoral immunity (23).In order to assess host immune responses after exposure to or infection with mycobacteria, the ex vivo whole-blood assay (WBA) is a helpful test. In the past, several variations of the WBA have been introduced in which unseparated heparinized blood is stimulated with antigen either overnight or for as long as 6 days, after which plasma or supernatant is analyzed for cytokines (8). Since WBAs are much simpler and faster than conventional assays using peripheral blood mononuclear cells (PBMC), they have been successfully applied in commercially available gamma interferon (IFN-γ) release assays (IGRAs) for diagnosis of tuberculosis (TB) (QuantiFERON-TB-Gold-In-Tube [QFT-G-IT]). These IGRAs have a number of advantages over skin tests (e.g., Mitsuda or Mantoux tests), as they can measure directly from whole blood within a single day, do not require repeated visits for reading of the test, and do not carry the inherent risk of boosting upon repeated testing. Furthermore, there are no exclusion criteria, as the test can also be performed for patients with skin disorders.To measure responses to recombinant M. leprae proteins and whole-cell sonicate of M. leprae, 6-day WBAs measuring IFN-γ in diluted blood samples have proved successful at field sites in various countries such as Nepal (33) and Malawi (4, 34). Since 6-day WBAs require access to a CO₂ incubator, our investigations focused on the development of a practical diagnostic assay on 24-h WBA. Furthermore, as (mixtures of) M. leprae peptides proved to induce more specific IFN-γ release by PBMC than did recombinant M. leprae proteins (16, 29), we previously used M. leprae peptides for the 24-h WBA (17) in analogy to the successful peptide-based QuantiFERON-TB-Gold-In-Tube (11). However, the shortened incubation period hampered the use of M. leprae peptides in WBA, as IFN-γ production induced was weak in comparison to that when M. leprae proteins were used (17, 29). Therefore, in the present study we analyzed other cytokines/chemokines as potential readout for WBA and tested possible enhancement of IFN-γ production in response to M. leprae peptides by addition of several cytokines, antibodies against factors that block the optimal release of IFN-γ, or antibodies that can trigger costimulation.     

Interleukin-10-dependent down-regulation of interferon-gamma response to Leishmania by Mycobacterium leprae antigens during the clinical course of a coinfection

We have described a case of a patient with an intriguing association of mucocutaneous leishmaniasis with lepromatous leprosy, two opposite polar forms of these spectral diseases. In the present follow-up study, we investigated the effect of the addition of Mycobacterium leprae antigens on interferon-gamma (IFN-γ) production in Leishmania antigen-stimulated cultures of peripheral blood mononuclear cells (PBMC) from this patient. For this purpose, PBMC cultures were stimulated with crude L. braziliensis and/or M. leprae whole-cell antigen extracts or with concanavalin A. In some experiments, neutralizing anti-human interleukin (IL)-10 antibodies were added to the cultures. IFN-γ and IL-10 levels in culture supernatants were measured by ELISA. During active leprosy, M. leprae antigens induced 72.3% suppression of the IFN-γ response to L. braziliensis antigen, and this suppression was abolished by IL-10 neutralization. Interestingly, the suppressive effect of M. leprae antigen was lost after the cure of leprosy and the disappearance of this effect was accompanied by exacerbation of mucosal leishmaniasis. Considered together, these results provide evidence that the concomitant lepromatous leprosy induced an IL-10-mediated regulatory response that controlled the immunopathology of mucosal leishmaniasis, demonstrating that, in the context of this coinfection, the specific immune response to one pathogen can influence the immune response to the other pathogen and the clinical course of the disease caused by it. Our findings may contribute to a better understanding of the Leishmania/M. leprae coinfection and of the immunopathogenesis of mucosal leishmaniasis.     

Leprosy patients with lepromatous disease recognize cross-reactive T cell epitopes in the Mycobacterium leprae 10-kD antigen

T cell responses play a critical role in determining protective responses to leprosy. Patients with self-limiting tuberculoid leprosy show high T cell reactivity, while patients with disseminated lepromatous form of the disease show absent to low levels of T cell reactivity. Since the T cell reactivity of lepromatous patients to purified protein derivative (PPD), a highly cross-reactive antigen, is similar to that of tuberculoid patients, we queried if lepromatous patients could recognize cross-reactive epitopes in Mycobacterium leprae antigens as well. T cell responses were analysed to a recombinant antigen 10-kD (a heat shock cognate protein) which is available from both M. tuberculosis (MT) and M. leprae (ML) and displays 90% identity in its amino acid sequence. Lymphoproliferative responses were assessed to ML and MT 10 kD in newly diagnosed leprosy patients (lepromatous, n = 23; tuberculoid, n = 65). Lepromatous patients showed similar, but low, lymphoproliferative responses to ML and MT 10 kD, while tuberculoid patients showed much higher responses to ML 10 kD. This suggests that the tuberculoid patients may be recognizing both species-specific and cross-reactive epitopes in ML 10 kD, while lepromatous patients may be recognizing only cross-reactive epitopes. This was further supported by linear regression analysis. Lepromatous patients showed a high concordance in T cell responses between ML and MT 10 kD (r = 0.658; P < 0.0006) not observed in tuberculoid patients (r = 0.203; P > 0.1). Identification of cross-reactive T cell epitopes in M. leprae which could induce protective responses should prove valuable in designing second generation peptide-based vaccines.     

Gamma Interferon Responses Induced by a Panel of Recombinant and Purified Mycobacterial Antigens in Healthy, Non-Mycobacterium bovis BCG-Vaccinated Malawian Young Adults

We have previously shown that young adults living in a rural area of northern Malawi showed greater gamma interferon (IFN-γ) responses to purified protein derivatives (PPD) prepared from environmental mycobacteria than to PPD from Mycobacterium tuberculosis. In order to define the mycobacterial species to which individuals living in a rural African population have been exposed and sensitized, we tested T-cell recognition of recombinant and purified antigens from M. tuberculosis (38 kDa, MPT64, and ESAT-6), M. bovis (MPB70), M. bovis BCG (Ag85), and M. leprae (65 kDa, 35 kDa, and 18 kDa) in >600 non-M. bovis BCG-vaccinated young adults in the Karonga District of northern Malawi. IFN-γ was measured by enzyme-linked immunosorbent assay (ELISA) in day 6 supernatants of diluted whole-blood cultures. The recombinant M. leprae 35-kDa and 18-kDa and purified native M. bovis BCG Ag85 antigens induced the highest percentages of responders, though both leprosy and bovine tuberculosis are now rare in this population. The M. tuberculosis antigens ESAT-6 and MPT64 and the M. bovis antigen MPB70 induced the lowest percentages of responders. One of the subjects subsequently developed extrapulmonary tuberculosis; this individual had a 15-mm-diameter reaction to the Mantoux test and responded to M. tuberculosis PPD, Ag85, MPT64, and ESAT-6 but not to any of the leprosy antigens. We conclude that in this rural African population, exposure to M. tuberculosis or M. bovis is much less frequent than exposure to environmental mycobacteria such as M. avium, which have antigens homologous to the M. leprae 35-kDa and 18-kDa antigens. M. tuberculosis ESAT-6 showed the strongest association with the size of the Mantoux skin test induration, suggesting that among the three M. tuberculosis antigens tested it provided the best indication of exposure to, or infection with, M. tuberculosis.     

Protection against Mycobacterium leprae Infection by the ID83/GLA-SE and ID93/GLA-SE Vaccines Developed for Tuberculosis

Despite the dramatic reduction in the number of leprosy cases worldwide in the 1990s, transmission of the causative agent, Mycobacterium leprae, is still occurring, and new cases continue to appear. New strategies are required in the pursuit of leprosy elimination. The cross-application of vaccines in development for tuberculosis may lead to tools applicable to elimination of leprosy. In this report, we demonstrate that the chimeric fusion proteins ID83 and ID93, developed as antigens for tuberculosis (TB) vaccine candidates, elicited gamma interferon (IFN-γ) responses from both TB and paucibacillary (PB) leprosy patients and from healthy household contacts of multibacillary (MB) patients (HHC) but not from nonexposed healthy controls. Immunization of mice with either protein formulated with a Toll-like receptor 4 ligand (TLR4L)-containing adjuvant (glucopyranosyl lipid adjuvant in a stable emulsion [GLA-SE]) stimulated antigen-specific IFN-γ secretion from pluripotent Th1 cells. When immunized mice were experimentally infected with M. leprae, both cellular infiltration into the local lymph node and bacterial growth at the site were reduced relative to those of unimmunized mice. Thus, the use of the Mycobacterium tuberculosis candidate vaccines ID83/GLA-SE and ID93/GLA-SE may confer cross-protection against M. leprae infection. Our data suggest these vaccines could potentially be used as an additional control measure for leprosy.     

T-Cell Recognition of Mycobacterial GroES Peptides in Thai Leprosy Patients and Contacts

We report here the mapping of T-cell-stimulatory determinants of the GroES 10-kDa heat shock protein homologues from Mycobacterium leprae and Mycobacterium tuberculosis, which are known as major immunogens in mycobacterial infections. Peripheral blood mononuclear cells (PBMC) from treated tuberculoid leprosy or lepromatous leprosy patients and from healthy household or hospital staff contacts of the patients were cultured with 20 16-mer peptides covering the entire sequences of both M. leprae and M. tuberculosis GroES. The total number of recognized peptides was found to be the largest in family contacts, while responder frequencies to the individual tested peptides varied (5 to 80%) with specificity between the patient and contact groups. Proliferative responses to some peptides showed positive or negative associations of low statistical significance with DR and DQ alleles, though responses to most GroES peptides were genetically permissive. Notably, the sequence of the 25–40 peptide of M. leprae, but not that of M. tuberculosis, was more frequently stimulatory in tuberculoid leprosy patients than in either group of sensitized healthy contacts. This peptide bound to a number of HLA-DR molecules, of which HLA-DRB5*0101 had the strongest affinity. The epitope core binding to this allele was localized to the 29-to-37 sequence, and its key residue was localized to the M. leprae-specific glutamic acid at position 32. This epitope may be of interest for the development of a blood test- or skin test-based diagnostic reagent for tuberculoid leprosy, subject to further clinical evaluation in untreated 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.     

An immunodominant 30-kDa antigen of a candidate anti-leprosy vaccine,Mycobacterium w,shares T and B cell determinants withM. leprae andM. tuberculosis

Earlier we reported that vaccination of leprosy patients withMycobacterium w induces an immune response directed predominantly against low molecular weight antigens. One of these antigens, with a molecular mass of 30-kDa, was recognized by a majority of the vaccinated subjects as well as the tuberculoid leprosy patients and healthy contacts. In the present communication we report further characterization of this antigen. Immunofluorescence and Western blot studies with antibodies raised against this antigen demonstrate that it is associated with the cell surface and has homologues present inM. leprae andM. tuberculosis. Delayed-type hypersensitivity studies carried out in guinea pigs immunized with the 30-kDa antigen show that in addition to sharing B cell determinants, this immunodominant antigen ofM. w also shares T cell determinants withM. leprae andM. tuberculosis.     

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.     

Cellular Immune Responses to ESAT-6 Discriminate between Patients with Pulmonary Disease Due to Mycobacterium avium Complex and Those with Pulmonary Disease Due to Mycobacterium tuberculosis

ESAT-6 (for 6-kDa early secreted antigenic target) is a secreted antigen found almost exclusively in organisms of the Mycobacterium tuberculosis complex. We compared in vitro gamma interferon (IFN-γ) responses by peripheral blood mononuclear cells to this antigen in patients with pulmonary disease due to either Mycobacterium avium complex (MAC) or Mycobacterium tuberculosis with those in healthy, skin test-negative, control subjects. Significant IFN-γ responses to ESAT-6 were detected in 16 (59%) of 27 M. tuberculosis pulmonary disease patients, 0 (0%) of 8 MAC disease patients, and 0 (0%) of 8 controls. Significant IFN-γ responses to M. tuberculosis purified protein derivative were detected in 23 (85%) of 27 M. tuberculosis disease patients, 2 (25%) of 8 MAC disease patients, and 5 (63%) of 8 healthy controls. M. avium sensitin was recognized in 24 (89%) of 27 M. tuberculosis disease patients, 4 (50%) of 8 MAC disease patients, and 1 (13%) of 8 controls. IFN-γ responses to ESAT-6 are specific for disease due to M. tuberculosis and are not observed in patients with MAC disease or in healthy controls.     

Inhibition of the Multiplication of Mycobacterium leprae by Vaccination with a Recombinant M. bovis BCG Strain That Secretes Major Membrane Protein II in Mice

The ability of a recombinant Mycobacterium bovis BCG strain that secretes major membrane protein II (MMP-II) of Mycobacterium leprae (BCG-SM) to confer protection against leprosy was evaluated by use of a mouse footpad model. C57BL/6J mice intradermally inoculated with BCG-SM produced splenic T cells which secreted significant amounts of gamma interferon (IFN-γ) in response to either the recombinant MMP-II, the M. leprae-derived membrane fraction, or the BCG-derived cytosolic fraction in vitro more efficiently than those from the mice infected with the vector control BCG strain (BCG-pMV, a BCG strain containing pMV-261). A higher percentage of CD8⁺ T cells obtained from BCG-SM-inoculated mice than those obtained from BCG-pMV-inoculated mice produced intracellular IFN-γ on restimulation with the M. leprae antigens. BCG-SM inhibited the multiplication of M. leprae in the footpads of C57BL/6J mice more efficiently than BCG-pMV. These results indicate that a BCG strain that secretes MMP-II could be a better vaccine candidate for leprosy.Leprosy, which is caused by Mycobacterium leprae, is an infectious disease that still affects thousands of people worldwide. According to WHO''s weekly epidemiological report, 254,525 new cases were detected in 2007 (25). One reason why leprosy is still prevalent may be due to the inherent characteristics of M. leprae, i.e., slow growth and weak pathogenicity. It takes 12 to 14 days for M. leprae to replicate, so it is predicted that 2 to 5 years are necessary for the clinical manifestations to appear after an infection (1, 18). Likewise, it takes 6 to 8 months for the recognizable swelling of the footpad to appear in nude mice (22).Leprosy is clinically divided into two major categories: multibacillary (MB) leprosy and paucibacillary (PB) leprosy. In the lesions of patients with PB leprosy, dendritic cells (DCs) and activated T cells are involved with confining M. leprae to a localized area. These pathological observations indicate that cell-mediated reactions are triggered and that the activation of both CD4⁺ and CD8⁺ T cells is closely associated with inhibition of the spread of the bacilli. In contrast, abundant foamy macrophages loaded with bacilli but not DCs appear in the lesions of MB patients (11). It can be speculated that antigen (Ag)-presenting cells such as DCs recognize the immunodominant Ags of M. leprae and express those derivatives on their surfaces, thereby activating T cells. Previously, using T cells from patients with PB leprosy, we have identified major membrane protein II (MMP-II), also known as bacterioferritin (ML2038), as one of the immunodominant Ags (8). We found that MMP-II activates DCs through Toll-like receptor 2, leading to higher levels of expression of major histocompatibility complex class I and class II, CD86, and CD83 Ags and increased levels of production of interleukin-12 p70. Furthermore, MMP-II-pulsed DCs derived from patients with PB leprosy activated both autologous CD4⁺ T cells and CD8⁺ T cells to produce gamma interferon (IFN-γ) in amounts larger than the amounts produced by T cells from patients with MB leprosy and M. bovis BCG-vaccinated healthy individuals, indicating that T cells from patients with PB leprosy may be primed with MMP-II in vivo.The BCG vaccine has been used for the prevention of tuberculosis, although its role in the prevention of leprosy is still being debated. The protective efficacy of BCG against leprosy has been tested in several trials, including studies in the Karonga District of northern Malawi, in which 50% protection was observed (17). Through combined systematic analyses of experimental studies, Setia et al. found that the BCG vaccine had an overall level of protective efficacy of 26% against human leprosy (19). Their observational studies overestimated the protective effect at 61%. In another review of 29 studies, Zodpey reported that 44.8% of the reports indicated that the BCG vaccine had a level of efficacy of 50% or more (26). These observations indicate that improvements to the BCG vaccine are necessary to increase its protective effect. Recently, we produced a recombinant BCG strain that secretes MMP-II (strain BCG-SM, where SM indicates secreting MMP-II). Since MMP-II has the ability to ligate Toll-like receptor 2, we expected BCG-SM to highly activate human T cells. In fact, BCG-SM activated not only naïve CD4⁺ T cells but also naïve CD8⁺ T cells through DCs (9). The fact that BCG-SM was more efficient than the parental BCG strain at the activation of both subsets of naïve T cells led us to seek further insights into the protective activity of BCG-SM. In the present study, we investigated the effect of vaccination of BCG-SM on the multiplication of M. leprae in mice.     

Gene Expression Profile and Immunological Evaluation of Unique Hypothetical Unknown Proteins of Mycobacterium leprae by Using Quantitative Real-Time PCR

The cell-mediated immunity (CMI)-based in vitro gamma interferon release assay (IGRA) of Mycobacterium leprae-specific antigens has potential as a promising diagnostic means to detect those individuals in the early stages of M. leprae infection. Diagnosis of leprosy is a major obstacle toward ultimate disease control and has been compromised in the past by the lack of specific markers. Comparative bioinformatic analysis among mycobacterial genomes identified potential M. leprae-specific proteins called “hypothetical unknowns.” Due to massive gene decay and the prevalence of pseudogenes, it is unclear whether any of these proteins are expressed or are immunologically relevant. In this study, we performed cDNA-based quantitative real-time PCR to investigate the expression status of 131 putative open reading frames (ORFs) encoding hypothetical unknowns. Twenty-six of the M. leprae-specific antigen candidates showed significant levels of gene expression compared to that of ESAT-6 (ML0049), which is an important T cell antigen of low abundance in M. leprae. Fifteen of 26 selected antigen candidates were expressed and purified in Escherichia coli. The seroreactivity to these proteins of pooled sera from lepromatous leprosy patients and cavitary tuberculosis patients revealed that 9 of 15 recombinant hypothetical unknowns elicited M. leprae-specific immune responses. These nine proteins may be good diagnostic reagents to improve both the sensitivity and specificity of detection of individuals with asymptomatic leprosy.     

Use of the polymerase chain reaction to detect Mycobacterium leprae in urine

Leprosy is an infectious disease caused by Mycobacterium leprae. The polymerase chain reaction (PCR) has been applied to detect M. leprae in different clinical samples and urine seems to be attractive for this purpose. PCR was used to improve the sensitivity for diagnosing leprosy by amplifying a 151-bp PCR fragment of the M. leprae pra gene (PCR-Pra) in urine samples. Seventy-three leprosy patients (39 males and 34 females, 14 to 78 years old) were selected for leprosy diagnosis at a reference laboratory in Maringá, PR, Brazil. Of these, 36 were under anti-leprosy multidrug therapy with dapsone and rifampicin for tuberculoid (TT) and dapsone, rifampicin and clofazimine for borderline (BB) and lepromatous (LL) forms. The control group contained 50 healthy individuals without any clinical history of leprosy. DNA isolated from leprosy patients'' urine samples was successfully amplified by PCR-Pra in 46.6% (34/73) of the cases. The positivity of PCR-Pra for patients with the TT form was 75% for both patients under treatment and non-treated patients (P = 0.1306). In patients with the LL form, PCR-Pra positivity was 52 and 30% for patients under treatment and non-treated patients, respectively (P = 0.2386). PCR-Pra showed a statistically significant difference in detecting M. leprae between the TT and LL forms of leprosy in patients under treatment (P = 0.0033). Although the current study showed that the proposed PCR-Pra has some limitations in the detection of M. leprae, this method has the potential to be a useful tool for leprosy diagnosis mainly in TT leprosy where the AFB slit-skin smear is always negative.     

Molecular and Immunological Analyses of the Mycobacterium avium Homolog of the Immunodominant Mycobacterium leprae 35-Kilodalton Protein

The analysis of host immunity to mycobacteria and the development of discriminatory diagnostic reagents relies on the characterization of conserved and species-specific mycobacterial antigens. In this report, we have characterized the Mycobacterium avium homolog of the highly immunogenic M. leprae 35-kDa protein. The genes encoding these two proteins were well conserved, having 82% DNA identity and 90% identity at the amino acid level. Moreover both proteins, purified from the fast-growing host M. smegmatis, formed multimeric complexes of around 1000 kDa in size and were antigenically related as assessed through their recognition by antibodies and T cells from M. leprae-infected individuals. The 35-kDa protein exhibited significant sequence identity with proteins from Streptomyces griseus and the cyanobacterium Synechoccocus sp. strain PCC 7942 that are up-regulated under conditions of nutrient deprivation. The 67% amino acid identity between the M. avium 35-kDa protein and SrpI of Synechoccocus was spread across the sequences of both proteins, while the homologous regions of the 35-kDa protein and the P3 sporulation protein of S. griseus were interrupted in the P3 protein by a divergent central region. Assessment by PCR demonstrated that the gene encoding the M. avium 35-kDa protein was present in all 30 M. avium clinical isolates tested but absent from M. intracellulare, M. tuberculosis, or M. bovis BCG. Mice infected with M. avium, but not M. bovis BCG, developed specific immunoglobulin G antibodies to the 35-kDa protein, consistent with the observation that tuberculosis patients do not recognize the antigen. Strong delayed-type hypersensitivity was elicited by the protein in guinea pigs sensitized with M. avium.     

Effects of Thalidomide on Intracellular Mycobacterium leprae in Normal and Activated Macrophages

Thalidomide is an effective drug for the treatment of erythema nodosum leprosum (ENL). ENL is an inflammatory reaction that may occur in multibacillary leprosy patients. Its cause(s) as well as the mechanism of thalidomide in arresting this condition are not fully understood. It has been suggested that ENL is an immune complex-mediated hypersensitivity precipitated by the release of Mycobacterium leprae from macrophages. The released antigen may complex with precipitating antibodies, initiating complement fixation and the production of inflammatory cytokines like tumor necrosis factor alpha (TNF-α). Thalidomide has been shown in vitro to reduce antigen- or mitogen-activated macrophage production of TNF-α. We investigated if thalidomide could also influence the viability of intracellular M. leprae. Mouse peritoneal macrophages were infected with M. leprae, activated with gamma interferon and endotoxin, or nonactivated, and treated with thalidomide. Intracellular bacilli were recovered, and metabolic activity was assessed by a radiorespirometric procedure. Thalidomide did not possess antimicrobial action against M. leprae in normal and activated host macrophages. This suggests that thalidomide does not retard the release of mycobacterial antigens, a possible prelude or precipitating factor for ENL. A distinct sequence of events explaining the mechanism of action for thalidomide's successful treatment of ENL has yet to be established.     

Specific Serological Diagnosis of Leprosy with a Recombinant Mycobacterium leprae Protein Purified from a Rapidly Growing Mycobacterial Host

In this report we demonstrate the utility of an monoclonal antibody inhibition enzyme-linked immunosorbent assay based on the Mycobacterium leprae 35-kDa protein, purified from the rapidly growing host Mycobacterium smegmatis, for the serodiagnosis of multibacillary leprosy. The assay proved highly specific (97.5%) and sensitive (90%) and compared favorably with two other established methods routinely utilized for leprosy serodiagnosis.     

rBCG30-Induced Immunity and Cross-Protection against Mycobacterium leprae Challenge Are Enhanced by Boosting with the Mycobacterium tuberculosis 30-Kilodalton Antigen 85B

Leprosy remains a major global health problem and typically occurs in regions in which tuberculosis is endemic. Vaccines are needed that protect against both infections and do so better than the suboptimal Mycobacterium bovis BCG vaccine. Here, we evaluated rBCG30, a vaccine previously demonstrated to induce protection superior to that of BCG against Mycobacterium tuberculosis and Mycobacterium bovis challenge in animal models, for efficacy against Mycobacterium leprae challenge in a murine model of leprosy. rBCG30 overexpresses the M. tuberculosis 30-kDa major secretory protein antigen 85B, which is 85% homologous with the M. leprae homolog (r30ML). Mice were sham immunized or immunized intradermally with BCG or rBCG30 and challenged 2.5 months later by injection of viable M. leprae into each hind footpad. After 7 months, vaccine efficacy was assessed by enumerating the M. leprae bacteria per footpad. Both BCG and rBCG30 induced significant protection against M. leprae challenge. In the one experiment in which a comparison between BCG and rBCG30 was feasible, rBCG30 induced significantly greater protection than did BCG. Immunization of mice with purified M. tuberculosis or M. leprae antigen 85B also induced protection against M. leprae challenge but less so than BCG or rBCG30. Notably, boosting rBCG30 with M. tuberculosis antigen 85B significantly enhanced r30ML-specific immune responses, substantially more so than boosting BCG, and significantly augmented protection against M. leprae challenge. Thus, rBCG30, a vaccine that induces improved protection against M. tuberculosis, induces cross-protection against M. leprae that is comparable or potentially superior to that induced by BCG, and boosting rBCG30 with antigen 85B further enhances immune responses and protective efficacy.     

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.