Comparative analysis of B- and T-cell epitopes of Mycobacterium leprae and Mycobacterium tuberculosis culture filtrate protein 10

Culture filtrate protein 10 (CFP-10) from Mycobacterium tuberculosis is a well-characterized immunodominant 10-kDa protein antigen known to elicit a very potent early gamma interferon response in T cells from M. tuberculosis-infected mice and humans. The sequence of the Mycobacterium leprae homologue of CFP-10 shows only 40% identity (60% homology) at the protein level with M. tuberculosis CFP-10 and thus has the potential for development as a T- or B-cell reactive antigen for specific diagnosis of leprosy. Antisera raised in mice or rabbits against recombinant M. leprae and M. tuberculosis CFP-10 proteins reacted only with homologous peptides from arrays of overlapping synthetic peptides, indicating that there was no detectable cross-reactivity at the antibody level. Sera from leprosy and tuberculosis patients were also specific for the homologous protein or peptides and showed distinct patterns of recognition for either M. leprae or M. tuberculosis CFP-10 peptides. At the cellular level, only 2 of 45 mouse T-cell hybridomas raised against either M. leprae or M. tuberculosis CFP-10 displayed a cross-reactive response against the N-terminal heterologous CFP-10 peptide, the region that exhibits the highest level of identity in the two proteins; however, the majority of peptide epitopes recognized by mouse T-cell hybridomas specific for each protein did not cross-react with heterologous peptides. Coupled with the human serology data, these results raise the possibility that peptides that could be used to differentiate infections caused by these two related microorganisms could be developed. Immunohistochemical staining of sections of M. leprae-infected nude mouse footpads resulted in strongly positive staining in macrophages and dendritic cells, as well as weaker staining in extracellular areas, suggesting that M. leprae CFP-10, like its homologue in M. tuberculosis, is a secreted protein.     

Immune profiling of leprosy and tuberculosis patients to 15-mer peptides of Mycobacterium leprae and M. tuberculosis GroES in a BCG vaccinated area: implications for development of vaccine and diagnostic reagents

Mycobacterium leprae (ML) GroES has been shown to induce strong T cell responses in tuberculoid as well as in exposed healthy contacts of leprosy patients, and therefore this antigen has been the focus of study as a potential vaccine candidate. Paradoxically, we have shown that ML GroES also induces extremely high titres of IgG1 antibody in leprosy patients across the disease spectrum, a response associated with disease progression. IgG1 antibodies in leprosy also show a negative association with interferon-gamma, a critical T cell cytokine responsible for macrophage activation and intracellular killing of mycobacteria. We therefore queried if antibody and T cell responses were being evoked by different epitopes in ML GroES proteins. To address the issue of epitope recognition in mycobacterial diseases, we have analysed 16 peptides (15-mer peptides) spanning the entire ML and M. tuberculosis GroES protein in leprosy (n = 19) and tuberculosis (n = 9) patients and healthy endemic controls (n = 8). Our analysis demonstrates clearly that the dominant peptides evokingT cell and IgG subclass antibodies were different. The target of both T and B cell responses were cross-reactive epitopes in all groups. Differences in disease and healthy states related to the strength (mean intensity) of the T cell and antibody response. IgG1 and IgG3 antibodies were associated with disseminated disease and IgG 2 and IgG4 with disease limitation. Such comprehensive immune profiling of antigen-specific responses is critical to understanding the disease pathogenesis and also if these reagents are to be exploited for either diagnostic or vaccine purposes.     

Identification and characterization of the ESAT-6 homologue of Mycobacterium leprae and T-cell cross-reactivity with Mycobacterium tuberculosis

In this paper we describe identification and characterization of Mycobacterium leprae ESAT-6 (L-ESAT-6), the homologue of M. tuberculosis ESAT-6 (T-ESAT-6). T-ESAT-6 is expressed by all pathogenic strains belonging to the M. tuberculosis complex but is absent from virtually all other mycobacterial species, and it is a promising antigen for immunodiagnosis of tuberculosis (TB). Therefore, we analyzed whether L-ESAT-6 is a similarly powerful tool for the study of leprosy by examining T-cell responses against L-ESAT-6 in leprosy patients, TB patients, and exposed or nonexposed healthy controls from areas where leprosy and TB are endemic and areas where they are not endemic. L-ESAT-6 was recognized by T cells from leprosy patients, TB patients, individuals who had contact with TB patients, and healthy individuals from an area where TB and leprosy are endemic but not by T cells from individuals who were not exposed to M. tuberculosis and M. leprae. Moreover, leprosy patients who were not responsive to M. leprae failed to respond to L-ESAT-6. A very similar pattern was obtained with T-ESAT-6. These results show that L-ESAT-6 is a potent M. leprae antigen that stimulates T-cell-dependent gamma interferon production in a large proportion of individuals exposed to M. leprae. Moreover, our results suggest that there is significant cross-reactivity between T-ESAT-6 and L-ESAT-6, which has implications for the use of ESAT-6 as tool for diagnosis of leprosy and TB in areas where both diseases are endemic.     

Mycobacterium tuberculosis genome-wide screen exposes multiple CD8 T cell epitopes

Mounting evidence suggests human leucocyte antigen (HLA) class I-restricted CD8(+) T cells play a role in protective immunity against tuberculosis yet relatively few epitopes specific for the causative organism, Mycobacterium tuberculosis, are reported. Here a total genome-wide screen of M. tuberculosis was used to identify putative HLA-B*3501 T cell epitopes. Of 479 predicted epitopes, 13 with the highest score were synthesized and used to restimulate lymphocytes from naturally exposed HLA-B*3501 healthy individuals in cultured and ex vivo enzyme-linked immunospot (ELISPOT) assays for interferon (IFN)-gamma. All 13 peptides elicited a response that varied considerably between individuals. For three peptides CD8(+) T cell lines were expanded and four of the 13 were recognized permissively through the HLA-B7 supertype family. Although further testing is required we show the genome-wide screen to be feasible for the identification of unknown mycobacterial antigens involved in immunity against natural infection. While the mechanisms of protective immunity against M. tuberculosis infection remain unclear, conventional class I-restricted CD8(+) T cell responses appear to be widespread throughout the genome.     

HLA and leprosy in the pre and postgenomic eras

Leprosy has intrigued immunologists for many decades. Despite minimal genetic variation between Mycobacterium leprae isolates worldwide, two completely different forms of the disease can develop in the susceptible human host: localized, tuberculoid, or paucibacillary leprosy, which can heal spontaneously, and disseminating, lepromatous, or multibacillary leprosy, which is progressive if untreated. The questions which host factors regulate these very different outcomes of infection, by what mechanisms, and whether these can be used to combat disease remain unanswered. Leprosy has been one of the very first human diseases in which human leukocyte antigen (HLA) genes were demonstrated to codetermine disease outcome. Jon van Rood was among the earliest researchers to recognize the potential of this ancient disease as a human model to dissect the role of HLA in disease. Decades later, it is now clear that HLA molecules display highly allele-specific peptide binding capacity. This restricts antigen presentation to M. leprae-reactive T cells and controls the magnitude of the ensuing immune response. Furthermore, specific peptide/HLA class II complexes can also determine the quality of the immune response by selectively activating regulatory (suppressor) T cells. All these factors are believed to contribute to leprosy disease susceptibility. Despite the global reduction in leprosy disease prevalence, new case detection rates remain invariably high, demonstrating that treatment alone does not block transmission of leprosy. Better tools for early detection of preclinical M. leprae infection, likely the major source of unidentified transmission, therefore is a priority. Newly developed HLA-based bioinformatic tools now provide novel opportunities to help combat this disease. Here, we describe recent work using HLA-DR peptide binding algorithms in combination with recently elucidated genome sequences of several different mycobacteria. Using this postgenomic HLA-based approach, we were able to identify 12 candidate genes that were unique to M. leprae and were predicted to contain T cell epitopes restricted via several major HLA-DR alleles. Five of these antigens (ML0576, ML1989, ML1990, ML2283, ML2567) were indeed able to induce significant T cell responses in paucibacillary leprosy patients and M. leprae-exposed healthy controls but not in most multibacillary leprosy patients, tuberculosis patients, or endemic controls. 71% of M. leprae-exposed healthy controls that did not have antibodies to the M. leprae-specific phenolic glycolipid-I responded to one or more M. leprae antigen(s), highlighting the potential added value of these unique M. leprae proteins in diagnosing early infection. Thus current state-of-the-art HLA immunogenetics can provide new tools for specific diagnosis of M. leprae infection, which can impact our understanding of leprosy transmission and can lead to improved intervention.     

A postgenomic approach to identification of Mycobacterium leprae-specific peptides as T-cell reagents

To identify Mycobacterium leprae-specific human T-cell epitopes, which could be used to distinguish exposure to M. leprae from exposure to Mycobacterium tuberculosis or to environmental mycobacteria or from immune responses following Mycobacterium bovis BCG vaccination, 15-mer synthetic peptides were synthesized based on data from the M. leprae genome, each peptide containing three or more predicted HLA-DR binding motifs. Eighty-one peptides from 33 genes were tested for their ability to induce T-cell responses, using peripheral blood mononuclear cells (PBMC) from tuberculoid leprosy patients (n = 59) and healthy leprosy contacts (n = 53) from Brazil, Ethiopia, Nepal, and Pakistan and 20 United Kingdom blood bank donors. Gamma interferon (IFN-gamma) secretion proved more sensitive for detection of PBMC responses to peptides than did lymphocyte proliferation. Many of the peptides giving the strongest responses in leprosy donors compared to subjects from the United Kingdom, where leprosy is not endemic, have identical, or almost identical, sequences in M. leprae and M. tuberculosis and would not be suitable as diagnostic tools. Most of the peptides recognized by United Kingdom donors showed promiscuous recognition by subjects expressing differing HLA-DR types. The majority of the novel T-cell epitopes identified came from proteins not previously recognized as immune targets, many of which are cytosolic enzymes. Fifteen of the tested peptides had > or =5 of 15 amino acid mismatches between the equivalent M. leprae and M. tuberculosis sequences; of these, eight gave specificities of > or =90% (percentage of United Kingdom donors who were nonresponders for IFN-gamma secretion), with sensitivities (percentage of responders) ranging from 19 to 47% for tuberculoid leprosy patients and 21 to 64% for healthy leprosy contacts. A pool of such peptides, formulated as a skin test reagent, could be used to monitor exposure to leprosy or as an aid to early diagnosis.     

Cross-reactive epitopes and HLA-restriction elements in human T cell recognition of the Mycobacterium leprae 18-kD heat shock protein

We have previously demonstrated that the Mycobacterium leprae 18-kD heat shock protein (HSP18) is represented among the antigenic targets of human T cell responses induced by M. leprae immunization and that the peptide 38-50 serves as an immunodominant epitope recognized by CD4+ T cell clones. By using peripheral blood mononuclear cells and T cell lines from the same donor group, we have in this study shown that the M. leprae HSP18 and peptide 38-50 were recognized by memory T cells 8 years after immunization with M. leprae. The finding that M. bovis BCG-induced T cell lines responded to M. leprae HSP18, but not to the peptide 38-50, suggested the existence of additional T cell epitopes of a cross-reactive nature. Consistent with this, testing of the T cell lines for proliferative responses to the complete HSP18 molecule, truncated HSP18 (amino acid (aa) residues 38-148) and overlapping synthetic peptides, made it possible to identify two cross-reactive epitope regions defined by aa residues 1-38 and 41-55. While peptide 38-50-reactive T cell clones showed limited cross-reactivity by responding to M. leprae, M. avium and M. scrofulaceum, the T cell lines specific to the epitopes 1-38 and 41-55 were broadly cross-reactive, as demonstrated by their response to M. leprae, M. tuberculosis complex, M. avium and other mycobacteria. MHC restriction analysis of the HSP18-responding T cell lines showed that the epitopes 1-38 and 38-50 were presented by one of the two HLA-DR molecules expressed from self HLA-DRB1 genes, whereas the epitope 41-55 was recognized in the presence of autologous as well as HLA-DR and HLA-DQ mismatched allogeneic antigen-presenting cells. The results obtained in this study made it possible to identify cross-reactive T cell epitopes of the M. leprae HSP18, and provide an explanation for T cell recognition of this antigen in individuals infected with species of the M. tuberculosis complex or environmental mycobacteria.     

Lsr2 peptides of Mycobacterium leprae show hierarchical responses in lymphoproliferative assays, with selective recognition by patients with anergic lepromatous leprosy

Lsr2 protein of Mycobacterium leprae was shown earlier to elicit B and T cell responses in leprosy patients (20, 28). Lymphoproliferation to M. leprae and Lsr2 antigens was observed in >70% of tuberculoid (T) patients and in 16 and 34% of lepromatous (L) patients, respectively. We focused on the M. leprae nonresponders in the lepromatous group using 22 synthetic Lsr2 peptides (end-to-end peptides A to F and overlapping peptides p1 to p16) in in vitro T cell responses. A total of 125 leprosy and 13 tuberculosis patients and 19 healthy controls from the area of endemicity (here, healthy controls, or HC) were investigated. The highest responses were observed (67 to 100%) in HC for all peptides except p1 to p3, and the lowest was observed in tuberculosis patients. Significant differences in lymphoproliferation were observed in T, L, and HC groups (analysis of variance [ANOVA], P = 0.000 to 0.015) for all end-to-end peptides except B and for p5 and p7 to p10. Hierarchical recognition between lepromatous and tuberculoid leprosy was noted for p8 (P < 0.05) and between the HC and L groups for p7 to p10, p15, and p16 (P < 0.005 to P < 0.02). Significant lymphoproliferation was observed to peptides A to F and p1 to p9, p11, p12, p15, p16 (P = 0.000 to 0.001) with 40% responding to peptides C and p16 in L patients. Lepromatous patients also showed significantly higher levels of a gamma interferon (IFN-γ) response to peptide C than to other peptides (P < 0.05). Major histocompatibility complex (MHC) class II bias for peptide recognition was not observed. These studies indicate that Lsr2 has multiple T cell epitopes that induce in vitro T cell responses in the highly infective lepromatous leprosy patients.     

Lysis of interferon-gamma activated Schwann cell by cross-reactive CD8+ alpha/beta T cells with specificity for the mycobacterial 65 kd heat shock protein.

Heat shock protein (hsp) 65 is a major T cell antigen of Mycobacterium leprae. The hsp 65 of M. leprae is nearly identical in M. bovis/M. tuberculosis (greater than 95% protein sequence homology) and surprisingly similar in man (65% protein sequence homology). Recently, we had provided evidence in a murine model that CD8+ T cells recognize and lyse Schwann cells presenting M. leprae antigen in the context of major histocompatibility (MHC) class I gene products. Because murine Schwann cells are class I negative, antigen presentation requires prior stimulation with interferon-gamma (IFN-gamma). CD8+ T cells were activated against tryptic fragments of mycobacterial hsp 65. These T cells recognized epitopes of hsp 65 which had been generated through the cytoplasmic class I processing pathway. They were also capable of lysing Schwann cells which had been activated by IFN-gamma and not primed with nominal hsp 65 peptides. In contrast, T cells activated against tryptic ova peptides only lysed Schwann cells which had been both stimulated with IFN-gamma and primed with ova peptides. Evidence is presented that class I (H-2D) restricted, CD8+ alpha/beta T lymphocytes with specificity for the mycobacterial hsp 65 recognize IFN-gamma-stimulated Schwann cells probably because they are specific for a(n) epitope(s) shared by the bacterial hsp and a host cognate. Activation of autoreactive T cells with specificity to shared epitopes could contribute to nerve damage in tuberculoid leprosy which is characterized by low to absent M. leprae in Schwann cells.     

Immunological crossreactivity of the Mycobacterium leprae CFP-10 with its homologue in Mycobacterium tuberculosis

Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) (Rv3874) is considered a promising antigen for the immunodiagnosis of tuberculosis (TB) together with early secreted antigens of M. tuberculosis (ESAT-6). Both ESAT-6 and CFP-10 are encoded by the RD1 region that is deleted from all tested M. bovis bacille Calmette-Guérin (BCG) strains but present in M. leprae, M. tuberculosis, M. bovis, M. kansasii, M. africanum and M. marinum. In this study, the homologue of CFP-10 in M. leprae (ML0050) is identified and characterized. Interferon-gamma production in response to this homologue by T cells from leprosy patients, TB patients and unexposed controls shows that CFP-10 of M. leprae is a potent antigen that crossreacts with CFP-10 of M. tuberculosis at the T-cell level. This crossreactivity has implications for the use of CFP-10 of these mycobacterial species as diagnostic tool in areas endemic for both the diseases.     

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.     

Identification of human T cell epitopes in the Mycobacterium leprae heat shock protein 70-kD antigen.

In a number of pathogens, heat shock proteins (hsp) stimulate humoral and cellular immune responses despite significant sequence identity with host hsp. The 70-kD hsp of Mycobacterium leprae, which shares 47% identity with human hsp70 at the protein level, elicited a T cell response in most Myco. bovis (bacille Calmette-Guérin (BCG)) vaccinees as well as leprosy and tuberculosis patients and their contacts. In order to locate T cell epitopes, DNA fragments encoding portions of the 70-kD hsp were expressed in the vector pGEX-2T and tested for T cell reactivity in an in vitro proliferative assay. Cultures of peripheral blood mononuclear cells (PBMC) from BCG vaccinees indicated that the C-terminal half of the molecule contained multiple T cell epitopes, as the T cells from a majority of Myco. leprae hsp70-reactive individuals responded to C-344. Lower proportions of patients with paucibacillary leprosy (36%) and tuberculosis patients (16%) responded to C-344. The smaller C-142 fragment which includes the terminal 70 residues unique to Myco. leprae and is the target for the human antibody response elicited a cellular response in few patients and no vaccinees. In order to map T cell epitopes, two series of synthetic peptides encompassing the region 278-502 were prepared. Using overlapping 12mer and 20mer peptides, this region of the molecule was found to contain several potential T cell epitopes. The longer peptides gave a clearer indication of reactive sequences including regions of the molecule which were not identified with the 12mer peptides. Fine mapping of reactive peptide pools using the 12mer peptides identified two T cell epitopes. Although both were located in regions of the molecule shared with Myco. tuberculosis, one appeared to be cross-reactive with the equivalent human sequence, and thus has the potential to initiate autoimmune responses.     

Enumeration of T cells reactive with Mycobacterium tuberculosis organisms and specific for the recombinant mycobacterial 64-kDa protein

The major goal of the present study was to develop a limiting dilution system for the enumeration of T cells which respond to mycobacterial antigens. Purified T cells from M. tuberculosis-immune mice were restimulated with mycobacterial antigens and accessory cells, and after 4 days expanded with antigen, accessory cells and T cell growth factor. After another 3 days, proliferative responses were determined. Similar cultures performed without antigen served as controls. Limiting dilution analysis revealed that approximately 1/2000 to 1/3000 T cells from M. tuberculosis-immune mice responded to whole M. tuberculosis organisms while T cells from normal mice did not respond. Similar T cell numbers reacted with several mycobacterial strains indicating expression of shared T cell antigens. Using a semi-purified recombinant 64-kDa protein from M. bovis the frequency of T cells generated after immunization with M. tuberculosis which reacted with a single mycobacterial protein could be estimated. We found that approximately 1/5 of the M. tuberculosis-reactive T cells recognized this particular antigen. Immunization with the recombinant 64-kDa protein in an adjuvant containing trehalose dimycolate, monophosphoryl lipid A and mycobacterial cell wall skeleton stimulated an equally high number of M. tuberculosis-reactive T cells (1/2000). These findings demonstrate that a high proportion of tuberculosis-responsive T cells are directed against the 64-kDa protein and that immunization with this antigen in an appropriate adjuvant system is capable of stimulating high numbers of M. tuberculosis-reactive T cells. Limiting dilution analysis with a panel of mycobacterial proteins or peptides may allow their ranking from immunodominant to immunosilent and facilitate identification of antigens or epitopes relevant to protection.     

Individuals from Different Populations Identify Multiple and Diverse T-Cell Determinants on Mycobacterial HSP70

The 70 kDa heat-shock protein (HSP) of Mycobacterium leprae stimulates both cellular and antibody responses in leprosy patients and subclinically infected individuals despite partial homology with host HSP70. Furthermore, mycobacterial HSP70 can act as a carrier protein in unprimed mice, suggesting the presence of widely shared T-cell determinants on this protein. In order to elucidate the frequency and genetic restriction of these T-cell epitopes, we have undertaken a systematic analysis of the proliferative responses to 20mer peptides encompassing the whole protein in different populations. Caucasian BCG vaccinees who responded to recombinant M. leprae HSP70 identilied multiple scattered T-cell determinants, four of which were recognized by 60% of subjects in association with a variety of HLA-DR haplotypes. When a group of Nepali leprosy and tuberculosis patients were tested, significant differences in the pattern of peptide recognition were observed. The dominant peptides recognized by Caucasian subjects were infrequently reactive and other peptides were stimulatory, again in association with a variety of HLA-DR phenotypes. The C-terminal 70 residues of the M. leprae HSP70 are specific to M. leprae and sera from lepromatous leprosy patients bind to this region. However, few T-cell determinants were identified in these residues, indicating that this region is unhelpful as a diagnostic tool for detecting M. leprae-specific T-cell responses. When compared with the equivalent regions of the human HSP70, the commonly recognized peptides showed significant differences in amino-acid sequence. When taken in conjunction with the failure of human HSP70 to stimulate M. leprae HSP70-reactive T-cell clones (E. Adams et al., unpublished observations), this finding indicates that the human T-cell response to this protein is largely directed at mycobacterial-specific determinants. The presence of multiple T-cell epilopes on M. leprae HSP70 with varied patterns of HLA-DR association suggests that the whole protein is required for stimulating effective T-cell responses in genetically diverse populations.     

The identification of a common pathogen-specific HLA class I A*0201-restricted cytotoxic T cell epitope encoded within the heat shock protein 65.

Bacterial antigens recognized by CD8(+) T cells in the context of MHC class I are thought to play a crucial role in protection against pathogenic intracellular bacteria. Here, we demonstrate the induction of HLA-A*0201-restricted CD8(+) T cell responses against six new high-affinity HLA-A*0201-binding CTL epitopes, encoded within an immunodominant and highly conserved antigen of Mycobacteria, the heat shock protein 65 (hsp65). One of these epitopes, Mhsp65(9(369)), is identical in a large number of pathogenic bacteria, and is recognized in a CD8-independent fashion. Mhsp65(9(369)) could be presented by either mycobacterial hsp65-pulsed target cells or BCG-infected macrophages. Interestingly, T cells specific for this epitope did not recognize the corresponding human hsp65 homologue, probably due to structural differences as revealed by modeling studies. Furthermore, in vitro proteasome digestion analyses show that, whereas the mycobacterial hsp65 epitope is efficiently generated, the human hsp65 homologue is not, thus avoiding the induction of autoreactivity. Collectively, these findings describe high-affinity HLA class I-binding epitopes that are naturally processed and are recognized efficiently by MHC class I-restricted CD8(+) T cells, providing a rational basis for the development of subunit vaccine strategies against tuberculosis and other intracellular infectious diseases.     

Specificity and function of immunogenic peptides from the 35-kilodalton protein of Mycobacterium leprae

We identified a T-cell determinant of the 35-kDa antigen of Mycobacterium leprae which is discriminatory against cross-sensitization by its closely related homologue in Mycobacterium avium. From synthetic peptides covering the entire sequence, those with the highest affinity and permissive binding to purified HLA-DR molecules were evaluated for the stimulation of proliferation of peripheral blood mononuclear cells (PBMCs) from leprosy patients and healthy sensitized controls. Responses to the peptide pair 206-224, differing by four residues between M. leprae and M. avium, involved both species-specific and cross-reactive T cells. Lymph node cell proliferation in HLA-DRB1*01 transgenic mice was reciprocally species specific, but only the response to the M. leprae peptide in the context of DR1 was immunodominant. Of the cytokines in human PBMC cultures, gamma interferon production was negligible, while interleukin 10 (IL-10) responses in both patients and controls were more pronounced. IL-10 was most frequently induced by the shared 241-255 peptide, indicating that environmental cross-sensitization may skew the response toward a potentially pathogenic cytokine phenotype.     

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

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

Recognition of mycobacterial epitopes by T cells across mammalian species and use of a program that predicts human HLA-DR binding peptides to predict bovine epitopes

Bioinformatics tools have the potential to accelerate research into the design of vaccines and diagnostic tests by exploiting genome sequences. The aim of this study was to assess whether in silico analysis could be combined with in vitro screening methods to rapidly identify peptides that are immunogenic during Mycobacterium bovis infection of cattle. In the first instance the M. bovis-derived protein ESAT-6 was used as a model antigen to describe peptides containing T-cell epitopes that were frequently recognized across mammalian species, including natural hosts for tuberculosis (humans and cattle) and small-animal models of tuberculosis (mice and guinea pigs). Having demonstrated that some peptides could be recognized by T cells from a number of M. bovis-infected hosts, we tested whether a virtual-matrix-based human prediction program (ProPred) could identify peptides that were recognized by T cells from M. bovis-infected cattle. In this study, 73% of the experimentally defined peptides from 10 M. bovis antigens that were recognized by bovine T cells contained motifs predicted by ProPred. Finally, in validating this observation, we showed that three of five peptides from the mycobacterial antigen Rv3019c that were predicted to contain HLA-DR-restricted epitopes were recognized by T cells from M. bovis-infected cattle. The results obtained in this study support the approach of using bioinformatics to increase the efficiency of epitope screening and selection.     

T cell repertoire in tuberculosis: selective anergy to an immunodominant epitope of the 38-kDa antigen in patients with active disease.

It is generally accepted that both host protection and pathogenic reactions in tuberculosis are mediated by T lymphocytes. However, little is known about the structures and discreet functions of epitopes stimulating the immune response. In this study, proliferative responses of blood T lymphocytes to synthetic peptides derived from the sequence of the 38-kDa antigen from Mycobacterium tuberculosis have been investigated in 41 healthy individuals and in 36 patients with active tuberculosis. Of the healthy purified protein derivative (PPD)-positive donors, 90% responded to a permissively recognized peptide, 38.G (residues 350-359), located at the carboxy terminus of the molecule. Four other permissively recognized epitopes of this molecule (38.A, 38.I, 38.E, 38.K) were stimulatory for more than 50% of healthy PPD-positive individuals. Patients with lymphatic tuberculosis responded to these peptides in a similar manner. In contrast, we observed a selective anergy to stimulation with peptide 38.G in the majority of patients with pulmonary (11% responders) and nonlymphatic extrapulmonary tuberculosis (25% responders). The lack of responsiveness to 38.G was epitope specific since the degree of responsiveness to the other four permissively recognized peptide epitopes was similar for patients and PPD-positive controls. Using the PEPSCAN technology and truncated peptides, the core epitope of 38.G was localized to a peptide 10 amino acids long (HFQPLPPAVV). This minimal structure was capable of inducing a proliferative response in all healthy 38.G responders tested. The mechanisms influencing this epitope-specific anergy in patients could give new insights into the immunopathogenesis of tuberculosis.