SummaryThe potential for development of mycobacteria as T helper type 1 (Th1) vaccines capable of induction of Th1 responses to recombinant antigens was explored in a model system based on an immunodominant peptide from house dust mite. Different recombinant mycobacterial preparations were compared for their ability to induce a Th1 response to the peptidea. It was found that mycobacterial viability was not a prerequisite for Th1 immunogenicity. A dominant interferon‐γ (IFN‐γ) response to peptide was observed in splenocytes from C57BL/6J mice immunized with live or heat‐killed preparations of recombinant Mycobacterium vaccae or with live attenuated bacillus Calmette–Guèrin (BCG) vaccine expressing the antigen. Interleukin‐5 (IL‐5), a marker of a Th2 response, was detected only in mice receiving live M. vaccae. A similar pattern was observed in BALB/b mice, although the magnitude of the IFN‐γ response was much lower. Control and immunized mice were subsequently exposed to allergen using a Th2‐inducing challenge protocol. A significant shift from a Th2 to a Th1 response was observed in immunized mice, as judged by cytokine expression by splenocytes and by subclass of circulating antibody. The effect was seen in three inbred mouse strains differing in their innate bias towards Th1 or Th2 responses. It was dependent on the presence of specific antigen in the mycobacterial preparation and, under the immunization conditions tested, was more pronounced with dead M. vaccae than with live BCG as carrier vaccine. The results demonstrate the potency of killed mycobacteria as Th1 adjuvants and suggest a potential application for recombinant mycobacteria in antigen‐specific immune modulation.

Recent evidence suggests that chronic exposure to lactobacilli, which are part of the normal intestinal flora, inhibits the development of allergic disorders. Allergy is mediated by Th2 cells, which produce high levels of IL4 and IL5, and suppressive effects of lactic acid bacteria on the development of allergy have been attributed to their Th1-inducing properties. On the other hand, lactic acid bacteria have also been shown to suppress autoimmune disorders which are mediated by Th1 cells producing high levels of IFNgamma. To study this apparent discrepancy, the immunomodulatory potential of lactobacilli was evaluated using recombinants that express an immunodominant T-cell epitope of Der p 1 of house dust mites. Mucosal immunization of C57BL/6 J mice with such recombinants resulted in the induction of T cells which produced low amounts of IFNgamma. Immunization with the house dust mite peptide followed by treatment with recombinant Lactobacillus plantarum resulted in the inhibition of both IFNgamma and IL5 production. The effect on IFNgamma production was shown to be a non-specific effect of L. plantarum. The effect on IL5 production, however, was only observed when the recombinant expressing the Der p 1 peptide, but not the control recombinant, was used for treatment. Neither of the recombinants had an effect on the antibody response. Taken together, these data suggest that recombinant L. plantarum may be a suitable candidate for the treatment of allergic disorders.

Following uptake by macrophages, live mycobacteria initially reside within an immature phagosome that resists acidification and retains access to recycling endosomes. Glycolipids are exported from the mycobacterial phagosome and become available for immune recognition by CD1-restricted T cells. The aim of this study was to explore the possibility that lipoproteins might similarly escape from the phagosome and act as immune targets in cells infected with live mycobacteria. We have focused on a 19-kDa lipoprotein from Mycobacterium tuberculosis that was previously shown to be recognized by CD8(+) T cells. The 19-kDa Ag was found to traffic separately from live mycobacteria within infected macrophages by a pathway that was dependent on acylation of the protein. When expressed as a recombinant protein in rapid-growing mycobacteria, the 19-kDa Ag was able to deliver peptides for recognition by MHC class I-restricted T cells by a TAP-independent mechanism. Entry into the class I pathway was rapid, dependent on acylation, and could be blocked by killing the mycobacteria by heating before infection. Although the pattern of 19-kDa trafficking was similar with different mycobacterial species, preliminary experiments suggest that class I presentation is more efficient during infection with rapid-growing mycobacteria than with the slow-growing bacillus Calmette-Guérin vaccine strain.

By expression of foreign antigens in attenuated strains derived from bacterial pathogens and in non-pathogenic commensal bacteria, recombinant vaccines are being developed that aim to stimulate mucosal immunity. Recent advances in the pathogenesis and molecular biology of these bacteria have allowed rational development of new and improved bacterial carriers and more effective gene expression systems. These advances have improved the performance and versatility of these delivery systems to induce mucosal immunity to recombinant antigens in animal models. Application of these (improved) technologies for development of human vaccines is still limited and awaits further exploration.

To date, only a limited number of antigens have been described as specific for Mycobacterium leprae, and in many cases, homologues have subsequently been shown to exist in mycobacteria such as M. avium and M. intracellulare. A Leprosy Synthetic Peptide Skin Test Initiative was established by the Steering Committee on the Immunology of Mycobacteria of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, to investigate the potential of synthetic peptides that encode T-cell epitopes as diagnostic tools, which could be used to develop a skin-test reagent specific for leprosy. Such M. leprae-specific peptides should have unique amino acid sequences, or significant sequence-dissimilarity from those in other mycobacteria. Synthetic peptides, 15 amino acids long, were synthesised from 33 genes or open reading frames within the M. leprae genome. Tuberculoid leprosy patients from four leprosy-endemic countries, Brazil, Ethiopia, Nepal and Pakistan, were tested as subjects known to have been infected with M. leprae, and to make good T-cell responses to antigens of M. leprae; UK blood donors were used as non-exposed or non-infected subjects. Peptides inducing potentially specific responses in leprosy patients and not in UK controls, and those inducing cross-reaction responses, present in both leprosy patients and non-exposed, non-infected controls, were identified. A difference from the equivalent M. tuberculosis sequence of five or more amino acid residues did not, by itself, identify peptides that were M. leprae-specific, suggesting that many of these peptides may have homologues in environmental mycobacteria. To date, this approach has identified a number of peptides with greater than 90% specificity and 19-47% sensitivity, which are undergoing further specificity-testing. Such peptides would have great potential as T-cell reagents with which to monitor exposure to M. leprae within communities, formulated either as skin-test reagents, or as antigens for tests in vitro.

The delivery of antigens to mucosal-associated lymphoid tissues in paediatric and immunocompromised populations by safe, non-invasive vectors, such as commensal lactobacilli, represents a crucial improvement to prevailing vaccination options. In this report, we describe the oral and nasal immunization of mice with vaccines constructed through an original system for heterologous gene expression in Lactobacillus in which the 50 000-molecular weight (MW) fragment C of tetanus toxin (TTFC) is expressed either as an intracellular or a surface-exposed protein. Our data indicate that L. plantarum is more effective in this respect than L. casei and that, under the experimental conditions investigated, delivery of TTFC expressed as an intracellular antigen is more effective than cell-surface expression. Immunization of mice with live recombinant lactobacilli induced significant levels of circulating TTFC-specific immunoglobulin G (IgG) following nasal or oral delivery of vaccine strains. In addition, following nasal delivery, secretory immunoglobulin A (sIgA) was induced in bronchoalveolar lavage fluids, as were antigen-specific antibody-secreting cells and antigen-specific T-cell activation in draining lymph nodes, substantiating their potential for safe mucosal delivery of paediatric vaccines.

ABSTRACTTo identifyMycobacterium leprae-specific human T-cell epitopes, which could be used to distinguish exposure toM. lepraefrom exposure toMycobacterium tuberculosisor to environmental mycobacteria or from immune responses followingMycobacterium bovisBCG vaccination, 15-mer synthetic peptides were synthesized based on data from theM. lepraegenome, 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-γ) 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 inM. lepraeandM. tuberculosisand 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 ≥5 of 15 amino acid mismatches between the equivalentM. lepraeandM. tuberculosissequences; of these, eight gave specificities of ≥90% (percentage of United Kingdom donors who were nonresponders for IFN-γ 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.

A genomic DNA region, designated RD1, that is present in virulent and clinical strains of Mycobacterium tuberculosis and M. bovis, has been shown to be deleted in bacillus Calmette Guérin (BCG). The DNA segments corresponding to three open reading frames (ORFs: ORF‐10, ORF‐14 and ORF‐15) of the RD1 region, that are deleted in BCG strains, were amplified from M. tuberculosis genomic DNA by polymerase chain reaction (PCR), subcloned into pGEX‐4T vector system and expressed in Escherichia coli as fusion proteins with glutathione‐S‐transferase (GST). The recombinant proteins appeared as major cellular proteins in SDS‐PAGE gels at the expected molecular mass. The identity of each fusion protein was confirmed by reactivity with anti‐GST antibodies in Western immunoblots. When pooled human sera from 11 tuberculosis (TB) patients were used as the source of antibodies, only GST‐ORF‐14 fusion protein reacted in Western immunoblots. The protein corresponding to ORF‐14 was then purified to near homogeneity and isolated free of its fusion partner (GST) by treating the purified GST‐ORF‐14 fusion protein with thrombin protease. In Western immunoblots, the purified ORF‐14 protein reacted with antibodies in 26 of 57 human sera (46%) from TB patients while no reactivity was seen with 11 sera from M. bovis BCG‐vaccinated healthy subjects. Interestingly, sera from nine of 15 (60%) long‐term contacts of TB patients also had antibodies reactive to the ORF‐14 protein. These results suggest that the ORF‐14 protein in combination with other immunodominant proteins could be useful in the serodiagnosis of individuals infected with M. tuberculosis.

The recognition of 16 mycobacterial Ags by a panel of T cell lines from leprosy patients and healthy exposed individuals from an endemic population was examined within the context of expressed HLA-DR molecules. Although overall no significant differences were found between the frequencies of Ag recognition in the different subject groups, when Ag-specific T cell responses were examined within the context of HLA-DR, a highly significant difference was found in the recognition of the 30/31-kDa Ag. HLA-DR3 appeared to be associated with high T cell responsiveness to the 30/31-kDa Ag in healthy contacts (p = 0.01), but, conversely, with low T cell responsiveness to this Ag in tuberculoid patients (p = 0.005). Within the group of HLA-DR3-positive individuals, differences in 30/31-kDa directed T cell responsiveness were highly significant not only between healthy individuals and tuberculoid patients (p < 0.0001), but also between healthy individuals and lepromatous patients (p = 0.009), and consequently between healthy individuals compared with leprosy patients as a group (p < 0.0001). A dominant HLA-DR3-restricted epitope was recognized by healthy contacts in this population. It has been proposed that secreted Ags may dominate acquired immunity early in infection. The low T cell response to the secreted, immunodominant 30/31-kDa Ag in HLA-DR3-positive leprosy patients in this population may result in retarded macrophage activation and delayed bacillary clearance, which in turn may lead to enhanced Ag load followed by T cell-mediated immunopathology.

T helper 1 cells play a major role in protective immunity against mycobacterial pathogens. Since the antigen (Ag) specificity of CD4(+) human T cells is strongly controlled by HLA class II polymorphism, the immunogenic potential of candidate Ags needs to be defined in the context of HLA polymorphism. We have taken advantage of class II-deficient (Ab0) mice, transgenic for either HLA-DRA/B1*0301 (DR3) or HLA-DQB1*0302/DQA*0301 (DQ8) alleles. In these animals, all CD4(+) T cells are restricted by the HLA molecule. We reported previously that human DR3-restricted T cells frequently recognize heat shock protein (hsp)65 of Mycobacterium tuberculosis, and only a single hsp65 epitope, p1-20. DR3.Ab0 mice, immunized with bacillus Calmette-Guérin or hsp65, developed T cell responses to M. tuberculosis, and recognized the same hsp65 epitope, p1-20. Hsp65-immunized DQ8.Ab0 mice mounted a strong response to bacillus Calmette-Guérin but not to p1-20. Instead, we identified three new DQ8-restricted T cell epitopes in the regions 171-200, 311-340, and 411-440. DR3.Ab0 mice immunized with a second major M. tuberculosis protein, Ag85 (composed of 85A, 85B, and 85C), also developed T cell responses against only one determinant, 85B p51-70, that was identified in this study. Importantly, subsequent analysis of human T cell responses revealed that HLA-DR3+, Ag85-reactive individuals recognize exactly the same peptide epitope as DR3.Ab0 mice. Strikingly, both DR3-restricted T cell epitopes represent the best DR3-binding sequences in hsp65 and 85B, revealing a strong association between peptide-immunodominance and HLA binding affinity. Immunization of DR3.Ab0 with the immunodominant peptides p1-20 and p51-70 induced T cell reactivity to M. tuberculosis. Thus, for two different Ags, T cells from DR3.Ab0 mice and HLA-DR3+ humans recognize the same immunodominant determinants. Our data support the use of HLA-transgenic mice in identifying human T cell determinants for the design of new vaccines.

ABSTRACT We have developed a novel epitope delivery system based on the insertion of peptides within a permissive loop of a bacterial superoxide dismutase molecule. This system allowed high-level expression of heterologous peptides in two mycobacterial vaccine strains, Mycobacterium bovis bacille Calmette-Guérin (BCG) and Mycobacterium vaccae. The broader application of the system was analyzed by preparation of constructs containing peptide epitopes from a range of infectious agents and allergens. We report detailed characterization of the immunogenicity of one such construct, in which an epitope from the Der p1 house dust mite allergen was expressed in M. vaccae. The construct was able to stimulate T-cell hybridomas specific for Der p1, and it induced peptide-specific gamma interferon responses when used to immunize naive mice. This novel expression system demonstrates new possibilities for the use of mycobacteria as vaccine delivery vehicles.

In our search for Mycobacterium leprae antigens that might specifically induce immunity or immunopathology, we have tested both humoral and cellular immune reactivity against purified recombinant M. leprae antigens in 29 paucibacillary (PB), 26 multibacillary (MB) leprosy patients, and 47 matched healthy contacts. The following M. leprae antigens were tested: 2L-1 (65L-1, GroEl-1), 2L-2 (65L-2, GroEl-2), 4L (SoDA), 43L, 10L (B) and 25L (Sra). The individuals were also typed for HLAD-RB1 and DQB1 in order to see whether leprosy status and/or immune reactivity to these antigens might be associated with certain HLA types. We also tested sera from another 48 patients before, during and after multidrug therapy (MDT) to study the relationship between antibody reactivity to recombinant M. leprae antigens and MDT. Antibody titers to the four recombinant M. leprae antigens tested and to D-BSA were higher in MB patients compared to PB patients and healthy controls, and declined with treatment. From a diagnostic or monitoring point of view none of the recombinant antigens seemed to be an improvement over D-BSA, mainly due to the lower sensitivity. IgG subclasses were measured in positive sera of untreated patients. These were mainly of the IgG1 and IgG3 subclasses, but subclass diversity was also observed and antigen dependent: all four subclasses could be detected against 10L (B), only IgG1 and IgG3 against 43L and only IgG1 against 25L and 2L-1. Cellular immune reactivity against the purified recombinant M. leprae antigens was measured in a lymphocyte stimulation test (LST). As for M. leprae, there was an inverse correlation between antibody and T-cell reactivity. However, the number of LST responders to recombinant antigens was much lower than to M. leprae. The 43L antigen was recognized most often (19%-24% of individuals tested) and more often than the 10L (B) antigen (10%-12%). No clear correlation was observed with leprosy type or protection and, in general, M. leprae nonresponders were also negative with recombinant antigens. Finally, we confirmed that HLA-DRB1*02 is associated with leprosy in this population, and we observed an association between DQB1*0601 and lepromatous leprosy. The number of positive individuals was too small to allow a meaningful analysis of the relationship between HLA type and immune reactivity. Although these data do not allow a conclusion as to one of these purified recombinant antigens being either protection or disease related, the antigen-dependent IgG subclass diversity warrants further study on antigen-specific qualitative differences in immune reactivity that may be relevant for the outcome of an infection with M. leprae.

Protein glycosylation has an important influence on a broad range of molecular interactions in eukaryotes, but is comparatively rare in bacteria. Several antigens from Mycobacterium tuberculosis, the causative agent of human tuberculosis, have been identified as glycoproteins on the basis of lectin binding, or by detailed structural analysis. By production of a set of alkaline phosphatase (PhoA) hybrid proteins in a mycobacterial expression system, the peptide region required for glycosylation of the 19 kDa lipoprotein antigen from M.tuberculosis was defined. Mutagenesis of two threonine clusters within this region abolished lectin binding by PhoA hybrids and by the 19 kDa protein itself. Substitution of the threonine residues also resulted in generation of a series of smaller forms of the protein as a result of proteolysis. In a working model to account for these observations, we propose that the role of glycosylation is to regulate cleavage of a proteolytically sensitive linker region close to the acylated N‐terminus of the protein.

A major focus of leprosy research in the last 10 years has been the identification and characterization of antigens of Mycobacterium leprae that interact with antibodies and T cells of the host's immune response. Through the combined efforts of many different laboratories, a substantial number of protein antigens have been identified and characterized. In this MicroReview we present an updated list of M. leprae protein antigens, and, with emphasis on recent developments, summarize what is known regarding their functional and immunological features.

The recognition of a panel of recombinant Mycobacterium leprae antigens by T cells and B cells from 29 borderline tuberculoid/tuberculoid (BT/TT) and 18 lepromatous leprosy (LL) patients and from 21 healthy controls (HC) in leprosy-endemic regions of Ethiopia was examined. All 11 antigenic molecules tested (including M. leprae hsp 10, hsp18, hsp65 and several novel M. leprae antigens) were shown to be recognized by T cells, but clear quantitative differences existed between reactivities induced by individual antigens. Similar quantitative differences were observed when antibody responses to hsp10 and hsp65 antigens were determined. No associations were found between the antigen-specific responses and the subject status of either BT/TT and LL patients or HC. Fifteen percent of the patients who were nonresponsive to sonicates of M. leprae showed significant T-cell responses to one or more individual M. leprae antigens. This indicates that M. leprae constituents other than the proteins tested are responsible for the M. leprae-specific nonresponsiveness in these patients, which may be exploited for the design of vaccines or immunotherapeutic modalities aimed at inducing M. leprae-specific immunity in nonresponders.

Crossed immunoelectrophoresis (CIE) has been used to develop a reference system for classifying mycobacterial antigens. The subsequent use of specific antibodies allowed further determination of antigens by molecular weight. The monoclonal antibody F126-2, originally raised against a 34-kDa antigen of Mycobacterium kansasii, reacted with antigen 84 (Ag84) in the CIE reference system for Mycobacterium bovis BCG and Mycobacterium tuberculosis. To characterize Ag84, we screened a lambda gt11 gene library from M. tuberculosis with antibody F126-2 and identified the encoding gene. The corresponding Mycobacterium leprae Ag84 gene was subsequently selected from a cosmid library, using the M. tuberculosis gene as a probe. Both genes were expressed as 34-kDa proteins in Escherichia coli, and the recombinant proteins indeed corresponded to Ag84 in the CIE reference system. The derived amino acid sequences of the M. tuberculosis and M. leprae proteins showed 85% identity, which indicates that Ag84 constitutes a group of highly conserved mycobacterial antigens. Antibodies of almost 60% of lepromatous leprosy patients responded to Ag84, indicating that the protein is highly immunogenic following infection in multibacillary leprosy.

By combining a DNA subclone and synthetic-peptide approach, we mapped epitopes of the immunogenic mycobacterial 70-kDa heat shock protein (HSP70) recognized by human CD4+ T-cell clones and lines. In addition, we identified the respective HLA-DR molecules used in antigen presentation. The donor groups used were healthy persons immunized with killed Mycobacterium leprae and tuberculoid leprosy patients. The results show that the N-terminal part of the HSP70 molecule contains three different T-cell epitopes, of which two were presented by DR7 (amino acids [aa] 66 to 82 and 210 to 226) and one was presented by DR3 (aa 262 to 274). The C-terminal part contains one epitope (aa 413 to 424) presented by HLA-DR2. The C-terminal epitope shows extensive homology to the corresponding region of the human HSP70 sequence. All of the T-cell epitopes identified were presented by only one particular HLA-DR molecule. We also found that HLA-DR5 and DRw53 can present HSP70 to T cells, demonstrating the presence of additional epitopes not yet defined at the peptide level. On the basis of the donors used in this study, recognition of HSP70 at the epitope level seems to be ruled by the restriction elements expressed by the donor rather than by any difference in reactivity between healthy individuals and patients. In conclusion, mycobacterial HSP70 is relevant to subunit vaccine design since it contains a variety of T-cell epitopes presented in the context of multiple HLA-DR molecules.

Sixty-three overlapping 15-oligomer peptides covering the 30-kDa protein antigen 85B of Mycobacterium leprae were tested by ELISA to identify epitopes recognized by human antibodies. Serum samples from patients with lepromatous leprosy (LL) reacted mainly with peptides comprising amino acid regions (AA) 206-230, 251-280, and 291-325. Sera of patients with active tuberculosis who responded to the native 30-kDa antigen did not recognize these peptides. The antibody-binding specificity to the defined B cell regions was evaluated in a blind study with 71 serum samples from patients and household contacts living in Ethiopia where leprosy is endemic. The peptide of AA 256-280 was recognized by 88% of LL patients, 15% of patients with tuberculoid leprosy, and none of the contacts. These findings suggest that there are major linear B cell epitopes on the M. leprae 30-kDa protein that are recognized by lepromin-negative LL patients, whereas lepromin-positive patients respond preferentially to conformational epitopes.

By screening a Mycobacterium leprae lambda gt11 expression library with a serum from an Ethiopian lepromatous leprosy (LL) patient a clone was isolated (LL4) belonging to hybridization group III of a panel of previously isolated M. leprae clones. Members of this hybridization group encode a serologically recognized 45 kDa protein. The complete DNA sequences of the partially overlapping clones LL4 and L1 (hybridization group III) are presented and these revealed the presence of an open reading frame (ORF) predicting a protein with a molecular size of 42 448 Da. Southern hybridizations on total genomic DNA of M. leprae, Mycobacterium tuberculosis and eight atypical mycobacteria showed that the LL4 DNA fragment is specific for M. leprae DNA even under low-stringency conditions. The M. leprae specificity of LL4 DNA was further confirmed by the polymerase chain reaction using four different sets of primers. Western blotting analyses showed that the M. leprae 45 kDa protein is frequently recognized by antibodies from leprosy patients and that this recognition is specific since no antibodies could be detected in sera of tuberculosis patients. T-cell proliferation assays also demonstrated T-cell recognition by leprosy patients and healthy contacts of the M. leprae 45 kDa protein. The specificity of the LL4 DNA region and the 45 kDa antigen that is encoded by hybridization group III could provide unique tools for the development of M. leprae-specific immunological and DNA reagents.

The genes for two novel members (designated 85A and 85C) of the Mycobacterium leprae antigen 85 complex family of proteins and the gene for the closely related M. leprae MPT51 protein were isolated. The complete DNA sequence of the M. leprae 85C gene and partial sequences of the 85A and MPT51 genes are presented. As in M. tuberculosis, the M. leprae 85A, 85C, and previously identified 85B component genes are not closely linked on the genome. However, the MPT51 genes of both species localize close to the respective 85A component genes. Like the 85B component, the M. leprae 85A-MPT51 and 85C antigens are recognized by T cells from healthy contacts and leprosy patients.