Permissive recognition of a mycobacterial T-cell epitope: localization of overlapping epitope core sequences recognized in association with multiple major histocompatibility complex class II I-A molecules.

Most T-cell epitopes are recognized in the context of a single or limited number of major histocompatibility complex (MHC) class II molecules. We have shown previously, however, that the immunodominant p61-80 epitope from the Mycobacterium tuberculosis 19,000 MW protein is recognized in a genetically permissive manner. In this study, permissive recognition of p61-80 was analysed in three murine MHC haplotypes (H-2b,d and k) with respect to: (i) T-cell-epitope core structure; (ii) I-A/I-E class II MHC restriction; and (iii) the identification of critical amino acid residues within the core region. Overlapping epitope core sequences composed of 6 to 8 amino acids were identified for each of the three H-2 haplotypes by T-cell epitope scanning (PEPSCAN) using peptide-specific T-cell lines. The epitope core sequences recognized by peptide and 19,000 MW protein-specific T cells were similar. In all three haplotypes, responses to p61-80 were restricted by class II MHC I-A molecules. To identify residues within the epitope core critically required for recognition, single substitution (alanine or leucine) analogue peptides were tested for their capacity to stimulate p61-80-specific T-cell hybridomas. A heterogeneous pattern of reactivity was observed, even among individual hybridomas derived from the same H-2 haplotype. Although every core residue could be defined as critical for at least one hybridoma, only one critical substitution (74Val-->Ala) was common to all hybridomas. The identification and structural analysis of genetically permissive epitopes of mycobacteria may be a useful strategy for the rational design of peptide-based vaccines for tuberculosis.     

Recognition of peptide epitopes of the 16,000 MW antigen of Mycobacterium tuberculosis by murine T cells.

The T-cell repertoire to a prominent immunogen of Mycobacterium tuberculosis has been investigated on the assumption that differences in epitope specificity could influence the protective and pathogenic host reactions. Proliferative responses of lymph node and spleen cells to overlapping peptides, spanning the entire sequence of the 16,000 MW protein antigen were analysed in C57BL/10 and B10.BR mice. Following footpad priming and in vitro challenge with homologous peptide, 12 out of the 14 peptides tested were found to be immunogenic. However, only two peptides of residues 31-40 and 71-91 stimulated strong proliferative responses of T cells from mice which had been presensitized with either killed or live M. tuberculosis organisms; another three peptides were only weakly stimulatory. These epitopes have been immunodominant in both H-2b and H-2k mouse strains, indicating the genetically permissive nature of their recognition. Furthermore, both major immunodominant epitopes were found to be species specific for the M. tuberculosis complex and therefore potentially suitable for the early diagnosis of tuberculous infection.     

Homologs of Mycobacterium leprae 18-kilodalton and Mycobacterium tuberculosis 19-kilodalton antigens in other mycobacteria.

Most of the antigens of Mycobacterium leprae and M. tuberculosis that have been identified are members of stress protein families, which are highly conserved throughout many diverse species. Of the M. leprae and M. tuberculosis antigens identified by monoclonal antibodies, all except the 18-kDa M. leprae antigen and the 19-kDa M. tuberculosis antigen are strongly cross-reaction between these two species and are coded within very similar genes. Studies of T cell reactivity against mycobacterial antigens have indicated that M. tuberculosis bears epitopes that are cross-reactive with the M. leprae 18-kDa antigen, but attempts to identify an 18-kDa antigen-like protein or protein coding sequence in M. tuberculosis have been unsuccessful. We have used a combination of low-stringency DNA hybridization and polymerase chain reaction techniques to identify, isolate, and sequence genes from M. avium and M. intracellulare that are very similar to the 18-kDa antigen gene of M. leprae and others that are homologs of the 19-kDa antigen gene of M. tuberculosis. Unlike M. leprae, which contains a single 18-kDa antigen gene, M. avium and M. intracellulare each have two 18-kDa antigen coding sequences. Although the M. leprae, M. avium, and M. intracellulare 18-kDa antigen genes are all very similar to one another, as are the M. tuberculosis, M. avium, and M. intracellulare 19-kDa antigen genes, we have been unable to detect any 18-kDa antigen-like coding sequences in DNA from M. tuberculosis.     

Members of the 30- to 32-kilodalton mycolyl transferase family (Ag85) from culture filtrate of Mycobacterium avium subsp. paratuberculosis are immunodominant Th1-type antigens recognized early upon infection in mice and cattle

The characterization of protective antigens is essential for the development of an effective, subunit-based vaccine against paratuberculosis. Surface-exposed and secreted antigens, present abundantly in mycobacterial culture filtrate (CF), are among the well-known protective antigens of Mycobacterium tuberculosis and Mycobacterium bovis. Culture filtrate, prepared from Mycobacterium avium subsp. paratuberculosis ATCC 19698 grown as a surface pellicle on synthetic Sauton medium, was strongly and early recognized in experimentally infected B6 bg/bg beige mice and cattle, as indicated by elevated spleen cell gamma interferon (IFN-gamma) secretion and lymphoproliferative responses of peripheral blood mononuclear cells, respectively. Strong proliferative and ex vivo IFN-gamma responses against antigen 85 (Ag85) complex (a major protein component from M. bovis BCG culture filtrate) could be detected in cattle as early as 10 weeks after oral M. avium subsp. paratuberculosis infection. Synthetic peptides from the Ag85A and Ag85B components of this complex were strongly recognized, whereas T-cell responses were weaker against peptides from the Ag85C protein. A promiscuous T-cell epitope spanning amino acids 145 to 162 of Ag85B (identical sequence in M. bovis and M. avium subsp. paratuberculosis) was identified in experimentally infected cattle. Finally, young calves, born from cows with confirmed paratuberculosis, demonstrated proliferative responses to purified, recombinant Ag85A and Ag85B from M. avium subsp. paratuberculosis. These results indicate that the M. avium subsp. paratuberculosis Ag85 homologues are immunodominant T-cell antigens that are recognized early in experimental and natural infection of cattle.     

Distinct differences in repertoires of low-molecular-mass secreted antigens of Mycobacterium avium complex and Mycobacterium tuberculosis

Antigens in a 4-week-old culture filtrate (CF) of Mycobacterium avium subsp. avium were separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by Western blotting. The culture had minimal lysis of bacilli, giving a CF preparation consisting mainly of secreted proteins. Comparison with a similar CF of Mycobacterium tuberculosis with almost no contamination with intracellular proteins showed the presence of cross-reactive antigens homologous to the four components of the antigen 85 complex, as well as MPT32. These were major constituents of the M. avium subsp. avium CF. In addition, there were several low-molecular-mass bands (<15 kDa) in both species that did not cross-react with polyclonal and polyvalent rabbit antibodies in Western blotting. Furthermore, these bands were not detected in corresponding sonicate preparations, indicating high localization indexes, which is typical of soluble secreted proteins. A 14-kDa protein was selected for purification and more detailed characterization. The N-terminal amino acid sequence was determined, and a matching gene was found within the genomic sequence of M. avium subsp. avium which was highly homologous to Rv0455c of M. tuberculosis. The gene encoded a signal peptide typical of secreted mycobacterial proteins. A rabbit antiserum was raised against the purified protein, and the antigen was demonstrated by Western blotting in CFs of M. avium subsp. avium, Mycobacterium avium subsp. paratuberculosis, Mycobacterium intracellulare, and Mycobacterium scrofulaceum but was not detected in M. tuberculosis. This is a new example of a highly homologous gene being differentially expressed by different mycobacterial species.     

M. tuberculosis-Complex Specific T-Cell Stimulation and DTH Reactions Induced With a Peptide from the 38-kDa Protein

An immunodominant T-cell-stimulatory epitope located near the carboxy terminus of the 38-kDa antigen from M. tuberculosis (38.G, residues 350-369) was found to be M. tuberculosis-complex specific. This was demonstrated by the presence of proliferative and delayed type hypersensitivity (DTH) responses in mice immunized with Mycobacterium tuberculosis and Mycobacterium bovis BCG, whereas mice immunized with M. avium or other non-tuberculous species of mycobacteria showed no such responses. Peptide 38.G stimulated the proliferation of peripheral blood lymphocytes from healthy purified protein derivative (PPD)-positive individuals but not from PPD-negative individuals. It also elicited DTH responses in M. tuberculosis sensitized mice and in PPD-positive healthy human volunteers. Peptide 38.G could therefore prove to be an important component in any new molecularly defined reagent used in the immunodiagnosis of tuberculous infection.     

Protection against virulent Mycobacterium avium infection following DNA vaccination with the 35-kilodalton antigen is accompanied by induction of gamma interferon-secreting CD4(+) T cells

Mycobacterium avium is an opportunistic pathogen that primarily infects immunocompromised individuals, although the frequency of M. avium infection is also increasing in the immunocompetent population. The antigen repertoire of M. avium varies from that of Mycobacterium tuberculosis, with the immunodominant 35-kDa protein being present in M. avium and Mycobacterium leprae but not in members of the M. tuberculosis complex. Here we show that a DNA vector encoding this M. avium 35-kDa antigen (DNA-35) induces protective immunity against virulent M. avium infection, and this protective effect persists over 14 weeks of infection. In C57BL/6 mice, DNA vaccines expressing the 35-kDa protein as a cytoplasmic or secreted protein, both induced strong T-cell gamma interferon (IFN-gamma) and humoral immune responses. Furthermore, the antibody response was to conformational determinants, confirming that the vector-encoded protein had adopted the native conformation. DNA-35 immunization resulted in an increased activated/memory CD4(+) T-cell response, with an accumulation of CD4(+) CD44(hi) CD45RB(lo) T cells and an increase in antigen-specific IFN-gamma production. The protective effect of the DNA-35 vectors against M. avium infection was comparable to that of vaccination with Mycobacterium bovis BCG and significantly greater than that for previous treated infection with M. avium. These results illustrate the importance of the 35-kDa protein in the protective response to M. avium infection and indicate that DNA vaccination successfully promotes a sustained level of protection during chronic M. avium infection.     

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.     

High frequency of CD4+ T cells specific for the TB10.4 protein correlates with protection against Mycobacterium tuberculosis infection

TB10.4 is a newly identified antigen of Mycobacterium tuberculosis recognized by human and murine T cells upon mycobacterial infection. Here, we show that immunization with Mycobacterium bovis BCG induces a strong, genetically controlled, Th1 immune response against TB10.4 in mice. BALB/c and C57BL/6 strains behave as high and low responders to TB10.4 protein, respectively. The TB10.4:74-88 peptide was identified as an immunodominant CD4+ T-cell epitope for H-2d mice. Since recent results, as well as the present study, have raised interest in TB10.4 as a subunit vaccine, we analyzed immune responses induced by this antigen delivered by a new vector, the adenylate cyclase (CyaA) of Bordetella pertussis. CyaA is able to target dendritic cells and to deliver CD4+ or CD8+ T-cell epitopes to the major histocompatibility complex class II/I molecule presentation pathways, triggering specific Th1 or cytotoxic T-lymphocyte (CTL) responses. Several CyaA harboring either the entire TB10.4 protein or various subfragments containing the TB10.4:20-28 CTL epitope were shown to induce TB10.4-specific Th1 CD4+ and CD8+ T-cell responses. However, none of the recombinant CyaA, injected in the absence of adjuvant, was able to induce protection against M. tuberculosis infection. In contrast, TB10.4 protein administered with a cocktail of strong adjuvants that triggered a strong Th1 CD4+ T-cell response induced significant protection against M. tuberculosis challenge. These results confirm the potential value of the TB10.4 protein as a candidate vaccine and show that the presence of high frequencies of CD4+ T cells specific to this strong immunogen correlates with protection against M. tuberculosis infection.     

MTC28, a novel 28-kilodalton proline-rich secreted antigen specific for the Mycobacterium tuberculosis complex.

Proteins that are actively secreted by Mycobacterium tuberculosis serve as major targets of immune responses in the infected host. To identify and purify novel proteins in the filtrates of M. tuberculosis cultures, a bacteriophage lambda library of M. tuberculosis H37Rv DNA was immunoscreened by using an anti-culture filtrate rabbit antiserum. Of 20 positive clones isolated, 6 were analyzed and found to express the genes for two known components of the early culture filtrate, the secreted 45/47-kDa antigen complex and the KatG protein, and two novel genes. Here we report the molecular cloning and nucleotide sequence of one of the new genes encoding a culture filtrate protein of 310 amino acid (aa) residues. We called this gene mtc28. The deduced polypeptide sequence contained an NH2-terminal, highly hydrophobic 32-aa region having properties of a secretion signal peptide. The putative 278-aa mature MTC28 protein was characterized at its NH2 and COOH termini by a high content of proline and alanine residues organized in an (AP)n motif. Thus, MTC28 is a new member of a group of proline-rich antigens found in M. tuberculosis and Mycobacterium leprae. As shown by DNA hybridization experiments, the mtc28 gene was present only in species of the M. tuberculosis complex. Purified recombinant MTC28 antigen evoked strong delayed-type hypersensitivity and antibody responses in guinea pigs immunized with Mycobacterium bovis BCG, but not in guinea pigs immunized with Mycobacterium avium. The strong immunological activity of MTC28 and the absence of B- and T-cell epitopes cross-reactive with a common environmental mycobacterial species, such as M. avium, make this novel antigen an attractive reagent for immunodiagnosis of tuberculosis.     

PPE protein (Rv3873) from DNA segment RD1 of Mycobacterium tuberculosis: strong recognition of both specific T-cell epitopes and epitopes conserved within the PPE family

Proteins encoded by DNA segment RD1 of Mycobacterium tuberculosis have recently been demonstrated to play important roles in bacterial virulence, vaccine development, and diagnostic reagent design. Previously, we characterized two immunodominant T-cell antigens, the early secreted antigen target (ESAT-6) and the 10-kDa culture filtrate protein (CFP10), which are encoded by the esx-lhp operon in this region. In the present study we characterized a third putative open reading frame in this region, rv3873, which encodes a PPE protein. We found that the rv3873 gene is expressed in M. tuberculosis H37Rv and that the native protein, Rv3873, is predominantly associated with the mycobacterial cell or wall. When tested as a His-tagged recombinant protein, Rv3873 stimulated high levels of gamma interferon secretion in peripheral blood mononuclear cells isolated from tuberculosis (TB) patients, as well as from healthy tuberculin purified protein derivative-positive donors. In contrast to other RD1-encoded antigens, Rv3873 was also found to be recognized by a significant proportion of Mycobacterium bovis BCG-vaccinated donors. Epitope mapping performed with overlapping peptides revealed a broad pattern of T-cell recognition comprising both TB-specific epitopes and epitopes also recognized by BCG-vaccinated donors. The immunodominant epitope (residues 118 to 135) for both TB patients and BCG-vaccinated individuals was found to be highly conserved among a large number of PPE family members.     

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.     

Predominant recognition of species-specific determinants of the GroES homologues from Mycobacterium leprae and M. tuberculosis.

The Mycobacterium leprae and M. tuberculosis 10,000 MW heat-shock protein homologues of GroES have previously been identified as major immunogens for human T cells. We used synthetic peptides to characterize the determinants recognized by murine T cells. The findings suggest that, despite 90% sequence identity between these two proteins, T cells recognize prominently the species-specific determinants localized within amino acid residues 21-40 and 49-72. Analysis of the molecular determinants of species-specificity for the M. leprae GroES sequence 25-40, using T-cell hybridomas and major histocompatibility complex (MHC)-binding assays, led to the identification of epitope cores and critical residues. Interestingly, closely overlapping epitope cores were found to be restricted by either H-2Ad (24-34) or H-2Ed (28-34). Furthermore, the site recognized by the M. leprae-specific monoclonal antibodies ML06 and ML10 was also localized in the overlapping sequences 25-31 and 25-29. In conclusion, we demonstrated that immunodominant species-specific T- and B-cell epitopes can be found in a mycobacterial heat-shock protein despite its highly conserved amino acid sequence. This finding suggests the feasibility of identifying a sufficient number of M. leprae-specific determinants for a composite T-cell immunodiagnostic reagent for tuberculoid leprosy.     

T-cell epitope mapping of the three most abundant extracellular proteins of Mycobacterium tuberculosis in outbred guinea pigs

The three most abundant extracellular proteins of Mycobacterium tuberculosis, the 30-, 32-, and 16-kDa major extracellular proteins, are particularly promising vaccine candidates. We have mapped T-cell epitopes of these three proteins in outbred guinea pigs by immunizing the animals with each protein and assaying splenic lymphocyte proliferation against a series of overlapping synthetic peptides covering the entire length of the mature proteins. The 30-kDa protein contained nine immunodominant epitopes, the 32-kDa protein contained two immunodominant epitopes, and the 16-kDa protein contained a highly immunodominant region at its N terminus. The immunodominant epitopes of the 30- and 32-kDa proteins in outbred guinea pigs were frequently identified in healthy purified-protein-derivative-positive or BCG-vaccinated individuals in previous studies. The immunodominant epitopes of these major extracellular proteins have potential utility in an epitope-based vaccine against tuberculosis.     

Synthetic peptides identify promiscuous human Th1 cell epitopes of the secreted mycobacterial antigen MPB70

MPB70 is a secreted protein of Mycobacterium bovis and Mycobacterium tuberculosis which stimulates both cellular and humoral immune responses during infection with bovine and human tubercle bacilli. In addition, vaccination with MPB70 has been shown to induce Th1 cell responses and protection in animal models of tuberculosis. The present study was carried out to map the dominant human Th1 cell epitopes of MPB70 in relation to major histocompatibility complex (MHC) class II restriction in healthy subjects showing strong T-cell responses to complex mycobacterial antigens. Peripheral blood mononuclear cells (PBMC) from HLA-DR-typed donors were tested with complex mycobacterial antigens (whole-cell M. tuberculosis and M. tuberculosis culture filtrates), with MPB70 purified from the culture filtrate of M. bovis BCG Tokyo, and with 13 synthetic peptides (25-mers overlapping by 10 residues) covering the sequence of MPB70. The donors that responded to the complex antigens and MPB70 also responded to the cocktail of synthetic MPB70 peptides. Testing of PBMC with individual peptides showed that peptides p5 (amino acids [aa] 61 to 85), p6 (aa 76 to 100), p8 (aa 106 to 130), p12 (aa 166 to 190), and p13 (aa 181 to 193) were most frequently recognized in proliferation and gamma interferon (IFN-gamma) assays. Testing of antigen-specific CD4(+) T-cell lines with the individual peptides of MPB70 confirmed that peptides p8, p12, and p13 contain immunodominant Th1 cell epitopes of MPB70. MHC restriction analysis with HLA-typed donors showed that MPB70 and its immunodominant peptides were presented to T cells promiscuously. The T-cell lines responding to MPB70 and peptides p8, p12, and p13 in IFN-gamma assays mediated antigen-peptide-specific cytotoxic activity against monocytes/macrophages pulsed with the whole-protein antigen or the peptides. In conclusion, the promiscuous recognition of MPB70 and its immunodominant peptide defined epitopes (aa 106 to 130 and 166 to 193) by IFN-gamma-producing Th1 cells supports possible application of this secreted antigen to subunit vaccine design.     

A comparison of the T cell delayed-type hypersensitivity epitopes of the 19-kD antigens from Mycobacterium tuberculosis and Myco. intracellulare using overlapping synthetic peptides.

Mycobacterial disease remains a serious international public health concern. Improved methods to rapidly and specifically detect mycobacterial infections would greatly enhance clinical management of these diseases. To define species-specific T cell epitopes that may be useful for the immunodiagnosis of mycobacterial infections, polymerized synthetic peptides from the 19-kD Mycobacterium tuberculosis and Myco. intracellulare protein homologues were tested in guinea pig DTH assays. Five Myco. tuberculosis and eight Myco. intracellulare peptides evoked skin test responses. Although all of the active Myco. tuberculosis and seven of the Myco. intracellulare peptides elicited non-specific DTH reactions, the peptide IN13 induced a Myco. intracellulare-specific skin test reaction, and thus represents a specific Myco. intracellulare T cell DTH epitope. This result suggests that the development of monospecific peptide-based immunodiagnostic reagents may be feasible for future clinical use.     

Duplex PCR for differential identification of Mycobacterium bovis, M. avium, and M. avium subsp. paratuberculosis in formalin- fixed paraffin-embedded tissues from cattle

We previously isolated and sequenced two genomic segments of Mycobacterium avium subsp. paratuberculosis, namely, f57, a species-specific sequence, and the p34 gene, coding for a 34-kDa antigenic protein. Comparison of sequences upstream of the p34 open reading frame (us-p34) from M. avium subsp. paratuberculosis and M. tuberculosis showed a 79-base deletion in M. tuberculosis. Sequence analysis of the p34 genes in another two species, M. bovis (strain BCG) and M. avium (strain D4), confirmed the differences observed between tuberculous and nontuberculous species. A duplex diagnostic PCR strategy based on coamplification of nonhomologous us-p34 and species-specific f57 sequences was therefore developed. Duplex PCR yielded three different patterns, specific either for tuberculous bacilli (M. tuberculosis, M. bovis, and M. africanum), for both nontuberculous mycobacteria M. avium and M. intracellulare, or for M. avium subsp. paratuberculosis. The specificity of this single-step DNA-based assay was assessed on DNA from cultured mycobacterial strains, as well as on a panel of formalin-fixed and paraffin-embedded tissues from cattle. Molecular assay results from tissular DNA were compared to conventional bacteriological and histological test results, including those obtained by Ziehl-Neelsen staining on tissue biopsy specimens. Molecular discrimination was successful and confirmed the value of duplex us-p34 and f57 sequence amplification for differential diagnosis of tuberculosis, paratuberculosis, or infections caused by other members of the M. avium complex.     

Evaluation of T-cell responses to peptides and lipopeptides with MHC class I binding motifs derived from the amino acid sequence of the 19-kDa lipoprotein of Mycobacterium tuberculosis

Cytotoxic T-lymphocyte (CTL) epitopes on the 19-kDa lipoprotein from Mycobacterium tuberculosis were identified by the use of lipopeptides and their cytokine profile studied. Selection of candidate CTL epitopes was based on synthetic peptides derived from the amino acid sequence of the 19-kDa lipoprotein showing major histocompatibility complex class I (MHC-I) binding motifs (H-2D(b) and H-2L(d)). Their ability to up-regulate and stabilize MHC-I molecules on the mouse lymphoma cell line RMA-S was studied. Similar studies were performed with peptides, in which the anchor amino acid of the H-2D(b) MHC-I motif was replaced by alanine. Three out of five peptides with H-2D(b) or H-2L(d) binding motifs and their corresponding lipopeptides as well, up-regulated and stabilized the H-2D(b) molecules on RMA-S cells. Replacement of the anchor amino acid residues of the H-2D(b) MHC-I motif by alanine revealed that the anchor amino acid asparagine at position 5, contributed more to binding of peptide to H-2D(b) molecules than leucine at position 11. The closely related lipopeptides LP19c and LP19d, in combination with incomplete Freund's adjuvant (IFA), induced CTL responses in C57BL/6 (H-2(b)) mice. These CTLs could recognize the naturally processed antigen, i.e. the 19-kDa antigen protein produced and processed by the EX-19 cell line. The capacity of the various lipopeptides to induce CTL correlated well with the ability of the (lipo)peptide to up-regulate and to stabilize H-2D(b) molecules. Lipopeptide LP19c primed spleen cells showed a T helper type one profile after in vitro stimulation with P19c and P19d 19 kDa peptides. The approach to characterize presumptive 19-kDa CTL epitopes might lead to selection of promising CTL epitopes, which can be applied in the development of subunit tuberculosis vaccines.     

H-2-associated effects of flanking residues on the recognition of a permissive mycobacterial T-cell epitope.

Previously we have identified an immunodominant, eight-residue, epitope core sequence (TAAGNVNI) from the 19,000 MW protein of Mycobacterium tuberculosis, which is recognized in the context of multiple H-2 I-A molecules. In this study, the role of residues flanking this T-cell epitope core was examined, using a series of 20 mer analogue peptides in which the native flanking residues were progressively replaced with L-alanine. Analogue peptides were tested for their capacity to stimulate a CD4+ 19,000 MW protein-specific T-cell line, revealing that all but one N-terminal flanking residue could be replaced collectively by alanine without significant loss of stimulatory activity. However, clear H-2-associated differences in the requirement for flanking residues were demonstrated with peptide-specific T-cell hybridomas. In particular, H-2d-derived hybridomas were much more stringent in their requirement for flanking residues than were H-2b hybridomas. All polyalanine-substituted peptides bound I-Ab molecules, with affinities similar to the native unsubstituted peptide. In contrast, significantly reduced binding to I-Ad was observed with several analogue peptides, although without a clear relationship to the degree of substitution. Furthermore, in H-2b mice, neither immunogenicity nor cross-reactivity with the native peptide showed a clear inverse relationship with respect to the degree of alanine substitution. The results presented in this paper indicate that flanking residues can influence T-cell specificity and that these effects may be controlled by major histocompatibility complex (MHC) haplotype.     

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.