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Previous Article | Next Article 
Infection and Immunity, October 2002, p. 5446-5453, Vol. 70, No. 10
0019-9567/02/$04.00+0 DOI: 10.1128/IAI.70.10.5446-5453.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Department of TB Immunology, Statens Serum Institute, Copenhagen, Denmark,1 Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands2
Received 30 January 2002/ Returned for modification 16 April 2002/ Accepted 17 July 2002
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The esat-6 family consists of 14 to 23 low-mass proteins, depending on the definition criteria used (18). Of the 23 proteins in this family at least 13 can be divided further into subfamilies due to high sequence relatedness of the molecules in the individual gene-protein subfamilies. One esat-6 subfamily is the Mtb9.9 family, consisting of five open reading frames (ORFs) (Rv1037c, Rv1198, Rv1793, Rv2346c, and Rv3619c), which was recently described by Alderson et al. (1). A second subfamily (the QILSS family [7]) consists of the five neighboring ORFs (Rv1038c, Rv1197, Rv1792, Rv2347c, and Rv3620c) that share individual identity on the protein level of >98%. Both of these families probably result from relatively recent gene duplications, as indicated by the high homology of the proteins (96 to 99% identity).
Here we analyze the T-cell epitopes on the TB10.4 antigen and describe the identification and characterization of two highly homologous proteins, TB10.3 and TB12.9, which together with TB10.4 constitute another subfamily within the esat-6 gene family. Although these molecules are highly homologous, our data indicate that the fine specificity of the T-cell responses to these molecules differ markedly, and very few T-cell epitopes are shared among the three family members. The possible role of these findings for antigenic variation in M. tuberculosis and as a potential mechanism for immune evasion is discussed.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preliminary experiments have shown that T-cell responses to TB10.4 and a mixture of all overlapping peptides are almost identical. The same has previously been demonstrated for two other esat-6 family members: ESAT-6 and CFP-10 (3). Therefore, we used a mixture of overlapping synthetic peptides of TB10.3 and TB12.9 as a substitute for whole protein.
Synthetic overlapping peptides (18- or 20-mer) covering the complete primary structure of the three proteins (see Fig. 4) were synthesized by standard solid-phase methods on an ABIMED peptide synthesizer (ABIMED, Langenfeld, Germany) at the Department of Infectious Diseases and Immunohematology/Bloodbank C5-P, Leiden University Medical Centre, Leiden, The Netherlands (TB10.4), or at Schafer-N, Copenhagen, Denmark. The peptides were purified by reverse-phase high-pressure liquid chromatography. Purified peptides were lyophilized and stored dry until reconstitution in phosphate-buffered saline (PBS).
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For this part of the study, the protocol (P136/97) was approved by the Ethics Committee of the Leiden University Medical Center.
Human donors. PBMC were obtained from 3 healthy nonvaccinated donors, 10 healthy BCG-vaccinated donors with a positive in vitro response to PPD, and 10 TB patients with microscopy- or culture-proven infection. Blood samples were drawn from TB patients 0 to 6 months after diagnosis. This part of the study was approved by the Local Ethical Committee for Copenhagen and Frederiksberg (RH 01-282/96 and KF 01-369/98).
Lymphocyte preparations and cell culture. PBMC were freshly isolated by gradient centrifugation of heparinized blood on Lymphoprep (Nycomed, Oslo, Norway) and stored in liquid nitrogen until use. The cells were resuspended in complete RPMI 1640 medium (Gibco-BRL/Life Technologies) supplemented with 1% penicillin-streptomycin (Gibco-BRL/Life Technologies), 1% nonessential amino acids (FLOW; ICN Biomedicals), and 10% heat-inactivated normal human AB serum. The viability and number of the cells were determined by Nigrosin staining. Cell cultures were established with 1.25 x 105 PBMC in 100 µl in microtiter plates (Nunc) and stimulated with 5 µg of PPD/ml, 5 µg of recombinant TB10.4 or single peptides/ml at final concentrations of 10, 5, or 0.5 µg/ml, or peptide pools corresponding to the full-length proteins in which each peptide was included at concentrations of 0.5, 0.1, or 0.05 µg/ml.
"No antigen" was included as a negative control, and PHA was used as a positive control (results not shown). Cell cultures were incubated for 5 days at 37°C (5% CO2, 95% air), and supernatants were harvested for cytokine analysis.
Cytokine analysis.
Gamma interferon (IFN-
) was detected with a standard sandwich enzyme-linked immunosorbent assay with a commercially available pair of monoclonal antibodies (Endogen) and used according to the manufacturer's instruction. Recombinant IFN- (also from Endogen) was used as a standard.
Enzyme-linked immunospot (ELISPOT) assay for IFN-.
We blocked 96-well polyvinylidene difluoride-packed plates (MAIPS 4510; Millipore) precoated with anti-human IFN- (Endogen) with 1% bovine serum albumin in PBS for 2 h at 37°C. Afterward, cells were added in different known concentrations, together with antigen (10 µg/ml), and then incubated 18 h at 37°C (5% CO2, 95% air). After being washed with 0.05% Tween 20 in PBS, anti-human IFN--Biotin (Endogen) secondary antibody was added, and the mixture was incubated for 2 h. Alkaline phosphatase-conjugated streptavidin (1:1,000 in PBS-1% BSA) was added and left for 1 h at 37°C. Plates were developed with nitroblue tetrazolium-BCIP (5-bromo-4-chloro-3-indolylphosphate; Sigma) and incubated for 15 min at 37°C in the dark.
Spots were counted under microscope and expressed as spot-forming units per 105 cells.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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response, and the number of TB10.4-specific cells was 40 to 45% of the frequency of cells responding to PPD (Table 1), thereby confirming the immuno-dominant nature of this antigen.
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The response induced in PBMC by the TB10.4-derived peptides were, as with the T-cell lines, highly heterogeneous (Fig. 3). The profile of T-cell responses to the TB10.4-derived peptides further demonstrated the presence of interindividual differences in the epitope recognition (Fig. 3).
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responses in at least one donor, and most donors recognized multiple peptides throughout the sequence (Fig. 3). The magnitude of the IFN- response to the dominant peptides was in many cases comparable to or even greater than the level induced by the peptide mixture and thus the recombinant protein.
The tendency found was that TB patients recognized a broader panel of epitopes than the BCG-vaccinated healthy donors. Donors in the BCG-vaccinated group had responses focused to one or two peptides (Fig. 3), whereas the TB patients often recognized several peptides and, for some donors, as many as six peptides. The overall dominant epitope for both groups was located at the N-terminal end of the protein (amino acids 1 to 18), and this region gave significant PBMC responses in the majority of the TB patients (eight of nine) and BCG-vaccinated donors (five of nine) (Fig. 3), whereas the recognition of this peptide was less pronounced when we analyzed T-cell lines (Fig. 2). In TB patients, p2 (amino acids 11 to 29) and p9 (amino acids 78 to 96) were also strongly recognized by several donors, with responses of >1,000 pg of IFN-/ml.
tb10.4 subfamily: homology and distribution. To identify other genes closely related to tb10.4 on the genome of M. tuberculosis and other mycobacterial species, a Southern blot analysis was performed. Genomic DNA from a number of mycobacterial strains was digested with PvuII and probed with tb10.4. This analysis resulted in three cross-hybridization bands in lanes corresponding to M. tuberculosis H37Rv, M. bovis, M. bovis BCG Danish, M. bovis BCG Pasteur, and M. kansasii (Table 2). In the more distantly related strains M. avium and M. intracellulare, we only observed one band, and in M. scrofulaceum, M. fortuitum, and M. szulgai no hybridizing bands were detected (Table 2).
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Furthermore, in agreement with the Southern blot analysis, a BLAST search of tb10.4 in the incomplete sequence databases of M. bovis (http://www.sanger.ac.uk) and the M. tuberculosis clinical isolate CSU#93 (http://www.tigr.org/tdb/) identified the presence of three TB10.4 family members (Tables 2 and 3). The corresponding protein sequences of TB10.4, TB10.3, and TB12.9 in CSU#93 and M. bovis were 100% identical to the H37Rv sequences except for the TB12.9 sequence of M. bovis, which was only 88.4% identical (Table 3).
Additionally, a search in the incomplete sequence database of the M. avium genome (http://www.tigr.org/tdb) identified only one ORF with high homology to tb10.4 (Table 2). This ORF was not identical to any of the three family members but had high homology to especially tb10.3 and tb10.4 (Table 3).
The results from the two approaches thus correlate well, and it is apparent that the TB10.4 family is conserved in the species belonging to the M. tuberculosis complex and species closely related to this one (e.g., M. kansasii), whereas only one candidate is present in the M. avium complex, and the rest of the strains lack this family completely.
T-cell recognition of TB10.4 family members. The fine specificity of the human T-cell recognition of the three closely related TB10.4 family members was analyzed by screening of a panel of overlapping peptides covering the complete sequences of TB10.3, TB10.4, and TB12.9 (Fig. 4).
PBMC from eight donors (four TB patients and four BCG-vaccinated donors) were used to compare the responses to the three sets of peptides (Fig. 5). Although the molecules exhibit a significant sequence homology, the recognition patterns found in the eight donors were quite different. The peptides from TB10.4 stimulated the largest responses, with IFN- levels to a single peptide in the range of 3,500 to 7,000 pg/ml (e.g., p1 [amino acids 1 to 18] in BCG3 and p3 [amino acids 21 to 38] in BCG1). For these donors with responses focused to one particular region, some recognition of the corresponding region in the other family members was found but, in general, minimal changes in the sequence translated into large differences at the stimulation level found with these peptides. In the highly conserved N-terminal region, the substitution of two amino acids—i.e., amino acids 12 and 13 (Leu-Gly substituted with Met-Ala)—in peptide 1 in TB10.3 reduced the IFN- release from BCG3 almost 10-fold, and changing four amino acids in the corresponding peptide from TB12.9 (Leu-Gly-His-Ala substituted with Trp-Ala-Asn-Val) completely ablated the recognition. Similarly, a substitution of three amino acids in peptide 6 (amino acids 54, 57, and 63) almost completely ablated the strong recognition of this epitope by PT3. The epitope mapping also resulted in the identification of a number of epitopes unique for TB10.3 such as peptide 8 (residues 71 to 90) in PT1 and BCG2. TB12.9 is the least similar among the three molecules, in particular due to the presence of an extra 51 C-terminal amino acids. This part of the molecule, which is lacking completely from TB10.4 and TB10.3, was also found to contain unique epitopes, especially peptide 8, recognized by two donors (BCG2 and PT4).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study we describe the identification and immunological evaluation of the TB10.4 family, consisting of TB10.3, TB10.4, and TB12.9, as a new subfamily of the esat-6 family. We used synthetic peptides to identify and compare the epitopes recognized in these proteins and found that especially TB10.4 contained a number of strongly recognized T-cell epitopes distributed throughout the protein sequence. The major epitopes recognized at the PBMC level were located in the N-terminal part of the molecule, but this region was less frequently recognized by T-cell lines, which may suggest that the skewing of responses occurs during in vitro propagation of a T-cell line. TB10.3 and TB12.9 were in general less antigenic than TB10.4 but contained T-cell epitopes, several of which were unique to these proteins. Sharing of the major T-cell epitopes by these proteins would be expected based on their highly homologous amino acid sequence, but the present study, together with recent observations from Alderson et al. (1), clearly demonstrate that the highly homologous proteins from the esat-6 family (Mtb9.9 and TB10.4 subfamilies) contain predominantly protein-specific epitopes and that shared epitopes are an exception. This finding underlines the highly specific binding of peptides to HLA-class II molecules and demonstrates that a difference of just one amino acid at critical positions may have dramatic consequences for T-cell recognition.
Expression of TB10.4 has been verified by N-terminal sequencing of the corresponding protein spot in a two-dimensional gel of a M. tuberculosis short-term culture filtrate fraction enriched in monoclonal antibody PV-2 (a monoclonal antibody that recognizes TB10.4) reactive protein (17). Although not formally demonstrated, the recognition of a number of unique epitopes on TB10.3 and TB12.9 also strongly suggests that these proteins are expressed by the bacteria during TB infection.
Pathogenic organisms will often use sequence polymorphism of highly immunogenic proteins to escape the host's immune defenses (9, 10, 16). This is not the case for M. tuberculosis as recently demonstrated by Musser et al. (11) and Streevatsan et al. (19), who found negligible sequence diversity in a large number of M. tuberculosis genes encoding important antigens recognized by the host immune system. This finding is in agreement with the recent finding of a lack of sequence diversity in the TB10.4 sequence originating from 13 clinical isolates of M. tuberculosis obtained from different geographical locations (unpublished observations).
An interesting possibility is that M. tuberculosis may compensate for this lack of sequence polymorphism by having duplicated the genes encoding major T-cell antigens, leading to several copies (homologues) of proteins that can substitute each other functionally but which differ in their immunodominant epitopes. Tightly controlled expression of these homologues may result in antigen variation and immune evasion. This hypothesis may be relevant for the whole esat-6 family, since the genomic organization of these proteins suggests that these proteins have similar functions. Apart from the esat-6 family, the immunodominant molecules within the antigen 85 complex in which the three molecules presumably have the same function in living bacteria could be examples of this phenomenon as well. In this regard, the TIGR database defines the three tb10.4 family genes plus esat-6 as one paralogous gene family (http://www.tigr.org), meaning that these genes possibly have been duplicated in M. tuberculosis during evolution. These duplication events may even involve whole clusters of genes, including regions surrounding the esat-6 family members such as PE and PPE genes as suggested by Cole et al. (7)
The lack of sequence homology to proteins and genes from other organisms illustrates that esat-6 family members have Mycobacterium-specific functions, and the need for several copies of the same gene may indicate that the genes encode function(s) of crucial importance for bacterial survival in the host. Using differential display for comparison of gene expression in M. tuberculosis H37Rv and the avirulent H37Ra, Rindi et al. (13) showed that TB10.4 was produced in the virulent strain but not in the avirulent strain, suggesting that this protein or gene may be involved in functions important for M. tuberculosis virulence. The presence of this gene in BCG (which is deleted of the esat-6-cfp10 operon) illustrates that attenuation can be obtained through the inactivation of various combinations of these different genes.
The availability of the M. tuberculosis genome and the current efforts to sequence a large number of additional mycobacterial genomes has set the stage for postgenomic approaches to the identification of novel antigens. The identification and characterization of protein-gene families is a very important outcome of these initiatives. If the gene families contain components that can substitute for each other functionally and represent mechanisms for immune evasion, the finding of markedly different epitope recognition of the individual components, as observed in the present study, may have important consequences for vaccine design. The extreme consequence may be that a successful TB vaccine will have to promote an immune response not only to a selected target antigen such as TB10.4 but also to epitopes characteristic for the rest of the closely related gene family. We are currently conducting experiments to address this important question.
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ACKNOWLEDGMENTS |
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We are grateful to Axel Kok-Jensen, Department of Pulmonary Diseases, Gentofte Hospital, Copenhagen, Denmark, for recruiting the patients and to Vita Skov for excellent technical assistance.
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FOOTNOTES |
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