Mycobacterium tuberculosis - Semantic Scholar

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Jul 30, 1997 - Xiaojiu Zhu, Hans J. Stauss1, Juraj Ivanyi and H. Martin Vordermeier ..... A. M., Deck, R. R., De Witt, C. M., Orme, I. M., Baldwin, S.,. D'Souza, C.
International Immunology, Vol. 9, No. 11, pp. 1669–1676

© 1997 Oxford University Press

Specificity of CD8F T cells from subunitvaccinated and infected H-2b mice recognizing the 38 kDa antigen of Mycobacterium tuberculosis Xiaojiu Zhu, Hans J. Stauss1, Juraj Ivanyi and H. Martin Vordermeier MRC Clinical Sciences Centre, Tuberculosis and Related Infections Unit, and 1Department of Immunology, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK Keywords: CD81, cytotoxic T lymphocyte, epitopes, Mycobacterium tuberculosis, tuberculosis

Abstract CD8F T cells have been implicated in protective anti-tuberculous immune responses, but little is known about the identity of mycobacterial antigens recognized by CD8F T cells. In this study we identified the Mycobacterium tuberculosis 38 kDa protein as a target for murine CD8F cytotoxic T lymphocytes (CTL) which were induced by vaccination of C57BL/6 mice with DNA delivered with a plasmid, with transfected tumour cells or by infection with tubercle bacilli. Using overlapping synthetic peptides covering the whole protein sequence, peptides predicted to contain H-2Kb or H-2Db motifs, as well as naturally processed peptides, we were able to identify CTL epitopes. Differences were demonstrated in peptide specificity between CTL from immunized or M. tuberculosis-infected mice. The identified CTL epitopes could be important for future analysis of the involvement of CD8F T cells in M. tuberculosis infections and for vaccine development. Introduction Pathogenic mycobacteria replicate intracellularly within macrophage phagosomes, which has for a long time led to the assumption that MHC class II-restricted CD41 T lymphocytes, producing type 1 cytokines, constitute the major protective effector population. However, a number of studies have made the case for an important role for CD81 T cells in antituberculous protective immunity in both mice and humans. Murine mycobacteria-specific CD81 T cell lines which were cytolytic in vitro for macrophages infected with tubercle bacilli (1) or protected mice against Mycobacterium lepraemurium infection have been described (2,3). Mice devoid of MHC class I-restricted CD81 T cells due to β2-microglobulin gene disruption died quickly after M. tuberculosis infection (4), whilst in vivo depletion of CD81 T cells with antibodies resulted in impaired resistance to M. tuberculosis infection (5) and decreased protection in Mycobacterium w vaccinated mice (6). Human mycobacteria-specific CD81 T cells have also been described (7,8), CD81 T cell clones have been established (9) and the case of a patient with isolated CD81 T cell

deficiency who suffered from frequent tuberculosis relapses has been reported (10). A protective role of CD81 T cells in human tuberculosis has been argued on the grounds that the number of CD81 T cells was significantly elevated in sections from patients with lymph node tuberculosis, a form of disease which can resolve without chemotherapy (11,12). CD81 T cells were not only present in large numbers within granulomata but also formed a sheath layer at the border between the granuloma and the lymphocytic mantle (11) which might indicate that CD81 T cells are instrumental in limiting the immunopathologic process. This is also supported by the description of hugely enlarged granulomata in M. tuberculosis infected β2-microglobulin knockout mice devoid of CD81 T cells (4). Data on the identity of mycobacterial antigens recognized by CD81 T cells is limited and very few cytotoxic T lymphocyte (CTL) epitopes have been characterized. Identification of such antigens and their respective peptide epitopes could be useful for investigating the precise role of CD81 T cell responses in tuberculosis and could also lead to the develop-

Correspondence to: H. M. Vordermeier Transmitting editor: P. C. L. Beverley

Received 28 April 1997, accepted 30 July 1997

1670 Mycobacteria-specific CD81 CTL ment of effective tuberculosis vaccines. In this study we identified the M. tuberculosis 38 kDa glyco-lipoprotein (13,14) as a target molecule for CD81 CTL responses and determined its peptide epitopes in H-2b mice. Our results indicate major variations in the epitope specificities recognized after different modes of immunization and infection.

(RPMI 1640 supplemented with 10% FCS, 50 µg/ml gentamicin, 2 mM L-glutamine and 5310–5 M 2-mercaptoethanol) in round-bottomed 96-well plates for 1 h at 37°C as described earlier (17). The cells were then washed and stained with the mAb HB11 (anti-H-2Kb) and HB27 (H-2Db). Bound mAb were visualized by a FITC-labelled rabbit anti-mouse IgG serum and analysed by FACS (Epics; Coulter, Dunstable, UK)

Methods

Immunization procedures

Mice

DNA vaccination. Mice were injected 3 times at intervals of 2 weeks with 100 µg of pXJ38 or control DNA vector in the quadriceps muscle. Spleen cells for CTL analysis were prepared 2 weeks after the last injection.

Female C57BL/6 (H-2b) mice were purchased from the Biological Services Unit, Royal Postgraduate Medical School, Hammersmith Hospital, London and used in the experiments when 7–10 weeks old. Synthetic peptides Peptides were synthesized by simultaneous multiple peptide synthesis using Fmoc technology as described earlier (15). Homogeneity was confirmed by analytical HPLC and, for the majority of peptides, by mass spectrometry. A set of pinsynthesized peptides (PEPSCAN, peptides: 10mers overlapping by six residues) covering the complete sequence of the 38 kDa protein was purchased from Chiron Mimotopes (Clayton, Australia). Plasmids The gene encoding the 38 kDa protein was cloned into the expression vector pcDNA3 (Invitrogen, San Diego, CA) under the control of the cytomegalovirus promoter. The plasmid pXJ38 was obtained by subcloning a 1.2 kb NdeI–BamHI fragment derived from pMS10.4 (16), that encoded the 38 kDa protein of M. tuberculosis, into the EcoRV site of pcDNA3. Cell transfection Transfectants EX38 and RX38 were generated by transfecting EL4, a chemically induced thymoma of C57BL/6 origin, and RMA, a Rauscher virus-induced T cell lymphoma of C57BL/ 6 origin with plasmid pXJ38. All transfectants were selected with G418 at a concentration of 1 mg/ml and cloned twice by limiting dilution before being used in the experiments. Expression of the 38 kDa protein was confirmed by RT-PCR and Western blotting of cell lysates using the 38 kDa antigenspecific mAb TB71 (13). Recombinant vaccinia virus A recombinant vaccinia virus expressing the 38 kDa protein (Vac38) was generated by inserting the coding sequence of the 38 kDa protein into the vaccinia virus recombinant plasmid pSC11 resulting in the plasmid pSC11/38. Recombinant vaccinia viruses were produced by transfection of pSC11/38 into 143 cells infected with wild-type vaccinia virus followed by selection for recombinant viruses. Vac38 was purified by repeated plating (4 times) and protein expression was confirmed by Western blot using the mAb TB71 (13). Peptide-binding assay RMA-S cells were incubated overnight at room temperature to enhance class I MHC molecule expression and then cultured in triplicates with 200 µM peptide in culture medium

Transfectant immunization. C57BL/6 mice were immunized by i.p. injection of 53106 EX38 transfectant cells in 100 µl PBS and boosted once or twice with the same dose. Spleen cells were harvested 2 weeks later. Infection with M. tuberculosis H37Rv. Animals were injected with 106 viable organisms by i.p. injection and spleen cells prepared 6 weeks later. Induction of CTL Splenocytes from immunized or vaccinated mice (see above) were cultured in 24-well plates at a density of 53106/ well. Complete culture medium contained RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine and 5310–5 M 2mercaptoethanol. Then, 53105 irradiated RX38/ well were added to the splenocytes from M. tuberculosis infected or nucleic acid vaccinated mice. Cells from mice immunized with tumour cells were stimulated with 53106 p.f.u. of Vac38/ well. After 5–7 days, responding cells were used in 51Cr-release assays. To produce CTL lines, T cells were restimulated every second week by culturing 53104 responder cells/well together with 53105 EX38/well. Each well also received 43106 irradiated syngeneic splenocytes as feeders and 10 U/ml rIL-2 (Genzyme, Cambridge, MA). To produce clones from these lines, T cells were then plated in 96-well round-bottom plates at 0.5, 5 and 20 cells/well with 104 EX38/ well, feeder cells (23105 cells/well) and rIL-2 (10 units/well). CTL assay Target cells EL4, RMA, RX38 or EX38 were labelled with 51Cr (50 µCi; Amersham, Amersham, UK) at 37°C for 1 h. Then, 53103 labelled target cells were plated into wells of 96well round-bottom plates which contained serial dilutions of effector T cells in a total volume of 200 µl/well. After 4–6 h incubation at 37°C, 100 µl supernatant/well was collected and radioactivity counted in a LKB γ-counter. Synthetic peptides derived from the sequence of the 38 kDa protein were tested by incubating 100 µM of each peptide with 53103 51Crlabelled EL4 cells for 1 h before effector cells were added. HPLC separated peptides from whole cell lysates were tested by incubating 10 µl peptide solutions/fraction with 53103 51Cr-labelled EL4 cells for 1 h before addition of the effector cells. In some assays, blocking anti-CD4 mAb (clone RM4-5) or anti-CD8 mAb (clone 53-6.7) (both from PharMingen, San Diego, CA) were added (5 µg/well) to the effector cell population 30 min before 51Cr-labelled target cells were added

Mycobacteria-specific CD81 CTL 1671

Fig. 1. Expression of mycobacterial 38 kDa protein in murine cell lines. Lysates of EX38, EL4, RX38, RMA and Vac38-infected cells were separated on a 12% SDS–PAGE, transferred onto a nitrocellulose membrane and stained with the mAb TB71 (13). Lane 1, mol. wt standards; lane 2, M. tuberculosis strain H37Rv culture filtrate; lane 3, EX38; lane 4, EL4; lane 5, RX38; lane 6, RMA; lane 7, Vac38-infected cells. Arrows indicate 38 kDa protein expressed in eukaryotic cells.

higher mol. wt (40 kDa) than that of the protein found in M. tuberculosis culture filtrates. When we stained these membranes for carbohydrate components we found that the 38 kDa protein, when expressed by eukaryotic cells, is not glycosylated (data not shown). This is in contrast to the protein expressed in mycobacteria, which has been described as glycoprotein (20). It is possible that the prokaryotic secretion signal peptide, which is removed during secretion into bacterial culture supernatants, is retained in the eukaryotically expressed protein, thus accounting for its higher mol. wt. The second, smaller, band of ~35 kDa could be a degradation product. We subsequently used these reagents to (i) immunize mice (EX38), (ii) to expand CTL effector cells in vitro (EX38, RX38 and Vac38) and (iii) as 51Cr-labelled target cells in CTL assays (EX38 and RX38). The 38 kDa protein is a target for CD81 CTL

(18). Specific lysis 5 (experimental release – spontaneous release/maximum release – spontaneous release)3100%. Spontaneous release was 10% or less. Peptide elution from cell lysates Peptides were eluted from EL4 and EX38 cells and separated by HPLC. Cells (2.53108) were suspended in 3 ml 0.7% trifluoroacetic acid (TFA) and ultrasonicated (Soniprep150) 3 times each for 30 s, followed by centrifugation at 27,000 g, 4°C for 1 h. The supernatants were subjected to centrifugation in Centricon filters (10 kDa cut-off) at 5000 g, 4°C for 3 h. The filtrates containing peptides ,10 kDa were separated on a reverse-phase HPLC column (SuperPac Pep-S, column 25034.0 mm, C18; Pharmacia LKB, Bromma, Sweden). Buffer A was 0.1% TFA in H2O and Buffer B was 0.1% TFA in acetonitrile. For peptide elution, the acetonitrile concentration was increased from 0 to 40% at a gradient of 1%/min. Dried fractions were dissolved in 100 µl PBS and sonicated for 5 min before used in CTL assays. Cytokine ELISA In vitro IFN-γ and IL-4 production in culture supernatants was determined by ELISA using mAb from PharMingen as described earlier (19). Results Tumour cell lines and recombinant vaccinia viruses expressing the mycobacterial 38 kDa protein Transfection of the MHC class II-negative tumour cell lines EL4 and RMA (both H-2b) with the plasmid pXJ38, carrying the gene encoding for the M. tuberculosis 38 kDa protein, produced stable transfectants designated EX38 and RX38 respectively. A recombinant vaccinia virus also expressed this protein (Vac38). In order to demonstrate protein expression, lysates of EX38, RX38 and of cells infected with Vac38 were subjected to SDS–PAGE and immunoblots developed with the 38 kDa protein-specific mAb TB71 (13). The results demonstrated that both EX38 and RX38, as well as cells infected with Vac38, express high levels of the mycobacterial protein (Fig. 1). The 38 kDa antigen expressed in eukaryotic cells was detected as two distinct bands, one with a slightly

We employed three immunization strategies to induce CTL in C57BL/6 mice, i.e. i.p. injection of the transfected line EX38, nucleic acid immunization with pXJ38 and i.p. infection of mice with tubercle bacilli. All three approaches were successful since we detected strong CTL responses in each case (Fig. 2A–C). Only mAb specific for CD8 effectively blocked specific lysis, whilst mAb to CD4 did not (Fig. 2D). In vitro stimulation of naive spleen cells with RX38 or EX38 for the same periods did not lead to significant cytolytic responses (data not shown). Identification of peptide epitopes recognized by CD81 CTL The specificity of long-term lines derived from the CTL populations described in the preceding section was tested using a collection of 92 10mer peptides, overlapping by six residues which covered the whole sequence of the 38 kDa protein (PEPSCAN).The specificity patterns of CTL derived from EX38and DNA-vaccinated mice showed differences (Fig. 3): EX38induced cells recognized peptides in the regions of amino acid residues 129–138 and 312–322 (Fig. 3A), whereas the nucleic acid vaccine-induced CTL recognized only peptides in the area between residues 129 and 138 (Fig. 3B). In view of possible structural limitations of this set of peptides, e.g. non-optimal length, insufficient quantities, gaps in sequence coverage due to six residue overlap, we examined also a set of 12 peptides which were predicted to contain H-2Kb or H2-Db binding motifs (21) (for sequences see Table 1). These predictions were validated using the RMA-S cellular binding assay which showed that all peptides bound to either H-2Kb or H2-Db or both (Table 1). These peptides were now used to sensitize EL4 cells in 51Cr-release assays employing effector cell lines derived from DNA vaccinated, EX38 vaccinated or infected mice. The results are shown in Table 1, and demonstrate that CTL from EX38-immunized mice recognized peptides with amino acid residues 129–137, 191–199, 225–234, 309–318, 312–319 and 317–325, whereas CTL from DNA-vaccinated mice recognized peptides 129–137, 243–250 and 312–319, but CTL from M. tuberculosis-infected mice recognized only one peptide (residues 225–234). The peptide recognition patterns of these CTL lines were established repeatedly over a period of several weeks and proved to be stable. Subsequently, 26 T cell clones were established from the

1672 Mycobacteria-specific CD81 CTL

Fig. 2. Induction of CD81 CTL. (A) CTL from EX38 immunized mice: 2 weeks after the last vaccination, spleen cells were stimulated in vitro with Vac38 for 5 days. 51Cr-release assay targets: RX38 and RMA. (B) CTL from DNA vaccinated mice: 2 weeks after last immunization, spleen cells were stimulated in vitro for 7 days with EX38. Targets: EX38 and EL4. (C) CTL from M. tuberculosis-infected mice: in vitro stimulation for 2 weeks with EX38 was carried out 6 weeks after infection. Targets: EX38 and EL4. (D) CTL activity can be blocked with mAb to CD8. Lines established from CTL induced by immunization with transfectant vaccination (EX38), DNA vaccination (DNA) and M. tuberculosis infection (TB) by two to three rounds of in vitro stimulation with EX38 were incubated with 51Cr-labelled EX38 in the presence of anti-CD4 or anti-CD8 mAb. E:T ratio: 5:1 or 3 :1.

CTL line derived from EX38 vaccination which recognized the widest range of tested peptides. These clones recognized EX38, but not EL4 cells alone. They could be divided broadly into three groups on the grounds of their peptide specificity and the results of three representative clones are given in Table 2. The vast majority (23 of 26, represented by clone 6R.7) recognized peptide 129–138, two of 26 recognized peptide 312–319 (represented by clone 6R.44), whereas one clone (6R.27) recognized only the EX38 transfectant, but none of the predicted motif peptides. Upon stimulation with EX38, all clones tested, regardless of specificity, secreted large amounts of IFN-γ, with no or only small amounts of IL-4 (Table 2), which corresponds to a Tc1 profile.

immunized animals (see above and Table 2). Results obtained with three representative clones are shown in Fig. 4. We found that the majority of clones (represented by clone 6R.7), with specificity for peptide 129–138, recognized HPLC fraction 29, whereas clones specific for peptide 312–319 (represented by clone 6R.44) reacted with fraction 31. This indicates that the peptides recognized by these clones were produced by natural processing. Interestingly, the clone 6R.27, which recognized none of the motif peptides (Table 2), was nevertheless able to lyse cells sensitized with HPLC fraction 29, thus indicating that more than one, yet to be described, 38 kDaderived peptide is present in this fraction (Fig. 4).

Recognition of naturally processed peptides

Discussion

To complement the epitope analysis, naturally processed peptides from EX38 cells were isolated. The cells were lysed in acid, which results in the denaturation of MHC molecules, and lower mol. wt peptides were separated by HPLC. HPLC fractions were collected (data not shown), dried by vacuum and tested with the T cell clones established from EX38

The results in this paper demonstrate that the M. tuberculosis 38 kDa glyco-lipoprotein is a target for murine MHC class Irestricted CD81 CTL and therefore suggest that this protein gains access to the MHC class I presentation pathway. The specificity of CD81 T cells in mycobacterial infections has previously not been determined aside from reports describing

Mycobacteria-specific CD81 CTL 1673 Table 1. Peptide recognition by effector CTL induced by immunization and infection Binding toa

Peptide sequence

74–81 129–137 166–175 191–199 225–234 243–250 264–273 291–300 309–318 312–319 317–325 350–357 Medium only

ERYPNVTI AQQVNYNLP IAALNPGVNL DTFLFTQYL ALGENGNGGM GCVAYIGI AQLGNSSGNF KTPANQAISM YPIINYEYAI INYEYAIV AIVNNRQKD DQVHFQPL

Specific lysis (%) of peptide-pulsed EL4b

H-2Kb

H-2Db

EX38/1

DNA/1

TB/1

11 – 11 – – 11 – – 11 111 – 11 –

11 11 111 1 1 11 11 11 11 – 11 1 –

7 35 2 33 30 1 2 4 45 30 20 3 2

9 47 8 9 7 27 3 4 10 28 3 5 8

10 7 7 3 24 2 –1 6 2 3 –2 –2 5

aRMA-S binding assay as described in Methods. Mean fluorescence with peptide/mean fluorescence medium 5 3–5.9, 1; 6–10, 11; .10, 111. b51Cr-release assay as described in Methods. C57BL/6-derived effector CTL populations: EX38/15 induced by immunization with transfected EX38; DNA/15 induced by nucleic acid vaccination; TB/15 induced by M. tuberculosis infection. E:T ratio 5 10:1. Positive values in bold.

Fig. 3. Mycobacterial epitopes recognized by CTL. (A) Effector CTL induced by EX38 immunization. (B) Effector CTL induced by DNA vaccination. Ninety-two synthetic peptides were used (10mers overlapping by six residues). E:T ratio 5 10:1. Targets: peptidepulsed 51Cr -labelled EL4.

the mycobacterial stress protein hsp65 and some of its epitopes (22,23). Indeed, this paper is the first study to conduct a comprehensive analysis of the constituent CTL epitopes. Antigen-specific CTL responses were induced by immunization with either transfected cells or DNA vaccination. These data corroborate the induction of CTL and protective immunity by mycobacterial hsp65 transfectant immunization (24,25), or nucleic acid vaccination with mycobacterial hsp65 or Ag85A (23,26). We have recently demonstrated that the 38 kDa DNA vaccine is inducing protective immunity in vaccinated mice

to challenge with virulent tubercle bacilli resulting in a reduction of bacterial loads by up to 1 log, which is of the same order of magnitude as that following BCG vaccination (27). Nucleic acid vaccination by intramuscular injection is characterized by antigen expression in muscle cells. However, recent studies have demonstrated that antigen is transferred from myocytes to bone marrow-derived antigen-presenting cells (APC) which are necessary to induce a CTL response after DNA vaccination (28–30). Antigen taken up by the APC may be processed via an exogenous pathway of class I presentation (31), which might result in the observed distinct CD8 peptide specificity induced by DNA immunization. Intracellular tubercle bacilli are contained in phagosomes (32,33) and do not enter the cytosol. Yet M. tuberculosis infection evokes transport of antigens like ovalbumin to the cytosol followed by TAP-dependent presentation to CD81 T cells (34). Restricted release of mycobacterial constituents out of phagosomal vacuoles results in limited intersection with the endocytic pathway (35,36) and antigens, like the 38 kDa protein, which are actively secreted by the pathogen, might be preferentially included in such vesicles. These mycobacterial antigens could also be processed in phagocytic compartments and enter a recently described phagosome-to-cytosol pathway (37). Alternatively, processed peptides could be regurgitated into the media to bind to surface class I molecules (38). Infection of macrophages with tubercle bacilli can inhibit to a certain degree MHC class II antigen processing (39,40), concomitant with facilitated MHC class I presentation to CD81 CTL (34). Our results demonstrating diverse CD8 peptide specificities suggest at least quantitative differences in peptide presentation between infected and transfected target cells. We used three approaches to identify the peptide sequences of CTL epitopes, i.e. (i) an empirical method using overlapping peptides, (ii) prediction of sequences on the basis of well established motifs (reviewed in 21) and (iii) elution of naturally processed peptides from transfected tumour cells.

1674 Mycobacteria-specific CD81 CTL

Fig. 4. Recognition of naturally processed peptides by CTL clones. CTL clones derived from EX38 vaccinated mice were tested with fractions 20–43 of HPLC purified naturally processed peptides. E:T ratio 5 10:1. Targets: peptide-pulsed 51Cr-labelled EL4. Lysis of EL4 without peptides 3 or less. Lysis of EL4 without effectors is indicated (no CTL).

Table 2. Specificity and lymphokine profile of CTL clones derived from EX38 immunization T cell clone

6R.7c 6R.27 6R.44

Specificity

129–138 EX38 312–319

Specific lysis (%) ofa

Lymphokine productionb

EX38

EL4/peptide

IFN-γ (pg/ml)

IL-4 (pg/ml)

45 76 32

53 (p129–138) ø3 (any peptide) 20 (p312–319)

5930 5880 5970

46 17 115

aPercent 51Cr-labelled EX38 or EL4 target cells pulsed with ‘motif’ peptides (see Table 1 for sequences) were incubated with the CTL clones at an E:T ratio of 5:1. Peptide specificities of clones are indicated in brackets. Lysis of EL4 cells without peptides was 3% or less for all clones. Lysis of EL-4 with any peptide alone was 1.5% or less. bIFN-γ and IL-4 in supernatants was determined by ELISA after 2 days of culture in the presence of EX38. cRepresentative of 23 out of 26 CTL clones.

The comprehensive procurement of overlapping peptides to cover all specificities would carry a prohibitive cost. Therefore, it is gratifying that prediction of epitopic peptides was successful. The predictive power of these motifs was proved by the fact that almost all predicted peptides used in this study did bind to H-2Kb or H2-Db molecules. Several peptides were found to bind to both H-2Kb and H-2Db. This is an interesting observation given the allele specificity of the predictive motifs (21), though such binding characteristics have been reported for peptides derived from the papilloma virus E6 and E7 proteins (17). We deliberately synthesized peptides which did not always have perfect motifs, e.g. p129–138, which proved to be the most prominently recognized determinant in this study. Hence, a strict adherence to the motif sequences would have missed this epitope. Other ligands not fitting to a standard motif have been recently described (41). The third approach, elution of peptides from target cell lines followed by HPLC separation, confirmed that the two major epitopes identified by motif peptides are also processed in vivo. Could peptides, such as those described in this study, be used in peptide-based vaccines? One precedent is a CTLinducing peptide from Listeria monocytogenes which protects mice against lethal infection (42). It is also encouraging that all T cell clones analysed in this paper, regardless of their

specificity, produced large amounts of IFN-γ, a cytokine believed to be of critical importance in the protective antituberculous immune responses (43,44). We are now testing the protective capacity of the peptides described in this study using ‘minigene’ constructs expressed either as DNA vaccine or as recombinant vaccinia virus (45–47). However, in view of the polymorphism of HLA alleles, it will almost certainly be necessary to include several different peptides to allow effective coverage of the whole population. In view of the importance of CD41 T cells in anti-tuberculous immunity, inclusion of epitopes recognized by CD41 T cells will also be mandatory. Most immunodominant mycobacterial CD4 epitopes are recognized in the context of multiple MHC class II alleles (48) and promiscuous peptides recognized in the context of multiple class I alleles have also been described (49,50). However, the extent of genetic restriction of CD81 T cell responses against the 38 kDa protein is yet to be determined. Recent comparative data on peptide binding to various human class I MHC molecules suggested that the majority of HLA-A and -B alleles world-wide can be grouped into four major HLA supertypes (supermotifs) defined by their broad peptide binding specificities (51). Thus, it is hoped that MHC class I polymorphism may be functionally much more limited, enabling broad population coverage using fewer

Mycobacteria-specific CD81 CTL 1675 peptides than originally forecast. Nevertheless, whilst the identification of the relevant peptides may contribute to the development of a potent subunit vaccine, it may be prudent to acknowledge that this objective is associated with a range of complexities concerning, e.g. the optimal structural formulation and modes of vaccine delivery.

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Abbreviations APC CTL TFA

antigen-presenting cell cytotoxic T lymphocyte trifluoroacetic acid

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