Identification of an HLA-A*0201-Restricted T-Cell Epitope on the MPT51 Protein, a Major Secreted Protein Derived fromMycobacterium tuberculosis, by MPT51 Overlapping Peptide Screening

Abstract

CD8؉ T cells play a pivotal role in protection against Mycobacterium tuberculosis infection. We identified a novel HLA-A*0201-restricted CD8؉ T-cell epitope on a dominant secreted antigen of M. tuberculosis, MPT51, in HLA-A*0201 transgenic HHD mice. HHD mice were immunized with plasmid DNA encoding MPT51 with gene gun bombardment, and gamma interferon (IFN-␥) production by the immune splenocytes was analyzed. In response to overlapping synthetic peptides covering the mature MPT51 sequence, the splenocytes were stimulated to produce IFN-␥ by only one peptide, p51-70. Three-color flow cytometric analysis of intracellular IFN-␥ and cell surface CD4 and CD8 staining revealed that the MPT51 p51-70 peptide contains an immunodominant CD8؉ T-cell epitope. Further analysis using computer algorithms permitted identification of a bona fide T-cell epitope, p53-62. A major histocompatibility complex class I stabilization assay using T2 cells Tuberculosis (TB) is still a major cause of death due to infectious disease worldwide. There were an estimated 8.8 million new cases in 2005, and 1.6 million people died of TB (37). The problem of TB is increasing worldwide due to several factors, including the prevalence of multi-drug-resistant strains and coinfection with human immunodeficiency virus (23). The only TB vaccine currently available is the attenuated Mycobacterium bovis strain bacillus Calmette-Guérin (BCG), yet its efficacy against pulmonary TB in adults has been controversial (32). Therefore, there is an urgent need for an improved vaccine for TB (16).Cell-mediated immunity plays a pivotal role in the control of Mycobacterium tuberculosis infection. There is mounting evidence that CD4 ϩ type 1 helper T lymphocytes (Th1) are involved in the development of resistance to this disease, primarily through the production of macrophage-activating cytokines, such as gamma interferon (IFN-␥) and tumor necrosis factor alpha. In addition, CD8 ϩ cytotoxic T lymphocytes (CTL) contribute to disease resistance since susceptibility to M. tuberculosis is increased in mice with a deficiency in CD8 ϩ T cells (17,18,31).To design a new generation of vaccines, information on the antigenic make-up of M. tuberculosis must be obtained in order to identify immunodominant proteins and epitopes. Secreted and surface-exposed cell wall proteins seem to play a pivotal role in the induction of protective cellular immunity against TB (2, 4). The mouse model of TB infection revealed that memory cells from immune mice produced substantial amounts of IFN-␥ in response to two fractions of culture filtrate of M. tuberculosis, represented by 6-to 10-kDa proteins and the antigen 85 (Ag85) complex, a 30-to 32-kDa protein family (3).The Ag85 complex (Ag85A, Ag85B, and Ag85C), which has mycolyltransferase activity in cell wall synthesis and in the biogenesis of cord factor (5) and the ability to bind to fibronectin (1), has been shown to be a major fraction of the secreted proteins of M. tuberculosis (35). Another major secreted protein, MPT51, was demonstrated t...

The diagnostic potential of MPT63‐derived HLA‐A*0201‐restricted CD8+T‐cell epitopes for active pulmonary tuberculosis

“…Firstly, we synthesized 10 MPT63-derived peptides which are potentially restricted by HLA-A Ã 0201 and measured their binding affinity for HLA-A Ã 0201 molecules on the basis of T2 cell line. In vivo immunization experiments revealed that among five high-affinity peptides, two (MPT63 [18][19][20][21][22][23][24][25][26] and MPT6 ) triggered cytotoxic T-cell response whereas the remaining three high-affinity peptides failed to induce IFN-g-secreting T-cell responses. Conversely, three low-affinity peptides (MPT63 [10][11][12][13][14][15][16][17][18][19] , MPT63 [20][21][22][23][24][25][26][27][28] and MPT63 [29][30][31][32][33][34][35][36][37] ) all exhibited immunogenicity in HLA-A Ã 0201 transgenic mice.…”

“…Interestingly, four identified overlapping epitopes (MPT63 [5][6][7][8][9][10][11][12][13][14] , MPT63 [10][11][12][13][14][15][16][17][18][19] , MPT63 [18][19][20][21][22][23][24][25][26] and MPT63 [20][21][22][23][24][25][26][27][28] ) are located in a signal peptide sequence (residues 1-29) of MPT63 and span a long peptide with 24 amino acids (residues 5-28, TMIKTAVAVVAMAAIATFAAPVAL). In our previous study, we identified four overlapping HLA-A Ã 0201-restricted CD8 + T-cell epitopes in an amino acid sequence of M. tuberculosis-specific antigen PPE68 (31).…”

“…As shown in Figure 4 and Table 2, all patients responded to one or more immunogenic peptides, the percentage of responding patients varying between 45% and 73%. The percentages of TB patients responding to MPT63 [5][6][7][8][9][10][11][12][13][14] , MPT63 [10][11][12][13][14][15][16][17][18][19] , MPT63 [18][19][20][21][22][23][24][25][26] , MPT63 [20][21][22][23][24][25][26][27][28] and MPT63 [29][30][31][32][33][34][35][36][37] were 63%, 73%, 45%, 60% and 68%, respectively. The combined diagnostic sensitivity of these five immunogenic peptides was 93%.…”

Immune Responses Against Mycobacterium

“…Most HLA-A*0201-restricted T-cell epitopes were nonamer peptides (Falk et al, 1991, Parker, 1994, but some epitopes were decamer peptides. We reported an immunodominant HLA-A*0201-restricted T-cell epitope in MPT51 antigen (Aoshi et al, 2008; Tables 1, 2). Main anchor amino acid positions are, position 2 (Leu) and position 9 (Val), which were conserved in MPT51 p53-62 (TLAGKGISVV) ( Table 2).…”

MHC Polymorphism and Tuberculosis Disease

“…More recently, using HLA-peptide tetramers derived from Mtb peptides predicted to bind to HLA-A*0201, Tang and his colleagues have found a very interesting Mtb epitopes activating polyfunctional CD8+ T cells in human TB (Tang et al, 2011). Moreover, some specific www.intechopen.com peptides to CD8 T Lymphocytes as HLA-B*35-restricted CD8(+) T-cell epitope in Mtb Rv2903c (Klein et al, 2002), HLA-A*0201-restricted T-cell epitope in the MPT51 protein (Aoshi et al, 2008) and HLA-B*35-restricted CD8 T cell epitopes in the antigen 85 (aa 204-212) WPTLIGLAM (Klein et al, 2001), were demonstrated to have a potential positive effect on Mtb -infected macrophages and produce significant level of gamma interferon and tumor necrosis factor alpha.…”