Mapping of Th1-Cell Epitope Regions of Mycobacterium tuberculosis Protein MPT64 (Rv1980c) Using Synthetic Peptides and T-Cell Lines from M. tuberculosis-Infected Healthy Humans

Objective: To identify T helper 1 (Th1)-cell stimulating and HLA-promiscuous peptides of MPT64 (Rv1980c), a major secreted antigen of Mycobacterium tuberculosis. Materials and Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from 35 healthy subjects and typed for HLA-DR molecules using genomic methods. To identify subjects infected with M. tuberculosis, PBMCs were tested in antigen-induced proliferation assays with whole cells and culture filtrate antigens of M. tuberculosis, M. tuberculosis-specific antigens ESAT-6 and CFP10, and MPT64. Culture filtrate-induced T-cell lines were established in vitro from 12 M. tuberculosis-infected and HLA-heterogeneous healthy subjects, and tested with 20 overlapping synthetic peptides covering the sequence of MPT64 in Th1-cell assays, i.e. antigen-induced proliferation and/or IFN-γ secretion. In addition, T-cell lines from three HLA-heterogeneous subjects were tested for cytotoxic activity against peptide-pulsed antigen-presenting cells. Results: PBMCs from 12 of 35 subjects responded to M. tuberculosis-specific antigens ESAT-6 and CFP10 as well as to MPT64, which suggested that they were infected with M. tuberculosis. Ten of twelve T-cell lines established from these donors responded to MPT64, and nine T-cell lines responded to 1 or more of the peptides of MPT64 in anti- gen-induced proliferation assays. Furthermore, 18 of the 20 peptides of MPT64 were recognized by the T-cell lines in 1 or more assay systems, and at least 5 peptides were recognized by T-cell lines from HLA-DR-heterogeneous subjects. Conclusion: Th1-cell-reactive epitopes are scattered throughout the sequence of MPT64, and at least 5 of its peptides are presented to Th1-cells in a HLA-promiscuous manner.

[1]  Harald G. Wiker,et al.  Synthetic Peptides Identify Promiscuous Human Th1 Cell Epitopes of the Secreted Mycobacterial Antigen MPB70 , 2003, Infection and Immunity.

[2]  A. Mustafa.  Recombinant and synthetic peptides to identify Mycobacterium tuberculosis antigens and epitopes of diagnostic and vaccine relevance. , 2005, Tuberculosis.

[3]  A. Mustafa,et al.  Mycobacterial Gene Cloning and Expression, Comparative Genomics, Bioinformatics and Proteomics in Relation to the Development of New Vaccines and Diagnostic Reagents , 2005, Medical Principles and Practice.

[4]  K. Lundin,et al.  Identification and HLA Restriction of Naturally Derived Th1-Cell Epitopes from the Secreted Mycobacterium tuberculosisAntigen 85B Recognized by Antigen-Specific Human CD4+T-Cell Lines , 2000, Infection and Immunity.

[5]  R. Al-Attiyah,et al.  Computer‐Assisted Prediction of HLA‐DR Binding and Experimental Analysis for Human Promiscuous Th1‐Cell Peptides in the 24 kDa Secreted Lipoprotein (LppX) of Mycobacterium tuberculosis , 2004, Scandinavian journal of immunology.

[6]  F. Berven,et al.  Comprehensive analysis of exported proteins from Mycobacterium tuberculosis H37Rv , 2007, Proteomics.

[7]  A. Mustafa,et al.  Amplification of Six Putative RD1 Genes of Mycobacterium tuberculosis for Cloning and Expression in Escherichia coli and Purification of Expressed Proteins , 2008, Medical Principles and Practice.

[8]  A. Mustafa,et al.  HLA-DR Binding Prediction and Experimental Evaluation of T-Cell Epitopes of Mycolyl Transferase 85B (Ag85B), a Major Secreted Antigen of Mycobacterium tuberculosis , 2005, Medical Principles and Practice.

[9]  R. Al-Attiyah,et al.  Whole Blood Assays to Identify Th1 Cell Antigens and Peptides Encoded by Mycobacterium tuberculosis-Specific RD1 Genes , 2008, Medical Principles and Practice.

[10]  A. Mustafa.  Development of new vaccines and diagnostic reagents against tuberculosis. , 2002, Molecular immunology.

[11]  A. Mustafa.  Biotechnology in the development of new vaccines and diagnostic reagents against tuberculosis. , 2001, Current pharmaceutical biotechnology.

[12]  F. Oftung,et al.  Human Th1 Cell Lines Recognize the Mycobacterium tuberculosis ESAT‐6 Antigen and its Peptides in Association with Frequently Expressed HLA Class II Molecules , 2003, Scandinavian journal of immunology.

[13]  Tianbing Ding,et al.  Expression and purification of Mycobacterium tuberculosis ESAT-6 and MPT64 fusion protein and its immunoprophylactic potential in mouse model. , 2008, Protein expression and purification.

[14]  H. Vordermeier,et al.  Immunogenicity of Mycobacterium tuberculosis RD1 region gene products in infected cattle , 2002, Clinical and experimental immunology.

[15]  R. Al-Attiyah,et al.  Efficient Testing of Large Pools of Mycobacterium tuberculosis RD1 Peptides and Identification of Major Antigens and Immunodominant Peptides Recognized by Human Th1 Cells , 2008, Clinical and Vaccine Immunology.

[16]  A. Mustafa,et al.  HLA-restricted immune response to mycobacterial antigens: relevance to vaccine design. , 2000, Human immunology.

[17]  Tobias Braun,et al.  Mycobacterial Lipopeptides Elicit CD4+ CTLs in Mycobacterium tuberculosis-Infected Humans1 , 2008, The Journal of Immunology.

[18]  A. Mustafa,et al.  Demonstration of In vivo Expression of a Hypothetical Open Reading Frame (ORF‐14) Encoded by the RD1 Region of Mycobacterium tuberculosis , 2007, Scandinavian journal of immunology.

[19]  M. Raviglione,et al.  Global epidemiology of tuberculosis: prospects for control. , 2008, Seminars in respiratory and critical care medicine.

[20]  J. Belisle,et al.  Peripheral Blood and Pleural Fluid Mononuclear Cell Responses to Low-Molecular-Mass Secretory Polypeptides of Mycobacterium tuberculosis in Human Models of Immunity to Tuberculosis , 2005, Infection and Immunity.

[21]  Andersen,et al.  Comparison of Antigen‐Specific T‐Cell Responses of Tuberculosis Patients using Complex or Single Antigens of Mycobacterium tuberculosis , 1998, Scandinavian journal of immunology.

[22]  A. Mustafa.  Th1 Cell Reactivity and HLA‐DR Binding Prediction for Promiscuous Recognition of MPT63 (Rv1926c), a Major Secreted Protein of Mycobacterium tuberculosis , 2009, Scandinavian journal of immunology.

[23]  R. Al-Attiyah,et al.  Assessment of in vitro immunity to Mycobacterium tuberculosis in a human peripheral blood infection model using a luciferase reporter construct of M. tuberculosis H37Rv , 2006, Clinical and experimental immunology.

[24]  R. Al-Attiyah,et al.  Mammalian Cell‐Entry Proteins Encoded by the mce3 Operon of Mycobacterium tuberculosis are Expressed During Natural Infection in Humans , 2004, Scandinavian journal of immunology.

[25]  Young,et al.  Identification of a Novel Protein Antigen Encoded by a Mycobacterium tuberculosis‐Specific RD1 Region Gene , 1999, Scandinavian journal of immunology.

[26]  A. Mustafa.  HLA-Promiscuous Th1-Cell Reactivity of MPT64 (Rv1980c), a Major Secreted Antigen of Mycobacterium tuberculosis, in Healthy Subjects , 2009, Medical Principles and Practice.

[27]  H. Vordermeier,et al.  Immunogenicity of Mycobacterium tuberculosis Antigens in Mycobacterium bovis BCG-Vaccinated and M. bovis-Infected Cattle , 2006, Infection and Immunity.

[28]  R. Al-Attiyah,et al.  Characterization of Human Cellular Immune Responses to Novel Mycobacterium tuberculosis Antigens Encoded by Genomic Regions Absent in Mycobacterium bovis BCG , 2008, Infection and Immunity.

[29]  R. Al-Attiyah,et al.  In vitro cellular immune responses to complex and newly defined recombinant antigens of Mycobacterium tuberculosis , 2004, Clinical and experimental immunology.

[30]  H. Wiker,et al.  Rapid and Specific Diagnosis of Tuberculous Pleuritis With Immunohistochemistry by Detecting Mycobacterium Tuberculosis Complex Specific Antigen MPT64 in Patients From a HIV Endemic Area , 2008, Applied immunohistochemistry & molecular morphology : AIMM.

[31]  P. Andersen,et al.  Restoration of mycobacterial antigen-induced proliferation and interferon-gamma responses in peripheral blood mononuclear cells of tuberculosis patients upon effective chemotherapy. , 2003, FEMS immunology and medical microbiology.

[32]  Alessandro Sette,et al.  Rational Design of a Multiepitope Vaccine Encoding T-Lymphocyte Epitopes for Treatment of Chronic Hepatitis B Virus Infections , 2007, Journal of Virology.

[33]  A. Mustafa,et al.  ProPred analysis and experimental evaluation of promiscuous T-cell epitopes of three major secreted antigens of Mycobacterium tuberculosis. , 2006, Tuberculosis.

[34]  R. Al-Attiyah,et al.  Cell-Mediated Immune Responses to Complex and Single Mycobacterial Antigens in Tuberculosis Patients with Diabetes , 2008, Medical Principles and Practice.