Analyzing Mycobacterium tuberculosis proteomes for candidate vaccine epitopes.

Secreted antigens of Mycobacterium tuberculosis (Mtb) induce strong T cell responses and interferon-gamma (IFN-gamma) secretion, both of which are integral in the defense against Mtb. We used web-based tools (SignaIP and Prosite) to identify putative secreted proteins from Mtb genomes CDC 1551 and H37Rv. We then used EpiMatrix, a proprietary pattern-matching algorithm, to do a preliminary analysis of these proteins for regions that contained a high number of class II MHC binding motif matches. The use of bioinformatics tools reduced the number of potential epitopes to be screened to 5% of the 1.3 million overlapping peptides. Peripheral blood mononuclear cells (PBMC) were obtained from healthy, asymptomatic tuberculin skin test-positive donors. Of the 17 highest-ranking peptide candidates that could be synthesized for this preliminary in vitro evaluation, 15 (88%) stimulated IFN-gamma response, and eight (47%) stimulated lymphocyte proliferation in vitro. IFN-gamma ELISpot assays were therefore a more sensitive test for T cell response to these peptides than were proliferation assays. One highly promiscuous epitope (MT2281-26-J, WRRRPLSSALLSFGLLLGGLPL) induced IFN-gamma secretion in PBMC from 11 of 25 Mtb immune subjects (44%). Overall, 15 epitopes, and MT2281-26-J in particular, are candidates for inclusion in a multi-epitope TB vaccine. These findings support the systematic application of bioinformatics tools to whole genomes when used in combination with in vitro methods for screening and confirming epitopes.

[1]  J. Flynn,et al.  An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection , 1993, The Journal of experimental medicine.

[2]  F. Mosteller,et al.  Efficacy of BCG Vaccine in the Prevention of Tuberculosis: Meta-analysis of the Published Literature , 1994 .

[3]  C. Dye,et al.  Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project. , 1999, JAMA.

[4]  Rolf Apweiler,et al.  A comparison of signal sequence prediction methods using a test set of signal peptides , 2000, Bioinform..

[5]  S. Ehlers,et al.  Inflammation and lymphocyte activation during mycobacterial infection in the interferon-gamma-deficient mouse. , 2001, Cellular immunology.

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

[7]  P. Andersen,et al.  ESAT-6 Subunit Vaccination againstMycobacterium tuberculosis , 2000, Infection and Immunity.

[8]  S Daugelat,et al.  Tuberculosis and Leprosy: Attempts to Identify T‐Cell Antigens of Potential Value for Vaccine Design , 1992, Scandinavian journal of immunology. Supplement.

[9]  M. Geletu,et al.  T-Cell Recognition of Mycobacterium tuberculosisCulture Filtrate Fractions in Tuberculosis Patients and Their Household Contacts , 1999, Infection and Immunity.

[10]  H. Vordermeier,et al.  T cell repertoire in tuberculosis: selective anergy to an immunodominant epitope of the 38‐kDa antigen in patients with active disease , 1992, European journal of immunology.

[11]  Sadie M. Johnson,et al.  Identification of Secreted Proteins ofMycobacterium tuberculosis by a Bioinformatic Approach , 2000, Infection and Immunity.

[12]  P. E. M. Fine,et al.  Variation in protection by BCG: implications of and for heterologous immunity , 1995, The Lancet.

[13]  M. Levin,et al.  Evaluation of human antimycobacterial immunity using recombinant reporter mycobacteria. , 2000, The Journal of infectious diseases.

[14]  J Stulík,et al.  Construction of a Francisella tularensis two‐dimensional electrophoresis protein database , 2001, Proteomics.

[15]  Christopher Dye,et al.  Global Burden of Tuberculosis: Estimated Incidence, Prevalence, and Mortality by Country , 1999 .

[16]  Amos Bairoch,et al.  The PROSITE database, its status in 2002 , 2002, Nucleic Acids Res..

[17]  D. Snider,et al.  Epidemiology of tuberculosis in the United States. , 1989, Epidemiologic reviews.

[18]  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.

[19]  G. Kaplan,et al.  Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-α/β , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[20]  M. Horwitz,et al.  Recombinant bacillus calmette-guerin (BCG) vaccines expressing the Mycobacterium tuberculosis 30-kDa major secretory protein induce greater protective immunity against tuberculosis than conventional BCG vaccines in a highly susceptible animal model. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Michael A. Gonzalez,et al.  From genome to vaccine: in silico predictions, ex vivo verification. , 2001, Vaccine.

[22]  P. Dierckx,et al.  Specific Lymphoproliferation, Gamma Interferon Production, and Serum Immunoglobulin G Directed against a Purified 32 kDa Mycobacterial Protein Antigen (P32) in Patients with Active Tuberculosis , 1988, Scandinavian journal of immunology.

[23]  A. Tomlinson,et al.  Strategy for isolating and sequencing biologically derived MHC class I peptides. , 1996, Journal of chromatography. A.

[24]  Julie McMurry,et al.  Immuno‐informatics: Mining genomes for vaccine components , 2002, Immunology and cell biology.

[25]  P. Andersen,et al.  Human T-cell responses to secreted antigen fractions of Mycobacterium tuberculosis , 1995, Infection and immunity.

[26]  C. Nacy,et al.  New tuberculosis vaccine development , 2002, Expert opinion on biological therapy.

[27]  Søren Buus,et al.  Tumor‐associated antigens identified by mRNA expression profiling induce protective anti‐tumor immunity , 2001, European journal of immunology.

[28]  P. Andersen.  Host Responses and Antigens Involved in Protective Immunity to Mycobacterium tuberculosis , 1997, Scandinavian journal of immunology.

[29]  G. Möller,et al.  Antibody‐coated Sheep Erythrocytes Suppress the Ability of Polyclonal B‐Cell Activators to Induce Plaque‐forming Cells against Sheep Erythrocytes , 1980, Scandinavian journal of immunology.

[30]  Ilan Beer,et al.  Analysis of endogenous peptides bound by soluble MHC class I molecules: a novel approach for identifying tumor‐specific antigens , 2002, European journal of immunology.

[31]  A. L. Sørensen,et al.  Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice. , 1995, Journal of immunology.

[32]  J. Ulmer,et al.  Vaccination with Plasmid DNA Encoding Mycobacterial Antigen 85A Stimulates a CD4+ and CD8+T-Cell Epitopic Repertoire Broader than That Stimulated byMycobacterium tuberculosis H37Rv Infection , 1998, Infection and Immunity.

[33]  M. Torres,et al.  Cytokine Profiles for Peripheral Blood Lymphocytes from Patients with Active Pulmonary Tuberculosis and Healthy Household Contacts in Response to the 30-Kilodalton Antigen ofMycobacterium tuberculosis , 1998, Infection and Immunity.

[34]  T. Eguale,et al.  Human T cell responses to the ESAT-6 antigen from Mycobacterium tuberculosis. , 1999, The Journal of infectious diseases.

[35]  E. Thiel,et al.  Flow cytometric determination of intracellular or secreted IFNgamma for the quantification of antigen reactive T cells. , 2001, Journal of immunological methods.

[36]  M. E. Villarino.  Prevention and treatment of tuberculosis among patients infected with human immunodeficiency virus; principles of therapy and revised recommendations , 1998 .

[37]  J. Abrams,et al.  Cytokine production at the site of disease in human tuberculosis , 1993, Infection and immunity.

[38]  G. Schoolnik,et al.  Comparative genomics of BCG vaccines by whole-genome DNA microarray. , 1999, Science.

[39]  J. Belisle,et al.  Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry , 1997, Infection and immunity.

[40]  H. Sbai,et al.  Use of T cell epitopes for vaccine development. , 2001, Current drug targets. Infectious disorders.

[41]  P. Andersen,et al.  Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins , 1994, Infection and immunity.

[42]  B. Barrell,et al.  Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence , 1998, Nature.

[43]  S. Kaufmann,et al.  How can immunology contribute to the control of tuberculosis? , 2001, Nature Reviews Immunology.

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

[45]  J. Venter,et al.  Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. , 2000, Science.

[46]  Zhu,et al.  38 000 MW antigen‐specific major histocompatibility complex class I restricted interferon‐γ‐secreting CD8+ T cells in healthy contacts of tuberculosis , 1998, Immunology.

[47]  K. Sepkowitz,et al.  Tuberculosis control in the 21st century. , 2001, Emerging infectious diseases.

[48]  J. Killion,et al.  Direct comparison of ELISPOT and ELISA-based assays for detection of individual cytokine-secreting cells. , 1994, Lymphokine and cytokine research.

[49]  U. Şahin,et al.  Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices , 1999, Nature Biotechnology.

[50]  J. Flynn,et al.  Major histocompatibility complex class I-restricted T cells are required for resistance to Mycobacterium tuberculosis infection. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[51]  J. Lang,et al.  Immune defence against HIV-1 infection in HIV-1-exposed seronegative persons. , 2001, Immunology letters.