Molecular Medicine

, Volume 9, Issue 3–4, pp 105–111 | Cite as

Identification of Early Secretory Antigen Target-6 Epitopes for the Immunodiagnosis of Active Tuberculosis

  • Donatella Vincenti
  • Stefania Carrara
  • Patrizia De Mori
  • Leopoldo P Pucillo
  • Nicola Petrosillo
  • Fabrizio Palmieri
  • Orlando Armignacco
  • Giuseppe Ippolito
  • Enrico Girardi
  • Massimo Amicosante
  • Delia Goletti


The early secretory antigenic target (ESAT)-6 purified protein and peptides from Mycobacterium tuberculosis were evaluated as antigens for the immunodiagnosis of tuberculosis (TB). Because the control of TB requires improved diagnostic procedures, efforts have increased to identify Mycobacterium tuberculosis-specific epitopes for the immunodiagnosis of active TB and to discriminate between active and latent states of infection. Two multiepitopic peptides from ESAT-6 protein were selected by computational analysis. Patients with active TB (7 HIV+ and 20 HIV) and control patients (17 HIV+ and 28 HIV) were enrolled. Enzyme-linked immunospot assay analysis for interferon-γ expression by peripheral blood mononuclear cells was quantified after stimulation with selected ESAT-6 peptides, purified protein derivative, or the intact ESAT-6 protein. During active TB, 20 of 27 patients responded in vitro to ESAT-6 peptides and 23 of 27 patients to purified protein derivative. None of the controls without active TB, including individuals with latent TB infection, recognized ESAT-6 peptides. By contrast, latently infected individuals did respond in vitro to both intact ESAT-6 protein and purified protein derivative. Thus, high T-cell response frequencies to ESAT-6 peptides are present only during active TB and can be used to discriminate between active and latent forms of infection.



The authors are grateful to all patients and the nursing staff who took part in this study. We thank V Colizzi and F Poccia for the helpful discussions, M Brescia for technical assistance, R Urso, M De Marco, R Maddaluno, and A Govoni for helping in the recruitment of patients, Jim Arthos, Zana Mariano, and Carla Nisii for editing the manuscript, and Massimo Carrara for helping with the graphics. This work was supported by Current Research Project of the Italian Ministry of Health grants.


  1. 1.
    Dye C, Scheele S, Dolin P, Pathania V, Raviglione MC. (1999) Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO global surveillance and monitoring project. JAMA 282:677–86.CrossRefGoogle Scholar
  2. 2.
    Di Perri G et al. (1989) Nosocomial epidemic of active tuberculosis among HIV-infected patients. Lancet 2:1502–4.PubMedGoogle Scholar
  3. 3.
    Jasmer RM, Nahid P, Hopewell PC. (2002) Latent tuberculosis infection. New Eng. J. Med. 347:1860–6.CrossRefGoogle Scholar
  4. 4.
    American Thoracic Society. (2000) Diagnostic standards and classification of tuberculosis in adults and children. Am. J. Respir. Crit. Care Med. 161:1376–95.CrossRefGoogle Scholar
  5. 5.
    Forbes BA. (1997) Critical assessment of gene amplification approaches on the diagnosis of tuberculosis. Immunol. Invest. 26:105–16.CrossRefGoogle Scholar
  6. 6.
    Fine PE et al. (1999) Tuberculin sensitivity: conversions and reversions in a rural African population. Int. J. Tuberc. Lung Dis. 3:962–75.PubMedGoogle Scholar
  7. 7.
    Huebner RE, Schein MF, Bass JB Jr. (1993) The tuberculin skin test. Clin. Infect. Dis. 17:968–75.CrossRefGoogle Scholar
  8. 8.
    Shafer RW, Edlin BR. (1996) Tuberculosis in patients infected with human immunodeficiency virus: perspective on the past decade. Clin. Infect. Dis. 22:683–704.CrossRefGoogle Scholar
  9. 9.
    Behr MA et al. (1999) Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science 284:1520–3.CrossRefGoogle Scholar
  10. 10.
    Arend SM et al. (2002) Tuberculin skin testing and in vitro T cell responses to ESAT-6 and culture filtrate protein 10 after infection with Mycobacterium marinum or M. kansasii. J. Infect. Dis. 186:1797–807.CrossRefGoogle Scholar
  11. 11.
    Mahairas GG, Sabo PJ, Hickey MJ, Singh DC, Stover CK. (1996) Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J. Bacteriol. 178:1274–82.CrossRefGoogle Scholar
  12. 12.
    Andersen P, Andersen AB, Sorensen AL, Nagai S. (1995) Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice. J Immunol 154:3359–72.PubMedGoogle Scholar
  13. 13.
    Pollock JM, Andersen P. (1997) Predominant recognition of the ESAT-6 protein in the first phase of interferon with Mycobacterium bovis in cattle. Infect. Immun. 65:2587–92.PubMedPubMedCentralGoogle Scholar
  14. 14.
    Ulrichs T et al. (1998) Differential T cell responses to Mycobacterium tuberculosis ESAT6 in tuberculosis patients and healthy donors. Eur. J. Immunol. 28:3949–58.CrossRefGoogle Scholar
  15. 15.
    Ravn P et al. (1999) Human T cell responses to the ESAT-6 antigen from Mycobacterium tuberculosis. J. Infect. Dis. 179:637–45.CrossRefGoogle Scholar
  16. 16.
    Doherty TM et al. (2002) Immune responses to the Mycobacterium tuberculosis-specific antigen ESAT-6 signal subclinical infection among contacts of tuberculosis patients. J. Clin. Microbiol. 40:704–6.CrossRefGoogle Scholar
  17. 17.
    Lalvani A et al. (2001) Enhanced contact tracing and spatial tracking of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells. Lancet 357:2017–21.CrossRefGoogle Scholar
  18. 18.
    Lalvani A et al. (2001) Enumeration of T cells specific for RD1-encoded antigens suggests a high prevalence of latent Mycobacterium tuberculosis infection in healthy urban Indians. J. Infect. Dis. 183:469–77.CrossRefGoogle Scholar
  19. 19.
    Chapman AL et al. (2002) Rapid detection of active and latent tuberculosis infection in HIV-positive individuals by enumeration of Mycobacterium tuberculosis-specific T cells. AIDS 16:2285–93.CrossRefGoogle Scholar
  20. 20.
    Lalvani A et al. (2001) Rapid detection of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells. Am. J. Respir. Crit. Care Med. 163:824–8.CrossRefGoogle Scholar
  21. 21.
    Pathan AA et al. (2001) Direct ex vivo analysis of antigen-specific IFN-γ-secreting CD4 T cells in Mycobacterium tuberculosis-infected individuals: association with clinical disease state and effect of treatment. J. Immunol. 167:5217–25.CrossRefGoogle Scholar
  22. 22.
    Elhay MJ, Oettinger T, Andersen P. (1998) Delayed-type hypersensitivity responses to ESAT-6 and MPT64 from Mycobacterium tuberculosis in the guinea pig. Infect. Immun. 66:3454–6.PubMedPubMedCentralGoogle Scholar
  23. 23.
    Cardoso FL et al. (2002) T-cell responses to the Mycobacterium tuberculosis-specific antigen ESAT-6 in Brazilian tuberculosis patients. Infect. Immun. 70:6707–14.CrossRefGoogle Scholar
  24. 24.
    Rammensee H, Bachmann J, Emmerich NP, Bachor OA, Stevanovic S. (1999) SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50:213–9. Refer to Scholar
  25. 25.
    Fleckenstein B, Jung G, Wiesmüller KH. (1999) Quantitative analysis of peptide-MHC class II interaction. Semin. Immunol. 11:405–16. Refer to Scholar
  26. 26.
    Sturniolo T et al. (1999) Generation of tissue-specific and promiscuous HLA ligand database using DNA microarrays and virtual HLA class II matrices. Nat. Biotechnol. 17:555–61. Refer to Scholar
  27. 27.
    Singh H, Raghava GP. (2001) ProPred: prediction of HLA-DR binding sites. Bioinformatics 17:1236–7.CrossRefGoogle Scholar
  28. 28.
    Proceedings of the 12th IHWC. (1997) HLA genetic diversity of HLA functional and medical implication. Charron D. (ed) EDK Medical and Scientific International Publisher, Paris.Google Scholar
  29. 29.
    Andersen AB, Brennan P. (1994) Proteins and antigens of Mycobacterium tuberculosis. In: Tuberculosis. Bloom B. (eds.) ASM Press, Washington DC. pp. 307–32.CrossRefGoogle Scholar
  30. 30.
    Orme IM. (1988) Induction of nonspecific acquired resistance and delayed-type hypersensitivity, but not specific acquired resistance in mice inoculated with killed mycobacterial vaccines. Infect. Immun. 56:3310–2.PubMedPubMedCentralGoogle Scholar
  31. 31.
    Andersen P, Heron I. (1993) Specificity of protective memory immune response against Mycobaterium tuberculosis. Infect. Immun. 61:844–51.PubMedPubMedCentralGoogle Scholar
  32. 32.
    Tanguay S, Killion JJ. (1994) Direct comparison of ELISPOT and ELISA-based assays for detection of individual cytokine-secreting cells. Lymphokine Cytokine Res. 13:259–63.PubMedGoogle Scholar
  33. 33.
    Favre N, Bordmann G, Rudin W. (1997) Comparison of cytokine measurements using ELISA, ELISPOT and semi-quantitative RT-PCR. J. Immunol. Methods 204:57–66.CrossRefGoogle Scholar
  34. 34.
    Harboe M, Oettinger T, Wiker HG, Rosenkrands I, Andersen P. Evidence for occurrence of the ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycobacterium bovis BCG. Infect. Immun. 1996; 64:16–22.PubMedPubMedCentralGoogle Scholar
  35. 35.
    Geluk A et al. (2002) Identification and characterization of the ESAT-6 homologue of Mycobacterium leprae and T-cell cross-reactivity with Mycobacterium tuberculosis. Infect. Immun. 70:2544–8.CrossRefGoogle Scholar
  36. 36.
    Shafer RW, Kim DS, Weiss JP, Quale JM. (1991) Extrapulmonary tuberculosis in patients with human immunodeficiency virus infection. Medicine 70:384–97.CrossRefGoogle Scholar
  37. 37.
    Chaisson RE et al. (1987) Tuberculosis in patients with the acquired immunodeficiency syndrome. Clinical features, response to therapy and survival. Am. Rev. Respir. Dis. 136:570–4.CrossRefGoogle Scholar
  38. 38.
    Divinagracia RM, Timothy JH, Stanley B, Schluger NW. (1998) Screening by specialists to reduce unnecessary test ordering in patients evaluated for tuberculosis. Chest 114:681–4.CrossRefGoogle Scholar

Copyright information

© Feinstein Institute for Medical Research 2003

Authors and Affiliations

  • Donatella Vincenti
    • 1
  • Stefania Carrara
    • 1
  • Patrizia De Mori
    • 2
  • Leopoldo P Pucillo
    • 2
  • Nicola Petrosillo
    • 3
  • Fabrizio Palmieri
    • 3
  • Orlando Armignacco
    • 4
  • Giuseppe Ippolito
    • 5
  • Enrico Girardi
    • 5
  • Massimo Amicosante
    • 2
  • Delia Goletti
    • 1
    • 3
  1. 1.Translational Research Unit of the National Institute for Infectious Diseases (INMI) “Lazzaro Spallanzani”RomeItaly
  2. 2.Laboratory of Clinical Pathology INMIRomeItaly
  3. 3.II Division of Health Department INMIRomeItaly
  4. 4.Division of Infectious Diseases of Belcolle Hospital of ViterboViterboItaly
  5. 5.Epidemiology Department INMIRomeItaly

Personalised recommendations