Enhancing sensitivity of detection of immune responses to Mycobacterium leprae peptides in whole-blood assays

Clin Vaccine Immunol. 2010 Jun;17(6):993-1004. doi: 10.1128/CVI.00046-10. Epub 2010 Apr 28.


Although worldwide leprosy prevalence has been reduced considerably following multidrug therapy, new case detection rates remain relatively stable, suggesting that transmission of infection still continues. This calls for new efforts, among which is development of assays that can identify subclinical/early-stage Mycobacterium leprae-infected subjects, a likely source of transmission. Areas in which leprosy is endemic often lack sophisticated laboratories, necessitating development of field-friendly immunodiagnostic tests for leprosy, like short-term whole-blood assays (WBA). In classical, peripheral blood mononuclear cell (PBMC)-based gamma interferon (IFN-gamma) release assays, M. leprae peptides have been shown to discriminate in a more specific fashion than M. leprae proteins between M. leprae-exposed contacts and patients as opposed to healthy controls from the same area of endemicity. However, peptides induced significantly lower levels of IFN-gamma than did proteins, particularly when whole blood was used. Therefore, possibilities of specifically enhancing IFN-gamma production in response to M. leprae peptides in 24-h WBA were sought by addition of various cytokines and antibodies or by mannosylation of peptides. In addition, other cytokines and chemokines were analyzed as potential biomarkers in WBA. We found that only interleukin 12 (IL-12), not other costimulants, increased IFN-gamma production in WBA while maintaining M. leprae peptide specificity, as evidenced by lack of increase of IFN-gamma in control samples stimulated with IL-12 alone. The IL-12-induced increase in IFN-gamma was mainly mediated by CD4+ T cells that did not produce IL-2 or tumor necrosis factor (TNF). Mannosylation further allowed the use of 100-fold-less peptide. Although not statistically significantly, macrophage inflammatory protein 1beta (MIP-1beta) and macrophage c protein 1 (MCP-1) levels specific for M. leprae peptide tended to be increased by IL-12. IP-10 production was also found to be a useful marker of M. leprae peptide responses, but its production was enhanced by IL-12 nonspecifically. We conclude that IFN-gamma-based WBA combined with IL-12 represents a more sensitive and robust assay for measuring reactivity to M. leprae peptides.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / immunology*
  • Cytokines / immunology
  • Humans
  • Interferon-gamma / blood*
  • Interleukin-12 / immunology*
  • Leprosy / blood
  • Leprosy / diagnosis
  • Leprosy / immunology*
  • Lymphocyte Activation
  • Molecular Sequence Data
  • Mycobacterium leprae / immunology*
  • Peptides / chemical synthesis
  • Peptides / chemistry
  • Peptides / immunology*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology*
  • Sensitivity and Specificity
  • T-Lymphocytes / immunology


  • Bacterial Proteins
  • Cytokines
  • Peptides
  • Recombinant Proteins
  • Interleukin-12
  • Interferon-gamma