Enhancing Sensitivity of Detection of Immune Responses toMycobacterium lepraePeptides in Whole-Blood Assays

Abstract

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-␥) 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-␥ than did proteins, particularly when whole blood was used. Therefore, possibilities of specifically enhancing IFN-␥ 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-␥ production in WBA while maintaining M. leprae peptide specificity, as evidenced by lack of increase of IFN-␥ in control samples stimulated with IL-12 alone. The IL-12-induced increase in IFN-␥ 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 1␤ (MIP-1␤) 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-␥-based WBA combined with IL-12 represents a more sensitive and robust assay for measuring reactivity to M. leprae peptides.Leprosy is a disabling and stigmatizing disease caused by infection with Mycobacterium leprae. The characteristic immunological and clinical leprosy spectrum, classified by Ridley and Jopling in 1966 (25), ranges from tuberculoid (TT) or paucibacillary (PB) leprosy to lepromatous (LL) or multibacillary (MB) leprosy. In between these poles the borderline states borderline lepromatous (BL), borderline borderline (BB), and borderline tuberculoid (BT) leprosy are positioned. TT/BT patients in general show high cellular responses to M. leprae antigens injected in the skin as well as in in vitro T-cell assays; have low antibody titers to M. leprae antigens, including phenolic glycolipid I (PGL-I); and develop localized granulomatous disease with few, if any, detectable bacilli in their lesions. At the opposite end of the spectru...

Beyond the IFN-γ horizon: biomarkers for immunodiagnosis of infection withMycobacterium tuberculosis

“…These findings are in line with expression patterns seen in other CMI assays e.g. following phytohaemagglutinin stimulation of whole blood from presumed healthy donors [32,60] and Mycobacterium leprae-specific peptide stimulation of whole blood from M. leprae-infected patients [135].…”

Evaluation of Gamma Interferon (IFN-γ)-Induced Protein 10 Responses for Detection of Cattle Infected with Mycobacterium bovis: Comparisons to IFN-γ Responses

“…In a study to develop diagnostic approaches for subclinical/early-stage leprosy, IP-10 was shown to augment the diagnostic potential of IFN-␥/M. leprae peptide-based tests (10). Most recently, a multicenter evaluation demonstrated that a Luminex-based IP-10 assay performed similarly to commercial IGRAs (17) for the diagnosis of TB in humans.…”

Gene Expression Profile and Immunological Evaluation of Unique Hypothetical Unknown Proteins of Mycobacterium leprae by Using Quantitative Real-Time PCR

“…Until now, a CMI-based test using some of M. leprae's unique proteins provided the greatest potential to detect HHCs, who are exposed to and/or infected by M. leprae (14)(15)(16)(17)(18)22) but do not exhibit obvious clinical symptoms, as well as TT/PB patients. However, several studies showed that most of the selected proteins or peptide antigens could not achieve a reasonable sensitivity of IGRAs, particularly in regions where leprosy is endemic (14)(15)(16)22).…”