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Synergistic antigen combinations for the development of interferon gamma release assays for paucibacillary leprosy
R M Oliveira1, E M Hungria, A de Araújo Freitas
1Tropical Pathology and Public Health Institute, Federal University of Goiás, Goiânia, GO, Brazil.
Insights
New combinations of Mycobacterium leprae antigens show promise for diagnosing paucibacillary leprosy. These novel combinations effectively stimulate cell-mediated immunity (CMI) and interferon-gamma (IFN-γ) production in whole blood assays, aiding in leprosy diagnosis.
Area of Science:
- Immunology
- Infectious Diseases
- Diagnostics
Background:
- Paucibacillary leprosy (PB) diagnosis relies on detecting Mycobacterium leprae-specific cell-mediated immunity (CMI), often measured by IFN-γ production.
- Novel M. leprae protein antigens stimulating CMI have recently been identified, offering potential for improved diagnostic tools.
Purpose of the Study:
- To evaluate the efficacy of different combinations of M. leprae antigens in whole blood assays (WBA) for diagnosing paucibacillary leprosy.
- To assess the potential of these antigen combinations for developing a new interferon gamma release assay (IGRA) for PB leprosy.
Main Methods:
- Whole blood assays (WBA) were performed on five groups: PB patients, multibacillary (MB) patients, household contacts (HHC), healthy endemic controls (EC), and tuberculosis (TB) patients.
- WBA were stimulated with individual M. leprae recombinant proteins (rML), five rML combinations, M. leprae cell sonicate (MLCS), PHA, and PBS.
- Interferon-gamma (IFN-γ) levels were measured using ELISA, with an arbitrary cut-off of 50 pg/ml.
Main Results:
- Three of five tested antigen combinations (46f+LID-1, ML0276+LID-1, ML2055+ML1632+ML2044) significantly increased IFN-γ production in both PB patients and contacts.
- The magnitude of IFN-γ response was higher in contacts compared to patients.
- The combination 46f+ML0276 specifically stimulated IFN-γ in symptomatic PB patients, not in asymptomatic contacts, and control groups showed minimal response, indicating specificity.
Conclusions:
- Synergistic effects observed with new M. leprae antigen combinations in WBA suggest their potential for developing an IGRA for paucibacillary leprosy diagnosis.
- These findings highlight the utility of novel antigen combinations in improving the sensitivity and specificity of leprosy immunodiagnostics.
Abstract:
The development of immunodiagnostic tests for paucibacillary leprosy (PB) is based on Mycobacterium leprae specific-cell mediated immunity (CMI)/IFN-γ production. Recently, novel M. leprae protein antigens that stimulate CMI have been described. This study evaluated different M. leprae antigen combinations in whole blood assay (WBA). Five study groups were tested (20 per group): newly diagnosed, untreated PB patients and multibacillary leprosy patients (MB); household contacts of MB patients (HHC); healthy endemic controls (EC); pulmonary tuberculosis patients (TB). WBA (heparinized, 24 h 37 °C 5% CO₂) were stimulated with: 10 μg/ml of each individual M. leprae recombinant protein (rML) and five combinations of rML (46f + LID-1, ML0276 + LID-1, ML2055 + ML1632 + ML2044, ML0276 + 46f, ML2055 + LID-1)-M. leprae cell sonicate (MLCS, 10 μg/ml), PHA (1 μg/ml), and PBS alone. Human IFN-γ ELISA (QuantiFERON-TB Gold/QFT-G, Cellestis) was performed using stimulated plasma (arbitrary cut-off = 50 pg/ml). Three out of five antigen combinations (46f + LID-1, ML0276 + LID-1, ML2055 + ML1632 + ML2044) were able to increase the levels of IFN-γ production in WBA in a larger number of responders among both PB leprosy and contacts. However, the magnitude of IFN-γ responses was higher among contacts. The antigen combination (46f + ML0276) stimulated IFN-γ only in symptomatic PB leprosy patients and not in asymptomatic contacts. Few controls (EC, TB) responded to combinations (0-15%), indicating the specificity of the response in an endemic area with high BCG coverage. The synergistic effect of new combinations of M. leprae proteins upon IFN-γ production in WBA indicates their potential use for the development of an interferon gamma release assay/IGRA for the diagnosis of PB leprosy.
