New biomarkers with relevance to leprosy diagnosis applicable in areas hyperendemic for leprosy

Abstract

Leprosy is not eradicable with currently available diagnostics or interventions, as evidenced by its stable incidence. Early diagnosis of Mycobacterium leprae infection should therefore be emphasized in leprosy research. It remains challenging to develop tests based on immunological biomarkers that distinguish individuals controlling bacterial replication from those developing disease. To identify biomarkers for field-applicable diagnostics, we determined cytokines/chemokines induced by M. leprae proteins in blood of leprosy patients and endemic controls (EC) from high leprosy-prevalence areas (Bangladesh, Brazil, Ethiopia) and from South Korea, where leprosy is not endemic anymore. M. leprae-sonicate-induced IFN-γ was similar for all groups, excluding M. leprae/IFN-γ as a diagnostic readout. By contrast, ML2478 and ML0840 induced high IFN-γ concentrations in Bangladeshi EC, which were completely absent for South Korean controls. Importantly, ML2478/IFN-γ could indicate distinct degrees of M. leprae exposure, and thereby the risk of infection and transmission, in different parts of Brazilian and Ethiopian cities. Notwithstanding these discriminatory responses, M. leprae proteins did not distinguish patients from EC in one leprosy-endemic area based on IFN-γ. Analyses of additional cytokines/chemokines showed that M. leprae and ML2478 induced significantly higher concentrations of MCP-1, MIP-1β, and IL-1β in patients compared with EC, whereas IFN-inducible protein-10, like IFN-γ, differed between EC from areas with dissimilar leprosy prevalence. This study identifies M. leprae-unique Ags, particularly ML2478, as biomarker tools to measure M. leprae exposure using IFN-γ or IFN-inducible protein-10, and also shows that MCP-1, MIP-1β, and IL-1β can potentially distinguish pathogenic immune responses from those induced during asymptomatic exposure to M. leprae.

Figure 1. IFN-γ responses in WBA from individuals in Bangladesh and South Korea

IFN-γ production in response to control stimuli (medium, PHA and M. leprae WCS) or to recombinant proteins (ML0840, ML1601 and ML2478) in 24 hour WBA of leprosy patients (TT/BT; n = 10), healthy household contacts (HHC; n =10) and endemic controls (EC; n=10) from Bangladesh (prevalence = 2.45/10,000) or healthy controls (EC; n=10) and tuberculosis patients (TB; n=10) from South Korea (prevalence <1/10,000). For each group the number of IFN-γ responders (>100 pg/ml) versus the total number of individuals in the group is indicated below the x-axis. Background values were <50 pg/ml. Median values for each group are indicated by horizontal lines. Significant differences between test groups are indicated by p-values.

Figure 2. IFN-γ responses to M. leprae antigens in PBMC from EC_high and EC_low in Brazil

IFN-γ production (corrected for background values) induced using PHA (A), M. leprae (B) or ML2478 recombinant protein (C) in 6 day cultures of PBMC from healthy individuals from an area of Fortaleza, Brazil with low (EC_low; prevalence <0.2/10,000; n=10) or high (EC_high; prevalence > 4/10,000; n=10) leprosy prevalence, healthy household contacts (HHC) of MB leprosy patients and TT/BT patients. Median values for each group are indicated by horizontal lines. Background values were <20 pg/ml.

Figure 3. IFN-γ responses to M. leprae proteins in WBA from EC_high and EC_low in Ethiopia

IFN-γ production (corrected for medium values) in response to PHA (A), M. leprae WCS (B) or recombinant protein ML2478 (C) in 24 hour WBA of healthy individuals from areas in Addis Ababa, Ethiopia with low (EC_low; prevalence = 0.36/10,000; n=17) and high (EC_high; prevalence =1.5/10,000; n=18) leprosy endemicity. Median values per test group are indicated by horizontal lines. For each group the number of IFN-γ responders versus the total number of individuals in the group is indicated below the x-axis.

Figure 4. Multiplex cytokine analyses in WBA from individuals in Bangladesh and South Korea

Concentrations (all corrected for background values) of IL-β (A), MIP-1β (B), MCP-1 (C) and IL-β, MIP-1β and MCP-1 combined (D) or IP-10 (E) induced by stimulation with M. leprae WCS in 24 hour WBA of leprosy patients (TT/BT; n = 10), healthy household contacts (HHC; n =10) and endemic controls (EC; n=10) from Bangladesh or healthy controls (EC; n=10) and tuberculosis patients (TB; n=10) from South Korea. Median values per test group are indicated by horizontal lines. Background values varied from <50 pg/ml for IFN-γ to <2000 pg/ml for MIP-1β. ns = not significant.

Figure 5. Multiplex cytokine analyses in whole blood cultures from EC in Ethiopia

Concentrations (all corrected for background values) of IL-β (A), MIP-1β (B), MCP-1 (C, E), IP-10 (D, F) induced by stimulation with M. leprae WCS (A–D) or ML2478 (E, F) in 24 hour WBA of leprosy patients (TT/BT; n = 10), healthy household contacts (HHC; n =10) and endemic controls (EC; n=10) from Bangladesh or healthy controls (EC; n=10) and tuberculosis patients (TB; n=10) from South Korea. Median values per test group are indicated by horizontal lines. Background values varied from <50 pg/ml for IFN-γ to <2000 pg/ml for MIP-1β.

Figure 6. IFN-γ/IL-10 ratio in M. leprae stimulated WBA

Ratios of IFN-γ concentrations (corrected for background values) with respect to IL-10 concentrations (corrected for background values) induced by stimulation with M. leprae WCS in 24 hour WBA in individuals from Bangladesh (A) and South Korea (B).