Peptides Derived from Mycobacterium leprae ML1601c Discriminate between Leprosy Patients and Healthy Endemic Controls

The stable incidence of new leprosy cases suggests that transmission of infection continues despite worldwide implementation of MDT. Thus, specific tools are needed to diagnose early stage Mycobacterium leprae infection, the likely sources of transmission. M. leprae antigens that induce T-cell responses in M. leprae exposed and/or infected individuals thus are major targets for new diagnostic tools. Previously, we showed that ML1601c was immunogenic in patients and healthy household contacts (HHC). However, some endemic controls (EC) also recognized this protein. To improve the diagnostic potential, IFN-γ responses to ML1601c peptides were assessed using PBMC from Brazilian leprosy patients and EC. Five ML1601c peptides only induced IFN-γ in patients and HHC. Moreover, 24-hour whole-blood assay (WBA), two ML1601c peptides could assess the level of M. leprae exposure in Ethiopian EC. Beside IFN-γ, also IP-10, IL-6, IL-1β, TNF-α, and MCP-1 were increased in EC from areas with high leprosy prevalence in response to these ML1601c peptides. Thus, ML1601c peptides may be useful for differentiating M. leprae exposed or infected individuals and can also be used to indicate the magnitude of M. leprae transmission even in the context of various HLA alleles as present in these different genetic backgrounds

Pathogen-Specific Epitopes as Epidemiological Tools for Defining the Magnitude of Mycobacterium leprae Transmission in Areas Endemic for Leprosy

During recent years, comparative genomic analysis has allowed the identification of Mycobacterium leprae-specific genes with potential application for the diagnosis of leprosy. In a previous study, 58 synthetic peptides derived from these sequences were tested for their ability to induce production of IFN-γ in PBMC from endemic controls (EC) with unknown exposure to M. leprae, household contacts of leprosy patients and patients, indicating the potential of these synthetic peptides for the diagnosis of sub- or preclinical forms of leprosy. In the present study, the patterns of IFN-γ release of the individuals exposed or non-exposed to M. leprae were compared using an Artificial Neural Network algorithm, and the most promising M. leprae peptides for the identification of exposed people were selected. This subset of M. leprae-specific peptides allowed the differentiation of groups of individuals from sites hyperendemic for leprosy versus those from areas with lower level detection rates. A progressive reduction in the IFN-γ levels in response to the peptides was seen when contacts of multibacillary (MB) patients were compared to other less exposed groups, suggesting a down modulation of IFN-γ production with an increase in bacillary load or exposure to M. leprae. The data generated indicate that an IFN-γ assay based on these peptides applied individually or as a pool can be used as a new tool for predicting the magnitude of M. leprae transmission in a given population

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

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-γ) 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-γ than did proteins, particularly when whole blood was used. Therefore, possibilities of specifically enhancing IFN-γ 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-γ production in WBA while maintaining M. leprae peptide specificity, as evidenced by lack of increase of IFN-γ in control samples stimulated with IL-12 alone. The IL-12-induced increase in IFN-γ 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 1β (MIP-1β) 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-γ-based WBA combined with IL-12 represents a more sensitive and robust assay for measuring reactivity to M. leprae peptides

Clonal Analysis of the T-Cell Response to <i>In Vivo</i> Expressed <i>Mycobacterium tuberculosis</i> Protein Rv2034, Using a CD154 Expression Based T-Cell Cloning Method

<div><p>Tuberculosis (TB), caused by <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), remains a leading cause of death worldwide. A better understanding of the role of CD4⁺ and CD8⁺ T cells, which are both important to TB protection, is essential to unravel the mechanisms of protection and to identify the key antigens seen by these T cells. We have recently identified a set of <i>in vivo</i> expressed <i>Mtb</i> genes (IVE-TB) which is expressed during <i>in vivo</i> pulmonary infection in mice, and shown that their encoded antigens are potently recognized by polyclonal T cells from tuberculin skin test-positive, <i>in vitro</i> ESAT-6/CFP10-responsive individuals. Here we have cloned T cells specific for one of these newly identified <i>in vivo</i> expressed <i>Mtb</i> (IVE-TB) antigens, Rv2034. T cells were enriched based on the expression of CD154 (CD40L), which represents a new method for selecting antigen-specific (low frequency) T cells independent of their specific function. An Rv2034-specific CD4⁺ T-cell clone expressed the Th1 markers T-bet, IFN-γ, TNF-α, IL-2 and the cytotoxicity related markers granzyme B and CD107a as measured by flow cytometry. The clone specifically recognized Rv2034 protein, Rv2034 peptide p81–100 and <i>Mtb</i> lysate. Remarkably, while the recognition of the dominant p81–100 epitope was HLA-DR restricted, the T-cell clone also recognized a neighboring epitope (p88–107) in an HLA-DR- as well as HLA-DQ1-restricted fashion. Importantly, the T-cell clone was able to inhibit <i>Mtb</i> outgrowth from infected monocytes significantly. The characterization of the polyfunctional and <i>Mtb</i> inhibitory T-cell response to IVE-TB Rv2034 at the clonal level provides detailed further insights into the potential of IVE-TB antigens as new vaccine candidate antigens in TB. Our new approach allowed the identification of T-cell subsets that likely play a significant role in controlling <i>Mtb</i> infection, and can be applied to the analysis of T-cell responses in patient populations.</p></div

Multi-center evaluation of a user-friendly lateral flow assay to determine IP-10 and CCL4 levels in blood of TB and non-TB cases in Africa

Objective: Multi-center evaluation of a user-friendly lateral flow test for detection of IP-10 and CCL4 levels in Mycobacterium tuberculosis (Mtb) antigen-stimulated whole blood samples from tuberculosis (TB) suspects. Design and methods: A quantitative lateral flow (LF)-based assay platform was applied to detect chemokines IP-10 and CCL4. Chemokine quantitation was achieved using interference-free, fluorescent up-converting phosphor (UCP) labels. The new assays allowed worldwide shipping and storage without requiring a cold chain and were tested at seven institutes (including Ethiopia, Malawi, The Gambia, South Africa, Uganda and Namibia) employing portable lightweight readers for detection of the UCP label. At each site, clinical samples, confirmed TB and non-TB (i.e. other respiratory diseases (ORD)) cases, were collected and analyzed simultaneously with quality control (QC) human IP-10 or CCL4 standards. Results: Performance of the UCP-LF assay in Africa using QC standards indicated high robustness allowing quantitative detection between 100 and 100,000 pg/mL. The optimized assays allowed successful determination of chemokine levels using 1 μL whole blood sample from the locally recruited subjects with TB or ORD. Conclusion: This African multi-center trial further demonstrated the applicability of the low-tech and robust UCP-LF platform as a convenient quantitative assay for chemokine detection in whole blood. Ambient shipping and storage of all assay reagents and the availability of lightweight standalone readers were acknowledged as essential requirement for test implementation in particular in remote and resource-limited settings

PBMC recognition of TB10.4 and IVE-TB antigen Rv2034.

<p>PBMC from a PPD⁺ donor were stimulated with different stimuli for 6 days and IFN-γ production (pg/ml) was determined in the supernatants. Both TB10.4 protein (10 µg/ml) (A) and Rv2034 protein (10 µg/ml) (B) were analyzed as well as control mitogen PHA and <i>Mtb</i> derived PPD (A and B). Medium values (unstimulated PBMC) were subtracted. IFN-γ concentrations were determined from triplicate-pooled supernatant. A cut-off value was set arbitrarily at 100 pg/ml.</p

Analysis of TB10.4 clonal cultures.

a<p>Single live CD14-CD19-CD3+ cells (population>100 cells).</p><p>Non-responding clonal cultures included CD4 (n = 9), CD8 (n = 2) and DN (n = 2) cells.</p><p>DN = Double negative.</p><p>+ = >1% positive cells.</p><p>+/− = <1% positive cells.</p