Immunologically reactive M. leprae antigens with relevance to diagnosis and vaccine development

<p>Abstract</p> <p>Background</p> <p>Leprosy is a chronic infectious disease caused by <it>Mycobacterium leprae </it>that can manifest a wide variety of immunological and clinical outcomes ranging from potent humoral responses among borderline lepromatous (BL) and lepromatous (LL) patients to strong cellular responses among tuberculoid (TT) and borderline tuberculoid (BT) patients. Until recently, relatively little has been known about the immune responses to individual proteins of <it>M. leprae </it>recognized during leprosy.</p> <p>Methods</p> <p>The immune reactivity to a panel of 33 <it>M. leprae </it>recombinant proteins was evaluated among leprosy patients and controls from a high endemic area for leprosy (Goiania/GO, Central Brazil). Serum IgG responses were measured by ELISA (45 participants/group) and T cell responses (20 participants/group) were evaluated by IFN-gamma production in 24 hours whole blood cultures with antigen (whole blood assay-WBA). Study groups were newly diagnosed, untreated TT/BT and BL/LL leprosy patients classified by Ridley Jopling criteria and household contacts of BL/LL patients (HHC). Control groups were HIV-1 negative pulmonary tuberculosis patients (TB) and healthy individuals from the same endemic area (EC). In silico predictions indicated the level of identity of <it>M. leprae </it>proteins with homologues in other mycobacteria and the presence of T cell and B cell epitopes.</p> <p>Results</p> <p>Despite the prediction that all proteins would be reactive, 16 of 33 (48%) of the single proteins tested were immunogenic (recognized in WBA or ELISA) and seventeen were non-immunogenic (not recognized in either assay). Among the 16 immunogenic proteins, 9 were considered leprosy specific in WBA inducing cell-mediated IFN-gamma secretion from TT/BT patients and HHC. Three of these proteins were also leprosy specific in serology being recognized by serum IgG from LL/BL patients. Seven of the immunogenic proteins were not leprosy specific.</p> <p>Conclusions</p> <p>New <it>M. leprae </it>antigens recognized by antibody responses of BL/LL patients and cellular responses of TT/BT leprosy patients were identified. An improved serological diagnostic test for leprosy could be developed by incorporating these IgG-reactive antigens to the current PGL-I based tests. Moreover our data indicate that the WBA is a robust, relatively simple and user friendly format for a T cell based diagnostic test. The field use of these test formats in leprosy endemic countries could contribute to early leprosy diagnosis before the development of deformities and disabilities.</p

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

Dissolved noble gases and stable isotopes as tracers of preferential fluid flow along faults in the Lower Rhine Embayment, Germany

Groundwater in shallow unconsolidated sedimentary aquifers close to the Bornheim fault in the Lower Rhine Embayment (LRE), Germany, has relatively low δ2H and δ18O values in comparison to regional modern groundwater recharge, and 4He concentrations up to 1.7 × 10−4 cm3 (STP) g–1 ± 2.2 % which is approximately four orders of magnitude higher than expected due to solubility equilibrium with the atmosphere. Groundwater age dating based on estimated in situ production and terrigenic flux of helium provides a groundwater residence time of ∼107 years. Although fluid exchange between the deep basal aquifer system and the upper aquifer layers is generally impeded by confining clay layers and lignite, this study’s geochemical data suggest, for the first time, that deep circulating fluids penetrate shallow aquifers in the locality of fault zones, implying  that sub-vertical fluid flow occurs along faults in the LRE. However, large hydraulic-head gradients observed across many faults suggest that they act as barriers to lateral groundwater flow. Therefore, the geochemical data reported here also substantiate a conduit-barrier model of fault-zone hydrogeology in unconsolidated sedimentary deposits, as well as corroborating the concept that faults in unconsolidated aquifer systems can act as loci for hydraulic connectivity between deep and shallow aquifers. The implications of fluid flow along faults in sedimentary basins worldwide are far reaching and of particular concern for carbon capture and storage (CCS) programmes, impacts of deep shale gas recovery for shallow groundwater aquifers, and nuclear waste storage sites where fault zones could act as potential leakage pathways for hazardous fluids

Evaluation of cytokine responses against novel Mtb antigens as diagnostic markers for TB disease.

OBJECTIVE: We investigated the accuracy of host markers detected in Mtb antigen-stimulated whole blood culture supernatant in the diagnosis of TB. METHODS: Prospectively, blood from 322 individuals with presumed TB disease from six African sites was stimulated with four different Mtb antigens (Rv0081, Rv1284, ESAT-6/CFP-10, and Rv2034) in a 24 h whole blood stimulation assay (WBA). The concentrations of 42 host markers in the supernatants were measured using the Luminex multiplex platform. Diagnostic biosignatures were investigated through the use of multivariate analysis techniques. RESULTS: 17% of the participants were HIV infected, 106 had active TB disease and in 216 TB was excluded. Unstimulated concentrations of CRP, SAA, ferritin and IP-10 had better discriminating ability than markers from stimulated samples. Accuracy of marker combinations by general discriminant analysis (GDA) identified a six analyte model with 77% accuracy for TB cases and 84% for non TB cases, with a better performance in HIV uninfected patients. CONCLUSIONS: A biosignature of 6 cytokines obtained after stimulation with four Mtb antigens has moderate potential as a diagnostic tool for pulmonary TB disease individuals and stimulated marker expression had no added value to unstimulated marker performance

Mycobacterium tuberculosis peptides presented by HLA-E molecules are targets for human CD8 T-cells with cytotoxic as well as regulatory activity

Tuberculosis (TB) is an escalating global health problem and improved vaccines against TB are urgently needed. HLA-E restricted responses may be of interest for vaccine development since HLA-E displays very limited polymorphism (only 2 coding variants exist), and is not down-regulated by HIV-infection. The peptides from Mycobacterium tuberculosis (Mtb) potentially presented by HLA-E molecules, however, are unknown. Here we describe human T-cell responses to Mtb-derived peptides containing predicted HLA-E binding motifs and binding-affinity for HLA-E. We observed CD8(+) T-cell proliferation to the majority of the 69 peptides tested in Mtb responsive adults as well as in BCG-vaccinated infants. CD8(+) T-cells were cytotoxic against target-cells transfected with HLA-E only in the presence of specific peptide. These T cells were also able to lyse M. bovis BCG infected, but not control monocytes, suggesting recognition of antigens during mycobacterial infection. In addition, peptide induced CD8(+) T-cells also displayed regulatory activity, since they inhibited T-cell proliferation. This regulatory activity was cell contact-dependent, and at least partly dependent on membrane-bound TGF-beta. Our results significantly increase our understanding of the human immune response to Mtb by identification of CD8(+) T-cell responses to novel HLA-E binding peptides of Mtb, which have cytotoxic as well as immunoregulatory activity

Cell-Mediated Immune Responses to in vivo -Expressed and Stage-Specific Mycobacterium tuberculosis Antigens in Latent and Active Tuberculosis Across Different Age Groups

A quarter of the global human population is estimated to be latently infected by Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). TB remains the global leading cause of death by a single pathogen and ranks among the top-10 causes of overall global mortality. Current immunodiagnostic tests cannot discriminate between latent, active and past TB, nor predict progression of latent infection to active disease. The only registered TB vaccine, Bacillus Calmette-Guérin (BCG), does not adequately prevent pulmonary TB in adolescents and adults, thus permitting continued TB-transmission. Several Mtb proteins, mostly discovered through IFN-γ centered approaches, have been proposed as targets for new TB-diagnostic tests or -vaccines. Recently, however, we identified novel Mtb antigens capable of eliciting multiple cytokines, including antigens that did not induce IFN-γ but several other cytokines. These antigens had been selected based on high Mtb gene-expression in the lung in vivo, and have been termed in vivo expressed (IVE-TB) antigens. Here, we extend and validate our previous findings in an independent Southern European cohort, consisting of adults and adolescents with either LTBI or TB. Our results confirm that responses to IVE-TB antigens, and also DosR-regulon and Rpf stage-specific Mtb antigens are marked by multiple cytokines, including strong responses, such as for TNF-α, in the absence of detectable IFN-γ production. Except for TNF-α, the magnitude of those responses were significantly higher in LTBI subjects. Additional unbiased analyses of high dimensional flow-cytometry data revealed that TNF-α+ cells responding to Mtb antigens comprised 17 highly heterogeneous cell types. Among these 17 TNF-α+ cells clusters identified, those with CD8+TEMRA or CD8+CD4+ phenotypes, defined by the expression of multiple intracellular markers, were the most prominent in adult LTBI, while CD14+ TNF-α+ myeloid-like clusters were mostly abundant in adolescent LTBI. Our findings, although limited to a small cohort, stress the importance of assessing broader immune responses than IFN-γ alone in Mtb antigen discovery as well as the importance of screening individuals of different age groups. In addition, our results provide proof of concept showing how unbiased multidimensional multiparametric cell subset analysis can identify unanticipated blood cell subsets that could play a role in the immune response against Mtb

Infliximab partially impairs the anti-Mycobacterium tuberculosis immune responses of severe psoriasis patients with positive tuberculin skin-test

Background Infliximab and etarnecept are now widely used for treating severe psoriasis. However, these drugs, especially infliximab, increased the risk of tuberculosis reactivation. Surprisingly, epidemiological data suggest that the tuberculosis rate in patients taking infliximab in Sao Paulo State, Brazil, is similar to that of some developed, non-endemic countries. Objective The aim of this study was to better understand the effect of infliximab on Mycobacterium tuberculosis (Mtb) immune responses of psoriasis patients in an endemic setting (Brazil). Methods We evaluated the tuberculosis-specific immune responses of severe psoriasis patients and healthy individuals, both tuberculin skin test (TST) positive, in the presence/absence of infliximab. Patients had untreated severe psoriasis, no co-morbidities affecting the immune responses and a TST &gt;10 mm. Healthy TST+ (&gt;10 mm) individuals were evaluated in parallel. PBMC cultures from both groups were stimulated with different Mycobacterium tuberculosis (Mtb) antigens (ESAT-6, 85B and Mtb lysate) and phytohemagglutinin, with or without infliximab (5 mu g/mL). Parameters evaluated were TNF-alpha, IFN-gamma and IL-10 secretion by ELISA, overnight IFN-gamma ELISpot and lymphocyte proliferative response (LPR). Results Infliximab almost abolished TNF-alpha detection in PBMC supernatants of both groups. It also significantly reduced the LPR to phytohemagglutinin and the Mtb antigens as well as the IFN-gamma levels secreted into day 5 supernatants in both groups. There was no concomitant exaggerated IL-10 secretion that could account for the decreases in these responses. ELISpot showed that, contrasting with the central-memory responses above, infliximab did not affect effector-memory INF-gamma-releasing T-cell numbers. Conclusions Infliximab affected some, but not all aspects of the in vitro antituberculosis immune responses tested. The preserved effector-memory responses, putatively related to exposure to environmental mycobacteria, may help to explain the lower than expected susceptibility to tuberculosis reactivation in our setting. Received: 29 December 2010; Accepted: 9 March 2011Fundacao de Amparo a Pesquisa do Estado de Sao Paulo [05/57761-7, 05/60075-8]Netherlands Leprosy Relief Foundation (NLR)European Commissio

A HILL TYPE ESTIMATOR OF THE WEIBULL TAIL-COEFFICIENT

International audienceWe present a new estimator of the Weibull tail-coefficient. The Weibull tail-coefficient is defined as the regular variation coefficient of the inverse cumulative hazard function. Our estimator is based on the log-spacings of the upper order statistics. Therefore, it is very similar to the Hill estimator for the extreme value index. We prove the weak consistency and the asymptotic normality of our estimator. Its asymptotic as well as its finite sample performances are compared to classical ones