HSP60 mimetic peptides from Mycobacterium leprae as new antigens for immunodiagnosis of Leprosy.

The early diagnosis of leprosy serves as an important tool to reduce the incidence of this disease in the world. Phage display (PD) technology can be used for mapping new antigens to the development of immunodiagnostic platforms. Our objective was to identify peptides that mimic Mycobacterium leprae proteins as serological markers using phage display technology. The phages were obtained in the biopanning using negative and positive serum from household contacts and leprosy patients, respectively. Then, the peptides were synthesized and validated in silico and in vitro for detection of IgG from patients and contacts. To characterize the native protein of M. leprae, scFv antibodies were selected against the synthetic peptides by PD. The scFv binding protein was obtained by immunocapture and confirmed using mass spectrometry. We selected two phase-fused peptides, MPML12 and MPML14, which mimic the HSP60 protein from M. leprae. The peptides MPML12 and MPML14 obtained 100% and 92.85% positivity in lepromatous patients. MPML12 and MPM14 detect IgG, especially in the multibacillary forms. The MPML12 and MPML14 peptides had positivity of 11.1% and 16.6% in household contacts, respectively. There was no cross-reaction in patient's samples with visceral leishmaniasis, tuberculosis and other mycobacteriosis for both peptides. Given these results and the easy obtainment of mimetic antigens, our peptides are promising markers for application in the diagnosis of leprosy, especially in endemic and hyperendemic regions.

Clinical, epidemiological, and laboratory prognostic factors in patients with leprosy reactions: A 10-year retrospective cohort study.

Introduction: Leprosy reactions, the main cause of neural damage, can occur up to 7 years after starting multidrug therapy. We aimed to approach the prognostic factors that may influence the leprosy reactions over the follow-up time. Methods: Retrospective cohort study, encompassing 10 years of data collection, composed of 390 patients, divided into 201 affected by reactions and 189 reaction-free individuals. Epidemiological, clinical, and laboratory variables were approached as prognostic factors associated with leprosy reactions. The association among variables was analyzed by a binomial test and survival curves were compared by the Kaplan-Meier and Cox proportional-hazards regression. Results: 51.5% (201/390) of patients were affected by leprosy reactions. These immunological events were associated with lepromatous leprosy (16.2%; 63/390; p < 0.0001) and multibacillary group (43%; 169/390; p < 0.0001). This study showed that survival curves for the prognostic factor anti-PGL-I, comparing positive and negative cases at diagnosis, differed in relation to the follow-up time (Log Rank: p = 0.0760; Breslow: p = 0.0090; Tarone-Ware: p = 0.0110). The median survival times (time at which 50% of patients were affected by leprosy reactions) were 5 and 9 months for those reactional cases with negative (26/51) and positive serology (75/150), respectively. The time-dependent covariates in the cox proportional-hazards regression showed anti-PGL-I as the main prognostic factor to predict leprosy reactions (hazard ratio=1.91; p = 0.0110) throughout the follow-up time. Conclusions: Finally, these findings demonstrated that anti-PGL-I serology at diagnosis is the most important prognostic factor for leprosy reactions after starting multidrug therapy, thus enabling prediction of this immunological event.

Biotechnological and Immunological Platforms Based on PGL-I Carbohydrate-Like Peptide of Mycobacterium leprae for Antibodies Detection Among Leprosy Clinical Forms.

Phenolic glycolipid I (PGL-I) is an abundant antigen on the Mycobacterium leprae cell wall, commonly used for operational classification of leprosy patients. Our aim was to develop PGL-I mimotopes with similar characteristics and functions of the native antigen. We have used a random peptide phage display (PD) library for selections against the monoclonal antibody anti-PGL-I. After three selection cycles, six peptides were identified. All sequences were interspersed by a spacer generating a chimeric peptide (PGLI-M3) that was artificially synthesized. The highly reactive peptide was submitted to a reverse PD selection with a single-chain Fv (scFv) antibody fragment combinatorial library. The most reactive scFv was then validated by enzyme-linked immunosorbent assay (ELISA) against both native PGL-I and two derived synthetic (NDO and ND-O-HSA). We have further proved the scFv specificity by detecting M. leprae bacilli in leprosy lesions through immunohistochemistry. We then described its applicability in ELISA for all clinical forms and household contacts (HC). Afterward, we showed differential binding affinities of PGLI-M3 to sera (anti-PGL-I IgM) from all leprosy clinical forms through surface plasmon resonance (SPR). ELISA IgM detection showed 89.1% sensitivity and 100% specificity, considering all clinical forms. Positivity for anti-PGL-I IgM was twofold higher in both HC and patients with paucibacillary forms in hyperendemic regions than in endemic ones. The SPR immunosensor was able to differentiate clinical forms with 100% accuracy. This is the first time that a PGL-I mimotope has efficiently mimicked the carbohydrate group of the M. leprae antigen with successful immunoassay applications and may become a substitute for the native antigen.

Differential Expression of IFN-γ, IL-10, TLR1, and TLR2 and Their Potential Effects on Downgrading Leprosy Reaction and Erythema Nodosum Leprosum.

Leprosy reactions are acute immunological events that occur during the evolution of chronic infectious disease causing neural damage and disabilities. A study using blood samples of 17 leprosy reaction patients and 17 reaction-free was carried out by means of associations between antigens, receptors, and expression of cytokines, using path analysis providing new insights into the immunological mechanisms involved in triggering leprosy reactions. Toll-like receptors (TLR) such as TLR1 and TLR2, presented balanced expression in the reaction-free multibacillary (MB) group (TLR1: 1.01 ± 0.23, TLR2: 1.22 ± 0.18; p = 0.267). On the other hand, downgrading type 1 reaction (T1R) (TLR1: 1.24 ± 0.17, TLR2: 2.88 ± 0.37; p = 0.002) and erythema nodosum leprosum (ENL) (TLR1: 1.93 ± 0.17, TLR2: 2.81 ± 0.15; p = 0.004) revealed an unbalance in relation to the expression of these receptors. When the path analysis was approached, it was noted that interleukin 10 (IL-10) expression showed a dependence relation with phenolic glycolipid I (PGL-I) in downgrading T1R (direct effect = 0.503 > residual effect = 0.364), whereas in ENL, such relationship occurred with lipoarabinomannan (LAM) (direct effect = 0.778 > residual effect = 0.280). On the contrary, in the reaction-free leprosy group, interferon-gamma (IFN-γ) levels were dependent on the association between TLR2 and TLR1 (0.8735). The high TLR2 expression associated with IL-10 levels, in the leprosy reaction groups, may be hypothetically related to the formation of TLR2/2 homodimers and/or TLR2/6 heterodimers linked to evasion mechanisms in downgrading reactions and pathophysiology of ENL.