Serodiagnosis of paucibacillary and multibacillary leprosy using a recombinant chimeric protein composed of specific B-cell epitopes derived from Mycobacterium leprae proteins.

Leprosy diagnosis is difficult due to the clinical similarity with other infectious diseases, and laboratory tests presents problems related to sensitivity and/or specificity. In this study, we used bioinformatics to assess Mycobacterium leprae proteins and formulated a chimeric protein that was tested as a diagnostic marker for the disease. The amino acid sequences from ML0008, ML0126, ML0308, ML1057, ML2028, ML2038, ML2498 proteins were evaluated, and the B-cell epitopes QASVAYPATSYADFRAHNHWWNGP, SLQRSISPNSYNTARVDP and QLLGQTADVAGAAKSGPVQPMGDRGSVSPVGQ were considered M. leprae-specific and used to construct the gene encoding the recombinant antigen. The gene was constructed, the recombinant protein was expressed, purified and tested in ELISA using 252 sera, which contained samples from multibacillary (MB) or paucibacillary (PB) leprosy patients, from their household contacts and healthy individuals, as well as from patients with Chagas disease, visceral and tegumentary leishmaniases (VL/TL), malaria, tuberculosis, and HIV. Sensitivity (Se) and specificity (Sp) for MB and PB samples compared to sera from both healthy subjects and individuals with cross-reactive diseases were 100%. The Se value for MB and PB samples compared to sera from household contacts was 100%, but Sp was 64%. In conclusion, data suggest that this protein could be considered in future studies for leprosy diagnosis.

Mitochondrial variants of complex I genes associated with leprosy clinical subtypes.

Leprosy is a chronic bacterial infection mainly caused by Mycobacterium leprae that primarily affects skin and peripheral nerves. Due to its ability to absorb carbon from the host cell, the bacillus became dependent on energy production, mainly through oxidative phosphorylation. In fact, variations in genes of Complex I of oxidative phosphorylation encoded by mtDNA have been associated with several diseases in humans, including bacterial infections, which are possible influencers in the host response to leprosy. Here, we investigated the presence of variants in the mtDNA genes encoding Complex I regarding leprosy, as well as the analysis of their pathogenicity in the studied cohort. We found an association of 74 mitochondrial variants with either of the polar forms, Pole T (Borderline Tuberculoid) or Pole L (Borderline Lepromatous and Lepromatous) of leprosy. Notably, six variants were exclusively found in both clinical poles of leprosy, including m.4158A>G and m.4248T>C in MT-ND1, m.13650C>A, m.13674T>C, m.12705C>T and m.13263A>G in MT-ND5, of which there are no previous reports in the global literature. Our observations reveal a substantial number of mutations among different groups of leprosy, highlighting a diverse range of consequences associated with mutations in genes across these groups. Furthermore, we suggest that the six specific variants exclusively identified in the case group could potentially play a crucial role in leprosy susceptibility and its clinical differentiation. These variants are believed to contribute to the instability and dysregulation of oxidative phosphorylation during the infection, further emphasizing their significance.

Exploring the Cell Wall and Secretory Proteins of Mycobacterium leprae as Biomarkers.

The bacterial cell wall is composed of a wide variety of intricate proteins in addition to lipids, glycolipids, and polymers. Given the diversity of cell wall proteins among bacterial species, they are a feasible target for biomarker identification and characterization in clinical research and diagnosis of the disease. The slow growth rate of Mycobacterium leprae poses a major hurdle in the accurate diagnosis of leprosy before the onset of peripheral neuropathy. The use of biomarker- based diagnostic methods can help in preventing the spread and manifestation of leprosy. Despite many advances in research methods and techniques, there remains a knowledge gap regarding the cell wall proteomes of M. leprae that can be used as biomarkers. The cell wall and secretory proteins of M. leprae are the major focus of this review article. This article enfolds the characteristics and functions of M. leprae cell wall proteins and gives an insight into those cell wall proteins that are yet to be established as biomarkers. Tools and techniques used in cell wall extraction and biomarker identification can also be explored in this article.

Challenges and advances in serological and molecular tests to aid leprosy diagnosis.

Leprosy is a neglected chronic infectious disease caused by obligate intracellular bacilli, Mycobacterium leprae and Mycobacterium lepromatosis. Despite multidrug therapy (MDT) success, leprosy accounts for more than 200,000 new cases yearly. Leprosy diagnosis remains based on the dermato-neurologic examination, but histopathology of skin biopsy and bacilloscopy of intradermal scraping are subsidiary diagnostic tests that require expertise and laboratory infrastructure. This minireview summarizes the state of the art of serologic tests to aid leprosy diagnosis, highlighting enzyme-linked immunosorbent assay (ELISA) and point-of-care tests (POCT) biotechnologies. Also, the impact of the postgenomic era on the description of new recombinantly expressed M. leprae-specific protein antigens, such as leprosy Infectious Disease Research Institute (IDRI) diagnostic (LID)-1 is summarized. Highly specific and sensitive molecular techniques to detect M. leprae DNA as the quantitative polymerase chain reaction (qPCR) and the loop-mediated isothermal amplification (LAMP) are briefly reviewed. Serology studies using phenolic glycolipid-I (PGL-I) semi-synthetic antigens, LID-1 fusion antigen, and the single fusion complex natural disaccharide-octyl (NDO)-LID show high sensitivity in multibacillary (MB) patients. However, serology is not applicable to paucibacillary patients, as they have weak humoral response and robust cell-mediated response, requiring tests for cellular biomarkers. Unlike ELISA-based tests, leprosy-specific POCT based on semi-synthetic PGL-I antigens and NDO-LID 1 antigen is easy to perform, cheaper, equipment-free, and can contribute to early diagnosis avoiding permanent incapacities and helping to interrupt M. leprae transmission. Besides its use to help diagnosis of household contacts or at-risk populations in endemic areas, potential applications of leprosy serology include monitoring MDT efficacy, identification of recent infection, especially in young children, as surrogate markers of disease progression to orient adult chemoprophylaxis and as a predictor of type 2 leprosy reactions. Advances in molecular biology techniques have reduced the complexity and execution time of qPCR confirming its utility to help diagnosis while leprosy-specific LAMP holds promise as an adjunct test to detect M. leprae DNA.

Comparison of antiphenolic glycolipid-1 antibody levels in seropositive contacts of leprosy after 2 years of single-dose rifampicin as postexposure prophylaxis.

Background: Leprosy is still a global problem, especially in developing countries, including Indonesia. Ineffective prevention of leprosy leads to active transmission of the disease. World Health Organization (WHO) recommend post-exposure prophylaxis (PEP) with single dose of rifampicin (SDR) for leprosy patients. Previous study showed protective effect of SDR against leprosy, especially for the first 2 years. Hence, the use of PEP and IgM anti PGL-1 examination are required to suspend the chain of leprosy transmission. This study evaluated the effectiveness of SDR administration by comparing IgM anti-PGL-1 antibody levels in seropositive household contacts before and after 2 years of SDR administration. Methods: Analytical observational laboratory study comparing IgM anti PGL-1 antibody levels before and after 2 years of SDR administration in leprosy contacts, with a prospective follow-up study design. We conducted this study from December 2022 to January 2023 at Dr. Mohammad Hoesin General Hospital Palembang. All seropositive household contacts of leprosy who had been administrated SDR 2 years ago were included, then PGL-1 antibody levels were examined. Results: The use of SDR showed significant improvement in leprosy contacts after 2 years (P=0.000). The median antibody level before SDR administration was 1,209.20 (615.81 - 4,353.60), which decrease to 146.03 (0 - 2,487.80) U/mL after 2 years. There was statistically significant relationship between history of BCG vaccination (P=0.003) and IgM PGL-1 antibody levels after 2 years of SDR administration. Conclusion: There is a significant decrease in IgM anti PGL-1 antibody levels among leprosy contacts after 2 years of SDR chemoprophylaxis administration.

Mycobacterium leprae's Infective Capacity Is Associated with Activation of Genes Involved in PGL-I Biosynthesis in a Schwann Cells Infection Model.

Peripheral nerves and Schwann cells (SCs) are privileged and protected sites for initial colonization, survival, and spread of leprosy bacillus. Mycobacterium leprae strains that survive multidrug therapy show a metabolic inactivation that subsequently induces the recurrence of typical clinical manifestations of leprosy. Furthermore, the role of the cell wall phenolic glycolipid I (PGL-I) in the M. leprae internalization in SCs and the pathogenicity of M. leprae have been extensively known. This study assessed the infectivity in SCs of recurrent and non-recurrent M. leprae and their possible correlation with the genes involved in the PGL-I biosynthesis. The initial infectivity of non-recurrent strains in SCs was greater (27%) than a recurrent strain (6.5%). In addition, as the trials progressed, the infectivity of the recurrent and non-recurrent strains increased 2.5- and 2.0-fold, respectively; however, the maximum infectivity was displayed by non-recurrent strains at 12 days post-infection. On the other hand, qRT-PCR experiments showed that the transcription of key genes involved in PGL-I biosynthesis in non-recurrent strains was higher and faster (Day 3) than observed in the recurrent strain (Day 7). Thus, the results indicate that the capacity of PGL-I production is diminished in the recurrent strain, possibly affecting the infective capacity of these strains previously subjected to multidrug therapy. The present work opens the need to address more extensive and in-depth studies of the analysis of markers in the clinical isolates that indicate a possible future recurrence.

High seropositivity against NDO-LID in a group of household contacts of leprosy patients. Are we close to leprosy elimination in Colombia?

INTRODUCTION: Leprosy is a chronic infectious disease caused by two mycobacteria (Mycobacterium leprae and Mycobacterium lepromatosis). The household contacts (HHC) of leprosy index cases are at higher risk of being infected with these mycobacteria. Therefore, serological testing in HHC would be an effective strategy to eliminate leprosy in Colombia. OBJECTIVE: To determine the seroprevalence and factors associated with the infection by M. leprae in HHC. METHODS: An observational study was conducted in 428 HHC located in the Colombian Caribbean, Andean, Pacific, and Amazonian regions. We evaluated the seropositivity and titrations of IgM, IgG, and protein A against NDO-LID. RESULTS: The evaluated HHC showed high seropositivity, precisely 36.9% anti-NDO-LID IgM, 28.3% anti-NDO-LID IgG, and 47.7% protein A. Furthermore, Protein A showed a greater capacity to detect infected individuals than other anti-NDO-LID conjugates (p < 0.0001). This study did not show differences in the seropositivity according to sex or age of the HHC (p > 0.05). Higher seropositivity for IgM was evidenced mainly in HHC located in the Colombian Pacific region (p 0.001). This research did not show differences in the seropositivity for these serological tests between HHC of PB or MB leprosy patients (p > 0.05). CONCLUSION: Leprosy transmission is still active between Colombian HHC. Consequently, controlling leprosy transmission in this population is fundamental to eradicating this disease.

Seroepidemiologic survey of the household contacts of leprosy patients.

OBJECTIVE: Leprosy is a disabling infectious disease caused by Mycobacterium leprae. This study aimed to investigate the prevalence of leprosy among household contacts of leprosy patients. METHODS: This study is a serological survey in household contacts of leprosy patients who had been treated or were undergoing treatment in the city of Presidente Prudente, São Paulo, Brazil, from 2006-2016, using clinical examination and screening for anti- Phenolic glycolipid-I antibodies with Mycobacterium leprae-flow serology. RESULTS: A total of 263 index cases of leprosy were identified during the study period. Of these, 53 were approached, and among their household contacts, 108 were examined. The ML-flow test was positive in 2 (1.85%) individuals, but clinical examination revealed no signs or symptoms of leprosy in them. Therefore, they were considered to have a subclinical infection. Leprosy was not confirmed in any household contacts. In this study, a lower percentage of household contacts, when compared to that in the literature, had a positive Mycobacterium leprae-flow test result. CONCLUSION: The use of Mycobacterium leprae-flow should be encouraged during the follow-up of at-risk populations, such as the household contacts of leprosy patients.

Factors associated with the development of leprosy in Brazilian contacts: a systematic review.

People who interact with leprosy patients in their environment, neighborhood, family, or social relationships are at risk to develop the disease. This systematic review investigated the risk and protective factors associated with the development of leprosy in Brazilian contacts. The studies were found in Cochrane Library, PubMed (MEDLINE), Embase, Virtual Health Library, grey literature and hand search until July 2021. The study selection, data extraction and quality assessment were independently performed by two investigators. The quality assessment was performed using the Newcastle-Ottawa Scale (NOS). This review was registered in PROSPERO (CRD42020160680). Seventeen articles fulfilled the inclusion criteria (n=544). The immunological and molecular factors, such as Anti-phenolic Glycolipid Antibodies (Anti-PGL-1) seropositivity, negative Mitsuda test, absence of Bacillus Calmette-Guérin (BCG) scar, positive Polymerase Chain Reaction (PCR) in blood; age and race; conviviality, education, contact time and type of contact, as well as elements related to the index case (bacilloscopic index; genetic conditions, family relationships), and some combined factors were shown to be relevant risk factors associated with the development of the disease in Brazilian leprosy contacts. The protective factors reported were the presence of one or more BCG scars, positive Mitsuda test, and education level. All selected studies were considered of high quality according to NOS. The knowledge of disease-related risk and protective factors provides the scientific basis for decision-making in the management of the disease in leprosy contacts.

Design of a specific peptide against phenolic glycolipid-1 from Mycobacterium leprae and its implications in leprosy bacilli entry.

BACKGROUND: Mycobacterium leprae, the causative agent of Hansen's disease, causes neural damage through the specific interaction between the external phenolic glycolipid-1 (PGL-1) and laminin subunit alpha-2 (LAMA2) from Schwann cells. OBJECTIVE: To design a LAMA2-based peptide that targets PGL-1 from M. leprae. METHODS: We retrieved the protein sequence of human LAMA2 and designed a specific peptide using the Antimicrobial Peptide Database and physicochemical parameters for antimycobacterial peptide-lipid interactions. We used the AlphaFold2 server to predict its three-dimensional structure, AUTODOCK-VINA for docking, and GROMACS programs for molecular dynamics simulations. FINDINGS: We analysed 52 candidate peptides from LAMA2, and subsequent screening resulted in a single 60-mer peptide. The mapped peptide comprises four ß-sheets and a random coiled region. This peptide exhibits a 45% hydrophobic ratio, in which one-third covers the same surface. Molecular dynamics simulations show that our predicted peptide is stable in aqueous solution and remains stable upon interaction with PGL-1 binding. In addition, we found that PGL-1 has a preference for one of the two faces of the predicted peptide, which could act as the preferential binding site of PGL-1. MAIN CONCLUSIONS: Our LAMA2-based peptide targeting PGL-1 might have the potential to specifically block this key molecule, suggesting that the preferential region of the peptide is involved in the initial contact during the attachment of leprosy bacilli to Schwann cells.

Arginase 1 is a marker of protection against illness in contacts of leprosy patients.

Leprosy household contacts are generally more prone to develop the disease compared to the general population. Previous studies have demonstrated that genes related to the alternative activation (M2) profile in macrophages are associated with the increased bacillary load in multibacillary leprosy patients (MB), and that contacts of MB patients have a higher risk of contracting the disease. In addition, positive serological responses to PGL-1 or LID-1 are associated with a higher risk of disease. We performed a 5-year follow-up of contacts of leprosy patients and evaluated the pattern of gene and protein expression in cells from contacts that developed leprosy during this period. Leprosy household contacts had decreased soluble CD163 and heme oxygenase 1 (HO-1) serum levels when compared with healthy donors and leprosy patients. In contrast, arginase 1 activities were higher in contacts when compared with both healthy donors and leprosy patients. Of the contacts, 33 developed leprosy during the follow-up. Gene expression analysis revealed reduced ARG1 expression in these contacts when compared with contacts that did not develop disease. Arginase activity was a good predictive marker of protection in contacts (sensitivity: 90.0%, specificity: 96.77%) and the association with serology for anti-PGL-1 and anti-LID-1 increased the sensitivity to 100%. Altogether, the data presented here demonstrate a positive role of arginase against leprosy and suggest that the evaluation of arginase activity should be incorporated into leprosy control programs in order to aid in the decision of which contacts should receive chemoprophylaxis.

Can anti-PGL-I antibody isotypes differentiate leprosy contacts and leprosy patients?

BACKGROUND: Serological tests for antibody measurement in leprosy have a series of limitations in discriminating contacts and patients. The present paper intends to evaluate if association of more than one antibody isotype in serum samples may be a useful tool in leprosy diagnosis. METHODS: This study evaluated 395 leprosy contacts and 71 leprosy index cases living in endemic municipalities in Northeastern Brazil. The participants were evaluated according to their anti-phenolic glycolipid antigen-I isotype (PGL-I) profile. Serum anti-PGL-I IgM, IgG, and IgA were measured by indirect ELISA. RESULTS: A strong association was found for antibody positivity in MB leprosy index cases. The odds ratios were 6.11 (95% CI 3.08 - 12.16) for IgM, 3.31 (1.66 - 6.61) for IgG, and 16.97 (8.39 - 34.2) for IgA. For IgM associated with one or more isotypes, the OR was 21.0 (95% CI 10.11 - 43.64), and for IgG + IgA, the OR was 17.58 (6.23 - 49.54). The highest diagnostic sensitivity of 76.0% (95% CI 61.8 - 86.9) was observed for IgM, and the lowest value was 24.1% (13.0 - 38.2), which was observed for IgG + IgA isotypes. Regarding presumptive positive predictive values, the lowest value was obtained for IgM at 24.7% (95% CI 18.1 - 32.3), and the highest values were observed for IgM+ one or more isotypes and for IgG + IgA isotype at 60.0% (44.3 - 74.3) and 66.7% (41.0 - 86.7), respectively. CONCLUSIONS: The present work demonstrated that by associating two or more positive antibody isotypes, the risk of facing a real case of leprosy may increase.

Interaction of constituents of MDT regimen for leprosy with Mycobacterium leprae HSP18: impact on its structure and function.

Mycobacterium leprae, the causative organism of leprosy, harbors many antigenic proteins, and one such protein is the 18-kDa antigen. This protein belongs to the small heat shock protein family and is commonly known as HSP18. Its chaperone function plays an important role in the growth and survival of M. leprae inside infected hosts. HSP18/18-kDa antigen is often used as a diagnostic marker for determining the efficacy of multidrug therapy (MDT) in leprosy. However, whether MDT drugs (dapsone, clofazimine, and rifampicin) do interact with HSP18 and how these interactions affect its structure and chaperone function is still unclear. Here, we report evidence of HSP18-dapsone/clofazimine/rifampicin interaction and its impact on the structure and chaperone function of HSP18. These three drugs interact efficiently with HSP18 (having submicromolar binding affinity) with 1 : 1 stoichiometry. Binding of these MDT drugs to the 'α-crystallin domain' of HSP18 alters its secondary structure and tryptophan micro-environment. Furthermore, surface hydrophobicity, oligomeric size, and thermostability of the protein are reduced upon interaction with these three drugs. Eventually, all these structural alterations synergistically decrease the chaperone function of HSP18. Interestingly, the effect of rifampicin on the structure, stability, and chaperone function of this mycobacterial small heat shock protein is more pronounced than the other two MDT drugs. This reduction in the chaperone function of HSP18 may additionally abate M. leprae survivability during multidrug treatment. Altogether, this study provides a possible foundation for rational designing and development of suitable HSP18 inhibitors in the context of effective treatment of leprosy.

Detection of Mycobacterium leprae infection in family clusters from six leprosy-endemic regions in Colombia.

BACKGROUND: Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which continues to be endemic in tropical countries, making it necessary to implement strategies for its elimination. The objective of the current article was to detect M. leprae infection and associated factors through serological and epidemiological evaluation in family clusters of leprosy patients. METHODS: Mycobacterium leprae infection was determined in 50 family clusters of leprosy patients from the departments of Bolívar, Atlántico, Santander, Boyacá, Chocó and Antioquia through the detection of antibodies (protein A, IgM, IgG) against anti-natural octyl disacharide-leprosy IDRI diagnostic (NDO-LID). RESULTS: Higher seroconversion and elevated titers of these antibodies against NDO-LID were observed in the population of Chocó and Atlántico (p<0.05). Additionally, a higher frequency of infection was observed in large family groups that consumed armadillo meat and belonged to a low socioeconomic stratum (p<0.05). Multivariate analysis established that the main associated factors for a family cluster experiencing this infection were belonging to a vulnerable economic stratum and a large family group. CONCLUSIONS: This study found that the set of social and demographic variables (i.e. armadillo consumption, geographic area, low socioeconomic status and belonging to a large family cluster) are related to the promotion of seropositivity in family clusters.

Laboratory diagnosis of leprosy: Two staining methods from bacilloscopy and rapid ml flow test.

Background: The diagnosis of leprosy is based on the characteristic signs and symptoms of the disease, subsidized by laboratory tests. When positive, the bacilloscopy closes the diagnosis for leprosy. Phenolic glycolipid-I, or PGL-I, is a molecule in the bacillus cell wall that confers a greater immune response. The ML Flow test is an immunochromatographic test for the detection of anti-PGL-I IgM in human blood or serum. Methods: A prospective study with data collection and biological materials in patients with suspected leprosy from August 2020 to May 2021. For microscopy, intradermal smears were stained with Auramine O, and after reading under a fluorescence microscope, reviewed by Ziehl-Neelsen. The ML flow test was performed according to the Bührer-Sékula protocol. To assess the agreement between the methods, the Kappa index was estimated. Results: Of the 94 suspected leprosy patients, 31 (32.9%) were diagnosed with leprosy. There was moderate agreement between the results of the ML Flow and Auramine O tests (Kappa = 0.58) and substantial agreement between the ML Flow and Ziehl-Neelsen microscopy (Kappa = 0.72). In paucibacillary cases, serology was positive in 100% of patients. Conclusions: This study concluded that the Ziehl-Neelsen technique remains the best option for standard leprosy staining, and the ML flow test is more positive among the three techniques evaluated and can be an effective tool in the early diagnosis of leprosy cases.

Mimicking B and T cell epitopes between Mycobacterium leprae and host as predictive biomarkers in type 1 reaction in leprosy.

Several Mycobacterial infections including leprosy and tuberculosis are known to evoke autoimmune responses by modulating homeostatic mechanism of the host. Presence of autoantibodies like, rheumatoid factor, anti-nuclear factor and antibodies to host, collagen, keratin, myelin basic protein (MBP) and myosin, have been earlier reported in leprosy patients. In the present study, we detected the role of mimicking epitopes between Mycobacterium leprae and host components in the induction of autoimmune response in leprosy. Based on our previous findings, we predicted and synthesized a total of 15 mimicking linear B cell epitopes (BCE) and 9 mimicking linear T cell epitopes (TCE) of keratin and MBP. Humoral and cell-mediated immune responses against these epitopes were investigated in Non-reaction (NR), Type 1 reaction (T1R) leprosy patients, and healthy controls. We observed significantly higher levels of antibodies against 8 BCE in T1R in comparison to NR leprosy patients. Further, we also found 5 TCE significantly associated with lymphocyte proliferation in the T1R group. Our results indicated that these epitopes play a key role in the induction of autoimmune response in leprosy and are also strongly associated with the inflammatory episodes of T1R. Conclusively, these molecules may be employed as a biomarker to predict the inflammatory episodes of T1R.

BCG-induced immunity profiles in household contacts of leprosy patients differentiate between protection and disease.

Leprosy is an infectious disease caused by Mycobacterium leprae leading to irreversible disabilities along with social exclusion. Leprosy is a spectral disease for which the clinical outcome after M. leprae infection is determined by host factors. The spectrum spans from anti-inflammatory T helper-2 (Th2) immunity concomitant with large numbers of bacteria as well as antibodies against M. leprae antigens in multibacillary (MB) leprosy, to paucibacillary (PB) leprosy characterised by strong pro-inflammatory, Th1 as well as Th17 immunity. Despite decades of availability of adequate antibiotic treatment, transmission of M. leprae is unabated. Since individuals with close and frequent contact with untreated leprosy patients are particularly at risk to develop the disease themselves, prophylactic strategies currently focus on household contacts of newly diagnosed patients. It has been shown that BCG (re)vaccination can reduce the risk of leprosy. However, BCG immunoprophylaxis in contacts of leprosy patients has also been reported to induce PB leprosy, indicating that BCG (re)vaccination may tip the balance between protective immunity and overactivation immunity causing skin/nerve tissue damage. In order to identify who is at risk of developing PB leprosy after BCG vaccination, amongst individuals who are chronically exposed to M. leprae, we analyzed innate and adaptive immune markers in whole blood of household contacts of newly diagnosed leprosy patients in Bangladesh, some of which received BCG vaccination. As controls, individuals from the same area without known contact with leprosy patients were similarly assessed. Our data show the added effect of BCG vaccination on immune markers on top of the effect already induced by M. leprae exposure. Moreover, we identified BCG-induced markers that differentiate between protective and disease prone immunity in those contacts.

Epitope mapping from Mycobacterium leprae proteins: Convergent data from in silico and in vitro approaches for serodiagnosis of leprosy.

Knowledge of immunodominant B-cell epitopes is essential to design powerful diagnostic strategies aiming for antibody detection. Outstanding progress in computational prediction has achieved a significant contribution to the biomedical fields, including immunodiagnosis. In silico analysis may have an even more important role when information concerning antigens from etiologic agents of neglected diseases, such as leprosy, is scarce. The aim of this study was to provide mapping of B-cell epitopes from two Mycobacterium leprae-derived antigens (Ag85B and ML2055), confirm their antigenicity, and to assess the ability of in silico immunoinformatics tools to accurately predict them. Linear B-cell epitopes predicted by ABCpred and SVMTrip servers were compared to antigenic regions of synthetic overlapping peptides that exhibited reactivity to antibodies from patients with leprosy. Our in vitro results identified several immunodominant regions that had also been indicated by in silico prediction, providing agreement between experimental and simulated data. After chemical synthesis, we used enzyme-linked immunosorbent assays to determine the effectiveness of the first identified sequence (GTNVPAEFLENFVHG) which had 72 % sensitivity and 78 % specificity (AUC = 0.79) while the second one (PVSSEAQPGDPNAPS) had 72 % sensitivity and 93.8 % specificity (AUC = 0.85). Using dot blotting, an easy-to-read visual test, both peptides could distinguish sera from patients with leprosy from those with tuberculosis and from sera of healthy volunteers. Our findings suggest that these synthetic peptides, with some refinement, may be useful as serological diagnostic antigens for leprosy. In addition, it was displayed that immunoinformatics provides reliable information for mapping potential B-cell epitopes for development of peptide-based diagnostic assays for neglected diseases.