Plant Expression of Hydrophobin Fused K39 Antigen for Visceral Leishmaniasis Immunodiagnosis.
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SUBMITTER: Silva BB
PROVIDER: S-EPMC8201991 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundVisceral leishmaniasis is the most severe form of leishmaniasis. Approximately 20% of zoonotic human visceral leishmaniasis worldwide is caused by Leishmania infantum, which is also known as Leishmania chagasi in Latin America, and disease incidence is increasing in urban and peri-urban areas of the tropics. In this form of disease, dogs are the main reservoirs. Diagnostic methods used to identify Leishmania infected animals are not able to detect all of the infected ones, which can compromise the effectiveness of disease control. Therefore, to contribute to the improvement of diagnostic methods for canine visceral leishmaniasis (CVL), we aimed to identify and test novel antigens using high-throughput analysis.Methodology/principal findingsImmunodominant proteins from L. infantum were mapped in silico to predict B cell epitopes, and the 360 predicted peptides were synthesized on cellulose membranes. Immunoassays were used to select the most reactive peptides, which were then investigated with canine sera. Next, the 10 most reactive peptides were synthesized using solid phase peptide synthesis protocol and tested using ELISA. The sensitivity and specificity of these peptides were also compared to the EIE-LVC Bio-Manguinhos kit, which is recommended by the Brazilian Ministry of Health for use in leishmaniasis control programs. The sensitivity and specificity of the selected synthesized peptides was as high as 88.70% and 95.00%, respectively, whereas the EIE-LVC kit had a sensitivity of 13.08% and 100.00% of specificity. Although the tests based on synthetic peptides were able to diagnose up to 94.80% of asymptomatic dogs with leishmaniasis, the EIE-LVC kit failed to detect the disease in any of the infected asymptomatic dogs.Conclusions/significanceOur study shows that ELISA using synthetic peptides is a technique with great potential for diagnosing CVL; furthermore, the use of these peptides in other diagnostic methodologies, such as immunochromatographic tests, could be beneficial to CVL control programs.
| S-EPMC3172188 | biostudies-literature
Project description:BackgroundRapid diagnostic test using rk39 antigen is widely used for visceral leishmaniasis. However it detects anti-rk39 antibodies in 20-32% of endemic healthy individuals. In search for a better biomarker of infection, we identified a protein of molecular weight 70 kDa (BHUP1), specifically recognized by sera of visceral leishmaniasis (VL) patients.MethodsThe protein was cloned as His-tagged fusion protein and purified. We evaluated the sensitivity and specificity of this protein in an enzyme linked immunosorbant assay (ELISA) format in comparison to the rk39 antigen using sera collected from various groups of individuals.ResultsThe sensitivity of rBHUP1 was 96.5% compared to 98.8% with rk39. For healthy controls from non endemic and endemic regions, the specificity of rBHUP1 was 100% and 95.6% compared to 100% and 84.9% for rk39, respectively. For other infectious diseases such as malaria, tuberculosis, viral fever, etc., specificity of rBHUP1 was as low as 74.5% when compared to 94% of rk39. At six month and one year follow-up, 74% and 22.5% patients tested positive with rBHUP1, respectively, compared to 97% and 77.4% with rk39 antigen.ConclusionThough the high sensitivity and specificity of rBHUP1 antigen for VL and healthy controls would have made it a good diagnostic biomarkers, however, its non-specific reaction with other infectious diseases limit its utility.
| S-EPMC4007064 | biostudies-literature
Project description:Gold standard serological diagnostic methods focus on antigens that elicit a strong humoral immune response that is specific to a certain pathogen. In this study, we used bioinformatics approaches to identify linear B-cell epitopes that are conserved among Leishmania species but are divergent from the host species Homo sapiens and Canis familiaris and from Trypanosoma cruzi, the parasite that causes Chagas disease, to select potential targets for the immunodiagnosis of leishmaniasis. Using these criteria, we selected heat shock protein 83.1 of Leishmania braziliensis for this study. We predicted three linear B-cell epitopes in its sequence. These peptides and the recombinant heat shock protein 83.1 (rHSP83.1) were tested in enzyme-linked immunosorbent assays (ELISAs) against serum samples from patients with tegumentary leishmaniasis (TL) and visceral leishmaniasis (VL) and from dogs infected with Leishmania infantum (canine VL [CVL]). Our data show that rHSP83.1 is a promising target in the diagnosis of TL. We also identified specific epitopes derived from HSP83.1 that can be used in the diagnosis of human TL (peptide 3), both human and canine VL (peptides 1 and 3), and all TL, VL, and CVL clinical manifestations (peptide 3). Receiver operating characteristic (ROC) curves confirmed the superior performance of rHSP83.1 and peptides 1 and 3 compared to that of the soluble L. braziliensis antigen and the reference test kit for the diagnosis of CVL in Brazil (EIE-LVC kit; Bio-Manguinhos, Fiocruz). Our study thus provides proof-of-principle evidence of the feasibility of using bioinformatics to identify novel targets for the immunodiagnosis of parasitic diseases using proteins that are highly conserved throughout evolution.
| S-EPMC4097446 | biostudies-other
Project description:A cDNA expression library of Clonorchis sinensis adult worm was constructed, and screened out immunologically. One clone, pBCs31, was selected in view of its predominant reactivity with an experimentally infected rabbit serum. Recombinant C. sinensis antigen with 28 kDa as a beta-galactosidase fusion protein produced in Escherichia coli was identified by immunoblot analysis. The cloned gene was composed of 16 copies of a 30 base pair repeat and an additional 320 bases. The deduced amino acid sequence of the tandem repeat was AQPPKSGDGG. On RNA slot blot analysis. C. sinensis adult worm RNA showed a positive reaction with the cloned gene. Enzyme-linked immunosorbent assay using a purified recombinant antigen of pBCs31 showed high specificity for diagnosis of clonorchiasis.
| S-EPMC2732929 | biostudies-literature
Project description:Introduction: The armamentarium of antileishmanial drugs is small. It is further being threatened by the development of resistance and decreasing sensitivity to the available drugs. The development of newer drugs is sorely needed. Areas covered: The authors have based their review on a literature search performed using PubMed. The article specifically looks at investigational drugs, which have demonstrated, at the very least, in vitro and in vivo activities against the leishmania species that cause visceral leishmaniasis. Specifically, the authors review the nitroimidazole compound fexinidazole, which is one of the few drugs which have reached Phase II trials. The article also discusses the R enantiomer of (S)-PA-824, which has shown good antileishmanial activity. Finally, the article also highlights the many novel delivery systems and oral formulations of amphotericin B, which are both cheap and less toxic and are currently under investigation. Expert opinion: Very few new drugs have reached the clinic for this neglected tropical disease and there is an urgent need for new efficacious therapeutics. The authors believe that support from public-private partnerships would help in enabling the prompt development of drug candidates that could potentially make the clinic.
| S-EPMC4262729 | biostudies-other
Project description:BackgroundVisceral leishmaniasis (VL) is a potentially fatal parasitic disease associated with fever, cachexia and impaired protective T-cell responses against the parasite.MethodsPeripheral blood leukocytes from 105 subjects with VL and healthy control subjects from the endemic region of Muzaffarpur, Bihar, India, were compared using flow cytometry and reverse-transcriptase quantitative polymerase chain reaction. Findings were correlated with clinical data.ResultsAn expanded population of low-density neutrophils that expressed HLA-DR, CD80 and CD86 was observed in subjects with VL. This neutrophil population contracted after successful treatment of disease. Plasma from patients with acute VL was able to induce similar high-level HLA-DR expression in neutrophils from healthy subjects. HLA-DR+ neutrophils from subjects with VL did not stimulate T-cell proliferation, but they did express higher programmed cell death ligand-1 (PDL1) than other neutrophils, and lymphocytes of the same subjects expressed high programmed cell death 1 (PD1).ConclusionsPatients with acute VL have expanded circulating low-density neutrophils expressing markers of antigen presentation, which diminish after treatment. Development of HLA-DR+ neutrophils is stimulated, at least in part, by components of plasma from patients with acute disease. Although we found no evidence that they act as antigen-presenting cells, these neutrophils expressed markers implicating a role in T-cell exhaustion.
| S-EPMC5091370 | biostudies-literature
Project description:IntroductionTo sustain the achievement of kala-azar elimination program (KEP), early detection and treatment of the visceral leishmaniasis (VL) cases and associated modalities such as treatment failure (TF), relapse VL (RVL), and Post-kala-azar dermal leishmaniasis (PKDL) is the cornerstone. A predictive biomarker for VL development and related complications could also play a crucial role in curtailing disease incidence and transmission. Investigations to find a biomarker with prospective capabilities are, however, scarce. Using samples and known clinical outcomes generated within two previous longitudinal cohort studies, we aimed to determine if fluctuations in serum anti-rK39 antibody levels could provide such predictive value.Materials and methodsSerum samples collected at four different time points (Baseline, 12, 18, and 24 months) from 16 patients who had developed VL within the monitoring period and 15 of their asymptomatic healthy controls counterparts were investigated. To investigate potential prediction of VL related complications, serum samples of 32 PKDL, 10 RVL, 07 TF, and 38 cured VL from a single dose AmBisome trial were analyzed. Of this second panel, all patients were monitored for 5 years and sera were collected at four time points (Baseline then 1, 6, and 12 months after treatment). The level of anti-rK39 antibodies in archived samples was measured by a semi-quantitative ELISA.ResultsThe mean antibody level was significantly higher in VL patients compared to their asymptomatic healthy counterparts at each time point. Likewise, we observed a trend toward elevations in antibody levels for PKDL, RVL, TF relative to the reducing levels observed in cured VL. Receiver operating characteristic (ROC) analysis found a promising predictive power of rK39 antibody levels to reveal progression from asymptomatic Leishmania donovani infection stage to VL, defined as 87.5% sensitive and 95% specific. Following treatment, rk39 antibody notably showed 100% sensitivity and 95% specificity in predicting TF.ConclusionOur data indicate that the relative quantity of serum anti-rK39 antibody has promise within either a predictive or prognostic algorithm for VL and VL-related modalities. These could enable VL control programs to implement more effective measures to eliminate the disease. Further research is, however, imperative to standardize the rK39 antibody ELISA between sites prior to broader use.
| S-EPMC6795025 | biostudies-literature
Project description:The unreliability of most of the existing antibody-based diagnostic kits to discriminate between active and treated VL cases, relapse situation and reinfection are a major hurdle in controlling the cases of Kala-azar in an endemic area. An antigen targeted diagnostic approaches can be an attractive strategy to overcome these problems. Hence, this study was focused on identifying the Leishmania antigens, lies in circulating immune complex (CICs), can be used for diagnostic as well as prognostic purposes. The present study was conducted on peripheral blood samples of 115 human subjects, based on isolation of CICs. The SDS-PAGE patterns showed an up-regulated expression of 55 kDa and 23 kDa fractions in an antigens obtained from CICs of all clinical and parasitologically proven untreated visceral leishmaniasis patients before treatment (VL-BT), which ensured absolute sensitivity. However, light expressions of these bands were observed in some VL treated cases. To ascertain the prognostic value, 2D expression profiles of circulating antigens were carried out, which revealed 3 upregulated and 12 induced immunoreactive spots. Out of these, ten prominent spots were excised and subjected for enzymatic digestion to generate peptides. Mass spectrometry (MS) analysis successfully explored 20 peptides derived from kinase, kinesin, acetyl Co-A carboxylase, dynein heavy chains (cytoplasmic and axonemal/flagellar), 60S ribosomal protein, nucleoporin protein, RNA polymeraseII, protease gp63, tubulin, DNA polymerase epsilon subunit, GTP-binding protein and tyrosyl-methionyl t-RNA synthetase-like protein and 19 hypothetical protein of unknown function. Presence of L. donovani proteins in circulating antigens were further validated using anti-Ld actin and anti-α tubulin antibody. Besides, MS derived peptides confirmed its reactivity with patients' sera. Therefore, these shortlisted potential antigens can be explored as antigen-based diagnostic as well as prognostic kit.
| S-EPMC5562322 | biostudies-literature
Project description:BACKGROUND:Visceral leishmaniasis is systematic serous parasitic disease with public health importance. Zoonotic form of visceral leishmaniasis is wide spread in Mediterranean basin and South America regions. Direct agglutination test (DAT) is an accurate, reliable and non-expensive serological test for the diagnosis of visceral leishmaniasis in human and canines but the antigen preparation involves some limitations. This study aimed to compare the conventional production of DAT antigen with our modified DAT antigen and then assessed on human and dog pooled sera. METHODS:Conventional DAT antigen has been prepared at the School of Public Health, Tehran University of Medical Sciences and some modifications were carried out on it, which named as modified DAT antigen. Three positive and one negative human and dog pooled serum were separately used for the comparison of modified DAT with conventional DAT antigen batches with one-month interval for a period of 9 months. RESULTS:A good concordance was observed between modified DAT compared to conventional DAT antigens for the detection of visceral leishmaniasis on human (100%) and dog (94.4%) pooled sera, respectively. CONCLUSION:Since the modified DAT antigen could be reduced the preparation time from 3 days to several hours and a good degree of agreement was found between modified DAT and convention DAT antigen batches, it can be used as a simple and easy tool for screening and serodiagnosis of human and canine L. infantum infection.
| S-EPMC4662736 | biostudies-literature
Project description:The direct agglutination test (DAT) for visceral leishmaniasis (VL) is the serodiagnostic test for VL that has the most robust sensitivity and specificity in the field across all endemic regions. It is based on trypsin-treated and formaldehyde-fixed whole promastigote cells from Leishmania donovani. The exact identity and nature of the epitopes on the DAT antigen that cause agglutination with VL patients' sera are currently unknown. In this study, we performed antigen-inhibition studies which revealed that lipophosphoglycan (LPG) and the DAT antigen share epitopes. Antibody inhibition with a monoclonal antibody directed against the phosphoglycan repeat epitope of LPG showed that this is not the epitope that reacts with human sera. Oxidation of carbohydrates by sodium metaperiodate did not alter the reactivity of human sera with the DAT antigen and LPG. This indicates that carbohydrates do not play a role in the reaction of the DAT antigen with antibodies in serum from VL patients, and that they also are not involved in the reaction of LPG with the same serum. We conclude that the noncarbohydrate moiety of LPG, that is, the core-anchor fragment, and potentially other noncarbohydrate epitopes on the surface of the DAT antigen are responsible for its agglutination with antibodies from VL patients. As LPG plays a role in the DAT reaction, this could facilitate the following: 1) incorporation of LPG, preferably the synthetic version of the core-anchor fragment, into an immunochromatographic test format that is more adapted as a point-of-care test (short incubation, little training, and equipment needed) than DAT and 2) enhancing the quality control for the production of the DAT antigen.
| S-EPMC7124922 | biostudies-literature