RESUMO
Opisthorchis viverrini is the etiological agent of the disease opisthorchiasis and related cholangiocarcinoma (CCA). It infects fish-eating mammals and more than 10 million people in Southeast Asia suffered from opisthorchiasis with a high fatality rate. The only effective drug against this parasite is Praziquantel, which has significant side effects. Due to the lack of appropriate treatment options and the high death rate, there is a dire need to develop novel therapies against this pathogen. In this study, we designed a multi-epitope chimeric vaccine design against O. viverrini by using immunoinformatics approaches. Non-allergenic and immunogenic MHC-1, MHC-2, and B cell epitopes of three candidate proteins thioredoxin peroxidase (Ov-TPx-1), cathepsin F1 (Ov-CF-1) and calreticulin (Ov-CALR) of O. viverrini, were predicted to construct a potent multiepitope vaccine. The coverage of the HLA-alleles of these selected epitopes was determined globally. Four vaccine constructs made by different adjuvants and linkers were evaluated in the context of their physicochemical properties, antigenicity, and allergenicity. Protein-protein docking and MD simulation found that vaccines 3 was more stable and had a higher binding affinity for TLR2 and TLR4 immune receptors. In-silico restriction cloning of vaccine model led to the formation of plasmid constructs for expression in a suitable host. Finally, the immune simulation showed strong immunological reactions to the engineered vaccine. These findings suggest that the final vaccine construct has the potential to be validated by in vivo and in vitro experiments to confirm its efficacy against the CCA causing O. viverrini.
Assuntos
Antígenos de Helmintos , Neoplasias dos Ductos Biliares , Colangiocarcinoma , Opistorquíase , Opisthorchis , Vacinas de Subunidades Antigênicas , Opisthorchis/imunologia , Animais , Colangiocarcinoma/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Opistorquíase/imunologia , Opistorquíase/prevenção & controle , Humanos , Neoplasias dos Ductos Biliares/imunologia , Antígenos de Helmintos/imunologia , Antígenos de Helmintos/química , Epitopos de Linfócito B/imunologia , Desenvolvimento de Vacinas , Biologia Computacional/métodos , Simulação de Acoplamento Molecular , Proteínas de Helminto/imunologia , Proteínas de Helminto/química , Epitopos de Linfócito T/imunologia , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Receptor 2 Toll-Like/imunologiaRESUMO
Helminths produce calreticulin (CRT) to immunomodulate the host immune system as a survival strategy. However, the structure of helminth-derived CRT and the structural basis of the immune evasion process remains unclarified. Previous study found that the tissue-dwelling helminth Trichinella spiralis produces calreticulin (TsCRT), which binds C1q to inhibit activation of the complement classical pathway. Here, we used x-ray crystallography to resolve the structure of truncated TsCRT (TsCRTΔ), the first structure of helminth-derived CRT. TsCRTΔ was observed to share the same binding region on C1q with IgG based on the structure and molecular docking, which explains the inhibitory effect of TsCRT on C1q-IgG-initiated classical complement activation. Based on the key residues in TsCRTΔ involved in the binding activity to C1q, a 24 amino acid peptide called PTsCRT was constructed that displayed strong C1q-binding activity and inhibited C1q-IgG-initiated classical complement activation. This study is the first to elucidate the structural basis of the role of TsCRT in immune evasion, providing an approach to develop helminth-derived bifunctional peptides as vaccine target to prevent parasite infections or as a therapeutic agent to treat complement-related autoimmune diseases.
Assuntos
Calreticulina , Complemento C1q , Evasão da Resposta Imune , Trichinella spiralis , Trichinella spiralis/imunologia , Complemento C1q/imunologia , Complemento C1q/metabolismo , Complemento C1q/química , Animais , Calreticulina/imunologia , Calreticulina/química , Calreticulina/metabolismo , Cristalografia por Raios X , Ligação Proteica , Simulação de Acoplamento Molecular , Proteínas de Helminto/imunologia , Proteínas de Helminto/química , Ativação do Complemento/imunologia , Imunoglobulina G/imunologia , Humanos , Antígenos de Helmintos/imunologia , Antígenos de Helmintos/química , Triquinelose/imunologia , Triquinelose/parasitologia , Via Clássica do Complemento/imunologia , Conformação ProteicaRESUMO
BACKGROUND: Fasciola hepatica is an important pathogen that causes liver fluke disease in definitive hosts such as livestock animals and humans. Various excretory/secretory products have been used in serological diagnosis and vaccination studies targeting fasciolosis. There are no commercial vaccines against fasciolosis yet. Bioinformatic analysis based on computational methods have lower cost and provide faster output compared to conventional vaccine antigen discovery techniques. The aim of this study was to predict B- and T-cell specific epitopes of four excretory/secretory antigens (Kunitz-type serine protease inhibitor, cathepsin L1, helminth defense molecule, and glutathione S-transferase) of Fasciola hepatica and to construct a multiepitope vaccine candidate against fasciolosis. METHODS AND RESULTS: Initially, nonallergic and the highest antigenic B- and T- cell epitopes were selected and then, physico-chemical parameters, secondary and tertiary structures of designed multiepitope vaccine candidate were predicted. Tertiary structure was refined and validated using online bioinformatic tools. Linear and discontinuous B-cell epitopes and disulfide bonds were determined. Finally, molecular docking analysis for MHC-I and MHC-II receptors was performed. CONCLUSION: This multi-epitope vaccine candidate antigen, with high immunological properties, can be considered as a promising vaccine candidate for animal experiments and wet lab studies.
Assuntos
Fasciola hepatica , Fasciolíase , Vacinas , Animais , Anticorpos Anti-Helmínticos , Antígenos de Helmintos/química , Epitopos de Linfócito B , Epitopos de Linfócito T , Fasciola hepatica/química , Fasciolíase/diagnóstico , Fasciolíase/prevenção & controle , Humanos , Simulação de Acoplamento MolecularRESUMO
Embryonated eggs are the infectious developmental stage of Trichuris trichiura and are the primary stimulus for the immune system of the definitive host. The intestinal-dwelling T. trichiura affects an estimated 465 million people worldwide with an estimated global burden of disease of 640 000 DALYs (Disability Adjusted Life Years). In Latin America and the Caribbean, trichuriasis is the most prevalent soil transmitted helminthiasis in the region (12.3%; 95% CI). The adverse health consequences impair childhood school performance and reduce school attendance resulting in lower future wage-earning capacity. The accumulation of the long-term effects translates into poverty promoting sequelae and a cycle of impoverishment. Each infective T. trichiura egg carries the antigens needed to face the immune system with a wide variety of proteins present in the shell, larvae's surface, and the accompanying fluid that contains their excretions/secretions. We used a proteomic approach with tandem mass spectrometry to investigate the proteome of soluble non-embryonated egg extracts of T. trichiura obtained from naturally infected African green monkeys (Chlorocebus sabaeus). A total of 231 proteins were identified, 168 of them with known molecular functions. The proteome revealed common proteins families which are known to play roles in energy and metabolism; the cytoskeleton, muscle and motility; proteolysis; signaling; the stress response and detoxification; transcription and translation; and lipid binding and transport. In addition to the study of the T. trichiura non-embryonated egg proteome, the antigenic profile of the T. trichiura non-embryonated egg and female soluble proteins against serum antibodies from C. sabaeus naturally infected with trichuriasis was investigated. We used an immunoproteomic approach by Western blot and tandem mass spectrometry from the corresponding SDS-PAGE gels. Vitellogenin N and VWD and DUF1943 domain containing protein, poly-cysteine and histidine tailed protein isoform 2, heat shock protein 70, glyceraldehyde-3-phosphate dehydrogenase, actin, and enolase, were among the potential immunoactive proteins. To our knowledge, this is the first study on the T. trichiura non-embryonated egg proteome as a novel source of information on potential targets for immunodiagnostics and immunomodulators from a neglected tropical disease. This initial list of T. trichiura non-embryonated egg proteins (proteome and antigenic profile) can be used in future research on the immunobiology and pathogenesis of human trichuriasis and the treatment of human intestinal immune-related diseases.
Assuntos
Antígenos de Helmintos/química , Proteínas de Helminto/química , Óvulo/química , Tricuríase/veterinária , Trichuris/química , Animais , Chlorocebus aethiops , Feminino , Proteínas de Helminto/imunologia , Proteínas de Helminto/metabolismo , Humanos , Proteoma , Tricuríase/sangue , Tricuríase/diagnóstico , Tricuríase/imunologiaRESUMO
Human trichinellosis can be diagnosed by a combination of medical history, clinical presentation, and laboratory findings, and through detection of anti-Trichinella IgG in the patient's sera. ELISA using excretory-secretory (E/S) antigens of Trichinella spiralis larvae is currently the most used assay to detect Trichinella spp. antibodies. Bead-based assay can detect antibodies to multiple antigens concurrently; the ability to detect antibody to T. spiralis using a bead assay could be useful for diagnosis and surveillance. We developed and evaluated a bead assay to detect and quantify total IgG or IgG4 Trichinella spp. antibodies in human serum using T. spiralis E/S antigens. The sensitivity and specificity of the assay were determined using serum from 110 subjects with a confirmed diagnosis of trichinellosis, 140 subjects with confirmed infections with other tissue-dwelling parasites, 98 human serum samples from residents of the United States with no known history of parasitic infection, and nine human serum samples from residents of Egypt with negative microscopy for intestinal parasites. Sensitivity and specificity were 93.6% and 94.3% for total IgG and 89.2% and 99.2% for IgG4, respectively. Twelve percent of sera from patients with confirmed schistosomiasis reacted with the IgG Trichinella bead assay, as did 11% of sera from patients with neurocysticercosis. The Trichinella spp. bead assay to detect IgG total antibody responses has a similar performance as the Trichinella E/S ELISA. The Trichinella spp. bead assay shows promise as a method to detect trichinellosis with a possibility to be used in multiplex applications.
Assuntos
Anticorpos Anti-Helmínticos/sangue , Imunoensaio/normas , Imunoglobulina G/sangue , Larva/imunologia , Trichinella spiralis/imunologia , Triquinelose/diagnóstico , Animais , Antígenos de Helmintos/química , Antígenos de Helmintos/metabolismo , Egito/epidemiologia , Humanos , Larva/patogenicidade , Sensibilidade e Especificidade , Suínos , Trichinella spiralis/patogenicidade , Triquinelose/sangue , Triquinelose/epidemiologia , Triquinelose/imunologia , Estados Unidos/epidemiologiaRESUMO
In this study, a key issue to be addressed is the safe disposal of hybridoma instability. Hybridoma technology was used to produce anti-O. viverrini monoclonal antibody. Previous studies have shown that antibody production via antibody phage display can sustain the hybridoma technique. This paper presents the utility of antibody phage display technology for producing the phage displayed KKU505 Fab fragment and using experiments in concomitant with molecular simulation for characterization. The phage displayed KKU505 Fab fragment and characterization were successfully carried out. The KKU505 hybridoma cell line producing anti-O. viverrini antibody predicted to bind to myosin was used to synthesize cDNA so as to amplify the heavy chain and the light chain sequences. The KKU505 displayed phage was constructed and characterized by a molecular modeling in which the KKU505 Fab fragment and -O. viverrini myosin head were docked computationally and it is assumed that the Fab fragment was specific to -O. viverrini on the basis of mass spectrometry and Western blot. This complex interaction was confirmed by molecular simulation. Furthermore, the KKU505 displayed phage was validated using indirect enzyme-linked immunosorbent assays (ELISA) and immunohistochemistry. It is worthy to note that ELISA and immunohistochemistry results confirmed that the Fab fragment was specific to the -O. viverrini antigen. Results indicated that the approach presented herein can generate anti-O. viverrini antibody via the phage display technology. This study integrates the use of phage display technology together with molecular simulation for further development of monoclonal antibody production. Furthermore, the presented work has profound implications for antibody production, particularly by solving the problem of hybridoma stability issues.
Assuntos
Anticorpos Anti-Helmínticos/biossíntese , Anticorpos Anti-Helmínticos/imunologia , Simulação de Dinâmica Molecular , Opisthorchis/imunologia , Biblioteca de Peptídeos , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/química , Antígenos de Helmintos/química , Antígenos de Helmintos/imunologia , Sistema Biliar/imunologia , Sistema Biliar/parasitologia , Bovinos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Camundongos , Simulação de Acoplamento Molecular , Miosinas/química , Reprodutibilidade dos Testes , Soroalbumina Bovina/análiseRESUMO
Hepatocellular carcinoma-associated antigen 59 (HCA59), one of significant excretory/secretory products of Haemonchus contortus (HcESPs), is identified to have immunomodulatory eï¬ ;ects on host cells. However, protection potential of the molecule in H. contortus remains poorly understood. In this study, H. contortus recombinant HCA59 protein amalgamated with poly (lactic-co-glycolic acid) (PLGA) nanoparticle adjuvant was tested for its protection against H. contortus infection in goats. Fifteen goats were allocated into three groups. On days 0 and 14, rHCA59 group was immunized with PLGA nanoparticles encapsulated with recombinant protein HCA59 (rHCA59-PLGA) respectively. Positive control group was unvaccinated, but challenged with H. contortus third stage larvae (L3). Negative control group was unvaccinated and unchallenged with L3. Goats in rHCA59 group and positive control group were challenged with 8000 H. contortus L3 after 14 days of the second immunization. Following immunization, high level of sera IgG, IgA, and IgE, as well as significantly high production of IL-4 and IL-9 was produced in rHCA59 group. After L3 challenge, the level of IL-17 and TGF-ß in rHCA59 group increased obviously. Meanwhile, the fecal eggs and the abomasal worm burdens in rHCA59 group was reduced by 44.1 % and 54.6 %, respectively. The studies suggested that rHCA59-PLGA nanoparticles conferred partial protection and could be a good candidate for the development of nanovaccines against H. contortus infection in goats.
Assuntos
Antígenos de Helmintos/imunologia , Carcinoma Hepatocelular/metabolismo , Hemoncose/prevenção & controle , Haemonchus/imunologia , Neoplasias Hepáticas/metabolismo , Vacinas/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/química , Fezes/parasitologia , Feminino , Doenças das Cabras/parasitologia , Doenças das Cabras/prevenção & controle , Cabras , Hemoncose/parasitologia , Nanoestruturas , Contagem de Ovos de Parasitas , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/químicaRESUMO
Antibodies have different chemical properties capable of targeting a diverse nature of antigens. Traditionally, immune antibody libraries are perceived to be disease-specific with a skewed repertoire. The complexity during the generation of a combinatorial antibody library allows for a skewed but diverse repertoire to be generated. Strongyloides stercoralis is a parasite that causes strongyloidiasis, a potentially life-threatening disease with a complex diagnosis that impedes effective control and treatment of the disease. This study describes the isolation of monoclonal antibodies against S. stercoralis NIE recombinant protein using an immune antibody phage display library derived from lymphatic filaria-infected individuals. The isolated antibody clones showed both lambda and kappa light chains gene usage, with diverse amino acid distributions. Structural analysis showed that electropositivity and the interface area could determine the binding affinity of the clones with NIE. The successful identification of S. stercoralis antibodies from the filarial immune library highlights the breadth of antibody gene diversification in an immune antibody library that can be applied for closely related infections.
Assuntos
Anticorpos Anti-Helmínticos/imunologia , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Biblioteca de Peptídeos , Anticorpos de Cadeia Única/imunologia , Strongyloides/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/química , Anticorpos Anti-Helmínticos/genética , Anticorpos Anti-Helmínticos/isolamento & purificação , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Antígenos de Helmintos/química , Antígenos de Helmintos/imunologia , Sítios de Ligação , Técnicas de Visualização da Superfície Celular , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/genética , Análise de Sequência de DNA , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
Angiostrongylus cantonensis is the main causative agent of eosinophilic meningoencephalitis (EoM) in humans. Molecular diagnostic methods are essential since the identification of larvae in cerebrospinal fluid (CSF) is extremely rare. To date, the detection of a 31 kDa antigen by Western blotting has been the primary immunodiagnostic method for EoM caused by A. cantonensis. However, cross-reactivity with other parasites has been observed. Therefore, we conducted a comparative analysis using sera from individuals with angiostrongyliasis. We also characterized proteins isolated from different cellular sources of A. cantonensis, Toxocara canis, Schistosoma mansoni, and Strongyloides stercoralis with mass spectrometry. A total of 115 cross-reactive proteins were identified. Three of these proteins, heat shock protein, an intermediate filament protein, and galectin 1, represent potential markers for cross-reactivity. In addition, synthetic peptides were generated from previously identified diagnostic targets and tested against sera from individuals infected with several other parasites. As a result, two other markers of cross-reactivity were identified: peptide #4 derived from the 14-3-3 protein and peptide #12 derived from the Lec-5 protein. In contrast, 34 proteins were exclusively present in the Angiostrongylus extracts and represent promising diagnostic molecules for specific identification of A. cantonensis infection. In particular, cytochrome oxidase subunit I is of great interest as a possible immunodiagnostic target for angiostrongyliasis.
Assuntos
Angiostrongylus cantonensis/imunologia , Antígenos de Helmintos/imunologia , Proteínas de Helminto/imunologia , Meningoencefalite/diagnóstico , Meningoencefalite/parasitologia , Infecções por Strongylida/diagnóstico , Sequência de Aminoácidos , Angiostrongylus cantonensis/química , Animais , Antígenos de Helmintos/sangue , Antígenos de Helmintos/química , Antígenos de Helmintos/isolamento & purificação , Western Blotting , Sequência Conservada , Reações Cruzadas , Eletroforese , Eletroforese em Gel Bidimensional , Proteínas de Helminto/química , Proteínas de Helminto/isolamento & purificação , Humanos , Imunoensaio , Testes Imunológicos , Espectrometria de Massas , Meningoencefalite/imunologia , Infecções por Strongylida/imunologia , Infecções por Strongylida/parasitologiaRESUMO
BACKGROUND: A urine antigen assay was applied to evaluate chemotherapeutic outcomes and reinfection patterns of opisthorchiasis in Thailand. METHODS: We used a prospective study design by following opisthorchiasis subjects at baseline and post-treatment using a urine antigen assay and faecal examination by the formalin-ethyl acetate concentration technique (FECT). RESULTS: The antigen of Opisthorchis viverrini in urine diminished within 4 weeks after praziquantel treatment. Concurrent faecal examinations by FECT showed that faecal eggs were negative at 4 weeks after treatment. In a subsequent study, reinfection rates and intensity patterns of O. viverrini were evaluated at 48 weeks after praziquantel treatment. Within a group of subjects with curative treatment (n=137), 16.8% became reinfected according to FECT and 27.7% according to the urine antigen assay (p<0.05). There were significant correlations in intensity of infection between pretreatment and at 48 weeks post-treatment in both faecal egg counts and antigen levels in urine. CONCLUSIONS: The results suggested that in addition to screening, the urine antigen assay is an efficient tool for monitoring outcomes of drug treatment and reinfection in opisthorchiasis. Due to the ease of urine sample collection and handling, the urine assay becomes an alternative method to faecal examination for diagnosis and monitoring of opisthorchiasis.
Assuntos
Antígenos de Helmintos/química , Antígenos de Helmintos/urina , Opistorquíase/tratamento farmacológico , Opistorquíase/parasitologia , Opisthorchis/efeitos dos fármacos , Praziquantel/uso terapêutico , Animais , Fezes/parasitologia , Humanos , Opistorquíase/diagnóstico , Opistorquíase/epidemiologia , Opisthorchis/isolamento & purificação , Praziquantel/farmacologia , Praziquantel/urina , Estudos Prospectivos , Reinfecção , Tailândia , Resultado do TratamentoRESUMO
We characterized Schistosoma japonicum HSP40 (Sjp40) and HSP90α (Sjp90α) in this study. Western blot analysis revealed both are present in soluble egg antigens and egg secretory proteins, implicating them in triggering the host immune response after secretion from eggs into host tissues. These observations were confirmed by immunolocalization showing both HSPs are located in the Reynolds' layer within mature eggs, suggesting they are secreted by miracidia and accumulate between the envelope and the eggshell. Both HSPs are present in the musculature and parenchyma of adult males and in the vitelline cells of females; only Sjp90α is present on the tegument of adults. Sjp40 was able to enhance the expression of macrophages, dendritic cells, and eosinophilic cells in mouse liver non-parenchymal cells, whereas rSjp90α only stimulated the expression of dendritic cells. T helper 1 (Th1), Th2, and Th17 responses were increased upon rSjp40 stimulation in vitro, but rSjp90 only stimulated an increased Th17 response. Sjp40 has an important role in reducing the expression of fibrogenic gene markers in hepatic stellate cells in vitro. Overall, these findings provide new information on HSPs in S. japonicum, improving our understanding of the pathological roles they play in their interaction with host immune cells.
Assuntos
Antígenos de Helmintos/imunologia , Proteínas de Choque Térmico HSP40/imunologia , Proteínas de Choque Térmico HSP90/imunologia , Proteínas de Helminto/imunologia , Schistosoma japonicum/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/química , Antígenos de Helmintos/metabolismo , Modelos Animais de Doenças , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Células Estreladas do Fígado/metabolismo , Imuno-Histoquímica , Fígado/imunologia , Fígado/metabolismo , Fígado/parasitologia , Fígado/patologia , Camundongos , Modelos Moleculares , Conformação Proteica , Schistosoma japonicum/metabolismo , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Clonorchiasis is endemic in East and Southeast Asian countries. For a preventive strategy against infectious diseases, vaccination is the most effective. Here, we evaluated the molecular characteristics and immune responses of CsAg17 protein from Clonorchis sinensis, and investigated its protective effects against C. sinensis challenge. METHODS: A cDNA clone encoding CsAg17 protein and containing a secretory signal peptide at the N-terminus was retrieved from the C. sinensis transcriptome bank. Recombinant CsAg17 B-cell epitope protein and cDNA vaccines were produced and their immune responses were evaluated in FVB mice. The proportional changes of CD3+/CD4+ and CD3+/CD8+ T cells were detected by flow cytometry, and immune effectors were measured by ELISA. RESULTS: The CsAg17 mRNA was transcribed at a higher level in C. sinensis adults than in metacercariae. The CsAg17 protein was distributed in the sperms, oral and ventral suckers, and mesenchymal tissues of C. sinensis adults. In mice challenged with C. sinensis metacercariae, vaccination with CsAg17 protein and cDNA resulted in a reduction to 64% and 69% in worm burden, respectively. Both CsAg17 protein and cDNA vaccines increased the proportion of CD3+/CD4+ and CD3+/CD8+ T cells and stimulated the production of Th1 type cytokines such as interleukin (IL)-2, IL-12, and interferon-γ, while maintaining minimum levels of Th2 cytokines. The levels of IgG specific to CsAg17 protein steeply increased in the two vaccinated groups from 2 weeks after immunization. The liver tissue retained good morphology in the mice vaccinated with CsAg17 protein or cDNA, whereas severe inflammation and large serous cysts were observed in the liver of the unvaccinated mice. CONCLUSIONS: Vaccination with CsAg17 protein and cDNA reduced the pathological changes in the bile duct and liver, and ameliorated the worm burden via cellular and humoral immune responses. Thus, they may serve as good vaccine candidates against C. sinensis infections.
Assuntos
Antígenos de Helmintos/imunologia , Clonorquíase/imunologia , Clonorchis sinensis/metabolismo , Proteínas Recombinantes/imunologia , Vacinas/imunologia , Animais , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/química , Antígenos de Helmintos/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Clonorquíase/parasitologia , Clonorquíase/prevenção & controle , Clonorchis sinensis/genética , Citocinas/imunologia , Feminino , Imunização , Imunoglobulina G/sangue , Fígado/parasitologia , Fígado/patologia , Metacercárias , Camundongos , Estrutura Secundária de Proteína , Proteínas Recombinantes/genética , Baço/imunologia , Vacinas de DNARESUMO
The enzyme-linked immunosorbent assay (ELISA) technique can play an important role in the early detection of fascioliasis. However, they have some diagnostic limitations, including cross-reaction with other helminths. It seems that the combination of recombinant parasite proteins as antigen can reduce these problems. Hence, the present study was aimed to design and confirm the antigenic recombinant multi-epitope (rMEP) construct of three protein epitopes (linear and conformational B-cell epitopes) of the parasite using immunoinformatic tools. For this purpose, the tertiary structures of Fasciola hepatica cathepsin-L1, saposin-like protein 2 and 16.5-kDa tegument-associated protein were predicted using the I-TASSER server. Validation of the modelled structures was performed by Ramachandran plots. The antigenic epitopes of the proteins were achieved by analysing the features of the IEDB server. The synthesized gene was cloned into the pET-22b (+) expression vector and transformed into the Escherichia coli BL21. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to verify and analyse the expression of the rMEP protein. Western blotting was utilized to confirm rMEP protein immunogenicity in two forms, one using an anti-His tag antibody and the other with human pooled sera samples (fascioliasis, non-fascioliasis and negative control sera). Our results demonstrated that the rMEP designed for the three proteins of F. hepatica was highly antigenic, and immune-detection techniques confirmed the antigen specificity. In conclusion, the presented antigenic multi-epitope may be very helpful to develop serodiagnostic kits such as indirect ELISA to evaluate the proper diagnosis of fascioliasis in humans and ruminants.
Assuntos
Antígenos de Helmintos/genética , Catepsinas/química , Fasciola hepatica/genética , Proteínas de Helminto/química , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/química , Western Blotting , Catepsinas/genética , Epitopos/imunologia , Escherichia coli/genética , Fasciola hepatica/química , Fasciolíase/diagnóstico , Proteínas de Helminto/genética , Humanos , Proteínas Recombinantes/químicaRESUMO
Galectins regulate growth and differentiation of immune cells and inflammation through their carbohydrate-binding function in humans, while also play a role in parasite survival. This study focused on the galectin of lymphatic filarial parasite Wuchereria bancrofti (Wb-Gal). The multiple sequence alignment with other galectins showed that the Wb-Gal belonged to galactoside binding lectin family, particularly tandem repeat type galectin-9. A homology model of Wb-Gal was developed in the I-TASser server using high similarity 3D structures with a quality score of 89.5. Molecular docking and dynamics studies revealed that the CCRD and NCRD of Wb-Gal bind with galactose and lactose. Further, Wb-Gal was cloned into the pET28 vector, expressed in E. coli Rosetta strain and purified by affinity chromatography. In the hemagglutination assays, the rWb-Gal bound to lactose, galactose, and glucose. Indirect Enzyme-Linked Immunosorbent Assay (ELISA) using different clinical filarial sera showed that the IgG and IgM response was against Wb-Gal x very high in all filarial clinical groups, whereas the IgA and IgG2 response was minimum to negligible. There was an enhanced response of IgG1 and IgG4 antibodies in Microfilaremics (MF) cases compared to Chronic Pathology (CP) and Endemic Normal (EN) individuals. Interestingly, the IgE response was comparatively higher in EN than MF and CP. These studies show that Wb-Gal is a member of the lectin family of proteins binding to different carbohydrates and may have an important role in the pathophysiology of filarial infection which needs to be investigated in greater detail.
Assuntos
Galectinas/química , Galectinas/fisiologia , Wuchereria bancrofti/fisiologia , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/química , Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica , Humanos , Imunomodulação , Modelos Moleculares , Filogenia , Conformação Proteica , Relação Estrutura-Atividade , Wuchereria bancrofti/classificaçãoRESUMO
BACKGROUND: Lymphatic Filariasis (LF) is one of the incapacitating and mosquito-borne sicknesses that on progression may prompt a few recognizable types of clutters like extreme lymphedema, hydrocele, and elephantiasis. METHODS: Antigenic preparations of B. malayi adult (BmA), S. cervi adult parasites and microfilariae (mf) total parasite extract were used to analyze the serological reactivity profile with human infectious sera collected from endemic areas of Bancroftian filariasis by performing Western blot and ELISA analysis. Sera from healthy human subjects were also included in the study to determine the variation incurred in the reactivity due to the filariasis infection. Gelelectrophoresis analysis of the crude-extract of BmA revealed seven protein bands while more than ten bands were recognized in S. cervi. RESULTS: our results represent a clear variation in protein patterns among the crude-antigens. ELISA results showed highest prevalence of IgG, IgM and IgG4 antibodies against all antigen preparations when recorded among microfilaraemic chronic infected patients. In both the antigenic preparations, the positive reactions were in the order of microfilaraemic>endemic normal>chronic>acute>nonendemic normal subjects. All sera of Mf+ patients were uniformly positive, while sera of both chronic and endemic normal subjects showed less reactivity. CONCLUSION: In the present study, we endeavoured to establish the extent of cross-reactivity of antigens derived from animal filarial parasites such as B. malayi and S. cervi with W. bancrofti filariasis sera of human patients. Besides, we further analyzed antibody-isotype profile of IgG, IgG4 and IgM in various human infection sera of bancroftian filarial subjects reactive to heterologous parasite antigens derived from adult worms of S. cervi from bovine and B. malayi from bovine and jirds.
Assuntos
Anticorpos Anti-Helmínticos , Filariose Linfática , Imunoglobulina G , Imunoglobulina M , Wuchereria bancrofti , Animais , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/química , Antígenos de Helmintos/imunologia , Reações Cruzadas , Filariose Linfática/sangue , Filariose Linfática/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Masculino , Wuchereria bancrofti/imunologia , Wuchereria bancrofti/metabolismoRESUMO
BACKGROUND: Trichinella spiralis muscle larval (ML) excretion/secretion (ES) antigen is the most widely used diagnostic antigen of trichinellosis, but preparation of ES antigen requires collecting worms from infected animals, and detection of specific IgG against ML ES antigen may result in a false negative at the early stage of infection. The aim of the study was to characterize T. spiralis elastase-1 (TsEla) and to evaluate its potential as diagnostic antigen for trichinellosis. METHODS: The complete cDNA sequences of the TsEla gene were cloned and expressed, and recombinant (rTsEla) was purified. TsEla transcription and expression in different T. spiralis life-cycle stages was investigated by qPCR and western blotting, and its location in the nematodes was evaluated using an immunofluorescence assay (IFA). The antigenicity of rTsEla was investigated by western blotting analysis and ELISA. Anti-Trichinella IgG, IgM and IgE of experimentally infected mice and specific IgG antibodies of trichinellosis patients were assayed by rTsEla-ELISA and ES-ELISA. RESULTS: The results of the qPCR and western blotting showed that TsEla was expressed in various T. spiralis life stages. Natural TsEla was detected in the soluble proteins and ES proteins of different life stages. IFA revealed that TsEla was identified in the whole nematodes of various stages, especially in the cuticle, stichosome and genital primordium of the parasite. Serum anti-Trichinella IgM, IgG and IgE in infected mice was first detected by rTsEla-ELISA at 6, 10 and 12 days post-infection (dpi), and reached 100% at 8, 14 and 14 dpi, respectively. When rTsEla-ELISA and ES-ELISA were used to detect anti-Trichinella IgG in sera of trichinellosis patients, the sensitivity was 97.37% (37/38) and 89.74% (34/38) (P > 0.05), and the specificity was 99.10% (220/222) and 98.20% (218/222), respectively (P > 0.05). The rTsEla cross-reacted with only one serum sample out of 20 samples from paragonimiasis patients and 7 samples from clonorchiasis patients. CONCLUSIONS: rTsEla is valuable to early diagnosis of trichinellosis and could be an alternative diagnostic antigen to the ML ES antigens.
Assuntos
Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Elastase Pancreática/química , Elastase Pancreática/imunologia , Trichinella spiralis/enzimologia , Triquinelose/diagnóstico , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/química , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Humanos , Estágios do Ciclo de Vida , Camundongos Endogâmicos BALB C , Elastase Pancreática/genética , Alinhamento de Sequência , Trichinella spiralis/genética , Trichinella spiralis/crescimento & desenvolvimento , Trichinella spiralis/imunologia , Triquinelose/sangue , Triquinelose/imunologia , Triquinelose/parasitologiaRESUMO
Dirofilaria immitis is a parasitic nematode that survives in the circulatory system of suitable hosts for many years, causing the most severe thromboembolisms when simultaneous death of adult worms occurs. The two main mechanisms responsible for thrombus formation in mammals are the activation and aggregation of platelets and the generation of fibrin through the coagulation cascade. The aim of this work was to study the anticoagulant potential of excretory/secretory antigens from D. immitis adult worms (DiES) on the coagulation cascade of the host. Anticoagulant and inhibition assays respectively showed that DiES partially alter the coagulation cascade of the host and reduce the activity of the coagulation factor Xa, a key enzyme in the coagulation process. In addition, a D. immitis protein was identified by its similarity to the homologous serpin 6 from Brugia malayi as a possible candidate to form an inhibitory complex with FXa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectrometry. These results indicate that D. immitis could use the anticoagulant properties of its excretory/secretory antigens to control the formation of blood clots in its immediate intravascular habitat as a survival mechanism.
Assuntos
Anticoagulantes/metabolismo , Dirofilaria immitis/metabolismo , Fator Xa/metabolismo , Animais , Anticoagulantes/química , Antígenos de Helmintos/química , Antígenos de Helmintos/metabolismo , Dirofilariose/parasitologia , Cães , Fator Xa/química , Interações Hospedeiro-Parasita , Serpinas/química , Serpinas/metabolismo , Tromboembolia/parasitologiaRESUMO
Schistosomes are parasitic blood flukes that infect approximately 250 million people worldwide. The disease known as schistosomiasis, is the second most significant tropical parasitic disease after malaria. Praziquantel is the only effective drug currently licensed for schistosomiasis and there are concerns about resistance to the drug. There has been much effort to develop vaccines against schistosomiasis to produce long-term protection in endemic regions. Surface-associated proteins, and in particular, those expressed in the body wall, or tegument, have been proposed as potential vaccine targets. Of these, annexins are thought to be of integral importance for the stability of this apical membrane system. Here, we present the structural and immunobiochemical characterization of four homologous annexins namely annexin B30, annexin B5a, annexin B7a and annexin B5b from S. mansoni. Bioinformatics analysis showed that there was no signal peptide predicted for any annexin in this study. Further analysis showed that each of all four annexin protein possesses a primary structure consisting of a short but variable N-terminal region and a long C-terminal core containing four homologous annexin repeats (I-IV), which contain five alpha-helices. The life cycle expression profile of each annexin was assessed using quantitative PCR. The results showed that the overall transcript levels of the each of four homologous annexins were relatively low in the egg stage, but increased gradually after the transition of cercariae (the invasive schistosome larvae) to schistosomula (the post-invasive larvae). Circular dichroism (CD) demonstrated that rAnnexin B30, rAnnexin B5a and rAnnexin 7a were folded, showing a secondary structure content rich in alpha-helices. The membrane binding affinity was enhanced when rAnnexin B30, rAnnexin B5a and rAnnexin 7a was incubated in the presence of Ca2+. All annexin members evaluated in this study were immunolocalized to the tegument, with immunoreactivity also occurring in cells and in muscle of adult parasites. All four recombinant annexins were immunoreactive and they were recognized by the sera of mice infected with S. mansoni. In conclusion, the overall results present the molecular characterization of annexin B30, annexin B5a, annexin B7a and annexin B5b from S. mansoni in host-parasite interactions and strongly suggest that the molecules could be useful candidates for vaccine or diagnostic development.
Assuntos
Anexinas/imunologia , Antígenos de Helmintos/imunologia , Proteínas de Helminto/imunologia , Schistosoma mansoni/imunologia , Esquistossomose mansoni/imunologia , Sequência de Aminoácidos/genética , Animais , Anexinas/química , Anexinas/genética , Anexinas/isolamento & purificação , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/química , Antígenos de Helmintos/genética , Antígenos de Helmintos/isolamento & purificação , Sequência de Bases/genética , Dicroísmo Circular , Biologia Computacional , Modelos Animais de Doenças , Feminino , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/isolamento & purificação , Humanos , Masculino , Camundongos , Microscopia Imunoeletrônica , Conformação Proteica em alfa-Hélice , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Schistosoma mansoni/genética , Schistosoma mansoni/ultraestrutura , Esquistossomose mansoni/sangue , Esquistossomose mansoni/parasitologia , Relação Estrutura-AtividadeRESUMO
Currently, three recombinant antigens based vaccines are under clinical trials against Schistosomiasis, but there is no vaccine available for prophylaxis or therapeutic. This study was conducted to construct a multi-epitope based vaccine against Schistosoma mansoni via utilizing Sm14, Sm21.7, Sm23, Sm29, Smp80, Sm-CB and SM-TSP-2 antigens. Helper T lymphocyte (HTL), cytotoxic T lymphocyte (CTL) and IFN-γ epitopes were predicted. Furthermore, Pan HLA DR-binding epitope was added to the vaccine. Moreover, 50S ribosomal protein L7/L12 of Mycobacterium tuberculosis as a novel TLR4 agonist was applied. The TAT peptide was added to the vaccine to augment intracellular delivery. The selected epitopes were linked together through appropriate linkers and chimeric vaccine was constructed with 617 amino acids with molecular weight of 65.43â¯kDa. Physico-chemical properties revealed a soluble protein with antigenic and non-allergic properties. Further analyses validated the stability of the construct that was able to interact with TLR4. Immunoinformatics analysis demonstrated the strong potential of constructed vaccine to stimulate T and B-cell mediated immune responses. In summary, obtained data indicated that the proposed vaccine can properly induce both T and B cells immune responses and could possibly be utilized for prophylactic or therapeutic aims in response to infection caused by S. mansoni.
Assuntos
Antígenos de Helmintos , Epitopos de Linfócito T , Schistosoma mansoni , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinas , Animais , Antígenos de Helmintos/química , Antígenos de Helmintos/imunologia , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Humanos , Schistosoma mansoni/química , Schistosoma mansoni/imunologia , Esquistossomose mansoni/imunologia , Esquistossomose mansoni/prevenção & controle , Vacinas/química , Vacinas/imunologiaRESUMO
Type 2 diabetes is a metabolic disorder characterized by persistently elevated glucose levels. There is no effective treatment strategy for this condition, and it poses a massive economic burden globally. Schistosoma soluble egg antigen (SEA)-induced immunomodulatory mechanisms have been reported in the treatment of autoimmune disease. This study aimed to determine the ability of Schistosoma japonicum SEA to protect against type 2 diabetes in Lepr db/db mice and understand the associated mechanisms. The mice were divided into four groups: C57BL/6 (the normal group), SEA (C57BL/6 mice treated with SEA), Lepr db/db , and SEA and Lepr db/db co-treatment groups. The mice in the SEA and co-treatment groups were injected with 50 µg of SEA (twice a week for 6 weeks), and the same volume of PBS was used as control. Blood glucose, insulin, and HOMA-IR levels were measured in all mice, which were sacrificed 6 weeks after the last SEA administration. Flow cytometry was used to detect the percentages of regulatory T cells in splenocytes. ELISA was used to detect the levels of IFN-γ, IL-2, IL-4, and IL-5 in cell culture supernatants. Compared with the mice in the Lepr db/db group, the mice in the SEA + Lepr db/db group exhibited significantly reduced insulin resistance, as evidenced by the enhancement of wound healing. The frequency of spleen regulatory T cells increased significantly after SEA administration; meanwhile, the secretion of IL-4 and IL-5 in spleen cells was elevated. These results indicate that SEA can reduce insulin resistance and provide new targets for the treatment of type 2 diabetes. The potential mechanisms might be associated with increases in regulatory T cells and Th2 cytokines in Lepr db/db mice, which warrants further investigation.