RESUMO
Schistosomiasis causes significant morbidity and mortality. Vaccine efforts to date indicate the need to increase the immunogenicity of Schistosoma antigens. The multiple antigen-presenting system, whereby proteins are genetically fused to rhizavidin and affinity linked to biotinylated templates, enables the generation of robust immune responses. The objective of this work was to express and purify the S. mansoni antigens, SmTSP-2 and SmCD59.2, in fusion with rhizavidin. The fusion with rhizavidin greatly decreased the expression level of rSmTSP-2, but not rSmCD59.2, and both were expressed in the insoluble fraction, requiring optimization of culture conditions. Evaluation of different E. coli strains and media showed that BL21-DE3 cultured in Terrific Broth provided the highest expression levels of both proteins. Investigation of a range of time and temperature of induction showed that E. coli strains expressing rRzv:SmTSP-2 and rRzv:SmCD59.2 showed the highest protein production at 23 °C for 15 h. Recombinant proteins were purified by a single step of affinity chromatography allowing isolation of these proteins in high concentration and purity. The optimization process increased final soluble protein yield of rRzv:SmTSP-2 by fourfold and rRzv:SmCD59.2 by tenfold, providing ~ 20 mg/L of each protein. Optimized fusion protein production will allow antigen use in biotin-rhizavidin affinity platforms.
Assuntos
Antígenos de Helmintos/biossíntese , Proteínas de Bactérias/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Schistosoma mansoni/metabolismo , Esquistossomose mansoni/imunologia , Animais , Antígenos de Helmintos/genética , Antígenos de Helmintos/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Cromatografia de Afinidade/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Schistosoma mansoni/química , Schistosoma mansoni/imunologia , Schistosoma mansoni/isolamento & purificação , Esquistossomose mansoni/metabolismo , Esquistossomose mansoni/parasitologiaRESUMO
Angiostrongylus cantonensis is a parasitic nematode and the main causative agent of human cerebral eosinophilic meningoencephalitis (EoM). A definitive diagnosis of EoM usually requires serologic or molecular analysis of the patient's clinical sample. Currently, a 31â¯kDa antigen is used in immunological tests for this purpose, however as a crude antigen preparation it may present cross-reactivity with other helminthic infections, especially echinococcosis. Heterologous expression studies using prokaryotic systems failed on producing antigenic proteins. The aim of this study was to express and purify three recombinant glycoproteins representing A. cantonensis antigens: ES-7, Lec-5, and 14-3-3, in Chinese hamster ovary (CHO) cells and ES-7 in human embryonic kidney (HEK) cells to develop a source of specific antigens to be used in the diagnosis of angiostrongyliasis. The potential diagnostic value of these three proteins was subsequently characterized in one- and two-dimensional electrophoresis and Western blot to dot blot analyses, with Angiostrongylus-positive sera, normal human sera (NHS), and a pool of Echinococcus-positive sera (included as a specificity control) used for detection. In addition, recognition of these three proteins following treatment with N-glycosidase F was examined. The ES-7 proteins that were expressed in HEK and CHO cells, and the Lec-5 protein that was expressed in CHO cells, were specifically recognized by A. cantonensis-positive sera in the 2D electrophoresis analysis. This recognition was shown to be dependent on the presence of glycidic portions, making mammalian cells a very promising source of heterologous expression antigenic proteins from Angiostrongylus.
Assuntos
Angiostrongylus cantonensis/genética , Antígenos de Helmintos/biossíntese , Proteínas de Helminto/biossíntese , Proteínas Recombinantes/biossíntese , Animais , Antígenos de Helmintos/genética , Western Blotting , Células CHO , Clonagem Molecular , Cricetulus , Expressão Gênica , Células HEK293 , Proteínas de Helminto/genética , Humanos , Immunoblotting , Proteínas Recombinantes/genética , Testes Sorológicos/métodos , Infecções por Strongylida/diagnósticoRESUMO
The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.
Assuntos
Expressão Gênica , Vetores Genéticos , Mycobacterium bovis/genética , Regiões Promotoras Genéticas , Animais , Antígenos de Helmintos/biossíntese , Antígenos de Helmintos/genética , Fusão Gênica Artificial , Clonagem Molecular , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Helminto/biossíntese , Proteínas de Helminto/genética , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Mutagênese , Micobacteriófagos/genética , Mycobacterium smegmatis/genética , Plasmídeos , Reação em Cadeia da Polimerase , Schistosoma mansoni/genética , Análise de Sequência de DNARESUMO
The Schistosoma mansoni Venom Allergen-Like proteins (SmVALs) are members of the SCP/TAPS (Sperm-Coating Protein/Tpx-1/Ag5/PR-1/Sc7) protein superfamily, which may be important in host-pathogen interactions. Whole mount in situ hybridisation demonstrated a distinct expression pattern in oral and ventral suckers of adult worms for SmVAL6 and in the oesophageal gland for SmVAL7 transcripts, respectively. Additionally, immunocytochemistry analysis corroborated SmVAL7 expression in the oesophageal gland. Analysis of protein expression across the parasite's life cycle revealed that the SmVAL6 protein is upregulated in cercariae and adult male worms. Furthermore, SmVAL6 protein was identified by mass spectrometry in tegument fractions of adult worms. Finally, we speculate on possible functions of these two SmVALs at the host-parasite interface.
Assuntos
Alérgenos/biossíntese , Antígenos de Helmintos/biossíntese , Expressão Gênica , Proteínas de Helminto/biossíntese , Schistosoma mansoni/crescimento & desenvolvimento , Estruturas Animais/química , Animais , DNA de Helmintos/química , DNA de Helmintos/genética , Perfilação da Expressão Gênica , Hibridização In Situ , Masculino , Espectrometria de Massas , Dados de Sequência Molecular , Schistosoma mansoni/química , Schistosoma mansoni/genética , Análise de Sequência de DNARESUMO
A cDNA encoding a 16.5-kDa protein termed FhTP16.5 was identified by immunoscreening of a cDNA library from Fasciola hepatica adult flukes using pooled sera from rabbits infected with F. hepatica for 4 weeks. Quantitative reverse transcriptase PCR (qPCR) analysis revealed that FhTP16.5 is not expressed in unembryonated eggs. It is poorly expressed in miracidia and highly expressed at the juvenile and adult stages; however, significant differences were found between the expression levels of FhTP16.5 in juveniles versus adult flukes. Recombinant FhTP16.5 was expressed at high levels in Escherichia coli, purified by affinity chromatography, and used to raise anti-FhTP16.5 polyclonal antibodies in rabbits. Immunoblot analysis using the anti-FhTP16.5 IgG antibody identified FhTP16.5 in crude and tegumental extracts and in excretory-secretory products of F. hepatica. The protein was not detected in crude extracts of Schistosoma mansoni or Schistosoma japonicum. Antibodies to FhTP16.5 were detected in the sera of rabbits at 3 to 12 weeks of F. hepatica infection as well as in the sera of humans with chronic fascioliasis; these findings suggest that FhTP16.5 could be a good antigen for serodiagnosis of fascioliasis. Immunohistochemistry demonstrated that FhTP16.5 localizes to the surface of the tegument of various developmental stages and in parenchymal tissues of the adult fluke. Such specific localization makes FhTP16.5 an attractive target for immunoprophylaxis or chemotherapy.
Assuntos
Antígenos de Helmintos/biossíntese , Fasciola hepatica/genética , Regulação da Expressão Gênica , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/química , Antígenos de Helmintos/genética , Cromatografia de Afinidade , Modelos Animais de Doenças , Escherichia coli/genética , Fasciola hepatica/química , Fasciola hepatica/imunologia , Fasciolíase/imunologia , Biblioteca Gênica , Humanos , Imuno-Histoquímica , Peso Molecular , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Schistosomiasis affects more than 200 million people worldwide; another 600 million are at risk of infection. The schistosomulum stage is believed to be the target of protective immunity in the attenuated cercaria vaccine model. In an attempt to identify genes up-regulated in the schistosomulum stage in relation to cercaria, we explored the Schistosoma mansoni transcriptome by looking at the relative frequency of reads in EST libraries from both stages. The 400 genes potentially up-regulated in schistosomula were analyzed as to their Gene Ontology categorization, and we have focused on those encoding-predicted proteins with no similarity to proteins of other organisms, assuming they could be parasite-specific proteins important for survival in the host. Up-regulation in schistosomulum relative to cercaria was validated with real-time reverse transcription polymerase chain reaction (RT-PCR) for five out of nine selected genes (56%). We tested their protective potential in mice through immunization with DNA vaccines followed by a parasite challenge. Worm burden reductions of 16-17% were observed for one of them, indicating its protective potential. Our results demonstrate the value and caveats of using stage-associated frequency of ESTs as an indication of differential expression coupled to DNA vaccine screening in the identification of novel proteins to be further investigated as potential vaccine candidates.
Assuntos
Perfilação da Expressão Gênica , Schistosoma mansoni/crescimento & desenvolvimento , Schistosoma mansoni/genética , Animais , Antígenos de Helmintos/biossíntese , DNA de Helmintos/química , DNA de Helmintos/genética , Etiquetas de Sequências Expressas , Proteínas de Helminto/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esquistossomose mansoni/prevenção & controle , Análise de Sequência de DNA , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologiaRESUMO
In search of reducing vaccine production costs', a recombinant M13 phage version of the anti-cysticercosis tripeptide vaccine (S3Pvac) was developed. The efficacy of S3Pvac-Phage vs. placebo was evaluated in a randomized trial that included 1,047 rural pigs in 16 villages of Central Mexico. Three to five months after vaccination 530 pigs were examined by tongue inspection. At 5-27 months of age, 331 pigs (197 vaccinated/134 controls) were inspected at necropsy. Vaccination reduced 70% the frequency of tongue cysticercosis and, based on necropsy, 54% of muscle-cysticercosis and by 87% the number of cysticerci.
Assuntos
Antígenos de Helmintos/imunologia , Bacteriófago M13/imunologia , Cisticercose/imunologia , Cisticercose/veterinária , Doenças dos Suínos/imunologia , Doenças dos Suínos/prevenção & controle , Taenia solium/imunologia , Vacinas/imunologia , Vacinas/uso terapêutico , Envelhecimento/imunologia , Animais , Antígenos de Helmintos/biossíntese , Bacteriófago M13/metabolismo , Cisticercose/prevenção & controle , México , População Rural , Suínos , Doenças dos Suínos/parasitologia , Vacinas de Produtos Inativados/imunologia , Aumento de Peso/efeitos dos fármacosRESUMO
Two different Echinococcus granulosus antigen B subunits (AgB8/1 and AgB8/2) were characterized and the structure of the genes encoding these two proteins were compared. DNA sequences were expressed in Escherichia coli and the antigens' diagnostic value was then assessed. The genomic sequence of AgB8/1 has a 92 bp intron in the position corresponding to amino acid 16; the AgB8/2 genomic sequence presents a 68 bp intron in the position corresponding to amino acid 20. Both introns are located between the putative N-terminal hydrophobic sequence and the secreted peptide. A comparison between the AgB8/1 and AgB8/2 nucleotide sequences showed a 53.5% identity among exons and a 50% identity between introns. According to the molecular diversity analysis, the elapsed time since both genes shared a common ancestor would be around 4.2x10(7) years. When the native AgB and the two recombinant antigens (rAgB8/1 and rAgB8/2) were tested in an anti-IgG ELISA, the sensitivity of the native antigen B was 77.41% and its specificity was 81.9%, while rAgB8/1 showed 54.84% of sensitivity and 80.17% of specificity and rAg138/2 had an 83.87% sensitivity and a 98.28% specificity. Statistical analysis confirms that rAgB8/2 has a better performance than rAgB8/1 and native AgB in ELISA.
Assuntos
Antígenos de Helmintos/genética , Echinococcus/genética , Proteínas de Helminto/genética , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/biossíntese , Sequência de Bases , Clonagem Molecular , Echinococcus/imunologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Genes de Helmintos , Vetores Genéticos , Proteínas de Helminto/biossíntese , Helmintíase/sangue , Humanos , Íntrons , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Sensibilidade e Especificidade , Alinhamento de SequênciaRESUMO
The aim of the present study was to compare the sensitivity, specificity and usefulness of the DIG-ELISA, DOT-ELISA and Indirect ELISA tests for determining the seroprevalence of fasciolosis in cattle under tropical conditions in Mexico. To standardize the tests, positive and negative sera to F. hepatica from 88 Holstein Freisian adult cows located in an enzootic area of fascioliosis and 88 crossbred adult cattle from a fluke-free area were used. For the epidemiological study, 85 crossbred cattle between 1 to 7 years of age were used. Animals were bled every two months, from March 1995 to September 1996 and the sera obtained were stored at -70 degrees C, until used. Indirect ELISA showed a sensitivity of 96.5% and a specificity of 98.8%, DIG-ELISA 97.5% and 80.0% and DOT-ELISA 93.1% and 95.4%, respectively. During 1995, Indirect ELISA yielded the highest levels of IgG anti-F. hepatica antibodies. However, in 1996, after animal treatment with triclabendazole, DIG-ELISA tended to show higher percentages of antibody-positive animals, but it was not significantly different (p>0.05) from the other tests. Comparisons made in parallel to the faecal sedimentation test demonstrated that all serological tests detected higher percentages of positive animals. Only one serum out of ten (10%) of Paramphistomum spp. cross-reacted with the DOT-ELISA test, but no cross-reaction was observed with sera from animals with other parasites. All ELISA tests were highly sensitive and specific; they may be recommended for use in seroepidemiological surveys for F. hepatica.
Assuntos
Anticorpos Anti-Helmínticos/sangue , Doenças dos Bovinos/epidemiologia , Ensaio de Imunoadsorção Enzimática/veterinária , Fasciola hepatica/imunologia , Fasciolíase/veterinária , Animais , Anti-Helmínticos/farmacologia , Anti-Helmínticos/uso terapêutico , Antígenos de Helmintos/biossíntese , Antígenos de Helmintos/química , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Bovinos , Doenças dos Bovinos/diagnóstico , Reações Cruzadas , Fasciolíase/diagnóstico , Fasciolíase/epidemiologia , Fezes/parasitologia , México/epidemiologia , Contagem de Ovos de Parasitas , Sensibilidade e Especificidade , Estudos Soroepidemiológicos , Triclabendazol , Clima TropicalRESUMO
A pool of 9 sera from Echinococcus granulosus infected patients (PSP) was used to screen an E. granulosus cDNA library constructed in the expression vector lambda gt11. Ten reactive phage clones were isolated and 8 were confirmed in spot-lysis arrays probed with PSP. The insert of 1 of these clones (lambda AgEg4) previously characterized as an E. granulosus cytosolic malate dehydrogenase encoding gene was subcloned into the plasmid vector pGEX-1 and expressed as a fusion with glutathione S-transferase. The fusion peptide (Ag4-GST) was produced in Escherichia coli and its antigenicity was confirmed in colony immunoassay and in immunoblot using nondenaturing conditions. The lack of antigenicity of Ag4-GST in immunoblot using denaturing conditions suggests that the recognized epitopes are conformational. Ag4-GST was purified by affinity chromatography and tested in ELISA and immunodots to access its sensitivity and specificity in the diagnosis of human cystic hydatid disease. An overall sensitivity of 53.6% was obtained. Cross-reactions were observed with some sera from patients infected with Schistosoma mansoni and Wuchereria bancrofti. Ag4-GST was not recognized by any of the sera from Taenia solium infected patients tested. These preliminary results suggest that Ag4-GST could be useful as an accessory antigen to discriminate some cross-reactions with sera from cysticercosis patients, especially in regions like southern Brazil, where schistosomiasis and filariasis are not prevalent.
Assuntos
Antígenos de Helmintos/análise , Clonagem Molecular/métodos , Equinococose/diagnóstico , Echinococcus/isolamento & purificação , Animais , Antígenos de Helmintos/biossíntese , Antígenos de Helmintos/sangue , DNA Complementar , Equinococose/parasitologia , Echinococcus/genética , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática/métodos , Biblioteca Gênica , Humanos , Immunoblotting , Plasmídeos , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossíntese , Sensibilidade e EspecificidadeRESUMO
The passive transfer of monoclonal antibodies, direct vaccination and in vitro assays have all shown that antigens associated with the tegumental membranes of Schistosoma mansoni are capable of mediating protective immune responses against the parasite in animal models. Furthermore, the principal antigens are highly antigenic during natural infection in man and stimulate strong humoral and cellular responses although, at present, their role in mediating protective immune responses in man remains equivocal. This presentation will review the current state of knowledge of the structure and expression of the major antigenic tegumental proteins of the schistosome and will attempt to relate the relevance of their structural features to possible function both in terms of protective immunity and the parasite's ability to survive within the definitive host. A focus will be the recent advances that have been made in the identification of means of anchoring of the antigenic proteins to the tegumental membrane. In addition, the implications of the structural complexity of the tegumental proteins in terms of their possible utility in vaccination and diagnosis will be considered.