RESUMO
With a view to producing peptides capable of inducing a protective immune response against Schistosoma mansoni and Fasciola hepatica, the sequence and structure of the protective antigens Sm14 and Fh15 were analyzed. Their C-termini showed a high level of sequence conservation which, together with models for their three-dimensional structures, aided in peptide selection. Vaccination trials in Swiss mice challenged with S. mansoni cercaria or F. hepatica metacercaria showed that peptides which included the sequences VTVGDVTA or EKNSESKLTQ were capable of inducing levels of protection equivalent to the recombinant form of Sm14. These peptides may represent an alternative to r-Sm14 for the development of a bivalent anti-helminth vaccine.
Assuntos
Fasciolíase/imunologia , Fasciolíase/prevenção & controle , Esquistossomose mansoni/imunologia , Esquistossomose mansoni/prevenção & controle , Vacinas Combinadas/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/análise , Anticorpos Anti-Helmínticos/biossíntese , Desenho de Fármacos , Fasciola hepatica/imunologia , Feminino , Imunização , Camundongos , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/imunologia , Conformação Proteica , Schistosoma mansoni/imunologiaRESUMO
Fasciola hepatica is the causative agent of fasciolosis in many areas in America, Europe, Africa, Asia and Australia. There is an urgent need for improved methods to control the parasite's transmission. We describe the use of an experimental vaccine based on a recombinant antigen cloned from another parasite, Schistosoma mansoni (Sm14), that induces high levels of cross protection in mice against both S. mansoni and F. hepatica. Sheep and mice vaccinated with Sm14 were significantly protected against challenge infection with metacercariae of Fasciola hepatica and were completely free of the histopathological hepatic damage related to liver fluke infection. The vaccine will provide a valuable new tool to aid in transmission control of this economically important disease.
Assuntos
Antígenos de Helmintos/imunologia , Proteínas de Transporte/administração & dosagem , Fasciola hepatica/imunologia , Fasciolíase/prevenção & controle , Proteínas de Helminto/administração & dosagem , Proteínas de Membrana Transportadoras , Schistosoma mansoni/imunologia , Vacinação , Animais , Proteínas de Transporte/imunologia , Fasciolíase/imunologia , Proteínas de Transporte de Ácido Graxo , Proteínas de Helminto/imunologia , Imunidade Ativa , Fígado/patologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Ovinos , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genéticaRESUMO
Sm14 was the first fatty acid-binding protein homologue identified in helminths. Thereafter, members of the same family were identified in several helminth species, with high aminoacid sequence homology between them. In addition, immune crossprotection was also reported against Fasciola hepatica infection, in animals previously immunized with the Schistosoma mansoni vaccine candidate, r-Sm14. In the present study, data on preliminary sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis of nine different helminth extracts focusing the identification of Sm14 related proteins, is reported. Out of these, three extracts - Ascaris suum (males and females), Echinostoma paraensei, and Taenia saginata - presented components that comigrated with Sm14 in SDS-PAGE, and that were recognized by anti-rSm14 policlonal serum, in Western blotting tests.
Assuntos
Proteínas de Transporte , Proteínas de Helminto/isolamento & purificação , Proteínas de Membrana Transportadoras , Schistosoma mansoni/química , Esquistossomose mansoni/imunologia , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Proteínas de Transporte de Ácido Graxo , Feminino , Proteínas de Helminto/química , MasculinoRESUMO
Sm14 was the first fatty acid-binding protein homologue identified in helminths. Thereafter, members of the same family were identified in several helminth species, with high aminoacid sequence homology between them. In addition, immune crossprotection was also reported against Fasciola hepatica infection, in animals previously immunized with the Schistosoma mansoni vaccine candidate, r-Sm14. In the present study, data on preliminary sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis of nine different helminth extracts focusing the identification of Sm14 related proteins, is reported. Out of these, three extracts - Ascaris suum (males and females), Echinostoma paraensei, and Taenia saginata - presented components that comigrated with Sm14 in SDS-PAGE, and that were recognized by anti-rSm14 policlonal serum, in Western blotting tests
Assuntos
Animais , Masculino , Feminino , Western Blotting , Eletroforese em Gel de Poliacrilamida , Proteínas de Helminto , Schistosoma mansoni , Esquistossomose mansoni , Proteínas de HelmintoRESUMO
In previous studies it was shown that the recombinant molecule, r-Sm14, induces high levels of protection against Schistosoma mansoni infection in two outbred animal models and immune crossprotection against infection by Fasciola hepatica in Swiss outbred mice. r-Sm14 was derived from a living worm extract, called SE, and is being developed as the molecular basis of an anti-helminth bivalent vaccine against the two parasites, for medical and veterinary application. Present data refer to SDS-PAGE and Western Blotting analysis of four different preparations of S. mansoni adult worms focusing Sm14 identification. The extracts correspond to the initial fraction of the SE extraction process, containing products released by living worms (SEi); SE2, reextraction of adult worms in PBS; and SE of separated male and female adult worms. In all extracts it was possible to detect the component of 14 kDa, that was recognized by specific anti-rSm14 antibody raised in rabbits
Assuntos
Animais , Masculino , Feminino , Camundongos , Proteínas de Helminto/análise , Schistosoma mansoni/química , Esquistossomose mansoni/imunologia , Anticorpos Anti-Helmínticos/imunologia , Western Blotting , Proteínas de Transporte , Eletroforese em Gel de Poliacrilamida , Ácidos Graxos , Proteínas de Helminto/imunologia , Schistosoma mansoni/imunologia , Vacinas/imunologiaRESUMO
Previous studies carried out with Sm14 in experimental vaccination against Schistosoma mansoni or Fasciola hepatica infections were performed with recombinant Sm14 (rSm14) produced in Escherichia coli by the pGEMEX system (Promega). The rSm14 was expressed as a 40 kDa fusion protein with the major bacteriophage T7 capsid protein. Vaccination experiments with this rSm14 in animal models resulted in consistent high protective activity against S. mansoni cercariae challenge and enabled rSm14 to be included among the vaccine antigens endorsed by the World Health Organization for phase I/II clinical trials. Since the preparation of pGEMEX based rSm14 is time consuming and results in low yield for large scale production, we have tested other E. coli expression systems which would be more suitable for scale up and downstream processing. We expressed two different 6XHis-tagged Sm14 fusion proteins in a T7 promoter based plasmids. The 6XHis-tag fusions allowed rapid purification of the recombinant proteins through a Ni+2-charged resin. The resulted recombinant 18 and 16 kDa proteins were recognized by anti-Sm14 antibodies and also by antiserum against adult S. mansoni soluble secreted/excreted proteins in Western-Blot. Both proteins were also protective against S. mansoni cercariae infection to the same extent as the rSm14 expressed by the pGEMEX system