RESUMEN
The Schistosoma mansoni fatty acid binding protein (FABP), Sm14, is a vaccine candidate against, S. mansoni and F. hepatica. Previously, we demonstrated the importance of a correct fold to achieve protection in immunized animals after cercariae challenge [[10]. C.R.R. Ramos, R.C.R. Figueredo, T.A. Pertinhez, M.M. Vilar, A.L.T.O. Nascimento, M. Tendler, I. Raw, A. Spisni, P.L. Ho, Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein: structural, functional and immunoprotection analysis. J. Biol. Chem. 278 (2003) 12745-12751.]. Here we show that the reduction of vaccine efficacy over time is due to protein dimerization and subsequent aggregation. We produced the mutants Sm14-M20(C62S) and Sm14-M20(C62V) that, as expected, did not dimerize in SDS-PAGE. Molecular dynamics calculations and unfolding experiments highlighted a higher structural stability of these mutants with respect to the wild-type. In addition, we found that the mutated proteins, after thermal denaturation, refolded to their active native molecular architecture as proved by the recovery of the fatty acid binding ability. Sm14-M20(C62V) turned out to be the more stable form over time, providing the basis to determine the first 3D solution structure of a Sm14 protein in its apo-form. Overall, Sm14-M20(C62V) possesses an improved structural stability over time, an essential feature to preserve its immunization capability and, in experimentally immunized animals, it exhibits a protection effect against S. mansoni cercariae infections comparable to the one obtained with the wild-type protein. These facts indicate this protein as a good lead molecule for large-scale production and for developing an effective Sm14 based anti-helminthes vaccine.
Asunto(s)
Femenino , Animales , Ratones , Esquistosomiasis , Schistosoma mansoni , Proteínas PortadorasRESUMEN
Os objetivos da presente investigação foram os de reavaliar resultados anteriores obtidos através de infecções experimentais de camundongos SW com cercarias e esquistossômulos da cepa LE de Schistosoma mansoni mantida em laboratório. Três grupos de camundongos foram considerados: animais do primeiro grupo foram infectados com cercarias pela via percutânea (método do anel), os do segundo inoculados pela via subcutânea com cercarias e os do terceiro inoculados pela mesma via com esquistossômulos obtidos "in vitro". Os dados obtidos mostraram que a via de infecção mais eficiente é a injeção subcutânea de esquistossômulos transformados "in vitro", com média de recuperação de vermes adultos de 54.1%, quando comparada às vias percutânea abdominal e subcutânea, com médias de 36,7% e 32,4%, respectivamente, sugerindo que em infecções experimentais de camundongos SW com S. mansoni da cepa LE, a pele pode ser considerada um eficaz sítio de atrito na via percutânea, enquanto no caso da inoculação de cercarias, um pequeno número de larvas não se transforma em esquistossômulos viáveis, talvez pelo fato de a barreira da pele haver sido evitada. Breves considerações a respeito dos sítios de atrito e eliminação de formas larvares do S. mansoni em camundongos, são apresentadas.
Asunto(s)
Animales , Masculino , Ratones , Modelos Animales de Enfermedad , Schistosoma mansoni/fisiología , Esquistosomiasis mansoni/parasitología , Schistosoma mansoni/crecimiento & desarrolloRESUMEN
The present investigation involves a reevaluation of previous results obtained after experimental infection of Swiss Webster mice with cercariae and schistosomula of the Schistosoma mansoni LE strain maintained under laboratory conditions. Three experimental groups of mice were considered: the animals of the first group were percutaneously (ring method) infected with cercariae, those of the second were subcutaneously inoculated with cercariae and the mice of the third were inoculated by the same route with schistosomula transformed in vitro. The data obtained so far indicated that the most effective method of infection is the subcutaneous injection with schistosomula, with a mean adult worm burden recovery of 54.1% when compared to the abdominal percutaneous and subcutaneous routes of infection with cercariae, in which the values were 36.7% and 32.4%, respectively. This suggests that, in experimental infections of SW mice with a LE S. mansoni strain, the skin is to be considered an effective attrition site in the percutaneous route, whereas in the case of inoculation with cercariae, a small amount of larvae fails to be transformed into viable schistosomula, possibly due to skin phase avoidance. A brief discussion about attrition sites and elimination of larval S. mansoni worms in mice is presented.
Asunto(s)
Modelos Animales de Enfermedad , Schistosoma mansoni/fisiología , Esquistosomiasis mansoni/parasitología , Animales , Masculino , Ratones , Schistosoma mansoni/crecimiento & desarrolloRESUMEN
The Schistosoma mansoni Sm14 antigen belongs to the fatty acid-binding protein family and is considered a vaccine candidate against at least two parasite worms, Fasciola hepatica and S. mansoni. Here the genomic sequence and the polymorphism of Sm14 have been characterized for the first time. We found that the conserved methionine at position 20 is polymorphic, being exchangeable with threonine (M20T). To evaluate the function of the amino acid residue at this position, we have also constructed the mutant Sm14-A20 besides the two native isoforms (Sm14-M20 and Sm14-T20). The three purified recombinant His6-tagged Sm14 proteins (rSm14-M20, rSm14-T20, and rSm14-A20) present a predominant â-barrel structure as shown by CD spectroscopy. Thermal and urea unfolding studies evidenced a higher structural stability of rSm14-M20 over the other forms (rSm14M20>rSm14-T20>rSm14-A20). All of the Sm14 proteins were able to bind 11-(dansylamino)undecanoic acid (DAUDA) without substantial difference in the binding affinity. However, rSm14-M20 exhibited a higher affinity for natural fatty acids than the rSm14-T20 and rSm14-A20 proteins as judged by competitive experiments against DAUDA (rSm14-M20>rSm14-T20> rSm14-A20). The rSm14-M20 or rSm14-T20 isoforms but not the rSm14-A20 mutant was able to induce significant protection against S. mansoni cercariae challenge in immunized mice. The level of protection efficacy correlates with the extent of structure stability of the recombinant Sm14 isoforms and mutant.
Asunto(s)
Humanos , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Proteínas del Helminto/química , Schistosoma mansoni/genética , Dicroismo Circular , Polimorfismo Genético , Cartilla de ADN , Proteínas de Unión a Ácidos Grasos , Proteínas de Neoplasias , Proteínas de Transporte de Ácidos Grasos , Secuencia de AminoácidosRESUMEN
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
Asunto(s)
Animales , Fasciola hepatica/inmunología , Fascioliasis/inmunología , Fascioliasis/prevención & control , Hígado/patología , Schistosoma mansoni/inmunología , Ovinos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Proteínas del Helminto/administración & dosificación , Proteínas del Helminto/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , VacunaciónRESUMEN
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.
Asunto(s)
Proteínas del Helminto/análisis , Proteínas de Transporte de Membrana , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Schistosoma mansoni/química , Esquistosomiasis mansoni/inmunología , Animales , Anticuerpos Antihelmínticos/inmunología , Western Blotting , Proteínas Portadoras/inmunología , Electroforesis en Gel de Poliacrilamida , Proteínas de Transporte de Ácidos Grasos , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Femenino , Proteínas del Helminto/inmunología , Masculino , Ratones , Schistosoma mansoni/inmunología , Vacunas/inmunologíaRESUMEN
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.
Asunto(s)
Anticuerpos Antihelmínticos/inmunología , Proteínas Portadoras , Proteínas del Helminto/inmunología , Proteínas de Transporte de Membrana , Schistosoma mansoni/inmunología , Vacunas/inmunología , Animales , Western Blotting , Electroforesis en Gel de Poliacrilamida , Escherichia coli/inmunología , Escherichia coli/metabolismo , Proteínas de Transporte de Ácidos Grasos , Femenino , Proteínas del Helminto/metabolismo , Ratones , Modelos Animales , Plásmidos/inmunología , Plásmidos/metabolismo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Schistosoma mansoni/metabolismo , VacunaciónRESUMEN
Molecular cloning of components of protective antigenic preparations has suggested that related parasite fatty acid-binding proteins could form the basis of the protective immune crossreactivity between the parasitic trematode worms Fasciola hepatica and Schistosoma mansoni. Molecular models of the two parasite proteins showed that both molecules adopt the same basic three-dimensional structure, consisting of a barrel-shaped molecule formed by 10 antiparallel beta-pleated strands joined by short loops, and revealed the likely presence of crossreactive, discontinuous epitopes principally derived from amino acids in the C-terminal portions of the molecules. A recombinant form of the S. mansoni antigen, rSm14, protected outbred Swiss mice by up to 67% against challenge with S. mansoni cercariae in the absence of adjuvant and without provoking any observable autoimmune response. The same antigen also provided complete protection against challenge with F. hepatica metacercariae in the same animal model. The results suggest that it may be possible to produce a single vaccine that would be effective against at least two parasites, F. hepatica and S. mansoni, of veterinary and human importance, respectively.
Asunto(s)
Antígenos Helmínticos/inmunología , Proteínas Portadoras , Proteínas del Helminto/inmunología , Proteínas de Transporte de Membrana , Schistosoma mansoni/inmunología , Secuencia de Aminoácidos , Animales , Reacciones Cruzadas , Mapeo Epitopo , Fasciola hepatica/inmunología , Proteínas de Transporte de Ácidos Grasos , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Conejos , Schistosoma mansoni/química , Esquistosomiasis mansoni/prevención & control , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Vacunas SintéticasRESUMEN
Molecular cloning of components of protective antigenic preparations have suggested that related parasite fatty acid binding proteins could form the basis of the well documented protective, immune cross reactivity between the parasitic trematode worms Fasciola hepatica and Schistosoma mansoni. We have now confirmed the cross protective potential of parasite fatty acid binding proteins and suggest that it may be possible to produce a single vaccine that would be effective against at least two parasites, F. hepatica and S. mansoni of veterinary and human importance respectively.