A combined proteomic and immunologic approach for the analysis of Schistosoma mansoni cercariae and adult worm protein extracts and the detection of one of the vaccine candidates, Sm28GST, from a Venezuelan parasite isolate / Aproximación al análisis proteómico e inmunológico de extractos proteicos de cercaria y verme adulto de Schistosoma mansoni y detección de uno de los candidatos a vacuna, Sm28GST, de un aislado venezolano

Understanding the mode of Schistosoma mansoni larval invasion and the mechanism of immune evasion utilized by larvae and adult worms is essential for a rational development of vaccines or drugs to prevent or cure the disease. This parasite has a very complex molecular organization in all parasite stages, and identifying the major parasite proteins would give clues to schistosome metabolism and to the interaction of the parasite with the host immune system. Our goal was the evaluation of the protein parasite repertoire using a proteomic approach, and the characterization of protein extracts from two different parasite stages of a Venezuelan isolate, such as cercariae and adult worms, previously performed by other authors in some other strains. A comparison among authors was made. Besides, we aimed to identify different isoforms of one of the vaccine candidates, the gluthation-S-transferase protein (Sm28GST), by 2D SDS-PAGE and mass spectrometry, and to achieve its immunologic detection using sera from rabbits immunized with synthetic peptides derived from the Sm28GST protein. These techniques allowed the identification of some of the target molecules of the protective immune response that are being evaluated as potential members of a multi-component and multi-stage anti-S. mansoni vaccine and to clarify if the selected peptides induce antibodies that are able to recognize different isoforms of the Sm28GST.

Es esencial comprender la forma como las larvas de Schistosoma mansoni invaden y los mecanismos de evasión inmune utilizados por larvas y adultos, para el desarrollo racional de vacunas o drogas para prevenir o curar la esquistosomiasis. Este parásito tiene una organización molecular muy compleja en todos sus estadíos, por lo que la identificación de las proteínas más importantes es clave para investigar el metabolismo del esquistosoma y la interacción del parásito con el sistema inmune del hospedero. El objetivo de este trabajo fue evaluar el repertorio proteico del parásito utilizando una aproximación proteómica y la caracterización de extractos proteicos de dos estadios parasitarios diferentes de un aislado venezolano, como la cercaria y el verme adulto, previamente realizado por otros autores en otras aislados. Se realizó una comparación entre autores. Además, se identificaron diferentes isoformas de uno de los candidatos a vacuna, la glutation S transferasa (Sm28GST) por 2D SDS-PAGE y espectrometría de masas y se logró su detección inmunológica, usando sueros de conejos inmunizados con péptidos sintéticos derivados de la proteína Sm28GST. Estas técnicas permitieron identificar algunas de las moléculas blanco de la respuesta inmune protectora que están siendo evaluados como miembros potenciales de una vacuna multi-estadio y multi-componente y aclarar si los péptidos seleccionados indujeron anticuerpos capaces de reconocer diferentes isoformas de la Sm28GST.

Field assessment of recombinant Schistosoma japonicum 26 kDa glutathione S-transferase in Chinese water buffaloes.

We have shown previously that anti-fecundity immunity can be induced experimentally against recombinant 26 kDa glutathione S-transferase (reSjc26GST) in Chinese water buffaloes (Bos buffelus), important reservoir hosts for Schistosoma japonicum in China. In the field study described here, we immunized buffaloes with reSjc26GST to induce protective immunity against S. japonicum and to evaluate its effectiveness in controlling schistosomiasis japonica. We selected two villages as test and control groups in inside-embankment areas endemic for schistosomiasis japonica. The buffaloes in the test village were vaccinated with reSjc26GST, whereas those in the control village were not. The indicators of the effect of the vaccine included the generation of specific IgG antibodies in the vaccinated buffaloes, changes in the prevalence and infection intensity in buffaloes and village children, changes in the density of infected snails, and changes in the infectivity of water bodies (assessed by sentinel mice) in transmission areas adjacent to both villages. Twenty months after vaccination, the infection rate of buffaloes in the test village was decreased by 60.4% (from an initial prevalence of 13.5% to 5.4%), and 67.9% when compared with that in the control village (initial prevalence of 16.7%). However, the infection rate in village children remained unchanged. The density of infected snails decreased by 71.4%, from 0.0049/0.11 m2 to 0.0014/0.11m2 in the high transmission area outside the embankment in the test village. There was no change in the infectivity of the water body transmission areas between the test and control villages. The levels of specific antibodies to reSjc26GST showed a continuous increase after vaccination. These results indicate that protective immunity was induced and maintained in buffaloes after vaccination with reSjc26GST. The vaccine could thus play a significant role in reducing S. japonicum transmission caused by water buffaloes in the Lake region of China.

Production and characterization of a monoclonal antibody against recombinant glutathione S-transferase (GST) of Fasciola gigantica.

A monoclonal antibody (MoAb) against a recombinant glutathione S-transferase (rGST) of F. gigantica was produced in BALB/c mice. Reactivity and specificity of this monoclonal antibody was assessed by ELISA and immunoblotting. Six stable clones, namely 3A3, 3B2, 3C6, 4A6, 4B1 and 4D6 were obtained, All these MoAb reacted with rGST and native GST at a molecular weight of 28 kDa and found to be IgG1, kappa-light chain isotypes. These MoAb cross-reacted with Schistosoma mansoni and Schistosoma japonicum antigens at molecular weights of 28 and 26 kDa, respectively, but no cross-reactions were detected with antigens of Eurytrema and Paramphistomum spp. The localization of GST in metacercaria, 7-week-old juvenile and adult F. gigantica was performed by immunofluorescence technique, using MoAb as well as polyclonal antibody (PoAb) to the native protein as probes. In general, all clones of MoAb gave similar results and the pattern was quite similar to staining by PoAb. The fluorescence was intense, which implied the presence of a high concentration of GST in the parenchymal tissue in all stages of the parasite. However, the parenchymal cells were not evenly stained which implied the existence of subpopulations of this cell type with regard to GST production and storage. In addition, in adult and juvenile stages a moderate fluorescence was present in the basal layer of the tegument, while light fluorescence was observed in the caecal epithelium, cells in the ovary, testis and vitelline gland of the adult. In the metacercaria stage, in addition to parenchymal tissue, the tegument and tegumental cells were stained relatively more intense with MoAb and PoAb than in other stages.

Glutathione S-transferases of 28kDa as major vaccine candidates against schistosomiasis

For the development of vaccines strategies to generate efficient protection against infections such as parasitic diseases, and more precisely schistosomiasis, controlling pathology could be more relevant than controlling the infection itself. Such strategies, motivated by the need for a cost-effective complement to existing control measures, should focus on parasite molecules involved in fecundity, because in metazon parasite infections pathology is usually linked to the output of viable eggs. In numerous animal models, vaccination with glutathione S-transferases of 28kDa has been shown to generate an immune response strongly limiting the worm fecundity, in addition to the reduction of the parasite burden. Recent data on acquired immunity directed to 28GST in infected human populations, and new development to draw adapted vaccine formulations, are presented.