Avaliação da resposta humoral estimulada por probiótico contendo a 'Proteína 1' de Superfície De Merozoíto de Plasmodium falciparum (pf-MSP1-19)

Abstract

Bacillus subtilis is a Gram-positive, non-pathogenic bacterium that receive the status GRAS (Generally Regarded As Safe) by the American FDA. When exposed to the adverse situation B. subtilis produces a morphologically distinct cell type, called the spore. Research has highlighted the potential of use spores as vaccines delivery, which are capable of stimulate systemic and localized immune responses, promoting the elevation of humoral response when coadministered with antigens coupled or integrated to the surface of spores. Currently, there is still no effective vaccine to combat malaria and among the antigens studied, the Pf-MSP1-19 protein of Plasmodium falciparum is one of the most promising and elucidated vaccine candidates. Against the foregoing, the main objective of this study was to evaluate the immune response against Plasmodium falciparum rPf-MSP1-19 antigen, present on the surface of Bacillus subtilis spores. Methods: For this, renografin separation and quantification by flow cytometry of Bacillus subtilis spores recombined with rPf-MSP1-19 and Wild Type (WT) spores were performed, followed by chemical coupling of rPf-MSP1-19 to the surface of the autoclaved WT spores. With the tools produced, Balb / c mice were immunized orally and intraperitoneally and the sera obtained were evaluated by indirect ELISA. Results and conclusion: In this work the chemical coupling using EDC and NHS proved to be the best method for the adsorption of the rPf-MSP1-19 protein at spore surface. A new approach to spore quantification was applied using Flow Cytometry and Trucount ™ Beads demonstrating high efficiency and sensitivity. It was observed that there was degradation of the rPf-MSP1-19 protein when expressed directly on the spore surface by the recombinant method. As for oral immunizations, it was not possible to obtain immune response against the antigen coupled to the spores chemically and to recombinant Bacillus subtillis. However, intraperitoneal immunizations induced elevated levels of anti- rPf-MSP1-19 IgG in the group immunized with rPf-MSP1-19 coupled to the surface of the autoclaved WT spores similar to the positive control. Suggesting the use of autoclaved wild type B. subtilis spores as an alternative delivery and antigen adjuvant for experimental immunizations, which may in the future be applied in immunizations in humans.