Avaliação de ROP2 recombinante de Neospora caninum como imunobiológico

Abstract

Neospora caninum is an intracellular apicomplex parasite, a major cause of miscarriage in cattle leading to significant economic losses. The search for the controlof neosporosis has motivated extensive studies, using as targets, recombinant proteinsderived from protozoan organelles, in animal models. Inoculation of subunit vaccines has demonstrated that Th1 and Th1/Th2 immune responses are protective to the challenge, however ineffective in blocking fetal infection. The objectives of this study were the expression of rNcROP2 and its fusion with LTB, resulting in rNcROP2 and rLTB/NcROP2 in Escherichia coli, evaluation of the antigenicity of proteins produced against naturally infected bovine sera, and identification of cytokine induction produced when used as a vaccine of subunit in BALB/c mice. The rNcROP2 and rLTB/NcROP2 proteins were expressed in an insoluble form, solubilized in only 8 M urea, yielding approximately 125 mg/L and 25 mg/L, respectively. The conformation adopted by the two constructs permitted the recognition of antigenic epitopes. The rNcROP2 protein used as a diagnostic in cattle showed sensitivity and specificity of 100 % with cutoff >0.044. As immunogenic vaccines, the two proteins at doses of 25 μg were immunogenic, since they stimulated the production of IgG antibodies after the second dose being maintained up to 60 days after inoculation, mainly of the IgG1 isotype. In the expression of cytokines using rLTB/NcROP2 a reduzed stimulation of cytokines IL-10 and IFN-γ it was observed, the inoculation did not bring significant increase of IL-10 and IFN- γ. However, IL-12 e IL-17 induction had a significant increase after inoculation. Responses to rNcROP2 and rLTB/NcROP2 inoculation in our study suggest that the targets are capable of producing a Th1/Th17 response. The results report a significant increase induction of IL-17 by subunit vaccines with ROP2, this effect suggests to be promisingin studies of protection against N. caninum.