Immunogenicity and protection of recombinant proteins from Neospora caninum formulated with ISCOMs in cattle

HECKER YP; CÓCERES V; WILKOWSKY S.; JARAMILLO ORTIZ J.; MORRELL E.; VERNA A.; GANUZA A.; CANO D.; LISCHINSKY L.; ÁNGEL S.; ZAMORANO P.; ODEÓN A.; CAMPERO C.M.; MOREIN B.; MOORE D.P.

Georgia

Simposio; 24th Annual Molecular Parasitology and Vector Biology Symposium; 2014

Center for Tropical and Emerging Global Diseases and The University of Giorgia

The aim of the present study was to evaluate the immunogenicity and protective efficacy of rNcSAG1, rNcHSP20, rNcGRA7 recombinant proteins formulated with ISCOMs in pregnant heifers. Twelve pregnant heifers were divided in 3 groups of 4 heifers receiving different formulations before mating. Immunogens were administered twice subcutaneously (sc): group A were animals inoculated with three recombinant proteins (rNcSAG1, rNcHSP20, rNcGRA7) formulated with ISCOMs; group B received ISCOM-MATRIX (without antigen) and group C received just sterile phosphate-buffered saline (PBS). The recombinant proteins were engineered to be expressed in Escherichia coli and purified in nickel resin. All groups were intravenously challenged with the Neospora caninum NC-1 strain at 70 days of gestation. Serum and heparinized blood samples were collected on weeks 0, 5, 13 and 16 post-inoculation. Dams were slaughtered at the week 17 of the experiment (104 days of pregnancy) and placental and fetal tissue samples were collected. Immunogenicity and specific antibody responses in heifers were tested by indirect enzyme linked immunosorbent assays (iELISAs) and western blot (WB). The cellular immune response in dams was assessed by quantifying IFN-γ production and the percentages of T-cells (CD4+, CD8+ and γδ+) and monocytes from peripheral blood mononuclear cells (PBMC). Fetal fluids and tissue samples were tested using WB, histopathology, immunohistochemistry and nested-PCR. Heifers from group A developed specific antibodies against rNcSAG1, rNcHSP20 and rNcGRA7 previous to the challenge. Following the immunization, an increase of antibodies against rNcSAG1 and rNcHSP20 in all animals from group A was detected being statistically different from groups B and C at week 5, 13 and 16 (P <0.001). When the antibodies against  rNcGRA7 were evaluated by ELISA, there were statistical differences between group A, B and C at week 5 and 16 (P <0.001). There were neither specific nor differences in the response among the experimental groups A, B and C at week 0, 5, 13 y 16 (P > 0.05). Transplacental transmission could be determined in all fetuses from groups A, B and C by WB, IHC and nPCR. It is noteworthy that dams with lower CD4+/CD8+ ratios in PBMC, regardless of the experimental group, had lower pathology scores.