부산대학교 도서관

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.exppara.2005.03.029 Byline: Ana M. Espino (a), Antonio Osuna (b), Ramon Gil (b), George V. Hillyer (c) Keywords: Fasciola hepatica saposin-like protein family; Trematode; Humoral and cellular response; DNA vaccination; FhSAP-2; Fasciola hepatica saposin-like protein; IL-10; interleukin 10; IFN[gamma]; [gamma] interferon; Th1; T-helper cell response type-1; Th2; T-helper cell response type-2; NK; natural killer; CMV; cytomegalovirus; DBB; denaturing binding buffer; PCR; polymerase chain reaction; LB; Luria broth; E. coli; Escherichia coli; SDS; sodium dodecyl sulfate; EDTA; ethylenediaminotetracetic acid; DNA; deoxyribonucleic acid; PBS; phosphate-buffered saline; Con A; Concanavalin A; ELISA; enzyme linked immunosorbent assay; IFA; immunofluorescence assay; IM; intramuscular; TPA; tissue plasminogen activator; GST; glutathione S-transferase; CatL; cathepsin L; MHC; major histocompatibility complex; PMSF; paramethylsulfonilfluoride; HP; high performance Abstract: The humoral and cellular responses to DNA vaccination of BALB/c mice with a novel antigen from the Fasciola hepatica saposin-like protein family (FhSAP-2) have been investigated. Two constructs were produced containing the FhSAP-2 DNA sequence, one intended for extracellular secretion of FhSAP-2 protein, and one expressing FhSAP-2 in the cytoplasm of a transfected cell. The constructs were tested in HEK 293T cells, with the secretory construct producing less detectable FhSAP-2 relative to cytoplasmic construct when observed by fluorescence. The size of expressed protein was confirmed by Western blot of cell lysate, but FhSAP-2 was undetectable in cell supernatants. Both, secretory and cytoplasmic constructs as well as FhSAP-2 recombinant protein were tested in mice. The antibody response elicited in mice vaccinated with the rFhSAP-2 induced high levels of IgG.sub.1, IgG.sub.2, and IgE as well as high levels of IL-10 and IFN[gamma] indicating a mixed Th1/Th2 response. Vaccination of mice intramuscularly with the cytoplasmic FhSAP-2 construct resulted in a dominant IgG.sub.2a isotype antibody as well as a dominant IFN[gamma] cytokine, with significant IgE, IgG.sub.1, and IL-10 responses also present, suggesting a mixed Th1/Th2 profile. Isotype and cytokine profiles elicited by the FhSAP-2 secretory construct were similar to those obtained with the cytoplasmic construct but at levels that were significantly lower. The results demonstrate that FhSAP-2 can be delivered as a DNA vaccine construct and induces a stronger Th1 response than the recombinant protein alone. This could result in an improvement in the immunoprophylactic potential of this candidate vaccine against F. hepatica. Author Affiliation: (a) Laboratory of Molecular Parasitology and Immunology, Department of Microbiology, University of Puerto Rico, School of Medicine, Puerto Rico (b) Institute of Biotechnology, Department of Parasitology, University of Granada, Spain (c) Laboratory of Parasite Immunology and Pathology, Department of Pathology and Laboratory Medicine, University of Puerto Rico, School of Medicine, Puerto Rico Article History: Received 20 December 2004; Revised 23 March 2005; Accepted 29 March 2005 Article Note: (footnote) [star] Accession No. AF286903.