[Show abstract][Hide abstract] ABSTRACT: An indirect enzyme-linked immunosorbent assay (ELISA) was developed and evaluated for its diagnostic ability to detect human IgG antibodies against Fasciola hepatica saposin-like protein-2. The assay was compared with an indirect ELISA with excretory-secretory products (FhES) from adult F. hepatica. In an analysis of the sera of 37 patients infected with F. hepatica, 40 patients with other parasitic infections, and 50 healthy controls, the sensitivity of both ELISA assays was 100%. However, the FhSAP2-based ELISA was more specific (95.6%) than the FhES-ELISA (91.9%). These results demonstrated that FhSAP2 can be used in the serodiagnosis of chronic human fascioliasis with the additional advantage of being relatively cheap and easy to produce. Studies are in progress to evaluate this FhSAP2-ELISA assay in a large-scale prevalence surveys in endemic areas.
[Show abstract][Hide abstract] ABSTRACT: It is not unusual to find common molecules among parasites of different species, genera, or phyla. When those molecules are antigenic, they may be used for developing drugs or vaccines that simultaneously target different species or genera of parasite. In the present study, we used a proteomic-based approach to identify proteins that are common to adult Fasciola hepatica and Schistosoma mansoni. Whole-worm extracts from each parasite were separated by 2-dimensional electrophoresis (2-DE), and digital images of both proteomes were superimposed using imaging software to identify proteins with identical isoelectric points and molecular weights. Protein identities were determined by mass spectrometry. Imaging and immunoblot analyses identified 28 immunoreactive proteins that are common to both parasites. Among these molecules are antioxidant proteins (thioredoxin and glutathione-S-transferase), glycolytic enzymes (glyceraldehyde 6-phosphate dehydrogenase and enolase), proteolytic enzymes (cathepsin-L and -D), inhibitors (Kunitz-type, Stefin-1), proteins with chaperone activity (heat shock protein 70 and fatty acid-binding protein), and structural proteins (calcium-binding protein, actin, and myosin). Some of the identified proteins could be used to develop drugs and vaccines against fascioliasis and schistosomiasis.
Journal of Parasitology 03/2011; 97(5):852-61. DOI:10.1645/GE-2495.1 · 1.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We recently reported the biochemical characterization of a novel Fasciola hepatica recombinant antigen termed FhSAP2, which has previously shown to elicit protection to F. hepatica infection in rabbits. Further we reported that intramuscular (IM) injections of BALB/c mice with a pFLAG-CMV(-2) vector carrying cDNA encoding for FhSAP2 (cDNA-FhSAP2) induce high levels of immune response. The aim of the present study is to ascertain whether the immune response induced by this DNA construct may induce protection in mice against subsequent infection with F. hepatica metacercariae (mc). In addition, protection following subcutaneous (SC) injections with recombinant FhSAP2 was evaluated.
Mice received three IM injections with 100 microg of cDNA-FhSAP2 or three SC injections with 20 microg of FhSAP2. Four weeks after the last vaccination mice were challenged orally with 5 F. hepatica me and euthanized 45 days after challenge.
Mean worm burdens found in mice vaccinated with cDNA-FhSAP2 was reduced by 83.3% and the mean worm burdens found in mice vaccinated with the recombinant protein was reduced by 60% when compared with controls. All vaccinated animals had less liver damage than challenge controls. Vaccination with cDNA-FhSAP2 seems to favor a mixed Th1/Th2-antibody dependent with higher predominance of Th1-regulated antibody response.
The vaccination with cDNA-FhSAP2 or recombinant FhSAP2 may protect hosts against F. hepatica infections. The vaccination with cDNA form of FhSAP2 appeared to be a little more efficient preventingthe infection. The predominance of Th1-dependent antibodies in the vaccinated animals may be responsible for the protection but this should be confirmed by Th1-cytokines determinations.
[Show abstract][Hide abstract] ABSTRACT: Fasciola hepatica saposin-like protein (FhSAP-2) is a novel antigen expressed at an early stage of infection and has been shown to induce in rabbits a significant protection to infection with F. hepatica. There are no studies to identify the immunologically relevant regions of FhSAP-2. In this work the amino acid sequence of FhSAP-2 was analyzed to identify potential T-cell epitopes. A predictive algorithm identified four possible sites. Experimental determination of the T-cell epitopes was achieved using a panel of overlapping peptides spanning the entire sequence of FhSAP-2, which was evaluated for their ability to induce lymphoproliferative responses of spleen cells from 8 immunized BALB/c (H-2d) mice. Five different epitopes were identified. There was minimal agreement between theoretical and experimental approaches. It was found that peptides containing amino acid residues AVTFA and IDIDLCDICT as part of their structure induce high levels of IL-2 and IFNgammain vitro and was classified as Th1 epitopes. Peptides that contain the residues ADQTV, CIEFVQQEVD and YIIDHVDQHN induced significant amount of IL-4 and IL-2 were considered as containers of Th0 epitopes. Identification of prominent T-cell epitopes from FhSAP-2 offers the possibility of understanding how the CD4+ T-cell response is involved in protection against fasciolosis and how it is implicated in susceptibility to infection.