Combination Vaccines of Fasciola gigantica Saposin-like Protein-2 and Leucine Aminopeptidase.

Saposin-like protein-2 (SAP-2) and leucine aminopeptidase (LAP) are major proteins involved in the digestive process of Fasciola gigantica (Fg). Both SAP-2 and LAP are highly expressed in F. gigantica; therefore, they could be vaccine candidates for fasciolosis. The aims of this study are (1) to observe the tissue expression of F. gigantica SAP-2 (FgSAP-2) and F. gigantica LAP (FgLAP) in F. gigantica by indirect immunofluorescence technique under confocal microscopy and (2) to test the vaccine potentials of individual and combined recombinant (r) FgSAP-2 and rFgLAP against F. gigantica in Imprinting Control Region (ICR) mice (n = 10 per group). By indirect immunofluorescence-confocal microscopy, FgSAP-2 and FgLAP were localized in the caecal epithelium but at different sites: FgSAP-2 appeared in small granules that are distributed in the middle and lower parts of the cytoplasm of epithelial cells, while FgLAP appeared as a line or zone in the apical cytoplasm of caecal epithelial cells. For vaccine testing, the percent protection of combined rFgSAP-2 and rFgLAP vaccines against F. gigantica was at 80.7 to 81.4% when compared with aluminum hydroxide (alum) adjuvant and unimmunized controls, respectively. The levels of IgG1 and IgG2a in the sera were significantly increased in single and combine vaccinated groups compared with the control groups. Vaccinated mice showed reduced liver damage when compared with control groups. This study indicates that the combined rFgSAP-2 and rFgLAP vaccine had a higher vaccine potential than a single vaccine. These results support the further testing and application of this combined vaccine against F. gigantica infection in farmed livestock animals.

The combined recombinant cathepsin L1H and cathepsin B3 vaccine against Fasciola gigantica infection.

Protections against Fasciola gigantica infection in mice immunized with the individual and combined cathepsin L1H and cathepsin B3 vaccines were assessed. The vaccines comprised recombinant (r) pro-proteins of cathepsin L1H and B3 (rproFgCatL1H and rproFgCatB3) and combined proteins which were expressed in Pichia pastoris. The experimental trials were performed in ICR mice (n = 10 per group) by subcutaneous injection with 50 µg of the recombinant proteins combined with Alum or Freund's adjuvants. At two weeks after the third immunization, mice were infected with 15 F. gigantica metacercariae per mouse by oral route. The percents of protection of rproFgCatL1H, rproFgCatB3 and combined vaccines against F. gigantica were approximately 58.8 to 75.0% when compared with adjuvant-infected control. These protective effects were similar among groups receiving vaccines with Alum or Freund's adjuvants. By determining the levels of IgG1 and IgG2a in the immune sera, which are indicative of Th1 and Th2 immune responses, it was found that both Th1 and Th2 humoral immune responses were significantly increased in vaccinated groups compared with the control groups, with higher levels of IgG1 (Th2) than IgG2a (Th1). Mice in vaccinated groups showed reduction in liver pathological lesions when compared with control groups. This study indicates that the combined rproFgCatB3 and rproFgCatL1H vaccine had a high protective potential than a single a vaccine, with Alum and Freund's adjuvants showing similar level of protection. These results can serve as guidelines for the testing of this F. gigantica vaccine in larger economic animals.

Expression and characterization of glutathione peroxidase of the liver fluke, Fasciola gigantica.

Glutathione peroxidase (GPx) is a key member of the family of antioxidant enzymes in trematode parasites including Fasciola spp. Because of its abundance and central role as an anti-oxidant that helps to protect parasites from damage by free radicals released from the host immune cells, it has both diagnostic as well as vaccine potential against fasciolosis. In this study, we have cloned, characterized, and detected the expression of the GPx protein in Fasciola gigantica (Fg). FgGPx (582 bp) was cloned by polymerase chain reaction (PCR) from complementary DNA (cDNA) from an adult fluke. Its putative peptide has no signal sequence and is composed of 168 amino acids, with a molecular weight of 19.1 kDa, and conserved sequences at NVACKUG, FPCNQFGGQ, and WNF. Phylogenetic analysis showed that GPx is present from protozoa to mammals and FgGPx was closely related to Fasciola hepatica GPx. A recombinant FgGPx (rFgGPx) was expressed in Escherichia coli BL21 (DE3) and used for immunizing mice to obtain polyclonal antibodies (anti-rFgGPx) for immunoblotting and immunolocalization. In immunoblotting analysis, the FgGPx was expressed in all stages of F. gigantica (eggs, metacercariae, newly excysted juveniles (NEJ), 4-week-old juveniles, and adults). This mouse anti-rFgGPx reacted with the native FgGPx at a molecular weight of 19.1 kDa in adult whole body (WB) and tegumental antigens (TA) as detected by immunoblotting. The FgGPx protein was expressed at a high level in the tegument, vitelline glands, and eggs of the parasite. Anti-rFgGPx exhibited no cross-reactivity with the other parasite antigens, including Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gastrothylax crumenifer, Paramphistomum cervi, and Setaria labiato papillosa. The possibility of using rFgGPx for immunodiagnosis and/or as a vaccine for fasciolosis in animals of economic importance will be explored in the future.

Characterization and vaccine potential of Fasciola gigantica saposin-like protein 1 (SAP-1).

The recombinant Fasciola gigantica Saposin-like protien-1 (rFgSAP-1) was cloned by polymerase chain reaction (PCR) from NEJ cDNA, expressed in Escherichia coli BL21 (DE3) and used for production of a polyclonal antibody in rabbits (anti-rFgSAP-1). By immunoblotting and immunohistochemistry, rabbit IgG anti-rFgSAP-1 reacted with rFgSAP-1 at a molecular weight 12kDa, but not with rFgSAP-2. The rFgSAP-1 reacted with antisera from mouse infected with F. gigantica metacercariae collected at 2, 4, and 6 weeks after infection. The FgSAP-1 protein was expressed at a high level in the caecal epithelium of metacercariae and NEJs. The vaccination was performed in Imprinting Control Region (ICR) mice (n=10) by subcutaneous injection with 50µg of rFgSAP-1 combined with Alum adjuvant. Two weeks after the second boost, mice were infected with 15 metacercariae per mouse by the oral route. The percents protection of rFgSAP-1 vaccine were estimated to be 73.2% and 74.3% when compared with non vaccinated-infected and adjuvant-infected controls, respectively. The levels of IgG1 and IgG2a specific to rFgSAP-1 in the immune sera, which are indicative of Th2 and Th1 immune responses, were inversely and significantly correlated with the numbers of worm recoveries. The rFgSAP-1-vaccinated mice showed significantly reduced levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and liver damage. These indicated that rFgSAP-1 has strong potential as a vaccine candidate against F. gigantica, whose efficacy will be studied further in large economic animals including cattle, sheep, and goat.