Pornanan Kueakhai

Vaccine potential of recombinant cathepsin B against Fasciola gigantica.

In Fasciola gigantica, cathepsin Bs, especially cathepsin B2 and B3 are expressed in early juvenile stages, and are proposed to mediate the invasion of host tissues. Thus they are thought to be the target vaccine candidates that can block the invasion and migration of the juvenile parasite. To evaluate their vaccine potential, the recombinant cathepsin B2 (rFgCatB2) and cathepsin B3 (rFgCatB3) were expressed in yeast, Pichia pastoris, and used to immunize mice in combination with Freunds adjuvant to evaluate the protection against the infection by F. gigantica metacercariae, and the induction of immune responses. Mice immunized with both recombinant proteins exhibited high percent of parasite reduction at 60% for rFgCatB2 and 66% for rFgCatB3. Immunization by both antigens induced continuously increasing levels of IgG1 and IgG2a with a higher level of IgG1 isotype, indicating the mixed Th1/Th2 responses with Th2 predominating. When examined individually, the higher levels of IgG1 and IgG2a were correlated with the lower numbers of worm recoveries. Thus, both cathepsin B2 and cathepsin B3 are plausible vaccine candidates whose potential should be further tested in large economic animals.

Production and characterization of a monoclonal antibody against recombinant saposin-like protein 2 of Fasciola gigantica.

A monoclonal antibody (MoAb) against recombinant Fasciola gigantica saposin-like protein 2 (rFgSAP-2) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with rFgSAP-2. This MoAb is an IgG1, κ light chain isotype. By immunoblotting and indirect ELISA, the MoAb reacted specifically with rFgSAP-2, the natural FgSAP-2 at 10kDa in whole body (WB) and excretory-secretory (ES) fractions of F. gigantica. It did not cross react with antigens in WB fractions from other parasites, including Opisthorchis viverrini, Schistosoma mansoni which are human parasites, Haemonchus placei, Setaria labiato-papillosa, Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gigantocotyle explanatum, Gastrothylax crumenifer, and Paramphistomum cervi which are ruminant parasites. By immunohistochemistry, the FgSAP-2 protein was localized only in the cytoplasm of caecal epithelial cells of 4-week-old juvenile and adult stages, but not in metacercariae, newly excysted juvenile (NEJ), 2- and 3-week-old juveniles. This finding indicated that FgSAP-2 is an abundantly expressed parasite protein that is released into the ES, hence SAP-2 and its MoAb may be used for immunodiagnosis of ruminant and human fasciolosis.

Molecular cloning and characterization of leucine aminopeptidase from Fasciola gigantica

M17 leucine aminopeptidase (LAP) is one of a family of metalloexopeptidases, of which short peptide fragments are cleaved from the N-terminals. In this study, the full length of cDNA encoding Fasciola gigantica LAP (FgLAP) was cloned from adult parasites. The amino acid sequences of FgLAP showed a high degree of identity (98%) with that from Fasciola hepatica and a low degree of identities (11% and 9%) with those from cattle and human. Phylogenetic analysis revealed that the FgLAP was closely related and grouped with F. hepatica LAP (FhLAP). Northern analysis showed that FgLAP transcriptional products have 1800 base pairs. Analysis by RNA in situ hybridization indicated that LAP gene was expressed in the cecal epithelial cells of adult parasites. A polyclonal antibody to a recombinant FgLAP (rFgLAP) detected the native LAP protein in various developmental stages of the parasite. In a functional test, this rFgLAP displayed aminolytic activity using a fluorogenic Leu-MCA substrate, and was significantly inhibited by bestatin. Its maximum activity was at pH 8.0 and enhanced by Mn(2+) ions. Localization of LAP proteins by immunohistochemistry and immunofluorescence techniques indicated that the enzyme was distributed in the apical cytoplasm of cecal epithelial cells. Because of its important metabolic role and fairly exposed position, FgLAP is a potential drug target and a possible vaccine candidate against fasciolosis.

Protection against Fasciola gigantica infection in mice by vaccination with recombinant juvenile-specific cathepsin L.

Fasciola gigantica cathepsin L1H (FgCatL1H) is one of the major cathepsin L released by juveniles of F. gigantica to aid in the invasion of hosts tissues. Due to its high sequence similarity with other cathepsin L (CatL) isoforms of late stage F. gigantica, it was considered to be a good vaccine candidate that can block all CatL-mediated protease activities and affect juveniles as well as adult parasites. In this study, recombinant proFgCatL1H protein expressed in yeast, Pichia pastoris, system was mixed with Freunds adjuvants and used to subcutaneously immunize mice that were later challenged with metacercariae of F. gigantica. The percentage of worm protection in the rproFgCatL1H-vaccinated mice compared to the non-immunized and adjuvant control mice were approximately 62.7% and 66.1%, respectively. Anti-rproFgCatL1H antisera collected from vaccinated mice reacted specifically with rproFgCatL1H and other cathepsin L isoforms of F. gigantica, but the antibodies did not cross react with antigens from other trematode and nematode parasites, including Eurytrema pancreaticum, Opisthorchis viverrini, Fischoederius cobboldi, Cotylophoron cotylophorum, Gigantocotyle explanatum, Paramphistomum cervi, and Setaria labiato-papillosa. The levels of IgG1 and IgG2a in mouse sera increased significantly at two weeks after immunization and were highest during the sixth to eighth weeks after immunization. The IgG1 level was higher than IgG2a at all periods of immunization, implicating the dominance of the Th2 response. The levels of IgG1 and IgG2a in the immune sera were shown to be strongly correlated with the numbers of worm recovery, and the correlation coefficient was higher for IgG1. The levels of serum aspartate aminotransferase and alanine transaminase were significantly lower in the sera of rproFgCatL1H-vaccinated mice than in the infected control mice indicating a lower degree of liver damage. This study demonstrated a high potential of FgCatL1H vaccine, and its efficacy is currently being studied in the larger economic animals.

Vaccine potential of recombinant cathepsinL1G against Fasciola gigantica in mice

In this study, we characterized and investigated the vaccine potential of FgCatL1G against Fasciola gigantica infection in mice. Recombinant mature FgCatL1G (rmFgCatL1G) was expressed in Escherichia coli BL21. The vaccination was performed in Imprinting Control Region (ICR) mice (n=10) by subcutaneous injection with 50μg of rmFgCatL1G combined with Freunds adjuvant. Two weeks after the second boost, mice were infected with 15 metacercariae by the oral route. The percents of protection of rmFgCatL1G vaccine were estimated to be 56.5% and 58.3% when compared with non vaccinated-infected and adjuvant-infected controls, respectively. Antibodies in the immune sera of vaccinated mice were shown by immunoblot to react with the native FgCatL1s in the extract of all stages of parasites and rmFgCatL1H, recombinant pro - FgCatL1 (rpFgCatL1). By immunohistochemistry, the immune sera also reacted with FgCatL1s in the caecal epithelial cells of the parasites. The levels of IgG1 and IgG2a in the immune sera, which are indicative of Th2 and Th1 immune responses, were also increased with IgG1 predominating. The levels of serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) in rmFgCatL1G-immunized group showed no significant difference from the control groups, but pathological lesions of livers in rmFgCatL1G-immunized group showed significant decrease when compared to the control groups. This study indicates that rmFgCatL1G has a vaccine potential against F. gigantica in mice, and this potential will be tested in larger livestock animals.

The in vitro anthelmintic effects of plumbagin on newly excysted and 4-weeks-old juvenile parasites of Fasciola gigantica

The effect of plumbagin (PB, 5-hydroxy-2-methyl-1,4-naphthoquinone) against newly excysted juveniles (NEJs) and 4-weeks-old immature parasites of Fasciola gigantica were compared with triclabendazole (TCZ). The anthelmintic efficacy of 1, 10 and 100μg/ml of PB or TCZ following incubation in vitro for 1-24h was compared using a combination of relative motility (RM), survival index (SI) and larval migration inhibition (LMI) assays for parasite viability. The RM and SI values of the PB-treated group decreased at a more rapid rate than the TCZ-treated group. For NEJs, the decreased RM values were first observed at 1h incubation with 1μg/ml PB, and 90% of flukes were killed at 24h. In contrast, in TCZ-treated groups a 10-fold higher concentration of TCZ (10μg/ml) resulted in only 9% dead parasites after 24h incubation. In 4-weeks-old juvenile parasites, PB reduced the RM value at 10μg/ml with 100% of flukes dead after 3h, while TCZ decreased RM values at the concentration of 100μg/ml but with only 5% of flukes killed at 24h. NEJs treated with PB exhibited 88%, 99% and 100% of LMIs at the concentrations of 1, 10 and 100μg/ml, respectively. NEJs incubated with TCZ have an LMI of only 32% at the highest concentration of 100μg/ml. Similarly PB had a significantly greater killing of immature 4weeks juvenile stages than TCZ at all concentrations; however, 4-weeks-old juvenile parasites were more resistant to killing by PB or TCZ at all concentrations when compared to NEJs. Further studies were carried out to investigate the alterations of the parasite tegument by scanning electron microscope (SEM). PB caused similar tegumental alterations in 4-weeks-old juveniles as those observed in TCZ treatment but with greater damage at comparative time points, comprising of swelling, blebbing and rupture of the tegument, loss of spines, and eventual erosion, lesion and desquamation of the total tegument. These data indicate that PB had a greater fasciolicidal effect against immature stages of F. gigantica parasites than TCZ and warrant further studies for use as a potential new anthelmintic against Fasciola infections.

Saposin-like protein 2 has an immunodiagnostic potential for detecting Fasciolosis gigantica.

Saposin-like protein 2 (SAP-2) plays an important role in the digestive process of Fasciola gigantica (Fg). It is one of the major proteins synthesized by the caecal epithelial cells and released into flukes excretion-secretion. Therefore, FgSAP-2 is a plausible target for detecting fasciolosis. A polyclonal antibody (PoAb) against recombinant FgSAP-2 was produced by immunizing rabbits with the recombinant protein (rFgSAP-2), and used in sandwich ELISA assay to detect the circulating FgSAP-2 in sera of mice experimentally infected with F. gigantica metacercariae. The assay could detect rFgSAP-2 and the native FgSAP-2 in the excretory-secretory (ES) and whole body (WB) fractions of adult F. gigantica at the concentrations as low as 38 pg/ml, 24 ng/ml, and 102 ng/ml, respectively. As well, the sera from mice experimentally infected with F. gigantica were tested positive by this sandwich ELISA, which exhibited sensitivity, specificity, false positive rate, false negative rate and accuracy at 99.99, 98.67, 1.33, 0.01 and 99.32%, respectively. Therefore, this assay could be used for diagnosis of fasciolosis by F. gigantica.

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.