Fasciola hepatica vaccine based on Kunitz-Type Molecule reduces adult worm fecundity in experimentally infected sheep

Description

Background Fasciolosis is a cosmopolitan and continuously expanding helmintiasis produced by the trematode Fasciola hepatica. Globally, sheep and cattle are the main definitive hosts of F. hepatica. The economic losses caused by this disease in livestock production are calculated to be around US$ 3 billion annually. The reports of resistance to anthelmintics described in F. hepatica highlight the urgent need to develop an effective vaccine that reduce the impact and spread of the disease through a reduction in the number of eggs and their viability, as well as a decrease in the number of adults to allow a reduction in liver damage Methods Sheep were immunized with two doses, administered 21 days apart, of the F. hepatica Kunitz-type molecule (sFhKT) vaccine, formulated with a liquid crystal nanostructure composed of 6-O-ascorbyl palmitate ester (Coa-ASC16) and synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine motifs (CpG-ODN), and were subsequently infected with 100 metacercariae. Thirty days after the number of worms in the bile canaliculi and gallbladder, as well as the egg count and viability in feces, were determined. Sheep-specific anti-IgG antibodies for sFhKT were measured by ELISA Results The sFhKT/CpG-ODN/Coa-ASC16 vaccine formulation with the highest sFhKT dose was the most effective, significantly reducing fecal egg counts (FEC) by 81.6%. It also reduced worm burden by 55.7%, although this difference was not statistically significant. The addition of Cathepsin L3 (FhCL3) to the formulation further reduced FEC (89.1%) but resulted in a lower reduction in worm burden. Sheep vaccinated with sFhKT/CpG-ODN/Coa-ASC16 exhibited slightly less hepatic damage than non-vaccinated animals, with histological lesions characterized by increased inflammatory infiltrates. The experimental vaccine FhKT/CpG-ODN/Coa-ASC16 induced an IgG antibody response with higher titers in immunized sheep compared to those that did not receive the vaccine, although the difference was not statistically significant. Conclusions In this study, sFhKT/CpG-ODN/Coa-ASC16 was evaluated as a vaccine candidate against F. hepatica in sheep. The significant reduction in worm fertility in vaccinated animals suggests the possibility of continuing with further investigations that could improve the efficacy of the proposed vaccine .

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Thirty-two parasite-free lambs were randomly assigned to experimental groups. Each received 100 metacercariae (mtc), totaling 2,700 mtc, Metacercariae were stored at 4°C until use. The study was conducted over three independent trials following the same design. All procedures adhered to ethical guidelines, ensuring minimal animal distress. The sFhKT peptide was synthesized based on a sequence provided by CIBICI-CONICET, UNC. Peptide purity (>95%) was confirmed via RP-HPLC and MALDI-TOF. CpG-ODN adjuvant was supplied by Operon Technologies. Coa-ASC16 nanostructure was prepared by mixing 2% (w/v) 6-O-ascorbyl palmitate and 5% dextrose, followed by heating to 72°C and ultrasonic homogenization. FhKT and CpG-ODN were incorporated using a standardized protocol. ELISA plates were coated with 1 µg/mL sFhKT in carbonate buffer (pH 9.6) and incubated overnight at 4°C. Plates were washed with PBS-Tween, blocked with 5% PBS-FBS, and incubated with diluted samples (2% PBS-FBS). Secondary anti-IgG1 peroxidase-conjugated antibody was applied, and detection was performed using TMB. Absorbance was measured at 450 nm. Fecal samples were collected from week 10 post-infection,. The sedimentation method combined with the modified McMaster technique was used. Six serial determinations of ALT and AST were performed. Blood samples (2 mL) were drawn via jugular venipuncture, centrifuged, and analyzed using enzymatic methods. Parasite burden was assessed by counting adult worms from the gallbladder and liver parenchyma. Worms were retrieved via bile duct pressure and surgical resection, then preserved in 10% formalin. Egg hatch rate was analyzed by collecting bile, washing, and incubating eggs at 25°C in darkness for 15 days, followed by light exposure to induce miracidium hatching. A 10% formalin solution halted hatching. The first 100 eggs were classified as hatched (empty) or unhatched, and hatch rate was calculated. Data were processed in Excel, Infostat, and GraphPad Prism. Normality was assessed via the Shapiro-Wilk test, and variance homogeneity via Bartlett’s test. Count data were analyzed using a generalized linear mixed model (GLMM) with a Poisson distribution, log-link function, and Trial as a random effect. Mean values were adjusted using GLMM. Egg and worm reduction percentages were calculated as: % FECR = (IC - V(1,2,3)) / IC × 100, % AWCR = (IC - V(1,2,3)) / IC × 100, where IC is the infected control group and V1, V2, V3 are vaccinated groups. Histopathological data were analyzed using Principal Component Analysis (PCA) and logistic regression for cirrhosis-associated factors (fibrosis, inflammation, hydropic degeneration, canalicular regeneration). For antibody response (OD at 1/500 dilution), GLMM was applied with Trial as a random effect. Post hoc comparisons were conducted using Fisher’s LSD test (p < 0.05). Hatch rate effects were assessed using a linear mixed-effects model (LMM).

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