In vitro and in vivo studies for assessing the immune response and protection-inducing ability conferred by Fasciola hepatica-derived synthetic peptides containing B- and T-cell epitopes

Abstract

Fasciolosis is considered the most widespread trematode disease affecting grazing animals around the world; it is currently recognised by the World Health Organisation as an emergent human pathogen. Triclabendazole is still the most effective drug against this disease; however, resistant strains have appeared and developing an effective vaccine against this disease has increasingly become a priority. Several bioinformatics tools were here used for predicting B- and T-cell epitopes according to the available data for Fasciola hepatica protein amino acid sequences. BALB/c mice were immunised with the synthetic peptides by using the ADAD vaccination system and several immune response parameters were measured (antibody titres, cytokine levels, T-cell populations) to evaluate their ability to elicit an immune response. Based on the immunogenicity results so obtained, seven peptides were selected to assess their protection-inducing ability against experimental infection with F. hepatica metacercariae. Twenty-four B- or T-epitope-containing peptides were predicted and chemically synthesised. Immunisation of mice with peptides so-called B1, B2, B5, B6, T14, T15 and T16 induced high levels of total IgG, IgG1 and IgG2a (p<0.05) and a mixed Th1/Th2/Th17/Treg immune response, according to IFN-γ, IL-4, IL-17 and IL-10 levels, accompanied by increased CD62L+ T-cell populations. A high level of protection was obtained in mice vaccinated with peptides B2, B5, B6 and T15 formulated in the ADAD vaccination system with the AA0029 immunomodulator. The bioinformatics approach used in the present study led to the identification of seven peptides as vaccine candidates against the infection caused by Fasciola hepatica (a liver-fluke trematode). However, vaccine efficacy must be evaluated in other host species, including those having veterinary importance.

Figure 1. Boxplot showing IgG anti-peptide antibody levels in mice immunised with the synthetic peptides formulated in the ADAD vaccination system.

The bottom and the top of the box indicate the 25^th and 75^th percentiles, respectively. A). Mice immunised using PAL. B). Mice immunised using AA0029.

Figure 2. Linear regression comparing the effect of PAL and AA0029 immunomodulators on IgG subtype levels.

A). IgG1 related to IgG. B). IgG2a related to IgG. Red indicates the PAL immunomodulator and blue AA0029. Circles and squares represent B- and T-peptides, respectively.

Figure 3. Scatterplot comparing the effect of PAL and AA0029 immunomodulators on cytokine levels.

A). IL-4 related to IL-10. B). IL-5 related to IL-10. Red indicates PAL and blue AA0029. Circles and squares represent B- and T-peptides, respectively.

Figure 4. Boxplot showing IFN-γ cytokine levels in splenocyte mouse cell culture immunised with synthetic peptides.

A). Mice immunised with PAL. B). Mice immunised with AA0029.

Figure 5. Boxplot showing IL-17 cytokine levels in splenocyte mouse cell culture immunised with synthetic peptides.

A). Mice immunised with PAL. B). Mice immunised with AA0029.

Figure 6. Individual cytokine expression levels are represented by shades of green to red in the central heatmap (highest values shown in green and lowest in dark red).

The right-hand margin provides the names of peptide sets. Rows and columns represent clusters of interleukins and peptides having a similar immunological response. A list of cytokines grouped within each cluster is also provided. Groupings having shorter distances (as indicated by the distance to k-means nearest group) had greater similarity. Bicluster analysis for B- and T-cell peptides formulated with the PAL immunomodulator. Five major clusters can be discerned (1, 2, 3, 4 and 5), encompassing peptides having similar cytokine levels.

Figure 7. Individual cytokine expression levels are represented by shades of green to red on the central heatmap (highest values shown in green and lowest in dark red).

The right-hand margin provides the name of peptide sets. Rows and columns represent clusters of interleukins and peptides having a similar immunological response. A list of cytokines grouped within each cluster is also provided. Groupings having shorter distances (as indicated by the distance to k-means nearest group) had greater similarity. Bicluster analysis for B- and T-cell peptides formulated with the AA0029 immunomodulator. Seven major clusters can be discerned (1, 2, 3, 4, 5, 6 and 7), encompassing peptides having similar cytokine levels.

Figure 8. ELISA showing B-cell epitope-containing synthetic peptide reactivity against a pool of sera from mice infected with F. hepatica metacercariae.

Figure 9. F. hepatica excretory/secretory antigen reactivity against hyper-immune sera from mice immunised with synthetic peptides containing B-cell epitopes as assessed by ELISA.

A). B-cell peptides formulated with PAL. B). B-cell peptides formulated with AA0029.

Figure 10. Flow cytometry analysis for quantitation of T-lymphocyte population through CD62L⁺ T-cell memory marker in mice immunised with T-cell epitope-containing synthetic peptides.

A). T-cell peptides formulated with PAL. B). T-cell peptides formulated with AA0029.

Figure 11. Kaplan-Meier curves depicting survival rates in mice immunised with single peptides and infected with F. hepatica metacercariae.

A). Mice immunised with single peptides containing B-epitopes. B). Mice immunised with single peptides containing T-epitopes. Survival rates of mice belonging to both untreated and infected controls are also represented. Human endpoint was established when an indicator of severe pain, excessive distress, suffering or an impending death was observed in any of the animals and then euthanised with an intraperitoneal injection of pentobarbital at 60 mg/kg using 30 g needles.