Immune response of rats vaccinated orally with various plant-expressed recombinant cysteine proteinase constructs when challenged with Fasciola hepatica metacercariae

Abstract

Background: Cysteine proteinases of Fasciola hepatica are important candidates for vaccine antigens because of their role in fluke biology and host-parasite relationships. In our previous experiments, we found that a recombinant cysteine proteinase cloned from adult F. hepatica (CPFhW) can protect rats against liver fluke infections when it is administered intramuscularly or intranasally in the form of cDNA. We also observed considerable protection upon challenge following mucosal vaccination with inclusion bodies containing recombinant CPFhW produced in Escherichia coli. In this study, we explore oral vaccination, which may be the desired method of delivery and is potentially capable of preventing infections at the site of helminth entry. To provide antigen encapsulation and to protect the vaccine antigen from degradation in the intestinal tract, transgenic plant-based systems are used.

Methodology: In the present study, we aimed to evaluate the protective ability of mucosal vaccinations of 12-week-old rats with CPFhW produced in a transgenic-plant-based system. To avoid inducing tolerance and to maximise the immune response induced by oral immunisation, we used the hepatitis B virus (HBV) core protein (HBcAg) as a carrier. Animals were immunised with two doses of the antigen and challenged with 25 or 30 metacercariae of F. hepatica.

Conclusions: We obtained substantial protection after oral administration of the plant-produced hybrids of CPFhW and HBcAg. The highest level of protection (65.4%) was observed in animals immunised with transgenic plants expressing the mature CPFhW enzyme flanked by Gly-rich linkers and inserted into c/e1 epitope of truncated HBcAg. The immunised rats showed clear IgG1 and IgM responses to CPFhW for 4 consecutive weeks after the challenge.

Fig 1. Schematic representation of the construct encoding fusion proteins.

(A) mCPFhW::C construct consists of the sequence encoding mature CPFhW (nt 319–976) ligated to the 5' end of the sequence encoding HBcAg(T) (nt 1903–2451). (B) pCPFhW::C construct consists of the sequence encoding the propeptide of CPFhW (nt 46–318) ligated to the 5' end of the sequence encoding HBcAg(T). (C) mCPFhW::G::C construct consists of the sequence encoding HBcAg(T) with an insertion encoding the mature CPFhW flanked by Gly-rich linkers ((Gly)₄-Ser-(Gly)₄-Gln-(Gly)₂). The mature CPFhW flanked at both ends by glycine residues is ligated between nt 2120 and 2151 of the sequence encoding HBcAg(T). (D) U::mCPFhW construct consists of a ubiquitin sequence spanning nt 767–995 ligated to the 5' end of the sequence encoding mature CPFhW.

Fig 2. Post-challenge serum antibody isotype responses of vaccinated and control rats to recombinant cysteine proteinase.

(A) IgG1. (B) IgM. (C) IgA. (D) IgE. Group G–Lyophilised lettuce expressing the mature CPFhW enzyme flanked by Gly-rich linkers; Group U–Lyophilised lettuce expressing the mature CPFhW protein fused with ubiquitin; Group C–Lyophilised control lettuce; Group N–None. At each timepoint, four rats from each group were euthanised and dissected. *Indicates significantly increased numbers of cells (p<0.05). Error bars indicate standard deviation.

Fig 3. Post-challenge serum antibody isotype responses of vaccinated and control rats to ES products of adult flukes.

(A) IgG1. (B) IgM. (C) IgA. (D) IgE. Group G–Lyophilised lettuce expressing the mature CPFhW enzyme flanked by Gly-rich linkers; Group U–Lyophilised lettuce expressing the mature CPFhW protein fused with ubiquitin; Group C–Lyophilised control lettuce; Group N–None. At each timepoint, four rats from each group were euthanised and dissected. *Indicates significantly increased numbers of cells (p<0.05). Error bars indicate standard deviation.

Fig 4. Leukocyte responses in the blood of vaccinated and control rats to challenge infections.

(A) eosinophils. (B) monocytes. (C) CD4+ T lymphocytes. (D) CD8+ T lymphocytes. Group G–Lyophilised lettuce expressing the mature CPFhW enzyme flanked by Gly-rich linkers; Group U–Lyophilised lettuce expressing the mature CPFhW protein fused with ubiquitin; Group C–Lyophilised control lettuce; Group N–None. At each timepoint, four rats from each group were euthanised and dissected. *Indicates significantly increased numbers of cells (p<0.05). Error bars indicate standard deviation.

Fig 5. Leukocyte responses in the peritoneal cavity of vaccinated and control rats to challenge infections.

(A) eosinophils. (B) monocytes. (C) CD4+ T lymphocytes. (D) CD8+ T lymphocytes. Group G–Lyophilised lettuce expressing the mature CPFhW enzyme flanked by Gly-rich linkers; Group U–Lyophilised lettuce expressing the mature CPFhW protein fused with ubiquitin; Group C–Lyophilised control lettuce; Group N–None. At each timepoint, four rats from each group were euthanised and dissected. *Indicates significantly increased numbers of cells (p<0.05). Error bars indicate standard deviation.

Fig 6. T lymphocyte responses in the mesenteric lymph nodes of vaccinated and control rats to challenge infections.

(A) CD4+ cells. (B) CD8+ cells. Group G–Lyophilised lettuce expressing the mature CPFhW enzyme flanked by Gly-rich linkers; Group U–Lyophilised lettuce expressing the mature CPFhW protein fused with ubiquitin; Group C–Lyophilised control lettuce; Group N–None. At each timepoint four rats from each group were euthanised and dissected. *Indicates significantly increased numbers of cells (p<0.05). Error bars indicate standard deviation.