Induction of Protective Cellular and Humoral Responses against Fasciolosis in Rabbits using Immunoaffinity Fraction of Fasciola gigantica Excretory Secretory Products

Fasciolosis due to Fasciola gigantica or F. hepatica causes significant production losses in animals, as well as being a zoonotic disease of global importance. In an attempt to develop vaccine against fasciolosis in rabbits, an immunoaffinity fraction of F. gigantica excretory secretory products was isolated. The fraction possesses 87.67% of the initial antigenic activities with 2051.5 fold increase in specific activity compared to crude extract. It consists of two bands of molecular weight 27 kDa and 23.5 kDa as revealed by SDS-PAGE. The detailed structural analyses of the pure fraction showed O-glycan [Ser-Arg-Ser-Arg-Ser-GlucNAc] using mass spectrometry. Vaccination of rabbits twice with the fraction resulted in 85% reduction in worm burden. It is also resulted in high antibody IgG levels as proved by ELISA. The highest IgG response was observed in vaccinated rabbits at two weeks post infection and remained stable to the end of the experiment. A significant expression of IL-4 and INF-γ was observed in vaccinated rabbits starting one week until thirteen weeks post infection. The level of IL-4 was significantly higher than the level of INFγ throughout the experiment as measured by ELISA. Collectively, the current results suggest promising immunoprophylactic potentials of the immunoaffinity fraction of Fasciola gigantica excretory secretory products against fasciolosis in rabbits through induction of both cellular and humoral responses.


Introduction
Fasciolosis is a worldwide zoonotic disease caused by trematode parasite of the genus Fasciola. WHO estimates that at least 2.4 million people are infected in more than 70 countries worldwide, with several million people at risk. No continent is free from fasciolosis, and it is likely that where animal cases are reported, human cases also exist [1]. Recently, Fasciola spp. was added to the WHO list of neglected tropical diseases after decades of neglect [2].
In Egypt, fasciolosis becomes hyperendemic and problematic where animal reservoir and snail vector are available [3]. The emerging situation of both human and livestock fasciolosis in Egypt has increased significantly due to both F. gigantica and F. hepatica [4][5][6].
Nearly 800,000 Egyptians are suffering from fasciolosis and about 24 millions are at risk [7,8].
The liver fluke secretes molecules, known as excretory-secretory products (ESPs) that modulate or suppress host immune responses [9,10]. During early chronic infections, there is a predominance of a Th2 response, which decreases in advanced chronic infections characterized by a persistent immune suppression [11].
CD4 + T cells can be separated into 2 major subsets, Th1 and Th2, on the basis of their cytokine secretion patterns and function. Th1 cells produce many cytokines, including IFN-γ and TNF-α, and promote the activation of macrophages which lead to the production of opsonizing antibodies. Also, Th1 cells promote mediation of a delayed-type hypersensitivity reaction and inflammatory responses. Th2 cells produce many other cytokines, including IL-4, IL-6, and IL-10, and promote immediate type hypersensitivity reactions, involving IgE, eosinophils, and mast cells [10]. Generally, helminth infections are manifested by suppression of Th1 function and induction of T cells, which express cytokines characteristic of the Th2 subset [12].
To control fasciolosis, a significant data suggests that a number of Fasciola molecules, including cathepsins L, glutathione S-transferase, leucine aminopeptidase and fatty acid binding proteins have the potency of inducing a protective response against the infection in laboratory animals [13] and large animal models [14].
Vaccination studies with purified native or recombinant Fasciola antigens suggest that this approach, which diminished morbidity and mortality and reduced transmission, is a realistic goal [15].However, despite long-standing research, a vaccine against this parasite has not yet been developed to the level of commercialization [16]. This can be largely attributed to a fact that immune responses to vaccines are influenced by the route of administration, type of antigen and adjuvant [17,18].
The objective of the current study is to identify Fasciola gigantica ESPs fraction with potential for use as vaccine, and to evaluate the protective humoral and cellular immune responses in F. giganticainfected rabbits. In our laboratory, rabbit is often used as a model for experimental fasciolosis to evaluate the effectiveness of Fasciola vaccine candidates [19][20][21].

Animals
Thirty native rabbits, 1.25-1.5 Kg each, were used in the current study. They were proved to be free from any parasitic infections by examining them parasitologically using fecal egg analysis and serologically with indirect ELISA using antigens of most common parasites that infect rabbits (Eimeria steadi, Fasciola gigantica and Trichostrongylus colubriformis). Animals were housed, fed and kept under conventional germ-free conditions in the animal house of the Veterinary Division, National Research Centre. All procedures related to animal experimentation met the International Guiding Principles for Biomedical Research Involving Animals as issued by the International Organizations of Medical Sciences.

Parasites
Metacercariae of F. gigantica were purchased from the Schistosome Biology Supply Center (SBSC) of Theodor Bilharz Research Institute (TBRI), Giza, Egypt. Adult F. gigantica worms were collected from the biliary tracts and gall bladders of condemned bovine livers from local slaughter-house.

Preparation of ESPs
The live intact F. gigantica worms were washed several times with cold 0.01 M PBS (pH 7.4) to eliminate any traces of bile, blood, and contaminated microorganisms. They were then incubated for 16 hr at 37°C in RPMI 1640 medium (pH 7.4) containing 100 IU of penicillin and 100 mg streptomycin/ml of medium. Following incubation, the medium was removed and centrifuged at 12,074 xg for 30 min at 4°C [22]. The supernatant containing ESPs was collected, and the protein content was measured using the method of Lowry et al. [23]. It was then stored at -20°C until use.

Preparation of Rabbit Hyperimmune Serum
100 µg of ESPs was mixed with an equal volume of Freund's complete adjuvant and injected subcutaneously into each of 5 rabbits. A booster dose of 100 µg ESPs in Freund's incomplete adjuvant was injected two weeks later. Second and third booster doses were given on days 21 and 28, respectively [24]. Blood samples were collected 4 days post last injection from rabbit's ear vein. Prepared antiserum was stored at -20°C until use.

Purification of F. gigantica ESPs
The prepared rabbit hyperimmune serum was dialyzed against 100 mM NaHCO 3 buffer pH 8.3 containing 500 mM Nacl and 0.02% (w/v) NaN 3 and coupled to the cyanogen bromide (CNBr)-activated Sepharose-4B at the ratio of 2 mg/ml-swollen beads by strictly following the manufacturer instructions. Crude ES (FgESPs) was applied to the column. After washing the column with 0.15 M PBS pH 7.3 several times, the bound material was eluted with 50 mM glycine-500 mM Nacl-0.02 % (w/v) NaN 3 PH 2.3. The eluted fraction was immediately brought to PH 8.0 with solid NaHCO 3 and then dialyzed against 0.03 M PBS-0.02% (w/v) NaN 3 PH 8.0. The isolated ES fraction was assayed for protein content by the method of Lowry et al. [22] and its immunogenic activity was investigated compared to unbound fraction and crude extract by ELISA.

SDS-PAGE
Both crude and isolated fraction of ESPs were separately mixed with reducing sample buffer containing 5% 2-mercaptoethanol and electrophoresed on SDS-PAGE 10% slab gel (60 µg protein/ well)according to procedures of Laemmli [25]. After separation slab gel was stained with silver stain according to Wray et al. [26]. High and low molecular weight standards (Sigma) were electrophoresed on the same gel to calculate the relative molecular weights of the examined antigens. Gel was photographed wet using Kodak Tri-X-pan films.

Chemical elucidation
The analytical sample was homogenous by Thin-Layer Chromatography (TLC), which was performed on EM silica gel 60 F sheet (0.2 mm) with CHCl 3 /CH 3 OH (9:1), (v/v); as the developing elute livers nt, rate of flow (R f )=0.2. The spot was detected with U.V model UVGL-58 as a violet spot. EI Mass spectra recorded on a Varian MAT 311A spectrometer.

Vaccination protocol
Rabbits were divided into 3 groups. The first group (five rabbits) was a normal control group. The second group was the infected control group (ten rabbits). The third group was the immunizedchallenged group (ten rabbits). Rabbits were immunized intramuscularly twice at 2 weeks interval, with 40 µg of purified fraction diluted in 250 μl 0.01M PBS and 250 μl of complete and incomplete Freund adjuvants (CFA) separately. Two weeks after the last booster dose of immunization, rabbits were infected orally with 30 F. gigantica metacercariae [27]. Rabbits were bled at 2 weeks intervals after infection until the end of the experiment for the collection of sera. All rabbits were necropsied at 13 weeks post challenge for the determination of worm burdens. Whole blood was collected from each rabbit and centrifuged at 214 xg at 4°C for 10 min, and the obtained serum samples were stored at -20°C until analysis.

Assessment of the protective value of the vaccine candidate
Fluke count: Worms were recovered from the gall bladder and livers of both vaccinated and non-vaccinated infected rabbits. The mean total number of flukes/group was calculated. Worm burden reduction in immunized animals was calculated by the formula (C-E)/ C-100, where C is the average number of flukes in the control group and E is the average number of flukes in the vaccinated group of animals.
IgG response: ELISA was adopted to evaluate the success of the purification process by determination of the antigenic activities of the eluted fraction compared with unbound one and crude extract. The assay was also adopted to evaluate the protective IgG level due to the fraction in rabbit serum samples collected at different intervals of infection based on the method of Engvall and Perlmann [28]. Plates were coated, separately, with 5 μg /ml isolated fraction, unbound fraction or crude ES in carbonate buffer. Positive and negative rabbit serum samples diluted at 1:100 was added to the coated plates separately. Anti-rabbit IgG horse radish peroxidase labeled-conjugate (1:1000) and ortho-phenylenediamine (OPD) 1 mg/ml substrate buffer (Sigma) were used. The optimum antigen concentration, antibody and conjugate dilutions were determined by checkerboard titration. Plates were read spectrophotometrically at 450 nm and the cut off values of Optical Densities (OD) were calculated according to Hillyer et al. [29].
Assessment of cytokines level: Serum levels of IL-4 and IFN-γ were measured with double-antibody sandwich ELISA kit (Glory Science Co., Ltd. Del,Rio,Tx 78840,USA). The concentration was calculated from the standard curve that was performed in the same assay.

Statistical analysis
Data are expressed as mean ± SD. Comparison between the mean values of different parameters in the studied groups was performed using 1-way ANOVA test.

Purified vaccine candidate
As summarized in Table 1, 87.67% of the initial antigenic activities in F. gigantica ESPs were recovered in the bound and eluted fraction which represents only 0.65% of total protein in crude ES applied to the column giving 2051.5 fold increases in the specific activities compared to crude ESPs.

Electrophoretic profile of the candidate
Under reducing conditions in SDS-PAGE 10% slab gel, the vaccine candidate was resolved in only two bands of molecular weight 27 kDa and 23.5 kDa compared to multiple bands in the crude extract ranged from 205 kDa to 14.2 kDa.     Table 1: Quantitative summary of F. gigantica ESPs purification. a Protein was measured as described by Lowry et al. [22]. b A unit of activity defined as the amount of protein required to give one well of agglutination. c Specific activity which is the number of activity per μg of protein and is related to the starting crude ESPs.  Levels of IgG response measured by ELISA against vaccine candidate in vaccinated challenged rabbit serum samples ( ) and non-vaccinated normal rabbit samples ( ). Bars in the figure represent SD.

Protective Response of the Vaccine Candidate in Rabbits
Parasitological response: Mean number of recovered worms in vaccinated infected rabbits was reduced to only 3 ± 1.04 worms compared to 20 ± 1.019 worms in infected non vaccinated animals recording 85% reduction in worm burden in vaccinated rabbits.
Humoral response: The level of developed IgG response due to bound and eluted fraction increased to record OD values 0.885 ± 0.0013 two weeks post first dose of vaccination and reached to OD value 0.969 ± 0.0014 four weeks post vaccination and before challenge. This IgG profile is higher than that due to ESPs which recorded OD value 0.568 ± 0.0012 after two weeks of vaccination and increased to 0.586 ± 0.0013 after four weeks. After challenge the IgG in the vaccinated rabbits with the fraction was significantly higher than that in vaccinated rabbits with crude ESPs at all intervals of the experiment starting one week post challenge (0.15 9 ± 0.0012) and reached its maximum level (0.898 ± 0.0013) nine weeks post challenge (Figures 4  and 5).
Cellular response: Levels of IFN-γ were significantly higher in vaccinated infected rabbits than in non-vaccinated infected control throughout the experiment recording the highest value (189.94 ± 0.268) one week post infection ( Figure 6). Levels of IL-4 were also higher in vaccinated infected rabbits than non-vaccinated infected control except 13 weeks post infection recording the highest value (342.04 ± 0.261) 11 weeks post infection (Figure 7). Significant higher level of IL-4 than IFN-γ was observed throughout the experiment (Figures 6 and 7).

Discussion
Based on the importance of Fasciola ESPs in eliciting humoral and cellular host immune responses [9,[30][31][32], the main facet of the current study is to evaluate the protective responses to some of these products against rabbit fasciolosis. In the present research, a fraction with most antigenic activities of ESPs was isolated by immunoaffinity column chromatography with 2051.5 purification folds than whole products. The success of the purification process was further supported by the low antigenic activity in the unbound fraction which means that most antigenic activity was associated with the pure bound fraction. SDS-PAGE showed that the purified fraction consists of two bands of molecular weight 27 and 23.5 kDa. 27 kDa was previously isolated from Fasciola ESPs [33,34]. It was previously supposed that 27 kDa appeared to be released from cells lining the gut [15,35] and the 28 and 26-27 kDa molecules were shown to essentially consist of cathepsin CP [36,37].  [38]. O-GlcNAc is a widespread dynamic carbohydrate modification of cytosolic and nuclear proteins with features analogous to phosphorylation. O-GlcNAc acts critically in many cellular processes, including signal transduction, protein degradation, and regulation of gene expression. However, the study of its specific regulatory functions has been limited by difficulties in mapping sites of O-GlcNAc modification [39].The existence of O-GlcNAc was previously documented in parasites [40] Plasmodium falciparum expresses its own O-GlcNAc transferase during intraerythrocytic development [41]. So, O-GlcNAc glcosylation of enzymes is recorded which supports glycosylation of Fasciola ES fraction in the current study which probably includes Cathepsin based on the existence of 27 kDa.
Vaccination of rabbits with the purified ES fraction emulsified in friend's adjuvant resulted in 85% reduction in worm burden. Selection of rabbits in the current study was based on its success as laboratory animal model in different researches as assessment of drug against fasciolosis [42] and in vaccination studies against fasciolosis [27].In addition, rabbits are cheaper than goats and sheep, easy to handle, raise and infect with fasciolosis.
In previous vaccination trials in rabbits with F. hepatica saposin emulsified in TiterMax adjuvant resulted in 81.2% reduction in worm burden after challenge with F. hepatica metacercariae [43]. In later study, FhSAP2 emulsified in Freund's adjuvant induced an 83.3% reduction in liver fluke count in rabbits [13].Vaccination of sheep with F. hepatica Cysteine proteinases emulsified in Freund's adjuvant resulted in 56.9% reduction in worm count [34]. The authors in the previous trials attributed worm reduction due to vaccination with ES components to high level of IgG. This explanation could be assumed in the current study particularly high level of IgG was recorded. In the present research, the level of developed IgG response due to vaccine candidate in vaccinated challenged rabbits was higher than that in control non vaccinated rabbits and also higher than IgG response to crude extract at different intervals of infection. This is consistent with observations made by other authors who, used immunogens of Fasciola ESPs and found associations between protection and the levels of antibodies [44][45][46].
In our study, the serum levels of pro-inflammatory cytokine, IFN-γ showed lower level than the anti-inflammatory cytokine level, IL-4. So, the T-cell response due to vaccine candidate is mainly Th2 although, Th1 also exists.
Adoption of anther vaccine candidate, FhSAP2, against rabbit fasciolosis induced protection associated with high levels of IFN-γ, TNFα, IL-2 and IL-10, which suggests a mixed Th1/Th2 mechanism with dominance of Th1-cytokines [13]. Involvement of both Th1/Th2 responses in protection against fasciolosis was previously reported using a cathepsin L-based vaccine [14].
Although the mechanism of protection induced by the candidate is unknown, it is speculated that it may be related to the antibodies produced after vaccination, which may activate cellular mechanisms that directly affect the parasite. Moreover, the current vaccine candidate was emulsified with FCA, so it is likely that the protection was achieved through an effective stimulation of TLR2, which is important for the production of inflammatory cytokines and interferon stimulated genes.
In conclusion, F. gigantica releases a glcosylated protein that could be successfully utilized in eliciting protective cellular and humoral responses against rabbit fasciolosis resulted in significant reduction in worm burden. The candidate deserves further investigations to evaluate different vaccination protocols, other adjuvants and protective effect in other hosts.