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Protection of cattle against a natural infection of Fasciola hepatica by vaccination with recombinant cathepsin L1 (rFhCL1)
Vaccine 28 (2010) 5551–5557
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Protection of cattle against a natural infection of Fasciola hepatica by vaccination with recombinant cathepsin L1 (rFhCL1) O. Golden a,∗ , R.J. Flynn a,1 , C. Read a , M. Sekiya a , S.M. Donnelly b , C. Stack b,2 , J.P. Dalton b,3 , G. Mulcahy a a
School of Agriculture, Food Science and Veterinary Medicine, UCD, Belfield, Dublin 4, Ireland Institute for the Biotechnology of Infectious Diseases, University of Technology Sydney, Building 4, Corner of Thomas and Harris Street, Ultimo, Sydney, New South Wales 2007, Australia b
a r t i c l e
i n f o
Article history: Received 19 February 2010 Received in revised form 4 June 2010 Accepted 10 June 2010 Available online 25 June 2010 Keywords: Fasciola hepatica Vaccine Recombinant Cattle
a b s t r a c t The liver fluke, Fasciola hepatica causes liver fluke disease, or fasciolosis, in ruminants such as cattle and sheep. An effective vaccine against the helminth parasite is essential to reduce our reliance on anthelmintics, particularly in light of frequent reports of resistance to some frontline drugs. In our study, Friesian cattle (13 per group) were vaccinated with recombinant F. hepatica cathepsin L1 protease (rFhCL1) formulated in mineral-oil based adjuvants, MontanideTM ISA 70VG and ISA 206VG. Following vaccination the animals were exposed to fluke-contaminated pastures for 13 weeks. At slaughter, there was a significant reduction in fluke burden of 48.2% in the cattle in both vaccinated groups, relative to the control non-vaccinated group, at p ≤ 0.05. All vaccinated animals showed a sharp rise in total IgG levels to rFhCL1 post-vaccination which was maintained over the course of the 13-week challenge infection and was significantly higher than levels reached in the control group. Arginase levels in the macrophages of vaccinated cattle were significantly lower than those of the control cattle, indicating that the parasiteinduced alternative-activation of the macrophages was altered by vaccination. The data demonstrate the potential for recombinant FhCL1 vaccine in controlling fasciolosis in cattle under field conditions. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction The helminth parasite Fasciola hepatica is the causative agent of fasciolosis, a global disease of a wide range of mammals including ruminants, particularly sheep and cattle [1]. It is responsible for significant production losses estimated at over US$2000 million per year to the world agricultural community [2]. Infection is acquired by the ingestion of infective metacercariae that lie dormant in cysts on vegetation. The parasites excyst in the intestine, penetrate the intestinal wall and migrate across the peritoneal cavity to the liver. They tunnel through the liver parenchyma for 8–12 weeks, and then move to the bile ducts where they mature and commence egg production. The eggs are passed into the intestine with the bile juices and then out on to pasture in the faeces [3].
∗ Corresponding author: Tel.: +353 1 716 6135; fax: +353 1 716 6185. E-mail address: [email protected] (O. Golden). 1 Present address: School of Veterinary Medicine and Science, The University of Nottingham, Sutton Bonnington LE12 5RD, UK. 2 Present address: University of Western Sydney, Building 21, Campbelltown, Campus, New South Wales 2007, Australia. 3 Present address: Institute of Parasitology, McGill University, Sainte Anne de Bellevue, Quebec H9X 3V9, Canada. 0264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2010.06.039
F. hepatica can survive for long periods in susceptible species such as sheep [4]. Cattle exhibit some resistance to liver fluke infection, but they are still unable to produce a sufficiently strong immune response to resist all parasites and they remain susceptible to re-infection [5]. Chronic disease is associated with a Type 2 helper response (Th2) and a concurrent down-regulation of the Type 1 helper response (Th1) which compromises the host’s ability to mount an appropriate response against bacterial pathogens [5–7]. It has also been demonstrated that the parasite alternatively activates ruminant macrophages, cells that preferentially metabolise l-arginine with arginase instead of inducible nitric oxide synthase [8]. Alternatively activated macrophages (AAMФ) play an important role in suppressing Th1 responses [9] and in fibrosis and tissue repair [10]. These functions are important for minimising damaging inflammatory responses and for repair of liver parenchymal damage caused by fluke migration. Additionally in a murine model of nematode infection AAM were found to be protective [11].
An effective vaccine against fasciolosis is important for the future control or prevention of the disease. Triclabendazole, the drug of choice for treatment of fasciolosis, is effective at killing early immature and adult liver fluke. However resistance to triclabendazole has been reported in domestic animals in Australia
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[12] and Europe [13,14]. In addition, constant consumer pressure to decrease drug residues in the food chain makes anthelmintics a less desirable option for control of fasciolosis in domestic animals. A number of proteins from F. hepatica have shown potential as vaccine candidates [15] including cathepsin L1 (FhCL1). FhCL1 is a cysteine protease that is stored in its inactive form in secretory vesicles of the gastrodermal epithelial cells before secretion in large quantities by the parasite [16,17]. When the pro-enzyme is released and activated, the parasite uses it for a variety of essential functions. For example, it allows the liver fluke to acquire nutrients by breaking down proteins to peptides, facilitating absorption [18] and degrades interstitial matrix proteins, enabling the parasite to migrate from the intestine and through the liver [19]. Furthermore, FhCL1 is involved in the fluke’s immuneevasion mechanisms as it appears to be capable of cleaving host immunoglobulins [20]. It is also active in the immunomodulation process that occurs in fasciolosis as it has been shown to modulate macrophage activity and suppress Th1 responses in mice [21,22]. Previous vaccine trials using native FhCL1 have shown that this molecule can induce reductions in fluke burdens of 55–72%, following experimental challenge in cattle and sheep [23–26]. In addition to reducing fluke burden, fewer flukes developed to maturity in vaccinated animals than in controls. A marked reduction in egg production was also seen in these trials (55–98%) and the eggs that were produced showed a lower viability on hatching assays [23,26]. Recombinant FhCL1 was expressed in Pichia pastoris and exhibits similar biochemical properties to the native enzyme [17]. An inactive mutant (rFhCL1) was prepared by a single amino acid replacement that involved substituting the active site cysteine with a glycine. In the present study, this highly stable recombinant molecule was formulated in commercially acceptable adjuvants and used to vaccinate cattle before exposing them to liver fluke-infested pastures. Our results demonstrate the potential of rFhCL1 as a first-generation vaccine for bovine fasciolosis under natural field conditions.
2. Materials and methods 2.1. Experimental animals and vaccination protocol Thirty-nine male castrated Holstein–Friesian calves between 3 and 8 months of age at the time of vaccination were used in the study. Serological and faecal screening was carried out to rule out previous exposure to F. hepatica. They were kept under normal husbandry conditions on fluke-free pasture at University College Dublin (UCD), Lyons Research Farm during the vaccination period. Experimental procedures were carried out under licence from the Department of Health and Children and after review by the UCD Animal Ethics Committee. The animals were vaccinated twice, 3 weeks apart. The vaccine was prepared on the day of vaccination. MontanideTM ISA 70VG and ISA 206VG adjuvants were provided as a gift by SEPPIC. They are, respectively, adjuvants dedicated to produce water-in-oil (W/O) and water in oil in water (W/O/W) emulsions which contain mannide oleate and mineral oil that induce strong Th1 and Th2 responses in bovine models [27,28] For each animal 200 g of rFhCL1 was added to 2 ml of MontanideTM adjuvant and vortexed for 30 s. This was administered subcutaneously on the dorsal neck region. The animals were divided into three groups as outlined in Table 1. Three weeks after the second vaccine the cattle were moved to Teagasc Research Centre in Athenry (Galway, Ireland) and allowed to graze on fluke-contaminated pastures. The presence of metacercariae on the pasture had been verified by local abattoir data and by previous
field trials. Concentrate supplements were provided throughout the trial. 2.2. Assessment of protection All cattle were killed after 13 weeks of pasture exposure to fluke. The livers were removed and the number of fluke present in the bile ducts and parenchyma of each animal was estimated as previously described [29]. 2.3. Antibody responses Blood samples were taken pre-vaccination, 2 weeks post 1st vaccine, at start of pasture exposure and at weeks 4, 8 10 and 12 of pasture exposure (wpe). The samples were centrifuged at 1900 × g for 5 min and the serum removed. The sera samples were stored at −20 ◦ C. ELISA was used to measure parasite-specific antibody levels in sera. rFhCL1-specific Immunoglobulin G (IgG), IgG1 and IgG2 isotypes were measured. The method used was based on that described by Clery et al. [5]. Briefly, 96-well plates were coated with 1 g/well of rFhCL1 overnight. 0.05% PBS–Tween was used as blocking buffer and 1% bovine serum albumin in phosphate buffered saline (1% BSA–PBS) was used as blocking and dilution buffer. Serum dilutions (threefold starting at 1/200) were added in duplicate to the plate (100 l per well) and incubated for 30 min at 37 ◦ C. Bound antibody was detected by addition of rabbit anti-bovine IgG-peroxidase conjugated antibody (Sigma A7414), diluted 1/2000, followed by 3,3 ,5,5 -tetramethylbenzidine (TMB-Sigma T4319). For measurement of IgG1 and IgG2, mouse anti-bovine IgG1 or IgG2 (Cedi-Diagnostics, 7500820/7500830) was added instead of anti-bovine IgG. After 30 min incubation at 37 ◦ C, rabbit anti-mouse IgG-HRP (Dako P0260) was added and incubated at 37 ◦ C, for 30 min. Bound antibody was then detected as described above. Absorbances were read at 450 nm in an Expert 96 Microplate reader. Antibody titres were calculated as the reciprocal of that dilution of serum falling midway on the linear portion of the OD curve of a range of positive control sera. 2.4. Avidity assays Antibody avidity, defined as the binding strength of an antibody with an antigen, was measured for rFhCL1-specific IgG at 12 wpe. Avidity was estimated using the thiocyanate elution method, described by Mulcahy et al. [24]. Briefly, replicates of each serum sample, diluted to antibody titre level were added to the plate. After incubation and washing, various levels of potassium thiocyanate (0, 1, 2, 3, 4 and 5 Molar KSCN – Sigma 207799) were added to the plate for 10 min at room temperature. The plates were washed and bound antibody was detected as described above, using rabbit anti-bovine conjugate and TMB. Absorbances were read at 450 nm. Avidity was expressed as [KSCN]50 , which is the molarity of KSCN required to reduce the OD of a serum diluted to titre by 50%.
Table 1 Experimental design: 39 male, castrated friesian cattle were divided into three groups (n = 13). Group 1 received no treatment. Groups 2 and 3 were vaccinated twice with rFhCL1 formulated in a montanide adjuvant, either ISA 70VG or ISA 206VG. Group
Treatment
Control ISA 70VG ISA 206VG
Nil 200 g rFhCL1 + 2 ml montanide ISA 70VG 200 g rFhCL1 + 2 ml montanide ISA 206VG
O. Golden et al. / Vaccine 28 (2010) 5551–5557
2.5. Liver enzyme assays Serum gamma glutamyl transferase (GGT) and glutamate dehydrogenase (GLDH) levels were measured using a Randox imola Clinical Chemistry Analyzer. Liver enzymes were measured at 3, 4, 8 10 and 12 wpe.
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Table 2 Fluke burdens (mean ± SE and range) of vaccinated and control cattle at 13 weeks after commencement of natural challenge. Group
Mean fluke burden ± SE
Range
% Protection
Control ISA 70VG ISA 206VG
16.43 ± 5.11 8.67 ± 2.89 8.33 ± 4.43
4–32 2–16 1–26
– 47.2 49.2
2.6. Macrophage isolation Blood was collected into heparinised tubes for macrophage isolation at three time points – at start of pasture exposure, and at 4 and 12 wpe. Macrophages were isolated using a MACS Separator magnet, as described by Flynn et al. [30]. Briefly, peripheral blood mononuclear cells (PBMC) were obtained from heparinised blood using Histopaque (Sigma 1077). Cells were incubated with anti-human CD14 MACS microbeads (Miltenyi 130-050-201) and monocytes were collected by positive isolation over an LS separation column (Miltenyi 130-042-401) inserted into a MACS magnet (Miltenyi Biotec 130-042-302). The monocytes were counted using a haemocytometer and resuspended at 1 × 106 cells per ml. This cell solution was added to a sterile 96-well plate in two sets of triplicate (100 l per well). One set was left unstimulated. Sterile, endotoxinfree liver fluke homogenate (LFH) was added at a concentration of 1 g per well to the other set. The plates were incubated at 37 ◦ C, 5% CO2 for 48 h. The supernatant was removed and 1% Triton X100 (Sigma T8787) was added to the cells (50 l per well). Both the supernatant and the cells were stored at −20 ◦ C. 2.7. Liver fluke homogenate preparation Liver fluke homogenate (LFH) was prepared using liver fluke collected from infected livers as previously described by Smith et al. [31]. Briefly, fluke were freeze-dried, ground up and homogenized using a bead beater. The homogenised fluke was centrifuged and the supernatant was removed and filter sterilized. The protein content was checked using a BCA assay and endotoxins were removed as described below.
density values were compared to a standard curve and results were expressed as Nitrite M. 2.11. Arginase assays Levels of arginase in macrophage lysates were measured using a protocol described by Corraliza et al. [33]. Briefly, 50 l of cell lysate was added to 50 l of 10 mM manganese chloride (Sigma M-3634) in Tris–HCl, pH 7.5. This was incubated for 10 min at 55 ◦ C to allow enzyme activation. 25 l of activated lysate was added to 25 l of 0.5 M l-arginine substrate, pH 9.7 (Sigma A8094), and incubated for 60 min at 37 ◦ C. The reaction was stopped with 400 l of acid stop solution, containing H2 SO4 , H3 PO4 and H2 O in a ratio of 1:3:7. Colour was developed by the addition of 9% isonitrosopriophenone (Sigma I3502), dissolved in 100% ethanol, and heated to 103 ◦ C for 45 min. The samples were centrifuged at 9600 × g for 1 min to remove any cloudy material and plated out on a 96-well plate. The absorbance was read at 570 nm. Mean optical density was compared to a urea standard curve and the results expressed as mU of arginase activity per 1 × 106 cells, where 1 U of enzyme activity is equal to the amount of urea produced in the reaction. 2.12. Statistical analysis Differences between groups were tested using the Mann– Whitney U test. A one- or two-tailed test was used as appropriate. A p value of <0.05 was considered statistically significant.
2.8. Bicinchoninic acid (BCA) assay
3. Results
The protein content of the LFH was measured using a BCA assay (Pierce 23225), according to manufacturer’s instructions.
3.1. Assessment of protection
2.9. Endotoxin removal Endotoxin was removed from the LFH using Triton X-114 phase separation, described by Aida and Pabst [32]. Briefly, the concentration of the LFH was adjusted to 1 mg/ml in Dulbecco’s medium (Biosciences Ltd. 14190-094). This solution was vortexed with Triton X-114 and incubated on ice for 5 min. After a further incubation at 37 ◦ C for 5 min, the sample was centrifuged at 5000 × g for 7 s at 37 ◦ C. The upper phase of the solution, containing endotoxin-free protein was collected and the above process was repeated twice. LFH preparations were checked for endotoxin contamination using the Cambrex QCL-1000 Limulus Amebocyte Lysate assay (Cambrex 50-647U), according to manufacturer’s instructions. Results were expressed as EU/ml. Endotoxin levels were found to be below the minimum reading on the standard curve used in the Cambrex kit.
2.10. Nitric oxide assays Nitric oxide levels in the macrophage supernatant were measured in duplicate using the Griess Reagent system (Promega G2930), according to manufacturer’s instructions. Mean optical
The fluke burdens in each group were compared to determine the level of protection achieved by vaccinating with rFhCL1. The results, presented in Table 2, show that the fluke burdens in the ISA 70VG and ISA 206VG vaccine groups were 47.2 and 49.2% lower than the control non-vaccinated group; however there was no significant difference between individual vaccinated groups and the control group. However if the animals from the two vaccinated group are considered as one group, their fluke burdens were significantly lower that the control non-vaccinated group (p ≤ 0.05). The overall protection achieved was 48.2%. All flukes recovered were found in the liver parenchyma tissues and no parasites were found in the bile ducts. 3.2. Total and isotypic antibody responses The titres of total IgG reactive to rFhCL1 were determined in each experimental group (Fig. 1). rFhCL1-specific IgG in the sera of the animals in the control group was detected at 8 wpe (Fig. 1a). The titres of antibodies in the vaccinated groups were high at start of pasture exposure. This was maintained over the course of the trial but decreased approaching 8 wpe. However the titres in these animals increased again after 8 wpe, which coincided with the commencement of antibody production in the control group, and was therefore regarded as a boosting by the challenge infection.
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Fig. 2. Mean (±SEM) avidity values for vaccinated and control animals at 12 wpe. Avidity is expressed as [KSCN]50 , the molarity of KSCN required to reduce by 50% the OD of individual sera that has been diluted to titre. Both vaccinated groups showed significantly higher avidity compared to control animals (n = 13), denoted by *p ≤ 0.001. ISA 70VG cattle (n = 13) had higher mean avidity values than ISA 206VG (n = 13), but this was not statistically significant.
cantly greater than those observed in the control group at 12 wpe (p ≤ 0.001). rFhCL1-specific IgG1 titres were significantly higher in the ISA 70VG group compared to the ISA 206VG group at 12 wpe (p ≤ 0.05) (Fig. 1b). There were no detectable rFhCL1-specific IgG2 titres in the sera of the control animals, as we have reported previously [5,24,26]. rFhCL1-specific IgG2 titres were observed in the sera of both vaccinated groups and were higher in the ISA 70VG group compared to the ISA 206VG group, with a significant difference between the two groups at 12 wpe (p ≤ 0.05), (Fig. 1c). 3.3. Avidity assays The mean [KSCN]50 values of serum rFhCL1-specific IgG were significantly higher in both vaccinated groups compared to the control animals at 12 wpe (p≤0.001) (Fig. 2). The ISA 70VG group antibodies appeared to have a higher mean [KSCN]50 value than the ISA 206VG group, but on analysis this was not found to be significantly different.
Fig. 1. Mean (±SEM) rFhCL1-specific IgG, IgG1 and IgG2 titres in vaccinated and control animals (a, b and c, respectively). Antibody titres are expressed as Log10 . All vaccinated animals produced significantly higher titres of rFhCL1-specific IgG and IgG1 compared to controls at 12 wpe, denoted by *p ≤ 0.001. There was no rFhCL1-specific IgG2 production by the control cattle (n = 13). Cattle vaccinated with rFhCL1 + ISA 70VG (group ISA 70VG) (n = 13) showed significantly higher titres of rFhCL1-specific IgG, IgG1 and IgG2 at 12 wpe, compared to those vaccinated with rFhCL1 and ISA 206VG (group ISA206VG) (n = 13), denoted by **p ≤ 0.05. PreVacc, pre-vaccination; Exp, start of pasture exposure; Post 1st Vacc, post 1st vaccination (2 weeks post 1st vaccination); wpe, weeks of pasture exposure.
Serum rFhCL1-specific IgG titres were significantly higher in both of the vaccinated groups compared to the controls at 10 and 12 wpe (p ≤ 0.05 and p ≤ 0.001, respectively). In addition, serum levels of rFhCL1-specific IgG in animals given the vaccine formulated in ISA 70VG were significantly higher than those of the ISA 206VG group at 12 wpe (p ≤ 0.05). rFhCL1-specific IgG1 was detected in animals of the control group at 4 wpe. In contrast, rFhCL1-specific IgG1 titres were detected in the vaccinated cattle rapidly after vaccination and increased over the course of the trial. These titres were signifi-
3.4. Liver enzyme assays Serum levels of glutamate dehydrogenase (GLDH) increased above reference range levels by week three post-exposure in all animals. The levels remained high over the course of the challenge, but no significant differences between the control and vaccinated groups were observed. Although, gamma glutamyl transferase (GGT) serum levels increased slowly during the period of exposure these remained within the reference range for the course of the trial in all cattle and no significant difference was observed between the vaccinated and control groups. (Fig. 3) 3.5. Macrophage assays Nitric oxide levels were measured in the culture medium obtained from LFH-stimulated macrophages. These remained low in all animals throughout the trial (data not shown). However, when arginase activity was measured in the lysate of the macrophages of the same cultures, an increase was observed over the course of the trial. Most relevantly, the arginase activity levels in the macrophages taken from both vaccinated groups were
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Fig. 3. Mean (±SEM) serum levels of GLDH and GGT in vaccinated (n = 13/group) and control cattle (n = 13) (a and b, respectively). Values expressed as iu/L. Levels of GLDH were above the reference range (0–12 iu/L) by 3 wpe in all animals, but showed very little increase over the rest of the challenge period. Serum levels of GGT remained within the reference range (0–20 iu/L) in all animals throughout the challenge period, although levels did increase over the course of the trial. There were no significant differences between groups for serum levels of GLDH or GGT.
Fig. 4. Mean (±SEM) arginase activity levels in macrophage lysate of macrophages isolated from heparinised blood of vaccinated (n = 13/group) and control cattle (n = 13) at time of challenge, 4 and 12 wpe. Arginase activity expressed as mU of arginase activity per 1 × 106 cells. In macrophages isolated from the control group the levels were significantly higher at 12 wpe when compared to time of exposure, denoted by † p ≤ 0.05. There was an increase in arginase activity in the macrophages of all groups over the exposure period. However, both vaccinated groups had significantly lower levels of arginase activity compared to controls at 12 wpe, denoted by **p ≤ 0.05.
significantly lower (p ≤ 0.05) than those in macrophages of the control group at 12 wpe (Fig. 4).
4. Discussion Previous vaccine trials using native FhCL1 have shown reductions in fluke burdens in the range of 55–72% compared to
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unvaccinated controls, and a marked reduction in parasite fecundity and/or egg viability [23–26]. The next step towards the production of a commercially viable liver fluke vaccine is assessment of the efficacy of the recombinant forms of FhCL1 and finding an effective adjuvant which can be licensed for commercial use [17]. The majority of previous vaccine trials used experimental infection. While this has the advantage of reducing variability in infection levels across animals it does not replicate conditions found in the field. Therefore, in the present trial we chose to assess the efficacy of our vaccine against a naturally acquired infection, which allowed us to ascertain the efficacy of the vaccine at farm level. Moreover, we formulated the vaccines in the mineraloil based MontanideTM adjuvants ISA 70VG and ISA 206VG since these are acceptable for commercial use. The immune response induced by these vaccines were characterised and compared to those induced in non-vaccinated animals exposed to infection. Previous vaccine trials with FhCL1 have shown that the vaccine produces an immune response which has elements of both Th1 and Th2 phenotypes indicated by the production of both IgG1 and IgG2 antibody isotypes [24]. Infected, unvaccinated animals were shown to produce only IgG1 antibodies, which is indicative of a nonprotective Th2 response. Fluke-specific IgG titre in infected animals was positively correlated with fluke burden, which illustrates that the immune response to liver fluke in the field is not protective. In contrast, Mulcahy et al. [24] showed that fluke-specific IgG2 titre and avidity correlated with vaccine-induced protection. Thus, a high fluke-specific IgG2 titre and avidity in vaccinated animals correlated with a low fluke burden. The two vaccine groups in this trial, ISA 70VG and ISA 206VG reduced the fluke burden at similar levels (47.2 and 49.2%, respectively) but these groups did not show significant differences when compared to controls. However, when the animals in both vaccinated group were considered as a single group and compared to non-vaccinated animals, a significant reduction in fluke burden was observed. The fluke burden in the animals kept on the pasture was not high, control animals had a mean burden of 16 fluke per liver, and adult parasites were not seen in the bile ducts (and therefore no eggs appeared in the faeces). It is possible that with a longer challenge period, more significant differences between the vaccinated groups and the controls may have become apparent. The humoral immune response induced by the vaccine supported data observed in our previous trials [23,24]. The rFhCL1specific IgG titres in the sera of the infected control animals were lower than those of the vaccinated animals. When isotyping ELISA were carried out, this total rFhCL1-specific IgG titre was seen to consist of rFhCL1-specific IgG1, with no rFhCL1-specific IgG2 production, indicating the polarised Th2 response. The vaccinated animals produced both rFhCL1-specific IgG1 and IgG2 isotypes, indicating a mixed Th1/Th2 response. The vaccinated cattle produced higher total rFhCL1-specific IgG and IgG1 titres than the control group. Correlations between fluke burden and antibody responses were studied but there were no significant correlations. This may have been due to the relatively low infections levels in the trial animals. Antibody avidity is the binding strength of the antibody with an antigen. As an immune response matures, the avidity of secreted antibodies increases due to an improved ‘fit’, which occurs from repeated selection for antibody populations by the antigen [34]. The avidity of total rFhCL1-specific IgG at 12 wpe was measured and the antibody produced by the vaccinated animals was found to have a significantly higher avidity for rFhCL1 than the control animals. This would be expected, as the antibody response produced by vaccination has had time to mature by the time of exposure, so the antibodies in the vaccinated animals would have a higher avidity than those of the control animals.
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Serum GLDH levels rose above the reference range by 3 wpe. Raised serum GLDH is caused by parenchymal damage [35], so immature fluke entering the liver would account for the increase. However, due to the low infection in the animals after this point there was very little increase over the challenge period. Increased GGT is associated with bile duct damage [36], so serum levels of this enzyme increase when the fluke mature and enter the bile ducts. Therefore, it was not surprising that GGT levels did not rise above the reference values at any point in the trial since no adult flukes were observed. Nitric oxide is produced by classically activated macrophages (CAMФ) [37], which are generated during Th1 responses [38]. We found that nitric oxide levels in culture medium obtained from LFH-stimulated macrophages, taken from control and vaccinated animals, was consistently low throughout the trial indicating a lack of classical activation (data not shown). By contrast, the arginase activity levels measured in the macrophage lysates showed significant differences between the vaccinated animals and the controls. The levels of arginase activity in the macrophage lysate of the two vaccinated groups at 12 wpe were significantly lower than those of the control animals. Since arginase expression is a marker of alternative-activation in macrophages [39], it appears that the AAMФ response in the vaccinated cattle was less pronounced than that of the control animals. AAMФ are instrumental in suppressing Th1 responses and promoting Th2 responses [9], so the depression in the levels of AAMФ in vaccinated animals correlates with the mixed Th1/Th2 phenotype that was observed in the humoral response of the vaccinated groups. In contrast with murine models [11] this might suggest that AAM are not protective during F. hepatica infection. The antibody and macrophage data obtained in this trial show that the vaccine has produced a qualitatively and quantitatively different innate and adaptive response to infection. The question still remains as to what mechanisms are involved in reducing the fluke burden in the vaccinated animals. Cathepsin Ls are utilised by the parasite at all stages of its life cycle in the mammalian host [20]. Although the cysteine protease profile differs over this period, a vaccine that induces antibodies that successfully inactivate FhCL1 could potentially have numerous effects on the development of the fluke. For example, the ability of the newly excysted juveniles (NEJ) to penetrate the intestinal wall and reach the peritoneal cavity would be impaired, reducing the number of parasites that can reach the liver. Also, if these antibodies were to bind to the secretory vesicles which contain FhCL1 within the parasite gastrodermal epithelial cells, they could physically block enzyme secretion, resulting in significantly lower levels of available cathepsin for feeding and migration within the host. The fluke appears to be most vulnerable to the immune system of the host when crossing the peritoneal cavity [40]. Studies in rats, which show a level of resistance to F. hepatica that is comparable to cattle, have shown evidence of an antibody dependant cell cytotoxicity (ADCC) occurring. This mechanism appears to be dependent on nitrite production by peritoneal cell populations, which consist mainly of monocyte/macrophage cells [41]. Neutralizing or reducing secreted volumes of FhCL1 at this point could render the fluke more susceptible to immune attack. Firstly, cathepsins are capable of cleaving host immunoglobulins at the hinge region, thereby preventing ADCC [20]. Inhibition of FhCL1 activity would, therefore, facilitate effective ADCC. Secondly, the Th1 suppression caused by FhCL1 would also be blocked leading to a greater production of nitrite, promoting more efficient ADCC. FhCL1 activity participates in the immunomodulation of host responses that occurs during animal fasciolosis. O’Neill et al. [21] demonstrated that the protease could suppress Th1 responses in mice. The mixed immune response observed in the vaccinated animals in our trial may have been due to the reduction in activity of
FhCL1. This is reflected in the production of both IgG1 and IgG2 titres and also the lower levels of alternative-activation seen in the re-stimulated macrophages of the vaccinated cattle. Long term induction and activity of AAMФ is dependent on Th2 cells [42]. At the hepatic stage of the life cycle FhCL1 is vital to F. hepatica, both for migration and acquisition of nutrients. If FhCL1 is sufficiently inactivated to prevent the parasite from feeding, this would clearly be an efficient method of killing the maturing fluke. If migration of the fluke is inhibited it could affect not only the progression of the life cycle, but also its ability to evade immune attack. Meeusen et al. [43] noted during infection in sheep, that although lymphocytes and other immune effector cells infiltrated migratory tunnels in the liver parenchyma, the parasites themselves never had these cells around them. It was suggested that the fluke were capable of migrating quickly enough to ‘leave behind’ the immune response. If this is the case, any impairment of the fluke’s ability to migrate will increase its susceptibility to immune attack. Effective control of F. hepatica is of increasing importance because fasciolosis is increasing in both frequency and distribution [44,45]. The development of resistance to triclabendazole has shown anthelmintics to be an unsustainable means of controlling the disease. An effective vaccine represents a cost-efficient, long term, environmentally–friendly alternative. However, the vaccine must also be formulated and delivered in a suitable adjuvant. The montanide adjuvants used in this study likely played an important part in promoting the mixed protective Th1/Th2 response observed in the vaccinated cattle. They have been shown to induce both Th1 and Th2 phenotypes in cattle [27,28]. The most important factor influencing the outcome of infection is the number of metacercariae ingested [4], which will determine the number of fluke migrating through the hepatic parenchyma. In this trial we achieved an overall reduction of 48.2% in fluke burden with vaccination. Hope Cawdery et al. [46] demonstrated that fluke burdens as low as 54 could impair weight gain in cattle. Dargie [47] demonstrated that only 3% of cattle in the UK had burdens exceeding 50 fluke, suggesting that the level of protection achieved with our vaccine would be commercially viable. Although we were unable to demonstrate it in this trial, previous vaccine trials with FhCL1 have shown a reduction in egg production and viability [23,26]. This effect on transmission could lead to a cumulative benefit of vaccination over a number of grazing seasons. In addition to the reduced numbers of fluke in the livers of the animals on pasture, there would be a reduction in pasture contamination resulting in lower infection levels the following year. These lower transmission rates may also have important implications for the spread of F. hepatica to humans in areas where this zoonosis is of importance, e.g. South America, Egypt, Iran and South-East Asia [15]. rFhCL1 combined with a MontanideTM adjuvant shows excellent potential as a successful fluke vaccine in cattle. Further trials, with a longer challenge period and higher fluke burdens will establish its efficacy more definitively and will also allow us to determine the effect of this vaccine on parasite fecundity and egg viability. Acknowledgments This work was funded by the EU Commission under Framework 6, Project Ref. FOOD-CT-2005-02305-DELIVER. The authors are grateful to Mr. Eddie Jordan and the staff of the UCD Lyons Estate Research Farm and to Barbara Good, J.P. Hanrahan and the staff of Teagasc Research Centre Athenry, for their care of the experimental animals. References [1] Mas-Coma MS, Esteban JG, Bargues MD. Epidemiology of human fascioliasis: a review and proposed new classification. Bulletin of the World Health Organization 1999;77(4):340–6.
O. Golden et al. / Vaccine 28 (2010) 5551–5557 [2] Spithill TW, Smooker PM, Sexton JL, Bozas E, Morrison CA, Creaney J, et al. Development of vaccines against Fasciola hepatica. In: Dalton JP, editor. Fasciolosis. CABI Publishing; 1999. p. 377–410. [3] Andrews SJ. The life cycle of Fasciola hepatica. In: Dalton JP, editor. Fasciolosis. 1st ed. Dublin: CABI; 1999. p. 1–31. [4] Boray JC. Experimental fascioliasis in Australia. Advances in Parasitology 1969;7:95–210. [5] Clery D, Torgerson P, Mulcahy G. Immune responses of chronically infected adult cattle to Fasciola hepatica. Veterinary Parasitology 1996;62(1/2):71–82. [6] O’Neill SM, Brady MT, Callanan JJ, Mulcahy G, Joyce P, Mills KHG, et al. Fasciola hepatica infection downregulates Th1 responses in mice. Parasite Immunology 2000;22(3):147–55. [7] Brady MT, O’Neill SM, Dalton JP, Mills KHG. Fasciola hepatica suppresses a protective Th1 response against Bordetella pertussis. Infection and Immunity 1999;67(10):5372–8. [8] Flynn RJ, Irwin JA, Olivier M, Sekiya M, Dalton JP, Mulcahy G. Alternative activation of ruminant macrophages by Fasciola hepatica. Veterinary Immunology and Immunopathology 2007;120(1/2):31–40. [9] Herbert DR, Holscher C, Mohr M, Arendse B, Schwegmann A, Radwanska M, et al. Alternative macrophage activation is essential for survival during schistosomiasis and downmodulates T helper 1 responses and immunopathology. Immunity 2004;20(5):623–35. [10] Sunderkotter C, Steinbrink K, Goebeler M, Bhardwaj R, Sorg C. Macrophages and angiogenesis. Journal of Leucocyte Biology 1994;55(3):410–22. [11] Anthony RM, Urban Jr JF, Alem F, Hamed HA, Rozo CT, Boucher JL, et al. Memory Th2 cells induce alternatively-activated macrophages to mediate protection against nematode parasites. Nature Medicine 2006;12(8):955–60. [12] Overend DJ, Bowen FL. Resistance of Fasciola hepatica to triclabendazole. Australian Veterinary Journal 1995;72(7):275–6. [13] Moll L, Gaasenbeek CPH, Vellema P, Borgsteede FHM. Resistance of Fasciola hepatica against triclabendazole in cattle and sheep in The Netherlands. Veterinary Parasitology 2000;91(1–2):153–8. [14] Lane G. Anthelmintic resistance. The Veterinary Record 1998;143(8):232. [15] McManus DP, Dalton JP. Vaccines against the zoonotic trematodes Schistosoma japonicum, Fasciola hepatica and Fasciola gigantica. Parasitology 2006;133:S43–61. [16] Dalton JP, Heffernan M. Thiol proteases released in vitro by Fasciola hepatica. Molecular and Biochemical Parasitology 1989;35(2):161–6. [17] Collins PR, Stack CM, O’Neill SM, Doyle S, Ryan T, Brennan GP, et al. Cathepsin L1, the major protease involved in liver fluke Fasciola hepatica virulence: propeptide cleavage sites and autoactivation of the zymogen secreted from gastrodermal cells. Journal of Biological Chemistry 2004;279(17):17038–46. [18] Tort J, Brindley PJ, Knox D, Wolfe KH, Dalton JP. Proteinases and associated genes of parasitic helminths. Advances in Parasitology 1999;43:161–266. [19] Berasain P, Goni F, McGonigle S, Dowd A, Dalton JP, Frangione B, et al. Proteinases secreted by Fasciola hepatica degrade extracelullar matrix and basement membrane components. The Journal of Parasitology 1997;83(1):1–5. [20] Carmona C, Dowd AJ, Smith AM, Dalton JP. Cathepsin L proteinase secreted by Fasciola hepatica in vitro prevents antibody-mediated eosinophil attachment to newly excysted juveniles. Molecular and Biochemical Parasitology 1993;62(1):9–17. [21] O’Neill SM, Mills KHG, Dalton JP. Fasciola hepatica cathepsin L cysteine proteinase suppresses Bordetella pertussis-specific interferon-gamma production in vivo. Parasite Immunology 2001;23:541–7. [22] Donnelly S, O’Neill SM, Stack CM, Robinson MW, Turnbull L, Whitchurch C, et al. Helminth cysteine proteases inhibit TRIF-dependent activation of macrophages via degradation of TLR3. Journal of Biological Chemistry 2010;285(5):3383–92. [23] Dalton JP, McGonigle S, Rolph TP, Andrews SJ. Induction of protective immunity in cattle against infection with Fasciola hepatica by vaccination with cathepsin L proteinases and with hemoglobin. Infection and Immunity 1996;64(12):5066–74. [24] Mulcahy G, O’Connor F, McGonigle S, Dowd A, Clery DG, Andrews SJ, et al. Correlation of specific antibody titre and avidity with protection in cattle immunized against Fasciola hepatica. Vaccine 1998;16(9/10):932–9. [25] Piacenza L, Acosta D, Basmadjian I, Dalton JP, Carmona C. Vaccination with cathepsin L proteinases and with leucine aminopeptidase induces high levels of protection against fascioliasis in sheep. Infection and Immunity 1999;67(4):1954–61.
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Protection of cattle against a natural infection of Fasciola hepatica by vaccination with recombinant cathepsin L1 (rFhCL1) O. Golden a,∗ , R.J. Flynn a,1 , C. Read a , M. Sekiya a , S.M. Donnelly b , C. Stack b,2 , J.P. Dalton b,3 , G. Mulcahy a a
School of Agriculture, Food Science and Veterinary Medicine, UCD, Belfield, Dublin 4, Ireland Institute for the Biotechnology of Infectious Diseases, University of Technology Sydney, Building 4, Corner of Thomas and Harris Street, Ultimo, Sydney, New South Wales 2007, Australia b
a r t i c l e
i n f o
Article history: Received 19 February 2010 Received in revised form 4 June 2010 Accepted 10 June 2010 Available online 25 June 2010 Keywords: Fasciola hepatica Vaccine Recombinant Cattle
a b s t r a c t The liver fluke, Fasciola hepatica causes liver fluke disease, or fasciolosis, in ruminants such as cattle and sheep. An effective vaccine against the helminth parasite is essential to reduce our reliance on anthelmintics, particularly in light of frequent reports of resistance to some frontline drugs. In our study, Friesian cattle (13 per group) were vaccinated with recombinant F. hepatica cathepsin L1 protease (rFhCL1) formulated in mineral-oil based adjuvants, MontanideTM ISA 70VG and ISA 206VG. Following vaccination the animals were exposed to fluke-contaminated pastures for 13 weeks. At slaughter, there was a significant reduction in fluke burden of 48.2% in the cattle in both vaccinated groups, relative to the control non-vaccinated group, at p ≤ 0.05. All vaccinated animals showed a sharp rise in total IgG levels to rFhCL1 post-vaccination which was maintained over the course of the 13-week challenge infection and was significantly higher than levels reached in the control group. Arginase levels in the macrophages of vaccinated cattle were significantly lower than those of the control cattle, indicating that the parasiteinduced alternative-activation of the macrophages was altered by vaccination. The data demonstrate the potential for recombinant FhCL1 vaccine in controlling fasciolosis in cattle under field conditions. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction The helminth parasite Fasciola hepatica is the causative agent of fasciolosis, a global disease of a wide range of mammals including ruminants, particularly sheep and cattle [1]. It is responsible for significant production losses estimated at over US$2000 million per year to the world agricultural community [2]. Infection is acquired by the ingestion of infective metacercariae that lie dormant in cysts on vegetation. The parasites excyst in the intestine, penetrate the intestinal wall and migrate across the peritoneal cavity to the liver. They tunnel through the liver parenchyma for 8–12 weeks, and then move to the bile ducts where they mature and commence egg production. The eggs are passed into the intestine with the bile juices and then out on to pasture in the faeces [3].
∗ Corresponding author: Tel.: +353 1 716 6135; fax: +353 1 716 6185. E-mail address: [email protected] (O. Golden). 1 Present address: School of Veterinary Medicine and Science, The University of Nottingham, Sutton Bonnington LE12 5RD, UK. 2 Present address: University of Western Sydney, Building 21, Campbelltown, Campus, New South Wales 2007, Australia. 3 Present address: Institute of Parasitology, McGill University, Sainte Anne de Bellevue, Quebec H9X 3V9, Canada. 0264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2010.06.039
F. hepatica can survive for long periods in susceptible species such as sheep [4]. Cattle exhibit some resistance to liver fluke infection, but they are still unable to produce a sufficiently strong immune response to resist all parasites and they remain susceptible to re-infection [5]. Chronic disease is associated with a Type 2 helper response (Th2) and a concurrent down-regulation of the Type 1 helper response (Th1) which compromises the host’s ability to mount an appropriate response against bacterial pathogens [5–7]. It has also been demonstrated that the parasite alternatively activates ruminant macrophages, cells that preferentially metabolise l-arginine with arginase instead of inducible nitric oxide synthase [8]. Alternatively activated macrophages (AAMФ) play an important role in suppressing Th1 responses [9] and in fibrosis and tissue repair [10]. These functions are important for minimising damaging inflammatory responses and for repair of liver parenchymal damage caused by fluke migration. Additionally in a murine model of nematode infection AAM were found to be protective [11].
An effective vaccine against fasciolosis is important for the future control or prevention of the disease. Triclabendazole, the drug of choice for treatment of fasciolosis, is effective at killing early immature and adult liver fluke. However resistance to triclabendazole has been reported in domestic animals in Australia
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[12] and Europe [13,14]. In addition, constant consumer pressure to decrease drug residues in the food chain makes anthelmintics a less desirable option for control of fasciolosis in domestic animals. A number of proteins from F. hepatica have shown potential as vaccine candidates [15] including cathepsin L1 (FhCL1). FhCL1 is a cysteine protease that is stored in its inactive form in secretory vesicles of the gastrodermal epithelial cells before secretion in large quantities by the parasite [16,17]. When the pro-enzyme is released and activated, the parasite uses it for a variety of essential functions. For example, it allows the liver fluke to acquire nutrients by breaking down proteins to peptides, facilitating absorption [18] and degrades interstitial matrix proteins, enabling the parasite to migrate from the intestine and through the liver [19]. Furthermore, FhCL1 is involved in the fluke’s immuneevasion mechanisms as it appears to be capable of cleaving host immunoglobulins [20]. It is also active in the immunomodulation process that occurs in fasciolosis as it has been shown to modulate macrophage activity and suppress Th1 responses in mice [21,22]. Previous vaccine trials using native FhCL1 have shown that this molecule can induce reductions in fluke burdens of 55–72%, following experimental challenge in cattle and sheep [23–26]. In addition to reducing fluke burden, fewer flukes developed to maturity in vaccinated animals than in controls. A marked reduction in egg production was also seen in these trials (55–98%) and the eggs that were produced showed a lower viability on hatching assays [23,26]. Recombinant FhCL1 was expressed in Pichia pastoris and exhibits similar biochemical properties to the native enzyme [17]. An inactive mutant (rFhCL1) was prepared by a single amino acid replacement that involved substituting the active site cysteine with a glycine. In the present study, this highly stable recombinant molecule was formulated in commercially acceptable adjuvants and used to vaccinate cattle before exposing them to liver fluke-infested pastures. Our results demonstrate the potential of rFhCL1 as a first-generation vaccine for bovine fasciolosis under natural field conditions.
2. Materials and methods 2.1. Experimental animals and vaccination protocol Thirty-nine male castrated Holstein–Friesian calves between 3 and 8 months of age at the time of vaccination were used in the study. Serological and faecal screening was carried out to rule out previous exposure to F. hepatica. They were kept under normal husbandry conditions on fluke-free pasture at University College Dublin (UCD), Lyons Research Farm during the vaccination period. Experimental procedures were carried out under licence from the Department of Health and Children and after review by the UCD Animal Ethics Committee. The animals were vaccinated twice, 3 weeks apart. The vaccine was prepared on the day of vaccination. MontanideTM ISA 70VG and ISA 206VG adjuvants were provided as a gift by SEPPIC. They are, respectively, adjuvants dedicated to produce water-in-oil (W/O) and water in oil in water (W/O/W) emulsions which contain mannide oleate and mineral oil that induce strong Th1 and Th2 responses in bovine models [27,28] For each animal 200 g of rFhCL1 was added to 2 ml of MontanideTM adjuvant and vortexed for 30 s. This was administered subcutaneously on the dorsal neck region. The animals were divided into three groups as outlined in Table 1. Three weeks after the second vaccine the cattle were moved to Teagasc Research Centre in Athenry (Galway, Ireland) and allowed to graze on fluke-contaminated pastures. The presence of metacercariae on the pasture had been verified by local abattoir data and by previous
field trials. Concentrate supplements were provided throughout the trial. 2.2. Assessment of protection All cattle were killed after 13 weeks of pasture exposure to fluke. The livers were removed and the number of fluke present in the bile ducts and parenchyma of each animal was estimated as previously described [29]. 2.3. Antibody responses Blood samples were taken pre-vaccination, 2 weeks post 1st vaccine, at start of pasture exposure and at weeks 4, 8 10 and 12 of pasture exposure (wpe). The samples were centrifuged at 1900 × g for 5 min and the serum removed. The sera samples were stored at −20 ◦ C. ELISA was used to measure parasite-specific antibody levels in sera. rFhCL1-specific Immunoglobulin G (IgG), IgG1 and IgG2 isotypes were measured. The method used was based on that described by Clery et al. [5]. Briefly, 96-well plates were coated with 1 g/well of rFhCL1 overnight. 0.05% PBS–Tween was used as blocking buffer and 1% bovine serum albumin in phosphate buffered saline (1% BSA–PBS) was used as blocking and dilution buffer. Serum dilutions (threefold starting at 1/200) were added in duplicate to the plate (100 l per well) and incubated for 30 min at 37 ◦ C. Bound antibody was detected by addition of rabbit anti-bovine IgG-peroxidase conjugated antibody (Sigma A7414), diluted 1/2000, followed by 3,3 ,5,5 -tetramethylbenzidine (TMB-Sigma T4319). For measurement of IgG1 and IgG2, mouse anti-bovine IgG1 or IgG2 (Cedi-Diagnostics, 7500820/7500830) was added instead of anti-bovine IgG. After 30 min incubation at 37 ◦ C, rabbit anti-mouse IgG-HRP (Dako P0260) was added and incubated at 37 ◦ C, for 30 min. Bound antibody was then detected as described above. Absorbances were read at 450 nm in an Expert 96 Microplate reader. Antibody titres were calculated as the reciprocal of that dilution of serum falling midway on the linear portion of the OD curve of a range of positive control sera. 2.4. Avidity assays Antibody avidity, defined as the binding strength of an antibody with an antigen, was measured for rFhCL1-specific IgG at 12 wpe. Avidity was estimated using the thiocyanate elution method, described by Mulcahy et al. [24]. Briefly, replicates of each serum sample, diluted to antibody titre level were added to the plate. After incubation and washing, various levels of potassium thiocyanate (0, 1, 2, 3, 4 and 5 Molar KSCN – Sigma 207799) were added to the plate for 10 min at room temperature. The plates were washed and bound antibody was detected as described above, using rabbit anti-bovine conjugate and TMB. Absorbances were read at 450 nm. Avidity was expressed as [KSCN]50 , which is the molarity of KSCN required to reduce the OD of a serum diluted to titre by 50%.
Table 1 Experimental design: 39 male, castrated friesian cattle were divided into three groups (n = 13). Group 1 received no treatment. Groups 2 and 3 were vaccinated twice with rFhCL1 formulated in a montanide adjuvant, either ISA 70VG or ISA 206VG. Group
Treatment
Control ISA 70VG ISA 206VG
Nil 200 g rFhCL1 + 2 ml montanide ISA 70VG 200 g rFhCL1 + 2 ml montanide ISA 206VG
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2.5. Liver enzyme assays Serum gamma glutamyl transferase (GGT) and glutamate dehydrogenase (GLDH) levels were measured using a Randox imola Clinical Chemistry Analyzer. Liver enzymes were measured at 3, 4, 8 10 and 12 wpe.
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Table 2 Fluke burdens (mean ± SE and range) of vaccinated and control cattle at 13 weeks after commencement of natural challenge. Group
Mean fluke burden ± SE
Range
% Protection
Control ISA 70VG ISA 206VG
16.43 ± 5.11 8.67 ± 2.89 8.33 ± 4.43
4–32 2–16 1–26
– 47.2 49.2
2.6. Macrophage isolation Blood was collected into heparinised tubes for macrophage isolation at three time points – at start of pasture exposure, and at 4 and 12 wpe. Macrophages were isolated using a MACS Separator magnet, as described by Flynn et al. [30]. Briefly, peripheral blood mononuclear cells (PBMC) were obtained from heparinised blood using Histopaque (Sigma 1077). Cells were incubated with anti-human CD14 MACS microbeads (Miltenyi 130-050-201) and monocytes were collected by positive isolation over an LS separation column (Miltenyi 130-042-401) inserted into a MACS magnet (Miltenyi Biotec 130-042-302). The monocytes were counted using a haemocytometer and resuspended at 1 × 106 cells per ml. This cell solution was added to a sterile 96-well plate in two sets of triplicate (100 l per well). One set was left unstimulated. Sterile, endotoxinfree liver fluke homogenate (LFH) was added at a concentration of 1 g per well to the other set. The plates were incubated at 37 ◦ C, 5% CO2 for 48 h. The supernatant was removed and 1% Triton X100 (Sigma T8787) was added to the cells (50 l per well). Both the supernatant and the cells were stored at −20 ◦ C. 2.7. Liver fluke homogenate preparation Liver fluke homogenate (LFH) was prepared using liver fluke collected from infected livers as previously described by Smith et al. [31]. Briefly, fluke were freeze-dried, ground up and homogenized using a bead beater. The homogenised fluke was centrifuged and the supernatant was removed and filter sterilized. The protein content was checked using a BCA assay and endotoxins were removed as described below.
density values were compared to a standard curve and results were expressed as Nitrite M. 2.11. Arginase assays Levels of arginase in macrophage lysates were measured using a protocol described by Corraliza et al. [33]. Briefly, 50 l of cell lysate was added to 50 l of 10 mM manganese chloride (Sigma M-3634) in Tris–HCl, pH 7.5. This was incubated for 10 min at 55 ◦ C to allow enzyme activation. 25 l of activated lysate was added to 25 l of 0.5 M l-arginine substrate, pH 9.7 (Sigma A8094), and incubated for 60 min at 37 ◦ C. The reaction was stopped with 400 l of acid stop solution, containing H2 SO4 , H3 PO4 and H2 O in a ratio of 1:3:7. Colour was developed by the addition of 9% isonitrosopriophenone (Sigma I3502), dissolved in 100% ethanol, and heated to 103 ◦ C for 45 min. The samples were centrifuged at 9600 × g for 1 min to remove any cloudy material and plated out on a 96-well plate. The absorbance was read at 570 nm. Mean optical density was compared to a urea standard curve and the results expressed as mU of arginase activity per 1 × 106 cells, where 1 U of enzyme activity is equal to the amount of urea produced in the reaction. 2.12. Statistical analysis Differences between groups were tested using the Mann– Whitney U test. A one- or two-tailed test was used as appropriate. A p value of <0.05 was considered statistically significant.
2.8. Bicinchoninic acid (BCA) assay
3. Results
The protein content of the LFH was measured using a BCA assay (Pierce 23225), according to manufacturer’s instructions.
3.1. Assessment of protection
2.9. Endotoxin removal Endotoxin was removed from the LFH using Triton X-114 phase separation, described by Aida and Pabst [32]. Briefly, the concentration of the LFH was adjusted to 1 mg/ml in Dulbecco’s medium (Biosciences Ltd. 14190-094). This solution was vortexed with Triton X-114 and incubated on ice for 5 min. After a further incubation at 37 ◦ C for 5 min, the sample was centrifuged at 5000 × g for 7 s at 37 ◦ C. The upper phase of the solution, containing endotoxin-free protein was collected and the above process was repeated twice. LFH preparations were checked for endotoxin contamination using the Cambrex QCL-1000 Limulus Amebocyte Lysate assay (Cambrex 50-647U), according to manufacturer’s instructions. Results were expressed as EU/ml. Endotoxin levels were found to be below the minimum reading on the standard curve used in the Cambrex kit.
2.10. Nitric oxide assays Nitric oxide levels in the macrophage supernatant were measured in duplicate using the Griess Reagent system (Promega G2930), according to manufacturer’s instructions. Mean optical
The fluke burdens in each group were compared to determine the level of protection achieved by vaccinating with rFhCL1. The results, presented in Table 2, show that the fluke burdens in the ISA 70VG and ISA 206VG vaccine groups were 47.2 and 49.2% lower than the control non-vaccinated group; however there was no significant difference between individual vaccinated groups and the control group. However if the animals from the two vaccinated group are considered as one group, their fluke burdens were significantly lower that the control non-vaccinated group (p ≤ 0.05). The overall protection achieved was 48.2%. All flukes recovered were found in the liver parenchyma tissues and no parasites were found in the bile ducts. 3.2. Total and isotypic antibody responses The titres of total IgG reactive to rFhCL1 were determined in each experimental group (Fig. 1). rFhCL1-specific IgG in the sera of the animals in the control group was detected at 8 wpe (Fig. 1a). The titres of antibodies in the vaccinated groups were high at start of pasture exposure. This was maintained over the course of the trial but decreased approaching 8 wpe. However the titres in these animals increased again after 8 wpe, which coincided with the commencement of antibody production in the control group, and was therefore regarded as a boosting by the challenge infection.
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Fig. 2. Mean (±SEM) avidity values for vaccinated and control animals at 12 wpe. Avidity is expressed as [KSCN]50 , the molarity of KSCN required to reduce by 50% the OD of individual sera that has been diluted to titre. Both vaccinated groups showed significantly higher avidity compared to control animals (n = 13), denoted by *p ≤ 0.001. ISA 70VG cattle (n = 13) had higher mean avidity values than ISA 206VG (n = 13), but this was not statistically significant.
cantly greater than those observed in the control group at 12 wpe (p ≤ 0.001). rFhCL1-specific IgG1 titres were significantly higher in the ISA 70VG group compared to the ISA 206VG group at 12 wpe (p ≤ 0.05) (Fig. 1b). There were no detectable rFhCL1-specific IgG2 titres in the sera of the control animals, as we have reported previously [5,24,26]. rFhCL1-specific IgG2 titres were observed in the sera of both vaccinated groups and were higher in the ISA 70VG group compared to the ISA 206VG group, with a significant difference between the two groups at 12 wpe (p ≤ 0.05), (Fig. 1c). 3.3. Avidity assays The mean [KSCN]50 values of serum rFhCL1-specific IgG were significantly higher in both vaccinated groups compared to the control animals at 12 wpe (p≤0.001) (Fig. 2). The ISA 70VG group antibodies appeared to have a higher mean [KSCN]50 value than the ISA 206VG group, but on analysis this was not found to be significantly different.
Fig. 1. Mean (±SEM) rFhCL1-specific IgG, IgG1 and IgG2 titres in vaccinated and control animals (a, b and c, respectively). Antibody titres are expressed as Log10 . All vaccinated animals produced significantly higher titres of rFhCL1-specific IgG and IgG1 compared to controls at 12 wpe, denoted by *p ≤ 0.001. There was no rFhCL1-specific IgG2 production by the control cattle (n = 13). Cattle vaccinated with rFhCL1 + ISA 70VG (group ISA 70VG) (n = 13) showed significantly higher titres of rFhCL1-specific IgG, IgG1 and IgG2 at 12 wpe, compared to those vaccinated with rFhCL1 and ISA 206VG (group ISA206VG) (n = 13), denoted by **p ≤ 0.05. PreVacc, pre-vaccination; Exp, start of pasture exposure; Post 1st Vacc, post 1st vaccination (2 weeks post 1st vaccination); wpe, weeks of pasture exposure.
Serum rFhCL1-specific IgG titres were significantly higher in both of the vaccinated groups compared to the controls at 10 and 12 wpe (p ≤ 0.05 and p ≤ 0.001, respectively). In addition, serum levels of rFhCL1-specific IgG in animals given the vaccine formulated in ISA 70VG were significantly higher than those of the ISA 206VG group at 12 wpe (p ≤ 0.05). rFhCL1-specific IgG1 was detected in animals of the control group at 4 wpe. In contrast, rFhCL1-specific IgG1 titres were detected in the vaccinated cattle rapidly after vaccination and increased over the course of the trial. These titres were signifi-
3.4. Liver enzyme assays Serum levels of glutamate dehydrogenase (GLDH) increased above reference range levels by week three post-exposure in all animals. The levels remained high over the course of the challenge, but no significant differences between the control and vaccinated groups were observed. Although, gamma glutamyl transferase (GGT) serum levels increased slowly during the period of exposure these remained within the reference range for the course of the trial in all cattle and no significant difference was observed between the vaccinated and control groups. (Fig. 3) 3.5. Macrophage assays Nitric oxide levels were measured in the culture medium obtained from LFH-stimulated macrophages. These remained low in all animals throughout the trial (data not shown). However, when arginase activity was measured in the lysate of the macrophages of the same cultures, an increase was observed over the course of the trial. Most relevantly, the arginase activity levels in the macrophages taken from both vaccinated groups were
O. Golden et al. / Vaccine 28 (2010) 5551–5557
Fig. 3. Mean (±SEM) serum levels of GLDH and GGT in vaccinated (n = 13/group) and control cattle (n = 13) (a and b, respectively). Values expressed as iu/L. Levels of GLDH were above the reference range (0–12 iu/L) by 3 wpe in all animals, but showed very little increase over the rest of the challenge period. Serum levels of GGT remained within the reference range (0–20 iu/L) in all animals throughout the challenge period, although levels did increase over the course of the trial. There were no significant differences between groups for serum levels of GLDH or GGT.
Fig. 4. Mean (±SEM) arginase activity levels in macrophage lysate of macrophages isolated from heparinised blood of vaccinated (n = 13/group) and control cattle (n = 13) at time of challenge, 4 and 12 wpe. Arginase activity expressed as mU of arginase activity per 1 × 106 cells. In macrophages isolated from the control group the levels were significantly higher at 12 wpe when compared to time of exposure, denoted by † p ≤ 0.05. There was an increase in arginase activity in the macrophages of all groups over the exposure period. However, both vaccinated groups had significantly lower levels of arginase activity compared to controls at 12 wpe, denoted by **p ≤ 0.05.
significantly lower (p ≤ 0.05) than those in macrophages of the control group at 12 wpe (Fig. 4).
4. Discussion Previous vaccine trials using native FhCL1 have shown reductions in fluke burdens in the range of 55–72% compared to
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unvaccinated controls, and a marked reduction in parasite fecundity and/or egg viability [23–26]. The next step towards the production of a commercially viable liver fluke vaccine is assessment of the efficacy of the recombinant forms of FhCL1 and finding an effective adjuvant which can be licensed for commercial use [17]. The majority of previous vaccine trials used experimental infection. While this has the advantage of reducing variability in infection levels across animals it does not replicate conditions found in the field. Therefore, in the present trial we chose to assess the efficacy of our vaccine against a naturally acquired infection, which allowed us to ascertain the efficacy of the vaccine at farm level. Moreover, we formulated the vaccines in the mineraloil based MontanideTM adjuvants ISA 70VG and ISA 206VG since these are acceptable for commercial use. The immune response induced by these vaccines were characterised and compared to those induced in non-vaccinated animals exposed to infection. Previous vaccine trials with FhCL1 have shown that the vaccine produces an immune response which has elements of both Th1 and Th2 phenotypes indicated by the production of both IgG1 and IgG2 antibody isotypes [24]. Infected, unvaccinated animals were shown to produce only IgG1 antibodies, which is indicative of a nonprotective Th2 response. Fluke-specific IgG titre in infected animals was positively correlated with fluke burden, which illustrates that the immune response to liver fluke in the field is not protective. In contrast, Mulcahy et al. [24] showed that fluke-specific IgG2 titre and avidity correlated with vaccine-induced protection. Thus, a high fluke-specific IgG2 titre and avidity in vaccinated animals correlated with a low fluke burden. The two vaccine groups in this trial, ISA 70VG and ISA 206VG reduced the fluke burden at similar levels (47.2 and 49.2%, respectively) but these groups did not show significant differences when compared to controls. However, when the animals in both vaccinated group were considered as a single group and compared to non-vaccinated animals, a significant reduction in fluke burden was observed. The fluke burden in the animals kept on the pasture was not high, control animals had a mean burden of 16 fluke per liver, and adult parasites were not seen in the bile ducts (and therefore no eggs appeared in the faeces). It is possible that with a longer challenge period, more significant differences between the vaccinated groups and the controls may have become apparent. The humoral immune response induced by the vaccine supported data observed in our previous trials [23,24]. The rFhCL1specific IgG titres in the sera of the infected control animals were lower than those of the vaccinated animals. When isotyping ELISA were carried out, this total rFhCL1-specific IgG titre was seen to consist of rFhCL1-specific IgG1, with no rFhCL1-specific IgG2 production, indicating the polarised Th2 response. The vaccinated animals produced both rFhCL1-specific IgG1 and IgG2 isotypes, indicating a mixed Th1/Th2 response. The vaccinated cattle produced higher total rFhCL1-specific IgG and IgG1 titres than the control group. Correlations between fluke burden and antibody responses were studied but there were no significant correlations. This may have been due to the relatively low infections levels in the trial animals. Antibody avidity is the binding strength of the antibody with an antigen. As an immune response matures, the avidity of secreted antibodies increases due to an improved ‘fit’, which occurs from repeated selection for antibody populations by the antigen [34]. The avidity of total rFhCL1-specific IgG at 12 wpe was measured and the antibody produced by the vaccinated animals was found to have a significantly higher avidity for rFhCL1 than the control animals. This would be expected, as the antibody response produced by vaccination has had time to mature by the time of exposure, so the antibodies in the vaccinated animals would have a higher avidity than those of the control animals.
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Serum GLDH levels rose above the reference range by 3 wpe. Raised serum GLDH is caused by parenchymal damage [35], so immature fluke entering the liver would account for the increase. However, due to the low infection in the animals after this point there was very little increase over the challenge period. Increased GGT is associated with bile duct damage [36], so serum levels of this enzyme increase when the fluke mature and enter the bile ducts. Therefore, it was not surprising that GGT levels did not rise above the reference values at any point in the trial since no adult flukes were observed. Nitric oxide is produced by classically activated macrophages (CAMФ) [37], which are generated during Th1 responses [38]. We found that nitric oxide levels in culture medium obtained from LFH-stimulated macrophages, taken from control and vaccinated animals, was consistently low throughout the trial indicating a lack of classical activation (data not shown). By contrast, the arginase activity levels measured in the macrophage lysates showed significant differences between the vaccinated animals and the controls. The levels of arginase activity in the macrophage lysate of the two vaccinated groups at 12 wpe were significantly lower than those of the control animals. Since arginase expression is a marker of alternative-activation in macrophages [39], it appears that the AAMФ response in the vaccinated cattle was less pronounced than that of the control animals. AAMФ are instrumental in suppressing Th1 responses and promoting Th2 responses [9], so the depression in the levels of AAMФ in vaccinated animals correlates with the mixed Th1/Th2 phenotype that was observed in the humoral response of the vaccinated groups. In contrast with murine models [11] this might suggest that AAM are not protective during F. hepatica infection. The antibody and macrophage data obtained in this trial show that the vaccine has produced a qualitatively and quantitatively different innate and adaptive response to infection. The question still remains as to what mechanisms are involved in reducing the fluke burden in the vaccinated animals. Cathepsin Ls are utilised by the parasite at all stages of its life cycle in the mammalian host [20]. Although the cysteine protease profile differs over this period, a vaccine that induces antibodies that successfully inactivate FhCL1 could potentially have numerous effects on the development of the fluke. For example, the ability of the newly excysted juveniles (NEJ) to penetrate the intestinal wall and reach the peritoneal cavity would be impaired, reducing the number of parasites that can reach the liver. Also, if these antibodies were to bind to the secretory vesicles which contain FhCL1 within the parasite gastrodermal epithelial cells, they could physically block enzyme secretion, resulting in significantly lower levels of available cathepsin for feeding and migration within the host. The fluke appears to be most vulnerable to the immune system of the host when crossing the peritoneal cavity [40]. Studies in rats, which show a level of resistance to F. hepatica that is comparable to cattle, have shown evidence of an antibody dependant cell cytotoxicity (ADCC) occurring. This mechanism appears to be dependent on nitrite production by peritoneal cell populations, which consist mainly of monocyte/macrophage cells [41]. Neutralizing or reducing secreted volumes of FhCL1 at this point could render the fluke more susceptible to immune attack. Firstly, cathepsins are capable of cleaving host immunoglobulins at the hinge region, thereby preventing ADCC [20]. Inhibition of FhCL1 activity would, therefore, facilitate effective ADCC. Secondly, the Th1 suppression caused by FhCL1 would also be blocked leading to a greater production of nitrite, promoting more efficient ADCC. FhCL1 activity participates in the immunomodulation of host responses that occurs during animal fasciolosis. O’Neill et al. [21] demonstrated that the protease could suppress Th1 responses in mice. The mixed immune response observed in the vaccinated animals in our trial may have been due to the reduction in activity of
FhCL1. This is reflected in the production of both IgG1 and IgG2 titres and also the lower levels of alternative-activation seen in the re-stimulated macrophages of the vaccinated cattle. Long term induction and activity of AAMФ is dependent on Th2 cells [42]. At the hepatic stage of the life cycle FhCL1 is vital to F. hepatica, both for migration and acquisition of nutrients. If FhCL1 is sufficiently inactivated to prevent the parasite from feeding, this would clearly be an efficient method of killing the maturing fluke. If migration of the fluke is inhibited it could affect not only the progression of the life cycle, but also its ability to evade immune attack. Meeusen et al. [43] noted during infection in sheep, that although lymphocytes and other immune effector cells infiltrated migratory tunnels in the liver parenchyma, the parasites themselves never had these cells around them. It was suggested that the fluke were capable of migrating quickly enough to ‘leave behind’ the immune response. If this is the case, any impairment of the fluke’s ability to migrate will increase its susceptibility to immune attack. Effective control of F. hepatica is of increasing importance because fasciolosis is increasing in both frequency and distribution [44,45]. The development of resistance to triclabendazole has shown anthelmintics to be an unsustainable means of controlling the disease. An effective vaccine represents a cost-efficient, long term, environmentally–friendly alternative. However, the vaccine must also be formulated and delivered in a suitable adjuvant. The montanide adjuvants used in this study likely played an important part in promoting the mixed protective Th1/Th2 response observed in the vaccinated cattle. They have been shown to induce both Th1 and Th2 phenotypes in cattle [27,28]. The most important factor influencing the outcome of infection is the number of metacercariae ingested [4], which will determine the number of fluke migrating through the hepatic parenchyma. In this trial we achieved an overall reduction of 48.2% in fluke burden with vaccination. Hope Cawdery et al. [46] demonstrated that fluke burdens as low as 54 could impair weight gain in cattle. Dargie [47] demonstrated that only 3% of cattle in the UK had burdens exceeding 50 fluke, suggesting that the level of protection achieved with our vaccine would be commercially viable. Although we were unable to demonstrate it in this trial, previous vaccine trials with FhCL1 have shown a reduction in egg production and viability [23,26]. This effect on transmission could lead to a cumulative benefit of vaccination over a number of grazing seasons. In addition to the reduced numbers of fluke in the livers of the animals on pasture, there would be a reduction in pasture contamination resulting in lower infection levels the following year. These lower transmission rates may also have important implications for the spread of F. hepatica to humans in areas where this zoonosis is of importance, e.g. South America, Egypt, Iran and South-East Asia [15]. rFhCL1 combined with a MontanideTM adjuvant shows excellent potential as a successful fluke vaccine in cattle. Further trials, with a longer challenge period and higher fluke burdens will establish its efficacy more definitively and will also allow us to determine the effect of this vaccine on parasite fecundity and egg viability. Acknowledgments This work was funded by the EU Commission under Framework 6, Project Ref. FOOD-CT-2005-02305-DELIVER. The authors are grateful to Mr. Eddie Jordan and the staff of the UCD Lyons Estate Research Farm and to Barbara Good, J.P. Hanrahan and the staff of Teagasc Research Centre Athenry, for their care of the experimental animals. References [1] Mas-Coma MS, Esteban JG, Bargues MD. Epidemiology of human fascioliasis: a review and proposed new classification. Bulletin of the World Health Organization 1999;77(4):340–6.
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