Background
Fasciolosis is one of the most widespread food-borne trematode diseases around the world, causing significant economic losses in developing countries (estimated at over 3 billion dollars per year) and affecting a wide range of mammals, mainly ruminants [
1,
2].
F. hepatica also causes disease in human beings, most cases being reported in Andean countries, Egypt and Iran [
3‐
5]. Several reports have estimated that up to 17 million people are infected and 91 to 170 million people live in areas having a high risk of acquiring the disease [
6].
Chronic
F. hepatica infection causes hepatomegaly, gallbladder and biliary duct thickening and dilatation, leading to cholangitis, cholecystitis, usually accompanied by obstruction of the biliary ducts.
F. hepatica infection also causes hepatic tissue damage and parenchymal destruction by juvenile fluke migration until the biliary ducts are reached [
7]. The immune response induced by
F. hepatica infection has been well described in experimental models, typically being characterised by the presence of a dominant Th2 response with IL-4, IL-5 and IL-13 secretion by spleen cells and regulatory cytokines IL-10 and TGF-β by macrophages and dendritic cells and Th1 immune response suppression in murine models [
8‐
10]. A dominant Th2 immune response also occurs during the chronic phase of the disease in cattle, involving high levels of IgG1 and little or no IgG2a; it has also been shown that susceptibility to infection is correlated with the IgG1/IgG2a ratio, as well as IL-4/IFN-γ levels [
11‐
14]. Such immunomodulation/immunoregulation of the host’s immune response by helminthic parasites is a key factor for successful infection and parasite survival within host tissues. Besides the large body of knowledge concerning an immune response in
F. hepatica infection, the precise molecular mechanisms leading to protection are not yet well understood.
Fasciolosis control in animals and humans is currently based on the mass administration of triclabendazole which is active against both adult parasites located in the bile ducts and immature flukes migrating through the liver. This strategy has led to progressive drug-resistance in animals [
15‐
17]. Despite there is evidence that a number of drugs have activity against
F. hepatica, it seems to be only partial, acting only either on the immature or mature flukes. Also, the potential risk of secondary effects makes the replacement of triclabendazole an ineffective strategy for its control [
18‐
20]. Developing an effective vaccine represents one of the most appealing strategies for preventing this disease and reducing the risk of infection in humans. No commercial vaccines are yet available for
F. hepatica although significant progress has been made regarding attempts at developing effective vaccines against infection caused by other parasites, only a few vaccines have been successfully tested against helminthic infection in animals [
21‐
24]. Several antigens have been identified and tested as vaccine candidates against
F. hepatica infection. Most studies have focused on using cathepsins, leucine-aminopeptidase, haemoglobin, fatty-acid binding proteins and glutathione S-transferase as vaccine candidates. Such antigens have mainly been produced as recombinant proteins and administered in different adjuvant-type formulations. Vaccine efficacy has ranged from 13 to 97% in cattle, measured as liver-fluke burden reduction [
25,
26]. Subunit-based, chemically-synthesised vaccine candidates represent a promising vaccine development strategy. Using synthetic peptides offers a wide range of advantages: inexpensive production, induction of a strong immune response, avoid using living organisms thus minimising the risk of acquiring disease, sequences which can interfere with vaccination success become eliminated and peptides from different antigens can be conjugated to the same carrier [
27,
28].
A previous study by our group involving the use of bioinformatics tools showed the protection-inducing ability of
F. hepatica cathepsin B and amoebapore-derived B- and T-cell epitopes which had been chemically-synthesised and selected as vaccine candidates according to their induced immune response in a murine model, reaching up to 66.7% protection after immunisation [
29]. According to the results obtained in the above mentioned article, where we found reduction in hepatic damage and increase of survival rates, in the present study we hypothesised that a combination of peptides with different induced-immune responses could act in a synergic way in order to improve the immunoprotection in mice. In the present study we used the so-called ADAD vaccination system. Previous reports by our group have demonstrated the improvement in immunoprotection trials against
F. hepatica when using this vaccination system [
30].
Better understanding concerning the molecular mechanisms by which F. hepatica infection occurs, as well as the molecular basis involved in obtaining protection, is extremely important in designing and selecting new vaccine candidates. The present study involved using a microarray-based methodology for studying the gene expression profile in the spleen of mice immunised with a highly protective anti-F. hepatica vaccine candidate using a combination of three synthetic peptides containing T-cell epitopes, derived from F. hepatica cathepsin B and amoebapore proteins, and the possible molecular pathways involved in inducing protection.
Discussion
The present work involved using a microarray-based technology together with protection studies against F. hepatica to better understand the gene expression profile and immunological mechanisms leading to protection in mice immunised with a novel anti-F. hepatica vaccine candidate.
F. hepatica cathepsin B and amoebapore-like proteins were selected as vaccine candidates in the present study. According to the pertinent literature, it is well known that
F. hepatica cathepsins are secreted proteins which are differentially expressed during the life stages of flukes; they have different associated functions in each stage and are also considered significant vaccine candidates.
F. hepatica cathepsin B is mainly expressed in metacercariae and newly excysted juveniles whilst cathepsin L is mainly expressed in newly excysted juveniles and adult worms [
45].
F. hepatica cathepsins are considered important vaccine candidates; several studies have demonstrated their protection-inducing ability in different experimental models [
46‐
49].
F. hepatica amoebapore proteins have shown amino acid sequence similarity with saposin-like proteins. These proteins have been shown to be highly immunogenic and their inducing-protection ability has also been demonstrated in mice and rabbits. Concerning
F. hepatica saposin-like protein expression, up-regulation has been demonstrated in eggs, newly excysted juveniles and adults and down-regulation in miracidia [
50]. The recently published draft genome for
F. hepatica identified three proteins from amoebapore-like proteins referred to as saposin-like proteins. The gene expression profile has revealed up-regulation of the amoebapore-like protein (putative, uncharacterised; Uniprot blast hits Q4KSL8; Q24939; Q24938; Q9NAT2; B6ZBP3) in newly excysted juveniles, juvenile (21 days) and adult stages. The amoebapore-like protein (Uniprot blast hits Q9NAT2; B6ZBP3) is down-regulated in the newly excysted juveniles stage but up-regulated in the juvenile (21 days) and adult stages. The third amoebapore-like protein (Uniprot blast hits Q9NAT2; B6ZBP3; Q4KSL7; Q4KSL8; Q24939) is also down-regulated in newly excysted juveniles stages but highly up-regulated in the juvenile (21 days) and adult stages [
51].
To date, little is known concerning the development of subunit-based, chemically synthesised anti-
F. hepatica vaccines [
52,
53]. The present study involved selecting mouse survival rates (represented by Kaplan-Meier survival curves) and hepatic damage score as indicators for protection. Recovered worm count should also be included as protection indicator in an ideal scenario; however, the number of worms recovered per animal was too low when using the mouse model of infection for
F. hepatica experiments; drawing conclusions for protection based on worm count could thus be biased. It was also considered that worm count in infected mice was indeed a less reliable method for assessing protection. Mice immunised with a combination of synthetic peptides containing either B- or T-cellepitopes increased mouse survival rate in the present study. Despite the peptides tested were originally designed based on their good predicted binding to the H2-Ed MHC type displayed by the syngeneic BALB/c mice as a model to obtain a specific response against
F. hepatica, testing the protective immune response induced in an outbred model such as CD1 mice could represent better the variability observed in the target population for vaccination such as sheep and cattle.
The combination of peptides containing T-epitopes (T14 + T15 + T16; group 6) induced the highest survival rate, thereby supporting our hypothesis that a multi-epitope-based vaccine is necessary for obtaining higher immune-protective responses against helminthic infection. A previous study by our group demonstrated the immunoprotective efficacy of single peptides containing B- or T-cell epitopes against experimental infection with
F. hepatica metacercariae, reaching good immunoprotective levels with the so-called peptide T15 [
29]. Here, we have demonstrated that adding peptides T14 and T16 induced better protective levels than the single peptide T15, taking the Kaplan-Meier survival curves and hepatic damage into account.
Adding peptides containing B-cell epitopes to our vaccine candidate induced a loss of immunogenicity as measured by determining IgG levels and immunoprotection levels became reduced. It is worth noting that vaccinating mice with ADAD vaccination system components induced little protection, as indicated by Kaplan-Meier survival curves, and little reduction was observed regarding hepatic damage.
A microarray-based methodology was used to identify the gene expression profile in BALB/c mice immunised with our most protective antigen combination formulated in ADAD vaccination system (Qs + AA0029 + T14 + T15 + T16) and the uninfected control mice to gain fresh insight into the molecular and immunological basis leading to protection. Eosinophilia has been associated with helminth infection, although its precise role in conferring protection is still controversial and several studies have been concerned with the role of eosinophils in protection by using both in vitro and in vivo studies [
54‐
56]. CCR3 is a receptor for C-C type chemokines, including eotaxin, MCP-3, MCP-4 and RANTES, and is highly expressed in eosinophils. Our study found up-regulation of the CCR3 chemokine receptor in mice immunised with our vaccine candidate, thus supporting the hypothesis that eosinophils could be mediating mechanisms leading to protection in
F. hepatica infection. By contrast, other studies have shown that eosinophils suffer induced-apoptosis by excretory/secretory products from
F. hepatica, thus suggesting that proposing a role for such cells in inducing protection should be taken with caution [
57]. IL-8 receptor gene (CXCR2) up-regulation was also found. CXCR2 is mainly expressed on neutrophil surface, providing some information concerning the possible mechanisms involved in Th2 cell-mediated immune clearance pathways. Up-regulation of other types of C-X-C family chemokines (i.e. CXCR4) has been shown to be essential in animals which are genetically-resistant to nematode parasites [
58].
Up-regulation of the endogenous toll-like receptor-4 agonist was also achieved by the immunisation trial through expression of the S100A8/S100A9 complex, known as calprotectin. These two members of the damage associated molecular pattern family are secreted during phagocyte stress response [
59]. Evidence has been presented regarding neutrophil and S100A8 neutrophil chemokine localisation in fibrotic areas in a murine model of
Schistosoma japonicum, suggesting its implication in the induction of fibrosis [
60]. S100A8 and S100A9 are up-regulated in liver and spleen during
S. japonicum infection. It has also been suggested that S100A8 up-regulation may protect against oxidative tissue damage. There is no evidence to date regarding the precise role of the S100A8/S100A9 complex concerning
F. hepatica infection or acquiring protective immunity. The present study’s results suggested activation by innate immunity with up-regulation of both S100A8 and S100A9 in the spleen of mice immunised with our vaccine candidate, a key factor in inducing protective immunity, at least in a murine model.
Reports in the pertinent literature have investigated the role of C3 during schistosome infection in a murine model. C3 depletion has led to a significant reduction in Th2-associated cytokines without any correlation with worm development or liver pathology. C3-defficient mice have not been able to effectively clear adult worms after treatment with praziquantel [
61]. C3 depletion seems to be agree as complement activation do not represent an immunologic mechanism leading to immunoprotection against
F. hepatica infection. Also, nitric oxide is related to Th1 immune response with subsequent macrophage activation, unleashing nitric oxide liberation as an immunoprotector-associated mechanism. However, we could not provide any experimental evidence showing its correlation with hepatic damage or fluke burden reduction.
It has also been shown that nitric oxide production is enhanced by the synergic effect of both IL-12 and IL-8. IL-12 is considered to be a cytokine inducing a Th1 immune response, with IFN-γ overproduction. IL-12 plays a key role in helminthic infection by inhibiting Th2 immune responses which are essential for a parasite surviving inside a mammalian host [
62]. It was observed in our study that the IL-12 signalling pathway was significantly altered by the immunisation trial, being the genes S100A8, CD40LG and JUN differentially expressed, making it a key factor leading to protection. Significant alteration in IL-8 signalling was observed after mice were immunised with our vaccine candidate, involving up-regulation of receptors for IL-8, MMP9 and CXCR2. Nitric oxide production has been studied in vitro in infection caused by helminths involving
Trichinella spp, Dirofilaria immitis and
Ascaris suum antigens; a correlation has been found between antigen-dependent stimulation and nitric oxide levels [
63‐
65]. Nitric oxide could regulate inflammation produced by egg release, prevent hepatic cell damage, the spread of damage in the liver and reduce granuloma formation by
S. mansoni [
66]. Concerning
F. hepatica infection, nitric oxide production has been studied in infected rats, showing reduced nitric oxide levels 7 and 14 days p.i. Such reduction has been associated with excretory/secretory released antigens, constituting one of the defence mechanisms used by this parasite during its migration through the peritoneal cavity of a mammalian host [
67]. It could be hypothesised that changes in nitric oxide signalling pathway induced by the immunisation trial could have been involved in inducing protective immunity, thereby becoming a necessary condition for such immunoprotection. No previous reports are available concerning the expression levels of MMP9 during
F. hepatica infection. In
S. mansoni-infected mice there is evidence of changes in MMP gene family expression in the chronic phase of the disease, which is associated to degradation of collagen deposited in tissues. Up regulation of MMP2, MMP3 and MMP8 has been reported in
S. mansoni-infected mice but no significant differential expression was observed in MMP9 gene [
68].
S. mansoni-infected patients have had low CXCR1 and CXCR2 frequency during the acute phase of the disease but high IL-8 levels in sera [
69]. Besides high CXCR1 and CXCR2 expression in our study, we could not provide any evidence of IL-8 in conferring protective immunity; however, it provided a first insight concerning its implication in
F. hepatica protection-inducing mechanisms.
Acknowledgements
We would like to thank the University of Salamanca’s Cancer Research Centre Genomic and Bioinformatics group for their technical support and microarray data acquisition and Jason Garry for correcting the manuscript.