Introduction

Fasciolosis is an important emerging foodborne zoonotic disease caused by two trematode species, Fasciola hepatica (F. hepatica) and Fasciola gigantica, found in temperate and tropical zones, respectively. The parasites infect herbivorous animals including livestock of economic importance, such as cattle, buffalo, sheep, and goats, leading to substantial losses to livestock industry, especially with respect to milk, meat production, and labor (Spithill et al. 1999). In addition, humans can be accidentally infected by Fasciola spp. Human fasciolosis is recognized as a significant public health problem that may be underestimated in most endemic countries due to the sharing of habitats with other closely related parasites, inadequate treatment, and prevention. The World Health Organization (WHO) reported that approximately 2.4 million people have been infected by this disease worldwide, and 180 million people are at risk of new infection. Presently, the control strategy of Fasciola infection relies on anthelmintic drugs, and the most effective one in current use is triclabendazole (Fairweather 1999). However, due to prolonged administration of the drugs to obtain an effective outcome, drug resistance has been reported in many countries, including Australia, Ireland, and Argentina (Overend and Bowen 1995; Fairweather and Boray 1999; Olaechea et al. 2011). Therefore, researchers have sought alternative control strategies to overcome the drug resistance problem. Vaccines are thought to be a more sustainable; several have been experimentally tested and found to be effective with high levels of safety, cost-effectiveness, and environmentally friendly.

Up to now, several vaccine candidates have been identified and tested for their efficacies against F. hepatica and F. gigantica in experimental and economic animals, and they include proteinases, fatty acid-binding proteins, and antioxidant enzymes. These candidate vaccines exhibited varying levels of protection depending on several factors, including type of adjuvants, administration routes, and immunogenicity of the candidate antigens. Cathepsin L proteinases have been shown to induce specific immune responses in cattle and sheep with protection varying from 33 to 69 % (Dalton et al. 1996; Piacenza et al. 1999; Golden et al. 2010). Leucine aminopeptidase in native and recombinant forms showed protection in mice and sheep ranging from 50 to 89 % (Piacenza et al. 1999; Maggioli et al. 2011a; Changklungmoa et al. 2013). Fatty acid-binding proteins showed a 23 and 43 % reduction of the worm recoveries in calves and sheep, respectively (Kumar et al. 2012; López-Abán et al. 2007). Glutathione S-transferase showed 77–84 % protection in mice against F. gigantica infection (Preyavichyapugdee et al. 2008), while thioredoxin–glutathione reductase also reduced the worm burden up to 96.7 % in F. hepatica-infected rabbits (Maggioli et al. 2011b). However, most vaccine candidates targeted mainly the adult parasites that have fully developed immune evasion mechanism and reside in the bile duct which is not accessible to the host’s immune cells. Therefore, the prospect of killing the parasites and protecting the host from being infected may be higher if the vaccines are designed to target the newly excysted or early juveniles.

Cathepsins, members of the cysteine protease family, are abundantly expressed in Fasciola spp. (Carmona et al. 1993; Berasaín et al. 1997; Halton 1997). Cathepsin Ls were the major proteases expressed in adult Fasciola spp. (Grams et al. 2001; Jefferies et al. 2001; Morphew et al. 2007; Robinson et al. 2009), whereas cathepsin Bs were expressed predominantly in juvenile parasites (Wilson et al. 1998; Meemon et al. 2004). However, several cathepsin L and B isoforms were also found to differentially express during developmental stages of Fasciola spp. (Robinson et al. 2008, 2009; Cwiklinski et al. 2015). Recently, a study on the F. hepatica genome revealed that the differentially expressed cathepsin L family consists of five clades with a total of 17 members while the cathepsin B family consists of a single clade with seven members (Cwiklinski et al. 2015). Because of their abundant expressions and important roles, cathepsin proteinases are thought to be the major targets for vaccine development against fasciolosis. In this article, the genes encoding cathepsins L and B expressed in juvenile Fasciola spp. and their biological activities were reviewed. Moreover, the efficacies of vaccines developed from these antigens in protecting experimental animals were compared with the adult cathepsin L and B isoforms.

Cathepsin B

Cathepsin B-like proteases have been demonstrated to be one of the major proteases synthesized by newly excysted juvenile (NEJ) and immature Fasciola parasites (Tkalcevic et al. 1995; Wilson et al. 1998; Law et al. 2003; Meemon et al. 2004; Cancela et al. 2008). The first cathepsin B of Fasciola spp. namely FhCatB1 (also known as CB2) was identified in the somatic and excretory–secretory (ES) extracts of F. hepatica NEJs and 5-week-old immature parasites (Tkalcevic et al. 1995; Wilson et al. 1998; Law et al. 2003). Later, three cathepsin B isotypes were identified from stage-specific cDNA libraries of F. gigantica and named FgCatB1, FgCatB2, and FgCatB3 (Meemon et al. 2004). The orthologous cathepsin B isotypes (CB1, CB2, CB3) were then identified in juvenile F. hepatica (Cancela et al. 2008, 2010). RT-PCR and phylogenetic analyses demonstrated that these isotypes were stage-specific (Meemon et al. 2004; Cancela et al. 2008; Smooker et al. 2010). FgCatB2 and FgCatB3 were expressed in metacercariae and NEJ while FgCatB1 was expressed in all stages with the highest level in the adult parasites (Meemon et al. 2004; Cancela et al. 2008). Moreover, other cathepsin B isotypes including cathepsin B4, B5, and B6-10 have also been respectively detected in the secretomes of NEJ, immature, and adult F. hepatica (Robinson et al. 2009). Phylogenetic analysis of Fasciola cathepsin B sequences available in the NCBI public database showed that they could be classified into four clades based on their sequence similarities and identities (Fig. 1). Clades 1–3 have been identified in previous studies (Meemon et al. 2004; Cancela et al. 2008), while the last distinct clade 4 has only recently been determined (Fig. 1). However, only sequences of cathepsin B in clade 4 were available in the database, and no molecular or biochemical study has been performed. The phylogenetic tree should become more precise when more sequences are found and analyzed in the future.

Fig. 1
figure 1

Phylogenetic analysis of Fasciola cathepsin B family. The amino acid sequences of Fasciola cathepsin Bs from the public database, http://www.ncbi.nlm.nih.gov, were multiple aligned by ClustalX (Larkin et al. 2007) and used to construct the phylogenetic tree by the neighbor-joining method (PAUP program) in MEGA6 (Tamura et al. 2013). The accession numbers obtained from GenBank are as indicated: FG F. gigantica, FH F. hepatica. AAO73002, AAO73003, AAO73004 (Meemon et al. 2004); ABU62925 (unpublished); AIS22046 (unpublished); AIS22047 (unpublished); ABF85678, ABF85679, ABF85680 (Cancela et al. 2008); CAD32937 (unpublished); AAD11445 (unpublished). Asterisks indicate that the partial sequences were used for analysis

The function of cathepsin B-like proteases has not yet been clearly defined. However, the findings that they are mainly expressed in the early stages of parasites suggested that cathepsin Bs may play important roles in excystment, gut penetration, and invasion through liver tissues (Wilson et al. 1998; Law et al. 2003). Consistent with this hypothesis, recombinant FgCatB2 and FgCatB3 could digest type I collagen and fibronectin (Table 1) (Chantree et al. 2012; Sethadavit et al. 2009). Moreover, recombinant FgCatB2 was also able to digest immunoglobulins, suggesting another role of this isotype in evasion from the host’s immune attack (Chantree et al. 2012). A recent study on silencing of FhCatB1(CB2) by RNA interference (RNAi) in F. hepatica NEJs revealed a significant reduction in penetration through the intestinal wall of rats (McGonigle et al. 2008). NEJs were also paralyzed or exhibited abnormal movement after incubation with FhCatB1 double-stranded RNA (McGonigle et al. 2008). Furthermore, the cathepsin B-selective inhibitor (CA-074) caused significant reductions of the motility and viability of F. hepatica NEJs (Beckham et al. 2009). Biochemical study revealed that F. hepatica cathepsin B strongly reacted with substrates containing isoleucine or valine at the P2 position (Beckham et al. 2009). This activity was different from most cathepsin Bs found in other species that preferentially cleaved substrates with leucine, phenylalanine, and arginine at the P2 position, suggesting that F. hepatica cathepsin B can be exploited as an anti-fluke drug target (Smooker et al. 2010).

Table 1 Substrate specificities of juvenile-specific cathepsin Bs and cathepsin Ls of Fasciola spp.

Using in situ hybridization revealed that the messenger RNA (mRNA) transcripts of cathepsin B were expressed in the cecal epithelial cells, in cells underlining the proximal digestive tract, and in the tegumental cells underlining the tegument of 4-week-old juvenile and adult F. gigantica (Meemon et al. 2004). Moreover, in adult parasites, mRNA transcripts were also detected in cells of the reproductive organs, including the prostate gland, Mehlis gland, vitelline gland, testis, and ovary (Meemon et al. 2004). Immunolocalization revealed cathepsin B expression in the cecal epithelial cells of metacercariae, NEJ, and 2-week-old juveniles of F. gigantica (Chantree et al. 2012; Sethadavit et al. 2009). In contrast, cathepsin B expression was not detected in the tissues of 4-week-old juveniles and adult F. gigantica by using either specific monoclonal or polyclonal anti-cathepsin B antibodies (Anuracpreeda et al. 2011; Chantree et al. 2012; Sethadavit et al. 2009). This may be due to the low identity of adult-specific cathepsin B1 compared to other juvenile-specific cathepsin Bs and, also, the low amount of cathepsin B expressed in the late juvenile and adult stages. In F. hepatica, the localization using a fluorescent-labeled active site probe detected active cathepsin B1 in the digestive tract of NEJs (Beckham et al. 2009); however, its localization in the adult parasites has not been investigated.

Cathepsin L

Many isotypes of cathepsin L have been identified in Fasciola spp. They can be classified into five clades according to their sequence identities (Robinson et al. 2008; Sansri et al. 2013; Cwiklinski et al. 2015). Clades 1, 2, and 5 belonged to adult-specific cathepsin L, while clades 3 and 4 were classified as juvenile-specific cathepsin L (Robinson et al. 2008; Sansri et al. 2013; Cwiklinski et al. 2015). Two juvenile cathepsin L proteases have been identified in F. hepatica, namely FhCL3 and FhCL4 (Harmsen et al. 2004; Cancela et al. 2008), and their sequences were identical to cathepsin L1G (FgCatL1G) and L1H (FgCatL1H) of F. gigantica, respectively (Cancela et al. 2008; Sansri et al. 2013). FhCL3 exhibited optimal activity and stability at neutral pH and could cleave collagen, but not immunoglobulin, suggesting its role in parasite migration through the liver (Table 1) (Corvo et al. 2009; Robinson et al. 2011). However, its F. gigantica orthologue, FgCatL1G, could digest several extracellular matrix proteins including collagen, laminin, and fibronectin and also cleaved immunoglobulin, suggesting its other role in immune evasion (Norbury et al. 2011). These studies indicated the functional differences among orthologous proteases of the two Fasciola species. Knocking down F. hepatica cathepsin L by RNAi caused reduction in penetration ability of NEJs through the intestinal wall (McGonigle et al. 2008). The study indicated the function of cathepsin L in gut penetration of NEJs. Both FhCL3 and FgCatL1G preferentially cleaved substrates with proline residue at P2 position (Corvo et al. 2009; Norbury et al. 2011), which enabled these cathepsins to digest the host collagen that contains a repeating motif of Gly-Pro-Xaa (Robinson et al. 2011). On the other hand, FgCatL1H could cleave native substrates, including type I collagen, laminin, and IgG, suggesting its roles in tissue migration and immune evasion (Sansri et al. 2013). However, FgCatL1H cleaved synthetic substrates with phenylalanine residue at the P2 position more effectively than with proline residue, indicating the different substrate preferences of FgCatL1H and FgCatL1G that belong to different clades (Sansri et al. 2013).

By using antibodies against the native cathepsin L of adult Fasciola, cathepsin L was detected mainly in the epithelium lining the digestive tract and also in the glycocalyx coating the surface tegument (Collins et al. 2004; Meemon et al. 2010). Polyclonal and monoclonal antibodies against adult-specific cathepsin L1 could also detect the cathepsin L in the epithelium lining the cecum and cecal lumen of metacercariae; NEJ; 1-, 3-, and 5-week-old juveniles (Anuracpreeda et al. 2014). The cross-reactivities among antibodies to cathepsin L isotypes were due to the similarity and identity of their amino acid sequences. Localization of FgCatL1H by both polyclonal and monoclonal antibody showed its expression in the epithelial cells of intestinal tract of metacercariae, NEJ, 2- and 4-week-old juveniles, and adult F. gigantica (Sansri et al. 2013; Wongwairot et al. 2015). The positive result in the adult might be due to a high degree of cross-reaction between FgCatL1H, which is the juvenile isoform, with the adult-specific cathepsin L. By contrast, FhCL3 was also detected by using its corresponding antibody in oral and ventral suckers and tegument of F. hepatica NEJ (Zawistowska-Deniziak et al. 2013).

Vaccine potentials of juvenile-specific cathepsins B and L

The vaccine potentials of juvenile cathepsin B and L isoforms were recently demonstrated in rats where vaccinations with recombinant F. hepatica CatB and F. gigantica CatL1G showed significant reductions in worm burden, liver damage, and parasite mass, with high percentages of protection at 60 and 43 %, respectively (Table 2) (Jayaraj et al. 2009). Moreover, a combined vaccine of these recombinant proteins showed higher protection at 66 % (Table 3) (Jayaraj et al. 2009). In F. gigantica, a strong protection was also demonstrated by vaccination with recombinant FgCatB2 and FgCatB3 at 60 and 66 %, respectively (Table 2) (Chantree et al. 2013). Vaccination with recombinant F. gigantica CatL1H showed protection against the Fasciola infection at 66 % (Sansri et al. 2015).

Table 2 Efficacies of cathepsin B and L vaccines against fasciolosis
Table 3 Percentages of protection of combined vaccines using cathepsin Bs, Ls, and leucine aminopeptidase (LAP) of Fasciola spp.

In comparison to other cathepsin L and B isotypes that are specifically expressed in late juvenile or adult parasites, vaccination with native or recombinant adult-specific cathepsin L achieved the protection against F. hepatica ranging from 33 to 69 % (Table 2) (Dalton et al. 1996; Piacenza et al. 1999; Golden et al. 2010). These percentages of protection were related to amount of the protein used for immunization (Dalton et al. 1996). Moreover, combined vaccination of native cathepsin L and hemoglobin resulted in protection increased to 51.9–72.4 % compared to 42.5–43.8 % of protection by a single protein vaccination (Table 3) (Dalton et al. 1996). Vaccination with leucine aminopeptidase in combination with cathepsins L1 and L2 also improved protection from 60 to 79 % (Piacenza et al. 1999). However, in addition to the immunogenicity of the antigens themselves, other factors that may influence the effectiveness of cathepsin vaccines include the types of adjuvants being used, the routes of administration, and the experimental animals. In sheep, using Quil A as the adjuvant could significantly reduce the fecal egg count and higher anti-sera activity than using Freund’s incomplete adjuvant or TiterMax Gold (Haçariz et al. 2009). In addition, using Alum and Adyuvac 50 with recombinant leucine aminopeptidase vaccine also showed high protection against F. hepatica infection (Maggioli et al. 2011a). Our previous study found that hamsters are too susceptible to F. gigantica infection resulting in high mortality, while rats are too resistant to the infection. However, mice have shown moderate immune responses against fasciolosis; thus, this species was considered optimal for testing vaccine against F. gigantica. Large animals also showed different degrees of resistance to fasciolosis; e.g., cattle have been shown to resist fasciola infection (Haroun and Hillyer 1986), while sheep and goats are not resistant to the reinfection (Chauvin et al. 1995; Martínez-Moreno et al. 1997)

Vaccination with F. gigantica CatB2 and CatB3 in mice caused the total IgG to increase significantly after 2 weeks post-immunization and was maintained at high levels after infection with metacercariae until termination at 4 weeks (Chantree et al. 2013). Similar results were also obtained for vaccination with recombinant rproFgCatL1H (Sansri et al. 2015). The level of IgG in F. gigantica cathepsin vaccinations was found to be IgG1 dominant, indicating the Th2 response (Chantree et al. 2013; Sansri et al. 2015). There was a strong negative correlation between the levels of total IgG and the numbers of worm recoveries (Chantree et al. 2013: Sansri et al. 2015). However, vaccination of calves with adult-specific cathepsin L1 of F. hepatica revealed a significant increase of both IgG1 and IgG2 levels during the entire experimental period from immunization to infection (Golden et al. 2010), whereas immunization in rats with F. hepatica cathepsin L elicited a higher level of IgG2a than IgG1 responses (Bentancor et al. 2002).

In mice, vaccinations with cathepsins B and L of F. hepatica and F. gigantica reduced the liver damage as indicated by decreased histopathology and the levels of liver enzymes including aspartate aminotransferase (AST), alanine transaminase (ALT), and gamma glutamyl transferase (GGT) (Jayaraj et al. 2009; Sansri et al. 2015). Similar results were also found in sheep vaccinated with adult-specific F. hepatica cathepsin Ls (Piacenza et al. 1999). These results indicated that Fasciola vaccines using cathepsin proteases are feasible and safe in experimental animals and could be considered for use in large animals and humans upon further testing.

Conclusion

Isotype-specific cathepsins B and L are highly expressed in juvenile Fasciola spp. Apart from sequence difference from adult-specific cathepsins, their biochemical and physiological characteristics are also different from the adult isotypes and they may play major roles in the invasion and migration in the host’s tissues. Therefore, these juvenile cathepsins could be developed as vaccines that may block the invasion of the newly excysted and the migration of early juvenile parasites through the host intestinal wall and within the host liver, as well as killing them, resulting in a strong protection against fasciolosis, as has been reported in experimental animals. Furthermore, combining juvenile-specific with adult-specific cathepsins into candidate vaccines that may confer even better protection should be the focus of future studies.