Vaccination against fasciolosis by a multivalent vaccine of stage-specific antigens

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Abstract

Liver flukes produce cathepsin B and cathepsin L in their excretory–secretory material. These proteases are proposed to be key virulence factors for parasite infection, and are therefore targets for vaccination. Cathepsin B is predominately released in the juvenile stage of the life cycle, while different cathepsin L's are released throughout the cycle. Three proteases (cathepsin L5, cathepsin L1g and cathepsin B) were expressed in yeast from cDNA clones isolated from adult, metacercariae and newly excysted juvenile flukes respectively. Each was used singly or in combination to vaccinate rats that were subsequently challenged with Fasciola hepatica metercercariae. Each protein induced an immune response, and all groups vaccinated with recombinant protein yielded significantly fewer and smaller flukes than the control group. Maximal protection of 83% was seen in the group vaccinated with cathepsin B and cathepsin L5 in combination.

Introduction

The liver flukes, Fasciola hepatica and F. gigantica, cause infectious disease predominately in ruminants. Humans are often infected in communities where there is close human–ruminant interaction, such as in some South American communities, Egypt and Iran (Mas-Coma, 2005). The geographical range of these two parasite species (temperate and tropical, respectively) ensures worldwide infections. Although anthelmintic treatment is effective against disease, this is an expensive and non-sustainable measure, and drug resistant strains have been reported (Brennan et al., 2007, Overend and Bowen, 1995). The alternative to anthelmintics is the development of a vaccine. However, despite several decades of research, there is no commercial vaccine available for fasciolosis.

Cysteine proteases are important facilitators of virulence in flukes (Dalton et al., 2003). They are produced by all stages of the fluke life cycle and may facilitate biological functions such as excystment, tissue invasion and immune evasion (Sajid and McKerrow, 2002). Two classes of cysteine protease are found in the excretory/secretory material of liver flukes – these are cathepsin L (cat L) and cathepsin B (cat B). Among the secreted cysteine proteases, F. hepatica cat L has been the most extensively studied (see for example Dalton et al., 2003, Kesik et al., 2007, Mulcahy et al., 1999, Smooker et al., 2000, Wijffels et al., 1994). Cat B has also been studied more recently (Beckham et al., 2006, Kennedy et al., 2006, Law et al., 2003, Meemon et al., 2004, Wilson et al., 1998). What is striking about these proteases is their temporal expression – cat B is predominantly expressed in the juvenile stages of the parasite (Wilson et al., 1998), while different cat L's appear to be expressed at all stages, although there may be some stage-specific expression of the various cat L genes. Cat L is the major protease activity in adult E/S.

Most experimental vaccine trials with liver fluke proteases have been performed using recombinant antigens derived from adult parasites with various levels of protective efficacy (Hillyer, 2005). However, liver flukes have complex life cycles and gene-expression varies between the different developmental stages within the same host. This can be problematic for developing strategies to vaccinate animals against adult-stage-specific antigens, as the juvenile stages are recognised as causing the most severe damage and pathology in liver fluke infection. Although the relatively high percentage identity (46–96%) between the various members of the cathepsin L family should result in immunological cross-reactivity between these proteases at different developmental stages due to the conservation of epitopes, this is likely to be suboptimal. It is preferable to use an antigen that is expressed strongly throughout the life cycle, or perform multivalent vaccination with more than one antigen. Multivalent vaccination against fasciolosis has been attempted. Dalton et al. (1996) showed that combination vaccines (cat L2 plus hemoglobin) gave higher efficacy in cattle than either antigen alone; however, in sheep a combination of leucine aminopeptidase (LAP) and cat L vaccines were not more efficacious than LAP alone (Piacenza et al., 1999).

The cat L proteases from liver fluke form a large, monophyletic clade that can be subdivided into several smaller clades, which contain proteases with the same amino acid at position 69 (Irving et al., 2003). These clades also group the cat L's into enzymes expressed either in adult or juvenile parasites. There are over 50 entries for Fasciola cat L sequences deposited in GenBank, and over 10 for cat B. We and others have identified cat L and B sequences predominantly expressed in juvenile and adult parasites and have subsequently expressed the proteases as recombinant proteins in yeast (Beckham et al., 2006, Law et al., 2003, Roche et al., 1997, Smooker et al., 2000).

The yeast expression system has proven to be useful for the functional expression of cat L1 and L2 (Dowd et al., 1994, Dowd et al., 1997), cat L5 (Smooker et al., 2000) and cat B (Beckham et al., 2006, Law et al., 2003). Cat L5 is an adult-stage protease, while cat B is primarily juvenile stage (Wilson et al., 1998); note however that cat B transcripts have been detected in adult parasites, see for example Meemon et al. (2004). In this study we evaluate the vaccine potential of liver fluke multivalent vaccines using juvenile and adult-stage liver fluke derived recombinant protein proteases in the rat fasciolosis model. This model has been chosen as it is an ideal means of assessing the performance of potential vaccine candidates against fasciolosis, and has been widely used for such purposes (Kesik et al., 2007, Kofta et al., 2000, Reszka et al., 2005). It is particularly useful in this instance for comparing the relative efficacy of the various vaccine formulations.

Section snippets

Plasmids and strains

Yeast-derived recombinant proteins were expressed in the pFLAG vector system. The cloning and expression of cat L5 (Smooker et al., 2000) and the cat B pro-protein (construct pFCatB) (Law et al., 2003) has been described. A construct encoding cathepsin L1g was provided by Luke Norbury (manuscript in preparation). This construct was made in exactly the same way as for cat L5 (Smooker et al., 2000). Note that each of these proteins was expressed as pro-cathepsins, which were not activated prior

Humoral responses induced by vaccination

Rats were bled on the day of challenge and the sera examined for antigen-specific reactivity. The titres to individual recombinant proteins are shown in Fig. 1. For cat B and L5, the group vaccinated with the single, homologous protein yielded the highest mean titres. However, this was not the case for cat L1g, where the highest titre was observed in the group vaccinated with cat L5 and L1g. Generally, vaccination with a mixture of proteins reduced the titre to the individual proteins, most

Discussion

Cysteine proteases make up a significant proportion of the protein present in the E/S of liver flukes. These proteases are likely to facilitate virulence by this parasite, by participating in immune evasion, tissue digestion and feeding. Cathepsin proteases in liver flukes are known to be temporally regulated. Although protein (or mRNA) from both cat L and cat B has been found in both juvenile and adult parasites, cat B and cat L predominate in the E/S material, respectively, in these stages.

Conclusion

Liver fluke multivalent vaccines using juvenile and adult-stage liver fluke derived recombinant protein proteases may be more effective than single component vaccines. The observation that a cocktail vaccine containing cat B and cat L5 can increase the protective efficacy over that of either protein delivered alone indicates that multivalent vaccines against parasites may not only be desirable, but for good levels of protection with acceptable adjuvants, a necessity.

Acknowledgements

We thank Luke Norbury for provision of the L1g yeast expression clone, and Dr Ramya Ramamoorthi for assistance.

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