WO2003097679A1 - Cystic echinococcosis protein and uses therefor - Google Patents

Abstract

A novel protein and encoding nucleic acid of the parasite, Echinococcus granulosus, are provided. The novel protein is expressed by all life cycle stages of Echinococcus granulosus or tape worm. The novel protein and nucleic acid may be useful in the detection of Echinococcus granulosus, the cause of hydatid disease, in humans and animals. More particularly, it is proposed that the recombinant protein may be used for serodiagnosis of hydatid antibodies in humans and animals and as a novel vaccine for the prevention and treatment of hydatid disease.

Description

TITLE

CYSTIC ECHINOCOCCOSIS PROTEIN AND USES THERFOR

FIELD OF INVENTION

THIS INVENTION relates to a novel protein of the parasite, Echinococcus granulosus, and to an isolated nucleic acid encoding same. This invention also relates to a diagnostic test for cystic echinococcosis, also termed hydatid disease.

More particularly, this invention relates to a recombinant protein for use in serodiagnosis of hydatid antibodies in humans and animals and also to the creation of a novel vaccine for the prevention and treatment of hydatid disease. BACKGROUND OF THE INVENTION

Cystic echinococcosis (CE), termed hydatid disease, is a parasitic disease caused by infection with the larval form (metacestode) of the tapeworm, Echinococcus granulosus (E. granulosus). E. granulosus is a major zoonosis of worldwide distribution and the disease is characterized by long term growth of metacestode cysts in humans and domestic animals. About 2.7 million people and many millions of livestock are infected with the disease and it is responsible for significant economic loss of livestock in the endemic area which includes the countries of South America, Asia and North Africa, the countries bordering the Mediterranean basin and Australia. Hydatid cysts of E. granulosus develop in internal organs (mainly the liver and lungs) of humans and intermediate hosts (herbivores such as sheep, horses, cattle, pigs, goats and camels) as unilocular fluid-filled bladders. The fluid-filled bladders consist of two parasite-derived layers, an inner nucleated germinal layer and an outer acellular laminated layer surrounded by a host-produced fibrous capsule. The brood capsules and protoscoleces bud offfrom the germinal membrane. Sexual maturity of adult tapeworm does not occur in the intermediate hosts but within the small intestine of the definitive hosts which are carnivores, such as dogs, wolves and foxes. The resulting gravid proglottids or released eggs are shed in the faeces and, following their ingestion by a human or ungulate host, an oncosphere larva is released that penetrates the intestinal epithelium into the lamina propria. The larva is then transported passively through blood or lymph to the target organs where it develops into a hydatid cyst. Since the life cycle relies on carnivores eating infected herbivores, humans are usually a 'dead-end' for the parasite. The life-span of hydatid cysts of E. granulosus can be as long as 53 years in humans and 16 years in horses. The ability of the parasite to survive for such a long time in hosts with the potential to resist infection implies that the parasite possesses strategies for subverting or avoiding protective immune responses. The immunology of hydatid disease has been divided conceptually into pre- encystment and post-encystment phases that are differentiated by the formation of the laminated layer around the hydatid cyst. This occurs between 2-4 weeks post- infection in the animal intermediate or human host following ingestion of the egg and release of the oncosphere. At present, definitive clinical diagnosis for hydatid disease is based on symptomatology and is implemented by physical imaging methods, such as radiology, ultrasonography, computed axial tomography (CT scanning) and magnetic resonance imaging. The early stages of infection are asymptomatic, therefore the clinical symptoms of the disease are not detectable until the hydatid cysts have reached a certain size, which is typically a number of years after the primary infection. Early diagnosis could provide significant improvements in the quality of the management and treatment of the disease because surgery and chemotherapy are poorly effective when administered to patients with advanced infections. Early diagnosis is also vital for disease management in cattle and other livestock. Early diagnosis and subsequent treatment could prevent the spread of disease throughout whole herds of animals and would therefore limit huge economic loss that could result from a disease epidemic.

Diagnostic techniques that are relatively easy to use and cheap are required for large scale screening of populations at high risk. Immunodiagnosis is also thought to be the best way to screen domestic animal hydatid infections, such as for animal quarantine purposes. Detection of circulating E. granulosus antigens in sera is less sensitive than antibody detection in sera, therefore the discovery and synthesis of a selective E. granulosus antigen that would bind specifically to the E. granulosus antibodies in infected sera is the key factor in developing immunodiagnosis. Up to now, there is no commercial kit (at least based on a defined antigen) for the serodiagnosis of hydatid disease.

Attempts to identify hydatid cyst fluid antigens for immunodiagnosis have resulted in the biochemical characterisation of two important E. granulosus antigens, antigen B (AgB) and antigen 5 (Ag5).

AgB is a polymeric lipoprotein with a molecular weight of 120 kDa (Oriol and Oriol, 1975, Am. J. Trop. Med. Hyg., 24, 96- 100). It can be measured in patient blood as circulating antigen and it has been suggested that AgB has an important role in the biology of the parasite and its relationship with the host (Shepherd et al, 1991 , Mol. Biochem. Parasitol., 44, 81-90; Rigano et al, 2001, Infect. Immun., 69, 288- 296). AgB is a highly immunogenic molecule, a characteristic that underpins its value in serodiagnosis. To date, several AgB cDNAs have been cloned, expressed as recombinant proteins and used for diagnosis; in addition, a number of AgB peptides have been synthesised and used in ELISA for diagnostic purposes. Peptide antigens have been considered as a way to enhance specificity and efforts have been made to define discrete epitopes of AgB and other molecules that could be mimicked by synthetic peptides.

Ag5 is a very high molecular weight lipoprotein complex composed of 57- and 67-kDa components that under reducing conditions dissociate into 38- and 22- to 24-kDa subunits (Lightowlers etal, 1989, Mol. Biochem. Parasitol., 37, 171-182). Historically, one of the most used immunodiagnostic procedures for hydatid disease was the demonstration of serum antibodies precipitating Ag5 (arc 5) by immunoelectrophoresis or similar techniques. Early work suggested absolute diagnostic specificity for detecting E. granulosus infection, but subsequent studies showed that Ag5 is cross-reactive with human antibodies to other taeniid cestodes, most notably E. multilocularis and Taenia solium and, indeed, other helminths (Shepherd and McManus, 1987, Mol. Biochem. Parasitol., 25, 143-154).

Although AgB and Ag5 have proved to be diagnostically valuable, there are difficulties related to their lack of sensitivity and specificity and problems with the standardization of their use (Babba et al, 1994, A. J. Trop. Med. Hyg., 50, 64-68). Cross-reactivity with antigens from other parasites, notably other taeniid cestodes (Liu et al, 1993, Parasitology, 106, 75-81; Poretti et al, 1999, Am. Trop. Med. Hyg., 60, 193-198; Ortona etal, 2000, Parasite Immunol., 22, 553-559; Rott etal, 2000, Acta Trop., 75, 331 -340), is a major problem. In addition, results from single and two dimensional electrophoresis and micro-sequencing have suggested that AgB and Ag5 comprise a family of proteins in cyst fluid which may complicate their use in diagnosis (Zhang and McManus, 1996, Parasite Immunol., 18, 597-606). OBJECT OF THE INVENTION

There is a need for the development of a reliable, inexpensive, highly specific and selective diagnostic test for E. granulosus infections. The object of the invention is to isolate a hydatid protein and encoding nucleic acid, that may be useful in diagnosis and treatment of hydatid disease, and in prevention of hydatid infection.

SUMMARY OF INVENTION In a first aspect, the invention provides an isolated protein comprising the amino acid sequence set forth in SEQ ID NO: 1. According to this aspect, the invention also provides antigenic and immunogenic peptide fragments of the isolated protein set forth in SEQ ID NO: 1, variants, derivatives and homologues of the isolated protein. Particular embodiments of said fragments are amino acid sequences as set forth in SEQ ID NO: 2 and SEQ ID NO: 3. In a second aspect, the invention provides an isolated nucleic acid encoding the isolated protein of the first aspect.

Preferably, the isolated nucleic acid has a nucleotide sequence as set forth in SEQ ID NO: 4.

According to this aspect, the invention also provides an isolated nucleic acid encoding a fragment of the isolated protein set forth in SEQ ID NO: 1 , as set forth in SEQ ID NO: 5 and SEQ ID NO: 6.

In a third aspect, the invention provides an antibody or antibody fragment that binds to the isolated protein of the first aspect.

In a fourth aspect, the invention provides a method for detecting hydatid infection in animals, said method including the steps of:

(i) contacting a biological sample with the isolated protein of the first aspect to thereby form a complex between said isolated protein and an antibody if present in said sample, and (ii) detecting the complex formed at step (i). Preferably, the biological sample is a biological fluid.

More preferably, the biological fluid is blood serum. Preferably, the assay is an immunoenzymatic assay. More preferably, the immunoenzymatic assay is a Western blot or ELISA. In a fifth aspect, the invention provides a method for the detection of a hydatid infection in an animal, said method including the steps of:

(i) contacting a biological sample with the antibody of the third aspect to thereby form a complex between said antibody and an isolated protein according to the first aspect if present in said sample; and

(ii) detecting the complex formed at step (i).

In a sixth aspect, the invention provides a kit for detecting a hydatid infection, comprising the isolated protein of the first aspect and one or more reagents that assist the detection of a complex formed between said isolated protein and an antibody.

In a seventh aspect, the invention provides a method for detecting a hydatid infection, including the step of detecting the isolated nucleic acid of the second aspect, or one or more fragments thereof, in a biological sample. Preferably, the biological sample is blood serum. According to this aspect, the invention provides a method for detecting a hydatid infection, including the step of amplifying the isolated nucleic acid of the second aspect, or one or more fragments thereof, prior to detection.

Preferably, amplification is performed using one or more primers selected

In an eighth aspect, the invention provides an expression construct comprising an isolated nucleic acid of the second aspect operably-linked to one or more regulatory sequences in an expression vector.

In a ninth aspect, the invention provides a host cell transfected with the expression construct of the eighth aspect. In a tenth aspect, the invention provides a method of producing an isolated protein of the first aspect, said method including the steps of:

(i) culturing a host cell according to the ninth aspect such that said isolated protein is expressed in said host cell; and (iii) isolating said recombinant protein. In an eleventh aspect, the invention provides a pharmaceutical composition for the prophylactic and/or treatment of hydatid disease in an animal, said pharmaceutical composition comprising the isolated protein of the first aspect, or fragments thereof, and a pharmaceutically acceptable carrier, diluent or excipient. In a particular embodiment, said pharmaceutical composition is an immunotherapeutic composition.

Preferably, said immunotherapeutic composition is a vaccine. Preferably, said animal is a mammal. In a twelfth aspect, the invention provides a method of treating or preventing a hydatid infection in an animal including the step of administering the composition of the eleventh aspect to the animal.

Preferably, said animal is a mammal.

Throughout this specification, "comprise", "comprises" and "comprising" are used inclusively rather than exclusively, and will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1A The partial amino acid sequence of the antigenic protein referred to as EpC 1

(SEQ ID NO: 1). Fig. IB

The C-terminal amino acid sequence of the antigenic protein referred to as EpCl (SEQ ID NO: 2). Fig. 1C

The N-terminal amino acid sequence of the antigenic protein referred to as EpCl (SEQ ID NO: 3). Fig. 2A

The nucleotide sequence (SEQ ID NO: 4) of the nucleic acid encoding the EpCl protein (SEQ ID NO: 1). Fig. 2B

The nucleotide sequence (SEQ ID NO: 5) of the nucleic acid encoding the EpCl protein fragment (SEQ ID NO: 2). Fig. 2C The nucleotide sequence (SEQ ID NO: 6) of the nucleic acid encoding the

EpCl protein fragment (SEQ ID NO: 3). Fig. 3 A comparison of the amino acid sequences of EpCl, a protein (Ts76) from Taenia solium from China (Ts C) and a protein (NC-3) from Taenia solium from Madagascar (Ts M).

Fig. 4

An overview of the isolation procedure for EpCl. Fig. 5

Western blot illustrating the sensitivity of hydatid cyst fluid proteins to infected mouse sera. 200 μl of hydatid cyst proteins (4 mg/ml) were electrophoresed on 12% polyacrolamide gel and transferred to a nitrocellulose membrane (NCM). The stripped NCMs were then probed with serum of mice infected with oncospheres. 'B' denotes a mouse with both i.v. and i.p. infection. 'M' denotes male, and 'F' denotes female.

Fig. 6

Western blot demonstrating the selectivity of EpCl for human sera, using sera from the Center for Disease Control, Atlanta, USA from confirmed cystic hydatid disease patients (n=30). 'HCF' denotes hydatid cyst fluid antigens.

Fig. 7

EF-hand motifs (A) searched with BLAST from GenBank and the GCG program showing the predicted secondary structure of EpC 1 (B). The grey bar at the top of the figure denotes the amino acid scale for EpCl (77 amino acids). Fig. 8

Immunolocalisation of EpCl in a metacestode of Echinococcus granulosus. Native EpCl expression is visualized by indirect immunofluorescence (panels C and D) with murine antibody against recombinant EpCl in protoscoleces and hydatid cyst. The figure shows immunofluorescence images (panels on right) with corresponding bright field images (panels on left). MP denotes mature protoscolece, IP denotes immature protoscolece; GL denotes germinal layer; LL denotes laminated layer; and HT denotes host tissues. The areas bound by antibody, and the relevant positions in the corresponding bright field areas are arrowed.

Fig. 9 RT-PCR demonstrating that EpC 1 is expressed at all stages of E. granulosus development. E. granulosus actin was used as the quality and quantity control.

Molecular markers (m; base pairs) are shown on the left of the figure. T denotes immature adult worms, 'M' denotes mature adult worms, 'P' denotes protoscoleces and 'O' denotes oncospheres.

Table 1

Diagnostic performance of recombinant EpCl protein in human sera infected with cystic echinococcosis and other parasitic diseases. Sensitivity % = tp/(tp+fn) x 100% = 76/100 = 76%

Specificity % = tn/(tn+fp) x 100% = 214/223 = 96% tp denotes true positives; fh denotes false negatives; fp denotes false positives; tn denotes true negatives.

Table 2 Diagnostic performances of recombinant EpC 1 protein and hydatid cyst fluid

AgB in human sera infected with cystic echinococcosis and other parasitic diseases. Data was obtained from the Center for Disease Control, Atlanta, USA.

Sensitivity % = tp/(tp+fh) x 100% = 102/111 = 91.9% (pre-surgery sera)

Specificity % = tn/(tn+fp) x 100% = 311/327 = 95.1% tp denotes true positives; f denotes false negatives; fp denotes false positives; tn denotes true negatives.

Table 3

Comparative performances of commercial kit (ELISA) for serodiagnosis of cystic echinococcosis (CE). Sensitivity for pre-surgery sera = 57.1%; specificity = 84.1%.

Table 4

Primers used to amplify the EpC 1 protein encoding nucleic acid sequence in RT-PCR: up-stream primer 1 (SEQ ID NO: 7); up-stream primer 2 (SEQ ID NO: 8); and down-stream primer (SEQ ID NO: 9). DETAILED DESCRIPTION OF INVENTION

The inventors have isolated and expressed a novel, antigenic protein which is expressed by all life cycle stages of E. granulosus and therefore is potentially valuable for use in the diagnosis and treatment of hydatid disease.

The sera of mice infected with oncospheres of E. granulosus, was used to screen a cDNA library of protoscoleces and a 77 amino acid protein was discovered, called EpCl, that reacted positively to the serum. EpCl was expressed in E. coli. and the recombinant EpCl was used to probe the sera of humans infected with hydatid and other similar parasitic diseases, for example, alveolar echinococcosis, Taenia solium cysticercosis, and schistosomiasis. EpCl demonstrated an unexpectedly high sensitivity (91.9% for pre-surgery sera) and specificity (an overall value of 95.6%) for sera infected with E. granulosus and limited antigenic activity for sera infected with other taeniid cestodes. For the purposes of this invention, by "isolated" is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical, synthetic or recombinant form.

By "protei ^'' is meant an amino acid polymer. The amino acids may be natural or non-natural amino acids, as are well understood in the art.

A "peptide'^'' is a protein having less than fifty (50) amino acids. A "polypeptide" is a protein having fifty (50) or more amino acids.

The invention provides an isolated protein, hereafter referred to as "EpC I protein". The amino acid sequence of EpCl protein is shown in SEQ ID NO: 1.

It will be appreciated that SEQ ID NO: 2 represents a C-terminal sequence initially deduced from the encoding sequence isolated by the present inventors. The subsequently-obtained N-terminal sequence is depicted by SEQ ID NO: 3.

In one embodiment, a "fragment" includes an amino acid sequence that constitutes less than 100%, but at least 10%, preferably at least 25%, more preferably at least 50% or even more preferably at least 75% of an isolated protein of the invention. Two examples of fragments of the EpCl protein are shown in SEQ ID NO: 2 and SEQ ID NO: 3.

In an alternative embodiment, fragments specifically exclude SEQ ID NO: 2.

In another embodiment, a "fragmenf is a small peptide, for example of at least 6, preferably at least 10 and more preferably at least 20 amino acids in length. Larger fragments comprising more than one peptide are also contemplated, and may be obtained through the application of standard recombinant nucleic acid techniques or synthesized using conventional liquid or solid phase synthesis techniques. Alternatively, peptides can be produced by digestion of a polypeptide of the invention with proteinases such as endoLys-C, endoArg-C, endoGlu-C and staphylococcus V8-protease. The digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques.

As used herein, "variant^'' EpCl proteins of the invention are proteins of the invention in which one or more amino acids have been replaced by different amino acids. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide (conservative substitutions).

Substantial changes in function are made by selecting substitutions that are less conservative. Generally, the substitutions which are likely to produce the greatest changes in a polypeptide' s properties are those in which (a) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, He, Phe or Val); (b) a cysteine or proline is substituted for, or by, any other residue; (c) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp) or (d) a residue having a bulky side chain (e.g., Phe or Tip) is substituted for, or by, one having a smaller side chain (e.g., Ala, Ser) or no side chain (e.g., Gly).

The aforementioned variants may fall within the scope of the term "homologues". As generally used herein, a "homologue" shares a definable nucleotide or amino acid sequence relationship with an EpC 1 nucleic acid or protein of the invention as the case may be. In one embodiment, protein homologues share at least 80%, preferably at least 90% and more preferably at least 95% sequence identity with any of the amino acid sequences of the invention.

Included within the scope of homologues are "orthologues", which are functionally-related polypeptides and their encoding nucleic acids, isolated from parasitic worms, such as Echinococcus species other than E. granulosus.

Terms used herein to describe sequence relationships between respective nucleic acids and proteins include "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity". Because respective nucleic acids/polypeptides may each comprise (1) only one or more portions of a complete nucleic acid/polypeptide sequence that are shared by the nucleic acids/polypeptides, and (2) one or more portions which are divergent between the nucleic acids/polypeptides, sequence comparisons are typically performed by comparing sequences over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incoφorated herein by reference) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al. , 1997, Nucl. Acids Res. 25 3389, which is incoφorated herein by reference. A detailed discussion of sequence analysis can be found in Unit 19.3 of

CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel etal. (John Wiley & Sons Ine NY, 1995-1999).

The term "sequence identity" is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, "sequence identity" may be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA).

In this particular invention, sequence homology is preferably compared over at least 12 contiguous residues, more preferably at least 30 contiguous residues, even more preferably at least 50 contiguous residues and advantageously at least 100 contiguous residues, or substantially the entire length of the protein or nucleic acid of the invention.

As used herein, "derivative" proteins of the invention are proteins, such as set forth in SEQ ID NOS: 1-3, which have been altered, for example by conjugation or complexing with other chemical moieties or by post-translational modification techniques as would be understood in the art. Such derivatives include amino acid deletions and/or additions to proteins of the invention, or variants thereof.

Other derivatives contemplated by the invention include, but are not limited to, modification to side chains, incoφoration of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the polypeptides, fragments and variants of the invention. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by acylation with acetic anhydride; acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; amidination with methylacetimidate; carbamoylation of amino groups with cyanate; pyridoxylation of lysine with pyridoxal-5 -phosphate followed by reduction with NaBHj; reductive alkylation by reaction with an aldehyde followed by reduction with NaBH₄; and trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS).

The carboxyl group may be modified by carbodimide activation via O- acylisourea formation followed by subsequent derivitization, by way of example, to a corresponding amide.

The guanidine group of arginine residues may be modified by formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

Sulphydryl groups may be modified by methods such as performic acid oxidation to cysteic acid; formation of mercurial derivatives using 4- chloromercuriphenylsulphonic acid, 4-chloromercuribenzoate; 2-chloromercuri-4- nitrophenol, phenylmercury chloride, and other mercurials; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; carboxymethylation with iodoacetic acid or iodoacetamide; and carbamoylation with cyanate at alkaline pH. Tryptophan residues may be modified, for example, by alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides or by oxidation with N-bromosuccinimide.

Tyrosine residues may be modified by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

The imidazole ring of a histidine residue may be modified by N- carbethoxylation with diethylpyrocarbonate or by alkylation with iodoacetic acid derivatives.

Examples of incoφorating unnatural amino acids and derivatives during peptide synthesis include but are not limited to, use of 4-amino butyric acid, 6- aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3- hydroxy-6-methylheptanoic acid, t-butylglycine, norleucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine and/or D-isomers of amino acids. Isolated proteins of the invention (inclusive of fragments, variants, derivatives and homologues) may be prepared by any suitable procedure known to those of skill in the art.

For example, the isolated EpCl protein may be prepared as a recombinant protein by a procedure including the steps of: (i) preparing an expression construct which comprises an isolated nucleic acid of the invention, operably-linked to one or more regulatory nucleotide sequences in an expression vector; (ii) transfecting or transforming a suitable host cell with the expression construct; and (iii) expressing the recombinant protein in said host cell.

An "expression vector" may be either a self-replicating extra-chromosomal vector such as a plasmid, or a vector that integrates into a host genome.

By "operably-linked^'' is meant that said regulatory nucleotide sequence(s) is/are positioned relative to the recombinant nucleic acid of the invention to initiate, regulate or otherwise control transcription.

Regulatory nucleotide sequences will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, splice donor/acceptor sequences and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by the invention and include, for example, tetracycline-repressible and metallothionin-inducible promoters. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter. In a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.

Suitable host cells for expression may be prokaryotic or eukaryotic, such as Escherichia coli (DH5α for example), yeast cells, SF9 cells utilized with a baculovirus expression system, CHO cells, COS, CV-1 and 293 cells, without limitation thereto.

The recombinant EpCl protein may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook et al, MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), incoφorated herein by reference, in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. 1995-1999), incoφorated herein by reference, in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, Inc. 1995-1999) which is incoφorated by reference herein, in particular Chapters 1, 5 and 6.

Isolated nucleic acids

The invention provides an isolated nucleic acid that encodes an isolated EpCl protein of the invention, hereinafter referred to as "EpCl nucleic acid". Preferred nucleotide sequences of the encoding nucleic acids are set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

It will also be appreciated that the invention includes within its scope variations in nucleotide sequences of the invention on account of degeneracy in the genetic code. Furthermore, the invention includes nucleotide sequences where codon sequences are selected on the basis of preferred usage in a particular organism. The latter sequences are particularly useful when a high level of encoded protein expression is required in said particular organism. The term "nucleic acid^'' as used herein designates single-or double-stranded mRNA, RNA, cRNA and DNA inclusive of cDNA and genomic DNA.

A "polynucleotide" is a nucleic acid having eighty (80) or more contiguous nucleotides, while an "oligonucleotide" has less than eighty (80) contiguous nucleotides. A "probe" may be a single or double-stranded oligonucleotide or polynucleotide, suitably labeled for the puφose of detecting complementary sequences in Northern or Southern blotting, for example.

A "primer" is usually a single-stranded oligonucleotide, preferably having

15-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid "template" and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or Sequenase™.

The present invention also contemplates homologues of isolated nucleic acids of the invention as hereinbefore defined. In one embodiment, nucleic acid homologues encode isolated protein homologues of the invention, inclusive of orthologues, variants, fragments and derivatives thereof.

In another embodiment, nucleic acid homologues share at least 70%, preferably at least 80%, more preferably at least 85%, and even more preferably at least 90% sequence identity with the isolated nucleic acids of the invention.

In yet another embodiment, nucleic acid homologues hybridise to isolated nucleic acids of the invention under at least low stringency conditions, preferably under at least medium stringency conditions and more preferably under high stringency conditions. "Hybridise and Hybridisation" is used herein to denote the pairing of at least partly complementary nucleotide sequences to produce a DNA-DNA, RNA-RNA or DNA-RNA hybrid. Hybrid sequences comprising complementary nucleotide sequences occur through base-pairing between complementary purines and pyrimidines as are well known in the art.

In this regard, it will be appreciated that modified purines (for example, inosine, methylinosine and methyladenosine) and modified pyrimidines (for example, thiouridine and methylcytosine) may also engage in base pairing. "Stringency" as used herein, refers to temperature and ionic strength conditions, and presence or absence of certain organic solvents and/or detergents during hybridisation. The higher the stringency, the higher will be the required level of complementarity between hybridising nucleotide sequences.

"Stringent conditions" designates those conditions under which only nucleic acid having a high frequency of complementary bases will hybridise.

By way of example, high stringency conditions include and encompass:-

(i) from at least about 31 % v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridisation at 42°C, and at least about 0.01 M to at least about 0.15 M salt for washing at 42°C; (ii) 1% BSA, 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridisation at 65°C, and (a) 0.1 x SSC, 0.1% SDS; or (b) 0.5% BSA, lmM EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS for washing at a temperature in excess of 65°C for about one hour; and

(iii) 0.2 x SSC, 0.1% SDS for washing at or above 68°C for about 20 minutes.

In general, washing is carried out at T_m = 69.3 + 0.41 (G + C) % -12°C. In general, the T_m of a duplex DNA decreases by about 1°C with every increase of 1% in the number of mismatched bases.

Notwithstanding the above, stringent conditions are well known in the art, such as described in Chapters 2.9 and 2.10 of. Ausubel et al, supra, which are herein incoφorated be reference. A skilled addressee will also recognize that various factors can be manipulated to optimize the specificity of the hybridisation. Optimisation of the stringency of the final washes can serve to ensure a high degree of hybridisation.

Typically, complementary nucleotide sequences are identified by blotting techniques that include a step whereby nucleotides are immobilised on a matrix

(preferably a synthetic membrane such as nitrocellulose), a hybridization step, and a detection step. Southern blotting is used to identify a complementary DNA sequence; northern blotting is used to identify a complementary RNA sequence. Dot blotting and slot blotting can be used to identify complementary DNA/DNA, DNA/RNA or RNA/R A polynucleotide sequences. Such techniques are well known by those skilled in the art, and have been described in Ausubel et al, supra, at pages 2.9.1 through 2.9.20. According to such methods, Southern blotting involves separating DNA molecules according to size by gel electrophoresis, transferring the size-separated DNA to a synthetic membrane, and hybridising the membrane bound DNA to a complementary nucleotide sequence.

In dot blotting and slot blotting, DNA samples are directly applied to a synthetic membrane prior to hybridisation as above. An alternative blotting step is used when identifying complementary nucleic acids in a cDNA or genomic DNA library, such as through the process of plaque or colony hybridisation. Other typical examples of this procedure is described in Chapters 8-12 of Sambrook et al, supra which are herein incoφoated by reference.

Methods for detecting labeled nucleic acids hybridised to an immobilised nucleic acid are well known to practitioners in the art. Such methods include autoradiography, chemiluminescent, fluorescent and colorimetric detection.

Homologues of EpCl nucleic acids of the invention may also be prepared by methods utilising nucleic acid sequence amplification techniques. In one embodiment, the method includes the steps of: (i) obtaining a nucleic acid extract;

(ii) creating one or more primers which, optionally, are degenerate wherein each said primer corresponds to a portion of a nucleic acid of the invention set forth in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; and (iii) using said primers to amplify, via a nucleic acid amplification technique, one or more amplification products from said nucleic acid extract. Suitably, said one or more primers are designed to be capable of annealing to one or the other strands of a double-stranded nucleic acid of the invention under annealing conditions typically used for amplification. In the case of degenerate primers, sequence differences between the primer and the nucleic acid sequence are introduced so as to account for possible sequence variation, such as due to degeneracy in homologous coding sequences. Non-limiting examples of primers are set forth in SEQ ID NOS; 7-9 and Table 4.

According to this method, the nucleic extract could be obtained from a species other than E. granulosus, although the method is not limited thereto. Nucleic acid amplification techniques are well known to the skilled addressee, and include polymerase chain reaction (PCR) and ligase chain reaction

(LCR) as for example described in Chapter 15 of Ausubel et al. supra, which is incoφorated herein by reference; strand displacement amplification (SDA) as for example described in U.S. Patent No 5,422,252 which is incoφorated herein by reference; rolling circle replication (RCR) as for example described in International

Application WO 92/01813 and International Application WO 97/19193, which are incoφorated herein by reference; nucleic acid sequence-based amplification

(NASBA) as for example described by Sooknanan et α/.,1994, Biotechniques 17

1077, which is incoφorated herein by reference; and Q-β replicase amplification as for example described by Tyagi et al, 1996, Proc. Natl. Acad. Sci. USA 93 5395 which is incoφorated herein by reference.

A preferred nucleic acid sequence amplification technique is PCR.

As used herein, an "amplification product refers to a nucleic acid product generated by nucleic acid amplification techniques. Expression constructs

The invention also provides an expression construct which comprises an isolated nucleic acid of the EpCl protein or homologue thereof, operably-linked to one or more regulatory sequences in an expression vector.

Regulatory nucleotide sequences present in the expression vector (such as an enhancer, promoter, splice donor/acceptor signals, terminator and polyadenylation sequences) that will facilitate expression of the protein of the invention. Selectable markers are also useful whether for the puφoses of selection of transformed bacteria (such as bla, kanR and tetR) or transformed mammalian cells (such as hygromycin, G418 and puromycin). Both constitutive and inducible promoters may be useful for expression of the proteins of the invention. Examples of inducible promoters are metallothionine- inducible and tetracycline-repressible systems as are well known in the art.

An expression construct may also include a fusion partner sequence as hereinbefore defined (for example, GST) so that the recombinant polypeptide of the invention is expressed as a fusion polypeptide with said fusion partner. An example of a GST fusion expression construct is provided in detail hereinafter. Antibodies The invention also provides antibodies against the isolated EpC 1 proteins of the invention, inclusive of fragments, variants and derivatives. Antibodies of the invention may be polyclonal or monoclonal. Well-known protocols applicable to antibody production, purification and use may be found, for example, in Chapter 2 of Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons NY, 1991-1994) and Harlow, E. & Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory, 1988, which are both herein incoφorated by reference.

Generally, antibodies of the invention bind to or conjugate with a polypeptide, fragment, variant or derivative of the invention. For example, the antibodies may comprise polyclonal antibodies. Such antibodies may be prepared, for example, by injecting a polypeptide, fragment, variant or derivative of the EpCl protein into a production species, which may include mice or rabbits, to obtain polyclonal antisera. Methods of producing polyclonal antibodies are well known to those skilled in the art. Exemplary protocols which may be used are described for example in Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY, supra, and in Harlow & Lane, 1988, supra.

In lieu of the polyclonal antisera obtained in the production species, monoclonal antibodies may be produced using the standard method as for example, described in an article by Kohler & Milstein, 1975, Nature 256, 495, which is herein incoφorated by reference, or by more recent modifications thereof as for example, described in Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY, supra by immortalizing spleen or other antibody producing cells derived from a production species which has been inoculated with one or more of the polypeptides, fragments, variants or derivatives of the invention. The invention also includes within its scope antibodies which comprise Fc or

Fab fragments of the polyclonal or monoclonal antibodies referred to above. Alternatively, the antibodies may comprise single chain Fv antibodies (scFvs) against the EpCl proteins of the invention. Such scFvs may be prepared, for example, in accordance with the methods described respectively in United States Patent No 5,091,513, European Patent No 239,400 or the article by Winter & Milstein, 1991, Nature 349 293, which are incoφorated herein by reference. Labels may be associated with an antibody of the invention, or antibody fragment, as follows: (A) direct attachment of the label to the antibody or antibody fragment;

(B) indirect attachment of the label to the antibody or antibody fragment; i. e., attachment of the label to another assay reagent which subsequently binds to the antibody or antibody fragment; and

(C) attachment to a subsequent reaction product of the antibody or antibody fragment.

The label may be selected from a group including a chromogen, a catalyst, an enzyme, a fluorophore, a chemiluminescent molecule, a lanthanide ion such as Europium (Eu³⁴), a radioisotope and a direct visual label. In the case of a direct visual label, use may be made of a colloidal metallic or non-metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.

A large number of enzymes useful as labels is disclosed in United States Patent Specifications U.S. 4,366,241, U.S. 4,843,000, and U.S. 4,849,338, all of which are herein incoφorated by reference. Enzyme labels useful in the present invention include, for example, alkaline phosphatase, horseradish peroxidase, luciferase, β-galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like. The enzyme label may be used alone or in combination with a second enzyme in solution.

The fluorophore may, for example, be fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITL), allophycocyanin (APC), Texas Red, Cy5, Cy3, or R-Phycoerythrin (RPE) as are well known in the art. Detection methods and kits

The detection methods of the invention generally fall into two broad classes: (i) detection of the nucleic acids encoding the EpCl proteins, and (ii) detection of EpCl proteins or antibodies that bind said protein.

Detection of the nucleic acid encoding the EpCl protein, or fragments thereof, can be performed using a nucleic acid sequence amplification technique, such as PCR, which will be described in detail in the Examples. Identity of the amplification products produced according to this method can be determined by any of well known techniques such as nucleotide sequencing, agarose gel electrophoresis and hybridization as hereinbefore described.

There are a variety of well known detection techniques for detecting EpCl proteins and serum antibodies in biological samples.

EpCl proteins present in a biological sample may be detected, or antibodies present in a biological sample (such as serum) may be detected by binding to EpCl proteins or peptides derived therefrom.

Such methods are generally referred to herein as "immunodiagnostic" methods of the invention.

Immunodiagnostic detection of the EpC 1 protein may be performed by any of a number of techniques, such as immunoblotting, immunochromatography, Enzyme- Linked Immunosorbent Assay (ELISA) and immunohistochemistry as are well known in the art. A detailed discussion of ELISA can be found in Unit 11.2, CURRENT

PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. 1995-1999). Examples of uses of ELISA for the detection of hydatid antigen reactivity with sera samples can be found in Rott et al, 2000 (Acta Tropica, 75, 331 -340) and Gonzalez-Sapinenza et al, 2000 (Journal of Clinical Microbiology, 38, 3979-3983). ELISA may also be provided in kit form comprising magnetic beads to which are bound a plurality of peptides having EpCl -specific epitopes.

The kit may still further comprise a secondary antibody according to the animal species being tested, which antibody binds complexes formed between tested antisera and said peptide-coated beads. Preferably, said secondary antibody includes a label such as alkaline phosphatase or horseradish peroxidase, which enzyme labels are useful with typical ELISA colour reaction substrates as are well known in the art.

It will be appreciated that a preferred method detects antibodies, preferably in serum, obtained from an animal, such as a human. The presence of an antibody- EpCl protein complex indicates that said animal has had sufficient exposure to hydatid disease to result in production of anti-EpCl antibodies.

Preferably, detection of such complexes is performed by Western blot, or immunoblot, as is described in detail in Unit 10.8, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. 1995- 1999), and will be described in the Examples of this document. Nucleic acid detection kits may comprise: (i) PCR primers; (ii) hybridization probes; and

(iii) one or more reagents for detecting hybridized nucleic acids and/or PCR amplification products (preferably labeled with, for example, digoxigenin). Non-limiting examples of primers are set forth in SEQ ID NOS: 7-9 and Table 4.

Pharmaceutical compositions and vaccines

A further aspect of the invention is the use of EpCl for the therapeutic and prophylactic treatment of hydatid infection. Production of EpCl of the invention for use as a vaccine could prove invaluable both clinically and economically in the prophylactic treatment of humans and livestock in the endemic areas.

The invention therefore provides pharmaceutical compositions that comprise at least one EpCl isolated protein, nucleic acid and/or expression construct of the invention (immunogenic agents).

Pharmaceutical compositions comprising immunogenic agents are generally referred to herein as "immunotherapeutic compositions". Preferably, the immunotherapeutic composition elicits a protective immune response and is herein referred to as a "vaccine".

Suitably, the pharmaceutical composition comprises an appropriate pharmaceutically-acceptable carrier, diluent or excipient. By "pharmaceutically-acceptable carrier, diluent or excipient" is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of admimstration, a variety of carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates and pyrogen-free water. A useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. NJ. USA, 1991) which is incoφorated herein by reference.

Any safe route of administration may be employed for providing a patient with the composition of the invention. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed. Intra-muscular and subcutaneous injection is appropriate, for example, for administration of immunogenic compositions, vaccines and DNA vaccines.

Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this puφose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.

The above compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is pharmaceutically-effective, or immunogenically-effective to protect an animal or human from a hydatid infection. The dose administered to a patient, in the context of the present invention, should be sufficient to affect a beneficial response in a patient over an appropriate period of time. The quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner.

It is preferred that immunogenic compositions, such as vaccines, include an adjuvant. As will be understood in the art, an "adjuvant" means a composition comprised of one or more substances that enhances the immunogenicity and efficacy of a vaccine composition. Non-limiting examples of suitable adjuvants include squalane and squalene (or other oils of animal origin); block copolymers; detergents such as Tween®-80; Quil® A, mineral oils such as Drakeol or Marcol, vegetable oils such as peanut oil; Corynebacterium-deήved adjuvants such as Corynebacterium parvum; Propionibacterium-deήved adjuvants such as Propionibacterium acne; Mycobacterium bovis (Bacille Calmette and Guerin or BCG); interleukins such as interleukin 2 and interleukin 12; monokines such as interleukin 1 ; tumour necrosis factor; interferons such as gamma interferon; combinations such as saponin-aluminium hydroxide or Quil-A aluminium hydroxide; liposomes; ISCOM® and ISCOMATRLX® adjuvant; mycobacterial cell wall extract; synthetic glycopeptides such as muramyl dipeptides or other derivatives; Avridine; Lipid A derivatives; dextran sulfate; DEAE-Dextran or with aluminium phosphate; carboxypolymethylene such as Carbopol' EMA; acrylic copolymer emulsions such as Neocryl A640 (e.g. U.S. Pat. No. 5,047,238); vaccinia or animal poxvirus proteins; sub-viral particle adjuvants such as cholera toxin, or mixtures thereof.

So that the present invention may be more readily understood and put into practical effect, the skilled person is referred to the following non-limiting examples.

EXAMPLES Materials and Methods Dogs Dogs used for production of adult worms were purchased from villages close to Urumqi, Xinjiang, China. All dogs were a local strain, aged 8-12 months and maintained in a special designed animal house in the Veterinary Research Institute, Xinjiang Academy of Animal Science, Urumqi, China, and fed with boiled sheep bond and meat, lungs and livers, biscuits and given tap water. The dogs were treated with 10 mg/kg (body weight) of praziquantel and followed with 20 mg/kg (body weight) of albendazole after a week. The dogs were then used for infection two weeks later. Mice

Chinese Kunming white (CKW) mice were used for infection experiments. The CKW mice were bred and supplied by the Xinjiang Experimental Centre, Urumqi, China, and maintained in the animal house at Xinjiang Veterinary Research Institute. All mice were 6-8 weeks of age at the commencement of experiments and 5-6 males and 5-6 females were allocated to each group. BALB/c mice used for the production of antiserum were purchased from Animal Resources Centre, Willeton, Australia and housed in animal care facilities under specific pathogen-free conditions in the Queensland Institute of Medical Research, Queensland, Australia. Protoscoleces

Sheep hydatid cysts were collected from a slaughter house in Urumqi, Xinjiang, China. Brood capsules and protoscoleces were aspirated and washed 10 times with PBS, then the protoscoleces were aliquoted into cryotubes. After the parasites had sedimented, residual PBS was aspirated and the packed protoscoleces were stored in liquid nitrogen until used. Immature and mature adult worms To obtain the adult worms, dogs were orally fed with the protoscoleces suspended in about 50 ml of hydatid cyst fluid. The worms were collected on day 35 (immature adult worms) and 62 (mature adult worms with 37.5% harbouring eggs) post-infection. Dogs were not fed, but were allowed water one day before being sacrificed. Sections of parasitised intestines were opened and soaked in 37°C normal saline (0.9% w/v NaCl) until all worms had been released. Recovered worms were washed ten times by repeated decantation of excess warm saline, followed by two washes in warm 2% (w/v) sodium bicarbonate to dissolve residual mucus, then with final two washes in PBS. Worms were aliquoted into cryotubes and stored in liquid nitrogen until used. Eggs

Eggs were released from mature worms by homogenizing the worms in an electric blender. The homogenate was sieved through a 132 μm sieve and the sheared worm material was retained. The eggs were further washed and retained on a 20 μm mesh. The washed eggs were stored in PBS containing 1000 i.u./ml benzyl penicillin and 1000 μg/ml streptomycin sulphate at 4°C (Osborn and Heath, 1982, Res. Vet. Sci., 33, 132-133). Oncospheres

Eggs were incubated in 50 ml screw-capped tubes at 37°C for 45 min in a sterile solution of 1% (w/v) pepsin (Sigma, 1:2500) and 1% (v/v) HCI in 0.85% NaCl (w/v). After centrifugation (500g, 5 min), the pepsin solution was poured off. The eggs were washed once with PBS and incubated in a sterile solution of 1 % (w/v) pancreatin (Sigma, 4 U.S.Pharmacopeia.), 1% (w/v) NaHCO3 and 5% (v/v) sterile sheep bile. The oncospheres were checked every 2 min with a microscope until all the oncospheres had been released from the embryonic membranes. The oncospheres were centrifuged (l,000g, 5 min). The supernatant was discarded and the oncospheres were washed twice with Hank's balanced salts solution. After the final wash, activation of oncospheres was estimated by placing a drop of suspension on a slide and adding a drop of 0.1 % (w/v) Trypan blue (Osborn and Heath, 1982). Activated oncospheres were hatched from eggs by using the method described by Osborn and Heath (1982). The oncospheres were further isolated by density-gradient separation with 100% Percoll (Sigma, USA) (Rajasekariah et al, 1980, J. Parasitol., 66, 355-356). After being washed 3 times with PBS, the supernatant was discarded and the oncospheres were used immediately to infect mice or were frozen in liquid nitrogen for molecular studies. Sera collection Mouse serum

Chinese Kunming white (CKW) mice, aged 6-8 weeks at the start of the experiments, were divided into three groups, each group given a different route of infection with the activated oncospheres. The first group of 7 mice (3 male, 4 female) were administered the oncospheres by intraperitoneal (i.p.) injection, the second group of 13 mice (7 male, 6 female) were first given an i.p. injection and then a second intravenous (i.v.) injection was administrated 21 days after the first injection. The third group of 6 mice (3 male, 3 female) were administered the oncospheres by i.v. injection only. The mice were sacrificed using CO₂ at 26 weeks after the first infection and the fluid-filled cysts in the peritoneal and /or thoracic cavities were individually counted and measured for size. Sera were collected and stored at - 20 °C for further uses. 26 normal mouse sera samples were collected from normal CKW mice as serum controls for the experiment; 20 sera samples were taken from mice (B ALB/c) infected with 10⁴ parasited red blood cells of Plasmodiun yoelii rodent malaria and cured with pyrimethamine (3 x 0.2 mg) intraperitonally when the parasitemia went up 5-10%, and 20 sera samples were taken from mice infected with schistosoma japonicum. Human serum Example 1

A total of 323 human sera samples were used in the study. One hundred sera samples were obtained from surgically or ultrasound confirmed CE patients. 25 sera samples were obtained from patients with cystic hydatid disease, (a gift from Dr. Zhu Bin, Xinjiang People's Hospital, Urumqi, Xjinjang, China), and 20 sera samples were kindly supplied by Professor Wen Hao, (First Experimental Hospital of Xinjiang Medical University, Urumqi, Xinjiang, China). 23 samples were obtained from Australia and 32 from France (Professor Ito, Department of Parasitology Asahikawa Medical College, Asahikawa, Japan). 20 sera samples were obtained from healthy donors, but donors living in the high endemic area of Xinjiang, China. 203 sera samples were obtained from patients with other parasite diseases (provided by Professor Ito), including alveolar echinococcosis (AE) (89 individual sera samples; 60 from Japan, 20 from France, and 9 from China), Taenia solium cysticercosis (72 individual sera samples; 21 from Cameroon, 13 from South America, 27 from China, 6 from USA and 5 from France), fascioliasis (15), schistosomiasis (8), paragonimiasis (6), spirometra (sparganosis) (5), trichinosis (5), toxocariasis (2) and entamoebiasis (1). Example 2

In collaboration with Dr Marianna Wilson and Dr Peter Schantz at The Center for Disease Control, Atlanta, USA, the specificity and sensitivity of the EpCl diagnostic protein was tested on 646 human sera samples, including sera from individuals with confirmed cystic echinococcosis (CE) (pre- and post surgery; different country origins) (n=319), neurocysticercosis (NCC) (n=100), schistosomiasis (50), alveolar echinococcosis (n=29), other parasitic infections (13 different parasite species) (n=80), liver cancer (n=12), liver abscesses (n=7) and confirmed negative controls (n=50) (Table 2). Preparation ofanti-EpCl rabbit serum A rabbit was immunized subcutaneously with 200-300 μg of highly pure

EpC 1 in Freund's Complete Adjuvant. After two, four and six weeks, the rabbit was boosted with 200-300 μg EpCl in Freund's Incomplete Adjuvant, and then bled for serum two weeks after the third boost. RNA extraction Total RNA was isolated from the protoscoleces of E. granulosus of sheep hydatid origin using TRIzol Reagent (Gibco, BRL Life Technologies Inc., Gaithersbarg, MA) according to the manufacturer's instructions. mRNA was extracted using Oligotex mRNA Mini Kit (Qiagen). Briefly, 2 mg of total RNA was pipetted into an RNase-free 1.5 ml microcentrifuge tube, and RNase-free water was added to 500 μl. After the same volume of buffer OBB (supplied) and 45 μl of Oligotex suspension were added, the tube was incubated for 3 min at 70 °C in a water bath. The tube was then kept at room temperature for 20 min and centrifuged for 2 min at 14,000x g to pellet the Oligotex/mRNA. The supernatant was removed by pipetting. The Oligotex beads were suspended in 400 μl of buffer OW2 (supplied) and pipetted into a spin column and centrifuged for 1 min at 12,000x g. After one more wash with OW2, the mRNA was eluted by adding 2 x 50 μl hot elute buffer (supplied) and spinning down at 12,000x g for 1 min. The eluted mRNA was precipitated with 1/10 of 3 M potassium acetic acid and 0.8 volume of isopropanol. The pellet was washed with 75% ethanol and dissolved in 10 μl of water (DEPC- treated). Construction of a protoscolex cDNA library cDNA was synthesised using the Stratagene cDNA synthesis kit (Stratagene, La Jolla, CA) according to manufacturer's instructions commencing with 5 μg of poly(A)⁺ RNA. Immunoscreening of the cDNA library

The protoscolex cDNA library was immunoscreened using a pool of 20 mice sera samples (infection sera of mice, ISM) infected with oncospheres of E. granulosus. Briefly, XLl-blue MRF' host cells were incubated with 1 μl of an optional dilution of the library, and plated on NZY agar plates. The agar plates were incubated at 42 °C for 4.5 h, transferred to a 37°C incubator and overlaid with 10 mM IPTG-nitrocellulose membranes. The membranes were orientated on the plates using a code of punctures that penetrated the membrane and agar surface, and the plates were incubated at 37 °C for a further 4 h. Each membrane was removed, air dried, and a positive control of dilute HCF (hydatid cyst fluid) was applied before being washed in PBS/T (PBS containing 0.05% Tween 20) (3x15 min) and blocked overnight at 4 °C in 5% skim milk in PBS. Following the blocking step, all incubation steps were carried out at room temperature on a moving platform with the membranes being washed thoroughly in 3x15 min washes after each step. The membranes were washed in PBS/T, incubated for 2 h with ISM (diluted to 1 in 100), and further washed 3 times in PBS/T. The secondary antibody of horseradish peroxidase conjugated sheep anti-mouse Ig (SILENUS) was diluted 1 : 2000 in PBS/T, and incubated with the membranes for 2 h. The membranes were developed in 45 ml of PBS containing 30 μl of 30% (v/v) hydrogen peroxide and 5 ml of the chromogenic substrate 4-chloro-l-naphthol (60 mg stack solution of 4-chloro-l- naphthol in 20 ml of ice cold methanol) after three washes in PBS/T and a final wash in PBS only. Fusion proteins adsorbed to the membranes recognised by the infected mouse serum appeared as pink to puφle coloured spots, with colour intensity indicating potential immunoreactivity. Positive plaques were identified by aligning the membrane with the agar plate, and plaques of interest were removed using a sterile Pasteur pipette and transferred to 500 μl of sterile SM buffer (100 mM NaCl, 10 mM MgSO4, 50 mM Tris-HCl, (pH 7.5), 2% (w/v) of gelatin). Twenty microlitres of chloroform was added to the phage stock to lyse the host cells, and the stock was stored at 4°C. The positive cloned phages were re-cloned by the same methods as described above for immunoscreening. Subcloning EpCl fragments intopET-41(b+) The cloned phage particles were concerted into pBK-CMN phagemid vectors

(Stratagene) by in vitro excision using a suitable helper phage. The EpCl insert was removed from the pBK-CMN phagemid vector by digesting 1 μg of plasmid DΝA with 2 Units of BamHl (ΝEB) and Not I (ΝEB) at 37 °C for 1 h. The digests were run on a 0.8% agarose gel and the cDΝA insert excised and purified using a QIAquick gel extraction kit (Qiagen). The pET -41 (b+) expression vector (Novagen) was chosen for expressing the EpCl protein. The restriction enzymes, BamHl (5') and Not I (3'), were found suitable for subcloning EpCl into the pET-41(b+) expression vector. After the pBK-CMN phagemid vector containing the EpCl insert and pET-41(b+) was cut with BamH I and Not I, the EpCl and linearised pET41- (b+) were run on a 0.8% agarose gel and extracted using a Quantum Prep® Freeze 'N Squeeze DNA Gel Extraction Spin Column (Bio-Rad), and precipitated with ethanol, respectively. The insert and linearised plasmid concentration were determined spectrophotometrically and the ligation reactions were set up using an estimated ratio of six parts insert to one part vector. Each ligation reaction (1.5 μl) was transformed into 40 μl of competent BL21 (DE3) (Novagen) cells with aliquots plated on LB agar containing kanamycin (30μg/ml), and incubated overnight at 37 °C. Plasmid DNA was extracted from the transformants and the cDNA insert size was determined by digesting the plasmid DNA with BamH I and Not I, and visualising the digests on an agarose gel. The DNA inserts of potentially the correct size were sequenced using the pET41-(b+) forward direction primer, to determine whether the insert was in frame with the vector's start codon. Then, the clone was selected for the expression of recombinant protein. Expression of recombinant EpCl

EpCl was expressed as a GST fusion protein. A single colony was used to inoculate 50 ml of LB broth containing kanamycin (30 μg/ml) (LB+kan) and incubated at 37 °C overnight. Three 1 L bottles each containing 350 ml of LB+kan were incubated with 10 ml of overnight culture and incubated at 37 °C in an orbital shaker (220 φm) until the culture had reached an OD600 of 0.6-0.8. Protein production was induced by the addition of lmM IPTG (final concentration) at 30 °C for an overnight culture. Purification of recombinant protein

Recombinant fusion EpCl was purified using GST-Bind™ Kits (Novagen) under native conditions. Briefly, the cell culture was centrifuged at 5,000x g for 10 min at 4 °C. The pellet was resuspended with GST binding buffer, frozen and thawed for 3 times and ultrasonicated on ice for 4 min using a gentle pulse (30% duty cycle, Sonifier 250, Branson) to lyse the cells. The cell lysis was centrifuged at 12,000x g for 30 min. The supernatant containing the crude protein was diluted with a GST binding buffer to 3-5 mg/ml and loaded through a column containing GST affinity resin (Novagen). The column was then washed with GST binding buffer until no protein excited in the drops measured by protein assay dye (Bio-Rad) (Bradford, 1976, Anal. Biochem., 72, 248-254). The recombinant protein was eluted by elute buffer (supplied). The concentration of the protein was measured by Bio- Rad protein assay dye (Bradford, 1976, supra). Western blot to screen mouse and human sera

The purified GST fusion protein (100 μl of 0.1 mg/ml) was mixed with 1/6 volume of 6x loading buffer and boiled at 100 °C for 3 min and subjected to electrophoresis on 12% SDS-PAGE gels (Laemmli, 1970, Nature, 227, 680-685). The separated protein was then transferred to a sheet of nitrocellulose membrane at 190 mA for 2 h, in 190 mM glycine, 25 mM Tris and 20% methanol (Towbin et al. , 1979, Proc.Natl. Acad. Sci., 76, 4350-4354). The membrane was cut into strips and blocked in 5% Skim milk in PBS at 37 °C for 1 h or at 4°C overnight. Serum was added at 1/200 for mouse serum or 1/100 for human serum in PBS/T at 37°C for 1 h or at 4°C overnight. The strips were washed 3 times in PBS/T, then incubated in sheep against mouse Ig horseradish peroxidase (HRP) conjugate (1:1000, Silenus, Melbourne) or goat against human IgG HRP conjugate (1 : 1000, Sigma) at 37 °C for 1 h or 4 °C overnight. The blot was washed three times as before and put into 4- chloro-1-napthol substrate buffer for developing colour. Crude hydatid cyst fluid antigens were run on 12% SDS-PAGE, transferred to a nitrocellulose membrane and processed as above as a control for the Western blot analysis. RT-PCR

Total RNAs from different stages (protoscoleces, oncospheres, immature adult worms and mature adult worms) of E. granulosus were treated with RNase-free DNase (Promega, Madison, WI), then reverse transcribed to cDNA using the

TM

SUPERSCRIPT Preamplification System (Life Technologies Inc., Rockville, MD) for first strand cDNA synthesis. PCR reactions were performed with primers designed from the EpCl sequence (SEQ ID NOS: 7-9; Table 4).

The SMART RACE technique using a universal upstream primer and a specific downstream primer (SEQ ID NO: 9) was used to amplify the longer sequence of SEQ ID NO: 1 which codes 34 more amino acids at the N-amino terminus than SEQ ID NO: 2. Immunocytochemistry

The location of the EpCl protein in the hydatid cyst, larval brood capsule (containing protoscoleces) and cyst wall was investigated using immunocytochemistry. Results

The inventors used pooled sera of mice primarily infected with oncospheres of E. granulosus to screen a cDNA library of protoscoleces, constructed from the mRNA purified from a collection of protoscoleces taken from sheep. One clone, called EpCl , reacted positively to the mouse serum, and was shown to encode a 77 amino acid protein (SEQ ID NO: 2). SEQ ID NO: 2 shows the amino acid sequence of the nucleotide encoding the EpC 1 protein and Figure 3 shows a comparison of the amino acid sequences of EpC 1 , a protein from Taenia solium from China (Ts76) and a protein from Taenia solium (NC-3) from Madagascar.

The EpCl protein was subcloned into the expression vector pET-41 and the GST fusion protein was expressed by E. coli and purified through a column containing GST affinity resin. The recombinant protein was expressed at high levels (> 100 mg/litre culture) and was highly pure.

Western blot experiments were carried out using EpCl protein to screen mouse and human sera for hydatid disease (Figures 5 & 6). Sequence analysis

A BLAST search showed that EpCl has two EF-hand motifs (Fig 7). The EF-hand motif, which assumes a helix-loop-helix structure normally responsible for Ca²⁺ binding, is found in a large number of functionally diverse Ca²⁺ binding proteins collectively known as the EF-hand protein superfamily. In many superfamily members, Ca²⁺ binding induces a conformational change in the EF-hand motif, leading to the activation or inactivation of target proteins. It is involved in muscle development and movement (Kiewitz et al, 2000, Biochim Biophys Acta, 1498, 207-19). An intriguing feature is the presence of EF-hand Ca (2+) binding motifs in the C-terminal half of the protein in mammalian cells (Simonic et al, 2000, FEBS Lett., 469, 33-8). They functionally contribute not only to Ca(2+) binding and calcium-sensitive gating of this Ca(2+) channel (Braun and Sy, 2001, J. Physiol., 533, 681-95.), but also to regulatory roles (Busch etal, 2000, Biol. Chem., 275(33), 25508-25525) in activation of some enzymes, such as mitochondrial glycerol-3- phosphate dehydrogenase (Brown et al, 1994, J. Biol. Chem., 269, 14363-14366) and glycosyl hydrolases, which may function in the formation of the laminated layer of the Echinococcus hydatid cyst. Functional gel assays using radioactive Ca(2+)- show that the EpCl strongly binds to Ca(2+); the binding is at least 30 times more pronounced than a commercial control of calmodulin using similar amounts of the two proteins. Example 1

Experiments were carried out to probe human sera of 323 confirmed patients with hydatid disease (cystic echinococcosis) (n=100) and other parasitic diseases, for example, alveolar echinococcosis (89), Taenia solium cysticerosis (72), fascioliasis (15), schistosomiasis (8), paragonimiasis (6), spirometra (5), trichinosis (5), toxocariasis (2) and entamoeba (1), with the recombinant EpCl protein. The recombinant EpC 1 protein positively diagnosed 76% of the patients with hydatid disease (n=100) and only positively diagnosed 3.9% of the patients with the other parasitic diseases (n=203) (Table 1 ). The recombinant EpC 1 protein positively diagnosed 5% of the patients with normal health (n=20) with hydatid disease. The selectivity and sensitivity percentages were calculated using the following equations: Sensitivity % = tp/(tp + fh) x 100% = 76/100 = 76% Specificty % = tn/(tn + fp) x 100% = 214/223 = 96% tp = true positives, fh = false negatives, fp = false positives, tn = true positives. Therefore the recombinant EpC 1 protein has a sensitivity for hydatid disease (cystic echinococcosis) of 76% and a specificity for hydatid disease of 96%. EpCl has arguably the highest sensitivity specificity ratio over any other hydatid disease antigen probed on infected human sera. Example 2 A further study that tested the sensitivity and specificity of the EpCl diagnostic protein for hydatid disease on 646 human sera samples, demonstrated the EpCl diagnostic protein had 91.9% sensitivity and 95.1% specificity for hydatid disease (cystic echinococcosis; Table 2). A commercial kit (Bordier Affinity Products, Switzerland), comprising hydatid cyst fluid AgB, for the diagnosis of cystic echinococcosis was tested concurrently with the EpCl protein on 88 of the cystic echinococcosis sera samples and 88 of the other parasite infection sera samples. The commercial kit only demonstrated 57.1% sensitivity and 84.1% specificity for cystic echinococcosis (Table 3). Therefore the diagnostic performance of the EpCl protein is superior. In addition the EpCl diagnostic protein demonstrated 91.9% sensitivity when tested on 111 pre-surgery cystic echinococcosis sera samples.

If the results of the trials of Example 1 and Example 2 are combined, the EpCl diagnostic protein has been tested on 989 sera samples, including sera from 419 CE, 172 NCC, 87 AE, 241 other infections/liver cancer cases and 70 confirmed negative controls. The overall sensitivity was 84.5% and specificity was 95.6% of the EpC 1 diagnostic protein. The data demonstrating 91.9% sensitivity of the EpC 1 diagnostic protein on the pre-surgery CE sera samples indicates the sensitivity of the protein may be higher than the 84.5% sensitivity we report from the results of the trials in Example 1 and Example 2. The increase in sensitivity obtained in Example 2 compared to Example 1 may be due to higher quality storage conditions used at The Center for Disease Control. Immunocytochemistry The results indicate that EpCl is expressed in the germinal layer of the hydatid cyst, brood capsule membrane, clusters of germinal cells along the brood capsule membrane and in immature protoscoleces (Figure 8), suggesting that EpCl may have a function associated with cyst growth and cell differentiation.

The PCR experiments carried out to detect the nucleotides of E. granulosus at all stages of its development (protoscoleces, oncospheres, immature adult worms and mature adult worms) showed that EpCl is expressed at all stages of the life cycle of E. granulosus (Figure 9). This result suggests that EpCl nucleic acid should be invaluable as a diagnostic tool and that the EpCl protein may be useful as a vaccine for all stages of the lifecycle of the parasite. Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

All computer programs, algorithms, patent and scientific literature referred to herein is incoφorated herein by reference.

Disease No. of serum Patient origin Positive (%) samples

Cystic echinococcosis 45 China 34 (75.6) 23 Australia 18 (78.3) 32 French 24 (75)

Alveolar echinococcosis 60 Japan 4 (6.7) 20 French 0(0) 9 China 0(0)

Taenia solium cysticercosis 21 Cameroon 3 (14.3)

13 South America 0(0)

27 China 0(0)

6 USA 1 (16.7)

5 French 0(0)

Schistosomiasis 8 South America 0(0)

Paragonimiasis 6 Japan 0(0)

Spirometra 5 Japan 0(0)

Trichinosis 5 USA 0(0)

Fascioliasis 15 Japan 0(0)

Toxocariasis 2 Japan 0(0)

Entamoeba 1 Japan 0(0)

Normal healthy donors 20 China 1(5)

Table 1

Table 2

Disease No. of sera samples Positive(%)

CE (pre-surgery) 42 24 (57.1)

CE (post-surgery) 46 27 (58.7)

Taenia solium 44 5 (13.6) cysticercosis

Schistosomiasis 22 4(18.2)

Others 22 5(22.7)

Table 3

Table 4

Claims

1. An isolated protein comprising the amino acid sequence set forth in SEQ ID NO: 1.

2. An isolated fragment of the isolated protein of claim 1.

3. An isolated protein homologue of the isolated protein of claim 1, or a fragment thereof, that has at least 80% sequence identity to SEQ ID NO: 1.

4. An isolated fragment according to claim 2 comprising the amino acid sequence set forth in SEQ ID NO: 2.

5. An isolated fragment according to claim 2 comprising the amino acid sequence set forth in SEQ ED NO: 3.

6. An isolated nucleic acid encoding the isolated protein of claim 1, or a fragment thereof.

7. An isolated nucleic acid according to claim 6 having a nucleotide sequence set forth in SEQ ID NO: 4.

8. An isolated nucleic acid encoding a fragment of the isolated protein of claim 1, having a nucleotide sequence set forth in SEQ ID NO: 5.

9. An isolated nucleic acid encoding a fragment of the isolated protein of claim 1, having a nucleotide sequence set forth in SEQ ID NO: 6.

10. An isolated nucleic acid primer or probe derived from any one of SEQ ID

NOS: 4-6.

11. An isolated nucleic acid homologue that hybridizes to SEQ ID NO : 4, or a fragment thereof, under high stringency conditions.

12. An isolated nucleic acid homologue that has at least 70% nucleotide sequence identity with SEQ ID NO: 4.

13. An antibody that binds the isolated protein of claim 1, or a fragment thereof.

14. An expression construct comprising an isolated nucleic acid according to claim 6, or a fragment thereof, wherein said nucleic acid is operably- linked to one or more regulatory sequences in an expression vector.

15. A host cell transfected or transformed with the expression construct of claim 14.

16. A method of producing the isolated protein of claim 1, or a fragment thereof, said method including the steps of (i) culturing a host cell according to claim 15 such that said isolated protein is expressed in said host cell, and (ii) isolating said isolated protein.

17. A method for detecting a hydatid infection in an animal, said method including the steps (i) contacting a biological sample with the antibody of claim 13 to thereby form a complex between said antibody and an isolated protein according to claim 1 , or a fragment thereof, if present in said sample, and (ii) detecting the complex formed.

18. The method of claim 17 wherein said animal is a mammal.

19. The method of claim 17 wherein said mammal is a human.

20. The method of claim 17 wherein said biological sample is a blood sample.

21. The method of claim 17 wherein detection of the antibody-protein complex is performed by immunoblot.

22. A kit for detecting a hydatid infection, comprising the antibody of claim

13 and one or more reagents that assist the detection of a complex formed between said antibody and the isolated protein of claim 1, or fragments thereof.

23. A method for detecting a hydatid infection in an animal, said method including the steps of (i) contacting a biological sample with the isolated protein of claim 1, or fragments thereof, to thereby form a complex between said isolated protein and an antibody if present in said sample, and (ii) detecting the complex formed.

24. The method of claim 23 wherein said animal is a mammal.

25. The method of claim 23 wherein said mammal is a human.

26. The method of claim 23 wherein said biological sample is a blood sample.

27. The method of claim 23 wherein detection of the antibody-protein complex is performed by an immunoenzymatic assay.

28. A kit for detecting a hydatid infection, comprising the isolated protein of claim 1 , or one or more fragments thereof, and one or more reagents that assist the detection of a complex formed between said isolated protein and an antibody.

29. A method for detecting a hydatid infection in an animal, including the step of detecting the isolated nucleic acid of claim 6, or one or more fragment thereof, in a biological sample.

30. The method of claim 29 wherein said animal is a mammal.

31. The method of claim 29 wherein said mammal is a human.

32. The method of claim 29 wherein said biological sample is a blood sample.

33. The method of claim 29 including the step of amplifying the isolated nucleic acid of claim 6, or one or more fragments thereof, prior to detection.

34. The method of claim 33 wherein amplification is performed using one or more primers selected from SEQ ID NOS: 7 to 9.

35. A pharmaceutical composition for the prophylactic and/or therapeutic treatment of a hydatid infection in an animal, said pharmaceutical composition comprising the isolated protein of claim 1, or fragments thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

36. The composition of claim 35 wherein the pharmaceutical composition is an immunotherapeutic composition.

37. The composition of claim 36 wherein the immunotherapeutic composition is a vaccine.

38. The composition of claim 35 wherein said animal is a mammal.

39. The composition of claim 35 wherein said mammal is a human.

40. A method of treating or preventing a hydatid infection in an animal including the step of administering the composition of any one of claims 35 to 37 to the animal.

41. The method of claim 40 wherein said animal is a mammal.

42. The method of claim 40 wherein said mammal is a human.