WO2008032073A2

WO2008032073A2 - Novel nematode protein and its use in producing anthelminthic agents and vaccines

Info

Publication number: WO2008032073A2
Application number: PCT/GB2007/003474
Authority: WO
Inventors: Peter Bruno Geldhof; David Patrick Knox
Original assignee: Moredun Research Institute
Priority date: 2006-09-13
Filing date: 2007-09-13
Publication date: 2008-03-20
Also published as: EP2061499A2; CN101563103A; WO2008032073A3; AU2007297299A1; GB0617962D0

Abstract

The present invention relates to a screen for identification of new anthelmintic agents, in particular to anti-haemonchus agents, as well as the preparation of a protein/peptide for use as a vaccine or immunogenic agent against helminths, such as haemonchus.

Description

NOVEL NEMATODE PROTEIN AND ITS USE IN PRODUCING

ANTHELMINTHIC AGENTS AND VACCINES Field of the Invention

Background to the Invention

Helminth parasites are responsible for a wide range of diseases and infestations of domestic animals which, leading as they do to loss of production and even animal mortality, are of considerable economic importance.

The blood-feeding nematode Haemonchus infects the lining of the gastrointestinal tract of ruminants. World-wide it is of very considerable economic importance having effects which range from reduction in weight gain, loss of production and agalactia through to death of domesticated animals. Anaemia is the basic feature of infection. When large numbers of larvae infect sheep, deaths can occur suddenly while the sheep still appear to be in good health. This is termed "acute prepatent disease" when eggs are not seen with faecal examination. Chronic infections involving smaller numbers of worms may produce oedema (bottle jaw), iron-deficiency anaemia, progressive weakness, wool breaks, and death. Both fourth- stage larvae and adult worms puncture blood vessels in the stomach wall and feed on the blood that is released. Nutrients are therefore used by the host to replace lost blood elements rather than for growth and wool production. A related nematode Ostertagia has similar effects. The diseases haemonchosis and ostertagiasis are characterised by wasting due in part to anorexia associated with severe infections.

Related blood-ingesting nematodes, the hookworms, infect ruminants, dogs, cats, other carnivores, and humans. Over 700 million humans are infected with hookworms, especially Necator americanus, with 700-900,000 new cases per year and 50-60,000 deaths due to the ravages of these parasites.

Control of helminth parasites presently relies primarily on the use of anthelmintic drugs combined with pasture management. Such techniques have a number of drawbacks however - frequent administration of drugs and pasture management are often not practical, and drug-resistant helminth strains are becoming increasingly widespread.

Extracts containing the protein doublet HI lOD obtained from Haemonchus give protection against haemonchosis when injected into sheep. This doublet, HI lOD, is found at the luminal surface of the intestine of Haemonchus. The preparation and use of Hl 1OD have been described in WO-A-88/00835.

In WO-A-89/00163 a protein with a molecular weight on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of 41 Kd is described in larvae of the parasitic nematode Trichostrongylus colubriformis. This protein was extracted from homogenised third stage larvae of T. colubriformis. The gene for this protein has been cloned. A very similar DNA sequence was demonstrated in Haemonchus contortus although expression of the Haemonchus gene in vivo was not reported. This document also reports results of vaccination trials in sheep against Haemonchus contortus with an antigen expression by recombinant organisms. In these trials reduced egg counts were reported in the faeces, with an average overall of 40% reduced egg counts. At slaughter 63 days after vaccination the vaccinated group of sheep were found to contain, on average 52% fewer worms than the control group.

EP0434909 discloses proteins with anticoagulant activity which may be useful as a vaccine. Other proteins potentially useful as vaccine candidates are disclosed in WO9402169 and WO9526402.

There is nevertheless an urgent need to identify new anthelmintic agents and/or develop novel vaccines against such parasites.

It is amongst the objects of the present invention to provide a peptide or protein for use in developing new anthelmintics. It is also amongst the objects of the present invention to provide a peptide or protein for use in raising an immune response in a ruminant animal.

Summary of the Invention

The present invention is based in part on the inventors' identification and characterisation of a novel protein expressed in Haemonchus contortus. The inventors have identified a number of physiological and enzyme activities associated with said protein, opening up the possibility of said protein being used as a drug target, as well as a vaccine candidate for treating helminthic and in particular Haemonchus infections.

As will be described in more detail hereinafter, the inventors have identified a protein of apparent molecular weight in denaturing conditions, of about 55 KDa. Genomic and proteomic studies show that it comprises 2 highly homologous proteins from H. contortus which show homology to prolyl carboxypeptidase like proteins in C. elegans and comprise two serine carboxypeptidase S28 type domains. Physiological and enzymic studies on the proteins show that the proteins possess significant dipeptidyl peptidase IV (DPP IV) activity and weak DPPII activity, as well as being able to delay fibrin clot formation.

Thus, in a first aspect of the present invention, there is provided a screening assay for identifying anthelmintic agents, the assay comprising the step of contacting an agent with a helminth protein, the protein being characterised as displaying dipeptidyl peptidase IV activity and resulting in an increase in blood clotting time so as to detect if the agent is capable of modulating said activity or blood clotting effect of said protein.

Conveniently, the protein comprises either sequence as shown in Figure 2a, or the nucleotide sequence encoding such proteins as shown in Figures 2c and 2d, or is substantially homologous or functionally equivalent thereto. A fragment of said protein may also be used, providing said fragment displays said DPP IV activity and optionally said increased blood clotting time effect.

It is understood that the term "modulate activity" relates to the ability of an agent to increase or decrease the DPP IV and optionally said blood clotting activity displayed by said enzyme. Desirably, the agent will be able to inhibit or decrease DPP IV activity of said enzyme and optionally inhibit or decrease the ability of the enzyme to increase blood clotting time.

In accordance with the first aspect, the enzyme may be provided by recombinant means, as described in more detail hereinafter, or simply purified from the parasite itself. The enzyme can be isolated from the blood feeding L4 or adult stages of the parasite.

In terms of being able to assay for an agents effect on the DPP IV activity of the enzyme, many DPP IV activity assays are known in the art. One such assay is the DPPIV-Gb™ protease assay provided by Promega (Promega Group, USA), which utilises a proluminescent DPP IV substrate, Gly-Pro-aminoluciferin, which generates a "glow-type" luminescent signal following cleavage of the substrate by a DPP IV enzyme and subsequent action by luciferase. Another DPP IV assay utilises Rhodamnine 1 10-conjugated glycine-proline which upon cleavage emits light (via rhodamine 1 10) in the fluorescein isothiocynate (FITC) wavelength. Further DPP IV assays utilise a chromogenic substrate such as p-nitroaniline (pNA) which when cleaved from its dipeptide results in an increase in absorbance at 405 nm. One such kit is provided by BIOMOL International, PA, USA. Thus, it will be appreciated that there are a variety of DPP IV assays known in the art and the invention it should be understood is not limited to any particular DPP IV assay.

Typically, the agent to be tested may be e.g. a small chemical entity, antibody specific for the protein, RNAi molecule designed to target expression of the protein peptide, peptide mimetic or the like, may be contacted with the isolated enzyme and thereafter the DPP IV activity of the enzyme measured. By comparing a level of DPP IV activity of the enzyme without the addition of said agent, it is possible to detect whether or not said agent has any modulatory effect on the DPP IV activity of the enzyme.

Naturally, once an agent has been identified as having an effect on the enzyme, the agent may be administered to a live helminth, such as Haemonchus contortus, so as to test its effect on the organism. Thereafter, the agent may be administered to a host animal which is infected with said helminth, in order to test for its effect on the parasite when in a host organism and to ensure that the agent is not deleterious to the infected host animal. Thus, desirably the anthelmintic agents identified in accordance with the present invention are specific for the parasite and do not substantially affect DPP IV proteins with DPP IV activity which may be present in the host animal.

There are already many DPP IV inhibitors known in the art, such as Diprotin A & B, vildagliptin, Sitagliptin, metformin, Saxagliptin and MK-0431 valine pyrrolidide, isoleucine thiazolidide, FE99901 (Ferring Pharmaceuticals), NVP- DPP728 (Novartis), LAF237 (Norvartis), SYR322 (Syrrx, Inc.), SYR619 (Syrrx, Inc.) and any or all of these may show activity in inhibiting an enzyme according to the present invention. In fact, the present inventors have, for example, shown that Diprotin A partially inhibited DPP IV type activity of said enzyme according to the present invention.

Thus, in a further aspect, there is provided use of a DPP IV inhibitor in the manufacture of a medicament for treating helminth infections, especially Haemonchus contortus infections.

There is also provided a method of treating an animal, such as a ruminant, infected by a helminth, comprising the step of administering to the animal a DPP IV inhibitor.

Without wishing to be bound by theory, it is envisaged that any such DPP IV inhibitors, by modulating activity of the enzyme according to the present invention may sterilise the fecundity of, damage, inhibit or kill the helminth, thereby treating the infected animal host.

As well as the DPP IV activity, the present inventors have observed that the enzyme as described herein has the activity of increasing blood clotting time.

Thus, in a further embodiment of the present invention, the screening step may be used to identify agents which inhibit said enzyme's ability to increase clotting time. Again without wishing to be bound by theory, it is envisaged that any such agent may lead to the blood meal, as ingested by the helminth, clotting within the helminth, leading as before to damage or death to the helminth organism.

Many assays for detecting blood clotting time are known including, for example "prothrombin time" and the "activated partial thromboplastin time" tests. These tests measure the time it takes for blood to clot after certain activating chemicals are added to the blood sample. Thus, by carrying out tests including said protein of the invention in the presence and absence of a drug candidate, it is possible to tell if the drug candidate is able to modulate blood clotting time as affected by said protein.

Similar tests are possible which use blood components, rather than whole blood. Examples include fibrin clotting time or fibrin clot reaggregation tests. Generally clot formation is detected by using photometric means, as a clot displays different light properties to non-clot comprising test media.

In a further aspect the present invention also provides nucleic acid molecules comprising one or more nucleotide sequences which encode a helminth serine carboxypeptidase enzyme or enzymically reactive, or antigenic portions thereof substantially corresponding to all or a portion of the predicted amino acid sequences as shown in Figure 2a or a nucleotide sequence capable of encoding said enzyme or portion thereof as shown in Figures 2c or 2d or sequences coding for a helminth serine carboxypeptidase enzyme which is substantially homologous with or which can hybridise with any of said sequences.

A nucleic acid according to the invention may thus be single or double stranded DNA, cDNA or RNA. Variations in the carboxypeptidase-encoding nucleotide sequences may occur between different strains of helminth within a species, between different stages of a helminth life cycle (e.g. between larval and adult stages), between similar strains of different geographical origin, and also within the same helminth. Such variations are included with the scope of this invention.

"Substantially homologous" as used herein includes those sequences having a sequence identity of approximately 60% or more, e.g. 75%, 80%, 90% or 95% or more, and also functionally-equivalent allelic variants and related sequences modified by single or multiple base substitution, addition, inversion and/or deletion. By "functionally equivalent" is meant nucleic acid sequences which encode polypeptides having carboxypeptidase activities which are similar enzymically i.e. which are capable of catalysing the same biochemical or physiological reaction (e.g. display DPP IV activity and/or an ability to increase blood clotting time) or similarly immunoreactive ie. which raise host protection antibodies against helminths.

Nucleic acid molecules which hybridise with the sequences shown in Figures 2c or 2d or any substantially homologous or functionally equivalent sequences as defined above are also included within the scope of the invention. "Hybridisation" as used herein defines those sequences binding under moderate or high stringency e.g. 2 x SSC, 65°C (where SSC = 0.15M NaCl, 0.015M sodium citrate, pH 7.2).

Methods for producing such derivative related sequences, for example by site- directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids are well known in the art, as are methods for determining whether the thus-modified nucleic acid has significant homology to the subject sequence, for example by hybridisation or nucleic acid sequencing. Provision of a nucleic acid molecule according to the invention thus enables recombinant carboxypeptidase enzymes, or enzymic or immunogenic fragments thereof, to be obtained in large quantities, thereby permitting the carrying out of screening assays as described above and/or development of anti-helminth vaccines and/or for use as an anticoagulant.

In another aspect the present invention thus provides nucleic acid molecules comprising one or more nucleotide sequences encoding one or more polypeptides for use in drug screening assays and/or for raising protective antibodies against helminth parasites, which sequences incorporate one or more enzymic or antigenic regions from the carboxypeptidase-encoding sequence as shown in Figure 2a.

The present invention also extends to synthetic polypeptides comprising one or more amino acid sequences constituting an carboxypeptidase enzyme or enzymic or antigenic portions thereof, substantially corresponding to all or a portion of the amino acid sequences as shown in Figure 2a, or a functionally-equivalent variant thereof.

An enzymic portion of said enzyme is understood to be shorter than the full length protein sequence, but able to display at least one of the proteins biochemical or antigenic properties, such as its DPP IV or increase in blood clotting time activity.

The term "antibody" as referred to herein includes whole antibodies, including those of the IgG, IgM and IgA isotypes, and any antigen binding fragment (i.e., "antigen-binding portion") or single chain thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V_H) and a heavy chain constant region. The IgG heavy chain constant region is comprised of four domains, C_HI, hinge, C_H2 and C_H3- Each light chain is comprised of a light chain variable region (abbreviated herein as V_L) and a light chain constant region. The light chain constant region is comprised of one domain, C_L. The V_H and V_L regions can be further subdivided into regions of hyper variability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V_H and V_L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system.

The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to a protein or protein fragment of the present invention. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, V_H, CL and C_HI domains; (ii) a F(ab')₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V_H and C_HI domains; (iv) a Fv fragment consisting of the V_L and V_H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546), which consists of a V_H domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, V_L and V_H, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V_L and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242 : 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883) or via other means such as the use of disulphide bonds or through dimerization motifs. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" or an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Other known antibody fragments include nanobodies , the technology surrounding which is licensed to Ablynx in Belgium.

The present invention further embraces variants and equivalents which are substantially homologous to the antibodies and antibody fragments set forth herein. These may contain, e.g., conservative substitutions, i.e. the substitution of one or more amino acids by similar amino acids. For example, conservative substitution refers to the substitution of an amino acid with another within the same general class, e.g., one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid by another neutral amino acid. What is intended by conservative amino acid substitution is well known in the art.

As used herein, "specific binding" refers to antibody binding to a predetermined antigen. Typically, the antibody binds with a dissociation constant (K_D) of 10^~7 M or less, and binds to the predetermined antigen with a K_D that is at least two-fold less than its K_D for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The invention further extends to vaccine compositions for stimulating immune responses against helminth parasites in a human or non-human animal, comprising at least one synthetic polypeptide as defined above, together with a pharmaceutically acceptable carrier.

The term "polypeptide" as used herein includes both full length protein, and shorter peptide sequences.

The present invention also relates to antibodies specific for the proteins identified herein. Such antibodies may be used in a therapeutic sense to bind to the protein(s) and interfere with its activity. The antibodies may be monoclonal or polyclonal and may be prepared according to techniques well known to one of skill in the art.

"Functionally equivalent" as used above in relation to the polypeptide amino acid sequences defines polypeptides related to or derived from the abovementioned polypeptide sequences where the amino acid sequence has been modified by single or multiple amino acid substitution, addition, inversion or deletion, and also sequences where the amino acids have been chemically modified, including by glycosylation or deglycosylation, but which nonetheless retain protective antigenic (immunogenic) activity. Such functionally-equivalent variants may occur as natural biological variations or may be prepared using known techniques, for example functionally equivalent recombinant polypeptides may be prepared using the known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of amino acids.

The synthetic polypeptides according to the invention may represent protective antigenic sequences. The term "protective antigen" as used herein defines those antigens capable of generating a host-protective (immunogenic) immune response ie. a response by the host which leads to the generation of immune effector molecules, antibodies or cells which sterilise the fecundity of, damage, inhibit or kill the parasite and thereby "protect" the host from clinical or sub-clinical disease and loss of productivity. Such a protective immune response may commonly be manifested by the generation of antibodies which are able to inhibit the metabolic function of the parasite, leading to stunting, lack of egg production and/or death.

The synthetic polypeptides according to this aspect of the invention may be prepared by expression in a host cell containing a recombinant DNA molecule which comprises a nucleotide sequence as broadly described above operatively linked to an expression control sequence, or a recombinant DNA cloning vehicle or vector containing such a recombinant DNA molecule. Alternatively the polypeptides may be expressed by direct injection of a naked DNA molecule according to the invention into a host cell.

The synthetic polypeptide so expressed may be a fusion polypeptide comprising a portion displaying the biochemical/physiological immunogenic activity of all or a portion of a carboxypeptidase enzyme according to the invention and an additional polypeptide coded for by the DNA of the recombinant molecule fused thereto. For example, it may be desirable to produce a fusion protein comprising a synthetic carboxypeptidase or other polypeptide according to the invention coupled to a protein such as β-galactosidase, phosphatase, glutathione-S-transferase, urease, hepatitis B core antigen (Francis et al., 1989) and the like. Most fusion proteins are formed by expression of a recombinant gene in which two coding sequences have been joined together with reading frames in phase. Alternatively, polypeptides can be linked in vitro by chemical means. All such fusion or hybrid derivatives of carboxypeptidase-encoding nucleic acid molecules and their respective amino acid sequences are encompassed by the present invention. Such suitable recombinant DNA and polypeptide expression techniques are described for example in Sambrook et al., 1989. Alternatively, the synthetic polypeptides may be produced by chemical means, such as the well-known Merrifield solid phase synthesis procedure.

Further aspects of the invention include use of a nucleic acid molecule or a synthetic peptide or polypeptide as defined above, for the preparation of a vaccine composition for stimulating an immune response in a mammal, especially a ruminant mammal.

Alternatively viewed, the invention also provides a method of stimulating an immune response in a mammal, especially a ruminant, animal against a helminth parasite infection comprising administering to said animal a vaccine composition comprising one or more polypeptides encoded by a nucleotide sequence as defined hereinabove.

A vaccine composition may be prepared according to the invention by methods well known in the art of vaccine manufacture. Traditional vaccine formulations may comprise one or more synthetic polypeptides according to the invention together, where appropriate, with one or more suitable adjuvants, eg. aluminium hydroxide, saponin, QuilA, or more purified forms thereof, muramyl dipeptide, mineral oils, or Novasomes, in the presence of one or more pharmaceutically acceptable carriers or diluents. Suitable carriers include liquid media such as saline solution appropriate for use as vehicles to introduce the peptides or polypeptides into a mammal. Additionally components such as preservatives may be included. An alternative vaccine formulation may comprise a virus or host cell eg. a microorganism (e.g. vaccinia virus, adenovirus, bacteriophage Salmonella) having inserted therein a nucleic acid molecule (e.g. a DNA molecule) according to this invention for stimulation of an immune response directed against polypeptides encoded by the inserted nucleic acid molecule.

Administration of the vaccine composition may take place by any of the conventional routes, e.g. orally or parenterally such as by intramuscular injection, optionally at, but not limited to, intervals e.g. two injections at a 7 - 28 day interval.

Expression of the carboxypeptidase-encoding sequences according to the invention can, as mentioned above, be achieved using a range of known techniques and expression systems, including expression in prokaryotic cells such as E. coli and in eukaryotic cells such as yeasts of the baculo virus-insect cell system or transformed mammalian cells and in transgenic animals and plants. Particularly advantageously, the nucleotide sequences may be expressed using the transgenic nematode system such as the system for the nematode Caenorhabditis described for example in Fire, (1986); Fire et al., (1989); Spieth et al., (1988); Han et al., (1990).

A further aspect of the invention provides a method for preparing a synthetic polypeptide as defined above which comprises culturing a eukaryotic or prokaryotic cell containing a nucleic acid molecule as defined above, under conditions whereby said polypeptide is expressed, and recovering said polypeptide thus produced.

Further aspects of the invention thus including cloning and expression vectors containing nucleotide sequences according to the invention. Such expression vectors include appropriate control sequences such as for example translational (e.g. start and stop codes) and transcriptional control elements (e.g. promoter-operator regions, ribosomal binding sites, termination stop sequences) linked in matching reading frame with the nucleic acid molecules of the invention.

Vectors according to the invention may include plasmids and viruses (including both bacteriophage and eukaryotic viruses) according to techniques well known and documented in the art, and may be expressed in a variety of different expression systems, also well known and documented in the art. Suitable viral vectors include, as mentioned above, baculovirus and also adenovirus and vaccinia viruses. Many other viral vectors are described in the art.

A variety of techniques are known and may be used to introduce such vectors into prokaryotic or eukaryotic cells for expression, or into germ line or somatic cells to form transgenic animals. Suitable transformation or transfection techniques are well described in the literature.

Transformed or transfected eukaryotic or prokaryotic host cells or transgenic organisms containing a nucleic acid molecule according to the invention as defined above, form a further aspect of the invention.

Eukaryotic expression systems in general, and the nematode expression system in particular, have the advantage that post-translational processing, and particularly glycosylation can occur - in the case of the transgenic nematode system, a glycosylation corresponding to that found in the native protein may be expected.

Mammalian cell expression systems, also have a number of advantages. Mammalian host cells provide good reproduction of the native form and protective epitopes of the antigen since a eukaryotic expression system will give rise to more similar glycosylation patterns, disulphide bonding and other post-translational modifications that E. coli lacks, which may produce an insoluble protein requiring refolding and having poor reproduction of the native form. In addition mammalian glycosylation is unlikely to induce an immune response which distracts from a protection anti-protein response. For protection of humans and domestic animals, it is thus preferable to use human or animal fibroblast or myeloma cell lines such as HeLa - a human cell line; BHK-baby hamster kidney cells; VERO, a monkey kidney cell line; FR3T3, Fisher rate fibroblasts; NIH3T3, a mouse fibroblast cell line; C 1271, a mouse mammary tumour cell line; CV-I, African green monkey kidney fibroblasts: 3T6, mouse embryo fibroblasts; L cells, a mouse cell line; CHO, a Chinese Hamster Ovary cell line; NSO NSI, Sp2 and other mouse myeloma cell lines and rat myeloma cell lines such as YB2/0 and Y3.

Vectors appropriate for different classes of mammalian cell lines are well known in the art. In general, these will comprise a promoter and/or enhancer operably connected to a nucleotide sequence encoding the antigen or fragment thereof. Suitable promoters include SV40 early or late promoter, eg. PSVL vector, cytomegalovirus (CMV) promoter, mouse metallothionein I promoter and mouse mammary tumour virus long terminal repeat. The vector preferably includes a suitable marker such as a gene for dihydrofolate reductase or glutamine synthetase. Vectors of those types are described in WO 86/05807, WO 87/04462, WO 89/01036 and WO 89/10404.

Transfection of the host cells may be effected using standard techniques, for example using calcium phosphate, DEAE dextran, polybrene, protoplast fusion, liposomes, direct microinjection, gene cannon or electroporation. The latter technique is preferred and methods of transfection of mammalian cell lines using electroporation are described by Andreason et al., 1980. In general, linear DNA is introduced more readily than circular DNA. Detailed Description of the Invention

The present invention will now be further described by way of example and with reference to the Figures which show:

Figure 1 SDS-PAGE profile of the serine carboxypeptidase enriched protein fraction under reducing conditions. The prominent band around 55 kDa (bands A and B) was picked and analysed by tryptic digest, peptide mass fingerprint analysis and LC-MS/MS analysis;

Figure 2 (A) Predicted amino acid sequence of the Haemonchus contortus serine carboxypeptidase like proteins Hc-PCPl and Hc-PCP2. Cleavage sites of the N-terminal signal sequences in both proteins are marked with an arrow. Putative glycosylation sites are marked with an *. Both proteins contain two serine carboxypeptidase S28 type domains which are marked with []. (B) Diagram of the H. contortis PCP's containing a signal peptide (SP) and two carboxypeptidase S28 type domains. (C) Predicted nucleotide sequence of Hc-PCPl . (D) Predicted nucleotide sequence of Hc-PCP2.

Figure 3 Transcription of Hc-pcpl and Hc-pcp2 as revealed by RT-PCR. The lanes are independent RT-PCR reactions using target RNA from exsheathed L3, L4, 11 and 22 day old adult parasites. RT-PCR for a cytoplasmic superoxide dismutase (SODc) was used as a control to check the uniformity of the RNA purifications.

Figure 4 Dipeptidyl peptidase activity assays. (A) Dose dependent degradation of the DPP IV and DPP II specific substrates by the serine carboxypeptidases at pH 7.5. (B) Activity against the DPP IV specific substrate by the serine carboxypeptidases incubated at different pH. (C) Testing the effect of two DPP IV specific inhibitors on the DPP IV type activity of the serine carboxypeptidases. Figure 5 Inhibition of fibrin reaggregation by the addition of the serine carboxypeptidases (PCP's). (A) The reaggregation of control fibrin monomers compared to the reaggregation of fibrin monomers incubated with different concentrations of the serine carboxypeptidases. (B) Analysis of the effect of DiprotinA on the inhibition of fibrin reaggregation. All results are shown as changes in scattered light measured at 350 nm, which is a measure of the amount of reaggregated fibrin monomers.

Figure 6 Proteolytic degradation of the fibrin α-chain by the serine carboxypeptidases and the partial inhibition of this activity by DiprotinA. (A) 10 % reducing SDS-PAGE of a fibrin solution prior (lane 1 ) and after a 15 sec incubation with the serine carboxypeptidases (lane 2). (B) 10 % reducing SDS-PAGE gel of a control fibrin solution (lane 1), fibrin incubated with the serine carboxypeptidases for 15 sec (lane 2) and fibrin incubated with the serine carboxypeptidases and DiprotinA for 15 sec (lane 3).

Figure 7 The digest product of the α-chain co-migrates with the γ-chain and mass spectrometry analysis, indicated that digestion was from the C-terminal end. The underlined lettering indicates regions of the alpha-chain where positive hits were obtained with tryptic digest fragments. Note that no hits were obtained towards the C-terminus indicating that this region had been removed by the carboxypeptidase activity.

Materials and Methods

Preparation of the serine carboxypeptidase proteins

Adult Haemonchus contortus were harvested by opening and washing the abomasum from an infected sheep. Worms were homogenized in PBS and subsequently centrifuged for 5 min at 3000 rpm. The supernatant was collected and the pellet rehomogenized in PBS followed by centrifugation at 3000 rpm. The supernatants were pooled and centrifuged at 10000 g for 10 min. The pellet was washed once with PBS and the pooled supernatants were centrifuged for 90 min at 35000 rpm. The pellets, which contain the serine carboxypeptidases, were resuspended in 1 ml of PBS and stored at -80°C prior to analysis.

Gel electrophoresis and mass-spectrometry analysis

The serine carboxypeptidase enriched protein extract was analysed on a 10 % SDS-PAGE gel under reducing conditions. The protein components were visualized by Coomassie Blue staining. Protein bands were excised from the gel and used for the mass spectrometry analysis. In short, protein bands were in-gel digested using trypsin, subsequently purified and analysed by MALDI-TOF mass spectrometry. Where necessary remaining material was used for LC -MS/MS analysis. The Mascot search engine was used to analyse the mass spectrometry and LC -MS/MS data and to identify the proteins.

Isolation and cloning ofcDNAs

Full length cDNA sequences of the serine carboxypeptidases (from hereon named Hc-pcpl and Hc-pcp2) were isolated from a 11 day old H. contortus cDNA library. Based on the consensus sequences of EST clusters HCC00232 {Hc-pcpl) and HCC00298 (Hc-pcpl), specific primers were designed to isolate the 5' and 3' ends of each cDNA. The primer sequences are shown in table 1. Gene specific primers were used in combination with the T3 cDNA library vector primer. PCR products were cloned in pGEM-T (Promega) and sequenced. Sequence analyses and alignments were performed using the DNAstar software (DNAstar Inc.).

Bioinformatics

The EST datasets of H. contortus and other nematodes (available on www.nema.cap.ed.ac.uk/nematodeESTs/nembase.html) were analysed using the Partigene bioinformatics pipeline (Parkinson et al, 2004) which is made available on the Nembase website (www.nematodes.org/nematodeESTs/nembase.html). Further analyses of sequences and the deduced amino acid sequences were performed using the DNAstar software (DNAstar Inc.). Database searches were performed on the NCBI server (www. ncbi . nlm.nih. gov/blast). Amino acid sequences were analysed for signal peptides and glycosylation sites using the Signal P- (www.cbs.dtu.dk/services/SignalP) and NetNGlyc-programs

(www.cbs.dtu.dk/services/NetNGlyc).

Reverse transcriptase coupled PCR

Reverse transcriptase coupled PCR (RT-PCR) was used to determine the stage-specific transcription of Hc-pcpl and Hc-pcp2. Total RNA was extracted from exsheathed L3, L4 and adult parasites using Total RNA Isolation Reagent (Advanced Biotechnologies Ltd.) as described by Hashmi et al. (2002). The RT-PCRs were carried out using the Superscript One-Step RT-PCR System (Invitrogen). The specific primers used for detection of the Hc-pcpl of the Hc-pcp2 transcripts are shown in Table 1. After 35 cycles of amplification, the RT-PCR products were separated on 1 % agarose gels. An RT-PCR for the cytoplasmic superoxide dismutase gene (SODc), a gene expressed in all lifecycle stages, was used to control the uniformity of the RNA purifications.

Dipeptidyl peptidase activity assays

An initial screen with a panel of substrates with specificity for DPP I, II, III and IV respectively indicated that there was strong DPP IV activity and very slight DPP II activity. The chromogenic substrates H-Ala-Pro-pNitroanilide and H-Lys- Ala-pNitroanilide (Bachem) were then used to measure DPP IV and DPP II activity respectively. Stock solutions of both substrates were made in methanol (cone. 1 M). Different volumes of the serine carboxypeptidase preparation (2-6 μl) were mixed with the substrates (final cone. 1 mM) in 100 μl of buffer ranging from pH 3 to 8 (pH3-5: 0.1 M Sodium acetate buffer, pH6-7: 0.1 M phosphate buffer, pH8: 0.1 M Tris buffer). Samples were incubated for 45 minutes at 37°C and subsequently transferred to a microtitre plate. Optical density was measured at 405 nm. Each experiment was performed in duplicate. The effect of DPP IV inhibitors, Diprotin A and Diprotin B, on enzyme activity was determined by adding different concentration of the inhibitors (lOμM, 100 μM, 500 μM and ImM) to the reaction mixture described above.

Fibrin clotting/reaggregation assay

A bovine fibrin solution (concentration 15 mg/ml) was clotted by the addition of 1 μl of a thrombin stock solution (10 mg/ml) and incubated at 37°C for 15 minutes. The fibrin clot was collected and resuspended in 8 ml of 1 M NaBr/0.05 Na acetate buffer pH 5.3. This fibrin monomer solution was subsequently concentrated to 750 μl and stored at 4°C prior to use. _.

23

Reaggregation/clotting of the fibrin solution was effected by mixing 5 μl of the fibrin monomer solution with 95 μl of PBS. The reaggregation was monitored in a spectrophotometer by measuring the scattered light in a 90° angle at 350 nm. Results are shown as the change in scattered light over a 10 minute period. The effect of the serine carboxy peptidases on the fibrin reaggregation was assessed by incubating 5 μl of the fibrin monomer solution with different concentrations of the serine carboxypeptidases for 30 min at 37°C prior to the spectrophotometric analysis. The effect of the DPP IV inhibitor Diprotin A was determined by adding 0.4 μl of a 0.5 M inhibitor stock solution to the reaction mixture described above.

Fibrin degradation assay

Twenty five μg of fibrin monomers, prepared as described above, was mixed with different concentrations of the serine carboxypeptidases and incubated at 37°C for different lengths of time, ranging from 15 sec to 30 min. Samples were subsequently analysed on 10 % SDS-PAGE gels under reducing conditions and visualized by Coomassie Blue staining. The effect of the DiprotinA inhibitor was investigated by adding the inhibitor at a final concentration of 0.1 M to the reaction mixture described above.

Results

Characterization of the serine carboxypeptidases

The protein profile of the serine carboxypeptidase enriched fraction is shown is Figure 1. It comprises a prominent band around 55 kDa (arrow in Figure 1). This band was picked from a Coomassie stained gel and analysed by tryptic digest, peptide mass fingerprint analysis and LC-MS/MS analysis. The outcome was subsequently screened against the H. contortus EST dataset. The LC-MS/MS analysis of the 55 kDa band resulted in three peptide sequences which showed a 100 % match with clusters HCC00232 and HCC00298 (Table 2). Cluster HCC00232 and HCC00298 respectively contain 421 and 37 individual EST sequences. The consensus sequence of cluster HCC00232 is 2579 bp long. An additional 884 bp at the 5' end was isolated from a cDNA library by PCR approach using gene specific primers in combination with the T3 vector primer. This resulted in a full-length coding sequence which encodes a 122 kDa protein. Cluster HCC00298 has a consensus sequence of 3037 bp. Together with an additional 562 bp at the 3' end, which was isolated in a similar approach as described above, it codes for a 129 kDa protein. Both proteins show homology to prolyl carboxypeptidase like proteins in C. elegans (PCP-2 ace. NP 501599 and PCP-3 ace. NP_501598.1). The shared identity between the H. contortus and C. elegans proteins varied around 38 %. The two H. contortus proteins are from hereon named Hc-PCPl (cluster HCC00232) and Hc-PCP2 (cluster HCC00298). An alignment of Hc-PCPl and Hc-PCP2 is shown in Figure 3 panel A. The two proteins show 64 % identity to each other in amino acid sequence and both contain a signal peptide and multiple putative glycosylation sites (Marked in Figure 3 panel A). Both Hc-PCPl and Hc-PCP2 are comprised of two serine carboxypeptidase S28 type domains (pfamO5577) which are organized in a tandem repeat (Figure 3 panel B). This feature seems to be nematode specific, only the C. elegans PCP's show a similar structure. Each of the S28 domains encode a 51 kDa serine carboxypeptidase, which approximately matches the size of band B on SDS-PAGE gels. Therefore, it is likely that the two tandem structured PCP's are post- translationally processed in four individual serine carboxypeptidases. Transcription profile of the Hc-PCPs

Hc-pcpl and Hc-pcp2 specific primers were used in a RT-PCR on total RNA samples from exsheathed L3, L4, 1 1 day old and 22 day old parasites. The results of the RT-PCRs are shown in Figure 3. Transcripts of the two genes were present from the L4 stage onwards. Reverse transcriptase coupled PCR for the cytoplasmic superoxide dismutase gene (SODc) (ace. Z69621) was used to control the uniformity of the RNA purifications.

Dipeptidyl peptidase activity

The results of the dipeptidyl peptidase activity assays are shown in Figure 4. At pH 7.5, the serine carboxypeptidases hydrolysed the DPP IV specific substrate in a dose dependent manner. Very slight activity was found against the DPP II specific substrate (panel A). The peak activity against the DPP IV was detected at pH 6 (Figure 4 panel B). The effect on the DPP IV activity by specific inhibitors is shown in Figure 4 panel C. DiprotinA partially inhibited the DPP IV type activity in a dose dependent manner. No effect was visible with the DiprotinB inhibitor.

Fibrin reaggregation assay

The results of the fibrin reaggregation assay are shown in Figure 5 panel A. The addition of the serine carboxypeptidases to a fibrin monomers solution significantly inhibits the reaggregation in a dose dependent manner, as measured by the change in scattered light over a 10 min period. This effect is partially reversed by the addition of the DPP IV inhibitor DiprotinA (Figure 5 panel B). Fibrin degradation assay

The fractionation of a fibrin monomer solution on a 10% SDS-PAGE gel under reducing conditions is shown in Figure 6 panel A. It results in three protein bands of approximately 63 kDa, 56 kDa and 47 kDa, these are respectively the α- chain, the β-chain and the γ-chain. The addition of the serine carboxypeptidases results in the extremely rapid proteolytic degradation of the α-chain after 15 sec of incubation at 37°C (Figure 6 panel A). The addition of the DiprotinA inhibitor partially reverses this effect (Figure 6 panel B). The digest product of the α-chain co- migrates with the γ-chain and mass spectrometry analysis, (Figure 7) indicated that digestion was from the C-terminal end. The red lettering indicates regions of the alpha-chain where positive hits were obtained with tryptic digest fragments. Note that no hits were obtained towards the C-terminus indicating that this region had been removed by the carboxypeptidase activity.

Table 1 : PCR primer sequences used for RT-PCR and RACE experiments

Table 2: Peptide sequences from the LC-MS/MS analysis of the 55 kDa band.

Claims

1. A screening assay for identifying anthelmintic agents, the assay comprising the step of contacting an agent with a helminth protein or fragment thereof, the protein or fragment thereof being characterised as displaying dipeptidyl peptidase IV activity and resulting in an increase in blood clotting time, so as to detect if the agent is capable of modulating said activity or blood clotting effect of said protein.

2. The screening assay of claim 1, wherein the protein or fragment thereof comprises a sequence shown in Figure 2a.

3. The screening assay of claim 1, wherein the protein or fragment thereof is encoded by a sequence shown in Figures 2c or 2d.

4. The assay of any preceding claim, wherein the agent inhibits or decreases the DPP IV activity of the protein or fragment thereof and/or the ability of the protein or fragment thereof to increase blood clotting time.

5. The screening assay of any preceding claim, wherein the protein or fragment thereof is provided by recombinant means or is purified from the Helminth itself.

6. The assay of any preceding claim, wherein the protein or fragment thereof is isolated from the blood feeding L4 or adult stages of the Helminth.

7. The assay of any preceding claim, wherein the agent to be tested is selected from the group consisting of:

(i) small chemical entities;

(ii) antibodies specific for the protein;

(iii) RNAi molecules designed to target expression of the protein; and

(iv) peptide mimetics or the like.

8. The assay of any preceding claim, wherein the agent may be administered to a live helminth, such as Haemonchus contortus or to a host animal infected with said helminth.

9. The assay of any preceding claim, wherein identified anthelmintic agents are specific for the parasite and do not substantially affect host DPP IV proteins with DPP IV activity.

10. Use of a DPP IV inhibitor in the manufacture of a medicament for treating helminth infections, especially Haemonchus contortus infections.

11. The use of claim 10, wherein the inhibitor is selected from the group consisting of:

(i) Diprotin A & B;

(ii) vildagliptin;

(iii) Sitagliptin;

(iv) metformin;

(v) Saxagliptin; (vi) MK-0431 valine pyrrolidide;

(vii) isoleucine thiazolidide;

(viii) FE99901 ;

(ix) NVP-DPP728;

(x) LAF237;

(xi) SYR322; and

(xii) SYR619.

12. A method of treating an animal, such as a ruminant, infected by a helminth, said method comprising the step of administering to the animal a DPP IV inhibitor.

13. A nucleic acid molecule comprising one or more nucleotide sequences which encode a helminth serine carboxypeptidase enzyme and/or enzymically reactive and/or antigenic portions thereof.

14. The nucleic acid molecule of claim 13, wherein the one or more nucleotide sequences comprise a sequence shown in Figures 2c or 2d or encode an enzyme having a sequence which substantially corresponds to all or a portion of the amino acid sequences shown in Figure 2a.

15. A nucleic acid molecule comprising one or more nucleotide sequences which encode an enzyme which is substantially homologous to a helminth serine carboxypeptidase or which can hybridise to any of the sequences of claims 13 or 14.

16. A nucleic acid molecule comprising one or more nucleotide sequences encoding one or more polypeptides, for use in drug screening assays and/or for raising protective antibodies against helminth parasites, which sequences incorporate one or more enzymic and/or antigenic regions from the carboxypeptidase-encoding sequence as shown in Figure 2a.

17. A synthetic polypeptide comprising one or more amino acid sequences constituting a carboxypeptidase enzyme or enzymic and/or antigenic portions thereof, substantially corresponding to all or a portion of the amino acid sequences as shown in Figure 2a, or a functionally-equivalent variant thereof.

18. The synthetic peptide of claim 17, wherein the peptide provides a protective antigenic sequence(s).

19. A fusion polypeptide comprising a portion displaying the biochemical/physiological immunogenic activity of all or a portion of a helminth serine carboxypeptidase enzyme and an additional polypeptide.

20. A method for preparing the synthetic or fusion polypeptides of claims 17-19, said method comprising the steps of;

(a) culturing a cell containing a nucleic acid molecule according to claims 13- 16;

(b) maintaining the cell under conditions whereby said nucleic acid is expressed; and

(c) recovering the polypeptide thus produced.

21. A vaccine composition for stimulating immune responses against helminth parasites in a human or non-human animal, comprising at least one synthetic or fusion polypeptide of claims 17-19, together with a pharmaceutically acceptable carrier.

22. A vaccine formulation comprising a virus or host cell having inserted therein one or more nucleic acid molecule(s) of claims 13-16, for stimulation of an immune response directed against polypeptides encoded by the one or more inserted nucleic acid molecule(s).

23. An antibody specific for the proteins of claims 17-19.

24. Use of a nucleic acid molecule or a synthetic or fusion peptide/polypeptide according to claims 13-19, for the preparation of a vaccine composition for stimulating an immune response in a mammal, especially a ruminant mammal.

25. A method of stimulating an immune response in a mammal, especially a ruminant, against a helminth parasite infection comprising administering to said animal a vaccine composition comprising one or more polypeptides encoded by the nucleotide sequences of claims 13-16.

26. Use of a nucleic acid molecule or a synthetic or fusion peptide/polypeptide according to claims 13-16 for the preparation of a medicament for use as an anticoagulant.

27. A method of preventing and/or delaying coagulation of blood in a mammal, comprising administering to said animal a nucleic acid molecule or a synthetic or fusion peptide/polypeptide according to claims 13-19.