AU726774B2 - Novel parasite protease genes and proteins - Google Patents

Description

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AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT

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S. SO S Applicant(s): Address for Service: Heska Corporation 1825 Sharp Point Drive Fort Collins Colorado 80525 UNITED STATES OF AMERICA DAVIES COLLISON CAVE Patent Trade Mark Attorneys Level 10, 10 Barrack Street SYDNEY NSW 2000 Novel parasite protease genes and proteins S

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Invention Title: The following statement is a full description of this invention, including the best method of performing it known to me:- 5020 NOVEL PARASITE PROTEASE GENES AND PROTEINS Field of the Invention The present invention relates to novel parasite protease genes, proteins encoded by such genes, antibodies raised against such proteins, and protease inhibitors produced using such proteins. Particular proteases of the present invention include astacin metalloendopeptidases and cysteine proteases. The present invention also includes therapeutic compositions comprising such nucleic acid molecules, proteins, antibodies and inhibitors, as well as their use to protect animals from disease caused by parasites, such is by tissue-migrating helminths such as S heartworm.

Background of the Invention 15 Parasite infections in animals, including humans, are typically treated by chemical drugs, because -there are essentially no efficacious vaccines available. One disadvantage with chemical drugs is that they must be administered often. For example, dogs susceptible to 20 heartworm are typically treated monthly to maintain protective drug levels. Repeated administration of drugs to treat parasite infections, however, often leads to the development of resistant strains that no longer respond to treatment. Furthermore, many of the chemical drugs are harmful to the animals being treated, and as larger doses become required due to the build up of resistance, the side effects become even greater.

-2- It is particularly difficult to develop vaccines against parasite infections both because of the complexity of the parasite's life cycle and because, while administration of parasites or parasite antigens can lead to the production of a significant antibody response, the immune response is cypically not sufficient to protect the animal against infection.

As for most parasites, the life cycle of Dirofilaria immitis, the helminth that causes heartworm, includes a variety of life forms, each of which presents different targets, and challenges, for immunization. Adult forms of the parasite are quite large and preferentially inhabit the heart and pulmonary arteries of an animal. Males worms are typically about 12 centimeters (cm) to about 20 cm long and 15 about 0.7 millimeters (mm) to about 0.9 mm wide; female worms are about 25 cm to about 31 cm long and about 1.0 to about 1.3 mm wide. Sexually mature adults, after mating, produce microfilariae which are only about 300 micrometers (pm) long and about 7 Am wide. The microfilariae traverse 20 capillary beds and circulate in the vascular system of dogs in concentrations of about 10 3 to about 105 microfilariae per milliliter (ml) of blood. One method of demonstrating infection in dogs is to detect the circulating microfilariae.

If dogs are maintained in an insect-free environment, the life cycle of the parasite cannot progress. However, when microfilariae are ingested by female mosquitos during blood feeding on an infected dog, subsequent development of -3the microfilariae into larvae occurs in the mosquito. The microfilariae go through two larval stages (L1 and L2) and finally become mature third stage larvae (L3) of about 1.1 mm length, which can then be transmitted back to a dog through the bite of the mosquito. It is this L3 stage, therefore, that accounts for the initial infection. As early as three days after infection, the L3 molt to the fourth larval (L4) stage, and subsequently to the fifth stage, or immature adults. The immature adults migrate to S 10 the heart and pulmonary arteries, where they mature and reproduce, thus producing the microfilariae in the blood.

"Occult" infection with heartworm in dogs is defined as an Sinfection in which no microfilariae can be detected, but the existence of adult heartworms can be determined through 15 thoracic examination.

Both the molting process and tissue migration are likely to involve the action of one or more enzymes, including proteases. Although protease activity has been identified in a number of parasites (including in larval 20 excretory-secretory products) as well as in mammals, there apparently has been no identification of an astacin metalloendopeptidase gene in any parasite or of a cysteine protease gene in any filariid.

Astacin metalloendopeptidases, so-called because of I their similarity to the metalloendopeptidase astacin found in crayfish, are a relatively newly recognized class of metalloproteases that have be en found in humans, mice and rats as well as apparently in Drosophila fruit flies, -4- Xenopus frogs and sea urchins; see, for example, Gomis-Ruth et al., 1993, J. Mol. Biol. 229, 945-968; Jiang et al., 1992, FEES Letters 312, 110-114; and Dumermuth et al., 1991, J. Piol. Chem. 266, 21381-21385. Human intestinal and mouse kidney brush border metalloendopeptidases share about 82 percent homology in the amino-terminal 198 amino acids.

Both share about 30 percent homology with astacin and with the amino-terminal domain of human bone morphogenetic protein 1. Members of the astacin family share an extended zinc-binding domain motif, the consensus sequence of which was identified by Dumermuth et al., ibid., as being HEXXHXXGFXHE, wherein H is histidine, E is glutamic acid, G is glycine, F is phenylalanine and X can be any amino acid. Gomis-Ruth et al., ibid., define the zinc-binding 15 domain motif as His-Glu-Uaa-Xaa-His-Xaa-Uaa-Gly-Uaa-Xaa- His, wherein Uaa is a bulky apolar residue-containing amino acid. Jiang et al., ibid., disclose not only the extended zinc-binding domain motif, which they represent as HEIGHAIGFXHE (letters in bold being strictly conserved) but 20 also two other sequences conserved between astacin metalloendopeptidases, including RXDRD spanning amino acids from about 15 through about 19 and YDYXSIMHY spanning amino acids from about 53. through about 61, assuming that the fi: st histidine in the extended zinc-binding domain motif is amino acid position 1. The three histidines at positions 1, 5 and 11 appear to be responsible for zinc binding as is the tyrosine at position 61. The glutamic acid at position 2 is responsible for catalysis. Other key amino acids include the glycine at position 8 which is involved in secondary structure and the glutamic acid at position 12 which forms a salt bridge with the amino-terminus of the mature enzyme.

Consensus sequences, particularly around the active sites, have also been identified for serine and cysteine proteases; see, for example, Sakanari et al., 1989, Proc.

Natl. Acad. Sci. USA 86, 4863-4867. Although cysteine protease genes have been isolated from several mammalian sources and from the nematodes Haemonchus contortus Pratt et al., 1992, Mol. Biochem. Parasitol. 51, 209-218) and Caenorhabditis elegans (Ray et al., 1992, Mol. Biochem.

Parasitol. 51, 239-250), the cloning of such genes does not necessarily predict that the cloning of novel cysteine protease genes will be straight-forward, particularly since S. the sequences shared by different cysteine proteases are Ssuch that probes and .rimers based on the consensus sequences are highly degenerative.

Heartworm not only is a major problem in dogs, which typically are unable to develop immunity after infection dogs can become reinfected even after being cured by chemotherapy), but is also becoming increasingly widespread in other companion animals, such as cats and ferrets.

Heartworm infections have also been reported in humans.

Other parasite infections are also widespread, and all require better treatment, including preventative vaccine programs and/or targeted drug therapies.

Summary of the Invention The present invention relates to proteases including astacin metalloendopeptidases and cysteine proteases, therapeutic compositions comprising such nucleic acid molecules, proteins, antibodies and inhibitors thereof, and uses thereof to protect animals from disease caused by parasites.

In particular, the present invention as claimed provides an isolated protein encoded by a nucleic acid sequence that hybridizes under stringent hybridization conditions with a Dirofilaria immitis astacin metalloendopeptidase gene.

The invention as claimed also provides A therapeutic composition capable of protecting an animal from disease caused by a filariid susceptible to an inhibitor of an astacin metalloendopeptidase, said therapeutic composition comprising an excipient and at least one compound selected from the group consisting of: an isolated astacin metalloendopeptidase protein encoded by a nucleic acid sequence that hybridizes under stringent hybridization conditions with a D. immitis astacin metalloendopeptidase gene, or a mimetope thereof; an antibody that selectively binds to said astacin metalloendopeptidase protein; S and an inhibitor of said astacin metalloendopeptidase protein identified by its ability to inhibit filariid astacin metalloendopeptidase activity.

Various embodiments of the present invention are provided as follows: -7- One embodiment of the present invention is an isolated parasite nucleic acid molecule capable of hybridizing, under stringent conditions, with a Dirofilaria immitis astacin metalloendopeptidase gene. Such a nucleic acid molecule, namely a parasite astacin metalloendopeptidase nucleic acid molecule, can include a regulatory region of a parasite astacin metalloendopeptidase gene and/or encode at least a portion of a parasite astacin metalloendopeptidase protein. A preferred nucleic acid molecule of the present invention includes at least a portion of SEQ ID NO: 1 and/or of 10 SEQ ID NO:2. The present invention also includes recombinant molecules and recombinant cells that include parasite astacin metalloendopeptidase 9 nucleic acid molecules of the present invention. Also included are methods S. to produce such nucleic acids, recombinant molecules and recombinant cells of the present invention.

e. .94099 9 09 Another embodiment of the present invention is an isolated protein that includes a parasite astacin metalloendopeptidase protein or a mimetope of such a protein. A parasite astacin metalloendopeptidase protein of the present invention preferably has astacin metalloendopeptidase activity and/or comprises a protein that, when administered to an animal in an effective manner, is capable of eliciting an immune response against a natural parasite astacin metalloendopeptidase protein. The present invention also includes inhibitors of astacin -8 metalloendopeptidase activity as well as antibodies that recognize selectively bind to) a parasite astacin metalloendopeptidase protein and/or mimetope thereof of the present invention. Also included are methods to produce such proteins, inhibitors and antibodies of the present invention.

Yet another embodiment of the present invention is an isolated filariid nematode nucleic acid molecule capable of hybridizing, under stringent conditions, with a D. immitis cysteine protease gene. Such a nucleic acid molecule, namely a filariid cysteine protease nucleic acid molecule, can include a regulatory region of a filariid cysteine p: otease gene and/or encode at least a portion of a filariid cysteine protease protein. A preferred nucleic 15 acid molecule of the present invention includes at least a portion of SEQ ID NO:12. The present invention also includes recombinant molecules and recombinant cells that include filariid cysteine protease nucleic acid molecule.s of the present invention. Also included are methods to 20 produce such nucleic acids, recombinant molecules and recombinant cells of the present invention.

Another embodiment of the present invention is an isolated protein that includes a filariid cysteine protease protein or a mimetope of such a protein. A filariid cysteine protease protein of the present invention preferably has cysteine protease activity and/or comprises a protein that, when administered to an animal in an effective manner, is capable of eliciting an immune 9 response against a natural filariid cysteine protease protein. The present invention also includes inhibitors of cysteine protease activity as well as antibodies that recognize selectively bind to) a filariid cysteine protease protein and/or mimetope thereof of the present invention. Also included are methods to produce such proteins, inhibitors and antibodies of the present invention.

Yet another embodiment of the present invention is a 10 therapeutic composition capable of protecting an animal from disease caused by a parasite. Such a therapeutic composition includes at least one of the following protective compounds: an isolated parasite astacin metalloendopeptidase protein or a mimetope thereof; an 15 isolated parasite nucleic acid molecule capable of hybridizing under stringent conditions with a D. immitis astacin metalloendopeptidase gene; an anti-parasite astacin metalloendopeptidase antibody; an inhibitor of astacin metalloendopeptidase activity identified by its ability to 20 inhibit parasite astacin metalloendopeptidase activity; an isolated filariid nematode cysteine protease protein or a mimetope thereof; an isolated filariid nematode nucleic acid molecule capable of hybridizing under stringent conditions with a D. immitis cysteine protease gene; an anti-filariid nematode cysteine protease antibody; and an' inhibitor of cysteine protease activity identified by its ability to inhibit filariid nematode cysteine protease activity. Also included is a method to protect an animal 10 from disease caused by a parasite that includes administering to the animal in an effective manner a therapeutic composition of the present invention. A preferred therapeutic composition of the present invention is a composition capable of protecting an animal from heartworm.

The present invention also includes a method to identify a compound capable of inhibiting astacin metalloendopeptidase activity of a parasite. Such a method 10 includes contacting an isolated parasite astacin metalloendopeptidase protein with a putative inhibitory compound under conditions in which, in the absence of the compound, the astacin metalloendopeptidase protein has astacin metalloendopeptidase activity; and determining 15 if the putative inhibitory compound inhibits astacin metalloendopeptidase a( ivity. Also included is a test kit :i to identify a compound capable of inhibiting astacin metalloendopeptidase activity that incl..des an isolated parasite astacin metalloendopeptidase protein having 20 astacin metalloendopeptidase activity and a means for determining the extent of inhibition of astacin metalloendopeptidase activity in the presence of a putative inhibitory compound.

Also included in the present invention is a method to i identify a compound capable of inhibiting cysteine proteaseo activity of a parasite. Such a method includes (a) contacting an isolated filariid cysteine protease protein with a putative inhibitory compound under conditions in 11 which, in the absence of the compound, the filariid cysteine protease protein has cysteine protease activity; and determining if the putative inhibitory compound inhibits cysteine protease activity. Also included is a test kit to identify a compound capable of inhibiting cysteine protease activity of a parasite that includes an isolated filariid cysteine protease protein having cysteine protease activity and a means for determining the extent of inhibition of cysteine protease activity in the presence of 10 a putative inhibitory compound.

Detailed Description of the Invention The present invention includes the discovery that parasites such as D. immit.'s express astacin metalloendopeptidases. As such, the present invention includes nucleic acid molecules encoding such proteins as well as the proteins themselves. To the inventors' a knowledge, astacin metalloendopeptidases have been found previously only in humans, mice, rats, crayfish, Drosophila, Xenopus and sea urchins. The present invention also includes the first identification and isolation of nucleic acid molecules encoding filariid nematode cysteine proteases as well as the cysteine proteases themselves, after a difficult and time-consuming search by the inventors for such nucleic acid molecules. Isolated nucleic acid molecules and proteins of the present invention, including homologues of such nucleic acid molecules and proteins, are useful both in protecting animals from 12 parasite infections and in other applications, including those disclosed below.

One embodiment of the present invention is an isolated parasite (or parasitic) astacin metalloendopeptidase protein or a mimetope thereof a mimetope of a parasite astacin metalloendopeptidase protein). According to the present invention, an isolated, or biologically pure, protein, is a protein that has been removed from its natural milieu. As such, "isolated" and "biologically 10 pure" do not necessarily reflect the extent to which the protein has been purified. An isolated parasite astacin metalloendopeptidase protein can be obtained from its natural source. Such an isolated protein can also be produced using recombinant DNA technology or chemical 15 synthesis.

As u i. herein, an isolated parasite astacin metalloendopeptidase protein can be a full-length protein or any homologue of such a protein, such as a protein in which amino acids have been deleted a truncated 20 version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized by glycosylation, phosphorylation, acetylation, myristylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol) such that the homologue has astacin metalloendopeptidase activity and/or is capable of' eliciting an immune response against a natural D. immitis astacin metalloendopeptidase protein. As used herein, an astacin metalloendopeptidase protein, the full-length 13 version of which is a protein that includes an extended zinc-binding domain, has characteristics similar to astacin and other members of the "astacin family of metalloendopeptidases"; see, for example, Dumermuth et al., ibid. A protein having astacin metalloendopeptidase activity is a protein that can cleave proteins in a manner similar to the zinc-dependent protease astacin. Astacin activity is inhibited by metal chelators such as ethylene diamine tetraacetic acid (EDTA) and 1,10-phenanthroline but 10 not by phosphoramidon, an inhibitor of several other metalloproteases including thermolysin and neutral Sendopepti-lases. Such an activity can be detected by those skilled in the art (see, for example, Dumermuth et al., ibid.). A protein capable of eliciting an immune response 15 against a natural protein is a protein that includes at least one epitope capable of eliciting an immune response .(humoral and/or cellular) against that natural protein (such as a D. immitis astacin m.'talloendopeptidase or a D.

immitis cysteine protease) when the protein the 20 natural protein or a homologue thereof) is administered to an animal as an immunogen using techniques known to those skilled in the art. The ability of a protein to effect an immune response can be measured using techniques known to those skilled in the art. Examples of methods to measure an immune response antibody detection, resistance to' infection) are disclosed herein.

A parasite astacin metalloendopeptidase protein of the present invention, including a homologue of the full-length 14 protein, has the further characteristic of being encoded by a nucleic acid molecule that is capable of hybridizing, under stringent conditions, with to) a D. immitis astacin metalloendopeptidase gene. A preferred astacin metalloendopeptidase protein of the present invention is capable of hybridizing, under stringent conditions, with at least a portion of the nucleic acid sequence disclosed in SEQ ID NO:1 and/or SEQ ID NO:2. As used herein, the phrase "at least a portion of" an entity refers to an amount of S 10 the entity that is at least sufficient to have the functional aspects of that entity. For example, at least a portion of a nucleic acid sequence, as used herein, is an amount of a nucleic acid sequence capable of forming a stable hybrid under stringent hybridization conditions. As *9 S 15 used herein, stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules (or seq.ences), including oligonucleotides, are used to identify similar sequences. Such standard conditions are disclosed, for example, in Sambrook et al., 20 Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989. Examples of such conditions are provided in the Examples section. It should be noted that the extent of homology required to form a stable hybrid can vary depending on whether the homologous sequences are interspersed throughout the nucleic acid molecules or are clustered localized) in distinct regions on the nucleic acid molecules. Also in accordance with present invention, at least a portion of a astacin 15 metalloendopeptidase protein is a protein of sufficient size to have astacin metalloendopeptidase activity and/or to be able to elicit an immune response against a natural parasite astacin metalloendopeptidase.

SEQ ID NO:1 and SEQ ID NO:2 represent the deduced sequences of two cDNA (complementary DNA) nucleic acid molecules denoted nDiMPAl i2 and nDiMPA2 2 126 respectively, the production of which is disclosed in the Examples. It should be noted that since nucleic acid sequencing 10 technology is not entirely error-free, SEQ ID NO:1 and SEQ ID NO:2 represent, at best, apparent nucleic acid sequences of the respective nucleic acid molecules. As will be discussed in greater detail below, nucleic acid molecules nDiMPA1, 29 and nDiMPA2 212 6 apparently comprise overlapping open reading frames, as deduced from SEQ ID NO:1 and SEQ ID NO:2.

Nucleic acid molecule nDiMPA1,, g apparently comprises g three open reading frames, referred to herein as PDiMPAlo 0 p, PDiMPAlo 0 R and PDiMPAlo 0 R, the deduced amino acid sequences 20 of which are disclosed, respectively, in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5. Nucleic acid molecule nDiMPA2 226 apparently comprises five open reading frames, referred to herein as PDiMPA2 0 oi, PDiMPA2o2, PDiMPA2o3, PDiMPA2o 0 m and PDiMPA2o 5 the deduced amino acid sequences of which are.

disclosed, respectively, in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10. That the open reading frames on both nucleic acid molecules are overlapping raises the possibility that translation of a 16 functional astacin metalloendopeptidase in vivo may involve frameshifting. Both SEQ ID NO:1 and SEQ ID NO:2 contain nucleic acid sequences, including stem-loop structures, that, for frameshift viral gene expression, have been implicated in ribosome slowing and, hence, frameshift translation. Nucleic acid molecules nDiMPAl, and nDiMPA2 2 1 2 6 may also be the result of alternative splicing patterns.

SEQ ID NO:11 represents a composite amino acid 10 sequence derived from the five open reading frames encoded by nDiMPA2 21 26 As such, SEQ ID NO:11 is an example of a combination of disclosed open reading frames, in this case a combination of PDiMPA2o 0 R, PDiMPA2o 0 2, PDiMPA2 0 oR, PDiMPA2oW 4 and PDiMPA2o 0 5 The astacin domain of SEQ ID 15 NO:11 has about 29 percent amino acid sequence homology identity within comparable regions) with the amino acid sequence of crayfish astacin. As used herein, an astacin domain is an amino acid sequence of about 200 amino acids that shares significant homology with crayfish 20 astacin, which is a 200-amino acid protein. The astacin domain of SEQ ID NO:11 spans from about amino acid positions 99 through 296. The astacin domain of SI2 ID NO:11 also shares about 30 percent, 31 percent, 33 percent and 33 percent homology at the amino acid level with the astacin domains of, respectively, human bone morphogenetic protein 1, mouse kidney brush border metalloendopeptidase, human intestinal brush border metalloendopeptidases and 17 Xenopus laevis embryonic protein UVS.2 (using sequences provided in Dumermuth et al., ibid.).

A preferred astacin metalloendopeptidase protein of the present invention includes an amino acid sequence having at least about 35 percent, more preferably at least about 45 percent, even more preferably at least about percent and even more preferably at least about 75 percent, amino acid homology with the astacin domain of SEQ ID NO:11 with the corresponding regions of the astacin domain 10 of SEQ ID NO:11). A more preferred astacin etalloendopeptidase protein of the present invention /includes at least a portion of at least one of the following open reading frames: PDiMPAlonpj, PDiMPAlo1 0 PDiMPAloRm, PDiMPA2o 0 1 PDiMPA2,p 2 PDiMPA2o 0 R, PDiMPA2o 0 4 and 15 PDiMPA2oR 5 the deduced amino acid sequences of which are disclosed, respectively, in SEQ ID NO:3, SEQ ID NO:4, SEQ NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID Preferred astacin itetalloendopeptidase proteins of the 20 present invention include an extended zinc-binding domain motif. More preferred astacin metalloendopeptidase proteins also contain the tyrosine zinc binding amino acidcontaining domain as identified by Jiang et al., ibid, and disclosed above.

Parasite astacin metalloendopeptidase protein homologues can be the result of natural allelic variation or natural mutation. Homologues can also be produced using techniques known in the art including, but not limited to, 18 direct modifications to the protein or modifications to the gene encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis. Isolated astacin metalloendopeptidase proteins of the present invention, including homologues, can be identified in a straight-forward manner by the proteins' ability to effect astacin metalloendopeptidase activity and/or to elicit an immune response against parasite astacin metalloendopeptidase proteins. Examples of such 10 identification techniques are disclosed herein.

The minimum size of an isolated protein of the present invention is sufficient to form an epitope, a size that is typically at least from about 7 to about 9 amino acids. As is appreciated by those skilled in the art, an epitope can include amino acids that naturally are contiguous to each other as well as amino acids that, due to the tertiary structure of the natural protein, are in sufficiently close proximity to form an epitope.

Any parasite astacin metalloendopeptidase is a 20 suitable protein of the present invention. Suitable parasites from which to isolate proteins (including isolation of the natural protein or production of the protein by recombinant or synthetic techniques) include parasitic helminths, protozoa and ectoparasites such as, but not limited to nematodes, cestodes, trematodes, fleas, flies, ticks, lice, true bugs,' and protozoa of the genera Babesia, Cryptosporidium, Eimeria, Encephalitozoon, Hepatozoon, Isospora, Leishmania, Neospora, Nosema, -19 Plasmoodiurn, Pneumocystis, Theileria, Toxoplasma and Trypanosoma. Preferred parasites include tissue-migrating parasitic helminths, and particularly tissue-migrating nematodes. Preferred nematodes include filariid, ascarid, strongyle and trichostrongyle nematodes. Particularly preferred tissue-migrating nematodes include parasites of the genera Acanthocheilonema, Aelurastrongylus, Ancylostoma, Angiostrongylus, Ascaris, Brugia, Bunostomum, Dictyocaulus, Dioctophyne, Dipetalonema, Dirofilaria, Dr-acunculus, Filaroides, Lagochilascaris, Loa, Mansonella, Muellerius, Necator, Onchocerca, Parafilaria, Parascarls, Protostrongylus, Setaria, Ste phanofil aria, Strongyloides, Strongylus, Thelazia, Toxascaris, Toxocara, Tn chinel La, Uncinaria and Wuchereria. other particularly preferred 15 nematodes include parasites of the gcnera Capillania, Chabertia, C-operia, Enteroblus, Haemonchus, Nernatodirus, Oesophagostomum, Ostertagia, Tnichostrongylus and TrichuxnIs. Preferred filariid nematodes include Dirotilaria, Acanthocheilonema, Brugia, Dipetalonena, Loa, 20 Onchocerca, Parafilaria, Setaria, Stephanofilaria and Wuchereria filariid nematodes. A particularly preferred parasite is a nematode of the genus Dir-ofilaria, with D.

immzitis, the parasite that causes heartworm, being even more preferred.

In accordance with the present invention, a mimetope' of a protein refers to any compound that is able to mimic the activity of that protel~, often because the mimetope has a structure that mimics the protein. For example, a 20 mimetope of a parasite astacin metalloendopeptidase protein is a compound that has an activity similar to that of an isolated parasite astacin metalloendopeptidase protein of the present invention. Mimetopes can be, but are not limited to: peptides that have been modified to decrease their susceptibility to degradation; anti-idiotypic and/or catalytic antibodies, or fragments thereof; nonproteinaceous immunogenic portions of an isolated protein carbohydr..te structures); and synthe tic or natural organic molecules, including nucleic acids. Such mimetopes can be designed using computer-generated structures of proteins of the present invention. Mimetopes can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides or other organic molecules, and 15 screening such samples by affinity chromatography techniques using, for example, antibodies raised against a protein of the present invention.

A preferred parasite astacin metalloendopeptidase protein cr mimetope of the present invention is a compound 20 that when administered to an animal in an effective manner, is capable of protecting that animal from disease caused by a parasite that is susceptible to treatment to inhibit astacin metalloendopeptidase activity. As such, the parasite preferably is essentially incapable of causing disease in an animal that is immunized with a parasite astacin metalloendopeptidase protein, and preferably with a D. immitis astacin metalloendopeptidase protein, of the present invention. In accordance with the present 21 invention, the ability of a protein or mimetope of the present invention to protect an animal from disease by a parasite refers to the ability of that protein or mimetope to treat, ameliorate and/or prevent disease, including infection leading to disease, caused by the parasite, preferably by eliciting an immune response, against the parasite. Such an immune response can include humoral and/or cellular immune responses. Suitable parasites to target include any parasite that is essentially incapable 10 of causing disease in an animal administered a parasite astacin metalloendopeptidase protein of the present invention. As such, a p: rasite to target includes any parasite that produces a protein having one or more epitopes that can be targeted by a humoral and/or cellular 15 response against a parasite astacin metalloendopeptidase a protein of the present invention and/or that can be targeted by a compound that is capable of substantially inhibiting parasite astacin metalloendopeptidase activity, thereby resulting in the reduced ability of the parasite to 20 cause disease in an animal. Suitable and preferred parasites to target include those parasites disclosed above. A preferred class of parasites to target include tissue-migrating parasitic helminths. A particularly preferred nematode helminth to target is D. immitis, which causes heartworm.

One embodiment of the present invention is a fusion protein that includes a parasite astacin metalloendopeptidase domain attached to a fusion segment.

22 Inclusion of a fusion segment as part of a protein of the present invention can enhance the protein's stability during production, storage and/or use. Depending on the segment's characteristics, a fusion segment can also act as an immunopotentiator to enhance the immune response mounted by an animal immunized with a protein of the present invention that contains such a fusion segment. Furthermore, a fusion segment can function as a tool to simplify purification of a protein of the present invention, such as 10 to enable purification of the resultant fusion protein using affinity chromatography. A suitable- fusion segment can be a domain of any size that has the desired function imparts increased stability to a protein, imparts increased immunogenicity to a protein, and/or simplifies S 15 purification of a protein). It is within the scope of the present invention to use one or more fusion segments.

Fusion segments can be joined to amino and/or carboxyl 'termini of the astacin metalloendopeptidase-containing domain of the protein. Linkages between fusion segments and 20 astacin metalloendopeptidase-containing domains of fusion proteins can be susceptible to cleavage in order to enable straight-forward recovery of the astacin metalloendopeptidase-containing domains of such proteins.

Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid molecul that encodes a protein including the fusion segment attached to either the carboxyl and/or amino 23 terminal end of an parasite astacin metalloendopeptidasecontaining domain.

Preferred fusion segments for use in the present invention include a glutathione binding domain, such as Schistosoma japonicum glutathione-S-transferase (G T) or a portion thereof capable of binding to glutathione; a metal binding domain, such as a poly-histidine segment capable of binding to a divalent metal ion; an immunoglobulin binding domain, such as Protein A, Protein G, T cell, B cell, Fc 10 receptor or complement protein antibody-binding domains; a sugar binding domain such as a maltose binding domain from a maltose binding protein; and/or a "tag" domain at least a portion of f-galaotosidase, a strep tag peptide, other domains that can be purified using compounds that 15 bind to the domain, such as monoclonal antibodies). More preferred fusion segments include metal binding domains, such as a poly-histidine segment; a maltose binding domain; a strep tag peptide, such as that available from Biometra in Tampa, FL; and an S10 peptide. An example of a preferred 20 fusion protein of the present invention is PHIS-PDiMPA2,, the production of which is disclosed herein.

Another embodiment of the present invention is a parasite astacin metalloendopeptidase protein that also includes at least one additional protein segment that is capable of protecting an animal irom one or more diseases. Such a multivalent protective protein can be produced by culturing a cell transformed with a nucleic acid molecule comprising two or 24 more nucleic acid domains joined together in such a manner that the resulting nucleic acid molecule is expressed as a multivalent protective compound containing at least two protective compounds, or portions thereof, capable of protecting an animal from diseases caused, for example, by at least one infectious agent.

Examples of multivalent protective compounds include, but are not limited to, a parasite astacin metalloendopeptidase protein attached to one or more other 10 parasite proteins, such to a filariid nematode cysteine protease protein of the present invention. Other examples *of multivalent protective compounds include a parasite astacin metalloendopeptidase protein attached to one or more compounds protective against one or more other S 15 infectious agents, particularly an agent that infects cats or dogs, such as, but not limited to, calicivirus, distemper virus, feline herpesvirus, feline immunodeficiency virus, feline leukemia virus, feline infectious peritonitis, hepatitis, hookworm, leptospirosis, S. 20 panleukopenia virus, parvovirus, rabies and toxoplasmosis.

Suitable heartworm multivalent protective proteins include, but are not limited to, a D. immitis astacin metalloendopeptidase and/or a D. immitis cystein protease of the present invention attached to at least one other D.

immitis protein such as, but not limited to, a D. immitis Gp29 protein, a D. immitis P39 protein, a D. immitis P22U protein, a D. immitis P22L protein, a D. immitis P20.5 protein, a D. immitis P4 protein, a D. immitis Di22 protein 25 and/or a D. immitis protease expressed in L3 and/or L4 larvae, as well as other helminth proteins sharing significant homology with such D. immitis proteins. A protein sharing significant homology with another protein refers to the ability of the nucleic acid sequences encoding such proteins to form stable hybridization complexes with each other under stringent hybridization conditions, as described, for example, in Sambrook et al., ibid. U.S. Patent Application Serial No. 08/208,885, filed 10 March 8, 1994, entitled immitis Gp29 Proteins, Nucleic Acid Molecules and Uses Thereof", discloses D. immitis Gp29 proteins and nucleic acid molecules that encode them. U.S.

Patent Application Serial No. 08/003,389, filed January 12, 1993, entitled "Immunogenic Larval Proteins", discloses a 15 39-kD (kilodalton) D. immitis protein (size determined by Tris glycine SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis)), referred to herein as P39, and a nucleic acid sequence that encodes it. U.S.

Patent Application Serial No. 08/003,257, filed January 12, o 20 1993, entitled "Reagents and Methods for Identification of Vaccines", discloses 22-kD and 20.5-kD D. immitis proteins (sizes determined by Tris glycine SDS-PAGE), referred to herein as P22L and P20.5, and nucleic acid sequences that encode them. U.S. Patent Application Serial No. 08/109,391, filed August 19, 1993, entitled "Novel Parasitic Helminth' Proteins", discloses D. immitis P4 and D. immitis P22U, as well as nucleic acid sequences that encode them. U.S.

Patent Application Serial No. 08/060,500, filed May 26 1993, entitled "Heartworm Vaccine", discloses a D. immitis Di22 protein and a nucleic acid sequence encoding it (included in Genbank data base accession number M82811); Serial No. 08/060,500 is a continuation of U.S. Patent Application Serial No. 07/683,202, filed April 8, 1991.

U.S. Patent Application Serial No. 08/153,554, filed November 16, 1993, entitled "Protease Vaccine Against Heartworm", discloses D. immitis larval proteases; Serial No. 08/153,554 is a continuation of U.S. Patent Application 10 Serial No. 07/792,209, filed November 12, 1991. Each of these patent applications is incorporated by reference herein in its entirety.

A particularly preferred parasite astacin metalloendopeptidase protein is a protein encoded by at we 15 least a portion of SEQ ID NO:1 or SEQ ID NO:2, and, as such, is a protein having an amino acid sequence that includes at least a portion of at least one of the open reading frames represented by, respectively, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10. Preferred proteins can include combinations of such reading frames, such as SEQ ID NO:11, that result in a functional protein. A homolotl search using these open reading frames indicate that all but SEQ ID NO:10 share significant homology with known I members of the astacin metalloendopeptidase family and with' a Caenorhabditis elegans R151.5 gene product, Genbank accession number U00036 (Wilson et al., 1994, Nature 368, 32-38), suggesting that C. elegans also encodes a astacin 27 metalloendopeptidase. Particularly well conserved are the extended zinc-binding domain motif and the tyrosinecontaining domain motif, the overall sequence homology being about 24 percent.

Particularly preferred proteins of the present invention include proteins having the astacin domain of SEQ ID NO:11, proteins that include that domain (such as, but not limited to, full-length proteins, fusion proteins and proteins providing multivalent protection) and proteins 10 that are truncated homologues of that domain. Even more obe** preferred proteins include PDiMPA204 and PHIS-PDiMPA2 8 0.

s a .6 04 Another embodiment of the present invention is an isolated parasite nucleic acid molecule capable of hybridizing, under stringent conditions, with a D. immitis 15 astacin metalloendopeptidase gene. As used herein, a D.

immitis astacin metalloendopeptidase gene includes all nucleic acid sequences related to a natural D. immitis astacin metalloendopeptidase gene such as regulatory regions that control production of a D. immitis astacin o 20 metalloendopeptidase protein encoded by that gene (such as, but not limited to, transcription, translation or posttranslation control regions) as well as the coding region itself. A parasite astacin metalloendopeptidase nucleic acid molecule of the present invention can include any isolated natural parasite astacin metalloendopeptidase gene' or a homologue thereof. A nucleic acid molecule of the 4 present invention can include one or more regulatory regions, full-length or partial coding regions, or 28 combinations thereof. The minimal size of a parasite astacin metalloendopeptidase nucleic acid molecule of the present invention is the minimal size capable of forming a stable hybrid under stringent hybridization conditions.

Suitable and preferred parasites are disclosed above.

In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu that has been subject to human manipulation). As such, "isolated" does not reflect the extent to which the nucleic acid molecule has been purified. An isolated nucleic acid molecule can include DNA, RNA, or derivatives of either DNA or RNA.

An isolated parasite astacin metalloendopeptidase nucleic acid molecule of the present invention can be obtained from its natural source either as an entire complete) gene or a portion thereof capable of forming a stable hybrid with that gene. An isolated parasite astacin o metalloendopeptidase nucleic acid molecule can also be produced using recombinant DNA technology polymerase 20 chain reaction (PCR) amplification, cloning) or chemical synthesis. Isolated parasite astacin metalloendopeptidase nucleic acid molecules include natural nucleic acid molecules and homologues thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode a parasite 29 astacin metalloendopeptidase protein of the present invention or to form stable hybrids under stringent conditions with natural isolates.

A parasite astacin metalloendopeptidase nucleic acid molecule homologue can be produced using a number of methods known to those skilled in the art (see,. for example, Sambrook et al., ibid.). For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as sitedirected mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification 15 and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof. Nucleic acid molecule homologues can be selected from a mixture of modified nucleic acids by screening for the function of the protein encoded by the nucleic acid astacin metalloendopeptidase activity or ability to elicit an immune response against at least one epitope of a parasite astacin metalloendopeptidase protein) and/or by hybridization with isolated parasite astacin metalloendopeptidase nucleic acids under stringent conditions.

30 An isolated nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes at least one parasite astacin metalloendopeptidase protein of the present invention, examples of such proteins being disclosed herein. Although the phrase "nucleic acid *molecule" primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid sequence" primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a protein.

As heretofore disclosed, parasite astacin metalloendopeptidase proteins of the present invention include, but are not limited to, proteins having full- 15 length astacin metalloendopeptidase coding regions, proteins having partial astacin metalloendopeptidase coding regions, fusion proteins, multivalent protective proteins and combinations thereof.

One embodiment of the present invention is a parasite 20 astacin metalloendopeptidase nucleic acid molecule that is S. capable of hybridizing under stringent conditions with nucleic acid molecule nDiMPA1 1 2 and/or with nucleic acid molecule nDiMPA2 2 1 2. As such, preferred astacin metalloendopeptidase nucleic acid molecules are capable of forming stable hybrids with nucleic acid molecules represented by SEQ ID NO:1 and SEQ ID NO:2. Particularly preferred astacin metalloendopeptidase nucleic acid molecules comprise at least a portion of nucleic acid 31 molecule nDiMPA 1 2 and/or nucleic acid molecule nDiMPA2 21 26 As such, a preferred nucleic acid molecule of the present invention includes a nucleic acid sequence including at least a portion of SEQ ID NO:1 OR SEQ ID NO:2. Such a nucleic acid molecule can be nDiMPA1, or nDiMPA22ne, can include nucleotides in addition to nDiMPAl 1 or nDiMPA2 212 or can be a truncation fragment of nDiMPA1, or nDiMPA2 21

M.

Particularly preferred nucleic acid molecules include nDiMPA1 68 9 nDiMPA1,2, nDiMPA2 2 12 nDiMPA2g,, nDiMPA2., and BvMPA2, the production of which is disclosed in the o..

Example-.

One preferred embodiment of the present invention is a parasite astacin metalloendopeptidase nucleic acid molecule capable of hybridizing to a nucleic acid molecule 15 encoding at least one of the following open reading frames as denoted by the following amino acid sequences: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 AND SEQ ID NO:11.

Preferably, such a nucleic acid molecule encodes a protein 20 that shares at least about 35 percent, more preferably at least about 45 percent, even more preferably at least about percent and even more preferably at least about percent amino acid homology with SEQ ID NO:11. More preferred astacin metalloendopeptidase nucleic acid molecules encode at least a portion of at lea:st one of such open reading frames. Particularly preferred astacin metalloendopeptidase nucleic acid molecules are capable of forming stable hybrids with nucleic acid molecules encoding 32 an extended zinc-binding domain motif to the carboxyl terminus of the motif as well as to the general zinc-binding domain) and, more preferably, also with nucleic acid molecules encoding other disclosed conserved domains of astacin metalloendopeptidases, such as the motif that contains the tyrosine believed to bind to zinc.

Knowing the nucleic acid sequence of certain parasite astacin metalloendopeptidase nucleic acid molecules of the present invention allows one skilled in the art to make copies of those nucleic acid molecules as well as to obtain nucleic acid molecules including at least a portion of such nucleic acid molecules and other parasite astacin metalloendopeptidase nucleic acid molecule homologues. Such nucleic acid molecules can be obtained in a variety of ways 15 iicluding screening appropriate expression libraries with antibodies of the present invention; traditional cloning techniques using oligonucleotide probes of the present invention to screen appropriate libraries or DNA; and PCR amplification of appropriate libraries or DNA using 20 oligonucleotide primers of the prerent invention.

Preferred libraries to screen or from which to amplify include parasite larval (especially L3, L4) and adult cDNA libraries as well as genomic DNA libraries. Similarly, preferred DNA sources to screen or from which to amplify include parasite larval (especially L3, L4) and adult cDNA and genomic DNA. Techniques to clone and amplify genes are disclosed, for example, in Sambrook et al., ibid.

33 The present invention also includes nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent conditions, with complementary regions of other, preferably longer, nucleic acid molecules of the present invention, such as to complementary regions of a parasite astacin metalloendopepticd se gene, including complementary regions of a D. immitis astacin metalloendopeptidase gene. Such oligonucleotides can hybridize under strir.gent conditions with complementary 10 regions of nDiMPAl, or nDiMPA2 21 2, complementary regions of nucleic acid molecules that include at least a portion of nDiMPA1 1 or nDiMAA2 2 1 6 and complementary regions of nucleic acid molecules that hybridize under stringent conditions with nDiMPA1,2 or nDiMPA2 2 26 Such 15 oligonucleotides can be RNA, DNA, or derivatives of either.

The minimal size of such oligonucleotides is the size required to form a stable hybrid between a given oligonucleotide and the comIlententary sequence on another nucleic acid molecule of the present invention. As such, 20 the size is dependent on nucleic acid composition and percent homology between the oligonucleotide and complementary sequence as well as upon hybridization conditions per se temperature, salt concentration).

For AT-rich nucleic acid sequences, such as those of parasitic helminths, oligonucleotides typically are at k least about 15 to about 17 bases in length. The size of the oligonucleotide must also be sufficient for the use of the oligonucleotide in accordance with the present 34 invention. Oligonucleotides of the present invention can be used in a variety of applications including, but not limited to, as probes to identify additional nucleic acid molecules, as primers to amplify or extend nucleic acid molecules or in therapeutic applications to inhibit, for example, expression of astacin metalloendopeptidases by a parasite. Such therapeutic applications includethe use of such oligonucleotides in, for example, antisense-, triplex formation-, ribozyme- and/or RNA drug-based technologies.

S 10 The present invention, therefore, includes such oligonucleotides and methods to interfere with the production of astacin metalloendopeptidase proteins by use S0 of one or more of such technologies. Appropriate oligonucleotide-containing therapeutic compositions can be 15 administered to an animal, using techniques known to those skilled in the art, either prior to or after infection by a parasite such as D. immitis, in order to protect the animal from disease.

Another embodiment of the present invention is an S 20 isolated filariid nematode cysteine protease protein or a mimetope thereof. As used herein, an isolated filariid nematode, or filariid, cysteine protease protein can be a full-length filariid cysteine protease protein or any homologue of such a protein. Filariid nematode cysteine protease proteins, including homologues thereof, can be isolated and produced according to the methods disclosed herein for parasite astacin metalloendopeptidase proteins.

Homologues and mimetopes of filariid cysteine protease 35 proteins are defined in a similar manner as are homologues and mimetopes of parasite astacin metalloendopeptidase proteins. Filariid cysteine protease proteins (including homologues) and mimetopes thereof each are capable of eliciting an immune response having at least one epitope capable of eliciting an immune response) against a filariid cysteine protease protein and/or are capable of effecting cysteine protease activity. Cysteine protease activity, as well as the ability of a protein to effect an 10 immune response, can be measured using techniques known to those skilled in the art. Cysteine protease activity can be measured by its ability to cleave peptides having a cysteine protease cleavage site, such as z-Val-Leu-Arg-AMC; such activity can be inhibited by, for example, by the S: 15 cysteine protease inhibitor E-64 (available from Boehringer Mannheim, Indianapolis, IN). Preferred filariids are disclosed herein. A particularly preferred filariid is D.

immitis.

A filariid cysteine protease protein of the present 20 invention, including any homologue thereof, has the additional characteristic of being encoded by a nucleic acid molecule that is capable of hybridizing, under stringent conditions, with a nucleic acid molecule comprising at least a portion of a nucleic acid sequence encoding a filariid cysteine protease protein. Preferred proteins are encoded by a nucleic acid molecule that forms stable hybrids with at least a portion of nDiCP 3 the production of which is described in detail in the Examples.

36 SEQ ID NO:12 represents the deduced sequence of nDiCP, 43 the deduced translation product of which is a 47 amino acid sequence represented in SEQ ID NO:13, the protein being denoted PDiCP 43 It should be noted that since nucleic acid sequencing technology is not entirely error-free, SEQ ID NO:12, at best, represents an apparent nucleic acid sequence of a nucleic acid molecule encoding at least a portion of full-length D. immitis cysteine protease.

Furthermore, SEQ ID NO:13 apparently represents an internal 10 amino acid sequence of D. immitis cysteine protease since SEQ ID NO:12 apparently has neither a start nor stop codon.

SEQ ID NO:13, however, includes amino acid sequences that are conserved among a number of cysteine proteases.

A comparison of SEQ ID NO:13 with the corresponding 15 regions of known parasite cysteine protease genes indicates *99 that SEQ ID NO:13 shares about 16 percent, about 22 percent, about 24 percent, about 35 percent, about 39 percent, about 44 percent and about 49 percent homology at the amino acid level with cysteine proteases from, 20 respectively, H. contortus (a nematode), Schistosoma *9 mansoni (a trematode), C. elegans (a nimatode), Fasciola hepatica (a trematode), Entamoeba histolytica (a protozoa), Trypanosoma cruzi (a protozoa) and T. brucie. SEQ ID NO:13 also shares about 50 percent amino acid homology w.th human cathepsin L, about 45 percent amino acid homology with chicken cathepsin L and about 56 percent amino acid homology with a Paragonimus westermani (trematode) cysteine protease. The serine at about position 30 and the cysteine 37 at about position 37 of SEQ ID NO:13 are conserved in all of these cysteine proteases. Please see, for example, the following for listings of the above-referenced sequences: Heussler et al., 1994, Mol. Biochem. Parasitol. 64, 11-23; Eakin et al., 1990, Mol. Biochem. Parasitol. 39, 1-8; Ray et alt, ibid., Pratt et al., ibid., Sakanari et al., ibid.; European Patent Application Publication No. 0524834A2, by Hamajima et al., published January 27, 1993.

Pref:-red filariid cysteine protease proteins of the present invention include amino acid sequences that share at least about 60 percent, more preferably-at least about percent, and even more preferably at least about percent, homology with SEQ ID NO:13. Particularly preferred filariid cysteine protease proteins of the present invention include PDiCP 4 proteins that include PDiCP 4 (including, but not limited to full-length proteins, fusion proteins and multivalent protective proteins), and proteins that include at least a portion of PDiCP 42 A preferred filariid cysteine protease protein or mimetope thereof is a compound that when administered to an

A

animal in an effective manner, is capable of protecting that animal from disease caused by a parasite that is susceptible to treatment with a composition that inhibits cysteine protease activity. A suitable parasite to target is any parasite that produces a protein having one or more epitopes that can be targeted by a humoral and/or cellular response against a filariid nematode cysteine protez.se protein of the present invention and/or that can be 38 targeted by a compound that is capable of substantially inhibiting filariid cysteine protease activity, thereby resulting in the reduced ability of the parasite to cause disease in an animal. Suitable and preferred parasites are disclosed above. A preferred class of parasites to target include tissue-migrating parasitic helminths.

A

particularly preferred nematode to target is D. immitis, which causes heartworm.

Also included in the present invention are fusion proteins and multivalent protective proteins that include at least one filariid cysteine protease protein. Such proteins can comprise fusion segments and/or multiple protective domains similar as disclosed for parasite astacin metalloendopeptidase proteins and can be produced in a similar manner as described for parasite astacin metalloendopeptidase proteins of the present inventioi.

Particularly preferred fusion proteins include PHIS-PDiCP 42 Yet another embodiment of the present invention is an isolated filariid nematode nucleic acid molecule capable of hybridizing, under stringent conditions, with a D. immitis cysteine protease gene. Such a nucleic acid molecule is referred to as a filariid nematode, or filariid, cysteiine protease nucleic acid molecule. As used herein, a filariid cysteine protease gene includes all nucleic acid sequences' related to a natural filariid cysteine protease gene such as regulatory regions that control production of a D.

iimitis cysteine protease protein encoded by that gene as well as the coding region itself. A filariid cysteine 39 protease nucleic acid molecule of the present invention can include an isolated natural filariid cysteine protease gene or a homologue thereof. A nucleic acid molecule of the present invention can include one or more regulatory regions, full-length or partial coding regions, or combinations thereof. The minimal size of a filariid cysteine protease nucleic acid molecule of the present invention is the minimal size capable of forming a stable hybrid under stringent hybridization conditions with a D.

immitis cystei:.e protease gene. Filariid immitis cysteine protease nucleic acid molecules can be isolated and produced according to the methods taught herein for the production and isolation of parasite astacin metalloendopeptidase ni- leic acid molecules. Cysteine protease nucleic acid molecule homologues can be selected a from a mixture of modified nucleic acids by screening for the function of the protein encoded by the nucleic acid cysteine protease activity and/or ability to elicit an immune response against at least one epitope of a filariid cysteine protease protein) and/or by hybridization with isolated D. immitis cysteine protease nucleic acids under stringent conditions.

An isolated filariid cysteine protease nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes at least one filariid cysteine' protease protein of the present invention, examples of such proteins being disclosed herein. As heretofore disclosed, filariid cysteine protease proteins of the present 40 invention include, but are not limited to, proteins having full-length filariid cysteine protease coding regions, proteins having partial filariid cysteine protease coding regions, fusion proteins, multivalent protective proteins and combinations thereof. The present invention also includes nucleic acid molecules encoding filariid cysteine protease proteins that have been modified to accommodate codon usage properties of the cells in which such nucleic acid molecules are to be expressed.

One embodiment of the present invention is a filariid cysteine protease nucleic acid molecule that includes a nucleic acid sequence that is capable of hybridizing under stringent conditions with D. immitis nucleic acid molecule nDiCP 43 the deduced sequence of which is disclosed in SEQ ID NO:12. Preferred filariid cysteine protease nucleic acid a molecules encode proteins having at least about 60 percent, more preferably at least about 70 percent and even more preferably at least about 80 percent, amino acid homology with SEQ ID NO:13. More preferred is a nucleic acid 20 molecule that encodes a D. immitis cysteine protease protein that comprises at least a portion of SEQ ID NO:13.

A preferred nucleic acid molecule of the present invention includes at least a portion of D. immitis nucleic acid molecule nDiCP, 4 Such a nucleic acid molecule can be nDiCP, 4 can include nucleotides in addition to nDiCP 43 (such as, but not limited to, a nucleic acid molecule encoding a full-length protein, a nucleic acid molecule encoding a fusion protein, or a nucleic acid molecule 41 encoding a multivalent protective compound), or can be a truncation fragment of nDiCP, 43 Particularly preferred filariid cysteine protease nucleic acid molecules include nDiCP 43 and nDiCP, 2 The inventors of the present invention had difficulty isolating a D. immitis cysteine protease nucleic acid molecule despite the wide variety of cysteine protease genes that have been cloned. Primers designed by the inventors using consensus sequences derived from known cysteine protease genes, including from known parasite cysteine protease genes, had a degeneracy that was o essentially too great to pull out a D. immitis cysteine protease gene. The inventors discovered that use of primers S. that incorporated D. immitis codon usage bias enabled the identification of the first D. immitis cysteine protease nucleic acid molecule, namely nDiCP, 43 Having identified the nucleic acid molecule nDiCP, 43 it is likely that one skilled in the art can make copies of that nucleic acid molecule as well as obtain other filariid 20 nematode cysteine protease nucleic acid molecul 3 including full-length genes and homologues thereof. Such nucleic acid molecules can be obtained in a variety of ways such as those described for the isolation and production of parasite astacin metalloendopeptidase nucleic acid molecules of the present invention. Preferred libraries to screen or from which to amplify include filariid larval (especially L3, L4) and adult cDNA libraries and filariid genomic DNA libraries. Similarly, preferred DNA sources to 42 screen or from which to amplify include filariid larval (especially L3, L4) and adult cDNA, as well as filariid genomic DNA. Preferred primers and probes to use are codon-biased for the given filariid.

The present invention also includes nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent conditions, with complementary regions of other, preferably longer, nucleic acid molecules of the present invention, such as to complementary regions of a filariid cysteine protease gene. Such oligonucleotides can hybridize under stringent conditions with complementary regions of nDiCP 43 complementary regions of nucleic acid molecules that include at least a portion of nDiCP 43 and complementary regions of nucleic acid molecules that hybridize under stringent conditions with nDiCP 43 Such oligonucleotides can be RNA, DNA, or derivatives of either.

Other criteria, such as minimal size, as well as methods to produce and use such oligonucleotides are as disclosed for parasite astacin metalloendopeptidase oligonucleotides of 20 the present invention.

The present invention also includes a recombinant vector, which includes at least one nucleic acid molecule of the present invention a parasite astacin retalloendopeptidase nucleic acid molecule and/or a filariid cysteine protease nucleic acid molecule, examples of which are disclosed herein) inserted into any vector capable of delivering the nucleic acid molecule into a host cell. Such a vector contains heterologous nucleic acid 43 sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention and that preferably are derived from a species other than the species from which the nucleic acid molecule(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating of parasite, including D. immitis, nucleic acid molecules of the present invention. One type of recombinant vector, herein referred to as a recombinant molecule and described in more detail below, can be used in the expression of nucleic acid molecules of the present invention. Preferred recombinant vectors are capable of replicating in the transformed cell.

Prefer ed nucleic acid molecules to include in recombinant vectors of the present invention include at least one of the following: a nucleic acid molecule that includes at least a portion of nDiMPA1,, a nucleic acid molecule that includes at least a portion of nDiMPA2 21 2 and a nucleic acid molecule that includes at least a portion of nDiCP, 43 Particularly preferred nucleic acid molecules to include in recombinant vectors of the present invention include nDiMPAl, 9 nDiMPAl 1 nDiMPA2 2 26 nDiMPA2,,, nDiMPA227, BvMPA2, nDiCP 1 4 3 and nDiCP 42 Isolated proteins of the present invention can be produced in a variety of ways, including production and recovery of natural proteins, production and recovery of 44 recombinant proteins, and chemical synthesis of the proteins. In one embodiment, an isolated protein of the present invention is produced by culturing a cell capable of expressing the protein under conditions effective to produce the protein, and recovering the protein. A preferred cell to culture is a recombinant. cell that is capable of expressing the protein, the recombinant cell being produced by transforming a host cell with one or more nucleic acid molecules of the present invention.

Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfecticn, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed recombinant) cell in such a manner that

C

their ability to be expressed is retained. A preferred nucleic acid molecule with which to transform a cell is a nucleic acid molecule that includes a parasite astacin metalloendopeptidase nucleic acid molecule and/or a filariid cysteine protease nucleic acid molecule of the present invention. Particularly preferred nucleic acid molecules with which to tranfo cells include n A,, molecules with which to transform cells include nDiMPAI 89 45 nDiMPAl 12 9, nDiMPA2 21 26 nDiMPA28g4, nDiMPA227, BVMPA2, nDiCP, 43 and nDiCP, 42 Suitable host cells to transform include any cell that can be transformed. Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule. Host cells of the present invention either can be endogenously naturally) capable of producing parasite, including D. immitis, proteins of the present invention or can be capable of producing such proteins after being transformed with at least one nucleic acid molecule of the present invention.

Host cells of the present invention can be any cell capable of producing at least one protein of the present invention, including bacterial, fungal (including yeast), animal parasite (including helminth, protozoa and ectoparasite), *o a insect, animal and plant cells. Preferred host cells include bacterial, mycobacterial, yeast, helminth, insect and mammalian cells. More preferred host cells include Salmonella, Escherichia, Bacillus, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells (normal dog kidney cell line for canine herpesvirus cultivation), CRFK cells (normal cat kidney cell line for feline herpesvirus cultivation) and COS cells. Particularly preferred host cells are Escherichia coli, including E. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains such as- UK-1 x3987 and SR-11 x4072; Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCK 46 cells; CRFK cells and non-tumorigenic mouse myoblast G8 cells ATCC CRL 1246). Additional appropriate mammalian cell hosts include other kidney cell lines CV-1 monkey kidney cell lines), other fibroblast cell lines human, murine or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster. ovary cells and/or HeLa cells.

A recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively linked to an expression vector containing one or more transcription control sequences. The phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified nucleic acid molecule.

Preferably, the expression vector is also capable of replicating within the host cell. Expression vecto- s can be *a either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors of the present invention include any vectors that function direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, animal parasite, insect, animal, and plant cells. Preferred expression vectors of the present invention can direct gene expression in 47 bacterial, yeast, helminth or other parasite, insect and mammalian cells and more preferably in the cell types heretofore disclosed.

Expression vectors of the present invention may also contain secretory signals signal segment nucleic acid sequences) to enable 'an expressed protein of the present invention a parasite astacin metalloendopeptidase protein and/or a filariid cysteine protease protein) to be secreted from the cell that produces the protein and/or contain fusion sequences .which lead to the expression of inserted nucleic acid molecules of the present invention as fusion proteins.

Eukaryotic recombinant molecules may include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequences of nucleic acid molecules of the a present invention. Examples of suitable fusion segments encoded by fusion segment nucleic acids have been disclosed. Suitable signal segments include natural signal segments a parasite astacin metalloendopeptidase or S 20 cysteine protease signal segment) or any heterologous signal segment capable of directing the secretion of a protein of the present invention. Preferred signal segments include, but are not limited to, tissue plasminogen activator interferon, interleukin, growth hormone,.

histocompatibility and viral envelope glycoprotein signal segments.

Nucleic acid molecules of the present invention can be operatively linked to expression vectors containing 48 regulatory sequences such as promoters, operators, repressors, enhancers, termination sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention. In particular,. recombinant molequles of the present invention inc.lude transcription control sequences.

Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which function in bacterial, yeast, helminth or other parasite, insect and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (such as XAp and Xp, and fusions that include such promoters), bacteriophage T7, T7lac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha mating factor, Pichia alcohol oxidase, alphavirus\ subgenomic promoters (such as Sindbis virus subgenomic promoters), baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, poxvirus, adenovirus, simian 49 virus 40, retrovirus actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells. Additional suitable transcription control sequences include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters promoters inducible by interferons or interleukins). Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with a parasitic helminth, such as a D. immitis, molecule prior

P

to isolation.

A recombinant molecule of the present invention is a molecule that can include at least one of any nucleic acid molecule heretofore described operatively linked to at least one of any transcription control sequence capable of effectively regulating expression of the nucleic acid molecule(s) in the cell to be transformed, examples of which are disclosed herein. Particularly preferred recombinant molecules include pfgal-nDiMPAl, 299 pflgal-nDiMPA2 2 126 ptrcHis-nDiMPA2, 8 pXPRHis-nDiMPA2go, pBBIII-nDiMPA22, 2 and ptrcHis-nDiCP, 42 Details regardig the production of such recombinant molecules is disclosed herein.

A -ecombinant cell of the present invention includes any cell transformed with at least one of any nucleic acid molecule of the present invention. A preferred recombinant 50 cell is a cell transformed with a nucleic acid molecule that includes at least a portion of a parasite astacin metalloendopeptidase nucleic acid molecule, such as nDiMPA1, 9 nDiMPA1,,, nDiMPA2 21 2, nDiMPA2 8 nDiMPA227 or BvMPA2 and/or at least a portion of a filariid cysteine protease nucleic acid molecule, such as a nucleic acid molecule including nDiCP 43 or nDiCP 42 Particularly preferred recombinant cells include

E.

coli: pgal-nDiMPAl, 29 E. coli: pfgal-nDiMPA2 21 2, E.

coli :ptrcHis-nDiMPA2,, E. coli:pXPHis-nDiMPA2gs,

S.

frugiperda:pBBIII-nDiMPA2 2 Z and E. coli:ptrcHis-nDiCP.

2 Details regarding the production of these recombinant cells is disclosed herein.

Recombinant cells of the present invention can also be co-transformed with one or more recombinant molecules including nucleic acid molecules encoding one or more proteins of the present invention parasite astacin metalloendopeptid;se proteins and/or filariid cysteine protease proteins) and one or more other proteins useful in the production of multivalent vaccines which can include one or more protective compounds.

It may be appreciated by one skilled in the art that use of recombinant DNA technologies can improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the 51 efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals promoters, operators, enhancers), substitutions or modifications of translational control signals ribosome binding sites, Shine-Dalgarno sequences), modification of nuclc:ic acid molecules of the present invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant enzyme production during fermentation. The activity of an expressed recombinant protein of the present invention may be improved by fragmenting, modifying, or derivatizing nucleic acid molecules encoding such a protein.

In accordance with the present invention, recombinant cells of the present invention can be used to produce one or more proteins of the present invention by culturing such cells under conditions effective to produce such a protein, and recovering the protein. Effective conditions to produce a protein include, but a- not limited to, appropriate media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An appropriate medium 52 refers to any medium in which a cell of the present invention, when cultured, is capable of producing a parasite protein, including a D. immitis protein, of the present invention. An effective medium is typically an aqueous medium comprising assimilable carbohydrate, nitrogen and phosphate sources, as well as appropriate salts, minerals, metals and other nutrients, such as vitamins. The medium may comprise complex nutrients or may be a defined minimal medium. Cells of the present invention can be cultured in conventional fermentation bioreactors, which include, but are not limited to, batch, fed-batch, .cell recycle, and continuous fermentors. Culturing can also be conducted in shake flasks, test tubes, microtiter dishes, and petri plates. Culturing is carried out at a temperature, pH and oxygen content appropriate for the recombinant cell. Such culturing conditions are well within the expertise of one of ordinary skill in the art.

Examples of suitable conditions are included in the Examples section.

Depending on the vector and host system used for production, resultant proteins of the present invention may either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the periplasmic space in E.

coli; or be retained on the outer surface of a cell or viral membrane. The phrase "recovering the protein" refers simply to collecting the whole fermentation medium containing the protein and need not imply additional steps 53 of separation or purification. Proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, ele..crophoresis, hydrophobic interaction chromatography, geL filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization. Proteins of the present invention are preferably retrieved in "substantially pure" form. As used herein, "substantially pure" refers to a purity that allows for the effective use of the protein as a therapeutic composition or diagnostic.

A vaccine for animals, for example, should exhibit no substantial toxicity and should be capable of stimulating the production of antibodies in a vaccinated animal.

The present invention also includes isolated

C*

antibodies capable of selectively binding to a protein of :i the present invention or to a mimetope thereof. Antibodies capable of selectively binding to a parasite astacin metalloendopeptidase protein of the present invention are referred to as anti-parasite astacin metalloendopeptidase antibodies. A particularly preferred antibody of this embodiment is an anti-D. immitis astacin metalloendopeptidase antibody. Antibodies capable of selectively binding to a filariid cysteine protease protein v of the present invention are referred to as anti-filariid cysteine protease antibodies. A particularly preferred antibody of this embodiment is an anti-D. immitis cysteine 54 protease antibody. Isolated antibodies are antibodies that have been removed from their natural milieu. The term "isolated" does not refer to the state of purity of such antibodies. As such, isolated antibodies can include antisera containing such antibodies, or antibodies that have been purified to varying degrees. As used herein, the term "selectively binds to" refers to the ability of such antibodies to preferentially bind to specified proteins and mimetopes thereof of the present invention. Binding can be measured using a variety of methods known to those skilled in the art including immunoblot assays, immunoprecipitation assays, radioimmunoassays, enzyme immunoassays ELISA), immunofluorescent antibody assays and immunoelectron microscopy; see, for example, Sambrook et al., ibid.

Antibodies of the present invention can be either polyclonal or monoclonal antibodies. Antibodies of the present invention include functional equivalents such as antibody fragments and genetically-engineered antibodies, 20 including single chain antibodies, that are capable of selectively binding to at least one of the epitopes of the protein or mimetope used to obtain the antibodies.

Preferred antibodies are raised in response to proteins, or mimetopes thereof, that are encoded, at least in part, by a nucleic acid molecule of the present invention.

A preferred method to produce antibodies of the present invention includes administering to an animal an effective amount of a protein or mimetope thereof of the 55 present invention to produce the antibodies and (b) recovering the antibodies. Antibodies raised against defined proteins or mimetopes can be advantageous because such antibodies are not substantially contaminated with antibodies against other substances that might otherwise cause interference in a diagnostic assay o. side effects if used in a therapeutic composition.

Antibodies of the present invention have a variety of potential uses that are within the scope of the present invention. For example, such antibodies can be used as vaccines to passively immunize an animal in order to protect the animal from parasites susceptible to treatment by such antibodies, as reagents in assays to detect :infection by such parasites and/or as tools to recover desired proteins of the present invention from a mixture of proteins and other contaminants.

Furthermore, antibodies of the present invention can be used to target cytotoxic agents to parasites of the present invention in order to directly kill such parasites.

Targeting can be accomplished by conjugating stably joining) such antibodies to the cytotoxic agents using techniques known to those skilled in the art. Suitable cytotoxic agents include, but are not limited to: doublechain toxins toxins having A and B chains), such as diphtheria toxin, ricin toxin, Pseudomonas exotoxin, modeccin toxin, abrin toxin, and shiga toxin; single-chain toxins, such as pokeweed antiviral protein, a-amanitin, and ribosome inhibiting proteins; and chemical toxins, such as 56 melphalan, methotrexate, nitrogen mustard, doxorubicin and daunomycin. Preferred double-chain toxins are modified to include the toxic domain and translocation domain of the toxin but lack the toxin's intrinsic cell binding domain.

One embodiment of the present invention is a therapeutic composition that, when administered to an animal in an effective manner, is capable of protecting that animal from disease caused by a parasite that is susceptible to at least one of the following treatments: immunization with an isolated parasite astacin metalloendopeptidase of the present invention, immunization with an isolated filariid cysteine protease of the present invention, administration of an inhibitor of astacin metalloendopeptidase activity or administration of an inhibitor of cysteine protease activity. As used herein, a parasite that is susceptible to such a tre .tuent is a parasite that, if such treatment is administered to an animal in an effective manner, shows substantially reduced ability to cause disease in the animal. It is to be 20 understood that such parasite can be susceptible to S. treatments other than just those listed immediately above.

Such treatments can include, but are not limited to, additional treatments, or therapeutic compositions, disclosed herein.

Therapeutic compositions of the present invention include at least one of the following protective compounds: 4 an isolated parasite astacin metalloendopeptidase protein or a mimetope thereof; an isolated parasite 57 nucleic acid molecule capable of hybridizing under stringent conditions with a D. immitis astacin metalloendopeptidase gene; an anti-parasite astacin metalloendopeptidase antibody; an inhibitor of astacin metalloendopeptidase activity identified by its ability to inhibit parasite astacin metalloendopeptidase activity; (e) an isolated filariid nematode cysteine protease protein or a mimetope thereof; an isolated filariid nematode nucleic acid molecule capable of hybridizing under stringent conditions with a D. immitis cysteine protease gene; an anti-filariid nematode cysteine protease antibody; and an inhibitor of cysteine protease activity identified by its ability to inhibit filariid nematode cysteine protease activity. As used herein, a protective compound refers to a compound that, when administered to an animal in an effective manner, is able to treat, ameliorate, and/or prevent disease caused by a parasite of the present invention. Preferred parasites to target are heretofore disclosed. Examples of proteins, 20 nucleic acid molecules and antibodies of the present 4 invention are disclosed herein. Astacin metalloendopeptidase inhibitors and cysteine protease inhibitors of the present invention are described in more detail below.

The present invention also includes a therapeutic' composition comprising at least one astacin parasite metalloendopeptidase-based or filariid nematode cysteine protease-based protective compound of the present invention 58 in combination with at least one additional compound protective against one or more infectious agents. Examples of such compounds and infectious agents are heretofore disclosed.

Therapeutic compositions of the present invention can be administered to any animal susceptible.to such therapy, preferably to mammrls, and more preferably to dogs, cats, humans, ferrets, horses, cattle, sheep and other pets and/or economic food animals. Preferred animals to protect include dogs, cats, humans and ferrets, with dogs and cats being particularly preferred.

In one embodiment, a therapeutic composition of the present invention can be administered to the vector in which the parasite develops from a microfilaria into L3, such as to a mosquito in order to prevent the spread of heartworm. Such administration could be orally or by

,R

developing transgenic vectors capable of producing at least one therapeutic composition of the present invention. In another embodiment, a vector, such as a mosquito, can ingest therapeutic compositions present in the blood of a host that has been administered a therapeutic composition

S*

of the present invention.

Therapeutic compositions of the present invention can be formulated in an excipient that the animal. to be treated can tolerate. Examples of such excipients include water,\ saline, Ringer's solution,. dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame 59 oil, ethyl oleate, or triglycerides may also be used.

Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability.

Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc., to 15 which sterile water or saline can be added prior to administration.

In one embodiment of the present invention, the therapeutic composition can also include an immunopotentiator, such as an adjuvant or a carrier.

20 Adjuvants are typically substances that generally enhance the immune response of an animal to a specific antigen.

Suitable adjuvants include, but are not limited to, Freund's adjuvant; other bacterial cell wall components; aluminum-based salts; calcium-based salts; silica; polynucleotides; toxoids; serum proteins; viral coat\ proteins; other bacterial-derived preparations; gamma interferon; block copolymer adjuvants, such as Hunter's Titermax adjuvant (Vaxcel

T

Inc. Norcross, GA); Ribi 60 adjuvants (available from Ribi ImmunoChem Research, Inc., Hamilton, MT); and saponins and their derivatives, such as Quil A (available from Superfos Biosector A/S, Denmark).

Carriers are typically compounds that increase the halflife of a therapeutic composition in the treated animal.

Suitable carriers include, but are not limited to, polymeric controlled release formulations, biodegradable implants, liposomes, bacteria, viruses, oils, esters, and glycols.

S 1 0 In order to protect an animal from disease caused by r a parasite of the present invention, a therapeutic composition of the present invention is administered to the S animal in an effective manner such that the composition is capable of protecting that animal from a disease caused by 15 a parasite. For example, an isolated protein or mimetope thereof, when administered to an animal in an effective manner, is able to elicit stimulate) an immune response, preferably including both a humoral and cellular response, that is sufficient to protect the animal from the 20 disease. Similarly, an antibody of the present invention, when administered to an animal in an effective manner, is administered in an amount so as to be present in the animal at a titer that is sufficient to protect the animal from the disease, at least temporarily. Oligonucleotide nucleic acid molecules of the present invention can also be' administered in an effective manner, thereby reducing expression of astacin metalloendopeptidase or cysteine protease proteins in order to interfere with development of 61 parasites targeted in accordance with the present invention.

Therapeutic compositions of the present invention can be administered to animals prior to infection in order to prevent infection and/or can be administered to animals after infection in order to treat disease caused by the parasite. For example, proteins, mimetopes thereof, and antibodies thereof can be used as immunotherapeutic agents.

Acceptable protocols to administer therapeutic 1 0 compositions in an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protoc;ls can be accomplished by those skilled in the art. A suitable single dose is a dose that is capable of protecting an 15 animal from disease when administered one or more times over a suitable time period. For example, a preferred single dose of a protein, mimetope or antibody therapeutic composition is from about 1 microgram to about milligrams (mg) of the therapeutic composition per kilogram 20 body weight of the ;-nimal. Booster vaccinations can be administered from about 2 weeks to several years after the original administration. Booster vaccinations preferably are administered when the immune response of the animal becomes insufficient to protect the animal from disease.

A preferred administration schedule is one in which from about 10 gsg to about 1 mg of the vaccine per kg body weight of the animal is administered from about one to about two 62 times over a time period of from about 2 weeks to about 12 months. Modes of administration can include, but are not limited to, subcutaneous, intradermal, intravenous, nasal, oral, transdermal and intramuscular routes.

According to one embodiment, a nucleic acid molecule of the .present invention can be administered to an animal in a fashion to enable expression of that nucleic acid molecule into a protective protein or protective RNA antisense RNA, ribozyme or RNA drug) in the animal to be protected from disease. Nucleic acid molecules can be delivered to an animal in a variety of methods including, but not limited to, (a)direct injection as "naked" S DNA or RNA molecules, such as is taught, for example in Wolff et al., 1990, Science 247, 1465-1468) or packaged 15 as a recombinant virus particle vaccine or as a recombinant cell vaccine delivered to a cell by a vehicle selected from the group consisting of a recombinant virus particle vaccine and a recombinant cell vaccine).

A recombinant virus particle vaccine of the present 20 invention includes a recombinant molecule of the present invention that is packaged in a viral coat and that can be expressed in an animal after adm"nistration. Preferably, the recombinant molecule is packaging-deficient. A number of recombinant virus particles can be used, including, but not limited to, those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, and retroviruses. Preferred recombinant particle viruses are those based on alphaviruses (such as Sindbis virus), herpesviruses and 63 poxviruses. Methods to produce and use recombinant virus particle vaccines are disclosed in U.S. Patent Application Serial No. 08/015/414, filed February 8, 1993, entitled "Recombinant Virus Particle Vaccines", which is incorporated by reference herein in its entirety.

When administered to an animal, a recombinant virus particle vaccine of the present invention infects cells within the immunized animal and directs the production of a protective protein or RNA nucleic acid molecule that is 10 capable of protecting the animal from disease caused by a parasite of the present invention. For example, a recombinant virus particle comprising a D. immitis astacin metalloendopeptidase nucleic acid molecule and/or a D.

immitis cysteine protease nucleic acid molecule of the 15 present invention is administered according to a protocol that results in the animal producing a sufficient immune response to protect itself from heartworm. A preferred single dose of a recombinant virus particle vaccine of the present invention is from about 1 x 104 to about 1 x 10 7 oo* 20 virus plaque forming units (pfu) per kilogram body weight of the animal. Administration protocols are similar to those described herein for protein-based vaccines.

A recombinant cell vaccine of the present invention includes recombinant cells of the present invention that express at least one protein of the present invention.

Preferred recombinant cells include Salmonella, E. coli, Mycobacterium, S. frugiperda, baby hamster kidney, myoblast G8, COS, MDCK and CRFK recombinant cells, with Salmonella 64 recombinant cells being more preferred. Such recombinant cells can be administered in a variety of ways but have the advantage that they can be administered orally, preferably at doses ranging from about 108 to about 1012 bacteria per kilogram body weight. Administration protocols are similar to those described herein for protein-based vaccines.

Recombinant cell vaccines can comprise whole cells or cell lysates.

In common with most other enteric pathogens, Salmonella strains normally enter the host orally. Once in the intestine, they interact with the mucosal surface, normally to establish an invasive infection. Most Salmonella infections are controlled at the epithelial surface, causing the typical Salmonella-induced 15 gastroenteritis. Some strains of Salmonella, including S.

typhi and some S. typhimurium isolates, have evolved the ability to penetrate deeper into the host, 'causing a disseminated systemic infection. It appears such strains have the capacity to resist the killing actions of 20 macrophages and other immune cells. S. typhi can exist for long periods as a facultative intracellular parasite. Some of the live vaccine strains can also persist for long periods in the mononuclear phagocyte system. Hosts infected in such a manner develop, in addition to a mucosal immune response, systemic cellular and serum antibody responses to'' the Salmonella. Thus, invading Salmonella, whether virulent or attenuated, can stimulate strong immune responsds, unlike many other enteric pathogens which only set up 65 local, noninvasive gut infections. The potent immunogenicity of live Salmonella makes them attractive candidates for carrying nucleic acid molecules of the present invention, and the proteins they encode, to the immune system.

A preferred recombinant cell-based vaccine is one in which the cell is attenuated. Salmonella typhimurium strains, for example, can be attenuated by introducing mutations into genes critical for in vivo growth and 10 survival. For example, genes encoding cyclic adenosine monophosphate (cAMP) receptor protein or adenylate cyclase are deleted to produce avirulent, vaccine strains. Such a. strains can deliver antigens to lymphoid tissue in the gut but demonstrate reduced capacity to invade the spleen and 15 mesenteric lymph nodes. These strains are still capable of stimulating both humoral and cellular immunity in mammalian hosts.

Recombinant cell vaccines can be used to introduce proteins of the present invention into the immune systems 20 of animals. For example, recombinant molecules comprising nucleic acid molecules of the present invention operatively linked to expression vectors that function in Salmonella can be transformed into Salmonella host cells. The resultant recombinant cells are then introduced into the animal to be protected. Preferred Salmonella host cells are those for which survival depends on their ability to maintain the recombinant molecule a balanced-lethal host-vector system). An example of such a preferred host 66 recombinant molecule combination is a Salmonella strain UK-1 x 398 7 or SR-11 x4072) which is unable to produce aspartate f-semialdehyde dehydrogenase in combination with a recombinant molecule also capable of encoding the enzyme.

Aspartate f-semialdehyde dehydrogenase, encoded by the asd gene, is an important enzyme in the pathway to produce diaminopimelic acid (DAP). DAP is an essential component of the peptidoglycan of the cell wall of Gram-negative bacteria, such as Salmonella, and, as such, is necessary for survival of the cell. Thus, Salmonella lacking a functional asd gene can only survive if they maintain a recombinant molecule that is also capable of expressing a functional asd gene.

In one embodiment, a nucleic acid molecule of the 15 present invention is inserted into expression vector pTECH- 1 (available from Medeva, London, and the resulting recombinant molecule is transfected into a Salmonella strain, such as BRD 509 (available from Medeva), to form a recombinant cell. Such recombinant cells can be used to produce the corresponding encoded protein or can be used as recombinant cell vaccines.

One preferred embodiment of the present invention is the use of nucleic acid molecules and proteins of the present invention, and particularly D. immitis nucleic acid molecules and proteins of the present invention, to protect an animal from heartworm. Preferred therapeutic compositions are those that are able to inhibit at least one step in the portion of the parasite's development cycle 67 that includes L3 larvae, third molt, L4 larvae, fourth molt and immature adult prior to entering the circulatory system. In dogs, this portion of the development cycle is about 70 days. As such, preferred therapeutic compositions include D. immitis astacin metalloendopeptidase-based and D. immitis cysteine protease-based therapeutic compounds of the present invention. Such compositions are administered to animals in a manner effective to protect the animals from heartworm. Additional protection may be obtained by administering additional protective compounds, including other D. immitis proteins, nucleic acid molecules and.

antibodies as heretofore disclosed.

One embodiment of the present invention is the use of enzymatically active parasite astacin metalloendopeptidase 15 and/or filariid nematode cysteine protease proteins of the present invention to identify inhibitors of such enzyme activity. While not being bound by theory, it is believed that parasites use such proteases in a number of ways, including, but not limited to, to effect embryonic and 20 larval development, to effect molting and to effect tissue migration both as larvae and adults. Such proteases are capable of degrading cutaneous connective tissue macromolecules as well as other proteinaceous material to facilitate such functions. It is also of interest that astacin metalloendopeptidases identified in sea urchins, Drosophila and Xenopus have been linked with development and maturation of the respectively organisms. As such, inhibitors of astacin metalloendopeptidase and/or cysteine 68 protease activity could be particularly beneficial in disrupting embryonic and/or larval development or molting by parasites in general and tissue migration by those parasites capable of such migration. Use of parasite enzymes to develop such inhibitors is also advantageous because inhibitors can be identified that are highly selective for the parasite without causing undue side effects to the anima' being treated.

One therapeutic composition of the present invention includes an inhibitor of parasite astacin metalloendopeptidase activity, a compound capable of substantially interfering with the function of a parasite astacin metalloendopeptidase susceptible to inhibition by an inhibitor of D. immitis astacin metalloendopeptidase 15 activity. An inhibitor of astacin metalloendopeptidase can *.be identified using enzymatically active parasite and preferably D. immitis, astacin metalloendopeptidase proteins of the present invention.

One embodiment of the present invention is a method to 20 identify a compound capable of inhibiting astacin o* metalloendopeptidase activity of a parasite. Such a method includes the steps of contacting combining, mixirg) an isolated parasite, preferably D. immitis, astacin metalloendopeptidase protein with a putative inhibitory compound under conditions in which, in the\ absence of the compound, the protein has astacin metalloendopeptidase activity, and determining if the putative inhibitory compound inhibits the astacin 69 metalloendopeptidase activity. Putative inhibitory compounds to screen include organic molecules, antibodies (including functional equivalents thereof) and substrate analogs. Methods to determine astacin metalloendopeptidase activity are known to those skilled in the art; see, for example, Gomis-Ruth, et al. ibid., and referenced cited therein.

The present invention also includes a test kit to identify a compound capable of inhibiting astacin metalloendopeptidase activity of a parasite. Such a test kit includes an isolated parasite, preferably D. immitis, astacin metalloendopeptidase protein having astacin Smetalloendopeptidase activity and a means for determining *the extent of inhibition of astacin metalloendopeptidase 15 activity in the presence of effected by) a putative inhibitory compound.

Astacin metalloendopeptidase inhibitors isolated by such a method, and/or test kit, can be used to inhibit any astacin metalloendopeptidase that is susceptible to such an 20 inhibitor. Preferred astacin metalloendopeptidase enzymes to inhibit are those produced by parasites. A particularly preferred astacin metalloendopeptidase inhibitor of the present invention is capable of protecting an animal from heartworm. It is also within the scope of the present invention to use inhibitors of the present invention to target astacin metalloendopeptidase-related disorders in animals. Therapeutic compositions comprising astacin metalloendopeptidase inhibitory compounds of the present 70 invention can be administered to animals in an effective manner to protect niimals from disease caused by the targeted astacin metalloendopeptidase enzymes, and preferably to protect animals from heartworm. Effective amounts and dosing regimens can be determined using techniques known to those skilled in the art.

Another therapeutic composition of the present invention includes an inhibitor of parasite cysteine protease activity, a compound capable of substantially interfering with the function of a parasite cysteine protease susceptible to inhibition by an inhibitor of filariid nematode cysteine protease activity. A cysteine protease inhibitor can be identified using enzymatically "active filariid nematode, and preferably D. immitis, cysteine protese proteins of the present invention.

One embodiment of the present invention is a method to identify a compound capable of inhibiting cysteine protease activity of a parasite. Such a method includes the steps of contacting combining, mixing) an isolated 20 filariid nematode, preferably D. immitis, cysteine protease protein with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein has cysteine protease activity, and determining if the putative inhibitory compound inhibits the cysteine protease activity. Putative inhibitory compounds to screen' include organic molecules, antibodies (including functional equivalents thereof) and substrate analogs. Methods to 71 determine cysteine protease activity are known to those skilled in the art, as heretofore disclosed.

The present invention also includes a test kit to identify a compound capable of inhibiting cysteine protease activity of a parasite. Such a test kit includes an isolated filariid nematode, preferably D. immitis, cysteine protease protein having cysteine protease activity and a means for determining the extent of inhibition of cysteine protease activity in the presence of effected by) a putative inhibitory compound.

Inhibitors isolated by such a method, and/or test kit, can be used to inhibit any cysteine protease that is susceptible to such an inhibitor. Preferred cysteine protease enzymes to inhibit are those produced by parasites. A particularly preferred cysteine protease 0* *:inhibitor of the present invention is capable of protecting an animal from heartworm. It is also within the scope of the present invention to use inhibitors of the present "*00 invention to target cysteine protease-related disorders in 20 animals. Therapeutic compositions comprising cysteine protease-inhibitory compounds of the present invention can be administered to animals in an effective manner to protect animals from disease caused by the targeted cysteine protease. Effective amounts and dosing regimens can be determined using techniques known to those skilled' in the art.

The efficacy of a therapeutic composition of the present invention to protect an animal from disease caused 72 by a parasite can be tested in a variety of ways including, but not limited to, detection of protective antibodies (using, for example, proteins or mimetopes of the present invention), detection of cellular immunity within the treated animal, or challenge of the treated animal with the parasite to determine whether the treated animal is resistant to disease. Such techniques are known to those skilled in the art.

In accordance with the present invention, the inventors have shown that protease inhibitors can inhibit parasite larval development. For example, bestatin and phosphoramidon have been shown to inhibit molting of D.

immitis larvae, as described in more detail in the Examples.

Another embodiment of the present invention includes the isolation of proteases, including metalloproteases and cysteine proteases from parasitic larval excretory/secretory (ES) products. Using a modified version of the protocol first described in U.S. Patent Application Serial No. 08/153,554, ibid., the inventors have, for example, isolated a fraction comprising a protein of approximately 60 kD (as determined by Tris-g'ycine sodium dodecyl sulfate polyacrylamide gel electrophoresis

(SDS-

PAGE) that has metalloprotease activity as determined by the protein's ability to cleave H-Phe-AMC. When submitted' i size-exclusion chromatography, the active fraction elutes with bovine serum albumin, indicating an approximate molecular weight of from about 62 to about 66 kD. The 73 modified protocol, which is described in more detail in the Examples, includes submitting parasitic, preferably D.

immitis, larval ES, to anion exchange chromatography, followed by size exclusion chromatography and isoelectric focus ing. The active fraction has a pi of about 6.8.

In another embodiment, the inventors have identified at least one parasite metalloprotease from the ES that is capable of degrading collagen. For example, electrophoresis of D. immitis larval ES through a gelatin-based matrix leads to isolated active fractions that migrate with apparent molecular weights of about 60, 95 and at least about 200 kD. The proteolytic activity of such fractions is essentially completely inhibited by EDTA.

It is also within the scope of the present invention to use isolated proteins, mimetopes, nucleic acid molecules and antibodies of the present invention as diagnostic reagents to detect infection by parasites. Such diagnostic reagents can be supplemented with additional compounds that can detect other phases of the parasite's life cycle.

20 The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention.

Examples The following examples include a number of recombinant' DNA and protein chemistry techniques which are known to those skilled in the art; see, for example, Sambrook 'et al., ibid.

74 Example 1 This Example discloses the cloning and sequencing of two parasite astacin metalloendopeptidase nucleic acid molecules of the present invention. This Example also discloses the production of a recombinant molecule and recombinant cell of the present invention.

A D. immitis third stage larvae cDNA expression library was prepared in the following manner. Total RNA was extracted from D.immitis third stage larvae (L3) using an acid-guanidinium-phenol-chloroform method similar to that described by Chomczynski and Sacchi, 1987, Anal. Biochem.

162, p. 156-159. Approximately 230,000 L3 were used in the RNA preparation. Poly A+ selected RNA was separated from total RNA by oligo-dT cellulose chromatography using Oligo dT cellulose from Collaborative Research Inc., Waltham, MA, according to the method recommended by the manufacturer.

The expression library was constructed by inserting the L3 poly A+ RNA into the expression vector lambda (X) Uni-ZAP m XR (available from Stratagene Cloning Systems, La Jolla, CA) using Stratagene's ZAP-cDNA Synthesis Kit* protocol and about 6-7 pg of L3 poly A+ RNA. The resultant library was amplified to a titer of about 4.88 x 109 pfu/ml with about 96% recombinants. Ten minilibraries were generated by one further round of amplification of randomly selected aliquots of the original L3 cDNA amplified library. These minilibrary phage were collected in phage dilution buffer 10 mM Tris-HCl, pH 7.5, 10 nM magnesium sulfate) and stored at 4 0

C.

75 A D. immitis astacin metalloendopeptidase nucleic acid molecule of about 689 nucleotides, representing a partial D. immitis astacin metalloendopeptidase gene and denoted nDiMPA1 68 9 was PCR amplified from D. immitis L3 cDNA expression minilibraries using the following two primers: a 4-fold degenerate primer having SEQ ID NO:14, namely ACWCATGAAATIGSICAT 3' (denoted MP1; W can be A or T; S can be C or G; I is inosine) and an antisense oligonucleotide having SEQ ID NO:15, namely 5'AATACGACTCACTATAG 3' (denoted T7). Primer MP1 was designed from published sequences of the metalloprotease conserved zinc binding domain. Primer T7 is complementary to the pBluescript® vector (available from Stratagene).

A nucleic acid molecule amplified from minilibrary number 10, which was denoted nDiMPAl" 9 was gel-purified, a electroeluted and cloned into the cloning vector pCRII (available from Invitrogen, San Diego, CA) following manufacturer's instructions, thereby forming recombinant vector pCRII-nDiMPAl9,. The nucleotide sequence of nDiMPA1 68 9 was determined and found to include nucleotides spanning a from about nucleotide position 610 through the 3' end of SEQ ID NO:1, the production of which is described in more detail below.

The L3 cDNA minilibrary number 10 was screened with the radiolabeled MP1 oligonucleotide as a probe, using\ stringent standard) hybridization conditions as described in Sambrook et al., ibid. Plaques which hybridized to the probe were rescreened and plaque- 76 purified. The plaque-purified clone including D. immitis nucleic acid molecule nDiMPAl 12 9 was converted into a double stranded recombinant molecule, herein denoted as pigal-nDiMPAlI 1 2 using R408 helper phage and XL1-Blue

E.

coli according to the in vivo excision protocol described in the Stratagene ZAP-cDNA Synthesis Kit®. Double-stranded plasmid DNA was prepared using an alkaline lysis protocol, such as that described in Sambrook et al., ibid.

Recombinant molecule p3gal-nDiMPAl 1 was transformed into E. coli to form recombinant cell E. coli:plgal-nDiMPA 1 2 Recombinant molecule plgal-nDiMPA 2 was submitted to nucleic acid sequencing using the Sanger dideoxy chain termination method, as described in Sambrook et al., ibid.

An about 1299 nucleotide consensus sequence of nucleic acid 15 molecule nDiMPA1,w was determined and is presented as SEQ ID NO:1. SEQ ID NO:1 apparently encodes three overlapping open reading frames. The first open reading frame, denoted PDiMPAlon, is about 191 amino acids (presented in SEQ ID NO:3) and encompasses about nucleotide number 18-590 of SEQ 20 ID NO:1. The second open reading frame, denoted PDiMPAlomn, is about 141 amino acids (presented in SEQ ID NO:4) and encompasses about nucleotide number 508-930 of SEQ ID:1.

Open reading frame PDiMPA1 0 oR includes the extended zinc binding domain and hydrophilic region

HEIGHTLGIFHE

beginning at about amino acid 36 of SEQ ID NO:4, as well as o the domain YDTGSVMHY (beginning at about amino acid position 87) that includes the tyrosine that is thought to bind to zinc at about amino acid position 95. The third 77 open reading frame, denoted PDiMPAlo 0 is about 121 amino acids (presented in SEQ ID NO: 5) and encompasses nucleotide number 785-1147 of SEQ ID:1.

A homology search of the non-redundant protein sequence database was performed through the National Center for Biotechnology Information using the BLAST network.

This database includes +SwissProt PIR SPUpdate GenPept GPUpdate. The search, which was performed using SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, showed significant homology at the amino acid level by all three .open reading frames to members of the astacin family of.

metalloendopeptidases. Significant homology throughout all three amino acid open reading frames is also associated with a C. elegans R151.5 gene product, Genbank accession number U00036.

It was apparent from the pBluescript vector sequences at and near the 5' end of nucleic acid molecule nDiMPAlI that two cDNA fragments had ligated to each other via their ends to form that nucleic acid molecule. A comparison 20 of the nucleotide sequence of SEQ ID NO:1 to that of SEQ ID NO:2 (the apparent nucleotide sequence of independentlyisolated astacin metalloendopeptidase nucleic acid molecule nDiMPA2 21 26 described in Example 2) as well as to nucleotide sequences of other members the astacin family of metalloendopeptidases, showed that nucleotide number 56 of SEQ ID NO:1 corresponds to the first nucleotide on the end of nDiMPA2 22 6 suggesting that nucleotide number '56 represents the junction of the two different cDNA sequences 78 in nDiMPAl 1 Therefore, nucleotides 1 through 55 of nDiMPA1 12 9 most likely represent a non-astacin related cDNA sequence which ligated to the 5' end of the astacin metalloendopeptidase cDNA fragment prior to cloning.

Astacin homology at the amino acid level can be found starting with amino acid number 125 of the first open reading frame of nDiMPA1li, namely PDiMPAlou, Example 2 This Example discloses the cloning and sequencing of 10 an additional parasite astacin metalloendopeptidase nucleic acid molecule of the present invention. This Example also discloses the production of a recombinant molecule and recombinant cell of the present invention.

Due to the unusual sequences on the 5' end of astacin 15 metalloendopeptidase nucleic acid molecule nDiMPAl,2, a second astacin metalloendopeptidase nucleic acid molecule, denoted nDiMPA22,m, was isolated from the L3 cDNA expression library described in Example 1, as follows.

A D. immitis astacin metalloendopeptidase nucleic acid molecule of about 271 nucleotides, denoted nDiMPA2,, was PCR amplified from D. immitis L3 cDNA expression minilibrary number 9 using the following two primers: an oligonucleotide having SEQ ID NO:16, namely TGGTATTATATCACATGAAATTGGTCATAC 3' (denoted ZNSEN) and an\ antisense oligonucleotide having SEQ ID NO:17, namely CCCAATTGTGTACTGTTGAAATTTATCAC 3' (denoted MP14). Prirer ZNSEN was designed from the nucleotide sequence encoding 79 the metalloprotease conserved zinc binding domain in nDiMPA, 2 and spans from about nucleotide 600 through about nucleotide 629 of SEQ ID NO:1. Primer MP14 is an antisense primer complementary to a region spanning from about nucleotide 842 through about nucleotide 870 of nDiMPA 1 Nucleic acid molecule nDiMPA2v, was radiolabeled and used as a probe to screen L3 cDNA minilibrary number 9.

Plaques which hybridized under stringent hybridization conditions to the probe were rescreened and plaque purified. A plaque-purified clone including D. immitis :i nucleic acid molecule nDiMPA2 2 1 6 was converted into a S double-stranded reco-binant molecule, herein denoted as pfigal-nDiMPA2 21 2 6 using R408 helper phage and XL1-Blue E.

coli according to the in vivo excision protocol described in the Stratagene ZAP-cDNA Synthesis KitO. Double-stranded plasmid DNA was prepared using an alkaline lysis protocol, such as that described in Sambrook et al., ibid.

Recombinant molecule pfgal-nDiMPA2 22 was transformed into E. coli to form recombinant cell E. coli:ppgal-nDiMPA2 21 s.

Recombinant molecule plgal-nDiMPA2 2 M was submitted to

S

nucleic acid sequencing using the Sanger dideoxy chain termination method, as described in Sambrook et al., ibid.

An about 2126-nucleotide consensus sequence of nucleic acid molecule nDiMPA2 2 1 2, was determined and is presented as SEQ ID NO:2. SEQ ID NO:2 apparently encodes five overlapping open reading frames. The first open reading frame, denoted PDiMPA2oI, is about 178 amino acids (presented in SEQ ID NO:6) and encompasses about nucleotide numbers 2-535 of SEQ 80 ID:2. The second open reading frame, denoted PDiMPA2oR, is about 145 amino acids (presented in SEQ ID NO:7) and encompasses about nucleotide numbers 453-887 of SEQ ID:2.

PDiMPA2 0 oR includes the extended zinc binding domain, beginning at about amino acid 36 of SEQ ID NO:7 as well as the tyrosine-containing motif, beginning at about amino acid 87. The third open reading frame, denoted PDiMPA2omR, is about 134 amino acids (presented in SEQ ID NO:8) and encompasses nucleotide number 730-1131 of SEQ ID:2. The fourth open reading frame, denoted PDiMPA2o 0 4, is about 154 amino acids (presented in SEQ ID NO:9) and encompasses nucleotide number 1112-1573 of SEQ ID:2. The fifth open reading frame, denoted PDiMPA2o 0 s, is about 163 amino acids (presented in SEQ ID NO:10) and encompasses nucleotide number 1429-1917 of SEQ ID:2.

o A comparison of the deduced nucleic acid sequences of nDiMPAl 12 (SEQ ID NO:1) and nDiMPA2 21 2 (SEQ ID NO:2) indicates that nDiMPA2 2 1 does not contain the stretch of nucleotides from about positions 1 through 55 of nDiMPAl, 20 (as numbered in SEQ ID NO:1). As discussed above, it is believed that this stretch of nucleotides represents an unrelated cDNA clone that ligated to the 5' end of the astacin metalloendopeptidas nucleic acid molecule. The stretch of nucleotides spanning from about positions 56 through 907 of nDiMPA1 1 2 (as numbered in SEQ ID NO:1) share\' 100% homology with the stretch of nucleotides spanning from about positions 1 through 852 of nDiMPA2 22 (as numbered in SEQ ID NO:2). The stretch of nucleotides spanning from 81 about positions 908 through 970 of nDiMPA1 12 (as numbered in SEQ ID NO:1) are missing from nDiMPA2 21 26 The stretch of nucleotides spanning from about positions 971 through 1133 of nDiMPA1, (as numbered in SEQ ID NO:1) share 100% homology with the stretch of nucleotides spanning from about positions 853 through 1015 of nDiMPA22,2 (as numbered in SEQ ID NO:2). Nucleic acid molecule nDiMPA2 226 has a significantly longer 3' end than does nDiMPAl,2,.

A homology search of the non-redundant protein sequence database was performed through the National Center for Biotechnology Information using the BLAST network.

This database includes +SwissProt PIR SPUpdate GenPept GPUpdate. The search, which was performed using SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10 and SEQ ID NO:11, showed that SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10 each shared significant homology at the amino acid level with known members of the astacin family of metalloendopeptidases. SEQ ID NO:11, in contrast did not show significant homology to a known astacin metalloendopeptidase.

A composite D. immitis ORF of the five open reading frames encoded by nDiMPA2 2 2, was produced by lining up the five ORFs in relation to known astacin metalloendopeptidase sequences. The composite D. immitis ORF, which spans a region significantly larger than the 200-amino acid astacin' protein, is presented in SEQ ID NO:11. A comparison between the astacin domain of SEQ ID NO:11 and crayfish astadin showed about 29% homology at the amino acid level. The 82 astacin domain of SEQ ID NO:11 also shared about percent, 31 percent, 33 percent and 33 percent homology at the amino acid level with the astacin domains of, respectively, human bone morphogenetic protein 1, mouse kidney brush border metalloendopeptidase, human intestinal brush border metalloendopeptidases and Xenopus laevis embryonic protein UVS.2.

Comparison of SEQ ID NO:11 and the C. elegans R151.5 gene product, (Genbank accession number U00036) showed an about 24% homology between the two sequences. The C.

elegans gene product also includes a well-conserved extended zinc binding domain motif and tyrosine-containing motif. It is interesting that although the C. elegans R151.5 gene product was identified by Wilson et al., ibid., as an open reading frame, the authors of that publication describing a 2.2 megabase contiguous nucleotide sequence from the free-living nematode C. elegans failed to appreciate the homology between R151.5 and the family of astacin metalloendopeptidases. The present inventors are apparently the first to note such a homology and the likelihood that C. elegans encodes an astacin a.

metalloendopeptidase.

Example 3 This Example discloses the production of a recombinant cell of the present invention and its use to produce a parasitic astacin metalloendopeptidase protein of the present invention.

83 Recombinant molecule ptrcHis-nDiMPA2 8 m, containing nucleotides from about positions 119 through 922 of nDiMPA2 2 26 (as numbered in SEQ ID NO:2) operatively linked to trc transcription control sequences and to a fusion sequence encoding a poly-histidine segment comprising 6 histidines was produced in the following manner. An about 804-nucleotide DNA fragment containing nucleotides spanning from about 119 through about 922 of nDiMPA2 212 (as numbered in SEQ ID NO:2), denoted nDiMPA2, 8 was cleaved from recombinant molecule pfgal-nDiMPA2 21 2 6 with BamHI restriction endonuclease, gel purified and subcloned into expression vector pTrcHisB (available from Invitrogen) that had been cleaved with BamHI. The resulting recombinant molecule ptrcHis-nDiMPA2. was transformed into E. coli to form recombinant cell E. coli:ptrcHis-nDiMPA2 8 4.

Recombinant cell E. coli:ptrcHis-nDiMPA2 8 4 is cultured in shake flasks containing an enriched bacterial growth medium containing about 0.1 mg/ml ampicillin at about 370 When the cells reach an ODo of about 0.3, expression of E. coli:ptrcHis-nDiMPA24 is induced by addition of about 1 mM isopropyl-f-D-thiogalactoside (IPTG), and the cells cultured for about 3 hours at about 37°C. Protein production is monitored by SDS-PAGE of recombinant cell lysates, followed by Coomassie blue staining, using standard techniques. Recombinant cell coli:ptrcHis-nDiMPA2 8 4 produces a fusion protein, denoted herein as PHIS-PDiMPA2 8 4, that is not produced by ceils 84 transformed with the pTrcHisB plasmid lacking a parasite nucleic acid molecule insert.

Example 4 This Example discloses the production of another recombinant cell of the present invention capable of producing a parasitic astacin metalloendopeptidase protein of the present invention.

Recombinant molecule pXPRHis-nDiMPA2w, containing nucleotides from about positions 119 through 922 of 10 nDiMPA2 21 (as numbered in SEQ ID NO:2) operatively linked to XPR transcription control sequences and to a fusion sequence encoding a poly-histidine segment comprising 6 histidines was produced in the following manner. Nucleic acid molecule nDiMPA25g, produced as described in Example 3, 15 was ligated into the BamHI restriction site of the XP/T7/RSET-B expression vector. The vector, which is about 3455 base pairs contains an about 1990-bp pair PvuII *to AatII fragment from pUC19 containing the ampicillin resistance gene and E. coli origin of replication; an about 20 1100 bp BglII to BglII DNA fragment from vector pRK248cIts e. a (available as ATCC #33766) with a PvuII linker added to one end, containing the XP R promoter, the c' 5 X repressor gene and 22 amino acids of the cro gene regulating lytic growth; an about 55-bp BglII to XbaI segment from pGEMEX-l\ (available from Promega, Madison, WI) which contains the T7 promoter; an about 170-bp XbaI to EcoRI segment fiom pRSET-B (available from Invitrogen) which contains the T7-S10 translational enhancer, the His6 fusion,* the 11 amino acid S10 leader fusion, an enterokinase cleavage site and the multiple cloning site; and an about 140-bp fragment containing synthetic translational and transcription termination signals including the T translation terminators in all three reading frames, RNA stabilization sequence from Bacillus thurengiensis crystal protein and the T, rho-independent transcription terminator from the trpA operon. The resulting recombinant molecule, denoted pXPnHis-nDiMPA2 8 s, was transformed into E. coli to form :i recombinant cell E. Example This Example describes another recombinant cell of the present invention and its use to produce a parasitic 15 astacin metalloendopeptidase protein of the present invention.

Recombinant molecule pBBIII-nDiMPA2 2 2 6 containing nucleic acid molecule nDiMPA2 21 2 6 (produced as described in Example 2) operatively linked to baculovirus polyhedron 20 transcription control sequences was produced in the following manner. In order to produce a baculovirus recombinant molecule capable of directing the production of the protein encoded by nDiMPA2 2 1 2 6 recombinant molecule pfgal-nDiMPA2 2 2 6 produced as described in Example 2, was\ digested with XhoI, end-filled with Klenow DNA Polymerase, digested with PstI, gel purified and referred to as BvMPA2.

The baculovirus shuttle plasmid, BlueBacIII (BBIII) 86 (available from Invitrogen) was digested with NcoI, end-filled, digested with PstI and treated with calf intestinal phosphatase. The resulting vector fragment was gel purified and ligated to BvMPA2. The resultant recombinant molecule, denoted pBBIII-nDiMPA2 2 n 6 was verified for proper insert orientation by restriction mapping. This construct and linear Baculogold baculovirus DNA (Pharmingen) were cotransfected into Spodoptera frugiperda Sf9 host cells (donated by Colorado Bioprocessing Center, Fort Collins, CO). The resulting recombinant virus termed BvMPA, was cultivated for increased production of recombinant virus and to verify expression of nDiMPA2 226 by Western blot.

Example 6 15 This Example describes the cloning and sequencing of a filariid nematode cysteine protease nucleic acid molecule of the present invention.

A D. immitis cysteine protease nucleic acid molecule of about 143 nucleotides, denoted nDiCP 43 representing a 20 partial D. immitis cysteine protease gene, was PCR amplified from D. immitis genomic DNA that had been extracted from adult female D.immitis worms using standard protocols similar to that described in Sambrook et al, ibid. The two primers used in the PCR amplification,' reaction included a 4-fold degenerate primer having SEQ ID NO:18, namely 5' CGGGATCCTGTGGWTCATGYTGGGC 3' (denoted 254C; BamHI site in bold; W is A or T; Y is C or T) and an 8-fold 87 degenerate antisense primer having SEQ ID No:19, namely TAICCICCRTTRCAICCYTC 3' (denoted G65; R is A or G; Y is C or T; I is inosine). Both primers were designed from published sequence of cysteine proteases. Primer 25C was further refined in that D. immitis codon bias was incorporated into 25C to reduce the degeneracy. The inventors found such codon bias was necessary to effectively isolate D. immitis cysteine protease nucleic acid molecules of the present invention.

The amplified PCR fragment, namely nDiCP 43 was gel purified and cloned into the pCRII cloning vector (available from Invitrogen, San Diego, CA), following manufacturer's instructions. An about 143 nucleotide sequence of nDiCP 14 was determined and is presented as SEQ ID NO:12. SEQ ID NO:12 apparently encodes a protein of about 47 amino acids, which is presented as SEQ ID NO:13.

The translation initiation site of the protein and the translation termination codon are not contained within this genomic clone.

A homology search of the non-redundant protein sequence database was performed through the National Center for Biotechnology Information using the BLAST network.

This database includes +SwissProt PIR SPUpdate GenPept GPUpdate. The search was performed using SEQ ID NO:13 and showed significant homology to numerous cysteine'.

proteinases. The highest scoring matches at the amino acid level include soybean probable thiol protease precursbr (Genbank accession number P22895), barley cysteine 88 proteinase EP-B 1 precursor (Genbank accession number P25249) and barley cysteine proteinase EP-B 4 precursor (Genbank accession number P25250). Parasite specific cysteine proteases having homology to SEQ ID NO:13 include cysteine proteases from Trypanosoma brucei (Genbank accession numbers S07051 and S12099), Leishmania pifanoi (Genbank accession number B48566), L. mexicana (Genbank accession number S25003), T. congolense (Genbank accession number (37048), and Trichomonas vaginalis (Genbank accession number X77220). SEQ ID NO:13 shared about 16 percent, about 22 percent, about 24 percent, about percent, about 39 percent, about 44 percent and about 49 percent homology at the amino acid level with cysteine S* proteases from, respectively, H. contortus (a nematode), Schistosoma mansoni (a trematode), C. elegans (a nematode), Fasciola hepatica (a trematode), Entamoeba histolytica (a S" protozoa), Trypanosoma cruzi (a protozoa) and T. brucie.

SEQ ID NO:13 also shared about 50 percent amino acid homology with human cathepsin. SEQ ID NO:13 also shared about 56 percent amino acid homology with a Paragonimus westermani cysteine protease reported in European Patent Application Publication No. 0524834A2, by Hamajima et al., published January 27, 1993. The serine at about position and the cysteine at about position 37 of SEQ ID NO:13 were conserved in all of these cysteine proteases. Note' that these homology calculations did not include the amino acids encoded by DNA primers SEQ ID NO:18 and SEQ ID NO:29.

As such, the region used in the homology calculations C C 9*

C

C

a. 89 spanned from about amino acid position 6 through 41 of SEQ ID NO:13.

Example 7 This Example discloses the production of a recombinant cell of the present invention and its use to produce a filariid nematode cysteine protease protein of the present invention.

Recombinant molecule ptrcHis-nDiCP 42 containing nucleotides from about positions 2 through 143 of nDiCP 4 (as numbered in SEQ ID NO:12) operatively linked to trc transcription control sequences and to a fusion sequence encoding a poly-histidine segment comprising 6 histidines was produced in the following manner. An about 142-nucleotide DNA fragment containing nucleotides spanning from about 2 through about 143 of nDiCP 43 (as numbered in SEQ ID NO:12), denoted nDiCP, 4 was sequentially digested from nDiCP 3 with BamHI restriction endonuclease followed by digestion with EcoRI. Nucleic acid molecule nDiCP 4 was gel purified and directionally subcloned into expression vector 20 pTrcHisA (available from Invitrogen) that had been cleaved with BamHI and EcoRI and subsequently been gel purified.

The resulting recombinant molecule, namely ptrcHis-nDiCP,4, was transformed into E. coli to form recombinant cell E.

coli: ptrcHis-nDiCP, 2 Recombinant cell E. coli:ptrcHis-nDiCP, 42 is cultured in shake flasks containing an enriched bacterial growth medium containing about 0.1 mg/ml ampicillin at about 370 C. When C CCCC* C C C C C C 90 the cells reach an ODw of about 0.3, expression of E.

coli:ptrcHis-nDiCP, 42 is induced by addition of about 1 mM isopropyl-f-D-thiogalactoside (IPTG), and the cells cultured for about 3 hours at about 37 0 C. Protein production is monitored by SDS-PAGE of recombinant cell lysates, followed by Coomassie blue staining, using standard techniques. Recombinant cell E. coli:ptrcHisnDiCP 1 4 produces a fusion protein, denoted herein as PHIS- PDiCP 42 that is not produced by cells transformed with the pTrcHisA plasmid lacking a filariid nucleic acid molecule insert.

Example 8 This Example demonstrates that the protease inhibitors bestatin and phosphoramidon are able to inhibit D. immitis 15 larval development, particularly molting. Bestatin (available from Enzyme Systems Products, Livermore, CA) primarily, if not exclusively, inhibits amino peptidases and other exopeptidases. Phosphoramidon (also available from Enzyme Systems Products) specifically inhibits 20 thermolysin and collagenase as well as metalloendoproteases from Bacillus subtilis, Streptomyces griseus and Pseudomonas aeruginosa microorganisms.

D. immitis larvae were cultured in NI media as described, for example, in U.S. Patent Application Serial'\ No. 08/153,554, ibid. NI medium contains a 1:1 mixture of NCTC-135 a-d Iscove's modified Dulbecco medium (available from Sigma Chemical Co., St. Louis, MO), 20% SeruMax, 91 micrograms (jg) of amphotericin B per ml, 0.1 nanograms (ng) of gentamicin per ml, 50 ag of sulfadiazine per ml and Ag of trimethoprim per ml. Larvae, at a concentration of about 200 L3 per milliliter (ml) of NI medium, were distributed in 0.5-ml aliquots into the wells of a 24-well plate. The volume in each well was adjusted to 1 ml using NI medium and 10-millimolar (mM) stocks of bestatin or phosphoramidon, each of which was dissolved in NI media.

The final concentration of inhibitor in the culture wells was either 0, 1, 2.5 or 5 mM. Larvae were incubated at about 37 0 C, 5% C02 and 95% relative humidity. Larvae were oserved daily and the percent molt molt) was evaluated at 72 hours. The percent molt was calculated for each well B. by dividing the number of cuticles by the number of larvae per well and multiplying by 100. There were three wells for each inhibitor concentration and six wells for the untreated control. The results of this study. appear in Table 1.

Table 1 Effect of Protease Inhibitors on Larval Molting

BB*BB

B. B

B

Group Concentratio molt s.d. Fi Sc nr reductio n Control NA 82.6 15.8 NA NA NA Bestatin 1.0 mii 60.0 9.0 27.3 mM 34.8 4.3 57.9 mM 4.9 0.9 94.1 Phosphoramidon 1.0 mM 42.6 2.1 48.4 mM 12.3 1.2 85.1 mM 0.0 0.0 100.0 92 The results indicate that treatment of L3 by bestatin and phosphoramidon significantly reduces the ability of the larvae to molt. An analysis of variance was performed using molt. The difference overall was significant (P 0.0001). Fisher PLSD (Fi) and Scheffe F-test (Sc) multiple comparisons comparing each group to the control were done after the ANOVA represent significant differences of p 0.05, NA not applicable).

It was also observed that, in general, bestatintreated larvae moved much more slowly throughout the study :i *compared to controls whereas phosphoramidon-treated larvae were very active compared to controls. Cuticle separation appeared to be occurring in the phosphoramidon-treated larvae, but the larvae could not open up the old cuticle and escape. The phosphoramidon-treated larvae were in poor shape by the end of the study. While not being bound by theory, it is believed that these phenomena suggest two distinct effects and that the inhibitors may be targeting •.different enzymes.

20 Example 9 Example 9 describes the isolation and characterization of protein-containing fractions from excretory/secretory (ES) products of D. immitis larvae.

ES products from about 11,600 D. immitis L3/L4 were collected and concentrated as described, for example, in U.S. Patent Application Serial No. 08/153,554, ibid. The buffer was exchanged to 20 mM piperazine-HCl, pH 93 0.005% Brij 35 using a Centriprep 10 (available from Amicon Inc., Beverly, MA). The resulting mixture was separated on a Mono-Q column (anion exchange) (available from Pharmacia Biotech Inc., Piscataway, NJ) equilibrated in 20 mM piperazine-HCl, pH 6.0 using an increasing gradient of sodium chloride. Each fraction was then brought to 0.005% Brij 35. Buffer A was 20 mM piperazine-HCl, pH 6.0. Buffer B was 1 M sodium chloride in 20 mM piperazine-HCl, pH The chromatography program was: 0% B for 5 minutes; (b) 10 0% to about 50% B over 25 minutes; hold 3 minutes; (d) about 50% to 100% B over 5 minutes. The flow rate was ml per minute. Fractions were collected every minute.

o The collected fractions were assayed for metalloprotease activity using the fluorogenic compound H- 15 phenylalanine-7-amido-4-methylcoumarin (H-Phe-AMC). The assay was conducted as follows. A stock solution of 10 mM H-Phe-AMC in dimethyl sulfoxide (DMSO) wt.s diluted 1:200 with 100 mM Tris-HCl, pH 7.0. About 100 microliters (il) of the diluted stock solution was placed in each of a 20 desired number of wells in a 96-well microtiter plate. To each well was added 25 Al of the fractions, or control samples, to be tested. The resulting mixture was incubated at about 37 0 C for at least 2 hours. The microtiter plate was then placed on a UV light box and photographed to identify fractions in which AMC was cleaved (released AMC glows under such conditions).

Fractions 20 and 21 collected from the anion exchange both exhibited metalloproteolytic activity. An aliquot of 94 fraction 21 was concentrated and evaluated by SDS-PAGE (14% Tris-glycine). One major band was detected that migrated with an apparently molecule weight of about 60 kD.

Anion exchange fraction 21 was then applied to s.ze exclusion chromatography as described in U.S. Patent Application Serial No. 08/153,554, ibid. Specifically, Fraction 21 from the anion exchange column was applied to a TSK 3000 SW column (available from Beckman Instruments Inc., Fullerton, CA) in 50 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, at a flow rate of 0.5 ml per minute.

Fractions were collected every 0.5 minutes. When assayed using the microtiter plate fluorescent assay, fractions 21 and 22 were positive. The relative time of elution of these fractions was very close to the elution time of bovine 15 serum albumin, which has a molecular weight of about 62 to 66 kD.

Anion exchange fraction 21 was also submitted to isoelectric focussing under "native" conditions. The resultant gel was sliced into 1 mm fractions and the strips 20 assayed by the microtiter plate fluorescent assay. The active fraction was in a region having a pi of about 6.8.

In a separate study, ES from about 2500 larvae was submitted to electrophoresis through each of two lanes of Novex Zymogel (available from Novex, San Diego, CA).

Zymogel contains about 0.1 percent gelatin. The two lanes were soaked in 2.5% Triton X-100 for about 30 minutes and subsequently washed in reaction buffer (50 mM Tris-HCl, pH 5 mM calcium chloride, 0.02% Brij 35 and 200 mM sodium chloride) for about 30 minutes. One lane was then incubated in reaction buffer at about 37°C for 66 hours. The other lane was incubated in reaction buffer containing 2 mM EDTA for the same amount of time. Both lanes were then stained in 0.5% CBB-R250, 40% methanol, 10% acetic acid and destained in methanol, 10% acetic acid in order to detect collagenase activity. Activity was identified by a clear zone in a blue background. ES proteins displaying metalloprotease activity that was completely inhibited by EDTA migrated with apparent molecular weights of about 60 kD, about 95 kD and at least about 200 kD.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

a The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

a a Amendments have been made to the claims which follow and new claims have been proposed to more closely define the invention. The claim amendments are based on various preferred embodiments as described and exemplified in the description. However, for the purpose of maintaining integrity of the text of the disclosure, substantial amendments have not been made to the description in light of the claim amendments. It is submitted that there is no disconformity in the specification as a consequence of amendment of the claims given the limitation of claim scope to various preferments of the invention described.

96 SEQUENCE LISTING The following Sequence Listing is submitted pursuant to 37 CFR S1.821. A copy in computer readable form is also submitted herewith. The paper and computer readable forms of this Sequence Listing are the same.

GENERAL INFORMATION: APPLICANT: Tripp, Cynthia Ann Frank, Glenn R.

Grieve, Robert B.

(ii) TITLE OF INVENTION: NOVEL PARASITE PROTEASE GENES AND PROTEINS (iii) NUMBER OF SEQUENCES: 19 (iv) CORRESPONDENCE

ADDRESS:

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NAME: Verser, Carol Talkington 4* REGISTRATION NUMBER: 37,459 REFERENCE/DOCKET NUMBER: 2618-21 (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: 303/863-9700 TELEFAX: 303/863-0223 97 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1299 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: TTTTTTTTTT TTTTTTTTGT TTCATTGTTC AGTCAGTGOA AAATTATCGA aCGCAGAAAG CATCACGAAA TACGTTAGAT CACATCAAAC AACTTATCAC CTTGAACGTA CAAAGAGAGA 120 TTGGAAACAT AGATGATAAG ACATTAGCTG ATGAAATAGT ATTACAACGA CGGGATCCTG 180 aGGCAAAATG GCATCATAAT GAACTATTCA TTAATGATCC AGATGCATAC TATCAAGGCG 240 ATGTCGATTT GTCGGAAAAA-rAAGCCGAAA TTCTAAGCGA ACATTTTAAA AATGAAATTG 300 CTTTAACAGA GAAAGACGAC ACAATAATAC GGCGAAAAAA GAGCATTGGT CGTGAACCAT 360 TTTACGTAAG ATGGAATCAT AAACGTCCCA TTAGCTATGA ATTTGCGGAA AGTATTCCAT 420 TAGAAACACG TAGAAAAATT CCTTCAGCAA TAGCAATGTG GGAAGAACGA ACATGCATAC 480 *GATTCCAAGA AAATGGCCCA. AATGTAGATC GAATTGAATT TTACGACGGT GGCGGTTGTT 540 CAAGTTTTGT CGGCCGAAC!A GGAGGGAATT TCAATTTCAA CACCAGGATG TGATATTATT 600 GGTATTATAT CACATGAAAT TGGTCATACT TTAGGAATAT TTCATGAGCA AGCACGTCGT 660 GATCAAAAAA aTCATATTTT TATTAATTAC AACAATATTC CATCAAGCCG TTGGAACAAT 720 TTTTTTCCAT TATCAGAATA TGAAGCTGAT ATGTTTAATT TACCTTATGA TACAGGATCA. 780 GTAATGCACT ATGGTTCATA CGGATTTGCA AGAAATCCGT ATGAACCAAC TATTACAACA 840 CGTGATAAAT TTCAACAGTA CACAATTC ZO CAACGTGAAG GGCCATCATT TCTGGATTAT 900 GOATCTGTTA AGCTTTATCT ACAAACGCAT TAATGATATT GTTATCAAAT GGATGATAAT 960 25 TTCAATAAGT ATAAACAGCG CTTATCGTTG TACAGAACAA TGTGCTGATA TGCACTGCGA 1020 TCATAATGGT TATCCGGATC CTAATAATTG, CGCGAAATGC TTGTGTCCAG, ATGGTTTTGC 1080 TGGTCGTACC TGTCAATTTG TTCAATATAC ATCTTGCGGA GCTCTCATTA AGGTAAGTAT 1140 TGTCTTTTGA CCTCTTCTCT GACTAAAATA TAAGTTAAGC ATATGTATCT TCCGTCTAAT 1200 GATTTTCTTG ATTTTGATTT GTTCAATGCT CTTCTTGATA. ATAATATAAA AATTTTTGAA 1260 AATAAAGTTA ACTTTTGGTC AAAAAAAAAA AAAAAAAAA 1299 98 INFORMATION FOR SEQ ID NO:2; SEQUENCE CHARACTERISTICS: LENGTH: 2126 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCF DESCRIPTION: SEQ ID NO:2: GAAAGCATCA CGAAA

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ACGT TAGATCACAT CAAACAACTT ATCACCTTGA ACGTACAAAG AGAGATTGGA AACATAGATG ATAAGACATT AGCTGATGAA ATAGTATTAC AACGACGGGA 120 TCCTGAGGCA AAATGGCATC ATAATGAACT ATTCATTAAT GATCCAGATG CATACTATCA 180 AGGCGATGTC GATTTGTCGG AAAAACAAGC CGAAATTCTA AGCGAACATT TTAAAAATGA 240 AATTGCTTTA ACAGAGAAAG ACGACACAAT AATACGGCGA AAAAAGAGCA TTGGTCGTGA 300 ACCATTTTAC GTAAGATGGA ATCATAAACG TCCCATTAGC TATGAATTTG CGGAAAGTAT 360 TCCATTAGAA ACACGTAGAA AAATTCGTTC AGCAATAGCA ATGTGGGAAG AACGAAC4ATG 420 CATACGATTC CAAGAAAATG GCCCAAATGT AGATCGAATT GAATTTTACG ACGGTGGCGG 480 *TTGTTCAAGT TTTGTCGGCC GAACAGGAGG GAATTTCAAT TTCAACACCA GGATGTGATA 540 **TTATTGG AT TATATCACAT GAAATTGGTC ATACTTTAGG AATATTTCAT GAGCAAGCAC 600 GTCGTGATCA AAAAAATCAT ATTTTTATTA ATTACAACAA TATTCCATCA AGCCGTTGGA 660 ACAATTTTTT TCCATTATCA GAATATGAAG CTGATATGTT TAATTTACCT TATGATACAG "0 GATCAGTAAT GCACTATZ3GT TCATACGGAT TTGCAAGAAA TCCGTATGAA CCAACTATTA 780 CAACACGTGA TAAATTTCA CAGTACACAA TTGGGCAACG TGAAGGGCCA TCATTTCTGG 840 ATTATGCATC TGATAAACAG CGCTTATCGT TGTACAGAAC AATGTGCTGA TATGCACTGC 900 GATCATAATG, GTTATCCGGA TCCTAATAAT TGCGCGAAAT GCTTGTGTCC AGATGGTTTT 960 GCTGGTCGTA. CCTGTCAATT TGTTCAATAT ACATCTTGCG GAGCTCTCAT TAAGGCGAGG. 1020 :AAAATGCCTG TTACGATTTC GAGCCCAAAT TATCCAAACT TCTTCAATGT TGGTGATCAA 1080 TGTATTTGGT TGCTTACAGC TCCACGCGTG ATTCGTAAAT TTGcAGTTTG TTGAACAATT 1140 TCAATTACAA TGTGAAGATA CGTGTGATAA ATCCTATGTA GAAGTGAAAG CTGACGCTGA 1200 TTTTCGACCT ACTGGATATC GATTTTGTTG TTCGCGAGTG CCACGTCATA TTTTTCAATC 1260 TGCGACAAAC GAGATGGTAG TPATATTTCG CGGTTTTGGT GA' ZCGGGAA ATGGCTTTAA 1320 AGCTAAAATT TGGTCAAACG TAGATGATGA TATAGCTAAT ACAATTGTAA CAACTGAAAT 1380 GGCAAAAATT TCGGAAAAAA TACCGAAGCT AACAGTTCCA ATAGTTAAAA CTATTACCAC 1440,.

TCCTACAATA ACAACTACTA CTGCTTTCAT GATATCACCC AAGAAAGGCA ATGTCAICGC 1500 CACGAGAGTT GCTATCACTA CTACGCCGAC TACTACAATT ACTACGACTA TTGC-CGGTAC 1,660 GTACCAATCA CCGTAACTAA TAATACTACA CCTGTAGTAR GTGAAACTTT ACCATCATTG 1620 CCAGTCAAGA TTCGAAACAA AATAGGTGCA TGCGAATGTG GTGAATGGAC AGAATGGACA 1680 99 GGTCCATGCT CTCAAGAATG TGGCGGTTGC GG&&P.ACGTC TTCGAACACG TCAGTGTTCA 1740 TCAGATACGG AATGTAGAAC AGAAGAAAAA CGTGCGTG-TG CTTTTAAGTT TGCCCATACG 1800 GGACTAATTT CCTTATCART AATGGAGAGT TTCPATATACT TTGGAAGGGC TGCTGTGTTG 1860 GTCTATTCCG ATCGGGAGAT ATGTGTTCAG CACTTGATGA TAACGAGAAT CCATTTCTGA 1920 AATTTCTAGA ATCACTGTTG AACATGCAAG ATTCTCGAAA AA&CGATAAT TTGCCTGACT 1980 CGAAAAAGAA GTGATTGAAT GATTCOATAA TATTGATTAA TAAAACGGGT TGTATTCTCG 2040 TCATAGAGTA TCCGTTGATG TTTTTATCCA AAhMATTCTC TTGCTTTTAA TTATTGTGAA 2100 TAAAACTTTT GTTTACCCAA AAAA& 2126 -100 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHAR.ACTERISTICS: LENGTH: 191 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Cys Phe Ile Val Gin Ser Val Giu Asn Tyr Arg Thr Gin Lys Ala Ser 1 5 10 Arg Asn Thr Leu Asp His Ile Lys Gin Leu Ile Thr Leu Asn Val Gin 25 Arg Giu Ile Gly Aua Ile Asp Asp Lys Thr Leu Ala Asp Glu Ile Val 40 Leu Gin Arg Arg Asp Pro Giu Ala Lys Trp His His Asn Glu Leu Phe 50 55 Ile Asn Asp Pro Asp Ala Tyr Tyr Gin Gly Asp Val AL.- Leu Ser Glu 70 75 ~.:Lys Gln Ala Giu Ile Leu Ser Giu His Phe Lys Asn Giu Ile Ala Leu 90 20 Thr Giu Lys Asp Asp Thr Ile Ile Arg Arg Lys Lys Ser Ile Gly Arg 100 105 110 Glu Pro Plie Tyr Val Arg Trp Asn His Lys Arg Pro Ile Ser Tyr Glu .9.115 120 125 *Phe Ala Giu Ser Ile Pro Leu Giu Thr Arg Arg Lys Ile Arg Ser Ala 130 135 140 Ile Ala Met Trp Glu Glu Arg Thr Cys Ile Arg Phe Gin Glu Asn Gly 145 150 155 160 P *Pro Aen Val Asp Arg Ile Giu Phe Tyr Asp Gly Gly Gly Cys Ser Ser 165 170 175 30 Phe Vai Gly Arg Thr Gly Gly Aura Phe Asn Phe Asn Thr Arg Met 180 185 190 -101- INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 141 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Ile Glu Leu Aun Phe Thr Thr Val Ala Val Val. Gin Val Leu Ser Ala 1 510 1s Glu Gin Glu Gly Ile Ser Ile Ser Thr Pro Gly Cys Asp Ile Ile Gly 25 Ile Ile Ser His Gi Ile Gly His Thr Leu Gly Ile Phe His Glu Gin 40 Ala Arg Arg Asp Gin Lye Asn His Ile Phe Ile Asn Tyr Asn Aen Ile s0 55 Pro Ser Ser Arg Trp Aen Asn Phe Phe Pro Leu Ser Giu Tyr Glu Ala 70 75 *Asp Met Phe Asn Leu Pro Tyr Asp Thr Gly 5cr Val Met His Tyr Gly 90 20 Ser Tyr Gly Phe Ala Arg Asn Pro Tyr Glu Pro Thr Ile Thr Thr Arg *100 105 110 Asp Lys Phe Gin Gin Tyr Thr Ile Gly Gin Arg Giu Gly Pro 5cr Phe 115 120 125 *Leu Asp Tyr Ala Ser Val Lys Leu Tyr Leu Gin Thr His 130 135 140 -102 INFORMATION F'OR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 121 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Cys Thr Met Val His Thr Asp Leu Gin Glu 1 5 10 Leu Gin His Val Ile Asn Phe Asn Ser Thr 25 Gly His His Phe Trp Ile Met His Leu Leu 40 Ile Aen Asp Ile Val Ile Lys Trp Met Ile 50 55 Ser Ala Tyr Arg Cys Thr Giu Gin Cys Ala 65 70 Asn Gly Tyr Pro Asp Pro Asn Asn Cys Ala 85 90 Gly Phe Ala Gly Arg Thr Cys Gin Phe Vai 100 .105 Ala Leu Ile Lys Val Ser Ile Val Phe 115 120

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Ile Arg Met Aen Gin Leu Giy An Ser Phe Ile Tyr Ile Ser Ile Ser 60 Asp Met His Cys 75 Lye Cys Leu Cys Gin Tyr Thr Ser 110 Gin Leu Val Lye Lye Arg Ile An Asp His S0 Pro Asp Cys Gly 103 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 178 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Lys Ala Ser Arg Asn Thr Leu Asp H-s Ile Lys Gin Leu Ile Thr Leu 1 5 10 Asn Val Gin Arg Glu Ile Gly Asn Ile Asp Asp Lys Thr Leu Ala Asp 25 Glu Ile Val Leu Gin Arg Arg Asp Pro Glu Ala Lys Trp His His Asn 40 Glu Leu Phe Ile Asn Asp Pro Asp Ala Tyr Tyr Gin Gly Asp Val Asp 50 55 Leu Ser Glu Lye Gin Ala Glu Ile Leu Ser Glu His Phe Lys Asn Glu 65 70 75 Ile Ala Leu Thr Glu Lys Asp Asp Thr Ile Ile Arg Arg Lys Lys Ser 85 90 20 Ile Gly Arg Glu Pro Phe Tyr Val Arg Trp Asn His Lys Arg Pro Ile 100 105 110 Ser Tyr Glu Phe Ala Glu Ser Ile Pro Leu Glu Thr Arg Arg Lys Ile 115 120 125 Arg Ser Ala Ile Ala Met Trp Glu Glu Arg Thr Cys Ile Arg Phe Gin 130 135 140 Glu Asn Gly Pro Asn Val Asp Arg Ile Glu Phe Tyr Asp Gly Gly Gly o! 145 150 155 160 oo** Cys Ser Ser Phe Val Gly Arg Thr Gly Gly Asn Phe Asn Phe Asn Thr 165 170 175 b.ooo: 30 Arg Met

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ro t, 104 INFORMATION XFOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 145 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Ile Giu Leu Asn Phe Thr Thr Val Ala Val Val Gin Vai Leu Ser Ala 1 5 10 Giu Gin Giu Gly Ile Ser Ile Ser Thr Pro Gly Cys Asp Ile Ile Gly 25 Ile Ile Ser His Glu Ile Gly His Thr Leu Gly Ile Phe His Giu Gin 40 Ala Arg Arg Asp Gin Lys Asn His Ile -he Ile Aen Tyr Asn Asn Ile 50 55 Pro Ser Ser Arg Trp Asn Asn Phe Phe Pro Leu Ser Giu Tyr Giu Ala 70 75 Asp Met Phe Asn Leu Pro Tyr Asp Thr Gly Ser Val Met His Tyr Gly 90 Ser Tyr Gly Phe Ala Arg Asn Pro Tyr Giu Pro Thr Ile Thr Thr Arg 100 105 110 *fft*Asp Lys Phe Gin G.In Tyr Thr Ile Gly Gin Arg Giu Gly Pro Ser Phe 115 120 125 Leu Asp Tyr Ala Ser Asp Lys Gin Arg Leu Ser Leu Tyr Arg Thr Met 25 130 135 140 Cys 145 -105- INFORM4ATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 134 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Cys Thr Met Val His Thr Asp Leu Gin Glu Ile Arg Met Ann Gin Leu 1 5 10 Leu Gin His Val Ile Ann Phe Asn Ser Thr Gin Leu Gly Ann Val Lys 25 Gly His His Phe Trp Ile Met His Leu Ile Asn Ser Ala Tyr Arg Cys 40 Thr Glu Gin Cys Ala Asp Met His Cys Asp His Asn Gly Tyr Pro Asp 50 55 Pro Asn Asn Cys Ala Lys Cys Leu Cys Pro Asp Gly Phe Ala Gly Arg 70 75 ***Thr Cys Gin Phe Val Gin Tyr Thr Ser Cys Gly Ala Leu Ile Lys Ala 90 20 Arg Lys Met Pro Val Thr Ile Ser Ser Pro Asn Tyr Pro Ann Phe Phe ***100 105 110 Val Gly Asp Gin Cys Ile Trp, Leu Leu Thr Ala Pro Arg Val Ile 115 120 125 Lys Phe Ala Val Cys 130 -106 INFORMA~TION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 154 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Phe Val Asn Leu Gin Phe Val Giu Gin Phe Gin Leu Gin Cys Glu Asp 1 5 10 Thr Cys Asp Lys Ser Tyr Val Glu Val Lys Ala Asp Ala Asp Phe Arg 25 Pro Thr Gly Tyr Arg Phe Cys Cys Ser Arg Val Pro Arg His Ile Phe 40 Gin Ser Ala Thr Asn Giu Met Val Val Ile Phe Arg Gly Phe Gly Asp s0 55 Ala Gly Asn Gly Phe Lys Ala Lys Ile Trp Ser Aun Vai Asp Asp Asp 70 75 so .le Ala Asn Thr Ile Vai Thr Thr Glu Met Ala Lys Ile Ser Glu Lys 90 *720 Ile Pro Lys Leu Thr Val Pro Ile Val Lys Thr Ile Thr Thr Pro Thr *..100 105 110 I *le Thr Thr Thr Thr Ala Phe met Ile Ser Pro Lys Lys Gly Aen Val 115 120 125 Thr Ala Thr Arg Aex Ala Ile Thr Thr Thr Pro Thr Thr Thr Ile Thr 130 135 140 Thr Thr Ile Ala Gly Thr Tyr Gin Ser Pro 145 150 107- INFORM4ATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 163 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Ann Tyr Tyr His Ser Tyr Ann Aen Ann Tyr Tyr Cys Phe Him Asp Ile 1 5 10 Thr Gin Glu Arg Gin Cys His Arg His Giu Ser Cys Tyr His Tyr Tyr 25 Ala Asp Tyr Tyr Aen Tyr Tyr Asp Tyr Cys Arg Tyr Val Pro Ile Thr 40 Val Thr Ann Ann Thr Thr Pro Val Val Ser Glu Thr Leu Pro Ser Leu so 55 Pro Val Lys Ile Arg Ann Lys Ile Gly Ala Cys Glu Cym Gly Glu Trp 70 75 *Thr Giu Trp Thr Gly Pro (aye Ser Gin Glu Cym Gly Gly Cys Gly Lys 8590 Arg Leu Arg Thr Arg Gin Gym 5cr Ser Amp Thr Giu Gym Arg Thr Glu *100 .105 110 Lye Arg Ala Gym Ala Ph. Lye Ph. Ala His Thr Gly Leu Ile Ser 115 120 125 *Leu 5cr Ile Met Glu Ser Phe Ile Tyr Phe Gly Arg Ala Ala Vai Leu 25 130 135 140 Val Tyr Ser Asp Arg Giu Ile Gym Val Gin His Leu Met Ile Thr Arg 145 150 155 160 Ile His Phe -108 INFORMATION FOR SEQ ID NO;11: SEQUENCE CHARACTERISTICS: LENGTH: 638 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:. SEQ ID NO:11:.

Lys Ala Ser Arg Asn Thr Lou Asp His Ile Lys Gln Leu Ile Thr Leu 1 5 10 Asn Val Gln Arg Giu Ile Gly Aen Ile Asp Asp Lys Thr Leu Ala Asp 25 Glu Ile Val Lou Gln Arg Arg Asp Pro Glu Ala Lye Trp His His Ann 40 Glu Lou Phe Ile Ann Asp Pro Asp Ala Tyr Tyr Gin Gly Asp Val Asp 50 55 *Lou Ser Glu Lys Gin Ala Glu Ile Lou Ser Giu His Phe Lys Leu Asn 70 75 Giu Ile Ala Lou Thr Glu Lys Asp Asp Thr Ile Ile Arg Arg Lye Lys 90 20 Ser Ile Gly Arg Giu Pro Phe Tyr Val Arg Trp Ann His Lye Arg Pro I *le Ser Tyr Giu Phe Ala Giu 8cr Ile Pro Lou Giu Thr Arg Arg Lye 115 120 125 *le Arg Ser Ala Ile Ala Met Trp Glu Giu Arg Thr Cys Ile Arg Phe S 25 130 135 140 Gin Glu Ann Gly Pro Ann Val Asp Arg Ile Giu Phe Tyr Asp Gly Gly 145 150 155 160 Gly Cyn Ser Ser Phe Val Gly Arg Gin Giu Gly Ile Ser Ile Ser Thr 165 170 175 Pro Gly Cys Asp Ile Ile Gly Ile Ile Ser His Giu Ile Gly His Thr 180 185 190 *Leu Gly Ile Phe His Glu Gin Ala Arg Arg Asp Gin Lye Ann His Ile :195 200 205 Phe Ile Ann Tyr Asn Ann Ile Pro Ser Ser Arg Trp Ann Ann Ph. Phe 210 215 220 Pro Lou Ser Giu Tyr Giu Ala Asp Met Phe Ann Lou Pro Tyr Asp Thr 225 230 235 240 Giy Ser Vai Met His Tyr Gly Ser Tyr Gly Phe Ala Arg Ann Pro Tyr 245 250 255 Giu Pro Thr Ile Thr Thr Arg Asp Lye Phe Gin Gin Tyr Thr Ile Gly 260 265 270 Gin Arg Giu Gly Pro Ser Ph. Lou Asp Met His Lou Ile Ann Ser Ala 275 280 285 Tyr Arg Cys Thr Giu Gin Cyn Ala Asp Met His Cys Asp His Ann Gly 290 295 300 -109 9* 9* 9 9*9* 99 9 9999..

9* a 99 *9 Tyr 305 Ala Ile Asn Arg Glu 385 Phe Ile Gly Asp Glu 465 Pro Aen Ile Thr Asn 545 Pro Gin Al a Ser G iu 625 Pro Gly Lys Phe Val 370 Asp Arg Phe Asp Asp 450 Lye Thr Val Thr Pro 530 Lys Cys Cys Phe Phe 610 Ile Asp Pro Arg Thr Ala Arg 340 Ph. Asn 355 Phe Val Thr Cys Pro Thr Gin Ser 420 Ala Gly 435 Ile Ala Ile Pro Ile Thr Thr Ala 500 Thr Thr 515 Vai Val Ile Gly Ser Gin Ser Ser 580.

Lys Ph.

595 Ile Tyr Cys Val Asn Cys 325 Lys Tyr Aen Asp Gly 405 Ala An An Lys Thr 485 Thr Ile Ser Ala Glu 565 Asp Ala Phe Gin Asn Cys 310 Gin Phe Met Pro Gly Asp Leu Gin 375 Lys Ser 390 Tyr Arg Thr Aen Gly Phe Thr Ile 455 Leu Thr 470 Thr Thr Arg Val Ala Gly Giu Thr 535 Cys Glu 550 Cys Gly Thr Giu His Thr Giy Arg 615 His Leu 630 Ala Lys Val Gin Vai Thr 345 Gin Cys 360 Ph. Val Tyr Val Phe Cys Glu Met 425 Lye Ala 440 Vai Thr Val Pro Ala Ph.

Ala Ile 505 Thr Tyr 520 Leu Pro Cys Gly Gly Cys Cys Arg 585 Gly Leu 600 Ala Ala Met Ile Leu 315 Thr Ser Trp Gin Val 395 Ser Val Ile ,.iu Val 475 Ile Thr Ser Leu Trp 555 Lys Glu Ser Leu Arg 635 Cys Ser Ser Leu Ph.

380 Lys Arg Ile Trp, Met 460 Lys Ser Thr Val Pro 540 Thr Arg Giu Leu Val 620 Ile Asp Gly Asn 350 Thr Leu Asp Pro Arg 430 Aen Lys Ile Lye Thr 510 Asn Lye Trp Arg Arg 590 Ile Ser Ph.

Gly Ala 335 Tyr Ala Gin Ala Arg 415 Gly Val Ile Thr Lye 495 Th Asn Ile Thr Thr 575 Ala Met Asp Ph.

320 Leu Pro Pro Cys Asp 400 His Ph.

Asp Ser Thr 480 Gly Thr Thr Arg Gly 560 Arg Cys Glu Arg 110 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERIV'ICS: LENGTH: 143 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: l. .143 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GGA TCC TOT GOT TCA TOT TOG GCT TTT TCT OTT ACT GGC AAT ATT OCA 48 Gly Ser Cys Gly Ser Cys Trp Ala Phe Ser Val Thr Oly Asn Ile Ala 1 5 10 AGT CTC TOG GCT ATT AAA ACA GOT OAT TTO ATA TCG CTT TCC GAG CAA 96 Ser Leu Trp Ala Ile Lys Thr Gly Asp Leu Ile Ser Lou Ser 1lu Gln 20 25 20 OAA TTG ATT OAT TGT GAT OTG GTT GAT GAG GGC TGC AAC GGC GGC TA 143 Glu Leu Ile Asp Cys Asp Val Val Asp Glu Oly Cys Aen Oly Gly 35 S.

S

S. S S INFORMATION FOR SEQ ID NO:13: 25 SEQUENCE CHARACTERISTICS: LENGTH: 47 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Gly Ser Cys Oly Ser Cys Trp Ala Phe Ser 1 5 10 Ser Leu Trp, Ala Ile Lys Thr Gly Asp Lou 25 Glu Lou Ile Asp Cys Asp Val Val Asp Glu 40 NO: 13: Val Thr Gly Aen Ile Ala Ile Ser Leu Ser Glu Gin Oly Cys Asn Oly Gly 111 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: ACWCATGAAA TIGSICAT 18 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA primer (xi) SEQUENCE DESCRIPTION: SEQ ID AATACGACTC ACTATAG 17 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs .o TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: TGGTATTATA TCACATGAAA TTGGTCATAC INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE nucleic .acid STRA.DEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CCCAATTGTG TACTGTTGAA ATTTATCAC 112 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: CGGGATCCTG TGGWTCATGY TGGGC INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOP"LOGY: linear (ii) MOLECULE TYPE: DNA primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: •TAICCICCRT

TRCAICCYTC

While various embodiments of the p, esent invention 20 have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly e..o understood, however, that such modifications and adaptations are within the scope of the present invention, 25 as set forth in the following claims:

Claims (10)

1. An isolated protein encoded by a nucleic acid sequence that hybridizes under stringent hybridization conditions with a Dirofilaria immitis astacin metalloendopeptidase gene.

2. The protein of claim 1, wherein said protein comprises at least a portion of at least one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: wherein said portion is encoded by nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule selected from the group consisting of nDiMPA11299 and nDiMPA2 2126

3. The protein of claim 1, wherein said protein comprises an extended zinc-binding domain motif. a 4. The isolated protein of Claim 1, wherein said protein comprises an astacin domain having at least about 35 percent homology with the astacin domain of SEQ ID NO: 11. a The isolated protein of Claim 1, wherein said protein comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID

6. The isolated protein of Claim 1, wherein said protein is an isolated filariid astacin matalloendopeptidase protein selected from the group consisting of: Dirofilaria, Acanthocheilonema, Brugia, Dipetalonema, 114 Loa, Ochocerca, Parafilaria, Stearia, Stephanofilaria and Wuchereria proteins.

7. The isolated protein of Claim 1, wherein said protein is an isolated D. immitis protein.

8. An isolated antibody capable of selectively binding to a protein as set forth in any one of claims 1 to 7.

9. A therapeutic composition capable of protecting an animal from disease caused by a filariid susceptible to an inhibitor of an astacin metalloendopeptidase, said therapeutic composition comprising an excipient and at least one compound selected from the group consisting of: an isolated astacin metalloendopeptidase protein encoded by a i'":nucleic acid sequence that hybridizes under stringent hybridization S- conditions with a D. immitis astacin metalloendopeptidase gene, or a mimetope thereof; an antibody that selectively binds to said astacin metalloendopeptidase protein; and an inhibitor of said astacin metalloendopeptidase protein identified by its ability to inhibit filariid astacin metalloendopeptidase activity.

10. The therapeutic composition of claim 9, wherein said filariid is from a genus selected from the group consisting of Dirofilaria, Acanthocheilonema, Brugia, Dipetalonema, Loa, Onchocerca, Parafilaria, Setaria, Stephanofilaria and Wuchereria.

11. Use of a composition of claim 9 for the treatment or proplylaxis of disease caused by a filariid susceptible to an inhibitor of an astacin -Z metalloendopeptidase. 115

12. A method for the treatment or proplylaxis of disease caused by a filariid susceptible to an inhibitor of an astacin metalloendopeptidase which method including the step of administering a therapeutically effective amount of a composition of claim 9 to a subject in need thereof. DATED this 21st day of August 2000 HESKA CORPORATION By its Patent Attorneys DAVIES COLLISON CAVE *fl. C o *a *a C. C C