| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | April 2, 2002 |
| PATENT TITLE |
Filariid nematode cysteine protease nucleic acid molecules and uses thereof |
| PATENT ABSTRACT | The present invention provides for filariid nematode cysteine protease proteins; to filariid nematode cysteine protease nucleic acid molecules, in particular, Dirofilaria immitis L3 larval cysteine protease nucleic acid molecules and Onchocerca volvulus L3 larval cysteine protease nucleic acid molecules; to antibodies raised against such proteins, and to compounds that inhibit filariid nematode cysteine protease activity. The present invention also includes methods to obtain such proteins, nucleic acid molecules, antibodies and/or inhibitors. The present invention also includes therapeutic compositions comprising such proteins, nucleic acid molecules, antibodies and/or inhibitors, and the use of such compositions to protect an animal from disease caused by parasitic helminths |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | January 9, 1998 |
| PATENT REFERENCES CITED |
Dinman et al. Exp. Parasitol. Aug. 1990 vol. 71 (2), 176-88.* Basch in Vaccines and World Health Science, Policy & Practice 1994, p. 245, Oxford Univ. Press, Inc., New York.* Zeng et al. Mol. Cell. Biol. 1990 vol. 6(10), 2765-2773.* Sakanari et al. Proc. Natl. Acad. Sci. 1989 vol. 86, p. 4863-4867.* Arlot-Bonnemains et al., 1996, Biochem., 319:975-82. Abraham et al., 1990, Exp. Parasitol., 70:314-322. Abraham et al., 1987, J. Parasitol., 73(2):377-383. Aimri et al., 1988, Mol. Biochem. Parasitol., 28:113-120. Boulay et al., 1995, Comp. Biochem. Physiol., 111B(3):353-359. Boulay et al., 1996, J. Comp. Physiol B, 166:310-318. Chung et al., 1995, J. Parasitol., p. 137-142. Cox et al., 1990, Mol. Biochem. Parasitol., 41:25-34. Dalton et al., 1989, Mol. Biochem. Parasitol., 35:161-166. Dresden et al., 1985, Exp. Parasitol., 59:257-263. Gamble et al., 1989, Mol. Biochem. Parasitol., 33:49-58. Grieve et al., 1983, Epidem. Rev., 5:220-246. Ham et al., 1994, Trans. Royal Soc. Trop. Med. Hyg., 88:132-135. Heussler et al., 1994, Mol. Biochem. Parasitol., 64:11-23. Heussler et al., 1994, Trop. Med. Parasitol., 45(Supp. II):179. Hong et al., 1993, Exp. Parasitol., 76:127-133. Hotez et al., 1985, J. Biol. Chem., 260:7343-7348. Lackey et al., 1989, Exp. Parasitol., 68:176-185. Lustigman et al., 1992, J. Biol. Chem., 267(24):17339-17346. Lustigman, 1993, Parasitol. Today, 9(8):294-297. Lustigman et al., 1996, J. of Biological Chem., 271(47):30181-30189. Maizels et al., 1989, TIBTECH, 7(11):316-321. Maki et al., 1986, J. Helminthol., 60:31-37. McKerrow et al., 1985, J. Biol. Chem., 231:47-51. McKerrow et al., 1982, Exp. Parasitol., 53:249-254. Petralanda et al., 1986, Mol. Biochem. Parasitol., 19:51-59. Pratt et al., 1992, Mol. Biochem. Parasitol., 51:209-218. Richer et al., 1993, Exp. Parasitol., 76:1-11. Richer et al., 1992, Exp. Parasitol., 75:213-222. Robertson et al., 1989, Exp. Parasitol., 69:167-173. Rogers, 1982, J. Parasitol., 12:495-502. Swamy et al., 1983, Mol. Biochem. Parasitol., 9:1-14. Tomashiro et al., 1987, J. Parasitol., 73:149-154. Wijffels, 1994, Biochem. J., 299:781-790 (Abstract). Yamakami, 1995, Eur. J. Biochem., 233:490-497. Molecular SIGMA Biology catalog, A New Dimension, 1989, Oligonucleotide Products, p. 54. Feng et al., 1985, J. Mol. Evol., 21:112-125. Johnson et al., 1993, J. Mol. Biol., 233:716-738. Meinkoth et al., 1984, Analytical Biochemistry, 138:267-284. Selzer et al., 1997, Genbank Accession No. AF031819, Direct Genbank submission. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. An isolated nucleic acid molecule selected from the group consisting of: (a) a cDNA or a RNA encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:44, and SEQ ID NO:46; and (b) a nucleic acid molecule comprising a complement of a nuclei acid molecule as recited in (a). 2. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises a nucleic acid sequence that encodes a protein selected from the group consisting of a Dirofilaria immitis third-larval stage cysteine protease protein and an Onchocerca volvulus third-larval stage cysteine protease protein. 3. An isolated nucleic acid molecule selected from the group consisting of: a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:39. 4. An isolated rcombinant molecule comprising the nucleic acid molecule as set forth in claim 1 operatively linked to a transcription control sequence. 5. An isolated rcombinant virus comprising the nucleic acid molecule as set forth in claim 1. 6. An isolated recombinant cell comprising the nucleic acid molecule as set forth in claim 1, said cell being capable of expressing said nucleic acid molecule. 7. A composition comprising an excipient and an isolated nucleic acid molecule selected from the group consisting of: a Dirofilaria immitis cDNA molecule, a Dirofilaria immitis RNA molecule, an Onchocerca volvulus cDNA molecule, and an Onchocerca volvulus RNA molecule, wherein said nucleic acid molecule encodes a protein having cysteine protease activity and said nucleic acid molecule hybridzes to a nucleic acid sequence complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:37, under conditions comprising (1) hybridizing in a solution comprising 2X SSC in the absence of nucleic acid helix destabilizing agents, at a temperature of 37.degree. C., and (2) washing in 1X SSC in the absence of nucleic acid helix destabilizing agents at a temperature of 69.degree. C.; and a nucleic acid molecule comprising a complement of said isolated nucleic acid molecule. 8. The composition of claim 7, wherein said composition further comprises a component selected from the group consisting of an adjuvant, a carrier, and a mixture thereof. 9. The composition of claim 7, wherein said composition is selected from the group consisting of a naked nucleic acid vaccine, a recombinant virus vaccine a recombinant cell vaccine. 10. An isolated nucleic acid molecule selected from the group consisting of a Dirofilaria immitis cDNA molecule, Dirofilaria immitis RNA molecule, an Onchocerca volvulus cDNA molecule, and an Onchocera volvulus RNA molecule, wherein said nucleic acid molecule encodes a protein having cysteine protease activity and said nucleic acid molecule hybridizes to a nucleic acid sequence complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:37, under conditions comprising hybridization and wash conditions which allow 10% or less base-pair mismatch, wherein such conditions are determined by a formula: T.sub.m =81.5.degree. C.+16.6 log M+0.41(% G+C)-500/n-0.61(% formamide), wherein T.sub.m represents the temperature at which two complementary nucleic acid molecule stands will disassociate, assuming 100% complementary between the two stands, n represents the number of nucleotides in the shorter stand of the duplex being hybridized and log M represents the ionic strengths of the hybridization and wash solutions in moles/liter; wherein said wash is conducted at a temperature of T.sub.m minus 10.degree. C.; and a nucleic acid molecule fully complementary to said isolated nucleic acid molecule. 11. An isolated nucleic acid molecule selected from the group consisting of a Dirofilaria immitis cDNA molecule, a Dirofilaria immitis RNA molecule, an Onchocerca volvulus cDNA molecule, and an Onchocerca volvulus RNA molecule, wherein said nucleic acid molecule encodes a protein having cysteine protease activity and said nucleic acid molecule hybridizes to a nucleic acid sequence complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:37, under conditions comprising (a) hybridizing in a solution comprising 2X SSC in the absence of nucleic acid helix destabilizing agents, at a temperature of 37.degree. C., and (b) washing in 1X SSC in the absence of nucleic acid helix destabilizing agents at a temperature of 69.degree. C. and a nucleic acid molecule fully complementary to said isolated nucleic acid molecule. 12. The nucleic acid molecule claim 11, wherein said nucleic acid molecule comprises a nucleic acid sequence that encodes a protein selected from the group consisting of a Dirofilaria immitis third-larval stage cysteine protease protein and an Onchocerca volvulus third-larval stage cysteine protease protein. 13. An isolated recombinant molecule comprising the nucleic acid molecule as set forth in claim 11, operatively linked to a transcription control sequence. 14. An isolated recombinant virus comprising the nucleic acid molecule as set forth in claim 11. 15. An isolated recombinant cell comprising the nucleic acid molecule as set forth in claim 11, said cell being capable of expressing said nucleic acid molecule. |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The present invention relates to novel filariid nematode 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 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 helminth parasites, such as by tissue-migrating helminths, including Dirofilaria and Onchocerca. BACKGROUND OF THE INVENTION 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 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. 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 typically 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. Sexually mature adults, after mating, produce microfilariae which traverse capillary beds and circulate in the vascular system. The microfilariae are ingested by female mosquitos during blood feeding on an infected dog, subsequent development of the microfilariae into two larval stages (L1 and L2) occurs in the mosquito. The microfilariae go through and finally become mature third stage larvae (L3) 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 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 infection in which no microfilariae can be detected, but the existence of adult heartworms can be determined through 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 excretory-secretory products) as well as in mammals, there has been no identification of a cysteine protease gene in any filariid nematode. Cysteine protease genes have been isolated from several mammalian sources and from the nematodes Haemonchus contortus (e.g., Pratt et al., 1992, Mol. Biochem. Parasitol. 51, 209-218) and Caenorhabditis elegans (Ray et al., 1992, Mol. Biochem. Parasitol. 51, 239-250). In addition, 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. The determination of these sequences, however, does not necessarily predict that the cloning of novel cysteine protease genes will be straight-forward, particularly since the sequences shared by different cysteine proteases are such that probes and primers 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 (i.e., 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 One embodiment of the present invention relates to an isolated filariid nematode larval nucleic acid molecule that hybridizes, under stringent hybridization conditions, with a Dirofilaria immitis L3 larval cysteine protease gene and/or an Onchocerca volvulus L3 larval cysteine protease gene. A preferred nucleic acid molecule of the present invention includes at least a portion of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, or an allelic variant of one or more of those nucleic acid sequences. The present invention also includes recombinant molecules and recombinant cells that include filariid nematode cysteine protease nucleic acid molecules of the present invention. Also included are methods to produce such nucleic acid molecules, recombinant molecules and recombinant cells of the present invention. Another embodiment of the present invention is an isolated protein that includes a filariid nematode larval cysteine protease protein or a mimetope of such a protein. A filariid nematode cysteine protease protein of the present invention preferably has cysteine protease activity and/or comprises a protein that, when administered to an animal, is capable of eliciting an immune response against a natural helminth cysteine protease protein. The present invention also includes inhibitors of cysteine protease activity as well as antibodies that recognize (i.e., selectively bind to) a filariid nematode 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 therapeutic composition capable of protecting an animal from disease caused by a parasitic helminth. Such a therapeutic composition comprises at least one of the following protective compounds: an isolated parasitic filariid nematode larval nucleic acid molecule that hybridizes under stringent hybridization conditions with a Dirofilaria immitis L3 larval cysteine protease gene and/or an Onchocerca volvulus L3 larval cysteine protease gene; an isolated filariid nematode larval cysteine protease protein or a mimetope thereof; an isolated antibody that selectively binds to a filariid nematode L3 larval cysteine protease protein; and an inhibitor of cysteine protease activity identified by its ability to inhibit filariid nematode L3 larval cysteine protease activity. Also included is a method to protect an animal from disease caused by a parasitic helminth that includes administering to the animal 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 cysteine protease activity of a parasitic helminth. Such a method includes (a) contacting an isolated filariid nematode larval 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 (b) determining if the putative inhibitory compound inhibits the activity. Also included is a test kit to identify a compound capable of inhibiting cysteine protease activity that includes an isolated filariid nematode larval cysteine protease protein having cysteine protease activity and a means for determining the extent of inhibition of cysteine protease activity in the presence of a putative inhibitory compound |
| PATENT PHOTOCOPY | Available on request |
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