Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2000-01-04
2002-12-03
Minnifield, Nita (Department: 1645)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S388100, C530S387900, C530S388200, C530S388600, C530S389100, C536S023100, C536S023400, C424S130100, C424S136100, C424S139100, C424S151100, C514S002600
Reexamination Certificate
active
06489448
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to parasitic helminth thiol specific antioxidant (TSA) nucleic acid molecules, proteins encoded by such nucleic acid molecules, antibodies raised against such proteins, and inhibitors of such proteins. The present invention also includes therapeutic compositions comprising such nucleic acid molecules, proteins, antibodies, and/or inhibitors, as well as their use to protect animals from diseases caused by parasitic helminths, such as heartworm.
BACKGROUND OF THE INVENTION
Parasitic helminth infections in animals, including humans, are typically treated by chemical drugs. One disadvantage with chemical drugs is that they must be administered often. For example, dogs susceptible to heartworm are typically treated monthly. Repeated administration of drugs, however, often leads to the development of resistant helminth strains that no longer respond to treatment. Furthermore, many of the chemical drugs cause harmful side effects in the animals being treated, and as larger doses become required due to the build up of resistance, the side effects become even greater. Moreover, a number of drugs only treat symptoms of a parasitic disease but are unable to prevent infection by the parasitic helminth.
An alternative method to prevent parasitic helminth infection includes administering a vaccine against a parasitic helminth. Although many investigators have tried to develop vaccines based on specific antigens, it is well understood that the ability of an antigen to stimulate antibody production does not necessarily correlate with the ability of the antigen to stimulate an immune response capable of protecting an animal from infection, particularly in the case of parasitic helminths. Although a number of prominent antigens have been identified in several parasitic helminths, including in Dirofilaria, there is yet to be a commercially available vaccine developed for any parasitic helminth.
As an example of the complexity of parasitic helminths, the life cycle of
D. immitis,
the helminth that causes heartworm, includes a variety of life forms, each of which presents different targets, and challenges, for immunization. In a mosquito,
D. immitis
microfilariae go through two larval stages (L1 and L2) and become mature third stage larvae (L3), which can then be transmitted back to the dog. In a dog, the L3 molt to the fourth larval stage (L4), and subsequently to the fifth stage, or immature adults. The immature adults migrate to the heart and pulmonary arteries, where they mature to adult heartworms. Adult heartworms 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 of the dog.
In particular, heartworm is a major problem in dogs, which typically cannot even develop immunity upon infection (i.e., dogs can become reinfected even after being cured by chemotherapy), and is becoming increasingly widespread in other companion animals, such as cats and ferrets. Heartworm infections have also been reported in humans.
Prior investigators have identified yeast thiol specific antioxidants (TSAs), as well as cloning several mammalian TSA genes, a protozoan TSA gene and a partial nucleic acid sequence of an adult Onchocerca TSA gene; see, for example, Chandrashekar et al., Genbank Accession No. U09385; Yamamoto et al, 1989,
Gene
80, 337-343, Torian et al., 1990,
Proc. Natl. Acad. Sci. USA
87, 6358-6362, Reed et al., 1992,
Infection and Immunity.
60, 542-549, Ramussen et al, 1992,
Electrophoresis
13, 960-969, Tannich et al., 1993,
Trop. Med. Parasitol.
44, 116-118, Prosperi et al., 1993,
J. Biol. Chem.
268, 11050-11056, Ishii et al., 1993,
J. Biol. Chem.
268, 18633-18636, Chae et al., 1993,
J. Biol. Chem.
268, 16815-16821, Ishii et al., 1993,
J. Biol. Chem.
268, 18633-18636, Chae et al., 1994,
Proc. Natl. Acad. Sci. USA
91, 7022-7026, Kawai et al, 1994,
J. Biochem.
115, 641-643, Chae et al, 1994,
Proc. Natl. Acad. Sci. USA
91, 7017-7021 and Chae et al, 1994,
Biofactors
4, 177-180. Although yeast, human and bovine cortex TSAs has been shown to have thiol-dependent reductase activity (see, for example, Sauri et al, 1995,
Biochem. Biophys. Res. Comm.
208, 964-969; Watabe et al, 1995,
Biochem. Biophys. Res. Comm.
213, 1010-1016), the other TSA genes having been identified by nucleic acid sequence homology. The determination of these sequences, however, does not indicate or suggest the cloning of novel larval parasitic helminth TSA genes.
As such, there remains a need to identify an efficacious composition that protects animals against diseases caused by parasitic helminths and that, preferably, also protects animals from infection by such helminths.
SUMMARY OF THE INVENTION
The present invention relates to a novel product and process for prevention and treatment of parasitic helminth infection. According to the present invention there are provided parasitic helminth larval thiol specific antioxidant (TSA) proteins; parasitic helminth nucleic acid molecules, including those that encode such proteins; antibodies raised against such TSA proteins (anti-parasitic helminth TSA antibodies); and compounds that inhibit parasitic helminth larval TSA activity (i.e, inhibitory compounds or inhibitors).
The present invention also includes methods to obtain such proteins, nucleic acid molecules, antibodies and inhibitory compounds. Also included in the present invention are therapeutic compositions comprising such proteins, nucleic acid molecules, antibodies, and/or inhibitory compounds, as well as use of such therapeutic compositions to protect animals from diseases caused by parasitic helminths.
One embodiment of the present invention is an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a
Dirofilaria immitis
(
D. immitis
) L4 (i.e., fourth stage larval) TSA gene, such nucleic acid molecules are referred to as parasitic helminth larval TSA nucleic acid molecules. A
D. immitis
L4 TSA gene preferably includes nucleic acid SEQ ID NO:1 and/or SEQ ID NO:3.
The present invention also relates to recombinant molecules, recombinant viruses and recombinant cells that include a TSA nucleic acid molecule of the present invention. Also included are methods to produce such nucleic acid molecules, recombinant molecules, recombinant viruses and recombinant cells.
Another embodiment of the present invention includes a parasitic helminth larval thiol specific antioxidant (TSA) protein (i.e., a parasitic helminth TSA protein) or a protein that includes a parasitic helminth larval TSA protein. A preferred parasitic helminth larval TSA protein comprises amino acid sequence SEQ ID NO:2.
The present invention also relates to mimetopes of parasitic helminth larval TSA proteins as well as to isolated antibodies that selectively bind to parasitic helminth larval TSA proteins or mimetopes thereof. Also included are methods, including recombinant methods, to produce proteins, mimetopes and antibodies of the present invention.
Another embodiment of the present invention is a method to identify a compound capable of inhibiting TSA activity, comprising the steps of: (a) contacting an isolated parasitic helminth L4 TSA protein with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein has TSA activity; and (b) determining if the putative inhibitory compound inhibits the TSA activity. Also included in the present invention is a test kit to identify a compound capable of inhibiting TSA activity. Such a test kit includes an isolated parasitic helminth larval TSA protein having TSA activity and a means for determining the extent of inhibition of the TSA activity in the presence of a putative inhibitory compound.
Yet another embodiment of the present invention is a therapeutic composition that is capable of protecting an animal from disease caused by a parasitic helminth. Such a therapeutic composition includes one
Klimowski Laura
Tripp Cynthia Ann
Heska Corporation
Minnifield Nita
Sheridan & Ross P.C.
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