Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
2000-08-04
2002-03-05
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C424S184100, C424S265100, C435S183000, C514S04400A, C530S300000, C530S350000, C536S023500
Reexamination Certificate
active
06352836
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to Dirofilaria and Brugia thioredoxin peroxidase (TPx-2) 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 disease, elephantiasis, and hydrocele.
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 and Brugia, 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 disease, 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 when the mosquito takes a blood meal. 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 disease is a major problem in dogs, which typically do not develop immunity, even upon infection (i.e., dogs can become reinfected even after being cured by chemotherapy). In addition, heartworm disease is becoming increasingly widespread in other companion animals, such as cats and ferrets.
D. immitis
has also been reported to infect humans.
As such, there remains a need to identify an efficacious composition that protects animals against diseases caused by parasitic helminths, such as heartworm disease. Preferably, such a composition also protects animals from infection by such helminths.
Prior studies have shown that larval stages of parasitic helminths are susceptible to antibody dependent cellular cytotoxicity (ADCC) in vitro. ADCC reactions mainly involve phagocytes such as macrophages, eosinophils and neutrophils. These cells are known to generate reactive oxygen species, such as hydroperoxides and free radicals, which can damage parasites. As a defense, parasites have evolved a number of antioxidant enzymes to overcome the damaging effects of reactive oxygen species generated by the host. While not being bound by theory, such parasitic helminth antioxidant enzymes are attractive targets for vaccines and other chemotherapeutic agents useful in the prevention or treatment of parasitic diseases.
Thioredoxin peroxidases (TPx, previously called thiol-specific antioxidants, or TSA) are newly discovered antioxidant enzymes. Antioxidants are involved in detoxification of reactive oxygen and sulfur species. Recent studies indicate that TPx proteins are involved in reducing hydroperoxides and lipid peroxides with thioredoxin as an intermediate donor. Prior investigators have identified yeast TPx proteins; and have cloned several mammalian TPx genes and a protozoan TPx gene. See, for example, 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. In addition, the nucleic acid and deduced amino acid sequences of an adult
Onchocerca volvulus
TPx have been determined; see GenBank™ Accession No. U31052, and Chandrashekar, et al., Feb. 22, 1996, Abstract 203, “Molecular Helminthology: An Integrated Approach”, Keystone Symposia. A distantly-related larval thioredoxin peroxidase nucleic acid molecule (TPx-1) was recently isolated from
D. immitis
; see U.S. patent application Ser. No. 08/602,010, by Tripp, et al., filed Feb. 15, 1996, and Tripp, et al., Feb. 22, 1996, Abstract 214, “Molecular Helminthology: An Integrated Approach”, Keystone Symposia. patent application Ser. No. 08/602,010, ibid., is incorporated by reference herein in its entirety. Although yeast, human and bovine cortex TPx proteins have been shown to have thioredoxin peroxidase activity (see, for example, Sauri et al, 1995,
Biochem. Biophys. Res. Comm.
208, 964-969; and Watabe et al, 1995,
Biochem. Biophys. Res. Comm.
213, 1010-1016), the other TPx genes or proteins have been designated as such only by nucleic acid sequence homology or by the binding of specific antibodies.
SUMMARY OF THE INVENTION
The present invention relates to a novel product and process to protect animals against parasitic helminth infection (e.g., to prevent and/or treat such an infection). The present invention provides Dirofilaria and Brugia thioredoxin peroxidase type-2 (TPx-2) proteins and mimetopes thereof; Dirofilaria and Brugia TPx-2 nucleic acid molecules, including those that encode such proteins; antibodies raised against such TPx-2 proteins (anti-Dirofilaria and anti-Brugia TPx-2 antibodies); and compounds that inhibit TPx-2 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 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 which includes a Dirofilaria TPx-2 nucleic acid molecule or a Brugia TPx-2 nucleic acid molecule. Such nucleic acid molecules are referred to as TPx-2 nucleic acid molecules. A preferred isolated nucleic acid molecule of this embodiment includes a
Dirofilaria immitis
(
D. immitis
) TPx-2 nucleic acid molecule or a
Brugia malayi
(
B. malayi
) TPx-2 nucleic acid molecule. A
D. immitis
TPx-2 nucleic acid molecule preferably includes nucleic a
Chandrashekar Ramaswamy
Tsuji Naotoshi
Brown Stacy S.
Heska Corporation
Heska Corporation and Colorado State University Research Foundat
Park Hankyel T.
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