Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-12-18
2003-02-04
Martinell, James (Department: 1633)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091320, C436S063000, C436S094000
Reexamination Certificate
active
06514694
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to
Toxoplasma gondii
nucleic acid molecules, proteins encoded by such nucleic acid molecules, antibodies raised against such proteins and methods to identify such nucleic acid molecules, proteins or antibodies. The present invention also includes compositions comprising such nucleic acid molecules, proteins and antibodies, as well as their use for inhibiting oocyst shedding by cats infected with
T. gondii
and for protecting animals from diseases caused by
T. gondii.
BACKGROUND OF THE INVENTION
Various attempts to develop a vaccine to both the asexual systemic stage and the sexual entero-epithelial stage of the Toxoplasma life cycle have been reported over the last thirty years (Hermentin, K. and Aspock, H. (1988),
Zbl. Bakt. Hyg. A,
269:423-436). These attempts can be grouped into the following categories: 1) immunization with whole killed organism, 2) immunization with selected antigens, either purified native or recombinant protein, 3) immunization with attenuated strains, and 4) immunization with irradiated organisms. Little success has been achieved with immunizations using whole killed organism (Frenkel, J. K. and Smith, D. D. (1982),
Journal of Parasitology,
68:744-748). Partial success has been observed with the pure native protein P30 (Bulow, R., and Boothroyd, J. C. (1991),
J. Immunol.
147:3496) and with selected fractions of parasite lysates (Lunden, A. Lovgren, K. Uggla, A., and Araujo, F. G.; (1993)
Infection and Immunity,
61:2639-2643). However, attempts with purified recombinant antigens have not been successful (Lunden, A., Parmley, S. F., Bengtsson, K. L. and Araujo, F. G. (1997)
Parasitology Research,
83:6-9). Studies with irradiated organisms have reported 0-90% protection and are complicated by the uncertainty of truly inactivated irradiated preparations. Effective vaccines have been produced using attenuated strains. Two such mutant strains, ts-4 (Waldeland, H., Pfefferkom, E. R., and Frenkel, J. K. (1983),
Journal of Parasitology,
69:171-175) and S48 (Hartley, W. J. and Marshall, S. C. (1957),
New Zealand Veterinary Journal,
5:119-124), successfully protect animals against the asexual systemic disease. These strains are delivered in the tachyzoite form and do not protect cats from oocyst shedding. Another strain, T-263 (Frenkel, J. K.; Pfefferkom, E. R.; Smith, D. D.; and Fishback, J. L. (1991),
American Journal of Veterinary Research,
52:759-763) is an oocyst minus strain, but was shown to progress through most of the entero-epithelial stages in the cat intestine. Exposure to this strain induces immunity in the cat to oocyst shedding upon subsequent challenge. There remains a need for an effective vaccine for prevention of the diseases caused by infection with
Toxoplasma gondii.
SUMMARY OF THE INVENTION
The present invention relates to novel compositions and methods to inhibit
Toxoplasma gondii
(
T. gondii
) oocyst shedding by cats, thereby preventing the spread of
T. gondii
infection. According to the present invention there are provided isolated immunogenic
T. gondii
proteins and mimetopes thereof,
T. gondii
nucleic acid molecules, including those that encode such proteins; recombinant molecules including such nucleic acid molecules; recombinant viruses including such nucleic acid molecules; recombinant cells including such nucleic acid molecules; and antibodies that selectively bind to such immunogenic
T. gondii
proteins.
The present invention also includes methods to obtain and/or identify proteins, nucleic acid molecules, recombinant molecules, recombinant viruses, recombinant cells, and antibodies of the present invention. Also included are compositions comprising such proteins, nucleic acid molecules, recombinant molecules, recombinant viruses, recombinant cells, and antibodies, as well as use of such compositions to inhibit
T. gondii
oocyst shedding by cats infected with
T. gondii,
or for preventing
T. gondii
infection in an animal.
The present invention further includes the use of the nucleic acid molecules or proteins of the present invention as diagnostic reagents for the detection of
T. gondii
infection. In a preferred embodiment, the present invention includes a novel detection method and kit for detecting
T. gondii
oocysts in the feces of
T. gondii
infected cats.
One embodiment of the present invention is an isolated nucleic acid molecule encoding an immunogenic
T. gondii
protein that can be identified by a method that includes the steps of: a) immunoscreening a
T. gondii
genomic expression library or cDNA expression library with an antiserum, including an antiserum derived from intestinal secretions; and b) identifying a nucleic acid molecule in the library that expresses a protein that selectively binds to an antibody in the antiserum. Antisera to be used for screening include antiserum raised against
T. gondii
oocysts, antiserum raised against
T. gondii
bradyzoites, antiserum raised against
T. gondii
infected cat gut, and antiserum isolated from a cat immune to
T. gondii
infection. Another embodiment is an isolated immunogenic
T. gondii
protein that can be identified by a method that includes the steps of: a) immunoscreening a
T. gondii
genomic expression library or cDNA expression library with such an antiserum; and b) identifying a protein expressed by the library that selectively binds to antibodies in the antiserum. Also included are methods to identify and isolate such nucleic acid molecules and proteins.
The present application also includes an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene that includes a nucleic acid sequence cited in Table 1. Also included in the present invention is an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene that includes a nucleic acid molecule cited in Table 1. Preferred nucleic acid molecules encode immunogenic
T. gondii
proteins. More preferred nucleic acid molecules are those cited in Table 1.
The present invention also relates to recombinant molecules, recombinant viruses and recombinant cells that include an isolated 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 is an isolated immunogenic protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene (i.e., with either the coding strand or the non-coding strand) comprising a nucleic acid sequence cited in Table 1 and/or a nucleic acid molecule cited in Table 1. Note that the nucleic acid molecule hybridizes with the non-coding strand of the gene, that is, with the complement of the coding strand of the gene. A preferred protein is an immunogenic
T. gondii
protein. More preferred proteins are those encoded by nucleic acid molecules cited in Table 1. Also preferred are the proteins cited in Table 1.
The present invention also relates to: mimetopes of immunogenic
T. gondii
proteins and isolated antibodies that selectively bind to immunogenic
T. gondii
proteins or mimetopes thereof. Also included are methods, including recombinant methods, to produce proteins, mimetopes and antibodies of the present invention.
Yet another embodiment of the present invention is a composition to inhibit
T. gondii
oocyst shedding in a cat due to infection with
T. gondii.
Such a composition includes one or more of the following protective compounds: an isolated immunogenic
T. gondii
protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene comprising a nucleic acid sequence cited in Table 1, and specifically with the non-coding-strand of that gene; an isolated antibody that selectively binds to said immunogenic
T. gondii
protein; and an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene comprising a nucleic acid seq
Heska Corporation
Heska Corporation
Martinell James
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