Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Parasitic organism or component thereof or substance...
Reexamination Certificate
1998-12-02
2002-08-20
Navarro, Mark (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Parasitic organism or component thereof or substance...
C424S273100, C424S191100, C424S184100, C424S200100, C536S023100, C536S023700, C435S006120, C435S091400
Reexamination Certificate
active
06436410
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention is in the field of animal health, and is directed to vaccine compositions and diagnostics for disease. More particularly, the present invention relates to polynucleotide molecules comprising a nucleotide sequence encoding the dihydrofolate reductase-thymidylate synthase (DHFR-TS) protein of Neospora, which polynucleotide molecules are useful in the production of vaccines against neosporosis, and as diagnostic reagents.
2. BACKGROUND OF THE INVENTION
Neospora is a pathogenic protozoan parasite of animals that has been recognized as a major cause of abortion, neonatal death, congenital infection, and encephalitic disease in mammals. Dubey and Lindsay, 1996, Vet. Parasitol. 67:1-59; Dubey and Lindsay, 1993, Parasitology Today, 9:452458
. N. caninum
infects dogs, and congenitally infects pups, often leading to paralysis. Tachyzoites of
N. caninum
have been isolated from naturally infected pups. Lindsay and Dubey, 1989, J. Parasitol. 75:163-165. Neospora is a major cause of abortion in dairy cattle. Cases of Neospora-related disease, i.e., neosporosis, have also been reported in goats, sheep and horses.
Although
N. caninum
is superficially similar to the pathogen,
Toxoplasma gondii, N. caninum
and
T. gondii
have been distinguished from each other both antigenically and ultrastructurally. Dubey and Lindsay, 1993, above. In addition, Neospora-like protozoan parasites isolated from the brains of aborted bovine fetuses and continuously cultured in vitro were shown to be antigenically and ultrastructurally distinct from both
T. gondii
and
Hammondia hammondi
, and were most similar to
N. caninum
. Conrad et al., 1993, Parasitology 106:239-249. Furthermore, analysis of nuclear small subunit ribosomal RNA genes revealed no nucleotide differences between strains of Neospora isolated from cattle and dogs, but showed consistent differences between Neospora and
T. gondii
. Marsh et al., 1995, J. Parasitol. 81:530-535.
The etiologic role of a bovine isolate of Neospora in bovine abortion and congenital disease has been confirmed. Barr et al., 1994, J. Vet. Diag. Invest. 6:207-215. A rodent model of central nervous system neosporosis has been developed using inbred BALB/c mice infected with
N. caninum
. Lindsay et al., 1995, J. Parasitol. 81:313-315. In addition, models to study transplacental transmission of
N. caninum
in pregnant outbred and inbred mice have been described by Cole et al., 1995, J. Parasitol. 81:730-732, and by Long et al., 1996, J. Parasitol. 82:608-611, respectively. Furthermore, an experimental
N. caninum
pygmy goat model that closely resembles naturally acquired Neospora-induced cattle abortion has been demonstrated. Lindsay et al., 1995, Am. J. Vet. Res. 56:1176-1180.
In protozoans such as
T. gondii
and Neospora, the essential metabolic enzymes dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are known to reside on the same protein molecule in two distinct enzymatic domains, i.e., the “DHFR domain” and the “TS domain.” In
T. gondii
, the DHFR and TS domains are reported to be separated by a junctional region of ~70 amino acids. Roos, 1993, J. Biol. Chem. 268:6269-6280. This bifunctional protein has served in at least one parasitic species as a target for deletion to create an attenuated strain for use in a live vaccine. Thus, Titus et al., 1995, Proc. Natl. Acad. Sci. 92:10267-10271 describes the targeted deletion by homologous recombination of the DHFR-TS gene from the protozoan parasite Leishmania major to produce dhfr
−
-ts
−
null mutant cells for use in a vaccine against a virulent strain of
L. major.
3. SUMMARY OF THE INVENTION
The present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a Neospora DHFR-TS protein. In a preferred embodiment, the Neospora DHFR-TS protein comprises the amino acid sequence of SEQ ID NO:3 or the amino acid sequence of a DHFR-TS protein as encoded by the DHFR-TS gene as present in phage &lgr;NclDHFRTS (ATCC Accession No. 209512). In a non-limiting embodiment, the isolated polynucleotide molecule comprises the nucleotide sequence of the Neospora DHFR-TS gene. In a preferred embodiment, the isolated polynucleotide molecule comprising the nucleotide sequence of the Neospora DHFR-TS gene comprises the nucleotide sequence of SEQ ID NO:1 from about nt 2405 to about nt 8199, or a nucleotide sequence that is the same as the nucleotide sequence of the DHFR-TS gene as present in phage &lgr;NclDHFRTS (ATCC Accession No. 209512). In a further non-limiting embodiment, the polynucleotide molecule encoding the DHFR-TS protein comprises the nucleotide sequence of SEQ ID NO:2.
The present invention further provides an isolated polynucleotide molecule that is substantially homologous to a polynucleotide molecule comprising the nucleotide sequence of the DHFR-TS gene as shown in SEQ ID NO:1 from about nt 2405 to about nt 8199, or the nucleotide sequence of the DHFR-TS gene as present in phage &lgr;NclDHFRTS (ATCC Accession No. 209512), or the nucleotide sequence of SEQ ID NO:2.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is substantially homologous to a Neospora DHFR-TS protein having the amino acid sequence of SEQ ID NO:3, or the amino acid sequence of a DHFR-TS protein as encoded by the DHFR-TS gene as present in phage &lgr;NclDHFRTS (ATCC Accession No. 209512).
The present invention further provides a polynucleotide molecule consisting of a nucleotide sequence that is a substantial portion of any of the aforementioned polynucleotide molecules. In a preferred embodiment, the polynucleotide molecule consists of a nucleotide sequence that encodes a peptide fragment of any of the aforementioned Neospora DHFR-TS proteins or substantially homologous polypeptides, such as a polypeptide consisting of the DHFR domain or the TS domain of the DHFR-TS protein.
In addition to the nucleotide sequences of any of the aforementioned DHFR-TS-related polynucleotide molecules, polynucleotide molecules of the present invention can further comprise, or alternatively may consist of, nucleotide sequences that naturally flank the DHFR-TS gene in situ in
N. caninum
, such as, e.g., the flanking nucleotide sequences shown in SEQ ID NO:1, or portions thereof.
The present invention further provides compositions and methods for the cloning and expression of a polynucleotide molecule of the present invention, including cloning vectors, expression vectors, and transformed host cells comprising said vectors. In a non-limiting embodiment, the present invention provides a cloning vector comprising a polynucleotide molecule having the nucleotide sequence of the DHFR-TS gene of
N. caninum
strain NC-1, such as, e.g., a &lgr; phage cloning vector designated as &lgr;NclDHFRTS (ATCC Accession No. 209512).
The present invention further provides a partially or substantially purified protein comprising the amino acid sequence of the Neospora DHFR-TS protein. In a non-limiting embodiment, the protein comprises the amino acid sequence of SEQ ID NO:3, or an amino acid sequence of a DHFR-TS protein as encoded by the DHFR-TS gene present in phage &lgr;NclDHFRTS (ATCC Accession No. 209512). The present invention further provides polypeptides that are substantially homologous to a Neospora DHFR-TS protein. The present invention further provides peptide fragments of any of the aforementioned proteins or polypeptides, such as, e.g., a polypeptide consisting of an isolated Neospora DHFR or TS domain.
The present invention further provides antibodies raised against a Neospora DHFR-TS protein or against a peptide fragment of said protein.
The present invention further provides genetic constructs comprising any of the aforementioned polynucleotide molecules such as, e.g., a polynucleotide molecule comprising the nucleotide sequence of the DHFR-TS gene, as shown in SEQ ID NO:1 from about nt 2405 to about nt 8199, or as present in phage &lgr;NclDHFRTS (ATCC Accession N
Durtschi Becky A.
Krishnan B. Rajendra
Yoder S. Christine
Baskar Padmavathi
Navarro Mark
Pfizer Inc.
Scully Scott Murphy & Presser
LandOfFree
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