Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-03-25
2003-07-29
Minnifield, Nita (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023400, C536S023500, C536S024320, C435S320100, C435S252300, C435S069100
Reexamination Certificate
active
06600027
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 nucleotide sequences encoding GRA1, GRA2, SAG1, MIC1, and MAG1 proteins from Neospora, which polynucleotide molecules and proteins 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, 9:452-458.
Neospora 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 and beef 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 etal., 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. 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. An experimental N. caninum sheep model that closely resembles naturally acquired Neospora-induced cattle abortion has also been demonstrated. Buxton et al., 1997, J. Comp. Path. 117:1-16.
In
T. gondii
, electron dense granules comprising an excretory-secretory group of antigens are present in the cytoplasm of tachyzoites. These antigens have been designated as GRA proteins. The GRAL protein of
T. gondii
has been reported to have a molecular weight ranging from about 22-27 kDa, and the GRA2 protein of
T. gondii
has been reported to have a molecular weight of about 28 kDa. Sam-Yellowe, 1996, Parasitol. Today 12:308-315. Similar electron dense granules are present in the cytoplasm of
N. caninum
tachyzoites (Bjerkas et al., 1994, Clin. Diag. Lab. Immunol. 1:214-221; Hemphill et aL, 1998, Intl. J. Parasitol. 28:429-438).
T. gondii
cells are also known to comprise a group of major surface antigens that have been designated as SAG. The SAG1 protein of
T. gondii
is reported to have a molecular weight of about 30 kDa. Kasper et al., 1983, J. Immunol. 130:2407-2412. Monoclonal antibodies directed against
T. gondii
SAG1 protein significantly blocked the ability of
T. gondii
tachyzoites to invade bovine kidney cells under tissue culture conditions. Grimwood and Smith, 1996, Intl. J. Parasitol. 26: 169-173. Because
T. gondii
SAG1 appears to play a role in the invasion process, it has been hypothesized that SAG1 may be necessary to support the virulence phenotype. Windeck and Gross, 1996, Parasitol. Res. 82:715-719. Consistent with this hypothesis is the observation that mice immunized with T. gondii SAG1 protein and then challenged with
T. gondii
had reduced toxoplasma cyst formation in their brains than did control mice. Debard et al., 1996, Infect. Immun., 64:2158-2166.
T. gondii
SAG1 may be functionally related to a similar molecule in
N. caninum
designated as NC-p36 described by Hemphill et al., 1997, Parasitol. 115:371-380.
Micronemes are intracelluar organelles located at the apical end of tachyzoites of both
T. gondii
and Neospora, and may play a role in host cell recognition and attachment to the host cell surface during invasion. Formaux etal., 1996, Curr. Top. Microbiol. Immunol. 219:55-58. At least 4 different microneme-associated (MIC) proteins have been identified in
T. gondii.
The MIC1 protein of
T. gondii
is about 60 kDa, binds to the surface of host cells, and has been reported to have partial homology to thrombospondin-related adhesive protein (TRAP) from
Plasmodium falciparum
which binds to human hepatocytes. Robson et al. 1995 EMBO J. 14:3883-3894.
The conversion of parasites from tachyzoites to bradyzoites is critical for chronic infection and persistence of
T. gondii.
A gene expressing an immunodominant, bradyzoite-specific 65 kD antigen, designated as MAG1, has been identified in
T. gondii
. Parmley et a/., 1994, Mol. Biochem. Parasitol. 66:283-296. MAG1 has been reported to be specifically expressed in bradyzoite cysts, but not in the tachyzoite stage. This specificity of expression may indicate the involvement of MAG1 in the conversion between tachyzoite and bradyzoite stages of the life cycle of the parasite. Bohne et al., 1996, Curr. Topics Microbiol. Immunol. 219:81-91.
Identification in Neospora of protein homologs of
T. gondii
GRA1, GRA2, SAG1, MIC1, and MAG1 proteins, and the nucleotide sequence of polynucleotide molecules encoding said Neospora proteins, will serve to facilitate the development of vaccines against neosporosis, as well as diagnostic reagents.
3. SUMMARY OF THE INVENTION
The present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the GRA1 protein from
N. caninum
. In a preferred embodiment, the GRA1 protein has the amino acid sequence of SEQ ID NO:2. In a further preferred embodiment, the isolated GRA1-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO:1 from about nt 205 to about nt 777, the nucleotide sequence of the open reading frame (ORF) of the GRA1 gene, which is presented in SEQ ID NO:3 from about nt 605 to about nt 1304, and the nucleotide sequence of the GRA1-encoding ORF of plasmid pRC77 (ATCC 209685). In a non-limiting embodiment, the isolated GRA1-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO:1 and SEQ ID NO:3. The present invention further provides an isolated polynucleotide molecule having a nucleotide sequence that is homologous to the nucleotide sequence of a GRA1-encoding polynucleotide molecule of the present invention. The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to the GRA1 protein of
N. caninum
. The present invention further provides a polynucleotide molecule consisting of a nucleotide sequence that is a substantial portion of any of the aforementioned GRA1-related polynucleotide molecules.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the GRA2 protein from
N. caninum
. In a preferred embodiment, the GRA2 prot
Brake David A.
Durtschi Becky
Krishnan B. Rajendra
Madura Rebecca
Yoder Christine
Minnifield Nita
Pfizer Inc.
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