Polynucleotide molecules encoding Neospora proteins

Abstract

The present invention provides isolated polynucleotide molecules comprising nucleotide sequences encoding GRA1, GRA2, SAG1, MIC1 and MAG1 proteins from Neospora caninum, as well as recombinant vectors, transformed host cells, and recombinantly-expressed proteins. The present invention further provides a polynucleotide molecule comprising the nucleotide sequence of the bidirectional GRA1/MAG1 promoter of N. caninum. The present invention further provides genetic constructs based on the polynucleotide molecules of the present invention that are useful in preparing modified strains of Neospora cells for use in vaccines against neosporosis.

What is claimed is:  
     
         1 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a Neospora GRA1 protein, said nucleotide sequence selected from the group consisting of the nucleotide sequence of the open reading frame (ORF) of SEQ ID NO: 1 from about nt 205 to about nt 777; the nucleotide sequence of the ORF of SEQ ID NO: 3 from about nt 605 to about nt 1304; the nucleotide sequence of the GRA1-encoding ORF of plasmid pRC77 (ATCC 209685); and a nucleotide sequence that is homologous to any of the aforementioned nucleotide sequences.  
     
     
         2 . The isolated polynucleotide molecule of  claim 1 , comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.  
     
     
         3 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide that is homologous to a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.  
     
     
         4 . An isolated polynucleotide molecule consisting of a nucleotide sequence that is a substantial portion of the nucleotide sequence of the polynucleotide molecule of  claim 1 .  
     
     
         5 . An isolated polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 from about nt 1 to about nt 204; SEQ ID NO: 1 from about nt 778 to about nt 1265; SEQ ID NO: 3 from about nt 1 to about nt 604; SEQ ID NO: 3 from about nt 1305 to about nt 1774; and a substantial portion of any of the aforementioned nucleotide sequences.  
     
     
         6 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a Neospora GRA2 protein, said nucleotide sequence selected from the group consisting of the nucleotide sequence of the ORF of SEQ ID NO: 4 from about nt 25 to about nt 660; the nucleotide sequence of the GRA2-encoding ORF of plasmid pRC5 (ATCC 209686); and a nucleotide sequence that is homologous to any of the aforementioned nucleotide sequences.  
     
     
         7 . The isolated polynucleotide molecule of  claim 6 , comprising the nucleotide sequence of SEQ ID NO: 4.  
     
     
         8 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide that is homologous to a polypeptide comprising the amino acid sequence of SEQ ID NO: 5.  
     
     
         9 . An isolated polynucleotide molecule consisting of a nucleotide sequence that is a substantial portion of the nucleotide sequence of the polynucleotide molecule of  claim 6 .  
     
     
         10 . An isolated polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 from about nt 1 to about nt 24; SEQ ID NO: 4 from about nt 661 to about nt 1031; and a substantial portion of any of the aforementioned nucleotide sequences.  
     
     
         11 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a Neospora SAG1 protein, said nucleotide sequence selected from the group consisting of the nucleotide sequence of the ORF of SEQ ID NO: 6 from about nt 130 to about nt 1089; the nucleotide sequence of the SAG1-encoding ORF of plasmid pRC102 (ATCC 209687); and a nucleotide sequence that is homologous to any of the aforementioned nucleotide sequences.  
     
     
         12 . The isolated polynucleotide molecule of  claim 11 , comprising the nucleotide sequence of SEQ ID NO: 6.  
     
     
         13 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide that is homologous to a polypeptide comprising the amino acid sequence of SEQ ID NO: 7.  
     
     
         14 . An isolated polynucleotide molecule consisting of a nucleotide sequence that is a substantial portion of the nucleotide sequence of the polynucleotide molecule of  claim 11 .  
     
     
         15 . An isolated polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 6 from about nt 1 to about nt 129; SEQ ID NO: 6 from about nt 1090 to about nt 1263; and a substantial portion of any of the aforementioned nucleotide sequences.  
     
     
         16 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a Neospora MIC1 protein, said nucleotide sequence selected from the group consisting of the nucleotide sequence of the ORF of SEQ ID NO: 8 from about nt 138 to about nt 1520; the nucleotide sequence of the ORF of SEQ ID NO: 10; the nucleotide sequence of the MIC1-encoding ORF of plasmid pRC340 (ATCC 209688); and a nucleotide sequence that is homologous to any of the aforementioned nucleotide sequences.  
     
     
         17 . The isolated polynucleotide molecule of  claim 16 , comprising the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 10.  
     
     
         18 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide that is homologous to a polypeptide comprising the amino acid sequence of SEQ ID NO: 9.  
     
     
         19 . An isolated polynucleotide molecule consisting of a nucleotide sequence that is a substantial portion of the nucleotide sequence of the, polynucleotide molecule of  claim 16 .  
     
     
         20 . An isolated polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 8 from about nt 1 to about nt 137; SEQ ID NO: 8 from about nt 1521 to about nt 2069; and a substantial portion of any of the aforementioned nucleotide sequences.  
     
     
         21 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a Neospora MAG1 protein, said nucleotide sequence selected from the group consisting of the nucleotide sequence of the ORF of SEQ ID NO: 11 from about nt 1305 to about nt 2786; the nucleotide sequence of the ORF of SEQ ID NO: 12 from about nt 122 to about nt 1381 the nucleotide sequence of the MAG1-encoding ORF of plasmid bd304 (ATCC 203413); and a nucleotide sequence that is homologous to any of the aforementioned nucleotide sequences.  
     
     
         22 . The isolated polynucleotide molecule of  claim 21 , comprising the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 12.  
     
     
         23 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide that is homologous to a polypeptide comprising the amino acid sequence of SEQ ID NO: 13.  
     
     
         24 . An isolated polynucleotide molecule consisting of a nucleotide sequence that is a substantial portion of the nucleotide sequence of the polynucleotide molecule of  claim 21 .  
     
     
         25 . An isolated polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 11 from about nt 1 to about nt 1304; SEQ ID NO: 11 from about nt 2787 to about nt 4242; SEQ ID NO: 12 from about nt 1 to about nt 121; SEQ ID NO: 12 from about nt 1382 to about nt 1892; and a substantial portion of any of the aforementioned nucleotide sequences.  
     
     
         26 . An isolated polynucleotide molecule, comprising the nucleotide sequence of SEQ ID NO: 11 from about nt 127 to about nt 703.  
     
     
         27 . An oligonucleotide molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS: 14 to 26 and 28 to 34, and the complements thereof.  
     
     
         28 . A recombinant vector, comprising a polynucleotide molecule: (a) comprising a nucleotide sequence encoding a  N. caninum  GRA1, GRA2, SAG1, MIC1 or MAG1 protein; (b) comprising a nucleotide sequence that is homologous to the nucleotide sequence of (a); or (c) consisting of a nucleotide sequence that is a substantial portion of the nucleotide sequence of (a) or(b).  
     
     
         29 . The recombinant vector of  claim 28 , selected from the group consisting of plasmid pRC77 (ATCC 209685); plasmid pRC5 (ATCC 209686); plasmid pRC102 (ATCC 209687); plasmid pRC340 (ATCC 209688), and plasmid bd304 (ATCC 203413).  
     
     
         30 . The recombinant vector of  claim 28 , wherein the polynucleotide molecule further comprises a nucleotide sequence encoding a carrier or fusion partner such that expression of the recombinant vector results in production of a fusion protein comprising the carrier or fusion partner fused to a protein or polypeptide encoded by the recombinant vector of  claim 28 .  
     
     
         31 . A transformed host cell, comprising the recombinant vector of  claim 28 .  
     
     
         32 . A substantially purified or isolated polypeptide selected from the group consisting of: (a) an  N. caninum  GRA1, GRA2, SAG1, MIC1 or MAG1 protein; (b) a polypeptide having an amino acid sequence that is homologous to an  N. caninum  GRA1, GRA2, SAG1, MIC1 or MAG1 protein; (c) a polypeptide consisting of a substantial portion of an  N. caninum  GRA1, GRA2, SAG1, MIC1 or MAG1. protein, or polypeptide which is homologous thereto; (d) a fusion protein comprising the protein or polypeptide of (a), (b) or (c) fused to a carrier or fusion partner; and (e) an analog or derivative of the protein or polypeptide of (a), (b), (c) or (d).  
     
     
         33 . The polypeptide of  claim 32 , wherein the GRA1 protein comprises the amino acid sequence of SEQ ID NO: 2; the GRA2 protein comprises the amino acid sequence of SEQ ID NO: 5; the SAG1 protein comprises the amino acid sequence of SEQ ID NO: 7; the MIC1 protein comprises the amino acid sequence of SEQ ID NO: 9; and the MAG1 protein comprises the amino acid sequence of SEQ ID NO: 13.  
     
     
         34 . An isolated antibody that specifically reacts to a  N. caninum  protein selected from the group consisting of GRA1, GRA2, SAG1, MIC1 and MAG1.  
     
     
         35 . A genetic construct comprising a polynucleotide molecule that can be used to disable a Neospora gene, comprising: (a) a polynucleotide molecule comprising a nucleotide sequence that is otherwise the same as a nucleotide sequence of a GRA1, GRA2, SAG1, MIC1 or MAG1 gene from  N. caninum,  or that is otherwise the same as a nucleotide sequence that is homologous thereto, or a substantial portion of said nucleotide sequence, but which nucleotide sequence further comprises one or more mutations capable of disabling the GRA1, GRA2, SAG1, MIC1 or MAG1 gene from  N. caninum;  or (b) a polynucleotide molecule comprising a nucleotide sequence that naturally flanks in situ the ORF of a Neospora GRA1, GRA2, SAG1, MIC1, or MAG1 gene, or a nucleotide sequence that is homologous to said flanking sequence; such that transformation of a Neospora cell with the genetic construct of (a) or (b) results in disabling of the GRA1, GRA2, SAG 1, MIC1 or MAG1 gene.  
     
     
         36 . The genetic construct of  claim 35 , further comprising a selectable marker.  
     
     
         37 . The genetic construct of  claim 35 , wherein the respective gene is disabled as the result of a homologous recombination event.  
     
     
         38 . A Neospora cell that has been modified by transformation with the genetic construct of  claim 35  such that the GRA1, GRA2, SAG1, MIC1 or MAG1 gene, or a combination of said genes, has been disabled.  
     
     
         39 . A method of preparing modified Neospora cells, comprising transforming Neospora cells with the genetic construct of  claim 35 , and selecting transformed cells that express a mutant phenotype selected from the group consisting of GRA1 − , GRA2 − , SAG − , MIC1 − , and MAG −  as a result of said transformation.  
     
     
         40 . A vaccine against neosporosis, comprising an immunologically effective amount of a component comprising: 
 (a) a polypeptide selected from the group consisting of: (i) an  N. caninum  GRA1, GRA2, SAG1, MIC1 or MAG1 protein; (ii) a polypeptide having an amino acid sequence that is homologous to an  N. caninum  GRA1, GRA2, SAG1, MIC1 or MAG1 protein; (iii) a polypeptide consisting of a substantial portion of an  N. caninum  GRA1, GRA2, SAG1, MIC1 or MAG1 protein, or polypeptide which is homologous thereto; (iv) a fusion protein comprising the protein or polypeptide of (i), (ii) or (iii); and (v) an analog or derivative of the protein or polypeptide of (i), (ii), (iii) or (iv);    (b) a polynucleotide molecule comprising a nucleotide sequence encoding the polypeptide of (a); or    (c) a Neospora cell that has been modified by transformation with the genetic construct of  claim 35  such that the GRA1, GRA2, SAG1, MIC1 or MAG1 gene, or a combination of said genes, in said cell has been disabled;    and a veterinarily acceptable carrier.    
     
     
         41 . The vaccine of  claim 40 , wherein the modified Neospora cells are live cells.  
     
     
         42 . The vaccine of  claim 40 , wherein the modified Neospora cells are inactivated cells.  
     
     
         43 . The vaccine of  claim 40 , further comprising an adjuvant or a cytokine.  
     
     
         44 . The vaccine of  claim 43 , wherein the adjuvant is selected from the group consisting of the RIBI adjuvant system, alum, mineral gel, an oil-in-water emulsion, a water-in-oil emulsion, Block copolymer, QS-21, SAF-M, AMPHIGEN® adjuvant, saponin, Quil A, monophosphoryl lipid A, Avridine lipid-amine adjuvant, SEAM62, and SEAM½.  
     
     
         45 . The vaccine of  claim 40 , which further comprises an immunologically effective amount of a second component that is capable of inducing a protective response against a disease or pathological condition that afflicts a mammal.  
     
     
         46 . The vaccine of  claim 45 , wherein the second component is capable of inducing, or contributing to the induction of, a protective response against a pathogen selected from the group consisting of bovine herpes virus, bovine respiratory syncitial virus, bovine viral diarrhea virus, parainfluenza virus types I, II, or III, Leptospira spp., Campylobacter spp.,  Staphylococcus aureus, Streptococcus agalactiae,  Mycoplasma spp., Klebsiella spp., Salmonella, spp., rotavirus, coronavirus, rabies,  Pasteurella hemolytica, Pasteurella multocida,  Clostridia spp.,  Tetanus toxoid, E. coli,  Cryptosporidium spp., Eimeria spp., and Trichomonas spp.  
     
     
         47 . A method of preparing a vaccine against neosporosis, comprising combining an immunologically effective amount of: 
 (a) a polypeptide selected from the group consisting of: (i) an  N. caninum  GRA1, GRA2, SAG1, MIC1 or MAG1 protein; (ii) a polypeptide having an amino acid sequence that is homologous to an  N. caninum  GRA1, GRA2, SAG1, MIC1 or MAG1 protein; (iii) a polypeptide consisting of a substantial portion of an  N. caninum  GRA1, GRA2, SAG1, MIC1 or MAG1 protein, or polypeptide which is homologous thereto; (iv) a fusion protein comprising the protein or polypeptide of (i), (ii) or (iii); and (v) an analog or derivative of the protein or polypeptide of (i), (ii), (iii) or (iv);    (b) a polynucleotide molecule comprising a nucleotide sequence encoding the polypeptide of (a); or    (c) a Neospora cell that has been modified by transformation with the genetic construct of  claim 35  such that the GRA1, GRA2, SAG1, MIC1 or MAG1 gene, or a combination of said genes, in said cell has been disabled;    with a veterinarily acceptable carrier.    
     
     
         48 . A method of vaccinating a mammal against neosporosis, comprising administering to the mammal the vaccine of  claim 40 .  
     
     
         49 . A kit for vaccinating a mammal against neosporosis, comprising a container comprising an immunologically effective amount of: 
 (a) a polypeptide selected from the group consisting of: (i) an  N. caninum  GRA1, GRA2, SAG1, MIC1 or MAG1 protein; (ii) a polypeptide having an amino acid sequence that is homologous to an  N. caninum  GRA1, GRA2, SAG1, MIC1 or MAG1 protein; (iii) a polypeptide consisting of a substantial portion of an  N. caninum  GRA1, GRA2, SAG1, MIC1 or MAG1 protein, or polypeptide which is homologous thereto; (iv) a fusion protein comprising the protein or polypeptide of (i), (ii) or (iii); or (v) an analog or derivative of the protein or polypeptide of (i), (ii), (iii) or (iv);    (b) a polynucleotide molecule comprising a nucleotide sequence encoding the polypeptide of (a); or    (c) Neospora cells that have been modified by transformation with the genetic construct of  claim 35  such that the GRA1, GRA2, SAG1, MIC1 or MAG1 gene, or a combination of said genes, in said cells has been disabled.    
     
     
         50 . The kit of  claim 49 , further comprising a second container comprising a veterinarily acceptable carrier or diluent.