Cassette for expressing a Toxoplasma gondii P30 protein

Abstract

The present invention provides a reagent for the detection or monitoring of a Toxoplasma gondii infection, which includes as a reactive substance a truncated Toxoplasma gondii P30 protein in which all of the hydrophobic C-terminal region of the native protein starting with the amino acid positioned after the amino acid 299 (SEQ ID NO: 1) has been deleted and all of the region of the native protein having the sequence starting with amino acid 31 and ending with the amino acid 299 (SEQ ID NO: 1) is contained. The present invention further describes methods of screening for anti-Toxoplasma antibodies in a biological sample

1 . An expression cassette which is functional in a cell derived from a nonmammalian eukaryotic organism allowing the expression of a DNA fragment encoding a  Toxoplasma gondii  P30 protein, placed under the control of the elements necessary for its expression, said P30 protein being secreted from said cell derived from a eukaryotic organism and recognized by anti-Toxoplasma antisera.  
     
     
         2 . The expression cassette as claimed in  claim 1 , which is functional in an insect cell.  
     
     
         3 . The expression cassette as claimed in  claim 1 , which is functional in a cell derived from a lower eukaryotic organism.  
     
     
         4 . The expression cassette as claimed in  claim 3 , wherein the cell derived from a lower eukaryotic organism is a yeast or a fungus.  
     
     
         5 . The expression cassette as claimed in  claim 4 , wherein the cell derived from a lower eukaryotic organism is selected from the group consisting of  Saccharomyces cerevisiae, Schizosaccharomyces pombe, Schizosaccharomyces malidevorans, Schizosaccharomyces sloofiae, Schizosaccharomyces octosporus  and  Hasegawaea japonicus.    
     
     
         6 . The expression cassette as claimed in one of  claims 1  to  5 , wherein the DNA fragment encodes a P30 protein having the sequence as shown in the sequence identifier NO: 1 starting with the amino acid +1 and ending with the amino acid +299 or an immunological equivalent of said P30 protein.  
     
     
         7 . The expression cassette as claimed in one of  claims 1  to  5 , wherein the DNA fragment encodes a P30 protein having the sequence as shown in the sequence identifier NO: 1 starting with the amino acid +31 and ending with the amino acid +299 or an immunological equivalent of said P30 protein, said DNA fragment comprising, in addition, a sequence encoding a heterologous secretory signal.  
     
     
         8 . The expression cassette as claimed in  claim 7 , wherein said sequence encoding a heterologous secretory signal is derived from the  Schizosaccharomyces pombe  pho1 gene or from the  Saccharomyces cerevisiae  alpha sex pheromone (Mating Factor α, MFα) gene.  
     
     
         9 . The expression cassette as claimed in one of  claims 1  to  8 , in which the DNA fragment encodes a P30 protein comprising at least one mutation, said mutation being characterized by the presence of an amino acid residue different from the natural residue in position 241 and/or 243 of the sequence as shown in the sequence identifier NO: 1, provided however that said mutation is characterized by a residue other than a threonine in position 243.  
     
     
         10 . The expression cassette as claimed in  claim 9 , wherein the amino acid residue in position 241 is a glutamine residue.  
     
     
         11 . The expression cassette as claimed in one of  claims 1  to  10 , wherein the elements necessary for the expression of said DNA fragment comprise especially a promoter region which is functional in said cell derived from a nonmammalian eukaryotic organism.  
     
     
         12 . The expression cassette as claimed in  claim 11 , wherein the promoter region is selected from the group consisting of the promoter regions derived from the genes PGK of  Saccharomyces cerevisiae,  adh and pho4 of  Schizosaccharomyces pombe  and p12,5K and p39K of baculovirus.  
     
     
         13 . A vector comprising the expression cassette as claimed in one of  claims 1  to  12 .  
     
     
         14 . A cell derived from a nonmammalian eukarotic organism comprising the expression cassette as claimed in one of  claims 1  to  12  or the vector as claimed in  claim 13 .  
     
     
         15 . A unicellular fungus or a yeast according to  claim 14 .  
     
     
         16 . The yeast as claimed in  claim 15 , which is selected from the group consisting of  Saccharomyces cerevisiae, Schizosaccharomyces pombe, Schizosaccharomyces malidevorans, schizosaccharomyces sloofiae, Schizosaccharomyces octosporus  and  Hasegawaea japonicus.    
     
     
         17 . A P30 protein produced by the expression cassette as claimed in one of  claims 1  to  12 , the vector as claimed in  claim 13  or the cell derived from a nonmammalian eukaryotic organism as claimed in one of  claims 14  to  16 .  
     
     
         18 . A process for the preparation of the P30 protein as claimed in  claim 17 , according to which: 
 (i) the cell derived from a nonmammalian eukaryotic organism as claimed in one of  claims 14  to  16  is cultured under appropriate conditions; and (ii) said protein secreted from said cell derived from a nonmammalian eukaryotic organism is recovered.    
     
     
         19 . A reagent for the detection and/or the monitoring of a  Toxoplasma gondii  infection, which comprises, as reactive substance, the protein as claimed in  claim 17 .  
     
     
         20 . A process for the detection of anti-Toxoplasma antibodies in a biological sample, such as a blood sample, from an individual or from an animal likely to be or to have been infected by  Toxoplasma gondii,  which comprises at least the following steps: 
 a mixture is prepared comprising: 
 i) the reagent as claimed in  claim 19  which is or which will be immobilized on a solid support,  
 ii) the sample,  
 iii) a labeled anti-immunoglobulin;  
 the mixture is incubated for a predetermined time;  
 the solid phase is separated from the liquid phase; and  
 the possible presence of anti-Toxoplasma antibodies is revealed by measuring the level of labeling in the solid phase.  
   
     
     
         21 . The process as claimed in  claim 20 , wherein: 
 a mixture is prepared comprising: 
 i) the reagent immobilized on the solid support, and  
 ii) the sample;  
   the mixture is incubated for a predetermined time which allows the formation of an immune complex immobilized on the solid support;    an anti-immunoglobulin labeled under appropriate incubating conditions allowing its reaction with the immobilized immune complex is added;    the solid phase is separated from the liquid phase; and    the possible presence of anti-Toxoplasma antibodies is revealed by measuring the level of labeling in the solid phase.    
     
     
         22 . A monoclonal or polyclonal antibody which is obtained by immunological reaction of a human or animal organism to an immunogenic agent consisting of a protein as defined in  claim 17 .  
     
     
         23 . A reagent for detecting the presence of  Toxoplasma gondii,  which comprises, as reactive substance, the antibody as claimed in  claim 22  which is labeled.  
     
     
         24 . A process for the detection of anti-Toxoplasma antibodies in a biological sample, such as a blood sample, from an individual or an animal likely to be or to have been infected by  Toxoplasma gondii,  which comprises the following steps: 
 a mixture is prepared comprising: 
 i) the first reagent, as claimed in  claim 19 , which is immobilized on a solid support,  
 ii) the sample,  
 iii) the second labeled reagent, as claimed in claim  23 ;  
   the mixture is incubated for a predetermined time;    the solid phase is separated from the liquid phase; and    the possible presence of anti-Toxoplasma antibodies is revealed by measuring the level of labeling in the solid phase.    
     
     
         25 . A process for the detection of anti-Toxoplasma antibodies in a biological sample, such as a blood sample, from an individual or an animal likely to be or to have been infected by  Toxoplasma gondii,  which comprises at least the following steps: 
 a mixture is prepared comprising: 
 i) an anti-immunoglobulin which is or which will be attached onto a solid support;  
 ii) the sample;  
 iii) the reagent as claimed in  claim 19 , which is labeled;  
   the mixture is incubated for a predetermined time;    the liquid phase is separated from the solid phase; and    the possible presence of anti-Toxoplasma antibodies is revealed by measuring the level of labeling in the solid phase.    
     
     
         26 . The process as claimed in  claim 25 , wherein: 
 a mixture is prepared comprising: 
 i) an anti-immunoglobulin attached onto the solid support;  
 ii) the sample;  
 iii) the reagent as claimed in  claim 19 , which is labeled;  
   the mixture is incubated for a predetermined time allowing the formation of an immune complex immobilized on the solid support;    the liquid phase is separated from the solid phase;    the reagent as claimed in  claim 19 , which is labeled, is added; and    the possible presence of anti-Toxoplasma antibodies is revealed by measuring the level of labeling in the solid phase.    
     
     
         27 . A process for the detection of the  Toxoplasma gondii  P30 protein in a biological sample, such as a tissue sample, from an individual or an animal likely to be or to have been infected by  Toxoplasma gondii,  wherein the sample and the reagent as claimed in  claim 23  are brought into contact under appropriate conditions which allow a possible immunological reaction, and the possible presence of an immune complex formed with said labeled reagent is detected by measuring the level of labeling in the biological sample.  
     
     
         28 . A pharmaceutical composition intended for the treatment or for the prevention of a  Toxoplasma gondii  infection in an individual or animals, comprising a therapeutically effective quantity of the expression cassette as claimed in one of  claims 1  to  12 , the vector as claimed in  claim 13 , the cell derived from a nonmammalian eukaryotic organism as claimed in one of  claims 14  to  16  or the P30 protein as claimed in  claim 17  or prepared according to the process as claimed in  claim 18 .  
     
     
         29 . An active immunotherapeutic composition, especially a vaccinal preparation, which comprises, as active ingredient, the protein as claimed in  claim 17 , the active ingredient being optionally conjugated with an appropriate immunological support, and optionally a pharmaceutically acceptable excipient.