Chimeric protein comprising non-toxic pseudomonas exotoxin and type IV pilin sequences

Abstract

The invention provides chimeric proteins comprising a non-toxic Pseudomonas exotoxin A sequence and a Type IV pilin loop sequence, wherein the Type IV loop sequence is inserted within the non-toxic Pseudomonas exotoxin A. The invention also provides polynucleotides encoding the chimeric proteins, and compositions comprising the polynucleotides or the chimeric proteins. The invention also provides methods for using the chimeric proteins, polynucleotides and compositions of the invention.

1 . A chimeric protein comprising: a non-toxic  Pseudomonas  exotoxin A sequence and a Type IV pilin loop sequence, the Type IV pilin loop sequence being located within the non-toxic  Pseudomonas  exotoxin A sequence, wherein the chimeric protein is capable of reducing adherence of a microorganism expressing the Type IV pilin loop sequence to epithelial cells, and further wherein the chimeric protein, when introduced into a host, is capable of generating polyclonal antisera that reduce adherence of the microorganism expressing the Type IV pilin loop sequence to the epithelial cells.  
     
     
         2 . The chimeric protein of  claim 1 , wherein the chimeric protein, when introduced into the host, is also capable of generating polyclonal antisera that neutralize cytotoxicity of  Pseudomonas  exotoxin A.  
     
     
         3 . The chimeric protein of  claim 1 , wherein the non-toxic  Pseudomonas  exotoxin A sequence comprises: (a) a translocation domain sufficient to effect translocation of the chimeric protein to a cell cytosol; and (b) an endoplasmic reticulum retention domain that functions to translocate the chimeric protein from endosome to endoplasmic reticulum.  
     
     
         4 . The chimeric protein of  claim 3 , wherein the chimeric protein further comprises a cell recognition domain that functions as a ligand for a cell surface receptor and that mediates binding of the chimeric protein to a cell.  
     
     
         5 . The chimeric protein of  claim 4 , wherein the Type IV pilin loop sequence is located between the translocation domain and the endoplasmic reticulum retention domain.  
     
     
         6 . The chimeric protein of  claim 5 , wherein the Type IV pilin loop sequence comprises cysteine residues at both the N- and C-termini of the Type IV pilin loop sequence.  
     
     
         7 . The chimeric protein of  claim 5 , wherein the Type IV pilin loop sequence is from bacteria or yeast.  
     
     
         8 . The chimeric protein of  claim 7 , wherein the Type IV pilin loop sequence is from  Pseudomonas aeruginosa, Neisseria meningtidis, Neisseria gonorrhoeae, Vibro cholera, Pasteurella multocidam , or  Candida.    
     
     
         9 . The chimeric protein of  claim 8 , wherein the Type IV pilin loop sequence is from  Pseudomonas aeruginosa.    
     
     
         10 . The chimeric protein of  claim 9 , wherein the Type IV pilin loop sequence is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.  
     
     
         11 . The chimeric protein of  claim 5 , wherein the translocation domain comprises amino acids 280 to 364 of domain II of  Pseudomonas  exotoxin A.  
     
     
         12 . The chimeric protein of  claim 5 , wherein the translocation domain is domain II of  Pseudomonas  exotoxin A.  
     
     
         13 . The chimeric protein of  claim 5 , wherein the endoplasmic reticulum retention domain is domain III of  Pseudomonas  exotoxin A except that amino acid Glu at position of 553 is deleted.  
     
     
         14 . The chimeric protein of  claim 1 , wherein the chimeric protein comprises more than one Type IV pilin loop sequence.  
     
     
         15 . The chimeric protein of  claim 5 , wherein the cell recognition domain is domain Ia of  Pseudomonas  exotoxin A.  
     
     
         16 . The chimeric protein of  claim 5 , wherein the cell recognition domain binds to α2-macroglobulin receptor, epidermal growth factor receptor, transferrin receptor, interleukin-2 receptor, interleukin-6 receptor, interleukin-8 receptor, Fc receptor, poly-IgG receptor, asialoglycoprotein receptor, CD3, CD4, CD8, chemokine receptor, CD25, CD11B, CD11C, CD80, CD86, TNFalpha receptor, TOLL receptor, M-CSF receptor, GM-CSF receptor, scavenger receptor, VEGF receptor, or cytokine receptor.  
     
     
         17 . A chimeric protein comprising: 
 (a) a non-toxic  Pseudomonas  exotoxin A sequence comprising domain Ia, domain II, and domain III; and    (b) a Type IV pilin loop sequence, wherein the Type IV pilin loop sequence is located between domain II and domain III of the non-toxic  Pseudomonas  exotoxin A sequence.    
     
     
         18 . The chimeric protein of  claim 17 , wherein the non-toxic  Pseudomonas  exotoxin A sequence has the amino acid sequence of SEQ ID NO:2 with ΔE553.  
     
     
         19 . The chimeric protein of  claim 17 , wherein the Type IV pilin loop sequence is from  Pseudomonas aeruginosa, Neisseria meningtidis, Neisseria gonorrhoeae, Vibro cholera, Pasteurella multocidam , or  Candida.    
     
     
         20 . The chimeric protein of  claim 17 , wherein the Type IV pilin loop sequence is from  Pseudomonas aeruginosa.    
     
     
         21 . A polynucleotide encoding a chimeric protein, the chimeric protein comprising: a non-toxic  Pseudomonas  exotoxin A sequence and a Type IV pilin loop sequence, the Type IV pilin loop sequence being located within the non-toxic  Pseudomonas  exotoxin A sequence, wherein the chimeric protein is capable of reducing adherence of a microorganism expressing the Type IV pilin loop sequence to epithelial cells, and further wherein the chimeric protein, when introduced into a host, is capable of generating polyclonal antisera that prevent adherence of the microorganism expressing the Type IV pilin loop sequence to the epithelial cells.  
     
     
         22 . The polynucleotide of  claim 21 , wherein the chimeric protein, when introduced into the host, is also capable of generating polyclonal antisera that neutralize cytotoxicity of  Pseudomonas  exotoxin A.  
     
     
         23 . The polynucleotide of  claim 21 , wherein the non-toxic  Pseudomonas  exotoxin A sequence comprises: (a) a translocation domain sufficient to effect translocation of the chimeric protein to a cell cytosol; and (b) an endoplasmic reticulum retention domain that functions to translocate the chimeric protein from endosome to endoplasmic reticulum.  
     
     
         24 . The polynucleotide of  claim 23 , wherein the chimeric protein further comprises a cell recognition domain that functions as a ligand for a cell surface receptor and that mediates binding of the chimeric protein to a cell.  
     
     
         25 . The polynucleotide of  claim 24 , wherein the Type IV pilin loop sequence is located between the translocation domain and the endoplasmic reticulum retention domain.  
     
     
         26 . The polynucleotide of  claim 25 , wherein the Type IV pilin loop sequence comprises cysteine residues at both the N- and C-termini of the Type IV pilin loop sequence.  
     
     
         27 . The polynucleotide of  claim 25 , wherein the Type IV pilin loop sequence is from bacteria or yeast.  
     
     
         28 . The polynucleotide of  claim 27 , wherein the Type IV pilin loop sequence is from  Pseudomonas aeruginosa, Neisseria meningtidis, Neisseria gonorrhoeae, Vibro cholera, Pasteurella multocidam , or  Candida.    
     
     
         29 . The polynucleotide of  claim 28 , wherein the Type IV pilin loop sequence is from  Pseudomonas aeruginosa.    
     
     
         30 . The polynucleotide of  claim 29 , wherein the Type IV pilin loop sequence is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.  
     
     
         31 . The polynucleotide of  claim 25 , wherein the translocation domain comprises amino acids 280 to 364 of domain II of  Pseudomonas  exotoxin A.  
     
     
         32 . The polynucleotide of  claim 25 , wherein the translocation domain is domain II of  Pseudomonas  exotoxin A.  
     
     
         33 . The polynucleotide of  claim 25 , wherein the endoplasmic reticulum retention domain is domain III of  Pseudomonas  exotoxin A except that amino acid Glu at position of 553 is deleted.  
     
     
         34 . The polynucleotide of  claim 25 , wherein the cell recognition domain is domain Ia of  Pseudomonas  exotoxin A.  
     
     
         35 . The polynucleotide of  claim 25 , wherein the cell recognition domain binds to α2-macroglobulin receptor, epidermal growth factor receptor, transferring receptor, Fc receptor, poly-IgG receptor, asialoglycoprotein receptor, CD3, CD4, CD8, chemokine receptor, CD25, CD11B, CD11C, CD80, CD86, TNFalpha receptor, TOLL receptor, M-CSF receptor, GM-CSF receptor, scavenger receptor, VEGF receptor, or cytokine receptor.  
     
     
         36 . A polynucleotide encoding a chimeric protein, the chimeric protein comprising: 
 (a) a non-toxic  Pseudomonas  exotoxin A sequence comprising domain Ia, domain II, and domain III; and    (b) a Type IV pilin loop sequence, wherein the Type IV pilin loop sequence is located between domain II and domain III of the non-toxic  Pseudomonas  exotoxin A sequence.    
     
     
         37 . The polynucleotide of  claim 36 , wherein the non-toxic  Pseudomonas  exotoxin A sequence has the amino acid sequence of SEQ ID NO:2 with ΔE553.  
     
     
         38 . The polynucleotide of  claim 36 , wherein the Type IV pilin loop sequence is from  Pseudomonas aeruginosa, Neisseria meningtidis, Neisseria gonorrhoeae, Vibro cholera, Pasteurella multocidam , or  Candida.    
     
     
         39 . The polynucleotide of  claim 36 , wherein the Type IV pilin loop sequence is from  Pseudomonas aeruginosa.    
     
     
         40 . An expression cassette comprising the polynucleotide of  claim 21 .  
     
     
         41 . A cell comprising the expression cassette of  claim 40 .  
     
     
         42 . A composition comprising a chimeric protein, the chimeric protein comprising: a non-toxic  Pseudomonas  exotoxin A sequence and a Type IV pilin loop sequence, the Type IV pilin loop sequence being located within the non-toxic  Pseudomonas  exotoxin A sequence, wherein the chimeric protein is capable of reducing adherence of a microorganism expressing the Type IV pilin loop sequence to epithelial cells, and further wherein the chimeric protein, when introduced into a host, is capable of generating polyclonal antisera that prevent adherence of the microorganism expressing the Type IV pilin loop sequence to the epithelial cells.  
     
     
         43 . The composition of  claim 42 , wherein the chimeric protein, when introduced into the host, is also capable of generating polyclonal antisera that neutralize cytotoxicity of  Pseudomonas  exotoxin A.  
     
     
         44 . The composition of  claim 42 , wherein the composition further comprises a pharmacologically acceptable carrier.  
     
     
         45 . The composition of  claim 42 , wherein the composition is formulated as a nasal or oral spray.  
     
     
         46 . The composition of  claim 42 , wherein the non-toxic  Pseudomonas  exotoxin A sequence comprises: (a) a translocation domain sufficient to effect translocation of the chimeric protein to a cell cytosol; and (b) an endoplasmic reticulum retention domain that functions to translocate the chimeric protein from endosome to endoplasmic reticulum.  
     
     
         47 . The composition of  claim 46 , wherein the chimeric protein further comprises a cell recognition domain that functions as a ligand for a cell surface receptor and that mediates binding of the chimeric protein to a cell.  
     
     
         48 . The composition of  claim 47 , wherein the Type IV pilin loop sequence is from  Pseudomonas aeruginosa.    
     
     
         49 . A method for eliciting an immune response in a host, the method comprising the step of administering to the host an immunologically effective amount of a composition comprising a chimeric protein comprising: a non-toxic  Pseudomonas  exotoxin A sequence and a Type IV pilin loop sequence, the Type IV pilin loop sequence being located within the non-toxic  Pseudomonas  exotoxin A sequence, wherein the chimeric protein is capable of reducing adherence of a microorganism expressing the Type IV pilin loop sequence to epithelial cells, and further wherein the chimeric protein, when introduced into the host, is capable of generating polyclonal antisera that prevent adherence of the microorganism expressing the Type IV pilin loop sequence to the epithelial cells.  
     
     
         50 . The method of  claim 49 , wherein the chimeric protein, when introduced into the host, is capable of generating polyclonal antisera that neutralize cytotoxicity of  Pseudomonas  exotoxin A.  
     
     
         51 . The method of  claim 49 , wherein the host is a human.  
     
     
         52 . The method of  claim 49 , wherein the non-toxic  Pseudomonas  exotoxin A sequence comprises: (a) a translocation domain sufficient to effect translocation of the chimeric protein to a cell cytosol; and (b) an endoplasmic reticulum retention domain that functions to translocate the chimeric protein from endosome to endoplasmic reticulum.  
     
     
         53 . The method of  claim 52 , wherein the chimeric protein further comprises a cell recognition domain that functions as a ligand for a cell surface receptor and that mediates binding of the chimeric protein to a cell.  
     
     
         54 . The method of  claim 53 , wherein the Type IV pilin loop sequence is from  Pseudomonas aeruginosa.    
     
     
         55 . A method of eliciting an immune response in a host, the method comprising the step of administering to the host an immunologically effective amount of an expression cassette comprising a polynucleotide encoding a chimeric protein comprising: a non-toxic  Pseudomonas  exotoxin A sequence and a Type IV pilin loop sequence, the Type IV pilin loop sequence being located within the non-toxic  Pseudomonas  exotoxin A, wherein the chimeric protein is capable of reducing adherence of a microorganism expressing the Type IV pilin loop sequence to epithelial cells, and further wherein the chimeric protein, when introduced into the host, is capable of generating polyclonal antisera that reduce adherence of the microorganism expressing the Type IV pilin loop sequence to the epithelial cells.  
     
     
         56 . The method of  claim 55 , wherein the chimeric protein, when introduced into the host, is capable of generating polyclonal antisera that neutralize cytotoxicity of  Pseudomonas  exotoxin A.  
     
     
         57 . The method of  claim 55 , wherein the host is a human.  
     
     
         58 . The method of  claim 55 , wherein the non-toxic  Pseudomonas  exotoxin A sequence comprises: (a) a translocation domain sufficient to effect translocation of the chimeric protein to a cell cytosol; and (b) an endoplasmic reticulum retention domain that functions to translocate the chimeric protein from endosome to endoplasmic reticulum.  
     
     
         59 . The method of  claim 58 , wherein the chimeric protein further comprises a cell recognition domain that functions as a ligand for a cell surface receptor and that mediates binding of the chimeric protein to a cell.  
     
     
         60 . The method of  claim 59 , wherein the Type IV pilin loop sequence is from  Pseudomonas aeruginosa.    
     
     
         61 . A method of generating antibodies specific for a Type IV pilin loop sequence, comprising introducing into a host a composition comprising a chimeric protein comprising a non-toxic  Pseudomonas  exotoxin A sequence and a Type IV pilin loop sequence, the Type IV pilin loop sequence being located within the non-toxic  Pseudomonas  exotoxin A, wherein the chimeric protein is capable of reducing adherence of a microorganism expressing the Type IV pilin loop sequence to epithelial cells, and further wherein the chimeric protein, when introduced into the host, is capable of generating polyclonal antisera that reduce adherence of the microorganism expressing the Type IV pilin loop sequence to epithelial cells.  
     
     
         62 . The method of  claim 61 , wherein the chimeric protein, when introduced into the host, is capable of generating polyclonal antisera that neutralize cytotoxicity of  Pseudomonas  exotoxin A.  
     
     
         63 . The method of  claim 61 , wherein the host is a human.  
     
     
         64 . The method of  claim 61 , wherein the non-toxic  Pseudomonas  exotoxin A sequence comprises: (a) a translocation domain sufficient to effect translocation of the chimeric protein to a cell cytosol; and (b) an endoplasmic reticulum retention domain that functions to translocate the chimeric protein from endosome to endoplasmic reticulum.  
     
     
         65 . The method of  claim 64 , wherein the chimeric protein further comprises a cell recognition domain that functions as a ligand for a cell surface receptor and that mediates binding of the chimeric protein to a cell.  
     
     
         66 . The method of  claim 65 , wherein the Type IV pilin loop sequence is from  Pseudomonas aeruginosa.