Modified exotoxin a proteins

Abstract

The present invention relates to the field of modified proteins, immunogenic compositions and vaccines comprising the modified proteins, their manufacture and the use of such compositions in medicine. More particularly, it relates to a modified EPA (Exotoxin A of Pseudomonas aeruginosa) protein. The modified EPA can be used as a carrier protein for other antigens, particularly saccharide antigens or other antigens lacking T cell epitopes.

1 - 15 . (canceled) 
     
     
         16 . A modified EPA (Exotoxin A of  Pseudomonas aeruginosa ) protein having an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, modified in that the amino acid sequence comprises at least one consensus sequence(s) selected from: D/E-X-N-Z-S/T (SEQ ID NO: 2) and K-D/E-X-N-Z-S/T-K (SEQ ID NO: 3), wherein X and Z are independently any amino acid except proline, wherein the at least one consensus sequences have each been added next to or substituted for one or more amino acids, independently selected from: (i) one or more amino acids between amino acid residues 198-218, (ii) one or more amino acids between amino acid residues 264-284, (iii) one or more amino acids between amino acid residues 308-328, and (iv) one or more amino acids between amino acid residues 509-529 of SEQ ID NO: 1 or at equivalent position(s) within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1. 
     
     
         17 . The modified EPA (Exotoxin A of  Pseudomonas aeruginosa ) of  claim 16  having an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, modified in that the amino acid sequence comprises at least two consensus sequence(s) selected from: D/E-X-N-Z-S/T (SEQ ID NO: 2) and K-D/E-X-N-Z-S/T-K (SEQ ID NO: 3), wherein X and Z are independently any amino acid except proline, wherein the at least two consensus sequences have each been added next to or substituted for one or more amino acids, independently selected from: (i) one or more amino acids between amino acid residues 198-218, (ii) one or more amino acids between amino acid residues 264-284, (iii) one or more amino acids between amino acid residues 308-328, (iv) one or more amino acids between amino acid residues 509-529, and (v) one or more amino acids between amino acid residues 230-250, of SEQ ID NO: 1 or at equivalent position(s) within an amino acid 5 sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1. 
     
     
         18 . A modified EPA (Exotoxin A of  Pseudomonas aeruginosa ) protein of  claim 16  having an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, modified in that the amino acid sequence comprises at least three consensus sequence(s) selected from: D/E-X-N-Z-S/T (SEQ ID NO: 2) and K-D/E-X-N-Z-S/T-K (SEQ ID NO: 3), wherein X and Z are independently any amino acid except proline, wherein the at least three consensus sequences have each been added next to or substituted for one or more amino acids, independently selected from: (i) one or more amino acids between amino acid residues 198-218, (ii) one or more amino acids between amino acid residues 264-284, (iii) one or more amino acids between amino acid residues 308-328, (iv) one or more amino acids between amino acid residues 509-529 of SEQ ID NO: 1, and (v) one or more amino acids between amino acid residues 230-250 or at equivalent position(s) within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1. 
     
     
         19 . The modified EPA protein of  claim 16 , wherein the consensus sequence(s) selected from: D/E-X-N-Z-S/T (SEQ ID NO: 2) and K-D/E-X-N-Z-S/T-K (SEQ ID NO: 3) have each been independently substituted for one or more amino acids of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1. 
     
     
         20 . The modified EPA protein of  claim 16 , wherein a further consensus sequence selected from: D/E-X-N-Z-S/T (SEQ ID NO: 2) and J-D/E-X-N-Z-S/T-U (SEQ ID NO: 5), wherein X and Z are independently any amino acid except proline and J and U are independently 1 to 5 naturally occurring amino acid residues, has been added next to, or substituted for, one or more amino acids, at the N-terminus of SEQ ID NO: 1 or at an equivalent position within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1. 
     
     
         21 . The modified EPA protein of  claim 16 , wherein a further consensus sequence selected from: D/E-X-N-Z-S/T (SEQ ID NO: 2) and J-D/E-X-N-Z-S/T-U (SEQ ID NO: 5), wherein X and Z are independently any amino acid except proline and J and U are independently 1 to 5 naturally occurring amino acid residues, has been added next to, or substituted for, one or more amino acids, at the C-terminus of SEQ ID NO: 1 or at an equivalent position within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1. 
     
     
         22 . The modified EPA protein of  claim 16 , wherein at least one consensus sequence selected from: D/E-X-N-Z-S/T (SEQ ID NO: 2) and K-D/E-X-N-Z-S/T-K (SEQ ID NO: 3), wherein X and Z are independently any amino acid except proline, has been added next to, or substituted for: (i) one or more amino acids between amino acid residues 198-218, (ii) one or more amino acids between amino acid residues 308-328, or (iii) one or more amino acids between amino acid residues 509-529 of SEQ ID NO: 1 or at equivalent position(s) within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1. 
     
     
         23 . The modified EPA protein of  claim 16 , wherein the amino acid sequence comprises substitution of leucine 552 to valine (L552V) (or at a position equivalent to L552 of SEQ ID NO:1) and deletion of glutamine 553 (4E553) (or at a position equivalent to E553 of SEQ ID NO:1). 
     
     
         24 . A conjugate comprising a modified EPA protein of  claim 16  covalently linked to an antigen. 
     
     
         25 . The conjugate of  claim 24 , wherein the antigen is a saccharide. 
     
     
         26 . A host cell comprising:
 i) one or more nucleotide sequences comprising polysaccharide synthesis genes, for producing a bacterial polysaccharide antigen or a yeast polysaccharide antigen or a mammalian polysaccharide antigen, integrated into the host cell genome;
 ii) a nucleotide sequence encoding a heterologous oligosaccharyl transferase, within a plasmid; 
 iii) a nucleotide sequence that encodes a modified EPA protein according to  claim 16 , within a plasmid. 
   
     
     
         27 . A process for producing a bioconjugate that comprises a modified EPA protein linked to a polysaccharide, said process comprising (i) culturing the host cell of  claim 26  under conditions suitable for the production of glycoproteins and (ii) isolating the bioconjugate, isolating the bioconjugate from a periplasmic extract from the host cell. 
     
     
         28 . An immunogenic composition comprising a conjugate of  claim 24 , and at least one of a pharmaceutically acceptable excipient or carrier. 
     
     
         29 . A vaccine comprising the immunogenic composition of  claim 28  and an adjuvant. 
     
     
         30 . A method of inducing an immune response in a subject, the method comprising administering a therapeutically or prophylactically effective amount of the conjugate of  claim 24  to a subject in need thereof. 
     
     
         31 . A method of inducing an immune response in a subject, the method comprising administering a therapeutically or prophylactically effective amount of the immunogenic composition of  claim 28 , to a subject in need thereof.