Expression system

Abstract

Methods for producing a conjugate of a bacterial toxin, including diphtheria toxin or CRM197, are provided.

1 . A process for producing a conjugate of a bacterial toxin, comprising:
 (1) growing a culture of  Escherichia coli  ( E. coli ) host cells containing a polynucleotide comprising:
 (a) a 3′ toxin sequence encoding a mature bacterial toxin polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO:32, and 
 (b) a 5′ signal sequence encoding a signal peptide, wherein the signal peptide directs transport of said bacterial toxin polypeptide to the bacterial periplasm when expressed in said host cell, and wherein the 5′ signal sequence is not derived from  Corynebacterium diphtheriae  ( C. diphtheriae ); and 
 inducing expression of said polynucleotide such that said bacterial toxin polypeptide is expressed periplasmically; and 
   (2) harvesting cell paste from the culture and purifying said bacterial toxin polypeptide; and   (3) conjugating said purified bacterial toxin to an antigen;   wherein the process of (1) is carried out in a fermentor.   
     
     
         2 . The process of  claim 1  wherein the 3′ toxin sequence encodes a polypeptide selected from the group consisting of (a) a polypeptide having the amino acid sequence of SEQ ID NO: 32, (b) a polypeptide having at least 95% sequence identity to SEQ ID NO: 32, (c) a polypeptide comprising SEQ ID NO:31, and (d) CRM197. 
     
     
         3 . The process of  claim 1  wherein the polynucleotide encodes a polypeptide comprising any one of SEQ ID NOs: 33-45. 
     
     
         4 . The process of  claim 1  wherein the 5′ signal sequence is directly 5′ of the 3′ toxin sequence of the polynucleotide. 
     
     
         5 . The process of  claim 1  wherein the 5′ signal sequence encodes a signal peptide having an amino acid sequence selected from:
 (a) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, and 
 (b) variants of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 containing 1, 2 or 3 point mutations, insertions or deletions, which variants are capable of directing transport of said bacterial toxin polypeptide to the periplasm of said bacterial host cell, and 
 (c) fragments of at least 10 amino acids of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, which fragments are capable of directing transport of said bacterial toxin polypeptide to the periplasm of said bacterial host cell. 
 
     
     
         6 . The process of  claim 1  wherein the 5′ signal sequence comprises a nucleic acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25. 
     
     
         7 . The process of  claim 1  where said antigen of step (3) is a capsular bacterial saccharide from a bacterium selected from the group consisting of  Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis , group B  Streptococcus , group A  Streptococcus, Salmonella , Enterococci, and  Staphylococcus aureus.    
     
     
         8 . The process of  claim 7 , where the activated saccharide is conjugated to said bacterial toxin via a linker. 
     
     
         9 . The process of  claim 7 , where conjugation of said purified bacterial toxin to an antigen comprises a process selected from:
 (a) direct reductive amination,   (b) activation of the saccharide by 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP),   (c) activation of the saccharide by cyanogen bromide,   (d) activation of the saccharide by 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) followed by derivitisation of the activated saccharide with adipic acid dihydrazide (ADH), and   (e) activation of the saccharide by cyanogen bromide followed by derivitisation of the activated saccharide with ADH.   
     
     
         10 . A process for producing a conjugate of a bacterial toxin, comprising:
 (1) growing a culture of  Escherichia coli  ( E. coli ) host cells containing a polynucleotide comprising:
 (a) a 3′ toxin sequence encoding a mature bacterial toxin polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO:32, and 
 (b) a 5′ signal sequence encoding a signal peptide, wherein the signal peptide directs transport of said bacterial toxin polypeptide to the bacterial periplasm when expressed in said host cell, and wherein the 5′ signal sequence is not derived from  C. diphtheriae , and wherein the signal peptide has an amino acid sequence selected from:
 (i) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, and 
 (ii) variants of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 containing 1, 2 or 3 point mutations, insertions or deletions, which variants are capable of directing transport of said bacterial toxin polypeptide to the periplasm of said bacterial host cell, and 
 (iii) fragments of at least 10 amino acids of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, which fragments are capable of directing transport of said bacterial toxin polypeptide to the periplasm of said bacterial host cell; and 
 
 inducing expression of said polynucleotide such that said bacterial toxin polypeptide is expressed periplasmically; and 
   (2) harvesting cell paste from the culture and purifying said bacterial toxin polypeptide; and   (3) conjugating said purified bacterial toxin to an antigen;   wherein the process of (1) is carried out in a fermentor.   
     
     
         11 . The process of  claim 10  wherein the 3′ toxin sequence encodes encodes a polypeptide selected from the group consisting of (a) a polypeptide having the amino acid sequence of SEQ ID NO: 32, (b) a polypeptide having at least 95% sequence identity to SEQ ID NO: 32, (c) a polypeptide comprising SEQ ID NO:31, and (d) CRM197. 
     
     
         12 . The process of  claim 10  wherein the polynucleotide encodes a polypeptide comprising any one of SEQ ID NOs: 33-45. 
     
     
         13 . The process of  claim 10  wherein the 5′ signal sequence is directly 5′ of the 3′ toxin sequence of the polynucleotide. 
     
     
         14 . The process of  claim 10 , wherein the 5′ signal sequence encodes a signal peptide having an amino acid sequence selected from:
 (a) SEQ ID NO: 24, and 
 (b) variants of SEQ ID NO: 24, varying from the corresponding sequence by 1, 2, or 3 point mutations, amino acid insertions, or amino acid deletions, which variants are capable of directing transport of said bacterial toxin polypeptide to the periplasm of said bacterial host cell, and 
 (c) fragments of at least 10 amino acids of SEQ ID NO: 24, which fragments are capable of directing transport of said bacterial toxin polypeptide to the periplasm of said bacterial host cell. 
 
     
     
         15 . The process of  claim 10  wherein the 5′ signal sequence comprises a nucleic acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25. 
     
     
         16 . The process of  claim 10  where said antigen of step (3) is a capsular bacterial saccharide from a bacterium selected from the group consisting of  Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis , group B  Streptococcus , group A  Streptococcus, Salmonella , Enterococci, and  Staphylococcus aureus.    
     
     
         17 . The process of  claim 16 , where the activated saccharide is conjugated to said bacterial toxin via a linker. 
     
     
         18 . The process of  claim 16 , where conjugation of said purified bacterial toxin to an antigen comprises a process selected from:
 (a) direct reductive amination,   (b) activation of the saccharide by 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP),   (c) activation of the saccharide by cyanogen bromide,   (d) activation of the saccharide by 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) followed by derivitisation of the activated saccharide with adipic acid dihydrazide (ADH), and   (e) activation of the saccharide by cyanogen bromide followed by derivitisation of the activated saccharide with ADH.   
     
     
         19 . A process for manufacturing an immunogenic composition comprising the steps of:
 (a) producing a conjugate of a bacterial toxin according to  claim 1 ; and   (b) mixing the said conjugate with a component selected from a pharmaceutically acceptable excipient, a further antigen, and an adjuvant.   
     
     
         20 . A process for manufacturing an immunogenic composition comprising the steps of:
 (a) producing a conjugate of a bacterial toxin according to  claim 16 ; and   (b) mixing the said conjugate with a component selected from a pharmaceutically acceptable excipient, a further antigen, and an adjuvant.