Recombinant expression of chlamydia momp antigen

Abstract

The present invention relates to methods for the recombinant expression of chlamydia major outer membrane protein (MOMP) comprising transforming a population of E. coli host cells with an expression vector comprising a nucleic acid molecule that encodes chlamydia MOMP and encodes a leader sequence for targeting the MOMP to the outer membrane of the cell, wherein the nucleic acid molecule is operatively linked to a promoter. The method of the invention allows expression of MOMP in the outer membrane of the cell, which leads to protein folding that is more like native MOMP relative to a MOMP protein that is expressed intracellularly. Also provided by the invention are uses of the recombinant MOMP in pharmaceutical compositions and methods for the treatment and/or prophylaxis of chlamydia infection and/or the effects thereof.

What is claimed is: 
     
         1 . A method for the recombinant expression of  Chlamydia  major outer membrane protein (MOMP) comprising:
 (a) transforming a population of  E. coli  host cells with an expression vector comprising a nucleic acid molecule comprising a sequence of nucleotides that encode a leader sequence for targeting the MOMP to the outer membrane of the cell and a sequence of nucleotides that encode  Chlamydia  MOMP, wherein the nucleic acid molecule is operatively linked to a promoter;   (b) culturing the transformed cells under conditions that permit expression of the nucleic acid molecule and translocation to the outer membrane of the cells to produce a recombinant  Chlamydia  MOMP; and   (c) optionally purifying the MOMP.   
     
     
         2 . The method of  claim 1 , wherein the sequence of nucleotides that encodes the MOMP is codon harmonized or codon optimized for optimal expression in an  E. coli  host cell. 
     
     
         3 . The method of  claim 1  or  2 , wherein the leader sequence is operatively linked to the N-terminus of the MOMP. 
     
     
         4 . The method of any of  claims 1 - 3 , wherein the leader sequence is directly adjacent to the MOMP sequence. 
     
     
         5 . The method of any of  claims 1 - 4 , wherein the nucleic acid molecule further comprises a sequence of nucleotides that encodes a polypeptide attached to the MOMP, wherein the polypeptide is selected from the group consisting of: a linker, an additional antigen, a polypeptide having adjuvant properties, a polypeptide for facilitating purification, a polypeptide for enhancing stability of the MOMP, a carrier protein, and a marker protein. 
     
     
         6 . The method of any of  claims 1 - 5 , wherein the MOMP comprises a sequence of amino acids as set forth in any of SEQ ID NO:23 through SEQ ID NO:31. 
     
     
         7 . The method of any of  claims 1 - 5 , wherein the MOMP comprises a sequence of amino acids that shares at least 90% sequence identity with the MOMP amino acid sequences set forth in any of SEQ ID NO:23 through SEQ ID NO:31. 
     
     
         8 . The method of any of  claims 1 - 7 , wherein the leader sequence comprises the  Shigella flexneri  SopA sequence set forth in SEQ ID NO:8, the  Salmonella enterica  PgtE sequence set forth in SEQ ID NO:9, the  Yersinia pestis  Pla sequence set forth in SEQ ID NO:10, the  E. coli  OmpP sequence set forth in SEQ ID NO:11, the  E. coli  OmpA sequence set forth in SEQ ID NO:12, or the pectate lysase B (PelB) sequence set forth in or SEQ ID NO:13. 
     
     
         9 . The method of any of  claims 1 - 7 , wherein the leader sequence comprises a sequence of amino acids that shares at least 90% sequence identity with the amino acid sequence set forth in any of SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13. 
     
     
         10 . The method of any of  claims 1 - 4 , wherein the nucleic acid molecule comprises a sequence of nucleic acids as set forth in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:21. 
     
     
         11 . The method of  claim 1 , wherein the nucleic acid molecule shares 90% or more homology with the nucleotide sequence set forth in any of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:21. 
     
     
         12 . The method of any of  claims 1 - 11 , wherein the promoter is a low or moderate strength promoter. 
     
     
         13 . The method of any of  claims 1 - 12 , wherein the expression vector is a vector that is associated with a low to moderate transcription or translation rate. 
     
     
         14 . The method of any of  claims 1 - 13 , wherein the method further comprises a step of inducing the transformed host cell with IPTG for from about 4 hours to about 6 hours. 
     
     
         15 . The method of  claim 14 , wherein the induction step is carried out at about 30° C. 
     
     
         16 . The method of  claim 14  or  15 , wherein the cell density (OD590) is allowed to reach about 0.4 to about 0.8 before the induction step is carried out. 
     
     
         17 . A recombinant MOMP produced by the method of any of  claims 1 - 16 . 
     
     
         18 . A pharmaceutical composition comprising an immunologically effective amount of the rMOMP of  claim 17  and a pharmaceutically acceptable carrier. 
     
     
         19 . The pharmaceutical composition of  claim 18 , further comprising one or more additional rMOMP antigens. 
     
     
         20 . The pharmaceutical composition of  claim 18  or  19 , further comprising an adjuvant. 
     
     
         21 . A method of inducing an immune response against  Chlamydia  MOMP in a patient in need thereof, comprising administering a pharmaceutical composition of any of  claims 18 - 20  to the patient. 
     
     
         22 . A nucleic acid molecule comprising a sequence of nucleotides that encodes  Chlamydia  major outer membrane protein (MOMP) comprising a nucleotide sequence that is codon harmonized relative to the wild-type nucleotide sequence encoding the same MOMP. 
     
     
         23 . The nucleic acid molecule of  claim 22 , further comprising a nucleotide sequence that encodes a leader sequence for targeting the MOMP to the outer membrane of a cell, wherein the leader sequence is operatively linked to the N-terminus of the MOMP. 
     
     
         24 . The nucleic acid molecule of  claim 23  which comprises a sequence of nucleotides as set forth in SEQ ID NO:15, SEQ ID NO:18, or SEQ ID NO:21. 
     
     
         25 . Use of (a) the rMOMP of  claim 17  in the manufacture of a medicament for the treatment or prophylaxis of disease associated with  Chlamydia trachomatis  infection. 
     
     
         26 . Use of a pharmaceutical composition of any one of  claims 18 - 20  for the treatment or prophylaxis of disease associated with  Chlamydia trachomatis  infection.