Production of recombinant vaccine in e.coli by enzymatic conjugation

Abstract

Prokaryotic cells proficient to produce glycoconjugates in vivo are provided herein, as well as methods for generating these cell and methods of using these cells to produce glycoconjugates. The compositions of the mentioned glycoconjugates as well as their different uses are also included.

1 - 55 . (canceled) 
     
     
         56 . An engineered Gram-negative bacterium for the production of a polysaccharide, wherein the Gram-negative bacterium comprises a regulatory gene of a capsular polysaccharide gene cluster of a Gram-positive bacterium. 
     
     
         57 . The Gram-negative bacterium of  claim 56 , wherein the Gram-negative bacterium comprises at least 25%, 50%, 75%, 85%, 90%, or at least 95% of the open reading frames of the capsular polysaccharide gene cluster. 
     
     
         58 . The Gram-negative bacterium of  claim 56 , wherein said Gram-negative bacterium is selected from the group consisting of:  Escherichia  species,  E. coli, Shigella  species,  Klebsiella  species,  Salmonella  species,  Yersinia  species,  Neisseria  species,  Vibrio  species and  Pseudomonas  species. 
     
     
         59 . The Gram-negative bacterium of  claim 56 , wherein said regulatory gene is a  Streptococcus pneumoniae  regulatory gene. 
     
     
         60 . The Gram-negative bacterium of  claim 59 , wherein said  Streptococcus pneumoniae  regulatory gene is from  S. pneumoniae  Type 1 or  S. pneumoniae  Type 4. 
     
     
         61 . The Gram-negative bacterium of  claim 56 , wherein said regulatory gene is a  Staphylococcus aureus  regulatory gene. 
     
     
         62 . The Gram-negative bacterium of  claim 56 , wherein said regulatory gene is a  Staphylococcus agalactiae  regulatory gene. 
     
     
         63 . The Gram-negative bacterium of  claim 56 , wherein said regulatory gene is an  Enterococcus faecalis  regulatory gene. 
     
     
         64 . The Gram-negative bacterium of  claim 56 , further comprising an oligosaccharyl transferase. 
     
     
         65 . The Gram-negative bacterium of  claim 64 , wherein said oligosaccharyl transferase is heterologous to the Gram-negative bacterium. 
     
     
         66 . The Gram-negative bacterium of  claim 56 , wherein one or more genes native to the Gram-negative bacterium have been deleted or inactivated. 
     
     
         67 . The Gram-negative bacterium of  claim 66 , wherein said one or more deleted genes comprise the waaL gene or wherein said one or more deleted genes comprise all genes associated with O antigen biosynthesis in the Gram-negative bacterium. 
     
     
         68 . The Gram-negative bacterium of  claim 56 , wherein said regulatory gene is selected from the group consisting of: wzg, wzh, wzd, wze, capA, capB, and capC. 
     
     
         69 . The Gram-negative bacterium of  claim 56 , further comprising a nucleic acid encoding a carrier protein comprising a consensus sequence for glycosylation. 
     
     
         70 . The Gram-negative bacterium of  claim 69 , wherein said nucleic acid encoding a carrier protein comprising a consensus sequence for glycosylation is (i) heterologous to the Gram-negative bacterium or (ii) selected from the group consisting of: detoxified exotoxin A from  P. aeruginosa , CRM197, Diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of  S. aureus , clumping factor A, clumping factor B,  E. coli  FimH,  E. coli  FimHC,  E. coli  heat labile enterotoxin, detoxified variants of  E. coli  heat labile enterotoxin, Cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin,  E. coli  sat protein, the passenger domain of  E. coli  sat protein,  C. jejuni  AcrA,  C. jejuni  natural glycoproteins,  S. pneumoniae  pneumolysin,  S. pneumoniae  NOX,  S. pneumoniae  PspA,  S. pneumoniae  PcpA,  S. pneumoniae  PhtD,  S. pneumoniae  PhtE,  S. pneumoniae  Ply, and  S. pneumoniae  LytB. 
     
     
         71 . The Gram-negative bacterium of  claim 56 , wherein the capsular polysaccharide is:
 (a) a  S. pneumoniae  capsular polysaccharide selected from the group consisting of: CPS1, CPS2, CPS3, CP4, CPS5, CPS6 (A and B), CPS7 (A,B, C), CPS8, CPS9 (A, L,N, V), CPS10 (A,B,C,F), CPS11 (A, B,C,D,F), CPS12(A,B,F), CPS13, CPS14 CPS15(A,B,C,F), CPS16(A,F), CPS17(A,F), CPS18(A,B,C,F), CPS19(A,B,C,F), CPS20,CPS21, CPS22(A,F), CPS23(A,B,F), CPS24(A,B,F), CPS25(A,F), CPS26, CPS27,CPS28(A,F), CPS29, CPS31, CPS32(A,F), CPS33(A,B,C,D,F), CPS34, CPS35(A,B,C,D,F), CPS36, CPS37, CPS38, CPS39, CPS40, CPS41(A,F), CPS42, CPS43, CPS44, CPS45, CPS46, CPS47(A,F), and CPS48;   (b) a  Staphylococcus aureus  capsular polysaccharide selected from the group consisting of: CPS5 and CPS8;   (c) a  Streptococcus agalactiae  (group B, GBS) capsular polysaccharide selected from the group consisting of: CPSIa, CPSIb, CPSII, CPSIII, CPSIV, CPSV, CPSVI, CPSVII, and CPSVIII; or   (d) an  Enterococcus faecalis  capsular polysaccharide selected from the group consisting of: CPSA, CPSB, CPSC, and CPSD.   
     
     
         72 . A recombinant glycoprotein produced by the Gram-negative bacterium of  claim 56 . 
     
     
         73 . A method of producing a recombinant glycoprotein comprising culturing the Gram-negative bacterium of  claim 56  under conditions suitable for the production of proteins. 
     
     
         74 . The method of  claim 73 , further comprising purifying the recombinant glycoprotein. 
     
     
         75 . An engineered  E. coli  cell comprising a  Plesiomonas shigelloides  O antigen gene cluster wherein the wbgW gene of the O antigen gene cluster is inactivated and wherein the  E. coli  cell produces Und-PP-D-FucNAc4N. 
     
     
         76 . An engineered  E. coli  cell comprising a  Shigella sonnei  or  Plesiomonas shigelloides  O17 O antigen gene cluster wherein the wbgW gene of the O antigen gene cluster is inactivated and wherein the  E. coli  cell produces Und-PP-D-FucNAc4N.