Polysaccharide and uses thereof
Provided herein is an E. coli O polysaccharide, O25B. Also provided herein are prokaryotic host cells containing enzymes (e.g., glycosyltransferases) used in O25B production. The host cells provided herein produce O25B bioconjugates, wherein said bioconjugates contain O25B linked to a carrier protein. Further provided herein are compositions, e.g., pharmaceutical compositions, including O25B and/or bioconjugates containing O25B. Such compositions can be used as vaccines against infection with ExPEC, and may further include one or more additional bioconjugates.
1. A recombinant prokaryotic host cell for producing an N-glycosylated carrier protein, comprising:
(a) a nucleotide sequence encoding:
(i) dTDP-Glucose 4,6-dehydratase;
(ii) dTDP-6-Deoxy-D-glucose 3,5-epimerase;
(iii) Glucose-1-phosphate thymidylyltransferase;
(iv) dTDP-4-dehydrorhamnose 3,5-epimerase;
(v) O antigen flippase;
(vi) dTDP-Rha:Glc-Rha(Ac)-GlcNAc-UPP α-1,3-rhamnosyltransferase;
(vii) UDP-Glc:Glc-Rha(Ac)-GlcNAc-UPP α-1,6-glucosyltransferase;
(viii) O antigen polymerase;
(ix) O-acetyl transferase;
(x) UDP-Glc:Rha-GlcNAc-UPP α-1,3-glucosyltransferase; and
(xi) dTDP-Rha: GlcNAc-UPP α-1,3-rhamnosyltransferase;
(b) a nucleotide sequence encoding an oligosaccharyl transferase; and
(c) a nucleotide sequence encoding a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO:15).
2. The host cell of claim 1 , wherein at least one of the waaL gene, gtrA gene, gtrB gene, gtrS gene, or rfb cluster is deleted from or functionally inactivated in the genome of the host cell.
3. The host cell of claim 1 , wherein the carrier protein is selected from the group consisting of detoxified Exotoxin A of P. aeruginosa (EPA), CRM197, maltose binding protein (MBP), 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, Streptococcus pneumoniae Pneumolysin and detoxified variants thereof, C. jejuni AcrA, and C. jejuni natural glycoproteins.
4. The host cell of claim 1 , wherein said host cell is an E. coli host cell.
5. A method of making an N-glycosylated carrier protein, said method comprising:
(a) culturing the host cell of claim 1 under conditions for producing the N-glycosylated carrier protein; and
(b) purifying the N-glycosylated carrier protein.
6. The method of claim 5 , wherein the host cell is E. coli.
7. The method of claim 5 , wherein the carrier protein is detoxified Exotoxin A of P. aeruginosa (EPA).
8. The method of claim 5 , wherein the N-glycosylated carrier protein comprises an E. coli O25B antigen having the structure of Formula O25B′:
wherein n is an integer of 1 to 30.
9. The method of claim 5 , wherein the nucleotide sequence encoding the carrier protein further comprises a nucleotide sequence encoding a signal sequence for targeting the carrier protein into the periplasmic space of the host cell.
10. A method of making an N-glycosylated carrier protein that comprises an E. coli O25B antigen, the method comprising:
(a) culturing a recombinant prokaryotic host cell under conditions for producing the N-glycosylated carrier protein, wherein the prokaryotic host cell comprises:
(i) a nucleotide sequence comprising a rfb gene cluster encoding the E. coli O25B antigen;
(ii) a nucleotide sequence encoding an oligosaccharyl transferase; and
(iii) a nucleotide sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X is any amino acid except Pro (SEQ ID NO:14); and
(b) purifying the N-glycosylated carrier protein that comprises the E. coli O25B antigen.
11. The method of claim 10 , wherein the host cell is E. coli.
12. The method of claim 10 , wherein the consensus sequence comprises Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO:15).
13. The method of claim 10 , wherein the O25B antigen comprises the formula O25B′:
14. The method of claim 10 , wherein the carrier protein is selected from the group consisting of detoxified Exotoxin A of P. aeruginosa (EPA), CRM197, maltose binding protein (MBP), 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, Streptococcus pneumoniae Pneumolysin and detoxified variants thereof, C. jejuni AcrA, and C. jejuni natural glycoproteins.
15. The method of claim 10 , wherein the carrier protein is detoxified Exotoxin A of P. aeruginosa (EPA).
16. The method of claim 10 , wherein the nucleotide sequence encoding the carrier protein further comprises a nucleotide sequence encoding a signal sequence for targeting the carrier protein into the periplasmic space of the host cell.
17. The method of claim 15 , wherein the host cell is E. coli.
18. The method of claim 17 , wherein the consensus sequence comprises Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO:15).
19. The method of claim 18 , wherein the nucleotide sequence encoding the carrier protein further comprises a nucleotide sequence encoding a signal sequence for targeting the carrier protein into the periplasmic space of the host cell.
20. The method of claim 19 , wherein the O25B antigen comprises the formula O25B′: