Polysaccharide and uses thereof

1. A composition comprising a bioconjugate of an E. coli O25B antigen covalently coupled to a carrier protein, wherein the E. coli O25B antigen comprises the structure of Formula O25B′:

wherein n is an integer of 1 to 30.

2. The composition of claim 1 , wherein the E. coli 025B antigen is covalently bound to an Asn residue in the carrier protein.

3. The composition 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 composition of claim 3 , wherein the carrier protein is detoxified EPA.

5. The composition of claim 4 , wherein the O25B antigen is covalently bound to an Asn residue in the detoxified EPA, and the Asn residue of the detoxified EPA is positioned in the 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).

6. A method for (i) vaccinating a subject against extra-intestinal pathogenic Escherichia coli , (ii) inducing an immune response in a subject against extra-intestinal pathogenic Escherichia coli , or (iii) inducing the production of opsonophagocytic antibodies in a subject that are specific to extra-intestinal pathogenic Escherichia coli , wherein the method comprises administering to the subject an effective amount of the composition of claim 1 .

7. The method of claim 6 , wherein the subject is at risk of developing a urinary tract infection, bacteremia, or sepsis.

8. A method for (i) vaccinating a subject against extra-intestinal pathogenic Escherichia coli , (ii) inducing an immune response in a subject against extra-intestinal pathogenic Escherichia coli , or (iii) inducing the production of opsonophagocytic antibodies in a subject that are specific to extra-intestinal pathogenic Escherichia coli , wherein the method comprises administering to the subject an effective amount of the composition of claim 5 .

9. The method of claim 8 , wherein the subject is at risk of developing a urinary tract infection, bacteremia, or sepsis.

10. A recombinant prokaryotic host cell for producing a bioconjugate of an E. coli O25B antigen covalently coupled to a carrier protein, comprising:

(a) a nucleotide sequence comprising an rfb gene cluster encoding the E. coli O25B antigen, wherein the rfb gene cluster encodes:

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 β-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 the carrier protein, wherein the carrier protein comprises a consensus sequence Asn-X-Ser(Thr), and X is any natural amino acid except Pro (SEQ ID NO: 14).

11. The recombinant prokaryotic host cell of claim 10 , wherein the nucleotide sequence of (c) encodes the 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).

12. The recombinant prokaryotic host cell of claim 10 , wherein the nucleotide sequence of (a) comprises genes of rmlB, rmlD, rmlA, rmlC, wxz, wekA, wekB, wzy, wbbJ, wbbK, and wbbL.

13. The recombinant prokaryotic host cell of claim 10 , wherein the nucleotide sequence of (a) comprises the rfb gene cluster having the nucleotide sequence of SEQ ID NO: 12.

14. The recombinant prokaryotic host cell of claim 10 , wherein at least one of the waaL gene, gtrA gene, gtrB gene, gtrS gene, or rib cluster is deleted from or functionally inactivated in the genome of the prokaryotic host cell.

15. The recombinant prokaryotic host cell 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.

16. The recombinant prokaryotic host cell of claim 10 , wherein said host cell is an E. coli host cell.

17. A method of making the bioconjugate of the E. coli O25B antigen covalently coupled to the carrier protein, the method comprising:

(a) culturing the recombinant prokaryotic host cell of claim 10 under conditions for production of the bioconjugate; and

(b) purifying the bioconjugate.

18. A bioconjugate of an E. coli O25B antigen covalently coupled to a carrier protein,

wherein the bioconjugate is produced by a method comprising:

(a) culturing a recombinant prokaryotic host cell under conditions for production of the bioconjugate, the recombinant prokaryotic host cell comprising:

i. a nucleotide sequence comprising an rib gene cluster encoding the E. coli O25B antigen, wherein the rib gene cluster encodes:

a. dTDP-Glucose 4,6-dehydratase;

b. dTDP-6-Deoxy-D-glucose 3,5-epimerase;

c. Glucose-1-phosphate thymidylyltransferase;

d. dTDP-4-dehydrorhamnose 3,5-epimerase;

e. O antigen flippase;

f. dTDP-Rha:Glc-Rha(Ac)-GlcNAc-UPP α-1,3-rhamnosyltransferase;

g. UDP-Glc:Glc-Rha(Ac)-GlcNAc-UPP β-1,6-glucosyltransferase;

h. O antigen polymerase;

i. O-acetyl transferase;

j. UDP-Glc:Rha-GlcNAc-UPP α-1,3-glucosyltransferase; and

k. dTDP-Rha: GlcNAc-UPP α-1,3-rhamnosyltransferase;

ii. a nucleotide sequence encoding an oligosaccharyl transferase; and

iii. a nucleotide sequence encoding the carrier protein, wherein the carrier protein comprises a consensus sequence Asn-X-Ser(Thr), and X is any natural amino acid except Pro (SEQ ID NO: 14); and

(b) purifying the bioconjugate.

19. A composition comprising the bioconjugate of claim 18 .

20. A method for (i) vaccinating a subject against extra-intestinal pathogenic Escherichia coli , (ii) inducing an immune response in a subject against extra-intestinal pathogenic Escherichia coli , or (iii) inducing the production of opsonophagocytic antibodies in a subject that are specific to extra-intestinal pathogenic Escherichia coli , wherein the method comprises administering to the subject an effective amount of the composition of claim 19 .