Biosynthetic system that produces immunogenic polysaccharides in prokaryotic cells
The invention is directed to bioconjugate vaccines comprising N-glycosylated proteins. Further, the present invention is directed to a recombinant prokaryotic biosynthetic system comprising nucleic acids encoding an epimerase that synthesizes an oligo- or polysaccharide having N-acetylgalactosamine at the reducing terminus. The invention is further directed to N-glycosylated proteins containing an oligo- or polysaccharide having N-acetylgalactosamine at the reducing terminus and an expression system and methods for producing such N-glycosylated proteins.
1. A composition comprising a bioconjugate, said bioconjugate comprising a carrier protein linked to an oligosaccharide or polysaccharide, wherein said oligosaccharide or polysaccharide comprises N-acetylgalactosamine at the reducing terminus, and wherein said carrier protein comprises the amino acid sequence D/E-X-N-Z-S/T (SEQ ID NO:31), wherein X and Z can be any natural amino acid except proline; and
a recombinant prokaryotic host cell that comprises
(a) a heterologous nucleic acid encoding an epimerase that synthesizes N-acetylgalactosamine on undecaprenyl pyrophosphate, wherein said epimerase comprises the amino acid sequence of SEQ ID NO. 2;
(b) a heterologous nucleic acid encoding an oligosaccharyl transferase; and
(c) a heterologous nucleic acid encoding said carrier protein.
2. The composition of claim 1 , wherein said carrier protein is linked to an oligosaccharide.
3. The composition of claim 1 , wherein said carrier protein is linked to a polysaccharide.
4. The composition of claim 1 , wherein said oligosaccharide or polysaccharide is from a Gram-negative bacterium.
5. The composition of claim 1 , wherein said oligosaccharide or polysaccharide is from E. coli.
6. The composition of claim 5 , wherein said oligosaccharide or polysaccharide is from E. coli 0157.
7. The composition of claim 1 , wherein said oligosaccharide or polysaccharide is from Shigella flexneri.
8. The composition of claim 7 , wherein said oligosaccharide or polysaccharide is from Shigella flexneri 6.
9. The composition of claim 1 , wherein said oligosaccharide or polysaccharide comprises a structure:
10. The composition of claim 1 , wherein said oligosaccharide or polysaccharide comprises a structure, α-D-PerNAc-α-L-Fuc-β-D-Glc-α-D-GalNAc.
11. The composition of claim 1 , wherein said carrier protein has been modified to comprise the amino acid sequence D/E-X-N-Z-S/T (SEQ ID NO:31), wherein X and Z can be any natural amino acid except proline.
12. The composition of claim 4 , wherein said carrier protein has been modified to comprise the amino acid sequence D/E-X-N-Z-S/T (SEQ ID NO:31), wherein X and Z can be any natural amino acid except proline.
13. The composition of claim 5 , wherein said carrier protein has been modified to comprise the amino acid sequence D/E-X-N-Z-S/T (SEQ ID NO:31), wherein X and Z can be any natural amino acid except proline.
14. The composition of claim 6 , wherein said carrier protein has been modified to comprise the amino acid sequence D/E-X-N-Z-S/T (SEQ ID NO:31), wherein X and Z can be any natural amino acid except proline.
15. The composition of claim 7 , wherein said carrier protein has been modified to comprise the amino acid sequence D/E-X-N-Z-S/T (SEQ ID NO:31), wherein X and Z can be any natural amino acid except proline.
16. The composition of claim 8 , wherein said carrier protein has been modified to comprise the amino acid sequence D/E-X-N-Z-S/T (SEQ ID NO:31), wherein X and Z can be any natural amino acid except proline.
17. The composition of claim 1 , wherein said carrier protein is P. aeruginosa exoprotein that has been modified to comprise the amino acid sequence D/E-X-N-ZSIT, wherein X and Z can be any natural amino acid except proline.
18. The composition of claim 1 , wherein said carrier protein is the Campylobacter AcrA protein.
19. The composition of claim 1 , wherein said nucleic acid encoding an oligosaccharyl transferase encodes the oligosaccharyl transferase from Campylobacter jejuni.
20. The composition of claim 1 , wherein said nucleic acid encoding an oligosaccharyl transferase is heterologous to said host cell.