Methods of producing bioconjugates of
Methods of producing bioconjugates of O-antigen polysaccharides covalently linked to a carrier protein using recombinant host cells are provided. The recombinant host cells used in the methods described herein encode a particular oligosaccharyl transferase enzyme depending on the O-antigen polysaccharide bioconjugate to be produced. The oligosaccharyl transferase enzymes can be PglB oligosaccharyl transferase or variants thereof. Also provided are compositions containing the bioconjugates, and methods of using the bioconjugates and compositions described herein to vaccinate a subject against extra-intestinal pathogenic E. coli . (ExPEC).
1. A method of preparing a bioconjugate of an E. coli O x antigen polysaccharide covalently linked to a carrier protein, the method comprising:
(i) providing a recombinant host cell comprising:
a. a nucleotide sequence of an rfb gene cluster for the O x -antigen polysaccharide;
b. a nucleotide sequence encoding the carrier protein comprising at least one glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1; and
c. a nucleotide sequence encoding an oligosaccharyl transferase PglB y ; and
(ii) culturing the recombinant host cell under conditions for production of the bioconjugate,
wherein:
the O x -antigen is O1A antigen polysaccharide, the PglB y comprises the amino acid mutations of N311V, K482R, D483H, and A669V;
wherein the amino acid mutations are relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, and
wherein the O1A antigen polysaccharide has the structure of:
and n is an integer of 1 to 100.
2. The method of claim 1 , further comprising isolating the bioconjugate from the recombinant host cell.
3. The method of claim 1 , wherein the recombinant host cell is an E. coli cell.
4. The method of claim 1 , wherein the carrier protein is detoxified Exotoxin A of P. aeruginosa (EPA) comprising SEQ ID NO: 3.
5. The method of claim 1 , wherein the carrier protein is selected from the group consisting of detoxified Exotoxin A of P. aeruginosa (EPA), E. coli flagellin (FliC), CRM197, maltose binding protein (MBP), Diphtheria toxoid, Tetanus toxoid, detoxified hemolysin A of S. aureus , clumping factor A, clumping factor B, E. coli heat labile enterotoxin, detoxified E. coli heat labile enterotoxin, Cholera toxin B subunit (CTB), cholera toxin, detoxified cholera toxin, E. coli Sat protein, the passenger domain of E. coli Sat protein, Streptococcus pneumoniae Pneumolysin, Keyhole limpet hemocyanin (KLH), P. aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae.
6. The method of claim 1 , wherein the PglB y consists of the amino acid mutations of N311V, K482R, D483H, and A669V.