Bioconjugates of e. coli o-antigen polysaccharides, methods of production thereof, and methods of use thereof

Patent metadata

Field	Value
Publication number	US-2023118878-A1
Application number	US-202217815705-A
Country	US
Kind code	A1
Filing date	Jul 28, 2022
Priority date	Mar 18, 2019
Publication date	Apr 20, 2023
Grant date	—

Abstract

A bioconjugate of an E. coli glucosylated O4 antigen polysaccharide covalently linked to a carrier protein and compositions thereof are provided. Also provided are recombinant host cells for producing the bioconjugate, and methods of producing the bioconjugate using the recombinant host cells. The recombinant host cells contain a nucleic acid encoding a glucosyl transferase capable of modifying the E. coli O4 antigen with glucose branching to produce the glucosylated O4 antigen polysaccharide. Bioconjugates of an E. coli glucosylated O4 antigen polysaccharide described herein can be used alone or in combination with one or more additional E. coli O-antigen polysaccharides to induce antibodies against an E. coli glucosylated antigen, and to vaccinate a subject against extra-intestinal pathogenic E. coli (ExPEC).

First claim

1 - 20 . (canceled)

21 . A recombinant host cell for producing a bioconjugate of an E. coli glucosylated O4 antigen polysaccharide covalently linked to a carrier protein, wherein the glucosylated O4 antigen polysaccharide comprises the structure of Formula

wherein n is an integer of 5 to 40,
the host cell comprising:
(i) a nucleotide sequence of an rfb gene cluster for the E. coli O4 antigen polysaccharide;
(ii) a nucleotide sequence encoding a glucosyl transferase, wherein the glucosyl transferase is capable of modifying the E. coli O4 antigen polysaccharide to produce the E. coli glucosylated O4 antigen polysaccharide;
(iii) a nucleotide sequence encoding a translocase that is capable of translocating bactoprenol-linked glucose;
(iv) a nucleotide sequence encoding a glycosyltransferase that is capable of glucosylating bactoprenol;
(v) a nucleotide sequence encoding the carrier protein; and
(vi) a nucleotide sequence encoding an oligosaccharyl transferase capable of covalently linking the E. coli glucosylated O4 antigen polysaccharide to the carrier protein to produce the bioconjugate.

22 . The host cell of claim 21 , wherein the glucosyl transferase has at least 99% sequence identity to SEQ ID NO: 4.

23 . The host cell of claim 21 , wherein the translocase has at least 99% sequence identity to SEQ ID NO: 7, and the glycosyltransferase has at least 99% sequence identity to SEQ ID NO: 8.

24 . The recombinant host cell of claim 21 , wherein:
the glucosyl transferase comprises the amino acid sequence of SEQ ID NO: 4.

25 . The recombinant host cell of claim 24 , wherein:
the translocase comprises the amino acid sequence of SEQ ID NO: 7, and the glycosyltransferase comprises the amino acid sequence of SEQ ID NO: 8.

26 . The recombinant host cell of claim 21 , wherein:
the oligosaccharyl transferase comprises the amino acid sequence of SEQ ID NO: 6 having the amino acid mutation N311V, or the amino acid sequence of SEQ ID NO: 6 having amino acid mutations Y77H and N311V.

27 . The recombinant host cell of claim 21 , wherein the carrier protein comprises at least one glycosylation site comprising a glycosylation consensus sequence of Asn-X-Ser(Thr), wherein X can be any amino acid except Pro; or an optimized glycosylation consensus sequence of Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any amino acid except Pro.

28 . The recombinant host cell of claim 21 , wherein the carrier protein is a detoxified exotoxin A of Pseudomonas aeruginosa (EPA).

29 . The recombinant host cell of claim 28 , wherein the carrier protein comprises SEQ ID NO: 3.

30 . The recombinant host cell of claim 21 , which is an E. coli cell.

31 . The recombinant host cell of claim 30 , which is from an E. coli K-12 strain.

32 . The recombinant host cell of claim 31 , which is from strain W3110.

33 . The recombinant host cell of claim 21 , wherein
the glucosyl transferase comprises the sequence of SEQ ID NO: 4, the translocase comprises the sequence of SEQ ID NO: 7, the glycosyltransferase comprises the sequence of SEQ ID NO: 8, the oligosaccharyl transferase comprises the sequence of SEQ ID NO: 6, SEQ ID NO: 6 with the amino acid mutation N311V, or SEQ ID NO: 6 with the amino acid mutations Y77H and N311V, the carrier protein comprises SEQ ID NO: 3, and the E. coli cell is an E. coli K-12 strain.

34 . A recombinant host cell for producing a bioconjugate of an E. coli glucosylated O4 antigen polysaccharide covalently linked to a carrier protein, wherein the glucosylated O4 antigen polysaccharide comprises the structure of Formula

wherein n is an integer of 5 to 40,
the host cell comprising:
(i) a nucleotide sequence of an rfb gene cluster for the E. coli O4 antigen polysaccharide;
(ii) a gtrS gene encoding a glucosyl transferase enzyme specific for E. coli serotype O4 that glucosylates the O4 antigen polysaccharide;
(iii) a grtA gene encoding a bactoprenol-linked glucose translocase;
(iv) a gtrB gene encoding a bactoprenol glucosyl transferase;
(v) a nucleotide sequence encoding the carrier protein; and
(vi) a nucleotide sequence encoding an oligosaccharyl transferase capable of covalently linking the E. coli glucosylated O4 antigen polysaccharide to the carrier protein to produce the bioconjugate.

35 . The recombinant host cell of claim 34 , wherein the glucosyl transferase enzyme encoded by the gtrS gene comprises SEQ ID NO: 4.

36 . The recombinant host cell of claim 35 , wherein the bactoprenol-linked glucose translocase encoded by the gtrA gene comprises SEQ ID NO: 7 and wherein the bactoprenol glucosyl transferase encoded by the gtrB gene comprises SEQ ID NO: 8.

37 . The recombinant host cell of claim 34 , wherein the oligosaccharyl transferase comprises the amino acid sequence of SEQ ID NO: 6 having the amino acid mutation N311V, or the amino acid sequence of SEQ ID NO: 6 having amino acid mutations Y77H and N311V.

38 . The recombinant host cell of claim 34 , wherein the carrier protein comprises at least one glycosylation site comprising a glycosylation consensus sequence of Asn-X-Ser(Thr), wherein X can be any amino acid except Pro; or an optimized glycosylation consensus sequence of Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any amino acid except Pro.

39 . The recombinant host cell of claim 34 , wherein the carrier protein is a detoxified exotoxin A of Pseudomonas aeruginosa (EPA).

40 . The recombinant host cell of claim 34 , wherein the carrier protein comprises SEQ ID NO: 3.

41 . A method of producing a bioconjugate of an E. coli glucosylated O4 antigen polysaccharide covalently linked to a carrier protein, comprising the step of culturing the recombinant cell of claim 21 under conditions wherein the bioconjugate is produced.

42 . A method of producing a bioconjugate of an E. coli glucosylated O4 antigen polysaccharide covalently linked to a carrier protein, comprising the step of culturing the recombinant cell of claim 34 under conditions wherein the bioconjugate is produced.

Assignees

• Janssen Pharmaceuticals Inc
• Glaxosmithkline Biologicals Sa

Inventors

• Geurtsen Jeroen
• Poolman Jan Theunis
• Fae Kellen Cristhina
• Burghout Pieter Jan
• Weerdenburg Eveline Marleen
• Ibarra Yon Patricia
• Abbanat Darren Robert
• Kemmler Stefan Jochen
• Kowarik Michael Thomas
• Mally Manuela
• Gambillara Fonck Veronica
• Braun Martin Edward
• Carranza Sandmeier Maria Paula

Classifications

• Y02A50/30

  Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change · CPC title

• A61K39/385 Primary

  Haptens or antigens, bound to carriers · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• A61K2039/70

  Multivalent vaccine · CPC title

• A61K39/0258 Primary

  Escherichia · CPC title