Bioconjugates made from recombinant N-glycosylated proteins from procaryotic cells

Patent metadata

Field	Value
Publication number	US-10835592-B2
Application number	US-201414522254-A
Country	US
Kind code	B2
Filing date	Oct 23, 2014
Priority date	Feb 20, 2008
Publication date	Nov 17, 2020
Grant date	Nov 17, 2020

Abstract

The present invention is directed to a bioconjugate vaccine, such as an O1-bioconjugate vaccine, comprising: a protein carrier comprising a protein carrier containing at least one consensus sequence, D/E-X-N-Z-S/T, wherein X and Z may be any natural amino acid except proline; at least one antigenic polysaccharide from at least one pathogenic bacterium, linked to the protein carrier; and, optionally, an adjuvant. In another aspect, the present invention is directed to a method of producing an O1-bioconjugate in a bioreactor comprising a number steps.

First claim

The invention claimed is:

1. A method of producing a composition comprising
a bioconjugate separated from periplasmic proteins, wherein said bioconjugate comprises a carrier protein linked to an O antigen, said method comprising:
(i) culturing a prokaryotic host cell that comprises (A) a nucleic acid encoding a carrier protein comprising the amino acid sequence D/E-X-N-Z-S/T, wherein X and Z may be independently any natural amino acid except proline; (B) a nucleic acid encoding an oligosaccharyl transferase; and (C) one or more nucleic acids encoding one or more glycosyltransferases capable of assembling the O antigen on a lipid carrier, wherein said culturing is done for a period of time sufficient for the host cell to produce the bioconjugate;
(ii) extracting periplasmic proteins from the prokaryotic host cell; and
(iii) separating the bioconjugate from the extracted periplasmic proteins and unconjugated carrier protein by anionic exchange, wherein said O antigen is from extraintestinal pathogenic Escherichia coli (ExPEC), Shigella flexneri 2a, S. flexneri 3a, S. flexneri 3b, S. flexneri 6 or S. sonnei.

2. The method of claim 1 , wherein said 0-antigen is heterologous to said host cell.

3. The method of claim 1 , wherein said carrier protein is heterologous to said host cell.

4. The method of claim 1 , wherein said carrier protein is Pseudomonas aeruginosa exoprotein (EPA); AcrA (CjO367c), HisJ (CjO734c), or OmpH1 (CjO982c) from Campylobacter jejuni ; Diphteria toxin (CRM197); or Cholera toxin.

5. The method of claim 4 , wherein said carrier protein is Pseudomonas aeruginosa exoprotein (EPA).

6. The method of claim 1 , wherein said oligosaccharyl transferase is heterologous to said host cell.

7. The method of claim 1 , wherein said oligosaccharyl transferase is from Campylobacter.

8. The method of claim 7 , wherein said oligosaccharyl transferase is from Campylobacter jejuni.

9. The method of claim 1 , wherein said composition is at least 98% pure with respect to the bioconjugate.

Assignees

• Glaxosmithkline Biologicals Sa

Inventors

• Fernandez Fabiana
• Wetter Michael
• Kowarik Michael
• Wacker Michael

Classifications

• A61K39/0283 Primary

  Shigella · CPC title

• A61K2039/6037

  Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT] · CPC title

• A61P31/00

  Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics · CPC title

• C07K2319/034

  containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved · CPC title

• A61K2039/6068

  Other bacterial proteins, e.g. OMP · CPC title