# US 10,307,473 B2 · Capsular gram-positive bacteria bioconjugate vaccines

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- Number: US 10,307,473 B2
- Document ID: US10307473B2
- Status: Granted
- Kind: B2
- Application number: 15/414,900
- Filed: 2017-01-25
- Published: 2017-05-11
- Granted: 2019-06-04
- Earliest claimed: 2010-05-06
- Assignees: Glaxosmithkline Biologicals SA
- Applicants: Glaxosmithkline Biologicals S.A.
- Inventors: Michael Wacker; Michael Kowarik; Michael Wetter
- Web page: https://patentgrep.com/patent/US10307473B2

## CPC

- A61K 39/085: Staphylococcus
- A61P 31/04: Antibacterial agents
- A61P 37/02: Immunomodulators
- C07K 14/21: from Pseudomonadaceae (F)
- C07K 16/1271: Micrococcaceae (F); Staphylococcaceae (F), e.g. Staphylococcus (G)
- C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)}
- C12N 9/1048: {Glycosyltransferases (2.4)}
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- A61K 2039/505: {comprising antibodies}
- A61K 2039/575: {humoral response}
- A61K 2039/6037: {Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]}
- A61K 2039/6087: {Polysaccharides; Lipopolysaccharides [LPS]}
- A61K 2039/64: {characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units}
- A61K 2039/70: {Multivalent vaccine}
- C07K 2317/734: Complement-dependent cytotoxicity [CDC]
- C07K 2319/00: Fusion polypeptide
- C07K 2319/02: containing a signal sequence
- C07K 2319/034: containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved
- C07K 2319/21: containing a His-tag
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention encompasses a novel S. aureus bioconjugate vaccine. More generally, the invention is directed to Gram-positive and other bioconjugate vaccines containing a protein carrier, at least one polysaccharide such as a capsular Gram-positive polysaccharide, and, optionally, an adjuvant or pharmaceutically acceptable carrier. The instant invention also includes methods of producing Gram-positive and other bioconjugate vaccines. An N-glycosylated protein is also provided that contains one or more polysaccharides such as Gram-positive polysaccharides. The invention is additionally directed to engineered prokaryotic organisms comprising nucleotide sequences encoding a glycosyltransferase of a first prokaryotic organism and a glycosyltransferase of a second prokaryotic organism. The invention further includes plasmids and prokaryotic cells transformed with plasmids encoding polysaccharides and enzymes which produce an N-glycosylated protein and/or bioconjugate vaccine. Further, the invention is directed to methods of inducing an immune response in a mammal comprising administering said bioconjugate vaccines.

## Claims

### Claim 1

A Gram-negative host prokaryotic organism comprising:
(i) a nucleotide sequence encoding at least one glycosyltransferase from a Gram-positive bacterium;
(ii) a nucleotide sequence encoding at least one glycosyltransferase from a Gram-negative bacterium;
(iii) a nucleotide sequence encoding a carrier protein, wherein said carrier protein comprises at least one inserted consensus sequence D/E-X-N-Z-S/T, wherein X and Z may be any natural amino acid except proline; and
(iv) a nucleotide sequence encoding an oligosaccharyl transferase, wherein said oligosaccharyl transferase is a PglB;
wherein the Gram-negative host prokaryotic organism produces Gram-positive glycosylated proteins;
wherein said Gram-negative host prokaryotic organism is Escherichia coli.

### Claim 2 (depends on 1)

The host prokaryotic organism of claim 1, wherein the Gram-negative bacterium produces a polysaccharide that has structural similarity to a second polysaccharide produced by the Gram-positive organism.

### Claim 3 (depends on 2)

The host prokaryotic organism of claim 2, wherein the polysaccharide produced by the Gram-negative bacterium contains a repeating unit that is partially identical to a repeating unit produced by the Gram-positive organism.

### Claim 4 (depends on 1)

The host prokaryotic organism of claim 1, wherein the protein comprises at least one inserted consensus sequence D/E-X-N-Z-S/T, wherein X and Z may be any natural amino acid except proline.

### Claim 5 (depends on 1)

The host prokaryotic organism of claim 1, wherein the protein comprises at least two inserted consensus sequence D/E-X-N-Z-S/T, wherein X and Z may be any natural amino acid except proline.

### Claim 6 (depends on 1)

A method of producing a glycosylated protein in the host prokaryotic organism of claim 1.

### Claim 7 (depends on 6)

The method of claim 6 wherein the glycosylated protein is a protein N-glycosylated with capsular polysaccharide of a Gram positive bacterium.

### Claim 8 (depends on 7)

The method of claim 7 wherein the protein N-glycosyated with capsular polysaccharide of a Gram positive bacterium is synthesized by a combination of different glycosyltransferases from different organisms, for example a Gram positive bacterium and a Gram positive bacterium.