# US 2014/0194346 A1 · Pasteurellaceae vaccines

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- Number: US 2014/0194346 A1
- Document ID: US20140194346A1
- Status: Published application
- Kind: A1
- Application number: 14/237,377
- Filed: 2012-08-07
- Published: 2014-07-10
- Earliest claimed: 2011-08-08
- Assignees: ETH Zurich
- Inventors: Markus Aebi; Flavio Schwarz; Andreas Naegeli
- Web page: https://patentgrep.com/patent/US20140194346A1

## CPC

- G01N 33/56911: {Bacteria}
- A61K 39/102: {Pasteurellales, e.g. Actinobacillus}, Pasteurella; Haemophilus
- A61P 31/04: Antibacterial agents
- C07K 14/285: from Pasteurellaceae (F), e.g. Haemophilus influenza
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- 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)

## Abstract

The present invention relates to an N-glycosylated protein for treating and/or preventing bacterial Pasteurellaceae infection in a mammal or bird, wherein the protein is a Pasteurellaceae protein, a functional fragment or derivative thereof having at least one glycosylated N-X-S/T consensus sequence. In addition, the present invention is directed to corresponding pharmaceutical compositions for treating and/or protecting mammals or birds having or being prone to develop a bacterial Pasteurellaceae infection. Furthermore, the invention describes methods for producing said N-glycosylated proteins.

## Claims

### Claim at position 1

1-14. (canceled)

### Claim 15

N-glycosylated protein for the manufacture of a medicament for treating and/or preventing bacterial Pasteurellaceae infection in a mammal or bird, wherein the protein is a Pasteurellaceae protein, a functional fragment or derivative thereof having at least one glycosylated N-X-S/T consensus sequence, wherein X is not Pro, and wherein the Pasteurellaceae protein, functional fragment or derivative thereof is
(1) autotransporter adhesin [ataC] A. pleuropneumoniae serotype 7 (strain AP76) (accession no. B3GX20_ACTP7; SEQ ID NO: 1), or
(2) AT family autotransporter/adhesin [COK—1394] Mannheimia haemolytica serotype A2 str. BOVINE (accession number E2P8A5_PASHA; SEQ ID NO: 13), or
(3) a functional fragment or derivative having at least 40, preferably at least 50, more preferably at least 70, most preferably at least 80% amino acid sequence identity to (1) or (2).

### Claim 16 (depends on 15)

N-glycosylated protein according to claim 15, wherein the N-linked glycan is selected from the group consisting of β-Glc/β-Gal, (β-Glc/β-Gal)-1,6-(α-Glc/α-Gal)n, wherein n is at least 1, preferably 1 to 10, preferably 1 to 6, more preferably 2 to 5, more preferably β-Glc-α1,6-Glc-α1,6-Glc, β-Glc-α1,6-Glc-α1,6-Glc-α1,6-Glc, β-Glc-α1,6-Glc-α1,6-Glc-α1,6-Glc-α1,6-Glc, β-Glc-α1,6-Glc-α1,6-Glc-α1,6-Glc-α1,6-Glc-α1,6-Glc, β-Gal-α1,6-Glc-α1,6-Glc, β-Gal-α1,6-Glc-α1,6-Glc-α1,6-Glc, β-Gal-α1,6-Glc-α1,6-Glc-α1,6-Glc-α1,6-Glc and β-Gal-α1,6-Glc-α1,6-Glc-α1,6-Glc-α1,6-Glc-α1,6-Glc.

### Claim 17 (depends on 15)

N-glycosylated protein according to claim 15, wherein the N-glycosylated protein is autotransporter adhesin [ataC] [A. pleuropneumoniae serotype 7 (strain AP76) (accession no. B3GX20_ACTP7; SEQ ID NO: 1), a functional fragment or derivative thereof having at least 40, preferably at least 50, more preferably at least 70, most preferably at least 80% amino acid sequence identity to ataC, wherein at least 10%, preferably at least 30%, more preferably at least 50%, most preferably at least 70% of all N-X-S/T consensus sequences are glycosylated.

### Claim 18 (depends on 17)

N-glycosylated protein according to claim 17, wherein at least 10%, preferably at least 30%, more preferably at least 50%, most preferably at least 70% of all N-X-S/T consensus sequences are glucosylated, more preferably glucosylated by β-Glc-α1,6-Glc-α1,6-Glc.

### Claim 19 (depends on 15)

N-glycosylated protein according to claim 15, wherein the N-glycosylated protein is
(i) a fragment of autotransporter adhesin [ataC] A. pleuropneumoniae serotype 7 (strain AP76) (accession no. B3GX20_ACTP7; SEQ ID NO: 1), preferably comprising amino acids 1866 to 2516 (SEQ ID NO: 16);
(ii) a fragment of autotransporter adhesin [ataC] A. pleuropneumoniae serotype 7 (strain AP76) (accession no. B3GX20_ACTP7; SEQ ID NO: 1), preferably comprising amino acids 61 to 984 (SEQ ID NO: 17);
(iii) a fragment of autotransporter adhesin [ataC] A. pleuropneumoniae serotype 7 (strain AP76) (accession no. B3GX20_ACTP7; SEQ ID NO: 1), preferably comprising amino acids 51 to 2428 (SEQ ID NO: 18);
(iv) a fragment of autotransporter adhesin [ataC] A. pleuropneumoniae serotype 7 (strain AP76) (accession no. B3GX20_ACTP7; SEQ ID NO: 1), preferably comprising amino acids 1866 to 2428 (SEQ ID NO: 19);
wherein (i), (ii), (iii) and (iv) are N-glycosylated, in
(a) 1 to 14, preferably at least 2 to 10, more preferably 2 to 8 consensus sequence(s) N-X-S/T for fragment (i),
(b) 1 to 9, preferably at least 2 to 8, more preferably 2 to 5 consensus sequence(s) N-X-S/T for fragment (ii),
(c) 1 to 73, preferably at least 2 to 50, more preferably 5 to 20 consensus sequence(s) N-X-S/T for fragment (iii) and
(d) 1 to 13, preferably at least 2 to 10, more preferably 2 to 8 consensus sequence(s) N-X-S/T for fragment (iv).

### Claim 20 (depends on 19)

N-glycosylated protein according to claim 19, wherein the N-glycosylated protein is glucosylated, more preferably glucosylated by Glc-α1,6-Glc-α1,6-Glc.

### Claim 21 (depends on 15)

Pharmaceutical composition comprising a pharmaceutically effective amount of at least one N-glycosylated protein according to claim 15 and optionally one or more pharmaceutically acceptable carriers and/or adjuvants.

### Claim 22 (depends on 15)

Method for producing N-glycosylated proteins according to claim 15, comprising the following steps:
(i) providing a cell, preferably a prokaryotic cell, more preferably an E. coli cell expressing an N-glycosyltransferase (NGT) and a Pasteurellaceae protein, functional fragment or derivative thereof having at least one N-X-S/T consensus sequence(s), wherein X is not Pro;
(ii) culturing said cell under conditions that lead to the N-linked glycosylation, preferably glucosylation of said Pasteurellaceae protein,
(iii) optionally coexpressing an glycosyltransferase for glycosyl extension of N-linked glycosyl, preferably for extending glucosyl residues, and
(iv) optionally purifying the N-glycosylated proteins.

### Claim 23 (depends on 22)

Method according to claim 22, wherein the glycosyltransferase in step (iii) is selected from a Pasteurellaceae protein, preferably proteins of Actinobacillus, Haemophilus, Histophilus and Mannheimia, more preferably proteins from Actinobacillus pleuropneumoniae, Haemophilus influenza, Haemophilus parasuis, Histophilus somni and Mannheimia haemolytica, most preferably an α6GlcT from A. pleuropneumoniae.

### Claim 24 (depends on 15)

Method of diagnosing a bacterial Pasteurellaceae infection comprising the following steps:
(i) providing a sample, preferably selected from blood, saliva, lacrimal, urine and/or feces from a mammal or bird suspected of having a Pasteurellaceae infection,
(ii) contacting said sample, a functional component or derivative thereof with an N-glycosylated protein according to claim 15 under conditions that allow for antibody binding, and
(iii) determining binding of said sample, a functional component or derivative thereof to an N-glycosylated protein according to claim 15.

### Claim 25 (depends on 15)

A method of treating and/or protecting mammals or birds having or being prone to develop a bacterial Pasteurellaceae infection comprising the administration of a therapeutically effective amount of at least one N-glycosylated protein according to claim 15 to a patient in need thereof.

### Claim 26 (depends on 24)

The method of claim 24, wherein the N-glycosylated protein is a Pasteurellaceae protein, preferably a protein from the genera Actinobacillus, Aggregatibacter, Avibacterium, Basfia, Bibersteinia, Chelonobacter, Gallibacterium, Haemophilus, Histophilus, Lonepinella, Mannheimia, Nicoletella, Pasteurella, Phocoenobacter and Volucribater, more preferably a protein of Actinobacillus, Haemophilus, Histophilus and Mannheimia, most preferably a protein from Actinobacillus pleuropneumoniae, Haemophilus parasuis, Haemophilus influenza, Histophilus somni and Mannheimia haemolytica.

### Claim 27 (depends on 21)

A method of treating and/or protecting mammals or birds having or being prone to develop a bacterial Pasteurellaceae infection comprising the administration of a therapeutically effective amount of at least one N-glycosylated protein according to a pharmaceutical composition of claim 21 to a patient in need thereof.