Fermentation process for producing a virulence factor from bordetella

Patent metadata

Field	Value
Publication number	US-9475848-B2
Application number	US-201314376274-A
Country	US
Kind code	B2
Filing date	Jan 30, 2013
Priority date	Feb 1, 2012
Publication date	Oct 25, 2016
Grant date	Oct 25, 2016

Abstract

A process for fermenting Bordetella species comprising incubating a sample of bacteria of a Bordetella species in a first environment under at least one bvg ( Bordetella virulence genes) modulating condition, for at least 5 generations, to produce a mature culture, and then incubating the mature culture in a second environment in the absence of the at least one bvg modulating condition.

First claim

We claim:

1. A fermentation process for culturing a species of Bordetella , comprising the following steps:
a) providing a sample of bacterial cells of a Bordetella species in a first culture medium comprising at least one Bordetella virulence gene (bvg) modulator, wherein the bvg modulator is niacin at a concentration of greater than or equal to 0.2 g/L and wherein the sample predominantly comprises Bordetella cells having a bvg+ genotype;
b) incubating the sample in the first culture medium comprising the at least one bvg modulator for at least five generations of growth, thereby producing a mature culture; and
c) incubating the mature culture in a second culture medium in the absence of the at least one bvg modulator for at least five generations of growth; wherein step c) occurs after step b), and wherein in step (c) at least one virulence factor selected from the group consisting of Pertussis Toxin (PT), Filamentous Haemagglutinin (FHA), Pertactin (PRN) and fimbrial agglutinogen is expressed.

2. The fermentation process of claim 1 wherein the Bordetella species is a species selected from the group consisting of Bordetella pertussis, Bordetella parapertussis , and Bordetella bronchiseptica.

3. The fermentation process of claim 1 wherein step b) comprises less than 40 generations of growth.

4. A fermentation process for culturing a species of Bordetella , comprising the following steps:
a) providing a sample of bacterial cells of a Bordetella species in a first culture medium comprising at least one Bordetella virulence gene (lava) modulator, wherein the bvg modulator is a sulphate salt at a concentration of between 0.04 mM and 40 mM, and wherein the sample predominantly comprises Bordetella cells having a bvg+ genotype;
b) incubating the sample in the first culture medium comprising the at least one bvg modulator for at least five generations of growth, thereby producing a mature culture; and
c) incubating the mature culture in a second culture medium in the absence of the at least one bvg modulator for at least five generations of growth; wherein step c) occurs after step b), and wherein in step (c) at least one virulence factor selected from the group consisting of Pertussis Toxin (PT), Filamentous Haemagglutinin (FHA), Pertactin (PRN) and fimbrial agglutinogen is expressed.

5. A fermentation process for culturing a species of Bordetella , comprising the following steps:
a) providing a sample of bacterial cells of a Bordetella species in a first culture medium comprising at least one Bordetella virulence gene (lava) modulator, wherein the bvg modulator is a phosphate salt at a concentration of between 0.4 g/L and 20 g/L, and wherein the sample predominantly comprises Bordetella cells having a bvg+ genotype;
b) incubating the sample in the first culture medium comprising the at least one bvg modulator for at least five generations of growth, thereby producing a mature culture; and
c) incubating the mature culture in a second culture medium in the absence of the at least one bvg modulator for at least five generations of growth; wherein step c) occurs after step b), and wherein in step (c) at least one virulence factor selected from the group consisting of Pertussis Toxin (PT), Filamentous Haemagglutinin (FHA), Pertactin (PRN) and fimbrial agglutinogen is expressed.

6. A fermentation process for culturing a species of Bordetella , comprising the following steps:
a) providing a sample of bacterial cells of a Bordetella species in a first culture medium comprising at least one Bordetella virulence gene (bvg) modulator, wherein the bvg modulator is sucrose at a concentration of between 10 mM and 100 mM, and wherein the sample predominantly comprises Bordetella cells having a bvg+ genotype;
b) incubating the sample in the first culture medium comprising the at least one bvg modulator for at least five generations of growth, thereby producing a mature culture; and
c) incubating the mature culture in a second culture medium in the absence of the at least one bvg modulator for at least five generations of growth; wherein step c) occurs after step b), and wherein in step (c) at least one virulence factor selected from the group consisting of Pertussis Toxin (PT), Filamentous Haemagglutinin (FHA), Pertactin (PRN) and fimbrial agglutinogen is expressed.

7. A fermentation process for culturing a species of Bordetella , comprising the following steps:
a) providing a sample of bacterial cells of a Bordetella species in a first culture medium comprising at least one Bordetella virulence gene (bvg) modulator, wherein the bvg modulator is selected from the group consisting of yeast extract, tryptic soy agar, tryptose phosphate, infusions of brain and heart tissue, and peptones, and wherein the concentration of the bvg modulator is between 1 g/L and 40 g/L, and wherein the sample predominantly comprises Bordetella cells having a bvg+ genotype;
b) incubating the sample in the first culture medium comprising the at least one bvg modulator for at least five generations of growth, thereby producing a mature culture; and
c) incubating the mature culture in a second culture medium in the absence of the at least one bvg modulator for at least five generations of growth; wherein step c) occurs after step b), and wherein in step (c) at least one virulence factor selected from the group consisting of Pertussis Toxin (PT), Filamentous Haemagglutinin (FHA), Pertactin (PRN) and fimbrial agglutinogen is expressed.

8. A fermentation process for culturing a species of Bordetella , comprising the following steps:
a) providing a sample of bacterial cells of a Bordetella species in a first culture medium comprising at least one Bordetella virulence gene (bvg) modulator, wherein the bvg modulator is proline at a concentration of between 0.25 g/L and 50 g/L, and wherein the sample predominantly comprises Bordetella cells having a bvg+ genotype;
b) incubating the sample in the first culture medium comprising the at least one bvg modulator for at least five generations of growth, thereby producing a mature culture; and
c) incubating the mature culture in a second culture medium in the absence of the at least one bvg modulator for at least five generations of growth; wherein step c) occurs after step b), and wherein in step (c) at least one virulence factor selected from the group consisting of Pertussis Toxin (PT), Filamentous Haemagglutinin (FHA), Pertactin (PRN) and fimbrial agglutinogen is expressed.

9. A fermentation process for culturing a species of Bordetella , comprising the following steps:
a) providing a sample of bacterial cells of a Bordetella species in a first culture medium comprising at least one Bordetella virulence gene (bvg) modulator, wherein the bvg modulator comprises sodium ions at a concentration between 100 mM and 2000 mM, and wherein the sample predominantly comprises Bordetella cells having a bvg+ genotype;
b) incubating the sample in the first culture medium comprising the at least one bvg modulator for at least five generations of growth, thereby producing a mature culture; and
c) incubating the mature culture in a second culture medium in the absence of the at least one bvg modulator for at least five generations of growth; wherein step c) occurs after step b), and wherein in step (c) at least one virulence factor selected from the group consisting of Pertussis Toxin (PT), Filamentous Haemagglutinin (FHA), Pertactin (PRN) and fimbrial agglutinogen is expressed.

10. A fermentation process for culturing a species of Bordetella , comprising the following steps:
a) providing a sample of bacterial cells of a Bordetella species in a first cultur

Assignees

• Glaxosmithkline Biologicals Sa

Inventors

• Dehottay Philippe Marc Helene
• Goffin Philippe

Classifications

• A61P31/04

  Antibacterial agents · CPC title

• A61P31/00

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

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• C12N1/38

  Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound (C12N1/34 takes precedence) · CPC title

• C07K14/235 Primary

  from Bordetella (G) · CPC title