Method for producing outer membrane vesicles

Patent metadata

Field	Value
Publication number	US-10709776-B2
Application number	US-201515302279-A
Country	US
Kind code	B2
Filing date	Apr 7, 2015
Priority date	Apr 7, 2014
Publication date	Jul 14, 2020
Grant date	Jul 14, 2020

Abstract

The present invention relates to ex vivo method for producing outer membrane vesicles (OMVs) by expression or overexpression of the hemolysin F gene (hlyF) in gram-negative bacterium.

First claim

The invention claimed is:

1. An ex vivo method for producing outer membrane vesicles (OMVs) comprising
expressing or overexpressing a hemolysin F gene (hlyF) in gram-negative bacterium; and
recovering the OMVs from the gram-negative bacterium.

2. The ex vivo method according to the claim 1 , wherein said expressing or overexpressing step comprises:
a) transforming the gram-negative bacterium with the hlyF gene and/or overexpressing an endogenous hlyF gene of the gram-negative bacterium; and
b) cultivating the gram-negative bacterium in an appropriate culture medium;

and wherein said recovering step comprises:
c) centrifuging the culture medium to remove bacteria and obtain a supernatant comprising OMVs;
d) filtering the supernatant; and
e) centrifuging filtered supernatant to obtain a pellet of OMVs.

3. The ex vivo method according to claim 1 wherein the gram-negative bacterium is an Enterobacteriaceae.

4. The ex vivo method according to claim 3 wherein the Enterobacteriaceae is an Escherichia coli.

5. OMVs having a different proteomic profile than OMVs produced by wild type gram-negative bacteria, wherein the OMVs are obtained by
a) transforming a gram-negative bacterium with a hlyF gene;
b) cultivating the gram-negative bacterium in an appropriate culture medium;
c) centrifuging the culture medium to remove bacteria and obtain a supernatant comprising OMVs;
d) filtering the supernatant; and
e) centrifuging filtered supernatant to obtain a pellet of OMVs, wherein the OMVs
contain at least 50% more Tsx protein than OMVs produced by wild type gram-negative bacteria; and/or
contain at least twice as much OmpW protein than OMVs produced by wild type gram-negative bacteria; and/or
contain at least 50% less Lpp protein than OMVs produced by wild type gram-negative bacteria; and/or
contain at least 30% less Pal protein than OMVs produced by wild type gram-negative bacteria.

6. A vaccine composition comprising an adjuvant and OMVs having a different proteomic profile than OMVs produced by wild type gram-negative bacteria, wherein the OMVs are obtained by
a) transforming a gram-negative bacterium with a hlyF gene;
b) cultivating the gram-negative bacterium in an appropriate culture medium;
c) centrifuging the culture medium to remove bacteria and obtain a supernatant comprising OMVs;
d) filtering the supernatant; and
e) centrifuging filtered supernatant to obtain a pellet of OMVs, wherein the OMVs
contain at least 50% more Tsx protein than OMVs produced by wild type gram-negative bacteria; and/or
contain at least twice as much OmpW protein than OMVs produced by wild type gram-negative bacteria; and/or
contain at least 50% less Lpp protein than OMVs produced by wild type gram-negative bacteria; and/or
contain at least 30% less Pal protein than OMVs produced by wild type gram-negative bacteria.

7. The vaccine composition according to claim 6 wherein the adjuvant is a TLR4 agonist or a TLR9 agonist.

Assignees

• Inst Nat Sante Rech Med
• Centre Nat Rech Scient
• Inst Nat De La Rech Agronomique Inra
• Univ Toulouse 3 Paul Sabatier
• Ct Hospitalier Universitaire Toulouse
• Institut National De Rech Pour Lagriculture Lalimentation Et Lenvironnement

Inventors

• Oswald Eric
• Martin Patricia
• Murase Kazunori

Classifications

• C07K14/245 Primary

  Escherichia (G) · CPC title

• C12N1/06

  Lysis of microorganisms · CPC title

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• C12N1/066

  by physical processes · CPC title

• A61K2039/555

  characterised by a specific combination antigen/adjuvant · CPC title