Method for producing outer membrane vesicles
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.
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.