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Outer membrane vesicles

An outer membrane vesicle, heterologous protein technology, applied in allergic diseases, antibody medical ingredients, antibacterial drugs, etc., can solve the problems of no excess, no function, difficult periplasmic protein targeting to OMV, etc.

Active Publication Date: 2015-05-06
GLAXOSMITHKLINE BIOLOGICALS SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have drawbacks, in particular due to the difficulty in overexpressing large numbers of integral membrane proteins without detrimental effects on the transformed bacteria
[0006] It has also proven difficult to target periplasmic proteins to OMV
Fusion of GFP to the Tat (twin-arginine transporter) signal sequence resulted in overexpression of GFP targeted to the periplasm, but GFP fluorescence barely exceeded background fluorescence levels in OMVs [11], suggesting that GFP did not enter OMVs either due to Wrongly folded without function in OMV

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0229] Example 1-heterologous protein expression into Escherichia coli OMV

[0230] Production of ΔtolR and ΔompA ko Mutants in Escherichia coli BL21(DE3)

[0231] Recombination-prone BL21(DE3) cells were generated by using a highly proficient homologous recombination system (red operon) [122]. Briefly, electrocompetent bacterial cells were transformed by electroporation (5.9 ms at 2.5 kV) with 5 μg of pAJD434 plasmid. Bacteria were then grown in 1 ml of SOC broth for 1 hour at 37°C and then inoculated on LB medium containing trimethoprim (100 μg / ml). Expression of the red gene carried by pAJD434 was induced by adding 0.2% L-arabinose to the medium.

[0232] [Delta]tolR and [Delta]ompA E. coli BL21 mutants known to spontaneously produce large numbers of OMVs were generated by replacing the ompA and tolR coding sequences with kanamycin (kmr) and chloramphenicol (cmr) resistance cassettes, respectively. A three-step PCR protocol was used to fuse the upstream and downstream re...

Embodiment 2

[0238] Example 2 - Plasmid construction

[0239] Five heterologous proteins from different Gram-positive and Gram-negative bacterial species and belonging to different cellular compartments were selected as model proteins to determine whether the heterologous proteins could enter E. coli OMV in their native configuration. These proteins include: (1) periplasmic TEM1 β-lactamase (Bla) from Escherichia coli, (2) factor H-binding protein (fHbp) lipoprotein from Neisseria meningitidis, (3) pyogenic chain Extracellular cholesterol-dependent streptolysin O (Slo, also known as GAS25) from S. pyogenes, (4) the cell envelope serine protease SpyCep from S. pyogenes (also known as GAS57), and (5) S. pyogenes from The putative surface repelling protein Spy0269 (also known as GAS40). The nucleic acid coding sequences for each of these five proteins were cloned into the pET-OmpA plasmid using the polymerase incomplete primer extension (PIPE) cloning method [123]. The pET-OmpA plasmid (als...

Embodiment 3

[0241] Example 3 - Expression of heterologous proteins into ΔtolR and ΔompA mutants, OMVs and total lysate preparations

[0242] To investigate whether Bla, slo, SpyCEP, fHbp, and Spy0269 proteins were packaged into OMVs, ΔtolR and ΔompA E. coli BL21 strains were transformed with pET-21_Bla, pET-21_slo, pET-21_SpyCEP, pET-21_fHbp plasmids, and pET21_spy0269. As a negative control, pET-OmpA empty vector was used to transform ΔtolR and ΔompA E. coli BL21 strains.

[0243] All strains were grown to logarithmic phase in liquid medium and induced expression of genes was performed by adding 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside). figure 2 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of total lysates of these media before and after induction with 1 mM IPTG is shown. Bands corresponding to Bla, slo, SpyCEP, fHbp and Spy0269 proteins were present in all induced samples and indicated by arrows. Thus, all 5 tested heterologous proteins were successfully induced in E. col...

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Abstract

The present invention provides an outer membrane vesicle (OMV) from a Gram-negative bacterium, comprising at least one heterologous protein that is free in the lumen of the OMV, wherein the OMV is capable of eliciting an immune response to the heterologous protein when administered to a mammal. The invention also provides methods for preparing the OMVs of the invention, pharmaceutical compositions comprising the OMVs of the invention, especially immunogenic compositions and vaccines, and methods of generating an antibody immune response in a mammal using OMVs.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 61 / 702,296, filed September 18, 2012, and U.S. Provisional Application No. 61 / 799,311, filed March 15, 2013, the entire contents of which are incorporated herein by reference for all purposes . technical field [0002] The present invention relates to vesicles from Gram-negative bacteria. Vesicles contain heterologous proteins present in their lumen. The vesicles are particularly useful in immunogenic compositions, such as vaccines. Background technique [0003] Gram-negative bacteria can spontaneously release outer membrane vesicles (OMVs) during their growth due to the turgor of the cell envelope. Formation of such OMVs can be facilitated by the disruption of certain bacterial components, eg in refs 1 and 2 the E. coli Tol-Pal system was disrupted to obtain strains that release vesicles into the medium during growth. OMVs can also be produced by destroying intact bacteria. Known methods for ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00
CPCA61K39/092A61K39/095A61K2039/55555A61P31/04A61P31/10A61P31/12A61P33/00A61P35/00A61P35/02A61P37/04A61P37/08Y02A50/30A61K39/39
Inventor G·格兰迪I·马格里特 伊 若斯E·恰洛特
Owner GLAXOSMITHKLINE BIOLOGICALS SA
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