Method for the production of outer membrane vesicles and immunogenic compositions thereof
Inventors
Grandi, Guido • Grandi, Alberto • Fantappie', Laura • Zanella, Ilaria • Koenig, Enrico • Gagliardi, Assunta
Assignees
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Publication Number
US-12551544-B2
Publication Date
2026-02-17
Expiration Date
Abstract
The invention relates to gram-negative bacteria carrying gene-inactivating mutations that cause deletion of proteins belonging to the OMV proteome, to Outer Membrane Vesicles (OMVs) produced by these bacteria and immunogenic compositions thereof.
Core Innovation
The invention provides a method for preparing isolated bacterial outer membrane vesicles (OMVs) expressing heterologous proteins. The method cultures a gram-negative bacterium in conditions suitable for vesiculation, wherein the bacterium is E. coli, and wherein the bacterium carries gene-inactivating mutations at the ompA gene and at least 5 specified additional genes encoding proteins naturally present in the OMVs of E. coli.
The method further comprises expressing heterologous proteins in the OMVs, and then separating the OMVs from the bacterial culture, with an optional purification step. The genetic design targets OMV-associated proteins by inactivating ompA together with a defined set of OMV proteome genes, thereby producing engineered OMVs that express heterologous proteins after vesiculation and recovery.
The description additionally characterizes engineered progressive mutant OMV strains and associated modifications in OMV protein composition while maintaining or increasing OMV yield. It further demonstrates that heterologous antigens can efficiently accumulate in the engineered OMVs and that OMVs derived from further LPS-pathway mutants induce higher antigen-specific IgG titers and stronger functional immune responses, including in situ vaccination efficacy against CT26 tumors with distal tumor suppression.
Claims Coverage
The document contains one independent claim. It recites a genetic and process framework with a quantitative inventive constraint (ompA plus at least 5 selected OMV-associated gene inactivations) and encompasses separation and optional purification of isolated OMVs that express heterologous proteins.
Gene-inactivating mutations at ompA plus specified OMV proteome genes
A gram-negative bacterium, E. coli, carries gene-inactivating mutations at the ompA gene and at least 5 of the following genes which encode proteins naturally present in the OMVs of E. coli: ybis, ais, eco, glpQ, mltA, proX, ydcL, glnH, efeO, bglX, agp, ygdI, yncD, slp, artI, yiaD, ompX, borD, yhiJ, emtA, fecA, nmpC, fhuA, hisJ, lamB, malE, malM, ygiW, cirA, fepA, loiP, yjeI, ecnB, rcsF, phoE, oppA, fkpA, ybaY, tsx, yggE, osmE, ygdR, yceI, bhsA, nlpE, pldA, yghJ, ydeN, ushA, mdoD, treA, bcsC, ftsP, ptrA, fadL, artJ, mlaA.
Expressing heterologous proteins in the OMVs
The engineered E. coli expresses heterologous proteins in the OMVs.
Isolating OMVs by separating from bacterial culture with optional purification
After culturing under vesiculation-suitable conditions, the method separates the OMVs from the bacterial culture, and optionally purifies the OMVs.
Claim coverage is anchored by the combination of ompA inactivation together with inactivation of at least 5 specific OMV-associated genes, heterologous protein expression in the resulting OMVs, and separation of OMVs from culture with optional purification.
Stated Advantages
The engineered progressive mutant OMV strains maintain or increase OMV yield.
Heterologous antigens can efficiently accumulate in the engineered OMVs.
OMVs derived from further LPS-pathway mutants induce higher antigen-specific IgG titers and stronger functional immune responses, including in situ vaccination efficacy against CT26 tumors with distal tumor suppression.
Documented Applications
In situ vaccination efficacy against CT26 tumors with distal tumor suppression.
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