# US 11,027,004 B2 · Immunogenic compositions containing bacterial outer membrane vesicles

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- Number: US 11,027,004 B2
- Document ID: US11027004B2
- Status: Granted
- Kind: B2
- Application number: 15/790,472
- Filed: 2017-10-23
- Published: 2018-07-26
- Granted: 2021-06-08
- Earliest claimed: 2016-10-24
- Assignees: Biomvis Srl
- Applicants: Universita' Degli Studi Di Trento
- Inventors: Guido Grandi; Laura Fantappie'; Carmela Irene
- Web page: https://patentgrep.com/patent/US11027004B2

## CPC

- A61K 39/092: {Streptococcus}
- A61K 39/02: Bacterial antigens
- A61K 39/085: Staphylococcus
- C07K 14/195: from bacteria
- C07K 14/31: from Staphylococcus (G)
- C07K 14/315: from Streptococcus (G), e.g. Enterococci
- A61K 2039/60: {characteristics by the carrier linked to the antigen}
- A61K 2039/6018: {Lipids, e.g. in lipopeptides}
- C12N 1/20: Bacteria; Culture media therefor
- C12N 1/205: Bacterial isolates
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12R 2001/19: Escherichia coli
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

This invention relates to outer membrane vesicles (OMVs) from Gram-negative bacteria. The vesicles comprise heterologous proteins or immunogenic fragments thereof expressed as lipoproteins in their membrane. The OMVs of the invention are capable of eliciting an immune response to the heterologous protein or to a fragment thereof when administered to a mammal. Other aspects of the invention relate to methods of preparing the OMVs and immunogenic compositions containing the same.

## Claims

### Claim 1

A method of preparing an outer membrane vesicle (OMV) from a Gram-negative bacterium, wherein
said OMV comprises a lipoprotein consisting of a heterologous protein which is a bacterial, viral, parasitic, cancer protein or polypeptide-carrying an acylated N-terminal cysteine,
said heterologous protein is fused to a lipoprotein leader sequence, said lipoprotein leader sequence causing said heterologous proteins to become lipidated,
and
said OMV is capable of eliciting an immune response to the heterologous protein when administered to a mammal,
said method comprising the following steps:
(i) expressing, in a Gram-negative bacterium, the heterologous protein fused to said leader sequence carrying a C-terminal Cysteine, wherein the leader sequence comprises the sequence Leu-(Ala/Ser)-(Gly/Ala)-Cys (lipobox), SEQ ID NO:111; and
(ii) isolating the OMV containing the heterologous protein.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the leader sequence is the murine lipoprotein Lpp MKATKLVLGAVILGSTLLAGC, SEQ ID NO:83, according to the one-letter amino acid code.

### Claim 3 (depends on 1)

The method according to claim 1, wherein said Gram-negative bacterium is a hyperblebbing strain of the Gram-negative bacterium.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the Gram-negative bacterium carries mutations at the ompA, msbB and pagP genes causing inactivation or deletion thereof.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the Gram-negative bacterium is selected from the group consisting of Escherichia coli, Neisseria menigitidis, Salmonella sp. and Shigella sp.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the heterologous protein is expressed in the Gram-negative bacterium by means of an expression vector comprising a nucleic acid encoding the heterologous protein linked to a nucleic acid encoding a signal sequence of a lipoprotein.

### Claim 7 (depends on 6)

The method according to claim 6, wherein said vector is either a plasmid or a vector which is integrated into the genome of the host strain producing the OMV.

### Claim 8 (depends on 1)

The method of claim 1, wherein the heterologous protein is selected from: double mutant of extracellular cholesterol depending streptolysin O (Slo-dm) from Streptococcus pyogenes; HlaH35L from Staphylococcus aureus; SpaKKAA antigen from Staphylococcus aureus; and LukE antigen from Staphylococcus aureus.