# US 11,992,522 B2 · Attenuating bacterial virulence by attenuating bacterial folate transport

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- Number: US 11,992,522 B2
- Document ID: US11992522B2
- Status: Granted
- Kind: B2
- Application number: 18/316,499
- Filed: 2023-05-12
- Published: 2024-04-11
- Granted: 2024-05-28
- Earliest claimed: 2016-11-11
- Applicants: Boehringer Ingelheim Vetmedica GmbH
- Inventors: Alaina Ingebritson; Axel Neubauer; Hilda Elizabeth Smith; Astrid De Greeff
- Web page: https://patentgrep.com/patent/US11992522B2

## CPC

- A61K 39/092: {Streptococcus}
- A61P 31/04: Antibacterial agents
- C07K 14/315: from Streptococcus (G), e.g. Enterococci
- C12N 1/205: Bacterial isolates
- A61K 2039/522: {avirulent or attenuated}
- A61K 2039/542: {oral/gastrointestinal}
- A61K 2039/552: {Veterinary vaccine}
- C12R 2001/46: Streptococcus {; Enterococcus; Lactococcus}
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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

## Abstract

The invention relates to bacterial infections, vaccines directed against those infections and bacterial vaccines. More particularly, the invention relates to vaccines directed against Streptococcus infections in pigs. The invention provides a ΔFolT mutant of a bacterium having reduced capacity to transport folate, wherein said capacity has been reduced by functionally deleting folate transporter (FolT) function. The invention also provides a method to reduce virulence of a bacterium comprising reducing the capacity of said bacterium to transport folate.

## Claims

### Claim 1

A method to reduce virulence of a Streptococcus suis (S. suis) bacterium comprising reducing the capacity of said bacterium to transport folate compared to wild type; wherein said capacity has been reduced by deletion or inactivation of a gene of the S. suis encoding folate transporter (FolT) function.

### Claim 2 (depends on 1)

A method according to claim 1 wherein said bacterium is capable of de novo folate synthesis.

### Claim 3 (depends on 1)

A method according to claim 1 comprising attenuating expression and/or function of the folate substrate binding protein (FolT) of said bacterium.

### Claim 4 (depends on 1)

A method according to claim 1 wherein said virulence is attenuated by providing a mutation, deletion or insertion in the foltT gene of said bacterium.

### Claim 5 (depends on 4)

A method according to claim 4 wherein said virulence is attenuated by providing a mutation, deletion or insertion in the DNA of said bacterium encoding the promotor of the folT gene.

### Claim 6 (depends on 4)

A method according to claim 4 wherein said virulence is attenuated by providing a mutation, deletion or insertion in the DNA of said bacterium encoding a transmembrane region of folate substrate binding protein FolT.

### Claim 7 (depends on 4)

A method according to claim 4 wherein said virulence is attenuated by providing a mutation, deletion or insertion in the FolT encoding DNA region of said bacterium encoding a region crucial for substrate binding.

### Claim 8 (depends on 1)

A method according to claim 1 wherein said bacterium is a Firmicutes, preferably a Streptococcus, more preferably a Streptococcus suis, more preferably a ΔFolT mutant deposited as “CBS 140425 Streptococcus suis ΔFolT mutant” at the Centraalbureau voor Schimmelcultures at Aug. 19, 2015, most preferably a ΔFolT mutant deposited as “CBS 143192 Streptococcus suis ΔFolT2 mutant” at the Westerdijk Fungal Biodiversity Institute at Aug. 25, 2017.

### Claim 9 (depends on 1)

A bacterium with attenuated virulence obtainable or obtained with a method according to claim 1.

### Claim 10

A method of treating Streptococcus infection in swine comprising administering a composition comprising a recombinant ΔFolT mutant of a Streptococcus suis (S. suis) bacterium having reduced capacity to transport folate compared to wild type, wherein said capacity has been reduced by deletion or inactivation of a gene of the S. suis encoding folate transporter (FolT) function.

### Claim 11 (depends on 10)

The method of treating Streptococcus infection in swine of claim 10, wherein the composition comprising the recombinant ΔFolT mutant bacterium is administered intravenously.

### Claim 12 (depends on 11)

The method of claim 11, wherein the recombinant ΔFolT mutant bacterium is present in an amount of about 9×105 CFU to 1.5×107 CFU.

### Claim 13 (depends on 12)

The method of claim 12, wherein the recombinant ΔFolT mutant bacterium is present in an amount of 9×105 CFU.

### Claim 14 (depends on 12)

The method of claim 12, wherein the recombinant ΔFolT mutant bacterium is present in an amount of 1.5×107 CFU.