# US 11,433,127 B2 · Non-toxigenic Clostridium difficile spores for use in oral vaccination

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- Number: US 11,433,127 B2
- Document ID: US11433127B2
- Status: Granted
- Kind: B2
- Application number: 16/858,029
- Filed: 2020-04-24
- Published: 2020-08-13
- Granted: 2022-09-06
- Earliest claimed: 2017-02-24
- Assignees: University of South Florida
- Applicants: University of South Florida; Trustees of Tufts College
- Inventors: Xingmin Sun; Abraham Sonenshein
- Web page: https://patentgrep.com/patent/US11433127B2

## CPC

- A61K 39/08: Clostridium, e.g. Clostridium tetani
- C07K 14/33: from Clostridium (G)
- C12N 9/1048: {Glycosyltransferases (2.4)}
- C12N 9/52: derived from bacteria {or Archaea}
- A61K 2039/542: {oral/gastrointestinal}
- A61K 2039/575: {humoral response}
- C07K 2319/40: containing a tag for immunodetection, or an epitope for immunisation
- 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)

## Abstract

Described are non-toxigenic Clostridium difficile strains and spores. Also described are vaccines comprising the Clostridium difficile spores. Further described are methods of preventing or treating a Clostridium difficile infection in a subject in need thereof.

## Claims

### Claim 1

A method of treating or preventing Clostridium difficile bacterial infection in a subject in need thereof, comprising administering to the subject a vaccine that comprises a non-toxigenic Clostridium difficile strain comprising:
a) an immunogenic protein comprising
i) a glucosyltransferase domain of Clostridium difficile toxin TcdB;
ii) a cysteine proteinase domain of Clostridium difficile toxin TcdB;
iii) a receptor binding domain of Clostridium difficile toxin TcdA; and
iv) a receptor binding domain of Clostridium difficile toxin TcdB,
wherein the glucosyltransferase domain of Clostridium difficile toxin TcdB comprises a W102A amino acid substitution and a D288N amino acid substitution when compared to SEQ ID NO.: 5, and wherein the cysteine proteinase domain of Clostridium difficile toxin TcdB comprises a C698A amino acid substitution when compared to SEQ ID NO.: 5.

### Claim 2 (depends on 1)

The method of claim 1, wherein the glucosyltransferase domain of Clostridium difficile toxin TcdB is positioned immediately upstream of the cysteine proteinase domain of Clostridium difficile toxin TcdB, wherein the amino acid sequence of the linked glucosyltransferase domain of Clostridium difficile toxin TcdB and the cysteine proteinase domain of Clostridium difficile toxin TcdB is set forth in SEQ ID NO.: 9; wherein the receptor binding domain of Clostridium difficile toxin TcdA comprises the amino acid sequence of SEQ ID NO.: 6; and wherein the receptor binding domain of Clostridium difficile toxin TcdB comprises the amino acid sequence of SED ID NO.: 7.

### Claim 3 (depends on 1)

The method of claim 1, wherein the immunogenic protein comprises the amino acid sequence of SEQ ID NO.: 3.

### Claim 4 (depends on 1)

The method of claim 1, wherein the non-toxigenic Clostridium difficile strain is non-toxigenic Clostridium difficile strain 169 (NTCD_Tcd169).

### Claim 5 (depends on 1)

The method of claim 1, wherein the non-toxigenic Clostridium difficile strain forms spores.

### Claim 6 (depends on 1)

The method of claim 1, wherein the vaccine further comprises a pharmaceutically acceptable excipient or carrier.

### Claim 7 (depends on 1)

The method of claim 1, wherein the vaccine is administered orally or intramuscularly.

### Claim 8 (depends on 1)

The method of claim 1, wherein the vaccine increases levels of anti-TcdA and anti-TcdB IgG antibodies in the subject.

### Claim 9 (depends on 1)

The method of claim 1, wherein the Clostridium difficile bacterial infection is caused by a hyper-virulent strain of Clostridium difficile.