# US 10,501,504 B2 · Adhesin as immunogen against enterotoxigenic Escherichia coli

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- Number: US 10,501,504 B2
- Document ID: US10501504B2
- Status: Granted
- Kind: B2
- Application number: 15/991,443
- Filed: 2018-05-29
- Published: 2019-02-21
- Granted: 2019-12-10
- Earliest claimed: 2005-01-11
- Assignees: The United States of America as represented by the Secretary of the Navy
- Applicants: United States of America as Represented by the Secretary of the Navy
- Inventors: Stephen J. Savarino
- Web page: https://patentgrep.com/patent/US10501504B2

## CPC

- C07K 14/245: Escherichia (G)
- A61K 39/0258: {Escherichia}
- A61P 1/12: Antidiarrhoeals
- A61P 31/04: Antibacterial agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 16/1232: Escherichia (G)
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61K 2039/523: {expressing foreign proteins}
- A61K 2039/545: {characterised by the dose, timing or administration schedule}
- C07K 2317/55: Fab or Fab'
- C07K 2319/00: Fusion polypeptide
- C07K 2319/35: containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
- 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
- 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)
- 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
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The inventive subject matter relates to the methods for the induction of immunity and prevention of diarrhea resulting from Escherichia coli. The inventive subject matter also relates to the use Escherichia coli adhesins as immunogens and to the construction of conformationally stability and protease resistant Escherichia coli adhesin constructs useful for inducing immunity to Escherichia coli pathogenic bacteria. The methods provide for the induction of B-cell mediated immunity and for the induction of antibody capable of inhibiting the adherence and colonization of Escherichia coli, including enterotoxigenic Escherichia coli, to human cells.

## Claims

### Claim 1

A method of inducing an immune response against enterotoxigenic Escherichia coli comprising the steps:
a. administering a priming dose of an immunogen comprising whole or a polypeptide fragment of enterotoxigenic Escherichia coli fimbriae or whole or a polypeptide fragment of enterotoxigenic Escherichia coli fibrillae, with a unit dose range of said priming dose of 50 μg to 1 mg of said immunogen; and
b. administering boosting doses of said immunogen, with a first dose administered at least 1 week after said priming dose, with a unit dose range of said boosting doses of 50 μg to 1 mg of said immunogen, whereby an immune response is elicited;
wherein said immunogens are conformationally stable variants engineered using in cis donor strand complementation; wherein said whole or a polypeptide fragment of enterotoxigenic Escherichia coli fimbriae are conformationally-stable Escherichia coli fimbrial adhesin polypeptide constructs comprising a major structural fimbrial subunit; and wherein said whole or a polypeptide fragment of ore-said enterotoxigenic Escherichia coli fibrillae are conformationally-stable CstH constructs.

### Claim 2 (depends on 1)

The method of claim 1, wherein said enterotoxigenic Escherichia coli fimbriae is a class 5 fimbriae.

### Claim 3 (depends on 1)

The method of claim 1, wherein said enterotoxigenic Escherichia coli fimbriae is selected from the group consisting of colonization factor antigen I, CS4, CS14, CS1, PCF071, CS17, CS19 and CS2.

### Claim 4 (depends on 1)

The method of claim 1, wherein said enterotoxigenic Escherichia coli fimbrial adhesin is selected from the group consisting of CfaE, CsfD, CsuD, CooD, CosD, CsdD, CsbD and CotD.

### Claim 5 (depends on 1)

The method of claim 1, wherein said immune response is induction of immunoglobulin molecules capable of binding to said enterotoxigenic Escherichia coli fimbriae or fibrillae.

### Claim 6 (depends on 1)

The method of claim 1, wherein said immune response inhibits enterotoxigenic Escherichia coli fimbriae or fibrillae adherence to human cells.

### Claim 7 (depends on 6)

The method of claim 6, wherein said human cells are mucosal epithelial cells.

### Claim 8 (depends on 1)

The method of claim 1, wherein said immune response reduces diarrhea in humans.

### Claim 9 (depends on 1)

The method of claim 1, wherein said immunogen is administered subcutaneously, transdermally, intramuscularly, orally, transcutaneously or nasally.

### Claim 10 (depends on 1)

The method of claim 1 wherein said CstH construct is [His]10dsc16CstH (SEQ ID NO:21) or dsc16CstH-[His]6 (SEQ ID NO:22).

### Claim 11 (depends on 4)

The method of claim 4 wherein said enterotoxigenic Escherichia coli fimbrial adhesin is CfaE.

### Claim 12 (depends on 1)

The method of claim 1 wherein said enterotoxigenic Escherichia coli fimbrial adhesin is CfaE, and wherein said major structural fimbrial subunit is a major subunit donor β strand of CfaB.

### Claim 13 (depends on 1)

The method of claim 1 wherein said immunogen is dscCfaE or dsc19CfaE[His]6.

### Claim 14 (depends on 1)

The method of claim 1 wherein said priming and said boosting doses are administered with adjuvant.

### Claim 15 (depends on 14)

The method of claim 14 wherein said adjuvant is mLT.

### Claim 16 (depends on 14)

The method of claim 14 wherein said adjuvant is administered simultaneously with said priming and said boosting doses.

### Claim 17 (depends on 1)

The method of claim 1 wherein 2 to 4 boosting doses are administered.

### Claim 18 (depends on 1)

The method of claim 1 wherein said priming and said boosting doses are provided in a buffered aqueous solution.

### Claim 19 (depends on 12)

The method of claim 12 wherein said major subunit donor β strand of CfaB is encoded by SEQ ID No: 7.

### Claim 20 (depends on 1)

The method of claim 1 wherein said major structural fimbrial subunit is a major subunit donor β strand selected from the group consisting of SEQ ID Nos: 8-15.

### Claim 21 (depends on 1)

The method of claim 1 wherein said immunogen is administered as a whole cell vaccine.