# US 9,750,793 B2 · Multifunctional oral vaccine based on chromosome recombineering

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- Number: US 9,750,793 B2
- Document ID: US9750793B2
- Status: Granted
- Kind: B2
- Application number: 14/428,870
- Filed: 2013-09-16
- Published: 2015-08-27
- Granted: 2017-09-05
- Earliest claimed: 2012-09-17
- Assignees: The United States of America, as represented by the Secretary, Department of Health and Human Services
- Applicants: The United States of America, as represented by the Secretary, Dept. of Health and Human Services
- Inventors: Madushini Nirosha Dharmasena; Dennis J. Kopecko
- Web page: https://patentgrep.com/patent/US9750793B2

## CPC

- A61K 39/0283: {Shigella}
- A61K 39/0275: {Salmonella}
- A61P 31/04: Antibacterial agents
- C07K 14/25: Shigella (G)
- C07K 14/255: Salmonella (G)
- C12N 1/20: Bacteria; Culture media therefor
- A61K 2039/522: {avirulent or attenuated}
- A61K 2039/523: {expressing foreign proteins}
- A61K 2039/542: {oral/gastrointestinal}
- A61K 2039/70: {Multivalent vaccine}
- C07K 2319/40: containing a tag for immunodetection, or an epitope for immunisation
- 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)
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

A recombineered Salmonella typhi Ty21a, compositions and vaccines comprising such a Ty21a, and a method for recombineering comprising inserting a large antigenic region into a bacterial chromosome for the purpose of making multivalent vaccines to protect against one or more disease agents are described herein.

## Claims

### Claim 1

A Salmonella Typhi Ty21a comprising a Shigella sonnei form 1 O-antigen biosynthetic gene region inserted into the Salmonella Typhi Ty21a chromosome, wherein:
a) heterologous Shigella sonnei form 1 O-antigen is stably expressed together with or without homologous Salmonella Typhi O-antigen;
b) immune protection is elicited against virulent Shigella sonnei challenge; and
c) immune protection is elicited against virulent Salmonella Typhi challenge when heterologous Shigella sonnei form 1 O-antigen is stably expressed together with homologous Salmonella Typhi O-antigen; and
d) the region comprises a DNA sequence, wherein the DNA sequence comprises:
1) the DNA sequence as set out in SEQ ID NO:2, or
2) a DNA sequence that shares at least about 90% sequence identity with the DNA sequence set out in SEQ ID NO:2.

### Claim 2 (depends on 1)

The Salmonella Typhi Ty21a of claim 1, further comprising an O-antigen biosynthetic gene region from a bacterial strain selected from the group consisting of: Shigella species, Escherichia coli serotypes, Salmonella enterica serovars, Vibrio cholerae serotypes, Enterobacter species, Yersinia species, Plesiomonas species, and Pseudomonas species.

### Claim 3

A Salmonella Typhi Ty21a comprising a Shigella dysenteriae 1 O-antigen biosynthetic gene region inserted into the Salmonella Typhi Ty21a chromosome, wherein:
a) heterologous Shigella dysenteriae serotype 1 O-antigen is stably expressed together with or without homologous Salmonella Typhi O-antigen;
b) immune protection is elicited against virulent Shigella dysenteriae challenge; and
c) immune protection is elicited against virulent Salmonella Typhi challenge when heterologous Shigella dysenteriae serotype 1 O-antigen is stably expressed together with homologous Salmonella Typhi O-antigen; and
d) the region comprises a DNA sequence, wherein the DNA sequence comprises:
1) the DNA sequence as set out in SEQ ID NO:33, or
2) a DNA sequence that shares at least about 90% sequence identity with the DNA sequence set out in SEQ ID NO:33.

### Claim 4 (depends on 3)

The Salmonella Typhi Ty21a of claim 3, further comprising an O-antigen biosynthetic gene region from a bacterial strain selected from the group consisting of: Shigella species, Escherichia coli serotypes, Salmonella enterica serovars, Vibrio cholerae serotypes, Enterobacter species, Yersinia species, Plesiomonas species, and Pseudomonas species.

### Claim 5

A plasmid construct having i) a DNA sequence as set out in SEQ ID NO: 1 or ii) a DNA sequence that shares at least about 90% sequence identity with the DNA sequence set out in SEQ ID NO:1.

### Claim 6 (depends on 5)

The plasmid construct of claim 5, further comprising a Shigella sonnei O-antigen biosynthetic gene region or a Shigella dysenteriae 1 O-antigen biosynthetic gene region.

### Claim 7

A method of recombineering a large antigenic gene region into a bacterial chromosome, comprising:
i) cloning the region into a vector containing:
ia) a genetically selectable marker flanked 5′ and 3′ by an FRT site, respectively;
ib) a multiple cloning site downstream of the 3′ FRT site; and
ic) two sites of chromosome homology, one of the two located upstream of the 5′ FRT site, and one of the two located downstream of the multiple cloning site;
ii) integrating the region into the bacterial chromosome using X, red recombination;
iii) selecting for the genetically selectable marker; and
iv) removing the selectable marker,
thus recombineering the large antigenic gene region into the chromosome.

### Claim 8 (depends on 7)

The method of claim 7, wherein the large antigenic gene region is about 5 to about 20 kb long.

### Claim 9 (depends on 7)

The method of claim 7, wherein the vector is selected from the group consisting of a plasmid, phage, phasmid, and cosmid construct.

### Claim 10 (depends on 9)

The method of claim 9, wherein the plasmid construct has i) a DNA sequence as set out in SEQ ID NO: 1 or ii) a DNA sequence that shares at least about 90% sequence identity with the DNA sequence set out in SEQ ID NO:1.

### Claim 11 (depends on 7)

The method of claim 7, wherein the bacterial chromosome is from Salmonella Typhi Ty21a.

### Claim 12 (depends on 7)

The method of claim 7, wherein the genetically selectable marker is an antibiotic resistance marker.

### Claim 13 (depends on 12)

The method of claim 12, wherein the antibiotic resistance marker is kanamycin.

### Claim 14 (depends on 7)

The method of claim 7, wherein the large antigenic gene region is selected from the group consisting of a Shigella sonnei O-antigen biosynthetic gene region, a Shigella dysenteriae 1 O-antigen biosynthetic gene region, a Shigella flexneri 2a 0-antigen biosynthetic gene region, and a Shigella flexneri 3a O-antigen biosynthetic gene region.

### Claim 15 (depends on 7)

The method of claim 7, wherein the large antigenic gene region is engineered between the two sites of chromosome homology, each site comprising between about 500 to about 1000 bp regions of bacterial chromosome homology before step ii.

### Claim 16 (depends on 13)

The method of claim 13, wherein the kanamycin resistance gene is removed via recombination induced following transformation of chromosomal integrants of step ii with pCP20.

### Claim 17

A vaccine comprising a pharmaceutically acceptable solid carrier and Salmonella Typhi Ty21a comprising a Shigella flexneri 2a O-antigen biosynthetic gene region inserted into the Salmonella Typhi Ty21a chromosome, wherein:
a) heterologous Shigella flexneri 2a O-antigen is stably expressed together with or without homologous Salmonella Typhi O-antigen;
b) immune protection is elicited against virulent Shigella flexneri 2a challenge; and
c) immune protection is elicited against virulent Salmonella Typhi challenge when heterologous Shigella flexneri 2a O-antigen is stably expressed together with homologous Salmonella Typhi O-antigen.

### Claim 18

A vaccine comprising a pharmaceutically acceptable solid carrier and Salmonella Typhi Ty21a comprising a Shigella flexneri 3a O-antigen biosynthetic gene region inserted into the Salmonella Typhi Ty21a chromosome, wherein:
a) heterologous Shigella flexneri 3a O-antigen is stably expressed together with or without homologous Salmonella Typhi O-antigen;
b) immune protection is elicited against virulent Shigella flexneri 3a challenge; and
c) immune protection is elicited against virulent Salmonella Typhi challenge when heterologous Shigella flexneri 3a O-antigen is stably expressed together with homologous Salmonella Typhi O-antigen.

### Claim 19 (depends on 1)

A vaccine comprising the Salmonella Typhi Ty21a of claim 1 in combination with a physiologically acceptable carrier.

### Claim 20 (depends on 1)

A method of treating at least one bacterial infection comprising administering a prophylactically or therapeutically effective amount of the Salmonella Typhi Ty21a of claim 1 to a subject, thus treating the at least one bacterial infection.

### Claim 21 (depends on 1)

The Salmonella Typhi Ty21a of claim 1, wherein the Shigella sonnei form 1 O-antigen biosynthetic gene region is partially or wholly chemically synthesized.

### Claim 22 (depends on 5)

The plasmid construct of claim 5, wherein the DNA sequence is partially or wholly chemically synthesized.