# US 11,077,200 B2 · Combined enteropathogen recombinant construct

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- Number: US 11,077,200 B2
- Document ID: US11077200B2
- Status: Granted
- Kind: B2
- Application number: 16/132,948
- Filed: 2018-09-17
- Published: 2019-01-10
- Granted: 2021-08-03
- 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: Patricia Guerry; Mario Artur Monteiro; Stephen Savarino
- Web page: https://patentgrep.com/patent/US11077200B2

## CPC

- A61K 47/646: {the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines}
- A61K 39/0258: {Escherichia}
- A61K 39/0283: {Shigella}
- A61K 39/105: {Delta proteobacteriales, e.g. Lawsonia; Epsilon proteobacteriales, e.g. campylobacter, helicobacter}
- A61K 47/6415: {Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins}
- C07K 14/245: Escherichia (G)
- C07K 16/1232: Escherichia (G)
- C07K 16/1267: Gram-positive bacteria
- C07K 16/44: against material not provided for elsewhere {, e.g. haptens, metals, DNA, RNA, amino acids}
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- A61K 2039/55505: {Inorganic adjuvants}
- A61K 2039/6037: {Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]}
- A61K 2039/70: {Multivalent vaccine}
- C07K 2317/55: Fab or Fab'
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- 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
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The inventive subject matter relates to a construct comprising antigens derived from multiple enterobacteria including Campylobacter jejuni capsule polysaccharide polymer, enterotoxigenic Escherichia coli recombinant polypeptide construct and lipopolysaccharide from Shigella spp. The subject invention also relates to a method of inducing an immune response utilizing the inventive composition.

## Claims

### Claim 1

A multi-agent immunogenic construct comprising a Campylobacter jejuni capsule polysaccharide conjugated to a protein carrier, wherein said protein carrier comprises an Escherichia coli enterotoxigenic recombinant polypeptide construct, wherein said Escherichia coli recombinant polypeptide construct comprises a minor or major subunit connected to one or more major fimbrial subunits or immunogenic fragments, thereof of the same fimbrial type, via a polypeptide linker, and wherein each of the one or more major fimbrial subunits contain, a donor β strand and are also connected to each other via a polypeptide linker wherein the C-terminal Escherichia coli fimbrial major subunit is connected, via a linker, to a C-terminal donor β strand derived from a major Escherichia coli fimbrial subunit that is homologous or heterologous to the immediately N-terminal major subunit, or wherein said recombinant polypeptide construct is connected to one or more additional recombinant polypeptide constructs, wherein each of the constructs contain fimbrial subunits derived from a different fimbrial type than any of the other constructs and wherein the recombinant polypeptide construct can contain a C-terminal histidine tag at the C-terminus, wherein the Escherichia coli recombinant polypeptide construct comprises the amino acid sequence of SEQ ID 104.

### Claim 2 (depends on 1)

The multi-agent immunogenic construct of claim 1, wherein the molar ratio of Campylobacter jejuni to Escherichia coli recombinant protein carrier is 1:1 to 5:1.

### Claim 3 (depends on 1)

The multi-agent immunogenic construct of claim 1, wherein the multi-agent immunogenic construct comprises Shigella lipopolysaccharide in addition to the Campylobacter jejuni capsule polysaccharide.

### Claim 4 (depends on 1)

The multi-agent immunogenic construct of claim 1, wherein the Escherichia coli recombinant polypeptide construct is encoded by the nucleotide sequence of SEQ ID No. 103.

### Claim 5 (depends on 2)

The multi-agent immunogenic construct of claim 2, wherein said Campylobacter jejuni polysaccharide is a repeating trisaccharide structure, wherein the repeating trisaccharide has the formula selected from the group consisting of:
[→3)-α-D-Gal-(1→2)-6d-α-D-altro-Me-Hep-(1→3)-β-D-GlcNAc-(1→]n, wherein “n” represents 1 to 100,
[→3)-β-6-deoxy-D-ido-Heptose (1→4)-β-D-GlcNAc-(1→]n wherein “n” represents 1 to 100;
[→3)-α-Araf-(1→3)-6-d-α-gulo-Hepp-(1→)]n, wherein “n” represents 1 to 100;
wherein “n” represents 1 to 100;
wherein “n” represents 1 to 100
wherein “n” represents 1 to 100;
[→2)-D-glycero-α-D-manno-Hepp-(1-4)-α-D-Glep-(1→]n
, wherein “n” represents 1 to 100;
wherein “n” represents 1 to 100;
[→3)-L-beta-D-ido-Hep-(1->4)-beta-D-Glc-(1→]n, with non-stoichiometric substitution of O-methyl-phosphoramidate at position 2 of L-glyeero-beta-D-ido-heptose and, wherein “n” represents 1 to 100;
[→3)-6d-beta-D-ido-Hep-(1->4)-beta-D-Glc-(1→1]n, derived from HS13, with non-stoichiometric substitution of O-methyl-phosphoramidate at position 2 or/and 7 of 6-deoxy-beta-D-ido-heptose and wherein “n” represents 1 to 100;
[→3)-L-beta-D-ido-Hep-(1->4)-beta-D-Glc-(1→]n and wherein “n” represents 1 to 100;
[→3)-L-alpha-D-ido-Hep-(1->4)-alpha-Gal-(1→]n, with non-stoichiomoetric substitution O-methyl-phosphoramidate at position 2 of 6-alpha-D-ido-heptose and wherein “n” represents 1 to 100;
where “P” represents phosphate; and
[→3)-6d-alpha-D-ido-Hep-(1->4)-alpha-Gal-(1→]n, derived from HS3, HS13 and HS50 with non-stoichiometric substitution of O-methyl-phosphoramidate at position 2 of L-glycero-alpha-D-ido-heptose, wherein “n” is 1 to 100.

### Claim 6 (depends on 3)

The multi-agent immunogenic construct of claim 3, wherein said Shigella lipopolysaccharide has the structure:

### Claim 7 (depends on 2)

The multi-agent immunogenic construct of claim 2, wherein said donor β strand contains 12 to 16 amino acids.

### Claim 8 (depends on 2)

The multi-agent immunogenic construct of claim 2, wherein the N-terminus of said minor or major subunit contains an 18-22 amino acid signal peptide.

### Claim 9 (depends on 2)

The multi-agent immunogenic construct of claim 2, wherein the amino acid sequence of said polypeptide linker is the amino acid sequence of SEQ ID No. 5 or a tri-glycine.

### Claim 10 (depends on 2)

The multi-agent immunogenic construct of claim 2, wherein one or more major subunits contain a deletion of the 14 to 18 N-terminal amino acids.

### Claim 11 (depends on 1)

The multi-agent immunogenic construct of claim 1, wherein said amino add sequence of said Escherichia coli fimbrial major subunit is SEQ ID No. 91.

### Claim 12 (depends on 1)

A method of inducing an immune response against Campylobacter jejuni strains comprising the steps:
a. administering the multi-agent immunogenic composition of claim 1 at a dose range of 0.1 μg to 10 mg per dose;
h. administering a boosting dose of said capsule polysaccharide composition at a dose range of 0.1 μg to 10 mg per dose.

### Claim 13 (depends on 6, 12)

The method of claim 12, wherein said Campylobacter jejuni capsule polysaccharide comprises the polysaccharide structures of claim 6.

### Claim 14 (depends on 3, 12)

The method of claim 12, wherein said multi-agent immunogenic composition comprises the construct of claim 3.

### Claim 15 (depends on 12)

The method of claim 12, wherein said donor β strand contains 12 to 16 amino acids.

### Claim 16 (depends on 12)

The method of claim 12, wherein said N-terminus of said minor or major subunit contains an 18-22 amino acid signal peptide.

### Claim 17 (depends on 12)

The method of claim 12, wherein the amino acid sequence of said polypeptide linker is the amino acid sequence of SEQ ID No. 5 or a tri-glycine.

### Claim 18 (depends on 12)

The method of claim 12, wherein one or more major subunits contain a deletion of the 14 to 18 N-terminal amino acids.