# US 8,771,703 B2 · Soluble recombinant influenza antigens

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- Number: US 8,771,703 B2
- Document ID: US8771703B2
- Status: Granted
- Kind: B2
- Application number: 13/001,111
- Filed: 2009-07-07
- Published: 2011-05-05
- Granted: 2014-07-08
- Earliest claimed: 2008-07-08
- Assignees: Medicago Inc.
- Applicants: Manon Couture; Nathalie Landry; Louis-Philippe Vezina; Michéle Dargis
- Inventors: Manon Couture; Nathalie Landry; Louis-Philippe Vezina; Michéle Dargis
- Web page: https://patentgrep.com/patent/US8771703B2

## CPC

- C12N 15/8257: {for the production of primary gene products, e.g. pharmaceutical products, interferon}
- A61K 39/12: Viral antigens
- A61K 39/145: Orthomyxoviridae, e.g. influenza virus
- A61P 31/16: for influenza or rhinoviruses
- C07K 14/005: from viruses
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- C07K 2319/04: containing an ER retention signal such as a C-terminal HDEL motif
- C07K 2319/50: containing protease site
- C07K 2319/70: containing domain for protein-protein interaction
- C07K 2319/73: containing coiled-coiled motif (leucine zippers)
- C07K 2319/735: containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
- C12N 2760/16122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2760/16134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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
- 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)

## Abstract

The present invention provides a recombinant soluble trimeric hemagglutinin (rHA) protein comprising a hemagglutinin ectodomain and an oligomerization domain. The rHA is produced as a soluble homotrimer, and may further comprises a signal peptide and/or an endoplasmic reticulum (ER) retention signal. The invention is also directed to nucleic acids encoding the rHA of the invention, as well as vectors and chimeric constructs comprising the nucleic acid. Methods of producing the rHA are also provided. The rHA described herein may be used to formulate influenza vaccines, or may be used to enrich existing vaccines.

## Claims

### Claim 1

A recombinant hemagglutinin (rHA), comprising:
a) a hemagglutinin domain selected from the group consisting of hemagglutinin subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16; b) an oligomerization domain selected from the group consisting of a GCN4-pII peptide, a proline rich domain (PRD) of maize γ-zein, and a bacteriophage T4 fibritin;
wherein amino acid residues are added to the C-terminal end of the oligomerization domain such that the oligomerization domain from GCN4-pII or bacteriophage T4 fibritin does not terminate in an α-helix and the oligomerization domain from the PRD of maize γ-zein does not terminate in an amphipathic polyproline II helix;
c) a signal peptide; and
d) an endoplasmic reticulum (ER) retention signal;
wherein the rHA is produced as a chimeric soluble homotrimer.

### Claim 2 (depends on 1)

A nucleotide sequence encoding the rHA of claim 1.

### Claim 3

A nucleic acid comprising:
a) a nucleotide sequence encoding a hemagglutinin domain selected from the group consisting of hemagglutinin subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16;
b) a nucleotide sequence encoding an oligomerization domain selected from the group consisting of a GCN4-pII peptide, a proline rich domain (PRD) of maize γ-zein, and a bacteriophage T4 fibritin;
wherein amino acid residues are added to the C-terminal end of the oligomerization domain such that the oligomerization domain from GCN4-pII or bacteriophage T4 fibritin does not terminate in an α-helix and the oligomerization domain from the PRD of maize γ-zein does not terminate in an amphipathic polyproline II helix;
c) a nucleotide sequence encoding a signal peptide; and
d) a nucleotide sequence encodinng an endoplasmic reticulum (ER) retention signal;
wherein the nucleic acid encodes a soluble recombinant hemagglutinin (rHA) that forms a homotrimer.

### Claim 4 (depends on 2)

A vector comprising the nucleotide sequence of claim 2.

### Claim 5 (depends on 1)

A host cell expressing the rHA of claim 1.

### Claim 6 (depends on 2)

A host cell transformed with the nucleotide sequence of claim 2.

### Claim 7 (depends on 4)

A host cell transformed with the vector of claim 4.

### Claim 8 (depends on 3)

A method of producing rHA, comprising providing a host cell comprising the nucleic acid of claim 3, and expressing the rHA in the host cell.

### Claim 9 (depends on 4)

A method of expressing rHA within a plant comprising introducing a vector of claim 4 into a plant and expressing the rHA.

### Claim 10 (depends on 9)

The method of claim 9, wherein the nucleic acid is introduced into the plant in a transient manner.

### Claim 11 (depends on 9)

The method of claim 9, wherein the nucleic acid is introduced into the plant so that it is stably transformed.

### Claim 12 (depends on 3)

A method of producing a recombinant hemagglutinin (rHA) in a plant comprising:
a) introducing a nucleic acid sequence into the plant, or portion thereof, the nucleic acid sequence comprising a regulatory region operatively linked to the nucleic acid of claim 3 and
b) growing the transgenic plant, thereby producing the rHA.

### Claim 13 (depends on 12)

The method of claim 12, wherein the nucleic acid is introduced into the plant in a transient manner.

### Claim 14 (depends on 12)

The method of claim 12, wherein the nucleic acid is introduced into the plant so that it is stably transformed.

### Claim 15 (depends on 1)

The rHA of claim 1, wherein the added amino acid residues are Ser-Ala-Ala.

### Claim 16 (depends on 1)

The rHA of claim 1, wherein the ER retention signal is SEKDEL (SEQ ID NO:4) or HDEL (SEQ ID NO:5).

### Claim 17 (depends on 3)

The nucleic acid of claim 3, wherein the added amino acid residues are Ser-Ala-Ala.

### Claim 18 (depends on 3)

The nucleic acid of claim 3, wherein the ER retention signal is SEKDEL (SEQ ID NO:4) or HDEL (SEQ ID NO:5).

### Claim 19 (depends on 3)

A vector comprising the nucleic acid of claim 3.

### Claim 20 (depends on 3)

A host cell transformed with the nucleic acid of claim 3.

### Claim 21 (depends on 19)

A host cell transformed with the vector of claim 19.

### Claim 22 (depends on 1)

The rHA of claim 1, wherein the signal peptide is a protein disulfide isomerase (PDI) signal peptide.

### Claim 23 (depends on 3)

The nucleic acid of claim 3, wherein the signal peptide is a protein disulfide isomerase (PDI) signal peptide.