# US 7,608,272 B2 · Methods and compositions for vaccination of animals with PRRSV antigens with improved immunogenicity

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 7,608,272 B2
- Document ID: US7608272B2
- Status: Granted
- Kind: B2
- Application number: 12/064,877
- Filed: 2006-08-30
- Published: 2008-09-25
- Granted: 2009-10-27
- Earliest claimed: 2005-08-30
- Assignees: Board of Regents of the University of Nebraska
- Inventors: Israrul H. Ansari; Fernando A. Osorio; Asit K. Pattnaik
- Web page: https://patentgrep.com/patent/US7608272B2

## CPC

- A61K 39/12: Viral antigens
- A61P 31/12: Antivirals
- A61P 37/02: Immunomodulators
- C07K 14/005: from viruses
- A61K 2039/552: {Veterinary vaccine}
- C12N 2710/24143: viral genome or elements thereof as genetic vector
- C12N 2770/10022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2770/10034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- 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)

## Abstract

Pigs challenged with hypoglycosylated variants of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) major surface protein GP5 exhibited increased production of PRRSV-neutralizing antibodies relative to the levels of neutralizing antibodies produced by pigs immunized with wild type (wt) or glycosylated GP5. This invention provides for methods of obtaining improved immune responses in pigs to PRRSV, compositions useful for obtaining the improved immune responses as well as isolated polynucleotides that encode hypoglycosylated variants of PRRSV major surface protein GP5.

## Claims

### Claim 1

A composition comprising a polynucleotide encoding a hypoglycosylated North American PRRSV GP5 polypeptide variant wherein at least one N-linked glycosylation site corresponding to asparagine 51 in a reference GP5 protein of SEQ ID NO: 1 is inactivated by mutation, and a therapeutically acceptable carrier.

### Claim 2 (depends on 1)

The composition of claim 1, wherein said polynucleotide comprises an infectious North American PRRSV RNA molecule.

### Claim 3 (depends on 1)

The composition of claim 1, wherein said polynucleotide comprises a DNA molecule that encodes an infectious North American PRRSV RNA molecule.

### Claim 4 (depends on 1)

The composition of claim 1, wherein said polynucleotide comprises a DNA molecule wherein a promoter active in mammalian cells is operably linked to said polynucleotide encoding said hypoglycosylated North American PRRSV GP5 polypeptide variant.

### Claim 5 (depends on 4)

The composition of claim 4, wherein said promoter is a CMV promoter.

### Claim 6 (depends on 1)

The composition of claim 1, wherein said N-linked glycosylation site is inactivated by replacing a codon encoding said asparagine 51 with a codon encoding an amino acid other than asparagine.

### Claim 7 (depends on 6)

The composition of claim 6, wherein said codon encoding an amino acid other than asparagine encodes an alanine or a glutamine residue.

### Claim 8 (depends on 1)

The composition of claim 1, wherein said polynucleotide encodes a hypoglycosylated North American PRRSV GP5 polypeptide variant protein wherein both of said N-linked glycosylation sites corresponding to asparagine 34 and asparagine 51 in a North American reference GP5 protein of SEQ ID NO:1 are inactivated.

### Claim 9 (depends on 8)

The composition of claim 8, wherein both of said N-linked glycosylation sites are inactivated by replacing codons encoding said asparagine 34 and said asparagine 51 with codons encoding an amino acid other than asparagine.

### Claim 10 (depends on 9)

The composition of claim 9, wherein said codons encoding another amino acid encode either an alanine or a glutamine residue.

### Claim 11 (depends on 8)

The composition of claim 8, wherein one of said N-linked glycosylation sites is inactivated by replacing one codon encoding said asparagine 34 or said asparagine 51 with a codon encoding an amino acid other than asparagine.

### Claim 12 (depends on 1)

The composition of claim 1, wherein said therapeutically acceptable carrier is selected from the group consisting of a protein, a buffer, a surfactant, and a polyethylene glycol polymer, or any combination thereof.

### Claim 13 (depends on 1)

The composition of claim 1, wherein said composition further comprises at least one adjuvant.

### Claim 14 (depends on 13)

The composition of claim 13, wherein said adjuvant is selected from the group consisting of aluminum hydroxide, Quil A, an alumina gel suspension, mineral oils, glycerides, fatty acids, fatty acid by-products, mycobacteria, and CpG oligodeoxynucleotides, or any combination thereof.

### Claim 15 (depends on 13)

The composition of claim 13, wherein said composition further comprises a second adjuvant is selected from the group consisting of interleukin 1 (IL-1), IL-2, IL4, IL-5, IL6, IL-12, gamma interferon (g-IFN), cell necrosis factor, MDP (muramyl dipeptide), immuno stimulant complex (ISCOM), and liposomes.

### Claim 16 (depends on 1)

The composition of claim 1, wherein said polynucleotide comprises a viral vector selected from the group consisting of a vaccinia virus vector, a herpes simplex viral vector, an adenovirus vector, an alphavirus vector, and a TGEV vector.

### Claim 17

An isolated polynucleotide encoding a hypoglycosylated North American PRRSV GP5 polypeptide variant wherein at least one N-linked glycosylation site corresponding to asparagine 51 in a reference GP5 protein of SEQ ID NO:1 is inactivated by mutation.

### Claim 18 (depends on 17)

The isolated polynucleotide of claim 17, wherein both N-linked glycosylation sites corresponding to asparagine 34 and asparagine 51 in SEQ ID NO:1 are inactivated.

### Claim 19 (depends on 17)

The isolated polynucleotide of claim 17, wherein said polynucleotide comprises an infectious North American PRRSV RNA molecule.

### Claim 20 (depends on 17)

The isolated polynucleotide of claim 17, wherein said polynucleotide comprises a DNA molecule that encodes an infectious North American PRRSV RNA molecule.

### Claim 21 (depends on 17)

The isolated polynucleotide of claim 17, wherein said polynucleotide comprises a DNA molecule wherein a promoter active in mammalian cells is operably linked to said polynucleotide encoding said hypoglycosylated North American PRRSV GP5 polypeptide variant.

### Claim 22 (depends on 21)

The isolated polynucleotide of claim 21, wherein said promoter is a CMV promoter.

### Claim 23 (depends on 17)

The isolated polynucleotide of claim 17, wherein said N-linked glycosylation site corresponding to asparagine 51 is inactivated by replacing a codon encoding said asparagine 51 with a codon encoding an amino acid other than asparagine.

### Claim 24 (depends on 23)

The isolated polynucleotide of claim 23, wherein said codon encoding another amino acid encodes an alanine or a glutamine residue.

### Claim 25 (depends on 17)

The isolated polynucleotide of claim 17, wherein an N-linked glycosylation site corresponding to asparagine 34 in a reference GP5 protein of SEQ ID NO:1 is inactivated.

### Claim 26 (depends on 25)

The isolated polynucleotide of claim 25, by replacing a codon encoding said asparagine 34 with a codon encoding an amino acid other than asparagine.

### Claim 27 (depends on 25)

The isolated polynucleotide of claim 25, wherein said codon encoding another amino acid encodes an alanine or a glutamine residue.

### Claim 28 (depends on 25)

The isolated polynucleotide of claim 25, wherein one of said N-linked glycosylation sites is inactivated by replacing one codon encoding said asparagine 34 or said asparagine 51 with a codon encoding an amino acid other than asparagine.

### Claim 29 (depends on 17)

The isolated polynucleotide of claim 17, wherein said polynucleotide comprises a viral vector selected from the group consisting of a vaccinia virus vector, a herpes simplex viral vector, an adenovirus vector, an alphavirus vector and a TGEV vector.