# US 10,548,972 B2 · Gene-deleted variant strain of porcine pseudorabies virus, vaccine composition, method of making the same and use thereof

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- Number: US 10,548,972 B2
- Document ID: US10548972B2
- Status: Granted
- Kind: B2
- Application number: 14/901,981
- Filed: 2015-01-06
- Published: 2016-09-29
- Granted: 2020-02-04
- Earliest claimed: 2014-08-22
- Assignees: Pulike Biological Engineering, Inc.
- Applicants: Pulike Biological Engineering, Inc.
- Inventors: Kegong Tian; Xuke Zhang; Jinzhong Sun; Feifei Tan
- Web page: https://patentgrep.com/patent/US10548972B2

## CPC

- A61K 39/245: Herpetoviridae, e.g. herpes simplex virus
- A61K 39/12: Viral antigens
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61P 31/22: for herpes viruses
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 7/04: Inactivation or attenuation; Producing viral sub-units
- A61K 2039/5252: {inactivated (killed)}
- A61K 2039/5254: {avirulent or attenuated}
- A61K 2039/543: {intranasal}
- A61K 2039/545: {characterised by the dose, timing or administration schedule}
- A61K 2039/552: {Veterinary vaccine}
- C12N 2710/16011: Herpesviridae
- C12N 2710/16634: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2710/16721: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2710/16734: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2710/16762: by genetic engineering
- C12N 2710/16771: Demonstrated in vivo effect
- C12N 2770/20071: Demonstrated in vivo effect
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention provides an attenuated strain of porcine pseudorabies virus (PRV), in which said attenuated strain of PRV is a variant strain of PRV with inactivation of gI/gE/11K/28K proteins. In addition, the present invention also provides a vaccine composition comprising the attenuated strain of PRV as an antigen, a preparation method and use thereof. Proved by immunogenicity and pathogenicity testing of the live vaccine, said live PRV vaccine can provide a good protection for pigs with no clinical signs observed, indicating excellent immune protection.

## Claims

### Claim 1

An attenuated strain of porcine pseudorabies virus comprising: a first attenuated porcine pseudorabies virus strain, wherein the first attenuated porcine pseudorabies virus strain comprises a porcine pseudorabies virus strain characterized by inactivation of gI/gE/11K/28K protein, wherein the first attenuated porcine pseudorabies virus strain is a variant strain of pseudorabies virus, wherein the gE protein has 95% or greater homology with SEQ ID NO: 5, and wherein the first attenuated strain of porcine pseudorabies virus strain comprises a gene variation resulting in inactivation of TK protein, wherein at least a portion of a TK gene is deleted from a first nucleotide sequence to generate a second nucleotide sequence, and wherein the second nucleotide sequence encodes for SEQ ID NO: 4.

### Claim 2 (depends on 1)

The attenuated strain of porcine pseudorabies virus of claim 1, wherein the whole ORF of gI/gE/11K/28K gene is deleted from the first attenuated porcine pseudorabies virus strain.

### Claim 3 (depends on 1)

The attenuated strain of porcine pseudorabies virus of claim 1, wherein the first attenuated porcine pseudorabies virus strain is an HN1201 strain.

### Claim 4

A vaccine composition, comprising: an immunizing amount of an antigen of an attenuated, variant strain of porcine pseudorabies virus; and a carrier, wherein the attenuated, variant strain of porcine pseudorabies virus comprises: a first attenuated porcine pseudorabies virus strain, wherein the first attenuated porcine pseudorabies virus strain comprises a porcine pseudorabies virus strain characterized by inactivation of gI/gE/11K/28K protein, wherein the first attenuated porcine pseudorabies virus strain is a variant strain of pseudorabies virus, wherein the gE protein has 95% or greater homology with SEQ ID NO: 5, and wherein the first attenuated strain of porcine pseudorabies virus strain comprises a gene variation resulting in inactivation of TK protein, wherein at least a portion of a TK gene is deleted from a first nucleotide sequence to generate a second nucleotide sequence, and wherein the second nucleotide sequence encodes for SEQ ID NO: 4.

### Claim 5 (depends on 4)

The vaccine composition of claim 4, wherein the immunizing amount comprises at least 106.0TCID50/ml of the attenuated, variant strain of porcine pseudorabies virus.

### Claim 6 (depends on 4)

The vaccine composition of claim 4, wherein the antigen comprises a live attenuated, variant strain of porcine pseudorabies virus, and wherein the vaccine composition further comprises a cryoprotectant.

### Claim 7 (depends on 4)

The vaccine composition of claim 4, further comprising an inactivated pathogen or antigen.

### Claim 8 (depends on 7)

The vaccine composition of claim 7, wherein the inactivated pathogen or antigen is selected from the group consisting classical swine fever virus, antigen of porcine reproductive and respiratory syndrome virus, antigen of porcine circovirus, antigen of haemophilus parasuis, and antigen of mycoplasma.

### Claim 9

A method of treating and preventing pseudorabies infection, comprising immunizing a pig with an attenuated strain of porcine pseudorabies virus, wherein the attenuated strain of porcine pseudorabies virus comprises: a first attenuated porcine pseudorabies virus strain, wherein the first attenuated porcine pseudorabies virus strain comprises a porcine pseudorabies virus strain characterized by inactivation of gI/gE/11K/28K protein, wherein the first attenuated porcine pseudorabies virus strain is a variant strain of pseudorabies virus, wherein the gE protein has 95% or greater homology with SEQ ID NO: 5, and wherein the first attenuated strain of porcine pseudorabies virus strain comprises a gene variation resulting in inactivation of TK protein, wherein at least a portion of a TK gene is deleted from a first nucleotide sequence to generate a second nucleotide sequence, and wherein the second nucleotide sequence encodes for SEQ ID NO: 4.