PRRS VIRUS VARIANT, EUROPEAN PRRS VIRUS CDNA CLONE, AND USES THEREOF

• US 20160317642A1
• Filed: 12/19/2014
• Published: 11/03/2016
• Est. Priority Date: 12/20/2013
• Status: Active Grant

• Current Assignee: Boehringer Ingelheim Vetmedica GmbH (C.H. Boehringer Sohn AG & Co. KG)
• Original Assignee: Boehringer Ingelheim Vetmedica GmbH (C.H. Boehringer Sohn AG & Co. KG)
• Inventors: KELLER, Christoph, SCHACHT, Erik, HERREL, Marieke, GALLEI, Andreas
• Granted Patent: US 10,010,601 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 2039/5254 avirulent or attenuated
• CPC Class Codes: A61K 2039/552 Veterinary vaccine
• CPC Class Codes: A61K 39/12 Viral antigens
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2770/10021 Viruses as such, e.g. new isolates, mutants or their genomic sequences
• CPC Class Codes: C12N 2770/10034 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2770/10043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12Q 1/02 involving viable microorganisms
• CPC Class Codes: G01N 2333/08 RNA viruses

Abstract

The present invention belongs to the field of animal health and provides means to study Porcine Reproductive and Respiratory Syndrome (PRRS), a viral disease affecting swine, and for the development of vaccines, therapeutics and diagnostics for the prophylaxis, treatment and diagnosis of PRRS. in a first consideration, the invention relates to a new PRRS virus variant, and, in a second consideration, to a nucleic acid sequence which comprises the genome of an infectious genotype I (EU) PRRS virus clone. Based on this, new PRRS vaccine candidates with improved properties are provided.

Claims

1-90. -90. (canceled)

91. A nucleic acid molecule which encodes a genotype I PRRS virus and which is capable of producing live virus when transfected into cells, wherein said molecule comprises:

(a) a first nucleic acid sequence having at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO;

45,

(b) a second nucleic acid sequence flanking the 5′

end of the first nucleic acid sequence and having at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO;

46,

(c) a third nucleic acid sequence flanking the 3′

end of the first nucleic acid sequence and having at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO;

47, and

(d) a polyadenine nucleotide sequence flanking the 3′

end of the third nucleic acid sequence;

92. The nucleic acid molecule of claim 91, wherein said virus is attenuated or able to induce a protective immune response against respiratory and/or reproductive signs of disease after infection with Porcine Reproductive and Respiratory Syndrome (PRRS) virus in swine.

93. The nucleic acid molecule of claim 92, wherein said virus is able to reach titers of at least 5×

105 to 1×

106 tissue culture infectious dose 50 (TCID50) per milliliter (ml) within 24 hours post infection of MA104 cells at an multiplicity of infection (MOI) of 0.001 to 0.1.

94. The nucleic acid molecule of claim 91, wherein said molecule comprises a nucleic acid sequence having at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO:

48.

95. The nucleic acid molecule of claim 91, wherein said molecule encodes a PRRS virus selected from the group consisting of:

(a) an isolated PRRS virus comprising an ORF4 protein which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs;

1-12;

(b) an isolated genotype I PRRS virus, comprising an ORF4 protein having a deletion of at least 9 amino acid residues in the region between the first two predicted N-terminal β

-sheets, as compared to the ORF4 protein of a wild type genotype I PRRS virus;

(c) an isolated genotype I PRRS virus, comprising an ORF4 protein having a deletion of at least 9 amino acid residues between amino acid positions 50 to 71, wherein the numbering of the amino acid positions refers to the amino acid sequence of ORF4 protein of the Lelystad virus;

or

(d) an ORF4 protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

13-24;

(e) the first two predicted N-terminal β

-sheets being the two amino acid sequences set forth in SEQ ID NO;

25 and SEQ ID NO;

26,

(f) the two amino acid sequences set forth in SEQ ID NO;

29 and SEQ ID NO;

30,

(g) the isolated PRRS virus comprises an ORF4 protein having a deletion of from 11 to 17 amino acid residues between amino acid positions 50 to 71, wherein the numbering of the amino acid positions refers to the amino acid sequence of ORF4 protein of the Lelystad virus and/or wherein the amino acid sequence of ORF4 protein of the Lelystad virus is the sequence set forth in SEQ ID NO;

43;

(h) an isolated PRRS virus whose genome comprises a nucleic acid molecule which encodes an ORF4 protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

1-12;

(i) an isolated genotype I PRRS virus, whose genome comprises a nucleic acid molecule which encodes an ORF4 protein having a deletion of at least 9 amino acid residues in the region between the first two predicted N-terminal β

-sheets, as compared to the ORF4 protein of a wild type genotype I PRRS virus;

(j) an isolated genotype I PRRS virus, whose genome comprises a nucleic acid molecule which encodes an ORF4 protein having a deletion of at least 9 amino acid residues between amino acid positions 50 to 71, wherein the numbering of the amino acid positions refers to the amino acid sequence of ORF4 protein of the Lelystad virus;

(k) the ORF4 protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO;

13-24;

(l) the first two predicted N-terminal β

-sheets being the two amino acid sequences set forth in SEQ ID NO;

25 and SEQ ID NO;

26,

(m) the two amino acid sequences set forth in SEQ ID NO;

29 and SEQ ID NO;

30, wherein the wild type genotype I PRRS virus is the Lelystad virus;

(n) the ORF4 protein having a deletion of between 11 to 17 amino acid residues between amino acid positions 50 to 71, wherein the numbering of the amino acid positions refers to the amino acid sequence of ORF4 protein of the Lelystad virus;

(o) the ORF4 protein comprises at least 95% sequence identity with the amino acid sequence of SEQ ID NO;

36;

(p) the ORF4 protein comprises at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO;

37;

or

(q) an isolated genotype I PRRS whose genome comprises an RNA molecule encoded by a nucleic acid molecule having at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO;

38.

96. The nucleic acid molecule of claim 91, wherein said nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:

56, SEQ ID NO;

57, and SEQ ID NO;

58.

97. The nucleic acid molecule of claim 91, wherein said molecule is a DNA molecule.

98. A DNA construct comprising a DNA molecule according to claim 97.

99. An RNA transcript of the DNA construct of claim 98.

100. A composition comprising a nucleic acid molecule of claim 91 suspended in a suitable amount of a pharmaceutically acceptable diluent or excipient.

104. The use of the PRRS virus according to claim 91 as a detection marker for the differentiation between infected and vaccinated animals (DIVA).

101. A genotype I PRRS virus, whose genome is encoded by a nucleic acid molecule comprising the sequence selected from the group consisting of:

SEQ ID NO;

49, SEQ ID NO;

56, SEQ ID NO;

57 or SEQ ID NO;

58.

102. A method for the treatment of Porcine Reproductive and Respiratory Syndrome comprising administering the virus of claim 101 to swine.

103. The method of claim 102, wherein the molecule is administered, by intranasal, intramuscular, oral, or intrauterine route.