Infectious cDNA clone of north american porcine reproductive and respiratory syndrome (PRRS) virus and uses thereof

• US 20020172690A1
• Filed: 04/22/2002
• Published: 11/21/2002
• Est. Priority Date: 12/22/1998
• Status: Active Grant

• Current Assignee: Zoetis Services LLC (Zoetis Incorporated)
• Original Assignee: Jay G. Calvert, Michael G. Sheppard, Siao-Kun W. Welch
• Inventors: Sheppard, Michael G., Welch, Siao-Kun W., Calvert, Jay G.
• Granted Patent: US 7,232,680 B2
• Time in Patent Office: Days
• US Class Current: 424/204.1
• CPC Class Codes: A61K 2039/525 Virus
• CPC Class Codes: A61K 2039/5254 avirulent or attenuated
• CPC Class Codes: A61K 2039/5256 expressing foreign proteins
• CPC Class Codes: A61K 2039/53 DNA (RNA) vaccination
• CPC Class Codes: A61K 2039/552 Veterinary vaccine
• CPC Class Codes: A61K 39/12 Viral antigens
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 15/08 for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/14 for RNA viruses
• 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 2770/10061 Methods of inactivation or attenuation
• CPC Class Codes: C12N 2770/10062 by genetic engineering
• 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

Abstract

The invention provides isolated polynucleotide molecules, including plasmids; viral vectors; and transfected host cells that comprise a DNA sequence encoding an infectious RNA sequence encoding a North American PRRS virus; and also North American PRRS viruses encoded thereby. The invention further provides isolated infectious RNA molecules encoding a North American PRRS virus. The invention also provides isolated polynucleotide molecules, infectious RNA molecules, viral vectors, and transfected host cells encoding genetically-modified North American PRRS viruses; and genetically-modified North American PRRS viruses encoded thereby. The invention also provides vaccines comprising such plasmids, RNA molecules, viral vectors, and North American PRRS viruses, and methods of using these vaccines in swine and in other animals. Also provided are isolated polynucleotide molecules, viral vectors, and transfected host cells that comprise a nucleotide sequence encoding a peptide of a North American PRRS virus. These viral vectors and transfected host cell lines are useful in providing peptides to compensate for mutated peptide coding sequences of DNA sequences encoding genetically-modified North American PRRS viruses so that functional virions can be generated.

Claims

1. An isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus, wherein said DNA sequence is SEQ ID NO:

1 or a sequence homologous thereto.

2. An isolated infectious RNA molecule encoded by an isolated polynucleotide molecule according to claim 1, or an isolated infectious RNA molecule homologous thereto, which infectious RNA molecule encodes a North American PRRS virus.

3. An isolated polynucleotide molecule according to claim 1 in the form of a plasmid.

4. A transfected host cell comprising a DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus, wherein said DNA sequence is SEQ ID NO:

1 or a sequence homologous thereto, which transfected host cell is capable of expressing the encoded North American PRRS virus.

5. A plasmid capable of directly transfecting a suitable host cell and expressing a Nidovirales virus from the suitable host cell so transfected, which plasmid comprises a) a DNA sequence encoding an infectious RNA molecule encoding the Nidovirales virus, and b) a promoter capable of transcribing said infectious RNA molecule in said suitable host cell.

6. A method for generating a Nidovirales virus, which method comprises transfecting a suitable host cell with a plasmid according to claim 5 encoding the Nidovirales virus and obtaining Nidovirales virus generated by the transfected host cell.

7. An isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus that is genetically modified such that when it infects a porcine animal it is unable to produce PRRS in the animal, wherein said DNA sequence is SEQ ID NO:

1 or a sequence homologous thereto, except for that it contains one or more mutations that genetically disable the encoded PRRS virus in its ability to produce PRRS.

9. A vaccine for protecting a porcine animal from infection by a PRRS virus, which vaccine comprises a genetically modified North American PRRS virus encoded by an infectious RNA molecule encoded by a polynucleotide molecule according to claim 7, or said infectious RNA molecule, or said polynucleotide molecule in the form of a plasmid, or a viral vector comprising a nucleotide sequence corresponding to claim 7 encoding a North American PRRS virus that is able to elicit an effective immunoprotective response against infection by a PRRS virus, in an amount effective to produce immunoprotection against infection by a PRRS virus;

and a carrier acceptable for veterinary use.

8. A transfected host cell comprising a DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus that is genetically modified such that when it infects a porcine animal it is unable to produce PRRS in the animal, wherein said DNA sequence is SEQ ID NO:

1 or a sequence homologous thereto, except for that it contains one or more mutations that genetically disable the encoded PRRS virus in its ability to produce PRRS, which transfected host cell is capable of expressing the encoded genetically modified North American PRRS virus.

10. An isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus that is genetically modified such that it comprises one or more heterologous antigenic epitopes, wherein the DNA sequence encoding the RNA molecule encoding the North American PRRS virus is SEQ ID NO:

1 or a sequence homologous thereto except for that it comprises one or more further nucleotide sequences that each encode a heterologous antigenic epitope, and wherein each heterologous antigenic epitope is capable of inducing an effective immunoprotective response against a particular pathogen in a mammal or a bird.

11. An isolated polynucleotide molecule according to claim 10 in the form of a plasmid.

13. A vaccine for protecting a mammal or a bird from infection by a pathogen, which vaccine comprises a genetically modified North American PRRS virus encoded by an infectious RNA molecule encoded by a polynucleotide molecule of claim 10, or said infectious RNA molecule, or a plasmid according to claim 11, in an amount effective to produce immunoprotection against infection by the pathogen from which the heterologous antigenic epitope or epitopes thereof or encoded thereby are derived;

and a carrier acceptable for pharmaceutical or veterinary use.

14. An isolated polynucleotide molecule according to claim 10 wherein the DNA sequence encoding the infectious RNA molecule encoding the genetically modified North American PRRS virus comprises one or more further mutations that disable the encoded genetically modified North American PRRS virus in its ability to produce PRRS in a swine animal.

15. An isolated polynucleotide molecule according to claim 14 in the form of a plasmid.

16. A vaccine for protecting a porcine animal from infection by a PRRS virus and from infection by a swine pathogen other than a North American PRRS virus, which vaccine comprises a genetically modified North American PRRS virus encoded by an infectious RNA molecule encoded by a polynucleotide molecule according to claim 14, or said infectious RNA molecule, or a plasmid according to claim 15, in an amount effective to produce immunoprotection against infection by a PRRS virus and against infection by the pathogen from which the heterologous antigenic epitope or epitopes thereof or encoded thereby are derived;

and a carrier acceptable for veterinary use.

18. A vaccine for protecting a porcine animal from infection by a PRRS virus, which vaccine comprises a genetically modified North American PRRS virus encoded by an infectious RNA molecule encoded by a polynucleotide molecule according to claim 17, comprising one or more detectable heterologous antigenic epitopes, treated or further genetically modified so that it is unable to produce PRRS in a porcine animal yet able to elicit an effective immunoprotective response against a PRRS virus in the porcine animal;

or said infectious RNA molecule, wherein the genetically modified North American PRRS virus encoded thereby comprising one or more detectable heterologous antigenic epitopes comprises further genetic modifications so that it is unable to produce PRRS yet able to elicit an effective immunoprotective response against a PRRS virus;

or an isolated polynucleotide according to claim 17, in the form of a plasmid, wherein the genetically modified North American PRRS virus encoded thereby comprising one or more detectable heterologous antigenic epitopes comprises further genetic modifications so that it is unable to produce PRRS yet able to elicit an effective immunoprotective response against a PRRS virus;

or a viral vector comprising a nucleotide sequence encoding an infectious RNA molecule encoding such a genetically modified North American PRRS virus, wherein the genetically modified North American PRRS virus encoded thereby comprising one or more detectable heterologous antigenic epitopes comprises further genetic modifications so that it is unable to produce PRRS yet able to elicit an effective immunoprotective response against a PRRS virus;

in an amount effective to produce immunoprotection against infection by a PRRS virus; and

a carrier acceptable for veterinary use.

20. A vaccine for protecting a porcine animal from infection by a PRRS virus, which vaccine comprises a genetically modified North American PRRS virus encoded by an infectious RNA molecule encoded by an polynucleotide molecule according to claim 19, lacking one or more detectable antigenic epitopes, treated or further genetically modified so that it is unable to produce PRRS in a porcine animal yet able to elicit an effective immunoprotective response against a PRRS virus in the porcine animal;

or said infectious RNA molecule, wherein the genetically modified North American PRRS virus encoded thereby lacking one or more detectable antigenic epitopes comprises further genetic modifications so that it is unable to produce PRRS yet able to elicit an effective immunoprotective response against a PRRS virus;

or an isolated polynucleotide molecule, in the form of a plasmid, wherein the genetically modified North American PRRS virus encoded thereby lacking one or more detectable antigenic epitopes comprises further genetic modifications so that it is unable to produce PRRS yet able to elicit an effective immunoprotective response against a PRRS virus;

or a viral vector comprising a nucleotide sequence encoding an infectious RNA molecule encoding such a genetically modified North American PRRS virus, wherein the genetically modified North American PRRS virus encoded thereby lacking one or more detectable antigenic epitopes comprises further genetic modifications so that it is unable to produce PRRS yet able to elicit an effective immunoprotective response against a PRRS virus;

in an amount effective to produce immunoprotection against infection by a PRRS virus; and

a carrier acceptable for veterinary use.

12. A transfected host cell comprising a DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus that is genetically modified such that it comprises one or more heterologous antigenic epitopes, wherein the DNA sequence encoding the RNA molecule encoding the North American PRRS virus is SEQ ID NO:

1 or a sequence homologous thereto except for that it comprises one or more further nucleotide sequences that each encode a heterologous antigenic epitope, and wherein each heterologous antigenic epitope is capable of inducing an effective immunoprotective response against a particular pathogen in a mammal or a bird.

17. An isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus that is genetically modified such that it comprises one or more detectable heterologous antigenic epitopes, wherein the DNA sequence encoding the infectious RNA molecule is the same as or homologous to the sequence SEQ ID NO:

1 or a sequence homologous thereto except for that it comprises one or more further nucleotide sequences that each encode a detectable heterologous antigenic epitope.

19. An isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus that is genetically modified so that it lacks a detectable antigenic epitope, wherein said DNA sequence is SEQ ID NO:

1 or a sequence homologous thereto, except for that it lacks one or more DNA sequences encoding a detectable antigenic epitope.

21. An isolated polynucleotide molecule comprising one or more nucleotide sequences that each encode a peptide encoded by a North American PRRS virus, wherein the genome sequence of said North American PRRS virus is the same as or homologous to an RNA molecule corresponding to SEQ ID NO:

1.

22. A transfected host cell comprising one or more nucleotide sequences that each encode a peptide encoded by a North American PRRS virus, wherein the genome sequence of said North American PRRS virus is the same as or homologous to an RNA molecule corresponding to SEQ ID NO:

1, which transformed cell is capable of expressing said peptide or peptides.

23. A method for generating a functional virion of a genetically modified North American PRRS virus, encoded by an RNA sequence corresponding to SEQ ID NO:

1 or a sequence homologous thereto except comprising one or more mutations in one or more peptide coding sequences, which method comprises further transfecting a transfected host cell according to claim 22 with an infectious RNA molecule, plasmid, or viral vector encoding the genetically modified North American PRRS virus, wherein the North American PRRS virus peptide or peptides encoded by said nucleotide sequence or sequences of said transfected host cell comprise the peptides of the mutated peptide coding sequence or sequences of said genetically modified North American PRRS virus.

27. A vaccine for protecting a mammal or a bird from infection by a Nidovirales virus, which comprises a genetically modified Nidovirales virus according to claim 25 in an amount effective to elicit an effective immunoprotective response against the wild-type Nidovirales virus in a mammal or a bird vaccinated therewith, and a carrier acceptable for pharmaceutical or veterinary use.

24. A method for generating a functional virion of a genetically modified North American PRRS virus encoded by an RNA sequence corresponding to SEQ ID NO:

1 or a sequence homologous thereto except comprising one or more mutations in one or more peptide coding sequences, which method comprises transfecting a suitable cell with an infectious RNA molecule, plasmid, or viral vector encoding the genetically modified North American PRRS virus and with a helper virus that expresses the North American PRRS virus peptide or peptides encoded by the mutated peptide coding sequence or sequences of said genetically modified North American PRRS virus, and separating genetically modified North American PRRS virions expressed by the cell from said helper virus.

25. A genetically modified Nidovirales virus that is capable of eliciting an immunoprotective response in a mammal or a bird vaccinated therewith, which genetically modified Nidovirales virus is prepared by obtaining an isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule encoding a wild-type Nidovirales virus, genetically mutating the DNA sequence encoding the infectious RNA molecule encoding the wild-type Nidovirales virus so as to obtain an isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule encoding a genetically modified Nidovirales virus which virus remains able to elicit an effective immunoprotective response against infection by the wild-type Nidovirales virus in a mammal or a bird, and expressing the genetically modified Nidovirales virus from the isolated polynucleotide molecule so obtained.

26. A method for preparing a genetically modified Nidovirales virus that is capable of eliciting an immunoprotective response in a mammal or a bird vaccinated therewith, which method comprises obtaining an isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule encoding a wild-type Nidovirales virus, genetically mutating the DNA sequence encoding the infectious RNA molecule encoding the wild-type Nidovirales virus so as to obtain an isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule encoding a genetically modified Nidovirales virus which virus remains able to elicit an effective immunoprotective response against infection by the wild-type Nidovirales virus in a mammal or a bird, and expressing the genetically modified Nidovirales virus from the isolated polynucleotide molecule so obtained.