Methods and compositions for vaccination of animals with PRRSV antigens with improved immunogenicity
View Patent ↗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.
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.
2. The composition of claim 1 , wherein said polynucleotide comprises an infectious North American PRRSV RNA molecule.
3. The composition of claim 1 , wherein said polynucleotide comprises a DNA molecule that encodes an infectious North American PRRSV RNA molecule.
4. 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.
5. The composition of claim 4 , wherein said promoter is a CMV promoter.
6. 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.
7. The composition of claim 6 , wherein said codon encoding an amino acid other than asparagine encodes an alanine or a glutamine residue.
8. 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.
9. 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.
10. The composition of claim 9 , wherein said codons encoding another amino acid encode either an alanine or a glutamine residue.
11. 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.
12. 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.
13. The composition of claim 1 , wherein said composition further comprises at least one adjuvant.
14. 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.
15. 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.
16. 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.
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.
18. 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.
19. The isolated polynucleotide of claim 17 , wherein said polynucleotide comprises an infectious North American PRRSV RNA molecule.
20. The isolated polynucleotide of claim 17 , wherein said polynucleotide comprises a DNA molecule that encodes an infectious North American PRRSV RNA molecule.
21. 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.
22. The isolated polynucleotide of claim 21 , wherein said promoter is a CMV promoter.
23. 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.
24. The isolated polynucleotide of claim 23 , wherein said codon encoding another amino acid encodes an alanine or a glutamine residue.
25. 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.
26. The isolated polynucleotide of claim 25 , by replacing a codon encoding said asparagine 34 with a codon encoding an amino acid other than asparagine.
27. The isolated polynucleotide of claim 25 , wherein said codon encoding another amino acid encodes an alanine or a glutamine residue.
28. 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.
29. 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.