Immunostimulatory oligonucleotides
Compositions and methods for stimulating toll-like receptor 9 (TLR9) are provided. More particularly, immunostimulatory oligonucleotides, methods of enhancing immunostimulatory properties of oligonucleotides, and methods of eliciting immune responses are disclosed herein.
1. An immunostimulatory oligonucleotide comprising at least one CpG motif and a 3′ cholesteryl moiety,
wherein the 3′ terminal sequence of the oligonucleotide comprises a plurality of thymine nucleotides, and
wherein the 3′ terminal sequence of the oligonucleotide comprises the oligonucleotide sequence of SEQ ID NO: 9.
2. The immunostimulatory oligonucleotide of claim 1 comprising one or more phosphodiester linkages between nucleotides of the immunostimulatory oligonucleotide.
3. The immunostimulatory oligonucleotide of claim 1 comprising one or more phosphorothioate linkages between nucleotides of the immunostimulatory oligonucleotide.
4. The immunostimulatory oligonucleotide of claim 1 , wherein the cholesteryl moiety is covalently attached to the 3′-terminal nucleotide of the immunostimulatory oligonucleotide via a linker.
5. The immunostimulatory oligonucleotide of claim 1 , wherein the plurality of thymine nucleotides comprises consecutive thymine nucleotides.
6. The immunostimulatory oligonucleotide of claim 1 , wherein the plurality of thymine nucleotides comprises between 4 and 6 consecutive thymine nucleotides.
7. The immunostimulatory oligonucleotide of claim 1 , wherein the immunostimulatory oligonucleotide comprises SEQ ID NO:2, 3, 4, 5, 6, or 8.
8. The immunostimulatory oligonucleotide of claim 1 , wherein the 3′ terminal sequence is TTTT.
9. The immunostimulatory oligonucleotide of claim 1 , wherein the 3′ terminal sequence of the immunostimulatory oligonucleotide comprises a plurality of guanine nucleotides.
10. The immunostimulatory oligonucleotide of claim 9 , wherein the plurality of guanine nucleotides comprises consecutive guanine nucleotides.
11. The immunostimulatory oligonucleotide of claim 9 , wherein the 3′ terminal sequence is GGGGG.
12. The immunostimulatory oligonucleotide of claim 9 , wherein the immunostimulatory oligonucleotide comprises SEQ ID NO:7.
13. The immunostimulatory oligonucleotide of claim 1 , wherein the immunostimulatory oligonucleotide comprises (TCG) n , where n is between 3 and 10.
14. The immunostimulatory oligonucleotide of claim 4 , wherein the linker comprises a carbon chain.
15. The immunostimulatory oligonucleotide of claim 14 , wherein the carbon chain comprises between 3 and 12 carbon atoms.
16. The immunostimulatory oligonucleotide of claim 4 , wherein the linker comprises a hexanediol.
17. The immunostimulatory oligonucleotide of claim 14 , comprising a cholesteryl-linker moiety having the following structure:
18. The immunostimulatory oligonucleotide of claim 4 , wherein the linker comprises a repeated chemical unit.
19. The immunostimulatory oligonucleotide of claim 18 , wherein the repeated chemical unit is repeated between 2 and 12 times.
20. The immunostimulatory oligonucleotide of claim 18 , wherein the repeated chemical unit is an ethylene glycol.
21. The immunostimulatory oligonucleotide of claim 18 , wherein the linker comprises a hexaethylene glycol.
22. The immunostimulatory oligonucleotide of claim 18 , wherein the cholesteryl moiety is covalently bound to the linker to form a cholesteryl-linker moiety.
23. The immunostimulatory oligonucleotide of claim 22 , comprising a cholesteryl-linker moiety having the following structure:
24. An immunostimulatory composition comprising the immunostimulatory oligonucleotide of claim 1 .
25. The immunostimulatory composition of claim 24 further comprising a vaccine for preventing or treating an infectious disease.
26. The immunostimulatory composition of claim 24 further comprising a vector.
27. The immunostimulatory composition of claim 26 , wherein the vector is a viral vector.
28. The immunostimulatory composition of claim 27 , wherein the viral vector comprises the immunostimulatory oligonucleotide.
29. The immunostimulatory composition of claim 24 further comprising a pharmaceutically acceptable carrier.
30. The immunostimulatory composition of claim 29 , wherein the oligonucleotide and the pharmaceutically acceptable carrier are covalently coupled.
31. The immunostimulatory composition of claim 24 further comprising a hapten.
32. The immunostimulatory composition of claim 31 , wherein the oligonucleotide and the hapten are covalently coupled.
33. A method of enhancing the immunogenicity of a TLR9 ligand comprising attaching a cholesteryl moiety to the 3′ terminus of the TLR9 ligand via a linker,
wherein the TLR9 ligand is an oligonucleotide having at least one of CpG motif,
wherein the 3′ terminal sequence of the oligonucleotide comprises a plurality of thymine nucleotides, and
wherein the 3′ terminal sequence of the oligonucleotide comprises the oligonucleotide sequence of SEQ ID NO: 9.
34. The method of claim 33 , wherein the cholesteryl moiety is covalently bound to the linker to form a cholesteryl-linker moiety.
35. The immunostimulatory oligonucleotide of claim 34 , wherein the cholesteryl-linker moiety comprises:
36. The immunostimulatory oligonucleotide of claim 34 , wherein the cholesteryl-linker moiety comprises:
37. A method of eliciting a TLR9-mediated immune response in a subject comprising administering to the subject the immunostimulatory oligonucleotide of claim 1 or the immunostimulatory composition thereof.
38. The method of claim 37 , wherein the administering is performed intravenously, intramuscularly, intramammary, intradermally, intraperitoneally, subcutaneously, by spray, by aerosol, in ovo, mucosally, transdermally, by immersion, orally, intraocularly, intratracheally, or intranasally.
39. The method of claim 37 , wherein the subject is an animal.
40. The method of claim 37 , wherein the subject is a mammal.
41. The method of claim 37 , wherein the subject is an aquatic species.
42. The method of claim 37 , wherein the subject is a mouse, pig, cow, horse, sheep, or human.