Packaging of immunostimulatory substances into virus-like particles: method of preparation and use
View Patent ↗The invention relates to the finding that virus like particles (VLPs) can be loaded with immunostimulatory substances, in particular with DNA oligonucleotides containing non-methylated C and G (CpGs). Such CpG-VLPs are dramatically more immunogenic than their CpG-free counterparts and induce enhanced B and T cell responses. The immune response against antigens optionally coupled, fused or attached otherwise to the VLPs is similarly enhanced as the immune response against the VLP itself. In addition, the T cell responses against both the VLPs and antigens are especially directed to the Th1 type. Antigens attached to CpG-loaded VLPs may therefore be ideal vaccines for prophylactic or therapeutic vaccination against allergies, tumors and other self-molecules and chronic viral diseases.
1. A composition for enhancing an immune response in an animal comprising:
(a) a virus-like particle, wherein said virus-like particle is a virus-like particle of an RNA-phage Qβ consisting of coat proteins with the amino acid sequence of SEQ ID NO:10; and
(b) an immunostimulatory substance, wherein said immunostimulatory substance is an unmethylated CpG-containing oligonucleotide, wherein said unmethylated CpG-containing oligonucleotide consists of the sequence GGGGGGGGGGGACGATCGTCGGGGGGGGGG (SEQ ID NO:116), and wherein said unmethylated CpG containing oligonucleotide exclusively consists of phosphodiester connected deoxynucleotides;
wherein said immunostimulatory substance is packaged into said virus-like particle.
2. The composition of claim 1 further comprising at least one antigen, wherein said antigen is bound to said virus-like particle.
3. The composition of claim 2 , wherein said virus-like particle comprises at least one first attachment site, and wherein said antigen further comprises at least one second attachment site being selected from the group consisting of:
(a) an attachment site not naturally occurring with said antigen; and
(b) an attachment site naturally occurring with said antigen;
wherein said second attachment site associates with said first attachment site.
4. The composition of claim 3 further comprising an amino acid linker, wherein said amino acid linker comprises said second attachment site.
5. The composition of claim 2 , wherein said antigen is selected from the group consisting of:
(a) polypeptides;
(b) carbohydrates;
(c) steroid hormones; and
(d) organic molecules.
6. The composition of claim 2 , wherein said antigen is derived from the group consisting of:
(a) viruses;
(b) bacteria;
(c) parasites;
(d) prions;
(e) tumors;
(f) self-molecules;
(g) non-peptidic hapten molecules
(h) allergens; and
(i) hormones.
7. The composition of claim 6 , wherein said antigen is a tumor antigen.
8. The composition of claim 7 , wherein said tumor antigen is selected from the group consisting of:
(a) Her2;
(b) GD2;
(c) EGF-R;
(d) CEA;
(e) CD52;
(f) CD21;
(g) human melanoma protein gp100;
(h) human melanoma protein melan-A/MART-1;
(i) tyrosinase;
(j) NA17-A nt protein;
(k) MAGE-3 protein;
(l) p53 protein;
(m) HPV16 E7 protein; and
(n) antigenic fragments of any of the tumor antigens from (a) to (m).
9. The composition of claim 2 , wherein said antigen is bound to said virus-like particle by way of a linking sequence.
10. A method for enhancing an immune response in an animal comprising introducing into said animal a composition comprising:
(a) a virus-like particle, wherein said virus-like particle is a virus-like particle of an RNA-phage Qβ consisting of coat proteins with the amino acid sequence of SEQ ID NO:10; and
(b) an immunostimulatory substance, wherein said immunostimulatory substance is an unmethylated CpG-containing oligonucleotide, wherein said unmethylated CpG-containing oligonucleotide consists of the sequence GGGGGGGGGGGACGATCGTCGGGGGGGGGG (SEQ ID NO:116), and wherein said unmethylated CpG containing oligonucleotide exclusively consists of phosphodiester connected deoxynucleotides;
wherein said immunostimulatory substance is packaged into said virus-like particle.
11. The method of claim 10 , wherein said composition further comprises an antigen, wherein said antigen is bound to said virus-like particle.
12. The method of claim 10 , wherein said virus-like particle is produced in a bacterial expression system.
13. The method of claim 11 , wherein said at least one antigen is bound to said virus-like particle by at least one non-peptide covalent bond.
14. The method of claim 11 , wherein said virus-like particle comprises at least one first attachment site, and wherein said antigen further comprises at least one second attachment site selected from the group consisting of:
(a) an attachment site not naturally occurring with said antigen; and
(b) an attachment site naturally occurring with said antigen;
wherein said second attachment site associates with said first attachment site.
15. The method of claim 11 , wherein said antigen is selected from the group consisting of:
(a) polypeptides;
(b) carbohydrates;
(c) steroid hormones; and
(d) organic molecules.
16. The method of claim 10 , wherein said immune response is an enhanced T cell response, wherein said T cell response is a Th cell response, and wherein said Th cell response is a Th1 cell response.
17. A method of producing a composition for enhancing an immune response in an animal comprising a virus-like particle and an immunostimulatory substance packaged into said virus-like particle which comprises:
(a) incubating said virus-like particle with said immunostimulatory substance, wherein said virus-like particle is a virus-like particle of an RNA phage Qβ consisting of coat proteins with the amino acid sequence of SEQ ID NO:10, and wherein said immunostimulatory substance is an unmethylated CpG-containing oligonucleotide, wherein said unmethylated CpG-containing oligonucleotide consists of the sequence GGGGGGGGGGGACGATCGTCGGGGGGGGGG (SEQ ID NO:116), and wherein said unmethylated CpG containing oligonucleotide exclusively consists of phosphodiester connected deoxynucleotides;
(b) adding RNase; and
(c) purifying said composition.
18. The method of claim 17 , wherein said virus-like particle is produced in a bacterial expression system.
19. A method of producing a composition for enhancing an immune response in an animal comprising a virus-like particle and an immunostimulatory substance packaged into said virus-like particle which comprises:
(a) incubating said virus-like particle with RNase, wherein said virus-like particle is a virus-like particle of an RNA-phage Qβ consisting of coat proteins with the amino acid sequence of SEQ ID NO 10;
(b) adding said immunostimulatory substance, wherein said immunostimulatory substance is an unmethylated CpG-containing oligonucleotide, wherein said unmethylated CpG-containing oligonucleotide consists of the sequence GGGGGGGGGGGACGATCGTCGGGGGGGGGG (SEQ ID NO 116), and wherein said unmethylated CpG containing oligonucleotide exclusively consists of phosphodiester connected deoxynucleotides; and
(c) purifying said composition.
20. The method of claim 19 , wherein said virus-like particle is produced in a bacterial expression system.
21. A method of producing a composition for enhancing an immune response in an animal comprising a virus-like particle and an immunostimulatory substance packaged into said virus-like particle which comprises:
(a) disassembling said virus-like panicle, wherein said virus-like particle is a virus-like particle of an RNA-phage Qβ consisting of coat proteins with the amino acid sequence of SEQ ID NO:10;
(b) adding said immunostimulatory substance, wherein said immunostimulatory substance is an unmethylated CpG-containing oligonucleotide, wherein said unmethylated CpG-containing oligonucleotide consists of the sequence GGGGGGGGGGGACGATCGTCGGGGGGGGGG (SEQ ID NO:116), and wherein said unmethylated CpG containing oligonucleotide exclusively consists of phosphodiester connected deoxynucleotides; and
(c) reassembling said virus-like particle.
22. The method of claim 21 further comprising removing nucleic acids of said disassembled virus-like particle.
23. The method of claim 21 further comprising purifying said composition after reassembly (c).
24. A method of producing a composition for enhancing an immune response in an animal comprising a virus-like particle and an immunostimulatory substance packaged into said virus-like particle which comprises:
(a) incubating said virus-like particle with solutions comprising metal ions capable of hydrolyzing the nucleic acids of said virus-like particle, wherein said virus-like particle is a virus-like particle of an RNA-phage Qβ consisting of coat proteins with the amino acid sequence SEQ ID NO:10;
(b) adding said immunostimulatory substance, wherein said immunostimulatory substance is an unmethylated CpG-containing oligonucleotide, wherein said unmethylated CpG-containing oligonucleotide consists of the sequence GGGGGGGGGGGACGATCGTCGGGGGGGGGG (SEQ ID NO:116), and wherein said unmethylated CpG containing oligonucleotide exclusively consists of phosphodiester connected deoxynucleotides; and
(c) purifying said composition.
25. The method of claim 24 , wherein said metal ions are selected from the group consisting of:
(a) zinc (Zn) ions;
(b) copper (Cu) ions;
(c) iron (Fe) ions; and
(d) any mixtures of at least one ion of (a), (b) and/or (c).
26. An immunogenic composition comprising an immunologically effective amount of the composition of claim 1 together with a pharmaceutically acceptable diluent, carrier or excipient.
27. A method of immunizing an animal comprising administering to said animal an immunologically effective amount of the vaccine of claim 26 .
28. An immunogenic composition comprising an immunologically effective amount of the composition of claim 2 together with a pharmaceutically acceptable diluent, carrier or excipient.
29. A method of immunizing an animal comprising administering to said animal an immunologically effective amount of the vaccine of claim 28 .
30. The composition of claim 3 , wherein said at least one antigen is bound to said virus-like particle by at least one non-peptide covalent bond.
31. The composition of claim 30 , wherein said first attachment site is a lysine residue and said second attachment is a cysteine residue.
32. The composition of claim 31 , wherein said at least one antigen is bound to said virus-like particle by way of a hetero-bifunctional cross-linker.
33. The composition of claim 32 , wherein said hetero-bifunctional cross-linker is Succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH).