Packaging of immunostimulatory substances into virus-like particles: method of preparation and use
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 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, and wherein said unmethylated CpG-containing oligonucleotide comprises about 20 to about 300 nucleotides; and
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 by at least one non-peptide covalent bond.
3. The composition of claim 1 , wherein said virus-like particle is a recombinant virus-like particle.
4. The composition of claim 1 , wherein said unmethylated CpG-containing oligonucleotide comprises the sequence:
5′ X 1 X 2 CGX 3 X 4 3′
wherein X 1 , X 2 , X 3 , and X 4 are any nucleotide.
5. The composition of claim 4 , wherein at least one of said nucleotide X 1 , X 2 , X 3 , and X 4 has a phosphate backbone modification.
6. The composition of claim 2 , wherein said antigen is an organic molecule.
7. 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.
8. The composition of claim 7 , wherein said antigen is a tumor antigen, and 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. 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 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, and wherein said unmethylated CpG-containing oligonucleotide comprises about 20 to about 300 nucleotides; and
wherein said immunostimulatory substance is packaged into said virus-like particle.
10. The method of claim 9 , wherein said composition further comprises an antigen, wherein said antigen is bound to said virus-like particle by at least one non-peptide covalent bond.
11. 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 particle, wherein said virus-like particle is a virus-like particle of RNA-phage Qβ consisting of coat proteins with the amino acid sequence of SEQ ID NO:10, and wherein said disassembling comprises incubating said virus-like particle with a disassembling solution comprising at least one disassembling agent selected from urea, guanidine hydrochloride (GuHCl) and dithioerythol (DTT) in a concentration capable of effecting said disassembling;
(b) adding said immunostimulatory substance, wherein said immunostimulatory substance is an unmethylated CpG-containing oligonucleotide, and wherein said unmethylated CpG-containing oligonucleotide comprises about 20 to about 300 nucleotides; and
(c) reassembling said virus-like particle, wherein said reassembling comprises removing said disassembling agent by at least one buffer exchange and allowing said reassembling of said virus-like particle.
12. A vaccine comprising an immunologically effective amount of the composition of claim 1 together with a pharmaceutically acceptable diluent, carrier or excipient.
13. A method of immunizing or treating an animal comprising administering to said animal an immunologically effective amount of the vaccine of claim 12 .
14. A vaccine comprising an immunologically effective amount of the composition of claim 2 together with a pharmaceutically acceptable diluent, carrier or excipient.
15. A method of immunizing or treating an animal comprising administering to said animal an immunologically effective amount of the vaccine of claim 14 .
16. 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.
17. The composition of claim 16 , wherein said first attachment site is a lysine residue and said second attachment site is a cysteine residue.
18. The composition of claim 2 , wherein said at least one antigen is a polypeptide suited to induce an immune response against an infectious disease.
19. The composition of claim 2 , wherein said at least one antigen is a recombinant polypeptide of Hepatitis B virus.
20. A composition for enhancing an immune response in an animal consisting of:
(a) a virus-like particle, wherein said virus-like particle is a virus-like particle of 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, and wherein said unmethylated CpG-containing oligonucleotide comprises about 20 to about 300 nucleotides; and
wherein said immunostimulatory substance is packaged into said virus-like particle.