CA2244946A1 - Gene expression vectors which generate an antigen specific immune response and methods of using the same - Google Patents

Gene expression vectors which generate an antigen specific immune response and methods of using the same

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Publication number
CA2244946A1
CA2244946A1 CA002244946A CA2244946A CA2244946A1 CA 2244946 A1 CA2244946 A1 CA 2244946A1 CA 002244946 A CA002244946 A CA 002244946A CA 2244946 A CA2244946 A CA 2244946A CA 2244946 A1 CA2244946 A1 CA 2244946A1
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CA
Canada
Prior art keywords
gene expression
recombinant gene
expression vector
seq
adjacent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CA002244946A
Other languages
French (fr)
Other versions
CA2244946C (en
Inventor
Dennis A. Carson
Eyal Raz
Mark Roman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California
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Individual
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Application filed by Individual filed Critical Individual
Publication of CA2244946A1 publication Critical patent/CA2244946A1/en
Application granted granted Critical
Publication of CA2244946C publication Critical patent/CA2244946C/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

The invention consists of recombinant gene expression vectors and vaccines useful in immunization of a host against an antigen and methods for use of such vectors and vaccines. In particular, the recombinant gene expression vectors of the invention are plasmids, cosmids or viruses which include noncoding, palindromic regions of single or double-stranded DNA or RNA polynucleotides which include at least one cytosine-guanine dinucleotide motif in each palindrome. These polynucleotide regions of each expression vector are immunostimulatory and serve as adjuvants to vaccination protocols against target antigens. Most preferably, the recombinant gene expression vectors of the invention are naked; i.e., non-viral vectors not associated with a delivery vehicle such as a liposome. The invention also includes live viral vaccines wherein the viruses include immunostimulatory polynucleotides of the invention. According to a preferred method of the invention, a target protein antigen is administered through its expression by a recombinant gene expression vector which contains the non-coding, immunostimulatory polynucleotides of the invention. In the most preferred embodiment of the method of the invention, the recombinant gene expression vector is administered to tissues of the host which contain a relatively high concentration of antigen presenting cells (e.g., skin or mucosa) compared to other host tissues.

Claims (39)

1. A recombinant gene expression vector into which at least one non-coding, immunostimulatory polynucleotide has been inserted, wherein the immunostimulatory polynucleotide is comprised of at least one strand of a palindrome, wherein the palindrome includes at least one dinucleotide consisting of adjacent, unmethylated cytosine and guanine nucleotides.
2. The recombinant gene expression vector of Claim 1 wherein the vector is a plasmid or cosmid.
3. The recombinant gene expression vector of Claim 2 wherein the plasmid or cosmid is naked.
4. The recombinant gene expression vector of Claim 1 wherein the palindrome of the immunostimulatory polynucleotide is at least 6 nucleotides in length.
5. The recombinant gene expression vector of Claim 1 wherein the dinucleotide in the palindrome consists of a cytosine nucleotide in the 5' position and a guanine nucleotide in the 3' position.
6. The recombinant gene expression vector of Claim 1 wherein the dinucleotide in the palindrome consists of a cytosine nucleotide in the 3' position and a guanine nucleotide in the 5' position.
7. The recombinant gene expression vector of Claim 5 wherein at least two purine nucleotides are upstream and adjacent to the cytosine nucleotide of the dinucleotide and at least two pyrimidine nucleotides are downstream and adjacent to the guanine nucleotide.
8. The recombinant gene expression vector of Claim 5 wherein at least two pyrimidine nucleotides are upstream and adjacent to the cytosine nucleotide of the dinucleotide and at least two purine nucleotides are downstream and adjacent to the guanine nucleotide.
9. The recombinant gene expression vector of Claim 6 wherein at least two purine nucleotides are upstream and adjacent to the guanine nucleotide of the dinucleotide and at least two pyrimidine nucleotides are downstream and adjacent to the cytosine nucleotide.
10. The recombinant gene expression vector of Claim 6 wherein at least two pyrimidine nucleotides are upstream and adjacent to the guanine nucleotide of the dinucleotide and at least two purine nucleotides are downstream and adjacent to the cytosine nucleotide.
11. The recombinant gene expression vector of Claim 1 further comprising a polynucleotide which encodes a polypeptide.
12. The recombinant gene expression vector of Claim 11 wherein the polypeptide is an antigen or immunostimulatory antigen fragment.
13. The recombinant gene expression vector of Claim 11 wherein the polypeptide is a cytokine.
14. The recombinant gene expression vector of Claim 11 wherein the polypeptide is a T lymphocyte epitope.
15. The recombinant gene expression vector of Claim 11 wherein expression of the encoded polynucleotide is under the control of a nuclear receptor promoter.
16. The recombinant gene expression vector of Claim 1 wherein the immunostimulatory polynucleotide is selected from the group of polynucleotides having one strand which consists of AACGTT (SEQ.ID.No.1); GCGCGC
(SEQ.ID.No.3); GACGTC (SEQ.ID.No.4); AGCGCT
(SEQ.ID.No.5); ATCGAT (SEQ.ID.No.6); CGATCG
(SEQ.ID.No.7); CGTACG (SEQ.ID.No.8); CGCGCG
(SEQ.ID.No.9); TCGCGA (SEQ.ID.No.10); ACCGGT
(SEQ.ID.No.11); ACGT (SEQ.ID.No.12); GACGATCGTC
(SEQ.ID.No.13); ACGATCGT (SEQ.ID.No.14); CGACGATCGTCG
(SEQ.ID.No.15); CGACGACGATCGTCGTCG (SEQ.ID.No.16);
CAACGTTG (SEQ.ID.No.17); ACAACGTTGT (SEQ.ID.No.18);
AACAACGTTGTT (SEQ.ID.No.l9), or CAACAACGTTGTTG
(SEQ.ID.No.20).
17. A recombinant gene expression vector consisting essentially of pKCB-1aaZ.
18. A recombinant gene expression vector consisting essentially of pKCB-2aaZ.
19. A method for enhancing the immune response of a host to an antigen comprising introducing a recombinant gene expression vector into a tissue of the host, wherein the recombinant gene expression vector into which at least one non-coding, immunostimulatory polynucleotide has been inserted, wherein the immunostimulatory polynucleotide is comprised of at least one strand of a palindrome, wherein the palindrome includes at least one dinucleotide consisting of adjacent, unmethylated cytosine and guanine nucleotides.
20. The method according to Claim 19 wherein the recombinant gene expression vector comprises a plasmid or cosmid.
21. The method according to Claim 20 wherein the recombinant gene expression vector is naked.
22. The method according to Claim 19 wherein the host tissue into which the recombinant gene expression vector is introduced is a tissue which has a high concentration of antigen presenting cells relative to other host tissues.
23. The method according to Claim 22 wherein the host tissue is skin or mucosa.
24. The method according to Claim 19 wherein the recombinant gene expression vector encodes at least one polypeptide.
25. The method according to Claim 24 wherein at least one of the encoded polypeptides is the antigen or an immunostimulatory fragment of the antigen.
26. The method according to Claim 24 wherein at least one of the encoded polypeptides is a cytokine.
27. The method according to Claim 24 wherein at least one of the encoded polypeptides is a T lymphocyte epitope.
28. The method according to Claim 25 wherein the antigen is expressed in the antigen presenting cells of the host tissue.
29. A method according to Claim 19 wherein the recombinant gene expression vector is coated onto the tynes of a multiple tyne device and is administered by penetrating the skin of the host with the tynes.
30. A method according to Claim 19 wherein the recombinant gene expression vector is introduced by absorption through skin or mucosa treated with a keratinolytic agent.
31. A method according to Claim 24 wherein expression of the encoded polypeptide by the recombinant gene expression vector is under the control of a nuclear receptor promoter.
32. A DNA or RNA virus into which at least one non-coding, immunostimulatory polynucleotide has been inserted, wherein the immunostimulatory polynucleotide is comprised of at least one strand of a palindrome, wherein the palindrome includes at least one dinucleotide consisting of adjacent, unmethylated cytosine and guanine nucleotides.
33. The virus of Claim 32 wherein the palindrome of the immunostimulatory polynucleotide is at least 6 nucleotides in length.
34. The virus of Claim 32 wherein the dinucleotide in the palindrome consists of a cytosine nucleotide in the 5' position and a guanine nucleotide in the 3' position.
35. The virus of Claim 32 wherein the dinucleotide in the palindrome consists of a cytosine nucleotide in the 3' position and a guanine nucleotide in the 5' position.
36. The virus of Claim 34 wherein at least two purine nucleotides are upstream and adjacent to the cytosine nucleotide of the dinucleotide and at least two pyrimidine nucleotides are downstream and adjacent to the guanine nucleotide.
37. The virus of Claim 34 wherein at least two pyrimidine nucleotides are upstream and adjacent to the cytosine nucleotide of the dinucleotide and at least two purine nucleotides are downstream and adjacent to the guanine nucleotide.
38. The virus of Claim 35 wherein at least two purine nucleotides are upstream and adjacent to the guanine nucleotide of the dinucleotide and at least two pyrimidine nucleotides are downstream and adjacent to the cytosine nucleotide.
39. The virus of Claim 35 wherein at least two pyrimidine nucleotides are upstream and adjacent to the guanine nucleotide of the dinucleotide and at least two purine nucleotides are downstream and adjacent to the cytosine nucleotide.
CA2244946A 1996-01-30 1997-01-28 Gene expression vectors which generate an antigen specific immune response and methods of using the same Expired - Lifetime CA2244946C (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US59355496A 1996-01-30 1996-01-30
US08/593,554 1996-01-30
PCT/US1997/001277 WO1997028259A1 (en) 1996-01-30 1997-01-28 Gene expression vectors which generate an antigen specific immune response and methods of using the same

Publications (2)

Publication Number Publication Date
CA2244946A1 true CA2244946A1 (en) 1997-08-07
CA2244946C CA2244946C (en) 2010-04-13

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ID=24375188

Family Applications (1)

Application Number Title Priority Date Filing Date
CA2244946A Expired - Lifetime CA2244946C (en) 1996-01-30 1997-01-28 Gene expression vectors which generate an antigen specific immune response and methods of using the same

Country Status (7)

Country Link
US (1) US20030203861A1 (en)
EP (1) EP0879284B1 (en)
JP (1) JP4359654B2 (en)
AT (1) ATE437943T1 (en)
CA (1) CA2244946C (en)
DE (1) DE69739515D1 (en)
WO (1) WO1997028259A1 (en)

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