WO2007091624A1 - 新規ウイルスベクター - Google Patents
新規ウイルスベクター Download PDFInfo
- Publication number
- WO2007091624A1 WO2007091624A1 PCT/JP2007/052195 JP2007052195W WO2007091624A1 WO 2007091624 A1 WO2007091624 A1 WO 2007091624A1 JP 2007052195 W JP2007052195 W JP 2007052195W WO 2007091624 A1 WO2007091624 A1 WO 2007091624A1
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- WIPO (PCT)
- Prior art keywords
- acnpv
- promoter
- gene
- dua
- antigen
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- LCJVIYPJPCBWKS-NXPQJCNCSA-N thymosin Chemical compound SC[C@@H](N)C(=O)N[C@H](CO)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CO)C(=O)N[C@H](CO)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@H]([C@H](C)O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@H](CCC(O)=O)C(O)=O LCJVIYPJPCBWKS-NXPQJCNCSA-N 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
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- 238000005199 ultracentrifugation Methods 0.000 description 1
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- 229940126580 vector vaccine Drugs 0.000 description 1
- 210000001631 vena cava inferior Anatomy 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
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Classifications
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
- C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
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- C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/20—Vector systems having a special element relevant for transcription transcription of more than one cistron
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention provides a novel transfer vector, a recombinant baculovirus obtained by homologous recombination of the transfer vector and baculovirus DNA, and a method for producing the same.
- the present invention also relates to a medicament (for example, a preventive or therapeutic agent for a vaccine, malaria, influenza virus infection) containing the recombinant baculovirus as an active ingredient.
- a medicament for example, a preventive or therapeutic agent for a vaccine, malaria, influenza virus infection
- Patent Document 1 describes a promoter derived from an early gene of baculovirus.
- a recombinant baculovirus expression vector having a DNA region containing a gene encoding a viral nonstructural protein and a DNA region containing a gene encoding a viral structural protein in a promoter derived from a late gene is disclosed. ing.
- Patent Document 2 discloses a non-mammalian DNA virus containing a promoter that is controlled so that an exogenous gene of a vector in which a desired foreign gene is bound to each of a plurality of independent promoters is expressed. And a method for expressing a foreign gene in a mammalian cell is disclosed.
- Patent Document 3 discloses a method for producing a protein by genetic recombination technology using baculovirus, which encodes the gp64 gene of baculovirus and the desired protein.
- a fusion gene combined with a gene is expressed and produced in a form in which a desired protein is fused to a virus particle, virus particles fused with the desired protein are recovered, and the virus particle force
- Patent Document 4 encodes a detection marker linked to a first promoter that is active in a host cell and inactive in a non-permissive cell with respect to a baculovirus expression system.
- a plurality of independent promoters comprising a second nucleic acid sequence comprising a foreign nucleic acid sequence operably linked to a second promoter active in non-permissive cells.
- Recombinant baculovirus expression vectors have been disclosed.
- Patent Document 5 describes that influenza B. hemagglutin ( ⁇ ) antigen-expressing recombinant baculovirus vector linked to a CAG promoter derived from chicken ⁇ -actin has an influenza virus infection-preventing effect. Therefore, it is disclosed that it is useful as a vaccine preparation!
- Patent Document 6 discloses a plasmid in which a gene encoding a protein that can be expressed on the cell surface is linked to a baculovirus promoter and a promoter derived from a mammalian cell, or two sets of baculovirus promoters. There is disclosed a method for producing a baculovirus vector comprising a co-transformation step of co-transfecting each of these with a plasmid in which a gene encoding a protein that can be expressed on the cell surface is linked to insect cells.
- Patent Document 7 discloses examination of anti-influenza virus activity against influenza virus infection using a recombinant baculovirus in which the cDNA of influenza virus ⁇ is incorporated into the CAG promoter, and only the recombinant baculovirus is disclosed.
- wild-type baculoviruses are also disclosed to have this activity.
- baculovirus vector having a novel structure, which is effective for infectious diseases such as influenza virus, malaria and tuberculosis, or diseases such as cancer, is effective.
- infectious diseases such as influenza virus, malaria and tuberculosis, or diseases such as cancer.
- pharmaceutical preparations particularly vaccine preparations, as ingredients is desired.
- Patent Document 1 Patent No. 3366328: Multiple promoter baculovirus expression system and defects Particle products
- Patent document 2 WO98 / 011243: Non-mammalian DNA having a modified coat protein
- Patent Document 3 Japanese Patent Laid-Open No. 2002-253263: Protein production method
- Patent Document 4 Japanese Patent Laid-Open No. 2003-284557: New baculovirus transfer vector and recombinant baculovirus for expression of foreign genes
- Patent Document 5 WO02 / 062381: Baculovirus vector vaccine
- Patent Document 6 Baculovirus vector 1 'baculovirus vector production method and gene introduction method
- Patent Document 7 Japanese Patent Laid-Open No. 2005-15346: Baculovirus-containing antiviral agent
- An object of the present invention is to provide a novel recombinant transfer vector, a recombinant baculovirus in which the recombinant transfer vector and baculovirus DNA are homologously recombined, and a method for producing the same.
- Another object of the present invention is to provide a medicament, particularly a vaccine preparation, containing the recombinant baculovirus as an active ingredient.
- the present inventors have described a protein having a desired immunogenicity, or a partial protein or a partial protein or both in insect cells and in vertebrate (particularly mammals, birds, fish, etc.) cells.
- a transfer vector with a novel structure capable of expressing a fusion protein of an immunogenic protein and cytokinin, etc. and a recombinant baculovirus obtained by homologous recombination of the recombinant transfer vector and baculovirus DNA It was.
- a recombinant transfer vector having a novel structure a recombinant baculovirus recombined with the recombinant transfer vector and baculovirus DNA.
- Effective as preventive drugs for infectious diseases such as malaria and influenza virus, as a drug that can express a protein having a desired immunogenicity in the target cells This is the end of the invention.
- the present invention provides the invention shown in the following item 1 31.
- Item 1 Downstream of a linked dual 'promoter, one of which is a vertebrate promoter and the other is a baculovirus promoter, and at least one gene encoding a protein capable of constituting a viral particle component
- a method for producing a transfer vector comprising a structure incorporating a dual 'promoter and a fusion gene, comprising linking a fusion gene comprising an immunogenic foreign gene.
- Item 2 The method according to Item 1, wherein the vertebrate promoter is a mammalian promoter.
- Item 3 Genes encoding a protein that can be a constituent of at least one viral particle are the baculovirus gp64 gene, vesicular stomatitis virus glycoprotein gene, type 1 human immunodeficiency virus glycoprotein gene, human respiratory organs Endoplasmic reticulum virus membrane glycoprotein gene, Madalutun protein gene to influenza A virus, Maglchun protein gene to influenza B virus, Herpes simplex virus glycoprotein gene, mouse hepatitis virus S protein gene Item 3. The method according to Item 1 or 2, wherein the method is a deviation.
- Vertebrate promoters are cytomegalovirus promoter, SV40 promoter, retrowinoles promoter, metamouthonein promoter, heat shock protein promoter, CAG promoter, Elongation factor 1 ⁇ promoter, actin promoter, ubiquitin Item 3.
- Noculoinores promoter is polyhedrin promoter, plO promoter
- Item 5 The method according to any one of Items 1 to 4, which is selected from any of the following forces: IE1 promoter, IE2 promoter, p35 promoter, p39 promoter, and gp64 promoter.
- the immunogenic foreign gene is a malaria antigen, influenza virus antigen, or Mycobacterium tuberculosis Original, SARS virus antigen, West Nile virus antigen, Dengue virus antigen, HIV antigen, HCV antigen, leucmania antigen, trypanosomal antigen, leucocytozone antigen alone, or these antigen gene groups 6.
- Section 7. PDua to PfAMA to gp64, pDua to Pfs25- gp64, pDual-H5N1 / HA1-gp64, pDua ⁇ SARS / S-gp64, pCP-H1N1 / HA1-gp64, pCAP-H1N1 / HA 1-gp64, pCU-H1N1 / HA1- gp64, pDua HINl / NP- gp64, pDual-HlNl / M2-gp64, p Dual-HlNl / NAe-gp64, pDua M2e- gp64, pCP- HA1 / NC99- gp64, pCP- H1N1 / HA 0- gp64, Item 7.
- Item 8. A gene encoding a protein that can be a component of at least one viral particle, linked downstream of a linked dual promoter, one of which is a vertebrate promoter and the other is a baculovirus promoter.
- a gene encoding a protein that can be a constituent of at least one virus particle is a baculovirus gp64 gene, a vesicular stomatitis virus glycoprotein gene, a human immunodeficiency virus type 1 glycoprotein gene, or a human respiratory syncytial virus Membrane glycoprotein gene, Madalchun protein gene to influenza A virus, Maglchun protein gene to influenza B virus, simple herpesvirus glycoprotein gene, mouse hepatitis virus S protein gene Item 9. The method according to Item 8, wherein
- Vertebrate promoters are cytomegalovirus promoter, SV40 promoter, retrowinoles promoter, metamouthone promoter, heat shock Item 10.
- the promoter is selected from any one of a promoter, a CAG promoter, an elongation factor 1 ⁇ promoter, a actin promoter, a ubiquitin promoter, an anolebumin promoter, and an MHC class II promoter.
- Item 11 The method according to any one of Items 8 to 10, wherein the promoter of the noculovirus is selected from a polyhedrin promoter, a plO promoter, an IE1 promoter, a p35 promoter, a p39 promoter, and a gp64 promoter.
- the immunogenic foreign gene is malaria antigen, influenza virus antigen, Mycobacterium tuberculosis antigen, SARS virus antigen, West Nile virus antigen, Dengue virus antigen, HIV antigen, HCV antigen, leusimian antigen, trypanosomal antigen, Leukochitozone antigen alone, or a fusion antigen of at least one selected from these antigen gene groups and cyto force in!
- Recombinant baculovirus is AcNPV- Dua ⁇ Hsp65, AcNPV- Dua ⁇ PbCSP, Ac NPV- Dua ⁇ H1N1 / HA1, AcNPV- Dua P PbTRAMP ⁇ AcNPV- Dua P PbAMAlD123, Ac NPV- Dua P PbMSP129, AcNPV — Dua PfCSP ⁇ AcNPV— Dua PfAMAl, AcNPV— Dua Pfs25, AcNPV— Dua H5N1 / HA1, AcNPV— Dua SARS / S, AcNPV— H1N1 / HA1, AcN PV— CAP— H1N1 / HA1, AcNPV— CU—H1N1 / HA1, AcNPV— Dua HlNl / NP, AcNPV— Dua H1N1 / M2, AcNPV— Dua HlNl / NAe ⁇ AcNPV— Du
- Item 14 A gene encoding a protein that can be a component of at least one viral particle and downstream of a linked dual promoter, one of which is a vertebrate promoter and the other is a baculovirus promoter, and at least one A transfer vector containing a structure incorporating a fusion gene containing an immunogenic foreign gene.
- Item 15. A gene encoding a protein that can be a component of at least one viral particle linked downstream of a linked dual promoter, one of which is a vertebrate promoter and the other is a baculovirus promoter, and at least One immunogen Item 15.
- the gene encoding a protein that can be a constituent of at least one viral particle is a baculovirus gp64 gene, a vesicular stomatitis virus glycoprotein gene, a human immunodeficiency virus type 1 glycoprotein gene, or a human respiratory syncytial virus Membrane glycoprotein gene, Madalchun protein gene to influenza A virus, Maglchun protein gene to influenza B virus, simple herpesvirus glycoprotein gene, mouse hepatitis virus S protein gene Item 16.
- Vertebrate promoters are cytomegalovirus promoter, SV40 promoter, retrowinoles promoter, metamouthonein promoter, heat shock protein promoter, CAG promoter, Elongation factor 1 ⁇ promoter, actin promoter, ubiquitin Item 16.
- the promoter of the noculovirus is selected from among the polyhedrin promoter, plO promoter, IE1 promoter, IE2 promoter, p35 promoter, p39 promoter, and gp64 promoter.
- the transfer vector according to crab is selected from any one of a promoter, an anolevmin promoter, and an MHC class II promoter.
- the promoter of the noculovirus is selected from among the polyhedrin promoter, plO promoter, IE1 promoter, IE2 promoter, p35 promoter, p39 promoter
- the immunogenic foreign gene is malaria antigen, influenza virus antigen, Mycobacterium tuberculosis antigen, SARS virus antigen, West Nile virus antigen, Dengue virus antigen, HIV antigen, HCV antigen, leusimian antigen, trypanosomal antigen, Leukochitozone antigen alone, or a fusion antigen of at least one selected from these antigen gene groups and cyto force in!
- the transfer vector according to any one of Items 14 to 18, selected from the displacement force. Paragraph 20.
- Item 21 A recombinant baculovirus produced by the method for producing a recombinant baculovirus according to any one of Items 8 to 13.
- Item 23 A pharmaceutical composition comprising the recombinant baculovirus of item 21 or 22 as an active ingredient.
- Item 25 A pharmaceutical composition comprising the recombinant baculovirus according to claim 21 or 22 as an active ingredient, wherein the composition is administered by any of intramuscular, respiratory tract and nasal routes.
- Item 29 The therapeutic or prophylactic agent for influenza virus infection according to claim 28, wherein the agent is administered by any one of intramuscular, respiratory tract and nasal routes.
- Item 31 The influenza virus infection vaccine according to Item 30, wherein the vaccine is administered by intramuscular, respiratory tract or nasal route.
- a novel recombinant transfer vector a recombinant baculovirus obtained by homologous recombination of the recombinant transfer vector and baculovirus DNA, and methods for producing them.
- the medicine containing the recombinant baculovirus of the present invention as an active ingredient is used as a therapeutic and Z or prophylactic agent for infectious diseases such as malaria, influenza virus, tuberculosis and hepatitis, cancer, autoimmune diseases, etc., or cell medicine. It is useful as a vaccine formulation.
- FIG. 1 is a drawing showing the influenza virus infection prevention effect (virus infection titer) by recombinant baculovirus AcNPV-Dud-H1N1 / HA1.
- FIG. 2 is a drawing showing the preventive effect (survival period) of influenza virus infection by recombinant baculovirus AcNPV-Dua HINl / HAl.
- FIG. 3 Influenza virus antigen gene (H1N1 / HA1), Mycobacterium tuberculosis antigen gene (Hsp65) and surface antigen gene on the surface of malaria parasite sporozoite (PbCSP) in recombinant baculovirus-infected cells that have also produced recombinant transfer vector power ) Western blotting analysis showing the expression of each fusion.
- Lane 1 AcNPV-WT Lane 2: AcNPV— Dua ⁇ HA1N Lane 3: AcNPV— WT Lane 4: AcNPV— Dua ⁇ Hsp65 Lane 5: AcNPV-WT Lane 6: AcNPV-Dual-PbCSP
- FIG. 4 is a drawing of fluorescent labeling showing that the recombinant baculovirus produced in vertebrate cells shows fusion expression of tuberculosis antigen gene (HSP65) and gp64 gene.
- HSP65 tuberculosis antigen gene
- A HepG2 cells with AcNPV- Dua ⁇ Hsp65
- B HepG2 cells with AcNPV-WT
- FIG. 5 Recombinant transfer vector strength in vertebrate cells
- the immunoprecipitation method confirmed that the produced recombinant baculovirus expresses the influenza virus HA antigen gene, gp64 gene and fusion protein. is there.
- HepG2 cells were transfected with AcNPV-WT (lane 1), AcNP V-CMV-HA foil (lane 2) or AcNPV-Dua ⁇ HA1N (lane 3). Three hours after transduction, cells were radiolabeled with [35S] methionine for 12 hours.
- Cell lysates were immunoprecipitated with serum from mice infected with H1N1 influenza virus.
- FIG. 6 A drawing of Western blotting analysis showing expression of a fusion expression product of a malaria parasite CSP gene and a gp64 gene in a virus particle of a recombinant baculovirus that has also produced a recombinant transfer vector force in insect cells.
- Lane 1 AcNPV-WT
- Lane 2 AcNPV-CMV-PbCSP
- Lane 3 AcNPV-PbCSPsurf
- Lane 4 AcNPV- Dua ⁇ PbC SP;
- FIG. 7 is a drawing showing RT-PCR results for confirming that HA1 antigen recombinant baculoviruses with converted vertebrate promoters express HA1 and gp64 fusion genes in HeLa cells, respectively.
- FIG. 9 shows the number of IFN-y producing cells reactive to PbCSP CTL epitopes in spleen cells of mice inoculated with recombinant baculovirus.
- FIG. 10 is a drawing showing the influenza virus infection-preventing effect (virus infection titer) by recombinant baculovirus AcNPV-Dua DuM2e.
- FIG. 11 is a drawing showing the influenza virus infection-preventing effect (virus infection titer) by recombinant baculovirus AcNPV-DuaH ”IAl / NC99.
- FIG. 12 shows the production of influenza virus-specific IgG antibodies induced in the blood by recombinant baculovirus AcNPV-Dual-H1N1 / HA1 administered by four different administration routes.
- FIG. 13 shows the production of influenza virus-specific IgG and IgA antibodies induced by recombinant baculovirus AcNPV-Dual-H1N1 / HA1 administered by four different routes of administration in nasal and alveolar lavage fluids. It is a drawing.
- FIG. 14 is a drawing showing the preventive effect (virus infection titer) of intranasal influenza virus by recombinant baculovirus AcNPV-Dual-H1N1 / HA1 administered by four different administration routes.
- FIG. 15 is a drawing showing the preventive effect (virus infectivity value) of influenza virus in the lung by recombinant baculovirus AcNPV-Dual-H1N1 / HA1 administered by four different administration routes.
- DNA molecule is intended to encompass not only double-stranded DNA but also each single-stranded DNA comprising a sense strand and an antisense strand constituting the same, and its length. It is not limited to this. Therefore, the polynucleotide (DNA molecule) encoding the immunogenic foreign gene of the present invention includes double-stranded DNA including genomic DNA and c unless otherwise stated. Single-stranded DNA containing DNA (sense strand), single-stranded DNA (antisense strand) having a sequence complementary to the sense strand, and synthetic DNA, and fragments of these fragments are also included.
- the polynucleotide and the DNA molecule include at least one of an expression suppression region, a coding region, a leader sequence, an exon, and an intron that do not ask different functional regions. be able to.
- Polynucleotides include RNA and DNA.
- Polypeptides comprising a specific amino acid sequence and polynucleotides comprising a specific DNA sequence include fragments, homologues, derivatives and variants thereof.
- Polynucleotide variants such as variant DNA, include naturally occurring allelic variants; non-naturally occurring variants; mutants with deletions, substitutions, additions and insertions. However, these mutants shall encode a polypeptide having substantially the same function as that of the polypeptide encoded by the polynucleotide before mutation.
- two promoters one of which is a vertebrate promoter (mammalian promoter, avian promoter, fish promoter) and the other is a baculovirus promoter, are linked to the transfer vector.
- the immunogenic foreign gene is located downstream of the dual 'promoter and upstream of the gene encoding a protein that can be a constituent of a virus particle.
- the recombinant baculovirus of the present invention is used in vertebrates as an active ingredient of medicines or vaccines.
- the vertebrates include mammals including humans such as rabbits, horses, pigs, hidges, goats, monkeys, mice, dogs, cats, and the like. Examples include geese, ducks, pigeons, turkeys, guinea fowls, and rums.
- the invention provides that one is a vertebrate promoter and the other is A novel structure that incorporates a fusion gene containing a gene encoding a viral membrane protein that can be expressed in insect cells and one immunogenic exogenous gene under the control of a linked dual promoter that is a promoter of a neurovirus.
- a transfer vector that includes the body.
- This transfer vector is cotransfected with baculovirus DNA in insect cells to induce homologous recombination, and under the control of the baculovirus promoter, expressed in insect cells and germinated virus
- a recombinant baculovirus in which the fusion gene capable of producing a fusion protein that can be a constituent component of the particle is incorporated into a baculovirus can be obtained.
- a fusion protein of a protein that can be a component of germinated virus particles and an immunogenic protein probably functions as a component vaccine when a recombinant baculovirus is administered to a vertebrate. Furthermore, the recombinant baculovirus administered to the vertebrate invades the vertebrate cells, and a fusion antigen with the target immunogenic foreign antigen is produced in the vertebrate cells from the virus genome, and functions as a DNA vaccine.
- a fusion protein of a protein that can be a constituent of virus particles and an immunogenic protein is presented as an antigen.
- a fusion protein of a protein and an immunogenic protein that can be a constituent of virus particles is produced in mammalian cells, and its immunostimulatory action results in viral infections, protozoal infections, bacterial infections, etc. It is thought to function as a preventive or therapeutic agent.
- the baculovirus DNA to be co-transferred with the transfer vector may be any of wild-type, mutant, and thread-replaceable baculovirus DNA.
- Examples of the host cell to be cotransfected include insect cells such as Spodoptera frugiperda.
- An immunogen for immunotherapy including vaccine therapy for the prevention and treatment of infections such as malaria, influenza virus, tuberculosis, autoimmune diseases, cancer, etc.
- a gene encoding an amino acid sequence of an antigen protein such as a gene encoding an amino acid sequence of a protein such as malaria antigen, influenza virus antigen, or Mycobacterium tuberculosis antigen, is referred to as an immunogenic foreign gene.
- the "foreign" gene means a gene introduced with an external force and corresponds to the "foreign” gene even if the same gene is present in the cell.
- the gene encoding the amino acid sequence of the protein serving as the immunogen is a gene encoding the amino acid sequence of an antigen protein having immunogenicity against a disease-causing substance such as an infectious disease, cancer, or autoimmune disease.
- a disease-causing substance such as an infectious disease, cancer, or autoimmune disease.
- genes encoding the amino acid sequences of these immunogenic antigenic proteins include the following.
- Genes encoding the amino acid sequence of the malaria antigen include the surface antigen CSP (Circumsporozoite Protein) on the surface of the malaria parasite, MSPl (Merozoite Surface Protein 1), a membrane protein on the surface of the metrozoite, and secreted from malaria-infected erythrocytes.
- CSP Cercumsporozoite Protein
- MSPl Malozoite Surface Protein 1
- a membrane protein on the surface of the metrozoite and secreted from malaria-infected erythrocytes.
- genes encoding amino acid sequences of proteins such as malaria S antigen, PffiMPl protein, SERA protein, TRAMP protein, and AMA1 protein present in the knob of malaria-infected erythrocytes.
- Genes encoding the amino acid sequence of influenza virus antigen include proteins such as HA antigen (hemadalchun antigen), NA antigen (neuraminidase antigen), M2 antigen (matrix protein antigen), and NP antigen (nucleoprotein antigen). Examples thereof include genes encoding the amino acid sequences.
- Genes encoding the amino acid sequence of the antigen protein for tuberculosis include HSP65 (6 5-kDa heat shock protein), a antigen (Antigen85A, Antigen85B ⁇ Antigen85C), Mtb72f, MDP-1, ESAT-6, MPB51, Examples include genes encoding amino acid sequences of proteins such as Mtb8.8, Mtb9.9, Mtb32, Mtb39 and Mtbl1.
- examples of mammalian genes include genes encoding antigenic proteins for infectious diseases such as humans, horses, horses, pigs, hidges, monkeys, mice, dogs, and cats.
- examples of avian genes include:-Infectious disease antigen genes (eg, avian influenza S antigen) such as chicks, ducks, pigeons, turkeys, guinea fowls, and rums, and fish genes include hamachi, yellowtail, Thailand, Examples include infectious disease antigen genes such as amberjack, horse mackerel, striped horse mackerel, isaki, salmon, beet salmon, carp, crucian carp, zimus, hamame, and amago.
- infectious disease antigen genes such as amberjack, horse mackerel, striped horse mackerel, isaki, salmon, beet salmon, carp, crucian carp, zimus, hamame, and amago.
- Pathogen genes that have been reported to be associated with mammalian, avian and fish infections are as follows: Pathogen genes such as Genbank are registered, and public data is stored and can be easily obtained from such institutions.
- the immunogenic foreign gene is not only the above-described immune antigen existing outside the human body, but also a cytodynamic in gene present in the human body, such as IL-12. Gene, IL-6 gene, IL-6 receptor gene, IL-2 gene, IL-18 gene, IFN-y gene, M-CSF gene, etc., or any antigen with immunogenicity
- a fusion gene obtained by fusing a gene of the antigen protein with a gene recombination technique is treated as an immunogenic foreign gene in the present invention.
- a transfer vector having these immunogenic foreign genes and a homologous recombinant recombinant baculovirus can be provided, and the recombinant baculovirus containing the immunogenic foreign gene is used as an active ingredient.
- a vaccine preparation comprising the pharmaceutical composition can be provided.
- the baculovirus used in the present invention is an entomopathogenic virus that infects insects, and is a group of DNA viruses that possess circular double-stranded DNA as a gene (Baculovirus family).
- a group of viruses called Nuclear Polyhedorosis Virus (NPV) make inclusion bodies called polyhedra in the nucleus of infected cells in the late stage of infection. It was expressed in place of the polyhedron! / ⁇ Even if a foreign gene is inserted, the virus can infect and proliferate without problems and produce a large amount of the desired foreign gene product, which has recently been applied to the production of the desired protein. Yes.
- Examples of the baculovirus used in the present invention include Autographa Californica Nuclear Polyhedorosis Virus (AcNPV) and Bombyx mori Nuclear Polyhedorosis Virus (BmNPV), Examples include Orgyia pseudotsugata Nuclear Polyhedrosis Vinorus (ONPV) and Lymantria disper Nuclear Polyhedorosis Virus (LdNPV).
- AcNPV Autographa Californica Nuclear Polyhedorosis Virus
- BmNPV Bombyx mori Nuclear Polyhedorosis Virus
- OFPV Orgyia pseudotsugata Nuclear Polyhedrosis Vinorus
- LdNPV Lymantria disper Nuclear Polyhedorosis Virus
- the baculovirus DNA may be any DNA that can cause homologous recombination with the transfer vector of the present invention. Specifically, the phase with the transfer vector of the present invention.
- the viral gene of baculovirus DNA that can undergo this recombination is as large as 130 kbp, and an immunogenic foreign gene of 15 kbp or more can be inserted.
- the baculovirus gene itself is hardly expressed in vertebrate cells, it is considered that there is almost no cytotoxicity, and therefore no harmful immune response is induced.
- a method for producing an immunogenic foreign gene DNA that can be fused to a viral gene, which is one of the components of a baculovirus transfer vector comprises the amino acid sequence of the antigenic protein having the target immunogenicity disclosed in this specification.
- the DNA corresponding to the nucleic acid sequence of the coding region of the immunogenic foreign gene is synthesized based on the nucleic acid sequence information of the immunogenic foreign gene. It can be easily produced and obtained by synthesizing it as it is (the DNA synthesis method). For this production, general genetic engineering techniques can be applied. [For example, Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989); Secondary Biochemistry Laboratory “Gene Research Methods I, II, 111", Japan See the Biochemical Society (1986) etc.].
- DNA can be chemically synthesized by the phosphoramidite method or the ester method, or can be synthesized using a commercially available automated oligonucleotide synthesizer.
- Double-stranded fragments can be synthesized by synthesizing complementary strands and annealing the complementary strands to the synthesized single strands under appropriate conditions, or using DNA polymerase with an appropriate primer sequence. Can be obtained by attaching to a single strand synthesized.
- immunogenic foreign gene DNA produced in the present invention include a DNA sequence encoding the amino acid sequence of Mycobacterium tuberculosis antigen protein and a DNA sequence encoding the amino acid sequence of malaria antigen protein.
- influenza virus antigen protein examples thereof include DNA comprising a DNA sequence encoding the amino acid sequence of
- the DNA used in the present invention is not limited to the full-length DNA sequence of the DNA sequence encoding the amino acid sequence of the polypeptide of the antigenic antigen having the immunogenicity, and is encoded by the DNA sequence. As long as the protein having the amino acid sequence has antigenicity, it may be a DNA sequence encoding a partial sequence.
- the DNA used in the present invention includes a DNA sequence encoding the amino acid sequence of the antigenic protein having the above-mentioned immunogenicity and a cytodynamic in gene existing in a human body, such as IL-12.
- Gene, IL-1 gene, IL-6 gene, IL-6 receptor gene, IL-2 gene, IL-18 gene, IFN-a gene, IFN- ⁇ gene, IFN-y gene, TNF gene, TGF-j8 It may be a DNA sequence fused with the DNA sequence of the gene, the GM-CSF gene, and the coding region of the M-CSF gene! /.
- the fusion DNA sequence is not limited to the entire length of the coding region, and the DNA sequence of the site-force in gene fused to the DNA sequence encoding the amino acid sequence of the polypeptide of the antigenic protein having immunogenicity is not limited to the full length. It may be a target DNA sequence.
- the DNA of the immunogenic foreign gene used in the present invention is not limited to a DNA molecule having a strong specific DNA sequence, but a DNA sequence selected by combining arbitrary codons for each amino acid residue. It is also possible to have Codon selection can follow conventional methods. In this case, for example, the codon usage of the host to be used can be considered [Nucleic Acids Res., 9, 43 (1981)].
- the production of the DNA of the immunogenic foreign gene used in the present invention by a genetic engineering technique is more specifically performed according to a conventional method from an appropriate source from which the DNA of the immunogenic foreign gene is expressed.
- a library can be prepared, and the library can also be selected by selecting desired clones using antibodies against appropriate probes and expression products specific to the immunogenic foreign gene DNA [Proc. Natl. Acad. Sci USA, ⁇ 78, 6613 (1981); Science ⁇ 22 2, 778 (1983), etc.].
- examples of the origin of genomic DNA include various cells and tissues that express DNA of an immunogenic foreign gene, cultured cells derived from these, and the like.
- infected red blood cell extracts infected with Plasmodium and influenza virus-infected cell extracts Or tuberculosis extract or the like.
- Extraction and separation of total DNA and RNA from the source, separation and purification of mRNA, acquisition of cDNA, cloning thereof, and the like can all be performed according to conventional methods.
- the DNA of the immunogenic foreign gene is produced by extracting the mRNA of the immunogenic tissue / cell from the extract as described above, and using the cDNA library of each immunogen obtained by 'isolation' purification. For example, after extracting mRNA of each of the above immunogens, adding poly A to the RNA, collecting the poly A-added RNA, producing cDNA using reverse transcriptase, It can also be carried out using a fuzzy library prepared by adding restriction enzyme sites to both ends of the DNA and then incorporating it into a phage.
- the method for screening an immunogenic foreign gene DNA from a cDNA library is not particularly limited, and can be carried out in accordance with a usual method.
- a specific method for example, a method for selecting a corresponding cDNA clone by immunoscreening using a specific antibody (eg, antimalarial antibody, anti-influenza virus antibody, anti-tuberculosis antibody) against a protein produced by cDNA; Examples thereof include a plaque hybridization method using a probe that selectively binds to a DNA sequence, a colony hybridization method and the like, and combinations thereof.
- a DNA fragment chemically synthesized based on information on the DNA sequence of an immunogenic exogenous gene is generally used.
- the already obtained immunogenic foreign genes and the DNA sequences of fragments thereof can be advantageously used as the probe.
- sense primers and antisense primers set based on the DNA sequence information of the immunogenic foreign gene can also be used as the above-mentioned screening probe.
- the DNA (nucleotide) used as a probe is a partial DNA (nucleotide) corresponding to the DNA sequence of an immunogenic foreign gene, and is at least 15 consecutive DNAs, preferably at least 20 consecutive DNAs. More preferably at least 30 contiguous DNAs.
- the positive clone itself for the production of DNA can also be used as a probe!
- the PCR method [Science, 230, 1350 (1985 ;)] Can be suitably used.
- the RACE method [Rapid amplification of cDNA ends; experimental medicine, 12 (6), 35 (1994)], especially the 5'-RACE method [MA Frohman, et al., Proc. Natl. Acad. Sci. ⁇ USA., 8, 8998 (1988)].
- Primers used in the PCR method can be appropriately set based on DNA sequence information of an immunogenic foreign gene and synthesized according to a conventional method. As shown in the examples below, this primer includes a DNA portion (SP6 promoter one primer and T7 promoter) attached to both ends of the vector plasmid into which the DNA of the immunogenic foreign gene has been incorporated. Terminators (primers) can also be used.
- Isolation and purification of the DNA / RNA fragment amplified by the PCR method can be carried out according to a conventional method, for example, by gel electrophoresis.
- the DNA or various DNA fragments of the immunogenic foreign gene obtained as described above can be obtained by a conventional method such as the dideoxy method [Proc. Natl. Acad. Sci., USA., 74, 5463 (1977)],
- the DNA sequence can be determined according to the Maxam-Gilbert method [Methods in Enzymology, 65, 499 (1980)] or simply using a commercially available sequence kit.
- a gene encoding an amino acid of a protein that can be a constituent component of a virus particle it can be fused with the above-mentioned immunogenic foreign gene in a target cell as a fusion protein and can be a constituent component of a virus particle in an insect cell. Any gene encoding a protein that can be expressed as a protein may be used.
- genes encoding amino acids of proteins that can be constituents of the virus particles include gp64 protein (GenBank Accession No. L22858), vesicular stomatitis virus glycoprotein G (VSVG: GenBank Accession No. M21416), simple herpesvirus glycoprotein (KOS: GenBank Accession No. K01760), human immunodeficiency virus type 1 gpl20 (GenBank Accession No. U47783), human respiratory syncytial virus membrane glycoprotein (GenBank Accession No. M86651), genes such as influenza A virus hemaggluton protein (GenBank Accession No. U38242), and genes such as viral envelope proteins closely related to baculovirus.
- the gp64 gene shown in the Examples below can be preferably exemplified.
- the production of DNA of a gene encoding an amino acid of a protein that can be a constituent component of the virus particle is a constituent component of the target virus particle, similar to the preparation of the DNA of the immunogenic foreign gene. Based on the nucleic acid sequence information of the polynucleotide that encodes the amino acid sequence of the polypeptide of the gene encoding the amino acid of the protein to be obtained!
- the power that can be synthesized, or the protein that can be a component of the virus particle By easily synthesizing DNA corresponding to the nucleic acid sequence encoding the amino acid sequence on the basis of the amino acid sequence information of the gene encoding the amino acid of the gene (a biological DNA synthesis method), it is easily produced and obtained. be able to.
- the DNA sequence corresponding to the nucleic acid sequence encoding the amino acid of the protein that can be a constituent of the virus particle is not limited to the full length of the coding region, and may be a DNA consisting of a partial DNA sequence. Good.
- the gene encoding the amino acid of the protein that can be a constituent of the virus particle is obtained using a general genetic engineering technique.
- DNA can be produced. [See, for example, Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989); Secondary Biochemistry Experiment Course “Gene Research Methods I, II, III”, edited by the Japanese Biochemical Society (1986)].
- a part of a promoter that controls the expression of an immunogenic foreign gene described later is already incorporated, and a gene (part) encoding a protein amino acid that can be a constituent of a virus particle is included.
- a commercially available vector plasmid introduced in advance can be used.
- vertebrate promoter capable of functioning in vertebrate cells
- promoters such as mammalian promoters, avian promoters, and fish promoters.
- mammalian promoters include cytomegalovirus promoter, SV40 promoter, retrowinoles promoter, metamouthone imp Examples include mouth motors, heat shock protein promoters, CAG promoters, elongation factor 1 ⁇ promoters, actin promoters, ubiquitin promoters, rubumin promoters, MHC class II promoters, and the like.
- avian promoters include actin promoter, heat shock protein promoter, longion factor promoter, ubiquitin promoter, and anolebumin promoter.
- fish promoters examples include actin promoters, heat shock protein promoters, and elongation factor promoters.
- baculovirus promoter that is one of the components of the baculovirus transfer vector used in the present invention include polyhedrin promoter, plO promoter, IE1 promoter, p35 promoter, p39 promoter, and gp64 promoter.
- the present invention relates to a novel transfer vector having a novel structure capable of expressing a target immunogenic foreign gene as an antigen protein in both insect cells and vertebrate cells, particularly mammalian cells.
- the structure of a novel transfer vector to be produced is that one is a vertebrate promoter, particularly a mammalian promoter, and the other is a baculovirus promoter. It is characterized by having a structure in which a DNA sequence encoding the amino acid sequence of the original protein and a DNA sequence encoding the amino acid sequence of the protein that can be a constituent of virus particles are linked.
- the vertebrate promoters to be linked may be directly linked or intervened between each promoter's DNA sequences. DNA sequences may be present (in which case the promoters of each other must have promoter activity in insect and vertebrate promoters, particularly mammalian cells) There is).
- the vertebrate promoter to be linked particularly the mammalian promoter and the baculovirus promoter, may be arranged closer to the gene to be expressed in the promoter region. In the examples described later, the baculovirus promoter is used. Has a structure located closer to the gene to be expressed than the mammalian promoter.
- the DNA sequence of a fusion gene comprising a gene encoding a gene protein that can be a component of a virus particle in the structure and a desired immunogenic foreign gene directly connects these two genes. It may consist of DNA sequences, and there may be intervening DNA sequences between them (in this case, however, the downstream gene and the upstream gene undergo a frame shift). Don't wake up, need to be arranged like). It is preferable that the antigen-presenting region of a protein of a foreign gene having a desired immunogenicity is fused with a protein that can be a constituent component of a virus particle. It is necessary to use the protein of the gene in a form fused with the protein without being separated from the protein that can be a constituent of the virus particle.
- a fusion gene containing these two genes may be formed in advance and incorporated into a vector, or one of the genes may be incorporated into the vector first, and then the other gene may be incorporated into the vector. It can be integrated to form a fusion gene in a vector.
- the above operation is a part of the required structure of the transfer vector of the present invention.
- the above vertebrate promoters in particular, promoter regions such as mammalian promoters and baculovirus promoter motors, and constituents of virus particles
- a commercially available expression vector that already has a gene region that encodes the amino acid sequence of a protein that can be converted into a desired region in the cloning region of the vector by arbitrarily cutting it with a restriction enzyme and incorporating another promoter.
- Insert a DNA sequence fused with an immunogenic foreign gene and a gene encoding the amino acid sequence of a protein that can be a component of a virus particle, or the desired immunogenic foreign gene has already been incorporated into a plasmid.
- Amino acid sequences of proteins that can be constituents of roaring viral particles Such as by inserting the N-terminal side of the DNA region of the gene encoding, it is possible to insert the required components.
- Proteins that can be constituents of the desired immunogenic foreign gene and virus particles His 'Tag, HVS' Tag, etc. may be added to detect the protein before the poly-A-tille on the C-terminal side of the DNA sequence fused with the gene encoding the amino acid sequence of Or expression of a fused recombinant protein between the promoter region and a region where a desired immunogenic foreign gene and a gene encoding the amino acid sequence of a protein that can be a constituent of a virus particle are fused.
- a DNA sequence encoding a FLAG sequence consisting of an 8 amino acid sequence may be inserted as a peptide tag.
- some of these plasmid vectors having a structure capable of expressing a desired immunogenic foreign gene as an antigen protein in both insect cells and vertebrate cells, particularly mammalian cells, of the present invention are used. You may manufacture using the commercially available plasmid which has the structure already satisfied. An amino acid sequence of a peptide for cleaving the fusion protein with an enzyme in a vertebrate cell may be interposed.
- the transfer vector of the present invention may be arranged with an enzyme for increasing transcriptional activity in vertebrates, particularly mammalian cells, upstream of the two promoter regions.
- Signal for promoting host exocrine secretion 'A DNA sequence encoding the amino acid sequence of the peptide may be linked to a gene for fusion expression.
- a vertebrate terminator region such as a rabbit ⁇ globulin terminator effective in vertebrate cells, may be provided downstream of the gene to be fused and expressed to terminate transcription.
- a specific example of the transfer vector of the present invention and the method for producing the same is as shown in the examples below, as a vertebrate promoter, particularly a cytomegalovirus (CMV) promoter or CMV promoter as a mammalian promoter.
- CMV cytomegalovirus
- the improved CAG promoter, a promoter with the ubiquitin (UBB) promoter joined to the CMV enhancer, and the polyhedrin (Polh) promoter as the baculoinores promoter are linked, and the influenza virus antigen gene, Amino acid sequence of protein that can be a component of malaria antigen gene, tuberculosis antigen gene and virus particle PDuaH "Isp65-gp64, pDual-PbCSP-g p64, pDual-HlNl / HAl-gp64, transfer vectors consisting of a DNA sequence fused with the gp64 antigen gene as a gene encoding the sequence.
- the present invention can also be a component of at least one viral particle linked downstream of a linked dual 'promoter, one of which is a vertebrate promoter, particularly a mammalian promoter, and the other is a baculovirus promoter.
- a step of producing a transfer vector comprising a structure in which a fusion gene comprising a gene encoding the amino acid sequence of a protein and at least one immunogenic foreign gene is incorporated; and the transfer vector and baculovirus DNA in a host cell
- a method for producing a recombinant baculovirus comprising a step of isolating a recombinant baculovirus, which has a process power for cotransfusion.
- a method for introducing a desired recombinant DNA (transfer vector) into a host cell and a method for transformation thereby are not particularly limited and are well known in the general field.
- Various conventional methods can be employed, such as conventional gene recombination techniques (e.g. Science, 224, 1431 (1984); Biochem. Biophys. Res. Comm. ⁇ 130, 692 (1985) Proc. Natl. Acad. Sci. USA ⁇ 80, 5990 (198 3).
- Recombinant DNA (transfer vector) expression method is described in Ohno et al., “Protein Experiment Protocol 1 Functional Analysis, Cell Engineering separate volume, experimental protocol series, 1997, Shujunsha ”can be produced as a reference.Insect cell handling, general methods of genetic recombination and cotransfusion are well known. Insect cell set Zenji Kajiura, a protein nucleic acid enzyme that can be used in the same way as a method for producing a recombinant virus
- the obtained recombinant baculovirus can be cultured according to a conventional method, and the DNA of the immunogenic foreign gene and the protein that can be a constituent of virus particles in the present invention designed as desired by the culture. Fusion (expression product) that is fused with DNA encoding the amino acid sequence Force Expressed and produced (accumulated and secreted) in the intracellular, extracellular or cell membrane of insect cells.
- Various media commonly used according to the employed host cells can be appropriately selected and used as the medium used for the culture, and the culture can also be performed under conditions suitable for the growth of the host cells.
- the method for producing a recombinant baculovirus and more particularly, process and said transformer fur vector and baculovirus DNA to prepare baculovirus D NA for performing transfer one vector and homologous recombination prepared above In Spodoptera frugiperda-derived Sf-9 cells, Sf-21 cells, and Tn5 cells derived from Trichoplusia ni (High Five cells: Invitrogen) It consists of the process of co-transformation.
- the baculovirus DNA for homologous recombination with the transfer vector produced above may be any of wild-type, mutant, or recombinant baculovirus DNA.
- the noculovirus DNA contains the amino acid sequence of a protein that can be a component of a dual 'promoter region DNA, an immunogenic foreign gene, and a virus particle.
- the probability of homologous recombination can be increased as long as it has a DNA structure that is homologous to baculovirus-derived DNA sandwiching the DNA of the fusion gene fused with the encoding gene.
- the weight ratio of the transfer vector to the baculovirus DNA is preferably mixed at, for example, about 1: 1 to 10: 1.
- virus plaques are prepared from the culture supernatant, suspended in the culture medium, vortexed to elute the virus from the agar, and centrifuged. Isolate and obtain a solution containing the recombinant virus.
- the baculovirus DNA may be a commercially available product.
- BacVector-1000 DNA or BacVector-2000 DNA (N ovagen).
- transfer vectors and baculovirus DNA obtained above are cotransfections for recombination of homologous yarns in insect cells.
- commercially available vector transfer kits (Bac Vector Transfection Kits (manufactured by Novagen) can be carried out according to the instruction manual attached to the vector transfer kit.
- the transfer vector produced above can produce recombinant baculovirus that is co-transformed into insect cells such as Sf-9 cells together with baculovirus DNA.
- a method for producing the recombinant baculovirus described above other recombinant baculoviruses can be produced using a phagemid (bacmid) incorporating the entire baculovirus genome.
- a method that can efficiently insert a foreign gene in Escherichia coli using sposposon can also be used.
- a recombinant baculovirus can be easily produced and recovered simply by extracting the bacmid having the viral gene and also transforming it into insect cells.
- the recombinant baculovirus purification of the present invention obtained by the above-described method for producing a recombinant baculovirus can be performed using a known virus purification method.
- Recombinant baculovirus can be purified, for example, by using a stock virus (usually 1 x 10 7 dishes / 10 cm dish) obtained from the above recombinant baculovirus production method.
- a stock virus usually 1 x 10 7 dishes / 10 cm dish obtained from the above recombinant baculovirus production method.
- X 10 7 -8 pfo / ml inoculate 0.5-1.0 ml, collect the culture supernatant several days after infection (4 days), and then suspend the virus pellet obtained by centrifugation in a buffer solution such as PBS. To do.
- the obtained suspension is subjected to a sucrose density gradient of 10 to 60%, and further centrifuged (25000 rpm, 60 minutes, 4 ° C conditions) to recover the virus band.
- the collected product is further suspended in PBS, then centrifuged (same as above), and the resulting purified recombinant virus pellet is stored at 4 ° C in a buffer solution
- the infectivity titer of the purified recombinant virus obtained above was measured by plaque assay using insect cells such as Sf-9 cells (Fields VIROLOGY 4th Edition p29-32 2001). Monkey.
- the baculovirus protein gp64 has an N-terminal end exposed to the outside of the particle and a C-terminal exposed to the inside of the particle.
- the protein is fused to the N-terminal side of gp64, the whole of the virus protein particle is exposed to the insect cell as a component of the virus particle, which makes it easier to present the antigen. It can be said that it is suitable for the purpose as a vaccine preparation
- composition of the present invention (medicine comprising the recombinant baculovirus of the present invention as an active ingredient)
- the recombinant baculovirus of the present invention comprising an active ingredient in the pharmaceutical composition of the present invention is the genetic engineering technique described in (2) above. Can be obtained.
- the pharmaceutical composition of the present invention is used as a constituent of the immunogenic foreign gene and virus particles of the present invention. Homologous recombination of a transfer vector and a baculovirus DNA constructed so that a fusion fused with a gene encoding the amino acid sequence of a possible protein can be expressed in insect cells and vertebrates, particularly mammalian cells including humans. It is important to contain the recombinant baculovirus obtained as an active ingredient.
- the pharmaceutical yarn composition according to the present invention includes specific AcNPV- Dua HINl / HAl, AcNPV- Dua Hsp65, AcNPV- Dua PfCSP, AcNPV- Dua-PfAMAl, AcNPV- Dua- Pfs25, AcNPV- Dual--.
- blood cells, other target cells are hepatocytes, kidney cells, and the like can be used for the treatment of diseases related to infection of target cells or tissues.
- Tissues containing these cells include lung, liver, kidney, brain, arteriovenous blood vessel, stomach 'intestine, urethra, skin, muscle, etc. Can be mentioned.
- the pharmaceutical composition includes, for example, a surface antigen CSP on the surface of a malaria parasite sporozoite, MSP1 of a membrane protein on the surface of a metrozoite, a malaria-infected erythrocyte force secreted malaria S antigen, a P1EMP1 protein present in a knob of a malaria-infected erythrocyte , SERA protein, TRAMP Increases the anti-infective effect against these infectious antigens against malaria antigens such as protein, AMA1 protein or against influenza virus antigens such as HA antigen, NA antigen, M2 antigen, NP antigen, etc.
- the survival period and survival rate for mammals including infected humans are compared with those in which the pharmaceutical composition of the present invention was not administered. It is useful as a preventive or therapeutic agent for malaria and influenza virus infection.
- the pharmaceutical composition of the present invention uses the action of enhancing the infection prevention effect against infectious antigens and lowering the infectious titer, thereby causing influenza virus, papilloma virus, herpesvirus, AIDS virus, hepatitis C virus, Viral diseases such as SARS virus, West Nile fever virus, dengue fever virus, and protozoan diseases such as malaria, trypanosoma, and leushimania, dysentery, typhoid fever, cholera, pneumococci, MRSA, VRE, gonorrhea, chlamydia, syphilis, tuberculosis It is useful as an agent for the treatment or prevention of infectious diseases and complications caused by pathogen infection such as bacterial diseases.
- an immunogenic gene for a transfer vector for obtaining a recombinant baculovirus as an active ingredient in the pharmaceutical composition of the present invention, an immunogenic gene for vertebrates other than humans, for example, As a chicken flu vaccine, ushitorino V saumatosis vaccine, and cold water disease vaccine, it has the effect of increasing the infection prevention effect against infectious antigens of these diseases and lowering the infectious value.
- the activity can be used to treat diseases associated with infection of target cells or tissues.
- the pharmaceutical composition of the present invention can be prepared as a composition comprising a pharmaceutically effective amount of the recombinant baculovirus of the present invention and a pharmaceutically acceptable carrier.
- the infection-protecting effect of the recombinant baculovirus of the present invention in vertebrates, particularly mammals including mammals or mammalian cells depends on, for example, the recombinant baculovirus of the present invention and a composition that can be added for pharmaceutical administration. After the produced pharmaceutical composition is administered intramuscularly, nasally or via the respiratory tract to vertebrates, particularly mammals including humans, the recombinant baculovirus of the present invention is used as an active ingredient a plurality of times. Immunize with a pharmaceutical composition. It is particularly preferable to administer the pharmaceutical composition of the present invention preferably via the respiratory tract.
- the pharmaceutical agent of the present invention after a lapse of a fixed period when the target pathogen is administered to a vertebrate, particularly a mammal including a human, to cause infection.
- the effect of preventing infection can be measured by comparing the survival rate in vertebrates, particularly mammals including humans, to which a recombinant baculovirus which is an active ingredient of the composition is administered.
- Fusion product of DNA sequences fused with genes encoding amino acid sequences of proteins that can be components of foreign genes and virus particles Virus particles budding from insect cells The pharmaceutical composition of the present invention is then administered in the form of viral particles to vertebrates, particularly mammals including humans, thereby obtaining foreign antigenic proteins that are constituents of viral particles. Immunity (humoral immunity and cellular immunity) is further promoted, and the antigen protein, which is an expression product of the fusion DNA sequence, is further acquired in mammalian cells including vertebrates, particularly humans (humoral immunity and cellular immunity). ) And is useful as a vaccine.
- the vaccine of the present invention has a specific AcNPV- DuaH "IlNl / HAl, AcNPV- Dua Hsp65, AcNPV-Dual-P! CSP, AcNPV- Dua-PfAMAl, AcNPV- Dua-Pfs25, AcNPV- Dua-H5.
- a vaccine comprising one of the recombinant baculoviruses of N1 / HA0, AcNPV—CP—H1N1 / HA2, AcNPV—CP—H1N1 / HA1—vp39, and AcNPV—CP—HI Nl / NP-vp39 as an active ingredient.
- the vaccine may be, for example, a surface antigen (CSP) on the surface of a sporozoite of Plasmodium, MSP1 of a membrane protein on the surface of a metrozoite, a malaria-infected erythrocyte Antigen, against malaria antigens such as PffiMPl protein, SERA protein, TRAMP protein and AMA1 protein present in the knob of malaria-infected erythrocytes, or influenza virus HA antigen, influenza virus NA antigen, influenza virus M2 antigen, influenza virus NP antigen
- infectious titer e.g., viral infectious titer
- influenza antigens such as, it is possible to increase the survival time and survival rate for mammals including infected humans.
- the vaccine of the present invention utilizes the action of increasing the infection prevention effect against infectious antigen and lowering the infectious titer, thereby causing influenza virus, papilloma virus, herpesvirus, AIDS virus, hepatitis C virus, Viral diseases such as SARS virus, West Nile fever virus, dengue fever virus, and protozoan diseases such as malaria, trypanosoma, and leushimania, dysentery, typhoid fever, cholera, pneumococci, MRSA, VRE, gonorrhea, chlamydia, syphilis, tuberculosis It is useful as an agent for the treatment or prevention of infectious diseases and complications caused by pathogen infection such as bacterial diseases.
- an immunogenic gene for vertebrates other than humans can be used, for example.
- Avian influenza vaccine, ushitrypanosomiasis vaccine, fish chilled water disease vaccine has the effect of increasing the infection prevention effect against infectious antigens of these diseases and lowering the infectious titer. It can be used to treat diseases related to infection of target cells or tissues.
- the present invention AcNPV-DuaH "IlNl / HAl, A as an active ingredient in the vaccine of the present invention cNPV— Dua ⁇ Hsp65, AcNPV— Dua PfCSP ⁇ AcNPV— Dua ⁇ PfAMAl, AcNPV— Dua ⁇ Pfs 25, AcNPV— Dua H5Nl / HAl, AcNPV— Dua ⁇ SARS / S, AcNPV— H1N1 / HA1, AcNPV — CAP— H1N1 / HA1, AcNPV— CU—H1N1 / HA1, AcNPV— DuaH to I IN 1 / NP, AcNPV— Dua H1N1 / M2, AcNPV— Dua HlNl / NAe ⁇ AcNPV— Dua M2e, AcNPV— CP— HA1 / NC 99, AcNPV—CP—H1N1 / HA0, AcNPV— CP—
- target cells affected by intense infection include blood cells, and other target cells include hepatocytes, kidney cells, brain cells, lung cells, epithelial cells, muscle cells, and the like.
- tissues containing these cells include lung, liver, kidney, brain, arteriovenous blood vessel, stomach and intestine, urethra, skin and muscle.
- the vaccine of the present invention has a pharmaceutically effective amount of the recombinant baculovirus of the present invention (AcNPV—Dua ⁇ H1N1 / HA1, AcNPV—Dua ⁇ Hsp65, AcNPV—Dua) as the pharmaceutical composition of (3) above.
- G P1CSP AcNPV— Dua ⁇ PfAMAl, AcNPV— Dua ⁇ Pfs25, AcNPV— Dua ⁇ H5N1 / HA1, Ac NPV— Dua ⁇ SARS / S, AcNPV— H1N1 / HA1, AcNPV— CAP— H1N1 / HA1, AcNPV— CU— H1N1 / HA1, AcNPV-Dual-HlNl / NP, AcNPV— Dua HlNl / M2, AcNPV— Dua ⁇ H1N 1 / NAe ⁇ AcNPV— Dua M2e ⁇ AcNPV— CP— HA1 / NC99, AcNPV— CP— H1N1 / HA0, Ac NPV—CP—H1N1 / HA2, AcNPV—CP—H1Nl / HAl—vp39, AcNPV—CP—HlNl / NP—vp39)
- a vaccine can be prepared in the form of a pharmaceutical composition according to a conventional method using a pharmaceutically acceptable carrier similar to the pharmaceutical composition of (3) above.
- a pharmaceutically acceptable carrier similar to the pharmaceutical composition of (3) above.
- the carrier include physiologically acceptable solutions such as sterile physiological saline and sterile buffered saline.
- the vaccine (hereinafter referred to as the pharmaceutical composition is the same as the pharmaceutical composition) is the recombinant baculovirus of the present invention (AcNPV—Dua ⁇ H1N1 / HA1, AcNPV—Dua ⁇ Hsp65, AcNPV—Dual-PfCSP).
- a specific example of the vaccine (pharmaceutical composition) of the present invention is a ribosome preparation.
- the ribosome preparation can be obtained by retaining the recombinant baculovirus of the present invention in the ribosome having a force that uses acidic phospholipid as a membrane component or a membrane component of neutral phospholipid and acidic phospholipid.
- acidic phospholipids and neutral phospholipids as membrane constituents one kind of various lipids commonly used in ribosome preparations of the kind that is not limited is used alone, or two or more kinds are mixed. Can be used.
- the ribosome membrane is formed according to a conventional method using the force of using an acidic phospholipid alone or a combination of a neutral phospholipid and an acidic phospholipid.
- a neutral phospholipid combined proportion of acidic phospholipids, 0.1 to 100 mole 0/0 approximately ribosome membrane components in, preferably 1 to 90 molar 0/0, more preferably 10 to 50 it is preferable to the mole 0/0 about.
- preparing the ribosome for example, cholesterol or the like can be added. Thereby, the fluidity of the phospholipid can be adjusted, and the preparation of the ribosome can be made simpler.
- the cholesterol is preferably added and blended in an amount up to an equal amount with respect to the phospholipid, preferably in an amount of 0.5-fold to an equal weight.
- the mixing ratio of the active ingredient and the acidic phospholipid in the ribosome preparation is such that the acid phospholipid is about 0.5 to 100 equivalents, preferably about 1 to 60 equivalents, more preferably 1.5 to 20 equivalents with respect to the active ingredients. It is good to be about.
- the mol% of the recombinant baculovirus of the present invention used as an active ingredient to the total lipid is several mol% to several tens mol%, preferably about 5 to 10 mol%, usually around 5 mol%. It is out.
- the above ribosome preparation, concentration, particle size control, etc. were performed according to conventional methods. it can. In addition, the ribosome preparation can be blended with the above-mentioned various additives as required.
- the recombinant baculovirus of the present invention as an active ingredient includes a fatty acid (for example, behenic acid, stearic acid, palmitic acid, myristic acid, oleic acid, etc.), an alkyl group
- a cholesteryl group and the like can be bonded to each other.
- the production of ribosomes prepared by combining them can also follow conventional methods (Long Circulating Liposomes: old drugs, New therapeutics., M. and Woodle, G. Storm, Eds: Springer—Verlag Berlin ( 1998)).
- the vaccine (pharmaceutical composition) of the present invention can be preferably used as a vaccine composition. In its use, it is preferred to use in combination with a pharmaceutically effective amount of an adjuvant to enhance the anti-infective (anti-malarial or anti-influenza virus) effect.
- any of those commonly used for this type of vaccine can be used without limitation.
- examples include Freund's complete adjuvant, muramyl dipeptide, hydroxyaluminum hydroxide, BCG, IL-12, N-acetylmethylmuramine-L-ara-l-D-isoglutamine (MDP), thymosin 1, QS -21 and the like.
- the amount of adjuvant used depends on the extent of symptoms such as softening of the skin, pain, erythema, fever, headache, muscle pain, etc. that may appear as part of the immune response to humans or animals after administration. Can be determined as appropriate.
- the vaccine (pharmaceutical composition) of the present invention can be used in combination with other known pharmaceuticals such as an immune response promoting peptide and an antibacterial agent (synthetic antibacterial agent).
- the vaccine can contain arbitrary drugs, additives and the like.
- a drug that helps the cellular uptake of the recombinant baculovirus of the present invention such as calcium ion can be exemplified.
- ribosomes and agents or additives that facilitate transfection such as fluorocarbon emulsions, cochleates, tubules, gold particles, biodegradable microspheres, cationic polymers, etc. can do.
- the amount of the active ingredient contained in the vaccine (pharmaceutical composition) (formulation) of the present invention is not particularly limited as long as it is a pharmaceutically effective amount, and a wide range of power can be selected.
- vaccine medicine
- the dose of the composition is not particularly limited, and can be appropriately selected over a wide range according to the desired therapeutic effect, administration method (administration route), treatment period, patient age, sex, and other conditions.
- a recombinant baculovirus that is an active ingredient of the vaccine (composition) of the present invention when administered to humans, it is converted to PFU of the recombinant virus and is equivalent to 10 2 to 10 12 PFU per patient. More preferably, a recombinant baculovirus equivalent to 10 6 to 10 9 PFU is administered.
- the vaccine (pharmaceutical composition) of the present invention is administered locally (for example, in pulmonary tissue) of a recombinant baculovirus suspension suspended in PBS (phosphate buffered saline) or saline. (Intrahepatic, intramuscular, and intracerebral), or by intranasal 'inhalation via the airway' or by intravascular (eg, intraarterial, intravenous, and portal vein) .
- the administration of the vaccine of the present invention is preferably exemplified by administration via the respiratory tract.
- the vaccine of the present invention is preferably administered by administering one or more additional vaccines while observing the state after the first administration rather than by a single administration. .
- the desired effect can be further enhanced.
- the recombinant baculovirus of the present invention (AcNPV-DuaH "IlNl / HAl, AcNPV—Dua ⁇ Hsp65, AcNPV—Dua ⁇ PfCSP ⁇ AcNPV—Dua ⁇ PfAMAl, AcNPV—Du— Pfs25, AcNPV— Dua ⁇ H5N1 / HA1, AcNPV— Dua ⁇ SARS / S, AcNPV— H1N1 / HA1, AcN PV— CAP— H1N1 / HA1, AcNPV— CU— H1N1 / HA1, AcNPV— Dua HlNl / NP,
- the recombinant baculovirus that is one of the active ingredients of the vaccine (medicine composition) of the present invention is generally the desired immunity.
- a pharmaceutically acceptable carrier i.e., non-toxic to vertebrates including humans at the dosage and concentration used
- a formulation in the form which are compatible with the other ingredients.
- the formulation preferably does not contain oxidizing agents and other compounds known to be harmful to recombinant baculoviruses! /.
- the carrier suitably includes minor amounts of additives such as substances that enhance isotonicity and iodologic stability.
- additives such as substances that enhance isotonicity and iodologic stability.
- Such substances are nontoxic to vertebrates, particularly mammals including humans, at the dosages and concentrations used, and phosphoric acid, succinic acid, succinic acid, acetic acid, and other organic acids or Buffers such as their salts; anti-acid agents such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides (eg, polyarginine or tripeptide); proteins (eg, serum albumin) Hydrophilic polymers (eg, polyvinylpyrrolidone); amino acids (eg, glycine, glutamic acid, aspartic acid, or arginine); monosaccharides, disaccharides and other carbohydrates (cellulose or derivatives thereof) , Including glucose, mannose, or dextrin); chelating agents (eg, EDTA
- a pharmaceutical composition comprising a vaccine (composition) of the present invention is a clinical condition of an individual patient (eg, a condition to be prevented or treated) in a manner consistent with good medical practice.
- the vaccine (composition) containing the recombinant baculovirus is administered in consideration of the site of delivery, the target tissue, the method of administration, the dosing schedule and other factors known to those skilled in the art. Accordingly, the appropriate dosage of the vaccine (composition) herein is determined in view of the above.
- Example 1 Transfer vector plasmid of the present invention and production method thereof
- pcDNA-CS87 is constructed according to the method of Yoshida et al. (Yoshida, S., et al., BBRC, 271, 107-115 (2000)), and the genomic DNA of Plasmodium berghei ANKA strain and mouse Igk secretion signal sequence and FLAG sequence A Nhel-Notl fragment containing a fused sequence was obtained, and the Nhel-Notl fragment was inserted into the Nhel-Notl site of pcDNA3.1 (Invitrogen: Invitrogen).
- Newly created Nhel site is underlined, mouse Ig kappa secretion signal sequence is italic, FLAG sequence is double underlined) and PbCSP-Rl (5,-GGAGGGCGGCCGCATCCCGGGTTT ): PCR was performed using the newly created Not I site underlined). PCR was performed using PfoDNA polymerase (Stratagene) for 30 cycles (denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 1 minute, and extension at 72 ° C for 2 minutes). The PCR product encodes PbCSP fused to the mouse Ig kappa secretion signal sequence in place of its original signal sequence without the C-terminal glycosyl phosphatidylinositol (GPI) anchor.
- GPI glycosyl phosphatidylinositol
- the pcDNA-CS87 plasmid contains a CMV promoter, mouse Igk secretion signal sequence, P bCSP gene protein (corresponding to amino acid positions 21-299), poly-A signal derived from ushi growth hormone gene, and poly-A sequence! /
- the Hsp65 gene was extracted from the M. tuberculosis H37Rv strain using QIAamp DNA Midi Kit (Qiagen) and cloned by PCR. That is, the extracted genomic DNA of Mycobacterium tuberculosis H37Rv is the primer phsp65-Fl (primer (5 '
- Bglll site is underlined
- phsp65- R1 ((5'- AATCCAATGCGGCCGCGGGAAT
- pcDNA-hsp65 After the PCR product was purified, it was cleaved with the restriction enzyme BglllZNotl, ligated into the BamHlZNotl site of pcDNA3.1 (+) (manufactured by Invitrogen), and the resulting plasmid was named pcDNA-hsp65.
- the pcDNA-hsp65 plasmid is a fusion of mouse Igk secretion signal sequence and hsp65 gene. Is a construct.
- the plasmid constructed by force was named pBACsurf-Hsp65.
- Primer pPolh-F2 (5, -CACCCGGACCG GATAATTAAAATGATAACCATCTCGCAAATAAATAAG-3 '(SEQ ID NO: 7): RsrII site is underlined) and pgp64—R2 (5'-GGTACCATATTGTCTATTACGGTTTCTAAT CATAC-3 ' PCR was performed using (SEQ ID NO: 8): the Kpnl site is underlined, and the resulting gp64 gene DNA fragment (about 1700 bp) was inserted into pENTR / D-TOPO to construct plasmid pENTR-gp64.
- the plasmid constructed by force was named pENTR-gp64.
- pBACsurf-Hsp65 was cleaved with PstI / Cfr9I, and the hsp65 gene fragment (about 1660 bp) was inserted into the PstI / Cfr9I site of pENTR-gp64 to construct plasmid pENTR-Hsp65-gp64.
- pENTR-hsp65-gp64 was cleaved with Rsrll / Kpnl, and a DNA fragment consisting of the polyhedrin promoter and the hsp65-g p64 gene (approximately 3360 bp) was inserted into the Rsrll / Kpnl site of TriEx-3 (Novagen). Then, a transfer vector plasmid pDua ⁇ Hsp65-gp64 whose expression was controlled by the desired dual promoter was constructed.
- pCR-Blunt-HA as a saddle type primer pHA—F1 (5'-CACCGAATTCGACACAATATGTATAGGCTACCATGCG-3 '(SEQ ID NO: 11): EcoRI site is underlined) and pHA-R1 (5'-CCCGGGCACCTCTGGA TTGGATGGACGGAATG-3' (sequence No.
- the PbTRAMP gene was cloned by PCR by the following method. That is, primers pTRAMP- Fl (5,-CACCGAATTCAAAATTGATACGAAAA AAAATGAAG -3 '(SEQ ID NO: 13): EcoRI site is underlined) and pTRAMP-Rl (5'-CCCGGGCTTTTAATTTTGAGGAGTCTTTATTTTC-3, (SEQ ID NO: 14): Cfr9I site is PCR was performed using the underlined), and the resulting PbTRAMP DNA fragment (approximately 860 bp) was cloned into pENTR / D-TOPO (Invitrogen), cleaved with EcoRI / Cfr9I, and pBA Csurf-Hsp65 Inserted into the EcoRI / Cfr9I site.
- the constructed plasmid is called pBACsurf-PbTRA I named it MP.
- pBACsurf-PbTRAMP was cleaved with EcoRI / Cfr9I, and a PbTRAMP gene DNA fragment (about 860 bp) was inserted into the EcoRI / Cfr9I site of pDuaH "Isp65-gp64 to construct plasmid pDua ⁇ PbTRA MP-gp64.
- the PbAMAl gene domain 123 was cloned by PCR by the following method. That is, primers pAMA- Fl (5, -CACCGAATTCAATCCA TGGGAAAAGTATACGGAAAAATAT-3 '(SEQ ID NO: 15): EcoRI site is underlined;) and pAMA-to-R1 (5'-CCCGGGCTTCTCTGGTTTGATGGGCTTTCATATGCAC-3' (SEQ ID NO: 16): Cfr9I site PCR is performed using the PbAM A1D123 DNA fragment (approx.
- pBACsurf- PbAMAlD123 was cleaved with EcoRI / Cfr9I, and the PbAMAl D123 gene DNA fragment (about 1280 bp) was added to the EcoRI / Cfr9I site of pDuaH "Isp65-gp64 obtained in (1.4) above.
- the plasmids pDua and PbAMAlD123-gp64 were constructed by insertion.
- the PbMSP119 gene was cloned by PCR by the following method. That is, primer pMspl- Fl (5,-CACCCTGCAGGACTACAAGGACGACG
- pBACsurf-PbMSP-119 is cleaved with PstI / Cfr9I, the PbMSP-119 gene DNA fragment (about 450 bp) is inserted into the PstI / Cfr9I site of pDuaH "Isp65-gp64, and plasmid pDual-PbMSP-119-gp64 is inserted It was constructed.
- P. falciparum genomic DNA was extracted from human erythrocytes infected with P. falciparum 3D7 by using QIAamp DNA Midi Kit (Qiagen).
- the P1CSP gene was cloned by PCR using this genomic DNA as a template by the following method. That is, primer p
- P. falciparum genomic DNA was extracted from human erythrocytes infected with P. falciparum 3D7 by using QIAamp DNA Midi Kit (Qiagen).
- the PfAMAl gene was cloned by PCR using this genomic DNA as a template by the following method. That is, the primer pPfAMAl- Fl (
- Pstl site is underlined, FLAG sequence is italicized) and pPfAMAl-Rl (5,-CCCGGGCT TTCATTTTATCATAAGTTGGTTTATG-3 '(SEQ ID NO: 22): Cfr9I site is underlined)
- the obtained PfAMAl DNA fragment (about 3500 bp) was cloned into pENTR / D-TOPO (Invitrogen), cut with PstI / Cfr9I, and inserted into the PstI / Cfr9I site of pDual-PbAMAlD12 3-gp64.
- the constructed plasmid is named pDua ⁇ PfAMAl-gp64 I attached.
- P. falciparum genomic DNA was extracted from human erythrocytes infected with P. falciparum 3D7 by using QIAamp DNA Midi Kit (Qiagen).
- the Pfs25 gene was cloned by PCR using this genomic DNA as a template by the following method. That is, primer p
- the HA1 gene from avian influenza virus H5N1 is synthesized and inserted into the EcoRI / Cfr9I site of pDuaH "Isp65-gp64 to construct plasmid pDuaH" I5Nl / HAl-gp64.
- SRS gene of SARS virus is synthesized and inserted into EcoRI / Cfr9I site of pDua ⁇ Hsp65-gp64 to construct plasmid pDua ⁇ SARS / S-gp64.
- the gene fragment of HA1 and gp64 obtained by cleaving pCP-H1N1 / HA1-gp64 with restriction enzymes Rsrll and Dralll is transformed into a vector obtained by cleaving pTriEx-1.l (Novagen) with restriction enzymes Rsrll and Dralll.
- the plasmid pCAP-HlNl / HAl-gp64 was constructed by insertion.
- human genomic DNA is converted into a U-shaped UBBp- f Kpnl (5, -GGGGGTACCTCGAGGAAGGTTTCTTCAACTC -3 '(SEQ ID NO: 29): Kpnl site is underlined) and UBBp-r RsrII (5'-GGGCGGTCCGGA CCTAGTTTAAAAGTAAAACATAAG -3 '(SEQ ID NO: 30) : The RsrII site is underlined) and PCR was performed to amplify the UBB promoter region.
- the obtained two fragments were ligated with a vector obtained by digesting pCP-HlNl / HAl-gp64 with restriction enzymes Fsel and RsrII to construct pCU-HlNl / HA1-gp64.
- NP-f EcoRI 5,-ACGGAAT: mCATTCAATTCAAACTGGA-3 '(SEQ ID NO: 31): EcoRI site is underlined
- NP-r C fr9I 5'-GATCCCGGGCCTTGTCAATGCTGAATGGCAA -3 '(SEQ ID NO: 32): Cfr9I site is underlined
- RT-PCR was performed, and the resulting fragment was treated with restriction enzymes EcoRI and Cfr9 I, and similarly treated with restriction enzymes EcoRI and Cfr9I PDuaH "IlNl / NAe-gp64 was constructed by inserting into pCP-HlNl / HAl-gp64.
- M2-f EcoRI (5,-CGGAATT CATGAGTCTTCTAACCGAGG -3 '(SEQ ID NO: 33): EcoRI site is underlined) and M2-r Cfr9I (5'-GATCCCGGGCCTCCAGCTCTATGCTGAC -3 '(SEQ ID NO: 34): Cfr9I site is underlined), RT-PCR was performed, and the resulting fragment was treated with restriction enzymes EcoRI and Cfr9I.
- pCP treated with restriction enzymes EcoRI and Cfr9I PDuaH "IlNl / M2-gp64 was constructed by insertion into -HlNl / HAl-gp64.
- NAe-f EcoRI (5,-ACGGAA) RT-PCR using rmCATTCAATTCAAACTGGA-3 '(SEQ ID NO: 35): EcoRI site is underlined) and NAe-r Cfr9I (5'-GATCCCGGGCCTTGTCAATGCTGAATGGCAA-3 '(SEQ ID NO: 36): Cfr 91 site is underlined)
- the resulting fragment was treated with restriction enzymes EcoRI and Cfr9I, and similarly inserted into pCP-HlNl / HAl-gp64 treated with restriction enzymes EcoRI and Cfr9I, so that pDuaH "IlNl / NAe-gp64 was inserted.
- the obtained fragment and pCP-H1N1 / HA1-gp64 were treated with restriction enzymes Eco RI and CFr9I, and a NC99-derived HA1 gene fragment was newly inserted into the HA1 introduction region of pCP-H1N1 / HA1-gp64.
- the obtained plasmid was designated as pCP-HAl / NC99-gp64.
- the obtained fragment and pCP-H1N1 / HA to gp64 were treated with restriction enzymes EcoRI and Cfr9I, respectively, and a new HA0 gene fragment was inserted into the HA1 transduction region of pCP-HlNl / HAl-gp64.
- the obtained plasmid was designated as pCP-HlNl / HA0-gp64.
- the obtained fragment and pCP-H1N1 / HA to gp64 were treated with restriction enzymes EcoRI and Cfr9I, respectively, and a new HA2 gene fragment was inserted into the HA1 introduction region of pCP-HlNl / HAl-gp64.
- the obtained plasmid was designated as pCP-HlNl / HA2-gp64.
- Dralll site was underlined and Smal was double underlined)), and PCR was performed to amplify the vp39 gene region.
- the amplified fragment and pDuaH "IlNl / HAl-gp64 were cleaved with restriction enzymes Spel and Dralll and joined together to construct pDua ⁇ vp39.
- NP-f 5 EcoRI (5,-ACGGAATTCATGGCGTCCC AAGGCACC-3, (SEQ ID NO: 49): EcoRI site is underlined) and NP-r EcoRI (5,-ACGGA) PCR was performed using H ⁇ ATTGTCGTACTCCTCTGCATTG-3 (SEQ ID NO: 50): EcoRI site is underlined), and the resulting fragment was digested with EcoRI. The obtained fragment was inserted into pDua vp39 digested with the restriction enzyme EcoRI to construct pCPl-H1N1 / NP-vp39.
- pGL3-Enhancer Promega was cleaved with the restriction enzyme Hindlll / Xbal, and the Luciferase gene DNA fragment (about 1690 bp) was ligated into the Hindlll / Xbal site of pcDNA3.1 (Invitrogen). This plasmid was named pcDNA-GL3 (luc).
- the pcDNA-hsp65 obtained in (1.2) of Example 1 above was cleaved with the restriction enzyme BglllZNotl, inserted into the BamHlZNotl site of pcDNA-CS87 to produce pcDNA-Ighsp65, and the resulting plasmid was pcDNA -Named IgHsp65.
- pcDNA-IgHsp65 was cleaved with Bglll / Sphl, and a gene cassette (about 2850 bp) consisting of a polyspA signal derived from the CMV promoter, Hsp65 gene with mouse Igk signal sequence, and ushi growth hormone gene was pBACgus-1 Inserted into Bglll / Sphl site (Novagen).
- the constructed plasmid was named pBACgus-CMV-Hsp65.
- the plasmid pcDNA_GL3 (luc) obtained above is digested with the restriction enzyme NhelZXbal, and the luciferase gene DNA fragment (about 1690 bp) is inserted into the Nhel / Xbal site of the plasmid pBACgus-CMV-Hsp65.
- pBACgus-CMV-GL3 The plasmid pcDNA_GL3 (luc) obtained above is digested with the restriction enzyme NhelZXbal, and the luciferase gene DNA fragment (about 1690 bp) is inserted into the Nhel / Xbal site of the plasmid pBACgus-CMV-Hsp65.
- pCR-Blunt- HA is cleaved with BamHI / Sse8387I and the HA gene DNA fragment (about 1750 bp) is pBl
- the plasmid pBluescript-HA was constructed by inserting it into the BamHI / Pstl site of uescript II (KS-). Furthermore, pBluescript-HA was cleaved with Hindlll / Xbal, and the HA gene DNA fragment (about 1800 bp) was inserted into the Hindlll / Xbal site of pBACgus-CMV-GL3 obtained in (1.3), and plasmid pBACgus-CMV-HA-foll Built.
- Example 2 Recombinant baculovirus of the present invention and its production method
- a transfer vector constructed in Example 1 above using a recombinant baculovirus preparation kit (BacVector-2000 Transfection Kit, Novagen):
- the produced recombinant baculoviruses are AcNPV- Dua ⁇ Hsp65, AcNPV- Dua ⁇ PbCS P, AcNPV- Dua H1N1 / HA1, AcNPV- Dua PbTRAMP ⁇ AcNPV- Dua PbAMAlD 12 3, AcNPV- Dua PbMSP-119, AcNPV- CMV- PbCSP ⁇ AcNPV- CMV- H1N1 / HA1U11, AcNPV— H1N1 / HA1, AcNPV— CAP— H1N1 / HA1, AcNPV— CU—H1N1 / HA1, AcNPV — Dua ⁇ H1N1 / NP ⁇ AcNPV— Dua ⁇ H1N1 / M2, AcNPV— Dua ⁇ HlNl / NAe ⁇ AcNPV— Dua 1-M2e, AcNPV— CP— HA1 / NC99, AcNPV—
- S19 cells were cultured to a density of 2 ⁇ 10 7 cells per 150 mm cell culture plate (Sumilon: manufactured by Sumitomo Akita Beta), and each of the above-mentioned baculoviruses was infected at an infection multiplicity of about 5. After 5-6 days, the culture is collected by centrifugation at 10,000 xg, 4 ° C for 25 minutes, and further centrifuged at 25, 000 rpm, 4 ° C for 90 minutes with a Beckman ultracentrifuge (SW28 Swing Groter). Particles were obtained.
- the recombinant baculovirus is a transfer vector constructed in Example 1 above using a recombinant baculovirus production kit (BacVector-2000 Transfection Kit, Novagen) pDua ⁇ H5N1 / HA1—gp64 And pDua ⁇ SARS / S-gp64 can be prepared by cotransfecting BacVector-2000 DNA into S19 cells.
- the recombinant baculoviruses produced are named AcNPV-H5N1 / HA1 and AcNPV-Dua® SARS / S, respectively.
- S19 cells were cultured to a density of 2 ⁇ 10 7 cells per 150 mm cell culture plate (Sumilon: manufactured by Sumitomo Akita Beta), and each of the above-mentioned baculoviruses was infected at an infection multiplicity of about 5. After 5-6 days, the culture is collected by centrifugation at 10,000 xg, 4 ° C for 25 minutes, and further centrifuged at 25, 000 rpm, 4 ° C for 90 minutes with a Beckman ultracentrifuge (SW28 Swing Groter). Particles can be obtained.
- SW28 Swing Groter Beckman ultracentrifuge
- Example 3 Pharmacological effect test of recombinant baculovirus of the present invention
- BALB / c female mice were inoculated 3 times with the recombinant virus solution for vaccine at 3-week intervals.
- the amount of inoculum was 0.2 mL / body in the case of intramuscular thigh muscle injection, and the virus solution was prepared so that the virus amount was 5 ⁇ 10 6 pfo / body.
- mice were anesthetized with an anesthetic solution for mice 3 weeks after the third vaccination and stabbed with a mosquito (Anopheles stephensi S DA 500 strain) infected with mouse malaria (Plasmodium berghei ANKA 2.34 clone). Infected.
- Table 1 shows the pharmacological effect test results for the protective effect of malaria infection as a vaccine of the pharmaceutical composition of the present invention. The survival rate of each group of mice is shown in the right end of Table 1.
- mice with malaria-infected erythrocytes in the peripheral blood were infected 3 All patients died by day 8.
- the recombinant viruses inserted with the sporozoite antigen (CSP) gene the recombinant baculovirus obtained in Example 2 (including the transfer vector of Example 1 (2): AcNPV-Dua ⁇ PbCSP)
- the transfer vector of Example 1 (2): AcNPV-Dua ⁇ PbCSP In the group inoculated intramuscularly (Group No. 4), 100% infection protection was observed.
- the wild-type baculovirus (group number 2) showed no difference from the control group (group number 1), and the recombinant baculovirus obtained in Example 2 using a mammalian virus promoter (vector of reference example 1). Included: AcNPV-CMV-PbCSP) group (Group No. 3) has a slightly higher survival rate compared to the control group, and the effect of recombinant virus inoculation may be weakened.
- group number 2 The wild-type baculovirus (group number 2) showed no difference from the control group (group number 1), and the recombinant baculovirus obtained in Example 2 using a mammalian virus promoter (vector of reference example 1). Included: AcNPV-CMV-PbCSP) group (Group No. 3) has a slightly higher survival rate compared to the control group, and the effect of recombinant virus inoculation may be weakened.
- AcNPV-CMV-PbCSP AcNPV-CMV-P
- Example 4 Pharmacological effect test of recombinant baculovirus of the present invention
- Vaccine virus was inoculated twice at 2-week intervals. Vaccine virus was inoculated with 1 ( 5 PFU) per mouse using a 29G insulin syringe in the thigh muscle.
- influenza virus Influenza virus
- BSA urine serum albumin
- Dulbecco's phosphate buffer Dulbecco's Phosphate Buffer Saline: (D-PBS) diluted to lower lOOOTCID / 0.05mL for lower respiratory tract infection and lOOOTCID / 0.005mL for upper respiratory tract infection, challenge
- mice Two weeks after the second vaccination, the mouse was anesthetized by intramuscular administration of 0.05 mL of mouse anesthetic solution.
- the influenza virus solution prepared in 4.2 was inoculated into the nasal cavity of anesthetized mice using a micropipette in an amount of 0.005 mL for the upper respiratory infection system and 0.05 mL for the lower respiratory infection system.
- mice Three days after virus inoculation, each group of 4 mice was euthanized by administering 0.1 mL of mouse anesthetic solution intramuscularly and letting off the abdominal inferior vena cava force under anesthesia. Each group of mice was dissected and the lungs were removed aseptically.
- mice The survival of mice was confirmed and recorded once a day until 11 days after virus inoculation.
- MEM + 10% FBS Cell growth medium
- FEBS fetal bovine serum
- the cryopreserved MDCK cells were rapidly thawed in warm water, suspended in 10 mL of cell growth medium, and the supernatant was removed by centrifugation (1000 rpm, 5 minutes, 4 ° C). The cell sediment collected by centrifugation was suspended and suspended in a cell growth medium. Seed in culture flask and set at 37 ° C, 5% CO Cultured in an incubator. After the start of culture, observe the morphology and growth state of the cells with a microscope. Immediately before the MDCK cells become confluent, wash the cells with D-PBS (-), disperse the cells by treating with trypsin-EDTA, Suspended in cell growth medium. The cell suspension was seeded in a culture flask and subcultured with fresh cell growth medium.
- a medium supplemented with 0.1% BSA to MEM 500 mL dOmM HEPES buffer was used as a virus growth medium (MEM + 0.1% BSA), and stored refrigerated until use. Antibiotics were added at the time of use.
- the cells were dispersed by trypsin-EDTA treatment, the number of cells was counted, and a 600,000 cell / mL MDCK cell suspension was prepared using a maintenance medium. Dispense 0.05mL each into a 96-well plate, ⁇ , 5% CO, 37 °
- each prepared lung homogenate dilution was dispensed at 0.05 mL per well into 6 wells of a 96 well plate.
- a 96 well plate dispensed with lung homogenate dilution was placed in a CO incubator set at 5% CO, 37 ° C.
- Recombinant baculovirus of Example 2 (including the transfer vector obtained in Example 1 (3): AcNPV-DuaH "IlNl / HAl) and recombinant baculovirus in the control group AcNPV inoculated group and vaccine group (Including the vector obtained in Reference Example 1: AcNPV-CMV-HlNl / HA foil)
- each virus infectious titer was logarithmically converted and One-way analysis of variance with consideration of multiplicity was analyzed by Tukey test (Release 8.1, SAS Institute Japan Ltd).
- Fig. 1 Effect of each vaccine group on survival time after influenza virus infection Log rank test for survival period of control group AcNPV inoculation group and vaccine group AcNPV- Dua HINl / HAl and AcN PV-CMV-H1N1 / HA foil inoculation groups The results are shown in Fig. 2.
- the AcNPV- Dua HINl / HAl intramuscular group was significantly more significant than the AcNPV group (p value: 0.0
- the virus infection titer in the lungs on the 6th day of infection was significantly suppressed (p value: 0.0094) compared with the AcNPV-CMV-HlNl / HA foil inoculation group.
- the AcNPV- Dua HINl / HAl intramuscular group was significantly more significant than the AcNPV group (p value: 0.0
- the survival period was extended.
- the survival period of the AcNPV-CMV-HlNl / HA foil inoculated group was not different from that of the AcNPV inoculated group (p value: 0.7851).
- the survival period of the AcNPV-Dua HlNl / HAl intramuscular inoculation group was AcNPV-CMV-HlNl /
- mice develop influenza virus pneumonia and die, so AcN PV-Dua H1N1 / HA1 can be inoculated intramuscularly to suppress the growth of intrapulmonary virus and to suppress the death of mice due to pneumonia. Can be guessed.
- Example 5 Expression test of vaccine antigen from recombinant baculovirus of the present invention in insect cells
- S19 cells were cultured to 12 ⁇ 10 6 cells per well of 12-well plate, and baculovirus particles obtained in Example 2 AcNPV- Dua ⁇ PbCSP, AcNPV- Dua ⁇ Hsp65, AcNPV- Dua ⁇ H1N1 / HA1, As a control, wild-type baculovirus AcNPV-WT was infected at an infection frequency of about 5.
- Each baculovirus-infected S19 cell protein-transferred membrane has a mouse anti-FLAG monoclonal antibody (SIGMA) as the primary antibody and a goat anti-mouse IgG (H + L) piotin label (Vector) as the secondary antibody. Incubation was further performed, and an avidin alkaline phosphatase label (manufactured by GIBCO-BRL) was added, followed by color development with NBT / BCIP (manufactured by GIBCO-BRL). The results are shown in Fig. 3.
- SIGMA mouse anti-FLAG monoclonal antibody
- Vector goat anti-mouse IgG piotin label
- lane 1 in the left lane is wild-type baculovirus (AcNPV-WT)
- lane 2 is a recombinant baculovirus (influenza virus HA antigen gene inserted under the dual 'promoter of the present invention ( AcNPV- Dua HINl / HAl) band
- lane 3 in the middle lane is wild-type baculovirus (AcNPV-WT)
- lane 4 is Mycobacterium tuberculosis Hsp65 gene under the dual 'promoter of the present invention.
- Recombinant baculovirus (AcNPV-DuaH "Isp65) band, lane 5 in the right lane in the figure shows wild type baculovirus (AcNPV -WT), Over emissions 6 shows recombinant baculovirus dual 'under the promoter of the present invention malaria parasite CSP gene was inserted a (AcNPV-Dua Bok PbCSP) respectively.
- Example 6 Expression test of vaccine antigen from recombinant baculovirus of the present invention in mammals
- HepG2 cells were infected with AcNPV-Dua ⁇ Hsp65 and AcNPV-WT as a control. Multiple infections at about 1. After 24 hours, remove the culture supernatant, rinse with PBS three times, and then add acetone'ethanol solution (7: 3 mixture) cooled to -20 ° C and fix the cells at -20 ° C for 5 minutes. Went. A 5% normal goat serum solution (manufactured by SIGMA) was blocked at room temperature for 1 hour.
- mouse anti-Hsp65 antibody (Yoshida et al., Vaccine 2005) as the primary antibody and goat anti-mouse IgG (H + DFITC label (manufactured by BIOSOURCE) as the secondary antibody were incubated and incubated. The cells reacting with were detected.
- HepG2 cells were cultured to a density of 1 x 10 7 cells per 100 mm diameter cell culture plate, and baculovirus particles obtained in Example 2 AcNPV- Duato H1N1 / HA1, AcNPV- C MV- H1N1 / HA foil and AcNPV-WT as a control were infected at an infection multiplicity of about 5. After 2 hours, the culture supernatant was removed, rinsed 3 times with PBS, and then FBS dialyzed with PBS was added to 10% of methionine and cystine-free medium (Dulbecco's Modified Eagle medium (Invitrogen)).
- methionine and cystine-free medium Dulbecco's Modified Eagle medium (Invitrogen)
- the medium was cultured for 3 hours, and isotope-labeled methionine and cysteine solution (TRANS35S-LABEL MP Biomedicals® Inc.) was prepared to a final concentration of S 5 micro Ci / ml. After 12 hours, remove the culture supernatant, rinse 3 times with PBS, and then wash the cells with 0.5 mL of RIPA buffer (1% Sodium deoxycholate ⁇ 1% Triton X-100, 0.1% SDS ⁇ 10 mM TrisHCl [pH 7.5]). A melted sample was obtained.
- RIPA buffer 1% Sodium deoxycholate ⁇ 1% Triton X-100, 0.1% SDS ⁇ 10 mM TrisHCl [pH 7.5]
- FIG. 4 shows a drawing of cells stained with a fluorescent-labeled antibody showing expression of Mycobacterium tuberculosis Hsp65 gene recombinant baculovirus in HepG2 cells.
- FIG. 4 shows a drawing when wild-type baculovirus was added to HepG2 cells.
- the recombinant baculovirus using the dual promoter transfer vector of the present invention expresses the target antigen in mammalian cells. You can see that
- the virus particles enter the mammalian cells, and the mammals. It is suggested that the promoter is activated and produces a fusion of the target foreign antigen gene and the gp64 gene in mammalian cells to induce acquired immunity.
- FIG. 5 the expression of fusion antigen in mammalian cells of recombinant baculovirus in which an influenza virus HA antigen gene is incorporated under a dual promoter is analyzed by immunoprecipitation.
- Lane 1 in the left lane in the figure is wild-type baculovirus (AcNPV-WT), and lane 2 is a recombinant baculovirus (AcNPV-CMV-H1N1 / HA) that incorporates the influenza virus HA antigen gene under the CMV promoter.
- lane 3 shows a HepG2 cell extract containing a recombinant baculovirus (AcNPV-Dua to H1N1 / HA1) in which the influenza virus HA antigen gene is fused and expressed with the gp64 gene under a dual promoter. It was used.
- the recombinant baculovirus of the present invention expresses the antigen protein of a desired immunogenic foreign antigen gene even in mammalian cells, and the recombinant virus is administered to mammals including humans. Furthermore, it is considered that antigen-specific acquired immunity can be induced with the expression of the fusion antigen in human cells.
- Example 7 Confirmation test of fusion antigen in actin antigen displayed on virus particles (virion) of the recombinant baculovirus of the present invention
- Baculovirus particles obtained in Example 2 AcNPV-WT, AcNPV-CMV-PbCSP, AcNPV-P bCSPsurf, AcNPV-Dua ⁇ PbCSP collected by ultracentrifugation
- Each 0.005 mL of virus concentrate contains 0.005 mL Boil at 95 ° C for 5 minutes and then electrolyze with 6% SDS-PAGE Electrophoresis was performed. After electrophoresis, the protein was transferred to a PVDF membrane (Immobilon-P, manufactured by Millipore), immersed in Block Ace (Dainippon Pharmaceutical Co., Ltd.), and blocked at 4 ° C for 12 hours.
- Western blotting was performed by the following procedure.
- the membrane to which each baculovirus particle protein has been transferred has a mouse anti-FLAG monoclonal antibody (SIGMA) as the primary antibody and a goat anti-mouse IgG (H + L) piotin label (Vector) as the secondary antibody.
- SIGMA mouse anti-FLAG monoclonal antibody
- Vector goat anti-mouse IgG
- GBCO-BRL avidin 'alkaline phosphatase label
- Fig. 6 is a Western blotting drawing showing the expression of each malaria CSP gene (PbCSP) in a recombinant baculovirus virus particle prepared from a recombinant transfer vector.
- Lane 1 shows the wild-type baculovirus
- Lane 2 shows the recombinant baculovirus produced by the transfer vector with the PbCSP antigen gene inserted under the control of the mammalian promoter
- Lane 3 shows the baculovirus polyhedrin promoter.
- Recombinant baculovirus prepared with a transfer vector inserted so that the PbCSP antigen gene is fused with the gp64 gene under control
- lane 4 shows the fusion expression between the PbCSP antigen gene and the gp64 gene using the dual 'promoter of the present invention. Electrophoresis of the recombinant baculovirus inserted so that PbC A fusion expression product of SP gene and gp64 gene was confirmed.
- the recombinant baculovirus of the present invention produced from Example 7 having the ability of the recombinant transfer vector of the present invention is a fusion of a desired immunogenic foreign gene and a gp64 gene in a recombinant virus particle. It can be seen that the expression product can exist.
- AcNPV-C was used to confirm the persistence of antigen expression in cultured cells of the recombinant virus.
- HeLa cells were infected with P-H1N1 / HA1, AcNPV-CAP-H1N1 / HA1, and AcNPV-CU-H1N1 / HA1, and the expression of the antigen was confirmed.
- HALF01 5 '-GAGCTGAGGGAGCAATTGAG—3, (Self sequence number 51) and primer HA1—R01 (5, GGGTGATGAATACCCCACAG-3, (SEQ ID NO: 52) to generate amplified DNA.
- HALF01 5 '-GAGCTGAGGGAGCAATTGAG—3, (Self sequence number 51) and primer HA1—R01 (5, GGGTGATGAATACCCCACAG-3, (SEQ ID NO: 52) to generate amplified DNA.
- HALF01 5 '-GAGCTGAGGGAGCAATTGAG—3, (Self sequence number 51)
- primer HA1—R01 5, GGGTGATGAATACCCCACAG-3, (SEQ ID NO: 52)
- RNA from cells infected with AcN PV-CU-H1N1 / HA1 at MOI 100
- Example 9 Induction of antibody titer and cellular immunity by PbCSP antigen recombinant virus
- BALB / c female mice were inoculated 3 times with the recombinant virus solution for vaccine at 3-week intervals.
- Vaccination In the case of intramuscular injection of the thigh muscle, the liquid volume was 0.2 mL / body, and a virus liquid was prepared so that the viral volume was 1 ⁇ 10 8 pfo / body.
- the vaccine was inoculated with wild-type virus (AcNPV-WT), AcN PV-PbCSPsurf (Yoshida et al. Virology 316: 161-70, 2003), AcNPV- Dua ⁇ PbCSP.
- Euthanasia was performed 3 weeks after the final immunization, and mouse serum and spleen were collected. Serum was used to measure specific antibody titers, and spleen was used for ELISPOT assembly.
- the antibody titer was measured by ELISA using a plate on which the PbCSP recombinant protein that had been forcedly expressed in E. coli and purified and recovered was immobilized.
- the ELISA method was performed according to the standard method. As a result, an increase in antibody titer could not be confirmed in the non-inoculated group and the wild-type virus inoculated group, and specific antibody titers were confirmed in the AcNPV-PbCSPsur acid species group and the AcNPV-Dua-PbCSP inoculated group.
- FIG. 8 shows the titers of PbCSP-specific IgG antibodies in the non-inoculated group, wild type virus inoculated group, AcNPV-PbCSPsuri inoculated group, and AcNPV-Dual-PbCSP inoculated group.
- ELISPOT assembly was performed using the spleen cells of the immunized mice.
- Cells were prepared from mouse spleen cells, and an appropriate amount of cells was added to MultiScreen-IP (Millipore). Thereto was added a peptide (amino acid sequence: SYIPSAEKI, SEQ ID NO: 53) known as a CD8 epitope of PbCSP, and cultured. Thereafter, the reaction was carried out using ELISPOT Mouse IFN- ⁇ LISPOT Set (BD Sciences), and color development was carried out using AEC substrate set (BD Sciences). The number of cells that gave an antigen-specific response was measured by measuring the colored spots.
- the non-inoculation group, the wild-type virus inoculation group, AcNPV-Pb in CSPsur acid species group antigen-specific cell forces can not be confirmed AcNPV-Dua In PbCSP inoculation group 10 6 to about per spleen cells 350 of the reaction Cells were observed. This indicates that AcNPV-Dua and PbCSP can induce cellular immunity significantly more than AcNPV-PbCSPsurf.
- Fig. 9 uninoculated group, wild type virus inoculated group, AcNPV-PbCSPsur acid species group, AcNPV- Du a ⁇ Shows the number of IFN- ⁇ -producing cells specific for the CTL epitope of PbCSP in the group inoculated with PbCSP.
- Example 10 Antiviral efficacy confirmation test of vaccine containing recombinant baculovirus as active ingredient
- M2e recombinant baculovirus (AcNPV-Dua ⁇ M2e) was inoculated twice into the thigh muscles at a dose of 3.4 ⁇ 10 8 PFU per mouse at 2-week intervals. Two weeks after the final vaccination, influenza virus (Influenza virus) A / PR / 8/34
- viruses were inoculated with 0.005 mL of solution and infected. Six days after infection, the animals were euthanized, the lungs were removed, and the amount of virus in the lungs was detected using MDCK cells. As a result, no influenza virus was detected in all mice tested in mice inoculated with AcNP V-Dua ⁇ M2e. At the same time, this was the same effect as that of the intramuscular inoculation group (1.0 X 10 7 PFU per mouse) of HA1 thread and changeable baculovirus vaccine (AcNPV-DuaH "IlNl / HAl).
- FIG. 10 the viral load in the lungs 6 days after infection with the influenza virus in the PBS group, the AcNPV-DuaVM2e-inoculated group, and the AcNPV-DuaH ”IlNl / HAl-inoculated group are shown.
- Example 11 Confirmation test of preventive effect of a medicine containing HA1 / NC99 recombinant Bakiuro virus as an active ingredient
- HA1 / NC99 recombinant baculovirus (AcNPV-Dual-HAl / NC99) was inoculated twice into the thigh muscles at a dose of 1.0 ⁇ 10 8 PFU per mouse at 2-week intervals. Two weeks after the final vaccination, influenza virus A / NewCaledonia / 20/99 was inoculated into the mouse nasal cavity with 1000TCID virus in 0.05mL solution. 3 days after infection
- influenza virus was not detected in 3 out of 4 mice inoculated with AcNPV- Dua H1N1 / NC99.
- Fig. 11 shows the viral load in the lungs 3 days after influenza virus infection in the PBS group, the wild type virus inoculation (AcNPV-WT) group, and the AcNPV- Dua ⁇ HA1 / NC99 inoculation group.
- Example 12 According to the route of administration of a pharmaceutical composition containing recombinant baculovirus as an active ingredient Specific antibody confirmation test
- HA1 recombinant baculovirus (AcNPV- Dua HINl / HAl), 2.0 X 10 7 PFU per mouse, nasally (0.005 mL inoculated with virus solution in both noses), nasal (virus solution with nasal force, etc.) 0.05 mL inoculation), trans-respiratory tract (0.05 mL of viral fluid from the respiratory tract), intramuscular injection (inoculated with 0.05 mL of viral fluid to the thigh muscle), and inoculated twice at 2-week intervals. . Two weeks after the final vaccination, nasal lavage fluid, alveolar lavage fluid, and serum were collected to confirm the expression of influenza virus-specific antibodies.
- the antibody titer was measured by ELISA using a plate on which an extract of MDCK cells infected with influenza virus A / PR / 8/341 was immobilized.
- the ELISA method was performed according to the standard method.
- specific IgG antibodies were confirmed in the blood in the nasal, respiratory tract and intramuscular injection groups, and strong antibody induction was confirmed particularly in the respiratory tract administration.
- antigen-specific IgG antibodies were also confirmed in nasal and alveolar lavage fluids, and strong antibody induction was confirmed, particularly when administered via the respiratory tract.
- production of antigen-specific IgA antibody was also confirmed in the alveolar lavage fluid.
- Fig. 12 shows the results of ELISA for measuring IgG antibodies specific to influenza virus in the blood of the nasal inoculation group, the nasal inoculation group, the respiratory tract inoculation group, and the intramuscular inoculation group.
- ELIS A was measured for influenza virus-specific IgG and IgA antibodies in nasal lavage fluid and alveolar lavage fluid from nasal vaccination, nasal vaccination group, airway vaccination group, intramuscular vaccination group Shows the results.
- Example 13 Confirmation test of effect by administration route of pharmaceutical composition containing recombinant baculovirus as active ingredient
- HA1 recombinant baculovirus (AcNPV- Dua HINl / HAl) at a dose of 1.0 X 10 7 PFU per mouse by the route of nasal, nasal, respiratory tract and intramuscular injection, respectively, every 2 weeks Inoculated twice.
- Influenza virus A / PR / 8/34 was injected into the mouse nasal cavity with a virus of 1000TCID 0.005m
- Fig. 14 shows the amount of virus in the nasal washing solution 3 days after infection with influenza virus in the nasal inoculation group, the nasal inoculation group, the respiratory tract inoculation group, and the intramuscular inoculation group.
- Figure 15 shows the amount of virus in the lungs 6 days after infection with influenza virus in the nasal vaccination, nasal vaccination, airway vaccination, and intramuscular vaccination groups!
- SEQ ID NOs: 1 and 2 are primer sequences for PbCSP-F1 and PbCSP-Rl primers for PCR of genomic DNA of the P. berghei ANKA strain.
- SEQ ID NOs: 3 and 4 are primer sequences for PCR primers phsp65-F1 and phsp 65-R1 for genomic DNA of Mycobacterium tuberculosis H37Rv.
- SEQ ID NOs: 5 and 6 are primer sequences of PCR primers phsp65-F2 and phsp65-R2 using pcDNA-hsp65 as a saddle type.
- SEQ ID NOs: 7 and 8 are primer sequences of PCR primers pPolh-F2 and pgp64-R2 for obtaining a gp64 gene DNA fragment using pBACgus-l (Novagen) as a saddle type.
- SEQ ID NOs: 9 and 10 are primer sequences for PCR primers HA-f and HA-r for production of influenza virus HA gene fragment.
- SEQ ID NOs: 11 and 12 are primer sequences for PCR primers pHA-Fl and pHA-R 1 using pCR-Blunt-HA as a saddle type.
- SEQ ID NOs: 13 and 14 are primer sequences for PCR primers pTRAMP-F1 and pTRAMP-R1 for the PbTRAMP gene.
- SEQ ID NOs: 15 and 16 are primer sequences for PCR primers pAMA-F1 and pAMAl-R1 of the PbAMAl gene domain 123 (D123).
- SEQ ID NOs: 17 and 18 are primer sequences of PCR primers pMspl-F1 and pMspl-R1 for the PbMSP119 gene.
- SEQ ID NOs: 19 and 20 are primer sequences of PCR primers pPfCSP-F1 and pPfCSP-R1 for the PfCSP gene.
- SEQ ID NOS: 21 and 22 are PCR primers for PfAMAl gene of P. falciparum pPfAMAl-F 1 and pPfAMAl-Rl primer sequences.
- SEQ ID NOs: 23 and 24 are primer sequences of PR primers pPfs25-F1 and pPfs25-Rl for PlPfs25 gene of Plasmodium falciparum P. falciparum 3D7 strain.
- SEQ ID NOs: 25 and 26 are primer sequences for PCR primers Polh-f Rsrll and GP 64-r Dralll using pCR-Blunt-HA as a saddle.
- SEQ ID NOs: 27 and 28 are primer sequences of CMV enhancer region PCR primers CMVenh-f Fsel and CMV enh-r Kpnl.
- SEQ ID NOs: 29 and 30 are primer sequences for PCR primers UBBp-f Kpnl and UBB p-r Rsrll for the UBB promoter region.
- SEQ ID NOs: 31 and 32 are primer sequences for primers NP-f EcoRI and NP-r Cfr9I for RT-PCR of genomic RNA of influenza virus PR8 / 34 strain.
- SEQ ID NOs: 33 and 34 are primer sequences for M2-f EcoRI and M2-r Cfr9I primers for RT-PCR of genomic RNA of influenza virus PR8 / 34 strain.
- SEQ ID NOs: 35 and 36 are primer sequences of primers NAe-f EcoRI and NAe-r Cfr9I for RT-PCR of genomic RNA of influenza virus PR8 / 34 strain.
- SEQ ID NOs: 37 and 38 are primer sequences of PCR primers M2-f Eco RI and M2e-r Cfr9I using pDuaH "IlNl / M2-gp64 as a saddle type.
- SEQ ID NOs: 39 and 40 are primer sequences for HA1-f EcoRI and HA1-r CFr9I (NC99), primers for RT-PCR of genomic RNA of NewCaledonia99 / 20 (NC99).
- SEQ ID NOs: 41 and 42 are primer sequences for PCR primers HA0-f EcoRI and HA2-r Cfr9I using pCR-Blunt-HA as a saddle.
- SEQ ID NOs: 43 and 44 are primer sequences for PCR primers HA2-f EcoRI and HA2-r Cfr9I using pCR-Blunt-HA as a saddle type.
- SEQ ID NOs: 45 and 46 are primer sequences of PCR primers vp39-f and vp39-r for the vp39 gene region.
- SEQ ID NOs: 47 and 48 are primer sequences for PCR primers Polh-S1 and HA1-r EcoRI of the HA1 gene fragment.
- SEQ ID NOs: 49 and 50 are PCR primers NP-f 5 E using pDuaH "IlNl / NP-gp64 as a saddle type CoRI and NP-r EcoRI primer sequences.
- SEQ ID NOs: 51 and 52 are primers for confirming the expression of AcNPV-CP-H1N1 / HA1, AcNPV-CAP-H1N1 / HA1, and AcNPV-CU-H1N1 / HA1.
- SEQ ID NO: 53 is a peptide known as the CD8 epitope of PbCSP.
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Priority Applications (14)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12/278,916 US9023365B2 (en) | 2006-02-09 | 2007-02-08 | Recombinant baculovirus vaccine |
AP2008004576A AP2880A (en) | 2006-02-09 | 2007-02-08 | Novel viral factor |
CA2636632A CA2636632C (en) | 2006-02-09 | 2007-02-08 | Novel viral vector |
AU2007213030A AU2007213030B2 (en) | 2006-02-09 | 2007-02-08 | Novel viral vector |
EP07708219A EP1983047A4 (en) | 2006-02-09 | 2007-02-08 | NEW VIRUS VECTOR |
BRPI0707646-0A BRPI0707646A2 (pt) | 2006-02-09 | 2007-02-08 | vetor viral |
MX2008010299A MX2008010299A (es) | 2006-02-09 | 2007-02-08 | Vector viral novedoso. |
JP2007531528A JP4171930B2 (ja) | 2006-02-09 | 2007-02-08 | 新規ウイルスベクター |
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US12/192,927 US9333249B2 (en) | 2006-02-09 | 2008-08-15 | Recombinant baculovirus vaccine |
IL210742A IL210742A (en) | 2006-02-09 | 2011-01-19 | A viral vector derived from a killer virus used to express p.falciparum antigens |
IL210740A IL210740A (en) | 2006-02-09 | 2011-01-19 | A drug containing recombinant Kilvirus expressing antigen of influenza virus |
US13/617,825 US9327018B2 (en) | 2006-02-09 | 2012-09-14 | Recombinant baculovirus vaccine |
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WO2009020236A3 (en) * | 2007-08-07 | 2009-04-16 | Educational Foundation Jichi M | Baculoviral vectors with a dual vertebrate and baculovirus promoter controlling an immunogenic fusion gene |
WO2011099541A1 (ja) | 2010-02-12 | 2011-08-18 | 大塚製薬株式会社 | 組換えウイルスの製造方法 |
JP2011528901A (ja) * | 2008-07-25 | 2011-12-01 | インスティテュート・フォー・リサーチ・イン・バイオメディシン | 抗a型インフルエンザウイルス中和抗体およびその使用 |
US8871207B2 (en) | 2008-07-25 | 2014-10-28 | Humabs, LLC | Neutralizing anti-influenza A virus antibodies and uses thereof |
WO2016059911A1 (ja) * | 2014-10-17 | 2016-04-21 | 国立大学法人金沢大学 | マラリアワクチン |
US9587010B2 (en) | 2011-07-18 | 2017-03-07 | The Institute For Research In Biomedicine | Neutralizing anti-influenza A virus antibodies and uses thereof |
WO2023157880A1 (ja) * | 2022-02-18 | 2023-08-24 | 国立大学法人金沢大学 | マラリアワクチン及びマラリア予防・治療方法 |
CN117305365A (zh) * | 2023-11-28 | 2023-12-29 | 中国科学院生物物理研究所 | 昆虫细胞—哺乳动物细胞表达穿梭载体SmartBM-1及其应用 |
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US9340603B2 (en) | 2008-07-25 | 2016-05-17 | Institute For Research In Biomedicine | Neutralizing anti-influenza A virus antibodies and uses thereof |
JP2011528901A (ja) * | 2008-07-25 | 2011-12-01 | インスティテュート・フォー・リサーチ・イン・バイオメディシン | 抗a型インフルエンザウイルス中和抗体およびその使用 |
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WO2011099541A1 (ja) | 2010-02-12 | 2011-08-18 | 大塚製薬株式会社 | 組換えウイルスの製造方法 |
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US10815294B2 (en) | 2011-07-18 | 2020-10-27 | Institute For Research In Biomedicine | Neutralizing anti-influenza A virus antibodies and uses thereof |
US9587010B2 (en) | 2011-07-18 | 2017-03-07 | The Institute For Research In Biomedicine | Neutralizing anti-influenza A virus antibodies and uses thereof |
WO2016059911A1 (ja) * | 2014-10-17 | 2016-04-21 | 国立大学法人金沢大学 | マラリアワクチン |
WO2023157880A1 (ja) * | 2022-02-18 | 2023-08-24 | 国立大学法人金沢大学 | マラリアワクチン及びマラリア予防・治療方法 |
CN117305365A (zh) * | 2023-11-28 | 2023-12-29 | 中国科学院生物物理研究所 | 昆虫细胞—哺乳动物细胞表达穿梭载体SmartBM-1及其应用 |
CN117305365B (zh) * | 2023-11-28 | 2024-03-19 | 中国科学院生物物理研究所 | 昆虫细胞—哺乳动物细胞表达穿梭载体SmartBM-1及其应用 |
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