WO2004067040A1 - Vaccin genique contre les parasites intracellulaires - Google Patents

Vaccin genique contre les parasites intracellulaires Download PDF

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Publication number
WO2004067040A1
WO2004067040A1 PCT/JP2004/000975 JP2004000975W WO2004067040A1 WO 2004067040 A1 WO2004067040 A1 WO 2004067040A1 JP 2004000975 W JP2004000975 W JP 2004000975W WO 2004067040 A1 WO2004067040 A1 WO 2004067040A1
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nucleic acid
acid sequence
gene
ubiquitin
sequence encoding
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PCT/JP2004/000975
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English (en)
Japanese (ja)
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Kunihiro Himeno
Kazunari Ishii
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Kyushu Tlo Company, Limited
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Publication of WO2004067040A1 publication Critical patent/WO2004067040A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • 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 relates to a genetic vaccine against intracellular parasitic pathogens utilizing the ubiquitin proteasome system.
  • Pectin is one of the immune response mechanisms that utilizes the property of memorizing an antigen once invaded and inducing an extremely rapid and effective immune response when the same antigen is invaded again.
  • Vaccine technology effectively infects the immune response memorized in the body at the time of infection by inoculating inactivated infectious organisms or some of the components of infectious organisms into humans in advance to establish a simulated infection. It demonstrates resistance to drawer infections.
  • protective immunity against intracellular parasitic pathogens such as malaria, tuberculosis, HIV and the like include induction of cell-mediated immunity centered on CD8 + T cells (killer T cells).
  • antigen-specific CD8 + T cells In order for antigen-specific CD8 + T cells to be induced, it is essential that the antigen be processed by the cytosolic enzyme poreteasome and then presented to MHC class I molecules.
  • An object of the present invention is to construct a fusion gene of a ubiquitin gene with a gene derived from an intracellular parasitic pathogen such as Mycobacterium tuberculosis, malaria parasite, Toxoplasma gondii, Trypanosoma cruzi, HIV and cytomegalovirus.
  • an intracellular parasitic pathogen such as Mycobacterium tuberculosis, malaria parasite, Toxoplasma gondii, Trypanosoma cruzi, HIV and cytomegalovirus.
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, based on the ubiquitin proteasome system, the MPB51 gene derived from Mycobacterium tuberculosis, the MSP-1 gene derived from malaria parasite, and the S gene derived from Toxoplasma gondii
  • the present invention has been completed by constructing a fusion gene (fusion DNA) with the AG-1 gene or the like. That is, the present invention is specified by the matters described below.
  • a genetic vaccine comprising a nucleic acid sequence encoding ubiquitin and a nucleic acid sequence encoding an antigenic protein comprising a T cell target sequence.
  • the genetic vaccine according to item 1 wherein the T cell target sequence contains at least 8 amino acids.
  • the antigen protein includes a protein derived from an intracellular parasitic pathogen or a fragment or variant thereof.
  • the nucleic acid sequence encoding ubiquitin includes a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20, or a variant thereof.
  • Item 7. The genetic vaccine according to item 1.
  • nucleic acid sequence encoding ubiquitin and the nucleic acid sequence encoding the antigen protein are linked without any intervening sequence, and the C-terminal G1y of ubiquitin is replaced with an amino acid other than G1y.
  • the sequence encoding the ubiquitin is arranged at the C-terminus of the ubiquitin so that the antigen protein is fused, and G1y at the C-terminus of the ubiquitin is replaced with an amino acid other than Gly.
  • Item 7. The genetic vaccine according to item 1. 17.
  • the genetic vaccines listed above include malaria, tuberculosis, toxoplasmosis, trypanosomiasis, acquired immunodeficiency syndrome, cytomegalovirus disease, chlamydiasis, rickettsiosis, leishmaniasis, Ebola hemorrhagic fever, trypanosoma cruzi, and seagas.
  • a disease selected from the group consisting of Japanese encephalitis, influenza, measles dengue virus, polio, alpha herpesvirus infection, severe acute respiratory syndrome (SARS) and hepatitis C
  • SARS severe acute respiratory syndrome
  • a genetic vaccine comprising a nucleic acid sequence encoding ubiquitin and a nucleic acid sequence encoding an antigenic protein of an intracellular parasitic pathogen.
  • the genetic vaccine according to item 24, wherein the intracellular parasitic pathogen is selected from the group consisting of viruses, bacteria, fungi, and protozoa.
  • the intracellular parasitic pathogen belongs to a protozoan.
  • the nucleic acid sequence encoding the protein derived from the above intracellular parasitic pathogen includes MPB51, MDP1, Ag85A, Ag85B, HSP65, Mtb72f gene derived from Mycobacterium tuberculosis, and MSP-1 gene derived from malaria parasite. At least one nucleic acid sequence selected from the group consisting of SAG-1 gene derived from Toxoplasma gondii, TSA gene derived from Trypanosoma cruzi, HIV-related gene, and cytomegalovirus-related gene, or a fragment or variant thereof. 25.
  • a nucleic acid construct comprising a nucleic acid sequence encoding ubiquitin and a nucleic acid sequence encoding a protein from an intracellular parasitic pathogen.
  • a method for producing a genetic vaccine comprising a nucleic acid sequence encoding ubiquitin and a nucleic acid sequence encoding a protein derived from an intracellular parasitic pathogen, wherein the nucleic acid sequence encoding ubiquitin and the cell Operatively linking a nucleic acid sequence encoding a protein from an endophytic pathogen.
  • the method according to item 31 wherein the disease is a disease caused by an intracellular parasitic pathogen. 3 5.
  • the above diseases are malaria, tuberculosis, toxoplasmosis, trypanosomiasis, acquired immunodeficiency syndrome, cytomegalovirus disease, chlamydiasis, rickettsia, leishmaniasis, Ebola hemorrhagic fever, trypanosomiasis cruzi, Selected from the group consisting of Chagas, disease, Japanese encephalitis, influenza, measles dengue virus, polio, alpha herpesvirus infection, severe acute respiratory syndrome (SARS) and hepatitis C The method according to item 31.
  • SARS severe acute respiratory syndrome
  • composition comprising a nucleic acid sequence encoding ubiquitin and a nucleic acid sequence encoding a protein derived from an intracellular parasitic pathogen as a genetic vaccine.
  • composition comprising a nucleic acid sequence encoding ubiquitin and a nucleic acid sequence encoding a protein derived from an intracellular parasitic pathogen for producing a genetic vaccine.
  • a fusion gene of a ubiquitin gene and a gene derived from an intracellular parasitic pathogen such as Mycobacterium tuberculosis, malaria parasite and Toxoplasma parasite is constructed, and a gene vaccine for each infected host is used.
  • the ubiquitinated pathogen antigen is processed by the proteasome, it is possible to obtain a fusion D ⁇ vaccine that strongly induces CD8 + ⁇ cells specific to the pathogen.
  • the recombinant protein pectin has to be expensive because it is not easy to purify, but DNA vaccines are easy to purify and inexpensive, so in developing countries where infections caused by the above-mentioned pathogens are serious. Are also relatively easy to use.
  • Gene therapy often uses a viral vector.
  • a neutralizing antibody against the virus of the vector and an IgE antibody that induces anaphylaxis are produced, so that the antibody is ineffective or unusable.
  • This DNA vaccine uses naked DNA and does not use a viral vector, and thus does not cause such a problem.
  • the protein expressed according to the present invention has a sugar chain added to the expressed protein, the original protein structure is maintained, and the target protein is produced in a small amount and continuously for a certain period of time. It is safe because it is supplied, does not require daily administration, and is effective.
  • genes used in this gene vaccine are all naked DNA, and there is no need to use a virus vector.Therefore, gene therapy using a gene gun and DNA vaccines can be performed easily and safely with repeated administration to the living body. it can. In addition, gene construction is easy and the risk of infection due to viral vector poisoning is extremely low. It is also very unlikely that it will be integrated into the genome DNA. It is understood that these and other advantages of the present invention will become apparent to those of ordinary skill in the art upon reading and understanding the following detailed description, with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 shows a plasmid construct encoding the SAG-1 gene from Toxoplasma gondii. Left: p cDNA—SAG 1 (basic type), center: p JW4304 1 SAG1 (secreted), right: p cDNAUB—SAG1 (ubiquitin-fused).
  • FIG. 2 shows the expression of SAG1 mRNA in mouse lymph nodes into which pc DNA-SAG1 was introduced by a gene gun. Top: SAG1 mRNA, Bottom:) 3-actin mRNA.
  • FIG. 3 shows that pDNA, pcDNA-SAG1, pJW4304-SAG1 and pcDNAUB-SAG1 are introduced into cells to synthesize polyubiquitinated SAG-1 protein in the cells. This is a graph that proves that
  • FIG. 4 is a graph showing activation of killer T cells by the DNA vaccine.
  • Line only indicates a group immunized with pcDNA
  • ⁇ indicates pJW4304-SAG1
  • ⁇ indicates pcDNA-SAG1
  • ⁇ indicates a group immunized with pcDNAUB_SAG1.
  • FIG. 5 is a graph showing activation of killer T cells by a DNA vaccine.
  • ( ⁇ ) Indicates a group immunized with pcDNAUB-SAG1,
  • (garden) indicates pJW4304-SAG1,
  • (1) indicates a group immunized with pcDNA-SAG1,
  • ( ⁇ ) indicates a group immunized with pcDNA.
  • FIG. 6 is a graph demonstrating that the SAG-1 specific killer cell activating effect is through the ubiquitin proteasome system using the proteasome inhibitor MG-132.
  • FIG. 7 is a bar graph demonstrating that the SAG-1 specific killer cell activating effect is through the ubiquitin proteasome system using the proteasome inhibitor epoxomycin.
  • (Mouth) shows the group treated with pcDNA-SAG1
  • ( ⁇ ) shows the group treated with pcDNA UB-SAG1.
  • FIG. 8 is a graph showing the effect of a DNA vaccine using a fusion gene (fusion DNA) of the ubiquitin gene and the SAG-1 gene.
  • FIG. 9 is a graph showing the effect of a DNA vaccine derived from a fusion gene of the ubiquitin gene and the SAG-1 gene on cytoin force production.
  • FIG. 10 is a graph showing the effect of a DNA vaccine on IFN-0 production. From the left, the groups immunized with pcDNAUB-SAG1, pcDNA-SAG1, JW4304-SAG1, pcDNA are shown. (Garden) shows the group stimulated with APC treated with infectious radiation, and (Mouth) shows the control group using APC treated with non-infected radiation.
  • FIG. 11 is a graph showing the effect of a DNA vaccine on IFN- ⁇ mRNA production. From the left, the group immunized with pcDNA, pcDNA-SAG1, pJW4304-one SAG1, pcDNAUB-SAG1 is shown.
  • Figure 12 shows DN from the fusion gene of ubiquitin gene and SAG-1 gene.
  • FIG. 4 is a graph showing the effect of A vaccine on antibody production.
  • (Mouth) shows a group immunized with pcDNA
  • ( ⁇ ) shows a group immunized with pcDNA-SAG1
  • ( ⁇ ) shows a group immunized with pJW4304_SAG1
  • ( ⁇ ) shows a group immunized with pcDNAUB-SAG1.
  • FIG. 13 is a graph showing the need for CD8 + T cells for anti-Toxoplasma immunity induced by pcDNAUB-SAG1 vaccination.
  • FIG. 14A is a graph showing the erythrocyte infection rate 5 days after anti-PbCSP immunization against the groups immunized with pcDNA, pM, pT, pU and pC and the non-immunized group. Each symbol indicates each individual.
  • Figure 14-B is a graph showing the erythrocyte infection rate 7 B after anti-PbCSP immunization for the groups immunized with pcDNA, pM, pT, ⁇ U and ⁇ C and the non-immunized group. is there. Each symbol indicates each individual.
  • FIG. 14-C is a graph showing the erythrocyte infection rate 10 days after anti-PbCSP immunization for the groups immunized with pcDNA, pM, pT, ⁇ U, and ⁇ C and the non-immunized group. Each symbol indicates each individual.
  • FIG. 14-D is a graph showing the erythrocyte infection rate 12 days after anti-PbCSP immunization against the groups immunized with pcDNA, pM, pT, ⁇ U and ⁇ C and the non-immunized group. Each symbol indicates each individual.
  • FIG. 15 is a graph showing the survival rate against Pb CSP infection in a group immunized with ubiquitin-only vector, pM, pU, and a non-immunized group.
  • (Decree) indicates a non-immune group.
  • (Plot) shows ubiquitin-only vector
  • ( ⁇ ) shows pM
  • (X) shows pU-immunized group.
  • SEQ ID NO: 1 is a representative nucleic acid sequence of human ubiquitin.
  • SEQ ID NO: 2 is the amino acid sequence of human ubiquitin. Pepsi, chicks, mice, rabbits and Drosophila also have the same amino acid sequence as this sequence.
  • SEQ ID NO: 3 is the nucleic acid sequence of ubiquitin of Caenorhabditis elegans (Nematoda).
  • SEQ ID NO: 4 is the amino acid sequence of ubiquitin of Caenorhabditis elegans (nematode).
  • SEQ ID NO: 5 is an example of a nucleic acid sequence of ubiquitin derived from a plant (soy).
  • SEQ ID NO: 6 is the amino acid sequence of ubiquitin derived from a plant (soy). garden pea, common sunflower ⁇ potato ⁇ corn, oats, Arabidopsis thai i ana also have the same amino acid sequence as this sequence.
  • SEQ ID NO: 7 is an example of the nucleic acid sequence of ubiquitin derived from Tryp kinesoma Ance.
  • SEQ ID NO: 8 is It is the amino acid sequence of ubiquitin derived from ri / DC.
  • SEQ ID NO: 9 is an example of the nucleic acid sequence of ubiquitin derived from Leishmania are / jio e. .
  • SEQ ID NO: 10 is an example of the amino acid sequence of ubiquitin from Leishmania tarentolae.
  • SEQ ID NO: 11 is an example of the nucleic acid sequence of ubiquitin derived from Neurospora cra ⁇ a.
  • SEQ ID NO: 12 is the amino acid sequence of ubiquitin derived from Neurospora crassa.
  • System U number 13 is an example of the nucleotide sequence of ubiquitin derived from Saccharomyces cerevisiae (yeast).
  • the system! 1 number 14 is the amino acid sequence of ubiquitin derived from Saccharomyces cerevisiae (yeast).
  • SEQ ID NO: 15 is 3 ⁇ 4ric? It is an example of the nucleic acid sequence of ubiquitin derived from it.
  • SEQ ID NO: 16 is the amino acid sequence of ubiquitin derived from Phytophthora i 3 @ ste / 2s.
  • SEQ ID NO: 17 is an example of the nucleic acid sequence of ubiquitin derived from Euplotes euzio sio / s.
  • SEQ ID NO: 18 is the amino acid sequence of ubiquitin derived from Euplotes eurystomus.
  • Rooster 3 system! 13 ⁇ 4 ⁇ 19 is an example of the nucleic acid sequence of ubiquitin derived from Autographa californica nuclear polyhedrosis virus.
  • Rooster system! 13 ⁇ 4 ⁇ 20 is the amino acid sequence of ubiquitin of Autographa californica nuclear polyhedrosis virus.
  • SEQ ID NO: 21 shows the nucleic acid sequence of G76A modified ubiquitin.
  • SEQ ID NO: 22 shows the amino acid sequence of G76A modified ubiquitin.
  • SEQ ID NO: 23 is another example of a nucleic acid sequence of ubiquitin derived from human.
  • SEQ ID NO: 24 is the amino acid sequence of ubiquitin derived from human.
  • SEQ ID NO: 25 is another example of a nucleic acid sequence of ubiquitin of human origin.
  • SEQ ID NO: 26 is the amino acid sequence of ubiquitin derived from human.
  • SEQ ID NO: 27 is another example of a nucleic acid sequence of ubiquitin derived from human.
  • SEQ ID NO: 28 is the amino acid sequence of ubiquitin derived from human.
  • SEQ ID NO: 29 is another example of a nucleic acid sequence of ubiquitin derived from human.
  • SEQ ID NO: 30 is the amino acid sequence of ubiquitin derived from human.
  • SEQ ID NO: 31 is another example of a nucleic acid sequence of ubiquitin of human origin.
  • SEQ ID NO: 32 is the amino acid sequence of ubiquitin derived from human.
  • SEQ ID NO: 33 is another example of a nucleic acid sequence of ubiquitin derived from human.
  • SEQ ID NO: 34 is the amino acid sequence of ubiquitin derived from human.
  • SEQ ID NO: 35 is another example of a nucleic acid sequence of ubiquitin derived from human.
  • SEQ ID NO: 36 is the amino acid sequence of ubiquitin derived from human.
  • SEQ ID NO: 37 is another example of a nucleic acid sequence of ubiquitin derived from human.
  • SEQ ID NO: 38 is an amino acid sequence of ubiquitin derived from human.
  • SEQ ID NO: 39 is a nucleic acid sequence of ubiquitin derived from Escherichia coli.
  • SEQ ID NO: 40 is the amino acid sequence of ubiquitin derived from Escherichia coli.
  • SEQ ID NO: 41 is the nucleic acid sequence of ubiquitin derived from chicken.
  • SEQ ID NO: 42 is the amino acid sequence of ubiquitin derived from chicken.
  • SEQ ID NO: 43 is the nucleic acid sequence of ubiquitin derived from mouse.
  • SEQ ID NO: 44 is the amino acid sequence of ubiquitin derived from mouse.
  • SEQ ID NO: 45 is the nucleic acid sequence of ubiquitin derived from Drosophila.
  • SEQ ID NO: 46 is the amino acid sequence of ubiquitin derived from Drosophila
  • SEQ ID NO: 47 is another example of the nucleic acid sequence of ubiquitin derived from plants (garden pea) It is.
  • SEQ ID NO: 48 is an amino acid sequence (garden pea) of ubiquitin derived from a plant.
  • SEQ ID NO: 49 is another example of a nucleic acid sequence of ubiquitin derived from a plant (common sunflower).
  • SEQ ID NO: 50 is the amino acid sequence (common sunflower) of ubiquitin derived from a plant.
  • SEQ ID NO: 51 is another example (potato) of the nucleic acid sequence of ubiquitin derived from a plant.
  • SEQ ID NO: 52 is the amino acid sequence of ubiquitin derived from a plant (potato).
  • SEQ ID NO: 53 is another example of a nucleic acid sequence of ubiquitin derived from a plant (maize :).
  • SEQ ID NO: 54 is the amino acid sequence of ubiquitin derived from a plant (maize;).
  • SEQ ID NO: 55 is another example of a nucleic acid sequence for ubiquitin derived from a plant (oat).
  • SEQ ID NO: 56 is the amino acid sequence of ubiquitin derived from a plant (oat).
  • SEQ ID NO: 57 is another example of a nucleic acid sequence of ubiquitin derived from a plant (Arabidopsis thaliana) 0
  • SEQ ID NO: 58 is the amino acid sequence of ubiquitin derived from a plant (Arabidopsis thaliana) 0
  • SEQ ID NO: 59 shows a sense primer used in Example 1.
  • SEQ ID NO: 60 shows the antisense primer used in Example 1.
  • SEQ ID NO: 61 shows a sense primer used in Example 1.
  • SEQ ID NO: 62 shows a sense primer used in Example 10.
  • SEQ ID NO: 63 shows the antisense primer used in Example 10.
  • SEQ ID NO: 64 shows the nucleic acid sequence of mouse tissue plasminogen activator used in Example 10.
  • SEQ ID NO: 65 was used in Example 10 and shows the amino acid sequence of mouse thread and plasminogen activator beta.
  • SEQ ID NO: 66 shows a sense primer used in Example 10.
  • SEQ ID NO: 67 shows the antisense primer used in Example 10.
  • SEQ ID NO: 68 shows a sense primer used in Example 10.
  • SEQ ID NO: 69 shows the antisense primer used in Example 10.
  • SEQ ID NO: 70 to SEQ ID NO: 91 are antigen sequences of intracellular parasite. It is understood that the above sequences are exemplary and that the sequences can have one or more alterations (eg, substitutions, deletions and / or additions) as long as they have the same function.
  • ubiquitin has the broadest meaning commonly used in the art, is a protein that is widely present in common in all eukaryotic cells, and forms a correct structure in a cell. It is known to bind and ubiquitinate proteins that are not present or abnormally expressed. When ubiquitinated, it is recognized by the proteasome, which breaks down proteins that interfere with the normal functioning of cells. It is particularly important that ubiquitin in the present invention has a function such as a signal indicating the degradation of a protein.
  • the gene encoding ubiquitin is called ubiquitin gene.
  • a promoter of a gene encoding ubiquitin, a structural gene encoding ubiquitin, and a DNA sequence containing both of them may be collectively referred to as a “ubiquitin gene”.
  • Ubiquitin is a protein with about 76 amino acids, the primary structure of which is conserved in most organisms.
  • genes Either can be used in the present invention.
  • One of them is polyubiquitin in which several to about 100 ubiquitins are linked, has a heat-inducible promoter, and is induced to be expressed by stress such as heat shock.
  • the other gene is expressed linked to the C-terminus of a particular liposome protein. Usually, this ribosome-linked form is expressed.
  • glycine at the C-terminus of ubiquitin is activated and binds to the ⁇ -amino group of lysine of the target protein.
  • ubiquitin include, for example,
  • a polynucleotide consisting of a nucleotide sequence having at least 70% identity to the polynucleotide of any one of (a) to (e) or its complementary sequence, and encoding a polypeptide having biological activity.
  • Nucleic acids encoding ubiquitin may be "silently modified.” Examples of such silent conversion include, for example, the following in humans, but are not limited thereto. It is understood that such sequences can also be used in the present invention.
  • the codons at positions 4-6 in the nucleic acid sequence of SEQ ID NO: 1 can be silently modified, for example, from CAG to CAA, and these codons encode amino acid residue G1n (gnoletamine).
  • the codons at positions 19 to 21 can be silently modified from ACT to ACC, ACA, and these codons encode the amino acid residue Thr (threonine).
  • the codons at positions 22 to 24 are Can be silently modified to TC, these codons encode the amino acid residue Leu (leucine).
  • codons at positions 28-30 in this nucleic acid sequence can be silently modified from GGT to GGC, and these codons encode amino acid residue G1y (glycine).
  • codons 37-39 can be silently modified from ATC to ATT, and these codons encode amino acid residue I 1 e (isoleucine).
  • codons 40-42 can be silently modified from ACC to ACT, and these codons encode the amino acid residue Thr (threonine).
  • codons 43-45 can be silently modified from CTC to CTT, and these codons encode amino acid residue Leu (leucine).
  • codons 46-48 can be silently modified from GAG to GAA, and these codons encode amino acid residue G1u (daltamic acid).
  • codons 49 to 51 in this nucleic acid sequence can be silently modified from GTG to GTC and GTT, and these codons encode amino acid residue Va1 (valine).
  • the codon at positions 55 to 57 in this nucleic acid sequence is # 0. Can be silently modified to CG, these codons encoding the amino acid residue Pro (proline).
  • codons 64 to 66 can be silently modified from ACC to ACA, and these codons encode the amino acid residue Thr (threonine). Also, in this nucleic acid sequence, codons 67-69 can be silently modified from ATC to ATT, and these codons encode amino acid residue I 1 e (isoleucine).
  • codons 73-75 can be silently modified from AAT to AAC, and these codons encode amino acid residue Asn (asparagine).
  • the codons at positions 91-93 can be silently modified from CAA to CAG, and these codons encode amino acid residue G1n (glutamine). '
  • codons 94-96 can be silently modified from GAT to GAC, and these codons encode the amino acid residue Asp (aspartic acid).
  • codons at positions 106 to 108 in this nucleic acid sequence can be silently modified from ATT to ATC, and these codons encode amino acid residue I 1 e (isoleucine). I do.
  • the codons at positions 115 to 117 can be silently modified from GAT to GAC, and these codons are amino acid residues A sp (aspartic acid) Code.
  • codons at positions 136 to 138 in this nucleic acid sequence can be silently modified from GCC to GCT, and these codons encode amino acid residue A1a (alanine). .
  • the codons at positions 139 to 141 can be silently modified from GGA to GGG, and these codons encode amino acid residue G1y (dalicin) .
  • codons at positions 142 to 144 in this nucleic acid sequence can be silently modified from AAA to AAG, and these codons are replaced with amino acid residues Lys (lysine). Code).
  • codons at positions 157 to 159 can be silently modified from GGT to GGG or GGA, and these codons encode amino acid residue G1y (glycine).
  • codon # 160-# 162 is silent variation CGT or al CGC, the CGG obtained, these codons encoding for amino acid residues A r g (arginine).
  • codons at positions 181 to 183 in this nucleic acid sequence can be silently modified from ATC to ATT, and these codons encode amino acid residue I 1 e (isoleucine).
  • codons 193 to 195 can be silently modified from TCC to TCT, and these codons encode amino acid residue S er (serine).
  • codons 196 to 198 can be silently modified from ACC to ACT, and these codons encode the amino acid residue Thr (sleonin).
  • codons at positions 199-201 in this nucleic acid sequence can be silently modified from TTG to CTG, and these codons encode amino acid residue Leu (leucine).
  • codons 205-207 in this nucleic acid sequence can be silently modified from CTG to TTG, and these codons encode amino acid residue Leu (leucine).
  • codons 208-210 in this nucleic acid sequence can be silently modified from GTA to GTG, GTT, GTC, and these codons encode the amino acid residue Val (parin).
  • the codon at positions 211 to 213 is CTC or These codons encode the amino acid residue Leu (leucine).
  • the codons at positions 214 to 216 can be silently modified from CGT to CGC, and these codons encode amino acid residue Arg (arginine).
  • codons at positions 217-219 in this nucleic acid sequence can be silently modified to CTC, TTG, CTT, and these codons encode the amino acid residue Leu (leucine).
  • the codons at positions 220 to 222 can be silently modified from AGA to AGG, and these codons encode the amino acid residue Arg (arginine).
  • the codons at positions 223 to 225 can be silently modified from GGT to GGG, and these codons encode amino acid residue G1y (daricin).
  • codons at positions 226-228 in this nucleic acid sequence can be silently modified from GGG to GGT, and these codons encode amino acid residue G1y (daricin).
  • Ubiquitin (Ub) is an extremely highly conserved protein across species, as shown below.
  • Arab i dops is thaliana (SEQ ID NO: 6)
  • the term "mode in which ubiquitin is not removed” refers to a mode in which the expression product of the gene of the present invention containing ubiquitin is resistant to cleavage by a protease into monoubiquitin and an antigen.
  • modes include, for example, substituting the C-terminal G1y of the amino acid sequence of ubiquitin with a sequence other than G1y (for example, Ala), and adding another amino acid to the C-terminal Gly.
  • G1y for example, Ala
  • Such a ubiquitin-containing product is efficiently polyubiquitinated, and processing by the proteasome is efficiently induced.
  • the ubiquitin and the antigen are easily cleaved before the ubiquitin is polyubiquitinated and transported to the proteasome if the C-terminal sequence remains G1y.
  • the sequence is not Gly.
  • Such biological activities of ubiquitin include, for example, (A) targeting to the proteasome, (B) binding to the ⁇ -amino group of the lysine of the target protein by glycation at the C-terminus, and (C) ) Induction by stress such as heat shock, and (D) expression linked to the C-terminus of ribosomal proteins, but are not limited thereto.
  • Particularly preferred is targeting by the proteasome.
  • An assay for targeting by the proteasome can be confirmed by an experiment known in the art, and examples include an assay as described in Examples. (Proteasome)
  • proteasome refers to a giant ubiquitous in eukaryotes. It is a type of protease, and it is said that there are at least three types of proteasomes in cells: ATP-independent 20S, ATP-dependent 26S, and football.
  • a ubiquitin molecule is added to the ⁇ -amino group of the lysine side chain of the target protein by a ubiquitinating enzyme, and ubiquitin is recognized and sent to the proteasome for degradation. This is because the proteasome can be said to be an assembly of proteases. The proteins selected in this system are rapidly metabolized.
  • the 20S proteasome has a hollow structure in which an ⁇ ring consisting of seven ⁇ subunits and a j3 ring consisting of seven subunits are stacked in the order of ⁇ , ⁇ , ⁇ ⁇ ⁇ in a cylindrical shape. ⁇ Depending on ⁇ 700 above and below the 20S proteasome, it becomes a 26S proteasome. The football version has $ 28 instead of $ 700.
  • the antigen may undergo degradation in any proteasome. ( ⁇ Cell target sequence)
  • ⁇ cell target sequence refers to an amino acid sequence (eg, epitope) capable of inducing specific recognition by a cell, and is usually at least about 5 amino acids, and preferably at least about 8 amino acids.
  • CTL cytotoxic T-lymp hocytes.
  • CD8-positive CTLs recognize antigen peptides in a class I-restricted manner
  • CD4-positive CTLs recognize class II-restricted antigens.
  • the T cell targeting system U is transported to the endoplasmic reticulum (ER) when the intracellular parasitic pathogen is taken up by the infested cells, where the peptide that binds to HLA class I molecules Forms a complex with class I molecules. This complex is said to be expressed on the cell surface.
  • the sites of interest in binding to class I molecules are the second amino acid and the ninth (possibly 8th or 10th) amino acid in this peptide.
  • the second amino acid depends on the type of HLA. For example, in the case of 11 Otachiichi 24, the second amino acid is often tyrosine (Y) or phenylalanine (F), and in the case of HLA-A2, it is mouth insulin (L).
  • the ninth (or eighth or tenth) amino acid is often HLA — in the case of A24, it is leucine (L), isoleucine (I) or fenirarazine (F), and HLA — In the case of A24, it is leucine (L) or valine (V).
  • the T cell target sequence may vary depending on the HLA type of the patient of interest. Once formed, such a T cell target sequence is recognized by the T cell receptor of CTL and triggers T cell degradation.
  • Such a T cell target sequence can be identified using a method known in the art. Examples of such a method include, but are not limited to, a cDNA expression cloning method and a SEREX method.
  • a method known in the art include, but are not limited to, a cDNA expression cloning method and a SEREX method.
  • To identify the T cell target sequence of the pathogen of interest for example, transiently express a sample from a cDNA library prepared from the pathogen of interest in COS-7 cells together with HLA cDNA.
  • a mixed culture with the pathogen-specific CTL can be performed, and the amount of interferon in the supernatant can be identified by measuring the amount of interferon in the supernatant using, for example, an ELISA. it can.
  • a clone having a T cell target sequence can be identified, and thereby its coding sequence can also be identified.
  • a cDNA library is screened using IgG antibodies present in the serum of a patient.
  • target cells eg, infected Cell
  • -derived phage library is expressed in E. coli, the protein produced by E. coli is transferred to a filter, hybridized with patient serum, detected with an anti-IgG antibody, and The gene can be cloned.
  • the identification of T cell target sequences on cells related to intracellular parasitic pathogens and the like includes (1) a method using T cells that recognize pathogens, and (2) a method using T cells against antigen-trapping molecules. Is roughly divided into methods for inducing. However, at present, immunization with the killer T cell recognition epitopes identified in this manner has not yet effectively induced killer T cells in vivo. The reason for this is that immunization with these epitopes does not result in the processing of those epitope antigens by proteases that are intracellular enzymes, which is an essential step to induce killer T cells.
  • the antigen of the proteasome is processed by fusing the Tag (induction) ′ molecule to the proteasome with the ubiquitin gene of the molecule using the gene of the known T cell target sequence.
  • the search for epitopes that efficiently induce killer T cells is based on antigen-specific killer T cells by fusing an antigen gene to the ubiquitin gene, which is a tag molecule to the proteasome. It induces strong antitumor immunity.
  • the tag molecule such as ubiquitin, is constantly present in antigen-presenting cells, and non-selectively binds to foreign antigens produced in the cells or antigen transfer gene products injected into the cells. Transferred to the proteasome.
  • a T cell target sequence gene in the present invention a T cell target sequence is searched from a database, primers are synthesized at both ends of the sequence, and RNA is obtained from the pathogen or its infected cells by the phenol-chloroform method. Is extracted, and the RNA is used as a template to perform reverse transcription using the RT-PCR method to construct a gene.
  • the PC method polymerase chain reaction
  • the PC method is a reaction that repeatedly replicates only a specific site of a DNA chain, and can amplify a small amount of DNA up to about 1,000,000 times.
  • a synthetic oligonucleotide containing the nucleotide sequences at both ends of the amplification section is used, and the reaction is carried out using a heat-resistant DNA polymerase.
  • the RT-PCR method reverse-transcrlptpolymerase chain reaction
  • the RT-PCR method is a method in which DNA is synthesized from RNA by using reverse transcriptase in the first reaction, and then a specific DNR is obtained by the PCR method that is usually used. This is a method for amplifying A site.
  • Protective immunity (especially against intracellular parasitic pathogens such as tuberculosis, toxoplasma, and malaria) preferably induces cellular immunity centered on CD8 + T cells.
  • T cell target sequence consisting of about 8 amino acids, which can be recognized by the CD8 + T cell receptor (T cell receptor), from the vaccine antigen protein. This excision is performed by the proteasome, a cytoplasmic enzyme. As a prerequisite, the vaccine antigen protein must be bound (ubiquitinated) to ubiquitin in the cell.
  • a gene encoding a vaccine antigen protein and a gene encoding a ubiquitin gene into an antigen-presenting cell with a gene gun or the like, a fusion protein encoded by the binding gene in the cell is obtained. Are synthesized. Then, ubiquitin, which is a part of the fusion protein, leads the fusion protein to the proteasome.
  • the T cell target sequence is excised and recognized by the CD8 + ⁇ cell receptor. In order to induce antigen-specific CD 8 + T cells, it is preferable that the antigen is processed by the proteasome, which is a cytoplasmic enzyme, and then presented to the MHC class I molecule.
  • proteasome-specific inhibitor 1 actacystin or MG_132 during the culture of antigen-presenting cells does not result in the presentation of antigen to MHC class I molecules and the inactivation of CD8 + T cells. It is also evident from the fact that similar phenomena have been confirmed in experiments using proteasome knockout mice (Immunol. Rev. 163: 161-76, 1998).
  • the T cell target system IJ recognized by the killer T cell receptor is composed of 8 (usually 7 to 10) amino acids in most cases. Due to differences in HLA, the killer T cell receptor of the individual often recognizes a different epitope.
  • the peptide encoded by the DNA used in the present invention is preferably a peptide that can be recognized by various MHC killer T cells, or a DNA encoding a plurality of epitopes. In such a preferred embodiment, there is little need for tailor-made tailoring considerations in clinical applications, and populations with bespoke and diverse MHC may be the target of this vaccine. This is a major economic and technical benefit, and can be applied to developing countries with weak economic bases.
  • RNA is extracted from the tumor cells by the phenol-chloroform method.
  • reverse transcription is performed using the RT-PCR method to construct a gene.
  • the PCR method polimyrasecinreaction
  • the PCR method is a reaction in which only a specific portion of a DNA chain is repeatedly replicated, and a small amount of DNA can be amplified up to about 1,000,000 times.
  • RNA reverse transcriptase
  • a T cell target sequence should be displayed on the target cell's MHC class I molecule that is recognized by specific CTL.
  • polyubiquitin chains should be covalently bound to substrates by a multi-enzyme system.
  • the glycine residue at the C-terminus was replaced with another amino acid so that it was not cleaved and removed by the cell substrate ubiquitin C-terminal hydrolase.
  • the proteins fused with ubiquitin are more rapidly degraded than the unfused proteins in a manner dependent on the ubiquitin-proteasome system, which is specifically called the UFD pathway.
  • the fusion of ubiquitin to a protein that requires degradation by the proteasome provided by the present invention, is a useful strategy for effectively producing CTL epitopes.
  • the term “cell biologically active substance” or “physiologically active substance” refers to a substance that acts on cells or tissues. Such effects include, but are not limited to, for example, controlling or altering the cell or tissue.
  • Bioactive substances include cytokines and growth factors.
  • the physiologically active substance may be a naturally occurring substance or a synthetic substance. Preferably, the physiologically active substance is produced by a cell or has the same action as that of the bioactive substance, but may have a modified action.
  • the bioactive agent may be in the form of a protein or a nucleic acid or other forms, but at the time of actual action, cytoforce usually refers to the protein form.
  • Cytokines are generally proteins or polypeptides that regulate the immune response, regulate the endocrine system, regulate the nervous system, antitumor effects, antiviral effects, regulate cell proliferation, regulate cell proliferation, It has an action of regulating.
  • cytokines may be in the form of proteins or nucleic acids or other forms, but at the time of actual action, cytokines usually refer to the protein form.
  • growth factor or “cell growth factor” are used interchangeably herein and refer to a substance that promotes or controls cell growth. Growth factors are also called growth factors or growth factors. Growth factors can be added to the medium in cell cultures or tissue cultures to replace the action of serum macromolecules. Many growth factors have been found to function as regulators of differentiation status in addition to cell proliferation.
  • Cytokines typically include interleukins, chemokines, hematopoietic factors such as colonies, tumor necrosis factors, and interferons.
  • Typical growth factors include platelet-derived growth factor (PDGF), epithelial growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF )).
  • PDGF platelet-derived growth factor
  • EGF epithelial growth factor
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • VEGF vascular endothelial growth factor
  • Preferred exemplary cytokines in the present invention include, but are not limited to, those capable of inducing differentiation, proliferation, and activation of immune cells, such as interleukin 1, interleukin 2, and interleukin 4 , Interleukin 6, interleukin 7, IL-12, IL-15, IL-18, Interferon ⁇ , interferon, interferon, granulocyte macrophage, colony stimulating factor (GM-CSF), growth factors (GF) such as HGF, VEGF, FGF, tumor necrosis factor (TNF), tumor necrosis factor a ( TNF- ⁇ ), tumor death factor (TNF-i3), and the like, but are not limited thereto.
  • the site force in may be, but is not limited to, IL-12, IL-15, IL-18.
  • Physiologically active substances such as cytokines and growth factors generally have a dun- dancy phenomenon, and are known by other names (eg, cell killing ability, signal transduction ability, etc.).
  • a growth factor can be used in the present invention as long as it has the activity of the physiologically active substance used in the present invention.
  • the cytokine or growth factor may be used in a preferred embodiment of the vaccine or medicament of the present invention, as long as it has the preferred activity herein (eg, the ability to kill the desired pathogen). it can. (General biochemistry)
  • protein As used herein, the terms “protein”, “polypeptide”, “oligopeptide J and” “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. This polymer may be linear, branched, or cyclic. Amino acids may be natural or non-natural, and may be modified amino acids. As used herein, the term is preferably, but not limited to, linear and usually composed of naturally occurring amino acids, preferably in the form translated by a nucleic acid molecule. The term may also include those assembled into a complex of multiple polypeptide chains. The term also includes naturally or artificially modified amino acid polymers.
  • modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Modification (eg, conjugation with a labeling component).
  • This definition also includes, for example,
  • polypeptides containing one or more analogs eg, including unnatural amino acids and the like
  • peptidomimetic compounds eg, peptoids
  • the gene product of the present invention usually takes the form of a polypeptide.
  • the polypeptide of the present invention usually has a specific sequence (SEQ ID NO: 2, 4, etc. or a variant thereof).
  • Gene products of genes such as ubiquitin and antigens eg, antigens derived from intracellular pathogens
  • the modified sequence can be used for the purpose of prevention and treatment in the present invention.
  • polynucleotide As used herein, the terms “polynucleotide”, “oligonucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymer of a nucleotide of any length. The term also includes “derivative oligonucleotides” or “derivative polynucleotides”. The term “derivative oligonucleotide” or “derivative polynucleotide” refers to an oligonucleotide or polynucleotide containing a derivative of a nucleotide or having an unusual linkage between nucleotides, and is used interchangeably.
  • oligonucleotides include 2,1-O-methyl-ribonucleotide, a derivative in which a phosphoric diester bond in an oligonucleotide is converted to a phosphorothioate bond, and oligonucleotides; Derivative oligonucleotide in which the phosphoric diester bond of N is converted to N3, -P5 'phosphoramidate bond, Derivative in which ribose and phosphodiester bond in the oligonucleotide are converted to peptide nucleic acid bond Derivative oligonucleotide in which peracyl in nucleotide is substituted with C-5 propynyl peracyl, peracyl in oligo nucleotide is C-5, derivative oligonucleotide in which peracyl is substituted with thiazole peracyl, cytosine in oligonucleotide is C-5 Propynylcytosine In
  • oligonucleotides and the like. Unless otherwise indicated, a particular nucleic acid sequence may also have its conservatively modified variants (eg, degenerate codon substitutions) and complements, similar to the explicitly indicated sequence. It is contemplated to include the sequence. Specifically, degenerate codon substitutions create a sequence in which the third position of one or more selected (or all) codons has been replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell.
  • the gene vaccine of the present invention usually takes this polynucleotide form and usually needs to be translated into an amino acid form, so it is transcribed and translated in vivo. It is preferable, but not limited, to take the form (for example, consisting of natural nucleotides).
  • nucleic acid molecule as used herein is also used interchangeably herein with nucleic acids, oligonucleotides, and polynucleotides, and includes cDNA, mRNA, genomic DNA, and the like.
  • nucleic acids and nucleic acid molecules may be included in the concept of the term “gene”.
  • a nucleic acid molecule encoding a gene sequence also includes "splice variants (variants, variants).”
  • a particular protein encoded by a nucleic acid includes any protein encoded by a splice variant of the nucleic acid.
  • splice variants are the products of alternative splicing of a gene. After transcription, the initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. The production mechanism of splice variants changes
  • the gene of the present invention can also include a splice variant thereof. Such a mutant is useful in the prophylactic treatment of the present invention.
  • the term “gene” refers to a factor that determines a transgenic trait. Those that define the primary structure of a protein are called structural genes, and those that control its expression are called regulatory genes (eg, promoters). As used herein, a gene includes a structural gene and a regulatory gene unless otherwise specified. Therefore, reference to a gene such as ubiquitin usually includes both a structural gene such as ubiquitin and a transcriptional or translational regulatory sequence such as a promoter such as ubiquitin. In the present invention, these regulatory sequences can also be used for diagnosis. As used herein, “gene” generally refers to “polynucleotide”, “oligonucleotide” and “nucleoacid”.
  • gene product also refers to “polynucleotide”, “oligonucleotide” and “nucleic acid” expressed by a gene and / or “protein” “polypeptide”, “oligopeptide” and “ Peptide ". Those skilled in the art can understand what a gene product is, depending on the situation.
  • homology of a gene refers to the degree of identity between two or more gene sequences.
  • the higher the homology between two genes the higher the identity or similarity between their sequences.
  • Whether or not two genes have homology can be determined by direct sequence comparison or, in the case of nucleic acids, by a hybridization method under stringent conditions.
  • the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, more preferably at least 80% , 90%, 95%, 96%, 97%, 98% or 99% identical, the genes are homologous.
  • the term “congener” of a gene refers to the mutual identity of two or more gene sequences when conservative substitutions are regarded as positive in the above homology. Spasticity of identity to Thus, if there are conservative substitutions, identity and similarity will differ depending on the presence of the conservative substitution. If there is no conservative substitution, identity and similarity show the same numerical value.
  • sequences having high similarity or homology to the elements used such as ubiquitin and antigens derived from intracellular parasitic pathogens, can also be used.
  • amino acid may be natural or non-natural as long as the object of the present invention is satisfied.
  • “Derivative amino acid” or “amino acid analog” refers to one that is different from a naturally occurring amino acid but has the same function as the original amino acid. Such derivative amino acids and amino acid analogs are well-known in the art.
  • natural amino acid means the L-isomer of a natural amino acid. Natural amino acids are glycine, alanine, phos, phosphorus, leucine, isoleucine, serine, methionine, threonine, pheninolealanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamic acid, and glutamine. Lipoxyglutamic acid, arginine, ordinine, and lysine.
  • unnatural amino acid refers to an amino acid that is not normally found in nature in a protein.
  • unnatural amino acids include norleucine, para-nitrophenylenoalanine, homophenylealanine, parahue / leophenylenolaine, 3-amino-2-benzylpropionic acid, D-form and L-form of homoarginine, and D-fe Dilulanine.
  • amino acid analog refers to a molecule that is not an amino acid but is similar to the physical properties and Z or function of an amino acid.
  • amino acid analogs examples include, but are not limited to, etyonin, syrup, 2-methylglutamine and the like.
  • Amino acid mimetics as examples of amino acid analogs, are compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • nucleotide may be natural or non-natural.
  • derivative nucleotide or “nucleotide analog” refers to a nucleotide different from a naturally occurring nucleotide but having the same function as the original nucleotide.
  • derivative nucleotides and nucleotide analogs are well known in the art. Examples of such derivative nucleotides and nucleotide analogs include phosphorothioate, phosphoramidate, methyl phosphonate, chiral methyl phosphonate, 2-O-methyl liponucleotide, peptide-nucleic acid (PNA), It is not limited to these. In the present invention, any analog may be used as long as the gene product is expressed.
  • a genetic vaccine containing natural nucleotides is used. This is because the natural form is more likely to be translated into a peptide.
  • nucleic acid refers to a polypeptide and a nucleic acid molecule having the same action as a given amino acid and a nucleic acid in the polypeptide and the nucleic acid molecule as a reference for comparison.
  • amino acids and nucleic acids that are predicted to have, for example, in ubiquitin they are located at the same position as the sequence responsible for linkage to lysine (for example, C-terminal glycine) and have the same contribution to catalytic activity.
  • nucleic acids encoding the same For example, in the case of a nucleic acid sequence, it may be a portion that performs the same function as the nucleic acid sequence or the specific portion encoded by the nucleic acid sequence.
  • a “corresponding” gene refers to a gene that has, or is expected to have, the same effect as a given gene in a reference species for comparison. Gene that has such an effect When two or more genes exist, they have the same evolutionary origin. Thus, the corresponding gene of a gene may be the ortholog of that gene. Therefore, genes corresponding to genes such as human ubiquitin, T cell target sequences, and antigens derived from intracellular parasitic pathogens should be found in other animals (mouse, rat, pig, pig, etc.). Can be. Such corresponding genes can be identified using techniques well known in the field.
  • the corresponding gene in an animal can be obtained by querying the sequence of a reference gene for the corresponding gene (eg, a gene such as human ubiquitin, a T cell target sequence, or an antigen derived from an intracellular parasitic pathogen) as a query sequence.
  • a reference gene for the corresponding gene eg, a gene such as human ubiquitin, a T cell target sequence, or an antigen derived from an intracellular parasitic pathogen
  • search refers to the use of one nucleobase sequence to find another nucleobase sequence having a specific function and / or property, either electronically or by biological or other methods.
  • BLAST Altschul et al., J. Mol. Biol. 215: 403-410 (1990)
  • FASTA Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444- 2448 (1988)
  • Smith and Waterman method Smith and Waterman, J. Mol. Biol. 147: 195—197 (1981)
  • Needleman and Wunsch method Needleman and Wunsch, J. Mol. Biol. 8: 443
  • Bio searches include stringent hybridization, macroarray in which genomic DNA is affixed to a nylon membrane or the like, or microarray (a microarray assay) in which the DNA is affixed to a glass plate, PCR, and in situ hybridization. And the like, but are not limited thereto.
  • ubiquitin, T cell target sequence for example, an antigen derived from an intracellular parasitic pathogen
  • T cell target sequence should also include the corresponding gene identified by such electronic search or biological search. Is intended.
  • fragment refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 with respect to a full-length polypeptide or polynucleotide (length is n).
  • the length of the fragment can be appropriately changed depending on the purpose.
  • the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 9 for a polypeptide.
  • nucleotides 5, 6, 7, 8, 9, 10, 15, 20, 30, 25, 30, 40, 50, 75, 100, and more nucleotides Lengths represented by integers not specifically listed here (for example, 11 and the like) may also be appropriate as the lower limit.
  • the length of the polypeptide and the polynucleotide can be represented by the number of amino acids or nucleic acids, respectively, as described above.
  • the above-mentioned numbers are not absolute, and the upper limit is provided as long as they have the same function.
  • the above-mentioned number as a lower limit is intended to include a few above and below (or, for example, 10% above and below) the number.
  • polynucleotide that hybridizes under stringent conditions refers to well-known conditions commonly used in the art.
  • a polynucleotide selected from the polynucleotides of the present invention as a probe, a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like, such a polynucleotide can be used.
  • polynucleotides that hybridize under stringent conditions were obtained by immobilizing DNA derived from colonies or plaques. Using a filter, 0.7-1. OM in the presence of NaCl 65.
  • 0.1- to 2-fold concentration of 33 ⁇ (saline-sodium citrate) solution Means a polynucleotide that can be identified by washing the filter under 65 ° C conditions.
  • Hybridization is described in experimental books such as Molecular Cloning 2nd ed., Current Protocols m Molecular Biology, Supplement 138, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995). It can be done according to the method that is being done.
  • sequences containing only the A sequence or only the T sequence are preferably excluded from the sequences that hybridize under stringent conditions.
  • polynucleotide capable of hybridizing refers to a polynucleotide that can hybridize to another polynucleotide under the above-mentioned hybridization conditions.
  • a polynucleotide capable of hybridizing a polynucleotide having at least 60% or more homology with a nucleotide sequence of DNA encoding a polypeptide having the amino acid sequence specifically shown in the present invention, preferably 80% Polynucleotides having a homology of at least 95%, more preferably polynucleotides having a homology of at least 95%.
  • Amino acids may be referred to herein by either their commonly known three-letter symbols or by the IUPAC—IUB Biochemica1 Nomenclatru e and omm i s s io n one-letter symbols as recommended herein. Nucleotides may also be referred to by the generally recognized one-letter code.
  • variant refers to a substance in which a substance such as an original polypeptide or polynucleotide is partially changed.
  • variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like.
  • variants include one or more substitutions, additions and / or deletions, or one or more substitutions, additions and Z or deletions with respect to a reference nucleic acid molecule or polypeptide. But not limited to them.
  • Alleles refer to genetic variants that belong to the same gene locus and are distinct from each other.
  • an “allelic variant” refers to a variant that has an allelic relationship to a gene.
  • allelic variants usually have sequences that are identical or very similar to their corresponding alleles, usually have nearly the same biological activity, but rarely have different biological activities. May have.
  • “Species homologues or homologs (homomog) j” refer to homology (preferably 60% or more homology, more preferably 80% or more, homologous to a certain gene at the amino acid level or the nucleotide level) in a certain species. % Or more, 85% or more, 90% or more, 95% or more) The method for obtaining such a species homolog is apparent from the description of the present specification.
  • the term “ortho 1 og” is also called the orthologous gene, and refers to a gene derived from speciation from a common ancestor with two genes.
  • the human and mouse ⁇ -hemoglobin genes are orthologs, and the human ⁇ - hemoglobin gene and ⁇ -hemoglobin gene are paralogs. Gene generated by duplication).
  • orthologs may exist for genes of protozoa such as malaria. Orthoguchi is useful for estimating molecular phylogenetic trees.
  • the ortholog of the present invention can also be useful in the present invention, since the ortholog can usually perform the same function as the original species in another species.
  • “conservative (modified) variants” applies to both amino acid and nucleic acid sequences.
  • Conservatively modified breaks for a particular nucleic acid sequence A variant refers to a nucleic acid that encodes the same or essentially identical amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, refers to an essentially identical sequence. Due to the degeneracy of the transit code, a number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine.
  • nucleic acid variation is a "silent modification (mutation)," which is one type of conservatively modified mutation. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) Force Produces functionally identical molecules It will be understood that they can be modified to Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence. Silent mutations may also be diagnostic indicators of the present invention. Preferably, such modifications can be made to avoid substitution of cysteine, an amino acid that has a significant effect on the conformation of the polypeptide.
  • Such base sequence modification methods include cleavage with a restriction enzyme or the like, ligation by a treatment with a DNA polymerase, a Klenow fragment, a DNA ligase, or the like, site-specific bases using synthetic oligonucleotides, etc.
  • Substitution method specifically site-directed mutagenesis method; Mark Zoller and Michael Smith, Methods in Enzymology, 100, 468-500 (1983)). You can also do
  • Such a nucleic acid can be obtained by a well-known PCR method, and can also be chemically synthesized. These methods may be combined with, for example, a site-specific displacement induction method, a hybridization method, and the like.
  • substitution refers to the replacement, addition, or removal of an amino acid or its substitute, or nucleotide or its substitute, respectively, from the original polypeptide or polynucleotide.
  • Techniques for such substitution, addition or deletion are well known in the art, and examples of such techniques include site-directed mutagenesis techniques.
  • the number of substitutions, additions, or deletions may be any number as long as it is one or more, and such a number indicates the number of the desired function (for example, transfer to the proteasome) in the variant having the substitution, addition, or deletion. Can be increased as long as is maintained. For example, such a number may be one or several, and preferably is within 20%, within 10%, or 100 or less, 50 or less, 25 or less of the total length. Or less.
  • antibody refers to a polyclonal antibody, a monoclonal antibody, a human antibody, a humanized antibody, a multispecific antibody, a chimeric antibody, and an anti-idiotype antibody, and fragments thereof such as F (ab,) 2 and Fab fragments, as well as other recombinantly produced conjugates. Further, such an antibody may be covalently bound to an enzyme such as alkaline phosphatase, horseradish peroxidase, ⁇ -galactosidase, or the like, or fused by recombination.
  • an enzyme such as alkaline phosphatase, horseradish peroxidase, ⁇ -galactosidase, or the like, or fused by recombination.
  • neutralizing antibody refers to an antibody that neutralizes a biological activity of an enzyme, a toxin, a bacterium, a virus, or the like.
  • the present invention particularly refers to an antibody that neutralizes a biological activity associated with an antigen containing a cell target sequence.
  • antigen refers to any substrate that can be specifically bound by an antibody molecule.
  • immunogen refers to an antigen that can initiate lymphocyte activation that produces an antigen-specific immune response.
  • epitopes refers to a group constituting a structure that determines an antigen.
  • epitopes contain a set of amino acid residues involved in recognition by a particular immunoglobulin, or, in the case of T cells, T cell receptor proteins And / or the set of amino acid residues required for recognition by the major histocompatibility complex (MHC) receptor.
  • MHC major histocompatibility complex
  • epitopes are molecular features (eg, primary, secondary or tertiary peptide structures and charges), immunoglobulins, T cell receptors or Form a site recognized by the HLA molecule.
  • Epitopes containing peptides can contain more than two amino acids in a spatial conformation unique to the epitope.
  • an epitope is composed of at least five such amino acids, and typically is composed of at least six, seven, eight, nine, or ten such amino acids.
  • epitopes are generally preferred because they resemble the antigenicity of the original peptide, but may not always be so in view of the conformation.
  • Methods for determining the spatial conformation of amino acids include, for example, X-ray crystallography, and two-dimensional nuclear magnetic resonance spectroscopy.
  • identification of the epitope in a given protein is readily accomplished using techniques well known in the art. See, eg, Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81: 3998 (a general method for rapidly synthesizing peptides to determine the location of immunogenic epitopes on a given antigen); US Pat. 4, 7 0 8, 8 7 No.
  • an “isolated” substance eg, a biological agent such as a nucleic acid or protein
  • a biological agent such as a nucleic acid or protein
  • a biological factor for example, if it is a nucleic acid, a factor other than a nucleic acid and a nucleic acid containing a nucleic acid sequence other than the target nucleic acid; if a protein, a factor other than the protein and the target protein Etc.
  • proteins that have been substantially separated or purified from proteins Proteins that have been substantially separated or purified from proteins.
  • isolated nucleic acids and proteins include nucleic acids and proteins that have been purified by standard purification methods.
  • isolated nucleic acids and proteins include chemically synthesized nucleic acids and proteins.
  • the substance of the present invention can be an isolated substance.
  • a “purified” substance eg, a biological factor such as a nucleic acid or a protein refers to a substance from which at least a part of a factor naturally associated with the substance has been removed. Thus, typically, the purity of the purified material is higher (ie, more concentrated) than in the state in which it is normally present.
  • the substance of the present invention can be a purified substance.
  • purified and isolated preferably refer to at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably Preferably at least 98 weight. / 0 means that a substance of the same type exists.
  • expression of a gene product such as a gene, a polynucleotide, or a polypeptide means that the gene (usually a DNA form) or the like undergoes a certain action in vivo and changes to another form.
  • gene, polynucleotide And the like are referred to as being transcribed and translated into the form of a polypeptide, but transcription and production of mRNA can also be a form of expression.
  • such forms of the polypeptide may have undergone post-translational processing.
  • the term “specifically expresses” means that a gene is expressed at a particular site or stage of a plant at a different (preferably higher) level than at other sites or stages. Say. To be specifically expressed may be expressed only at a certain site (for example, a specific site such as a diseased site) or may be expressed at other sites. Preferably, specific expression means expression at only a certain site. Such specific expression can be achieved by using a promoter or the like that induces specific expression.
  • “detection” or “quantification” of gene expression can be achieved using a simple method including, for example, mRNA measurement and immunological measurement.
  • the molecular biological measurement method include Northern blotting, dot plotting, and PCR.
  • the immunological measurement method include, for example, an ELISA method, a RIA method, a fluorescent antibody method, a Western blot method, and an immunohistochemical staining method using a microtiter plate.
  • Examples of the quantification method include the £ 13 method and the scale 1A method. It can also be performed by a gene analysis method using an array (for example, a DNA array or a protein array).
  • biological activity refers to a factor (eg, a polypeptide or protein (eg, ubiquitin, a peptide containing a cell target sequence (eg, an antigen derived from an intracellular parasitic pathogen), etc.)).
  • a factor eg, a polypeptide or protein (eg, ubiquitin, a peptide containing a cell target sequence (eg, an antigen derived from an intracellular parasitic pathogen), etc.
  • An activity that can be possessed in a living body and includes activities that exhibit various functions (for example, transport to proteasome, ability to elicit antibodies, transcription promoting activity, etc.).
  • two factors eg, binding of ubiquitin and its partner to its partner
  • its biological activity will depend on the binding between ubiquitin and its partner and the resulting organism Biological changes (eg, transport to the proteasome, etc.).
  • a "vaccine” is defined as an infectious agent or a portion of an infectious agent, or an agent capable of producing such an agent or portion, which is administered into the body to generate active immunity.
  • a composition eg, a suspension or solution
  • the antigenic portion of the cutin may be a microorganism (eg, a virus or bacterium) or a natural product purified from the microorganism, a synthetic product or a genetically engineered protein, a peptide, a polysaccharide, or similar product. It can be an organism, or a nucleic acid molecule comprising a nucleic acid sequence encoding such a protein. Vaccines exert their effects by raising neutralizing antibodies.
  • the term “gene vaccine” refers to a composition of a vaccine that is expressed in a subject to which the vaccine is administered and that contains an agent (typically, a nucleic acid molecule) whose expression has the effect of the vaccine. (Eg, suspension or solution).
  • An exemplary genetic vaccine can be a nucleic acid molecule (eg, a vector, plasmid, naked DNA, etc.) containing a nucleic acid sequence encoding a gene product having antigenicity.
  • the immunological effect of the vaccine can be confirmed using any method known in the art.
  • CTL precursor cell frequency analysis was performed by limiting dilution of peripheral blood lymphocytes or lymphocytes cultured in the presence of antigenic peptides and IL-2, and coexisting with IL-12 and feeder cells. Stimulate with the vaccine or its target, and measure the presence or absence of IFN- ⁇ production by ELIS II or the like.
  • the positive cells can be used to calculate the frequency of CTL precursor cells according to Poisson analysis to evaluate the efficacy of the vaccine.
  • the number of positive cells is the number of antigen-specific CTLs, and the greater the number, the higher the efficacy as a vaccine.
  • an “adjuvant” is a substance that increases or otherwise alters an immune response when mixed with an administered immunogen.
  • Adjuvants are optionally classified, for example, as mineral, bacterial, plant, synthetic or host products.
  • pathogen refers to an organism or factor that can cause a disease or disorder in a host. Pathogens to humans include, but are not limited to, for example, viruses, bacteria, protozoa, rickettsia, chlamydia, fungi, neoplasms (such as cancer), and the like. Specific examples of intracellular parasitic pathogens include, but are not limited to, protozoa such as Mycobacterium tuberculosis, Malaria parasite, Toxoplasma parasite, Trypanosoma cruzi, and viruses such as HIV and cytomegalovirus. Absent.
  • Particularly effective antigens for such pathogens include MPB51, MDPl, Ag85A, Ag85B, HSP65, Mtb72f gene derived from Mycobacterium tuberculosis, MSP-1 gene derived from malaria parasite, Toxoplasma Protozoan-derived SAG-1 gene, TSA gene derived from Trypanosoma cruzi, HIV-related gene, cytomegalovirus-related gene, etc., but are not limited thereto.
  • Waku Tins are typically effective for, but are not limited to, viruses, bacteria, cancer, and the like.
  • a “disease” for which a vaccine using a nucleic acid encoding an antigen protein derived from an intracellular parasitic pathogen is effective is a disease caused by an intracellular parasitic pathogen.
  • diseases include, for example, malaria, tuberculosis, toxoplasmosis, trivanosomiasis, acquired immunodeficiency syndrome, cytomegalovirus disease, cladiemia, rickettsiosis, leishmaniasis, Ebola hemorrhagic fever, trypanosoma cruzi infection.
  • prevention refers to treating a disease or disorder before such condition is caused so that the condition does not occur.
  • treatment refers to preventing the deterioration of a disease or disorder when such a condition occurs, preferably maintaining the status quo, more preferably reducing the disease, More preferably, it refers to fluctuating.
  • Such therapeutic or prophylactic activity is preferably tested in vitro prior to use in humans and then in vivo.
  • in vitro assays for demonstrating the therapeutic or prophylactic utility of the gene vaccines of the present invention include the effect of specific binding of the vaccine to cell lines or patient tissue samples.
  • Such tests can be determined using techniques known to those of skill in the art (eg, immunological assays such as ELISA).
  • In vivo tests include, for example, the ability to elicit neutralizing antibodies. , But not limited thereto.
  • subject refers to an organism to which the treatment of the present invention is applied, and is also referred to as “patient”.
  • patient refers to an organism to which the treatment of the present invention is applied, and is also referred to as “patient”.
  • patient or subject can preferably be a human.
  • the present invention provides methods for treatment, inhibition and prevention by administering to a subject an effective amount of the gene vaccine of the present invention.
  • the gene of the present invention may be substantially purified (including, for example, conditions that limit its effect or are substantially free of substances that produce undesirable side effects). .
  • the animal targeted by the present invention may be any organism (eg, an animal (eg, a vertebrate, an invertebrate)) that has an immune system or a similar system.
  • vertebrates e.g., metal eels, smelt eel, chondrichthyes, teleosts, amphibians, reptiles, birds, mammals, etc.
  • mammals e.g. Bags, oligodonts, skin wings, winged fins, carnivores, carnivores, longnoses, equinoids, artiodactyla, dentates, squamous, sea cows, whales, primates, Rodents, ⁇ etc.
  • Illustrative subjects include, but are not limited to, animals such as, for example, horses, pigs, horses, chickens, cats, dogs, and the like. More preferably, primates (eg, chimpanzees, macaques, humans) are targeted. Most preferably, humans are targeted.
  • animals such as, for example, horses, pigs, horses, chickens, cats, dogs, and the like. More preferably, primates (eg, chimpanzees, macaques, humans) are targeted. Most preferably, humans are targeted.
  • such a composition may further include a pharmaceutically acceptable carrier and the like.
  • the pharmaceutically acceptable carrier contained in the medicament of the present invention includes any substance known in the art.
  • the vaccine according to the invention can also comprise any composition that achieves an immune system with a more immunologically favorable immune response in the event of a disease.
  • Vaccines usually contain an immune determinant and an immunostimulant that acts to rapidly enhance this immune determinant response, and the immunodeterminant is combined with the immunostimulant. It is preferable to use them.
  • Immunostimulants are commonly used, for example, Freund
  • Pharmaceutically acceptable carriers that can be used in the compositions, vaccines and the like of the present invention include antioxidants, preservatives, coloring agents, flavorings, and diluents, emulsifiers, suspending agents, solvents, Includes but is not limited to fillers, bulking agents, buffers, delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants.
  • the medicament of the present invention is administered in the form of a composition comprising a vaccine, or a variant or derivative thereof, together with one or more physiologically acceptable carriers, excipients or diluents.
  • a suitable vehicle may be water for injection, physiological solution, or artificial cerebrospinal fluid, which may be supplemented with other materials common in compositions for parenteral delivery. is there.
  • acceptable carriers, excipients, or stabilizers are non-toxic to the recipient, and are preferably inert at the dosages and concentrations employed, e.g., Phosphate, citrate, or other organic acid; ascorbic acid, monotocopherol; low molecular weight polypeptide; protein (eg, serum albumin, gelatin or immunoglobulin); hydrophilic polymer (eg, polybutylpyrroli) Amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (eg, EDTA); Sugar alcohols (eg mannitol or sorbitol ); Salt-forming counterions (eg, sodium); and / or non-ionic surface activators (eg, Tween, p1 uronic or polyethylene daricol (PEG)).
  • exemplary suitable carriers include neutral buffered saline or serum albumin. And physiological saline mixed with water.
  • the product is formulated as a lyophilizate using a suitable excipient (eg, sucrose).
  • suitable excipient eg, sucrose
  • Other standard carriers, diluents and excipients may be included as desired.
  • Other exemplary compositions include a Tris buffer at pH 7.0-8.5 or an acetate buffer at ⁇ 4.0-5.5, which further comprises sorbitol or a suitable alternative thereof. Can include.
  • animal drug compositions, quasi-drugs, marine drug compositions, food compositions, cosmetic compositions, and the like can also be manufactured by a known preparation method.
  • the vaccine and the like of the present invention can be administered parenterally in combination with a pharmaceutically acceptable carrier.
  • the medicament of the present invention may contain a physiologically acceptable carrier, excipient or stabilizing agent (Japanese Pharmacopoeia, 14th edition or its latest edition, Remington's Pharma ceutlca 1 Sciences, 18 lyophilized by mixing a sugar chain composition having a desired degree of purity with a sugar chain composition having a desired degree of purity (see, for example, th Edtion, AR Gennaro, ed., Mack Publishing Company, 1990). It can be prepared and stored in the form of a cake or aqueous solution.
  • a physiologically acceptable carrier excipient or stabilizing agent
  • aqueous solutions for example, aqueous solutions, liposomes, microparticles, microcapsules, and the like.
  • the peptides of the present invention can be administered orally or parenterally.
  • Such administration methods include oral administration, parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration (nasal administration, intravaginal administration, respiratory tract administration, oral cavity, rectal mucosa) And intestinal mucosa), topical administration to the affected area, dermal administration, etc.).
  • the vaccine used in the present invention preferably does not contain pyrogens.
  • Such drugs Preparation of biological acceptable composition, more to considering P H, isotonicity, stability and the like, one skilled in the art, can be carried out easily.
  • Formulations for such administration may be provided in any formulation.
  • Such preparation forms include, for example, liquid preparations, injections, and sustained release preparations.
  • Methods of introduction include, but are not limited to, oral administration, inhalation (eg, lungs), injection using syringes, catheters, and tubes, needleless injection, and gene guns. In this case, it can be co-administered with other biologically active agents.
  • the amount of pectin used in the prophylactic method of the present invention can be easily determined by those skilled in the art in consideration of the purpose of use, the target disease (eg, species), the age, weight, and medical history of the patient.
  • the frequency may be, for example, once every few months (eg, once a week, once a month) or once per year before the epidemic. It is preferable to administer once a week and once a month while observing the progress, and it is advantageous to perform booster immunization at intervals of at least about one week. More preferably, the interval between boosts can be at least about 3 weeks.
  • the dose of the vaccine or the like of the present invention varies depending on the age, body weight, symptoms or administration method of the subject, and is not particularly limited.
  • administering means that the vaccine or the like of the present invention or a pharmaceutical composition comprising the same is given to a host to be treated, alone or in combination with another therapeutic agent. .
  • the combination may be administered, for example, either simultaneously as a mixture, separately but simultaneously or concurrently; or sequentially. This includes the indication that the combined drugs are administered together as a therapeutic mixture, and that the combined drugs are separate but simultaneously (eg, separate mucosal The procedure to be administered is also included.
  • “Combination” administration further includes separately administering one of the compounds or agents given first, followed by the second.
  • the administration of the vaccine in the present invention may be performed by any method, but it is advantageous to use needleless injection or a gene gun. This is because administration can be performed without imposing an excessive burden on the patient.
  • the needleless syringe according to the present invention refers to a method in which a gaston is moved by gas pressure or elasticity of an elastic member to eject a drug solution to the skin without using a needle, and a drug component is injected subcutaneously, more preferably into a subcutaneous cell.
  • a medical device to be administered means a medical device to be administered.
  • ShimaJET TM manufactured by Shimadzu Corporation
  • Jiwekuta manufactured Elitemedical Co.
  • Single Vision Medi-Jector Vision
  • PenJet Co. Pen jet
  • TM Pen jet
  • a gene gun is a medical / experimental device capable of in vivo gene transfer by accelerating DNA-coated high-density particles such as gold and tungsten using the gas pressure of a helium or the like.
  • the advantages of a gene gun are that it can efficiently transfer cells with a small amount of DNA and that stable results can be obtained with different operators.
  • Helios Gene Gun manufactured by Bio-Rad in the United States is commercially available and can be used. (See below)
  • the “instruction” describes a method of administering or diagnosing the medicament or the like of the present invention to a doctor, a patient or the like who administers or diagnoses (possibly a patient). It is.
  • This instruction includes a statement indicating a procedure for administering the diagnostic agent, the prophylactic agent, the medicine, and the like of the present invention.
  • These instructions are prepared in accordance with the format prescribed by the competent authority of the country in which the invention is implemented (for example, the Ministry of Health, Labor and Welfare in Japan, and the Food and Drug Administration (FDA) in the United States), and are prepared in accordance with the competent authority. Specifies that approval has been obtained.
  • the instruction sheet is a so-called package insert, which is usually provided by a paper medium, but is not limited thereto.
  • a film affixed to a bottle an electronic medium (for example, a home page provided on the Internet (web Site), e-mail).
  • the end of the prophylactic treatment according to the method of the present invention is determined by confirming that the disease is not induced by exposure to an antibody (eg, a neutralizing antibody) induced by use of a commercially available assay or a device or a pathogen. Can be.
  • an antibody eg, a neutralizing antibody
  • the present invention also provides a pharmaceutical package or kit comprising a container containing the vaccine of the present invention.
  • a notice in the form of a government agency that regulates the manufacture, use or sale of pharmaceuticals or biological products may optionally accompany such containers, and may include such notice for production, use or administration for human administration. Represents government approval for sale. Description of the preferred embodiment
  • the present invention provides a genetic vaccine comprising a nucleic acid sequence encoding ubiquitin and a nucleic acid sequence encoding an antigenic protein of an intracellular parasitic pathogen.
  • the invention provides a nucleic acid sequence encoding ubiquitin
  • a nucleic acid sequence encoding an antigen protein having a T cell target sequence is provided.
  • Such vaccines comprise a nucleic acid molecule (fusion DNA) in which a nucleic acid encoding an antigen protein derived from an intracellular parasitic pathogen or an antigen protein containing a T cell target sequence and a nucleic acid encoding a ubiquitin gene are linked.
  • fusion DNA a nucleic acid molecule
  • Such a genetic vaccine has an antigen recognized by T cells (for example, an antigen derived from a pathogen such as an intracellular parasite or a T cell target sequence) due to the property of ubiquitin to direct toward the proteasome.
  • Antigen is efficiently decomposed by the efficient transport to the proteasome, and the effect of treating or preventing a disease or disorder with almost no side effects is achieved.
  • Protozoa such as malaria parasites have the effect of being able to cope with various mutations that conventional vaccines cannot cope with.
  • Such genetic actin designs and constructs a nucleic acid sequence encoding ubiquitin, and designs a nucleic acid sequence encoding an antigen protein derived from an intracellular parasitic pathogen or an antigen protein including a T cell target sequence. By building, It can be manufactured by connecting the same.
  • Such constructions can be performed using molecular biology or genetic engineering techniques well known in the art, and such techniques are described in the forces described herein or in the cited literature. It is described.
  • the vaccine of the present invention unlike recombinant protein vaccines and vaccines using pathogen proteins, is unlikely to cause anaphylactic shock even with frequent administration, and is excellent in safety. Further, purification and preparation are simpler and more economical than the recombinant protein vaccine / cytokine. It is also effective against pathogens that frequently mutate.
  • gene therapy using a naked DNA and a DNA vaccine without using the viral vector of the present invention can easily and safely perform repeated administration to a living body. Furthermore, the construction of the naked DNA gene is much easier than in the case of using a viral vector. In the present invention, since a virus vector is not used, the construction of the gene is easy, and the risk of infection due to the virulence of the virus vector is extremely small. In addition, since a virus vector is not used, the possibility of integration into genomic DNA is extremely low.
  • the gene vaccine of the present invention has the following advantageous features.
  • the recombinant protein vaccine may cause serious side effects such as anaphylaxis when administered multiple times, but this DNA vaccine has a low risk.
  • Recombinant protein vaccines have to be expensive because they are not easy to purify, but DNA vaccines are easy and inexpensive to purify. It is relatively easy to use even in developing countries.
  • gene therapy often uses viral vectors.
  • the epitope recognized by the killer T cell receptor is often composed of eight amino acids, but due to differences in the tissue antigen (MHC in humans and HLA in humans) of the infected host, The body often recognizes different epitopes.
  • the peptide encoded by the DNA used in the present invention is one that can be recognized by killer T cells of various types of MHC or DNA that encodes a plurality of epitopes. There is little need for tailor-made / tiler-made considerations, and populations with many different types of MHC can be targeted for this vaccine. This has great technical and economic benefits and can be applied to developing countries with weak economic bases.
  • the present invention provides a nucleic acid construct comprising a nucleic acid sequence encoding ubiquitin and a nucleic acid sequence encoding an antigen protein including a T cell target sequence.
  • the present invention provides a nucleic acid construct comprising a nucleic acid sequence encoding ubiquitin and a nucleic acid sequence encoding an antigenic protein derived from an intracellular parasitic pathogen.
  • Such a nucleic acid construct is prepared by designing and constructing a nucleic acid sequence encoding ubiquitin, and by designing and constructing a nucleic acid sequence encoding an antigen protein of interest, and linking as necessary. can do.
  • Such constructions can be performed using molecular biology or genetic engineering techniques well known in the art, and the techniques described herein may be based on the forces described herein or the literature cited. Is described. Containing the ubiquitin gene according to the present invention The amount can be appropriately changed depending on various factors, but is 1 ⁇ g to 20 // g, preferably 6 ⁇ to 9 g per mouse (20 g), but is not limited thereto.
  • the content of the antigen gene according to the present invention can be appropriately changed depending on various factors, it is 1 ⁇ to 20 per mouse (20 g), preferably 6 g to 9 tg, but is not limited thereto. As these amounts fall outside these ranges, vaccine efficacy tends to diminish, but does not disappear.
  • a nucleic acid encoding a ubiquitinated gene or an antigen gene (for example, a gene encoding an antigen protein derived from an intracellular parasitic pathogen) is constructed by the RT-PCR method described above, but is not limited to this method. is not. Specifically, DNA is synthesized from RNA by using reverse transcriptase in the first reaction, and then a specific portion of the DNA chain is repeatedly replicated by PCR to amplify a small amount of DNA. After transformation, transform into Escherichia coli and perform shaking culture in a 37 ° C constant temperature bath (about 16 hours).
  • the bacterial cell component as a precipitate is dissolved in a Tris-HCl / EDTA solution.
  • the bacterial cell components are lysed with an alkaline solution NaOH / SDS solution, and after lysis, neutralization is performed with a neutralizing solution acetic acid rim to stop the reaction.
  • the solution is added to an anion exchange resin column for nucleic acid purification for 1 oad, the column is washed with a NaC1 / acetic acid realm solution, and eluted with NaC1ZTris-HC1.
  • the fact that the gene is actually expressed in the cells can be confirmed by transfection into COS cells.
  • the nucleic acid sequence encoding the antigenic protein used in the present invention is usually the amino acid encoded by the nucleic acid sequence.
  • Peptides having an acid sequence are characterized by having CTL reactivity. Whether a factor has CTL reactivity can be determined by confirming whether CTL is activated when a candidate factor is administered to a host having a T cell immune system. it can.
  • Factors that activate such CTLs typically have a characteristic amino acid sequence having a length of 8 to 10 amino acids, and such sequences are exemplified herein as tables.
  • the present invention is not limited thereto, and any factor can be used as long as it activates CTL. Therefore, variants having at least one mutation selected from the group consisting of substitution, addition and deletion of one or several amino acids of the specific sequences exemplified in the table are also used in the present invention. be able to.
  • the nucleic acid sequence encoding an antigen protein used in the present invention usually has at least 7, more usually at least 7, It encodes a peptide consisting of 8 amino acids, more preferably a peptide consisting of ⁇ to 10 amino acids, but is not limited thereto.
  • amino acid sequences are exemplified elsewhere herein.
  • a nucleic acid sequence encoding such an amino acid sequence can be prepared by designing the code using common sense in molecular biology. Such a coding sequence may be any sequence as long as it can be translated into the amino acid sequence of interest in the host, but is preferably transcribed using the codon frequency often used in the host. It is advantageous to use sequences that translate more efficiently.
  • a preferred antigenic protein (an antigenic protein having a T cell target sequence or an antigenic protein derived from an intracellular parasitic pathogen) comprises at least 7, more preferably at least 8 amino acids.
  • the second, first or third (preferably second) amino acid sequence is a hydrophobic amino acid (eg, Va1, Leu, I1e, Tyr or Phe);
  • the eighth, ninth or tenth (preferably ninth) sequence is a hydrophobic amino acid (eg, Va1, Leu, Phe or I1e). Not limited to).
  • Such sequences are known to be susceptible to targeting by CTL in certain situations. However, it is understood that such preferred sequences may vary depending on the situation. Also, by understanding such variations and applying it to actual gene vaccine production, it is possible to produce a gene vaccine with fewer side effects. This is because it is known that the preferred sequences differ depending on the type of HLA.
  • the antigen protein containing the T cell target sequence used in the present invention is derived from a pathogen protein (for example, a protein derived from an intracellular pathogen such as protozoan or Mycobacterium tuberculosis) and an intracellular parasitic pathogen. And proteins selected from the group consisting of antigenic proteins.
  • a pathogen protein for example, a protein derived from an intracellular pathogen such as protozoan or Mycobacterium tuberculosis
  • proteins selected from the group consisting of antigenic proteins are selected from the group consisting of antigenic proteins.
  • the antigenic protein has remarkable properties as an epitope.
  • this antigen protein is not necessarily required to be epitope when present in its natural state. This is because any protein can be used as long as it has antigenicity when the antigen protein is actually expressed on a cell as a protein.
  • the antigenic protein used in the invention is MPB51, MDP1, Ag85A, Ag85B, HSP65, Mtb72 from M. tuberculosis.
  • f gene MSP-1 gene derived from malaria parasite, SAG-1 gene derived from Toxoplasma gondii, TSA gene derived from Trypanosoma cruzi, HIV-related gene, cytomegalovirus-related gene, or a fragment or variant thereof And the like, and specifically, for example, are described in the following table.
  • Such sequences can vary depending on the host to be administered. In such cases, the host intended for administration should be tested for HLA and other types in advance. May be advantageous. This is because a specific sequence can be selected.
  • the antigenic peptide of the present invention is advantageously, but not limited to, a full-length gene of a target antigenic protein, and any sequence can be used as long as it exhibits antigenicity.
  • Such full-length antigen genes for example, MPB51, MDP1, Ag85A, Ag85B, HSP65, Mtb72f gene derived from M. tuberculosis, MSP-1 gene derived from malaria parasite, SAG-1 derived from Toxoplasma gondii
  • Immunization with genes TSA gene from Trypanosoma cruzi, HIV-related genes, cytomegalovirus-related genes
  • ubiquitin-fused antigens are rapidly degraded by the ubiquitin-proteasome pathway, and include a variety of peptides containing T cell target sequences (also called CTL epitopes) that can be presented to many types of MHC class I. Produces effective production. In other words, this type of vaccination has multiple types of MCH Class I Available to patients.
  • the incidence of side effects is lower compared to vaccines that use vector viruses or heterologous peptide genes.
  • the latter type of vaccine causes certain side effects.
  • This side effect can be mediated, for example, by antibody neutralization, anaphylaxis mediated by allophilic antibodies, and CD4 + T cells that recognize foreign / heterologous antigens or vector virus antigens (including vaccines).
  • a systemic inflammatory disease is lower compared to vaccines that use vector viruses or heterologous peptide genes.
  • immune activators eg, complete Freund's adjuvant or IL-12 site-in
  • IL-12 site-in immune activators
  • DNA vaccines can be designed depending on the disease, the type of pathogen, and may be relevant for most types of disease.
  • an antigen gene having a T cell target sequence or an antigen protein resulting from an intracellular parasitic pathogen is a naturally occurring antigen gene and has a full-length sequence or a sequence close thereto (for example, , 80% or more of the whole, 90% or more).
  • the ubiquitin used in the present invention may be any ubiquitin. Because ubiquitin is widely conserved across species, sequences of different species of ubiquitin can also be used. Examples of such sequences include, for example, SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 18, 20, 22, 26, 28, 30, 32 when represented by nucleic acid sequences.
  • the ubiquitin used in the present invention is advantageously modified to be resistant to cleavage by proteases. By becoming resistant to cleavage by proteases, the efficiency of transport to the proteasome increases. Modification to have such resistance can be performed using a method known in the art. Modifications can be made using molecular biology or genetic engineering techniques.In the actual screening, the ability to confirm resistance to proteases by biochemical techniques or the proteasome The effect can be confirmed by observing the efficiency of transportation to the destination.
  • Examples of such resistance alterations include, but are not limited to, substitution of an amino acid other than G1y at the C-terminal G1y of ubiquitin and addition of an amino acid to the C-terminal G1y. Not done.
  • modification or addition of amino acids to the C-terminal Gly degrades more rapidly than unfused proteins in a manner dependent on the ubiquitin-proteasome system.
  • Gly at the C-terminus of ubiquitin is advantageously substituted with Ala, but is not limited thereto, and any natural amino acid to be substituted or added may be used. It should be understood that similar effects are expected with amino acids.
  • ubiquitin used in the genetic vaccine of the present invention has the sequence shown in SEQ ID NO: 22.
  • the nucleic acid sequence encoding ubiquitin and the nucleic acid sequence encoding the antigen protein are linked without any intervening sequence.
  • the absence of an intervening sequence has the effect of promoting polyubiquitination and increasing the efficiency of proteasome processing.
  • the nucleic acid sequence encoding ubiquitin and the nucleic acid sequence encoding the antigen protein are linked without any intervening sequence, and the C-terminal G1y of ubiquitin is substituted with an amino acid other than Gly.
  • the present invention has revealed that a ubiquitin-containing product in such a manner is efficiently polyubiquitinated, and processing by the proteasome is efficiently induced.
  • the sequence encoding ubiquitin is positioned at the N-terminus of ubiquitin such that the antigen protein is fused.
  • Such an arrangement promotes polyubiquitination and increases the efficiency of processing by the proteasome.
  • it has been considered preferable to interpose an intervening sequence between them (W098 / 45444, etc.), but in the present invention, conversely, it is better to fuse without intervening and achieve the desired effect. It turned out to be convenient.
  • the sequence encoding ubiquitin is arranged such that the antigen protein is fused to the N-terminus of ubiquitin, and Gy at the C-terminus of ubiquitin is G It is replaced by an amino acid other than 1 y. This is because such a mode of ubiquitin-containing product is even more efficiently polyubiquitinated, and processing by the proteasome is efficiently induced.
  • the nucleic acid sequence encoding ubiquitin is arranged such that it is fused in a manner that does not remove ubiquitin.
  • Representative examples of such modes include, but are not limited to, substituting Gly at the C-terminus of ubiquitin with a sequence other than Gly or adding an amino acid to Gly. Since Gly at the C-terminus of ubiquitin plays a role in the interaction with the target sequence, the same effect can be achieved by modifying the interaction to inhibit the interaction. Such inhibition includes, but is not limited to, adding a sequence of entirely different nature, or adding a bulky amino acid sequence.
  • the genetic vaccine is advantageously, but not limited to, naked DNA (and thus does not use a viral vector). Therefore, the gene vaccine of the present invention can be delivered using any vector.
  • the precursor cells in order to efficiently induce CD8 + -positive killer T cells specific for an antigen containing a T cell target sequence (eg, an antigen derived from an intracellular parasitic pathogen), the precursor cells must be located on APCs. Force required to recognize and activate an antigen containing a T cell target sequence bound to an MHC class I molecule Usually, the level of presentation of pathogen-derived antigens on APC taras I molecules is low.
  • the present invention provides a method for introducing a DNApactin to efficiently express an antigen containing a T cell target sequence on an APC class I molecule, and further comprising the step of transferring an antigen gene containing the T cell target sequence to a ubiquitin gene. It is possible to carry an antigen containing a T cell target sequence to the proteasome system by providing a gene vaccine containing the same (preferably, both are fused).
  • an antigen protein derived from an intracellular parasitic pathogen or an antigen containing a T cell target sequence is used (preferably, fusion) with a ubiquitin gene, which is a Tag molecule, to a proteasome to thereby provide a T cell target distribution.
  • a ubiquitin gene which is a Tag molecule
  • Induces potent anti-pathogens eg, anti-intracellular parasitic pathogens mainly based on antigen-specific killer T cells that contain rows.
  • the antigen peptide is used to activate CD8 + killer T cells specifically for antigens of pathogens including those derived from intracellular parasitic pathogens such as malaria antigens, toxoplasma antigens, and Mycobacterium tuberculosis-derived antigens. It is essential that antigen presenting cells present MHC class I molecules. For this purpose, it is premised that intracellular parasitic pathogens such as malaria antigen, toxoplasma antigen, and antigens derived from Mycobacterium tuberculosis are treated with proteasome which is an intracellular enzyme.
  • the gene encoding ubiquitin which is a tag molecule leading to the proteasome.
  • the antigens of intracellular parasitic pathogens such as malaria antigens, toxoplasma antigens, and M. tuberculosis antigens, are introduced into the cytoplasm by combining the gene with the antigen gene containing the sequence and introducing the binding gene directly into the cytoplasm with a gene gun.
  • a protein complex preferably a fusion
  • this operation allows intracellular parasitic pathogens such as malaria antigens, toxoplasma antigens, and M. tuberculosis antigens. It is possible to induce a strong anti-pathogen immunity mainly composed of CD8 + killer T cells specific for an antigen containing a T cell target sequence such as an antigen.
  • cytokine gene therapy method using a gene gun
  • the target protein is supplied in a small amount and continuously for a certain period of time, so that it is highly safe, does not require daily administration, and is effective.
  • the expressed protein differs from the recombinant protein synthesized in Escherichia coli in that preferable post-translational modification is performed in the host to which the protein is administered, so that sugar chains are added to the expressed protein, and the original protein is expressed. Structure is preserved.
  • the genetic vaccine of the invention is for treating or preventing a disease or disorder associated with a T cell target sequence, particularly an intracellular parasitic pathogen.
  • diseases or disorders include, for example, parasites (such as intracellular parasitic pathogens such as malaria, tuberculosis, toxoplasmosis, trypanosomiasis, acquired immunodeficiency syndrome, cytomegalovirus disease, cladiemia, rickettsiosis , Leishmaniasis, Ebola hemorrhagic fever, Trypanosoma cruzi infection, Chagas disease, Japanese encephalitis, influenza, measles and dengue virus, polio, a herpes virus infection, severe acute respiratory syndrome (SARS) ) And hepatitis C), viral diseases or disorders, bacterial diseases or Include, but are not limited to, disorders, neoplasms (such as cancer).
  • parasites such as intracellular parasitic pathogens such as malaria, tuberculosis, toxoplasmo
  • the present invention provides a genetic vaccine for treating a disease caused by a cell-regulated pathogen including a protozoan and the like.
  • a genetic vaccine for treating a disease caused by a cell-regulated pathogen including a protozoan and the like.
  • a phenomenon of efficient degradation of cancer cells by the proteasome was unexpectedly confirmed.
  • the present invention since it has become possible to prevent toxoplasmosis, which is an important cause of death and seriousness in AIDS-related diseases, the present invention is practically also effective as a preventive drug for AIDS.
  • the genetic vaccine of the invention confers protective immunity against diseases caused by antigenic proteins.
  • the genetic vaccines of the present invention provide protection against such diseases. Whether to provide protective immunity can be determined by observing the production of neutralizing antibodies and the like.
  • the genetic vaccine of the present invention confers a therapeutic effect on diseases caused by antigenic proteins.
  • the genetic vaccine of the invention induces CD8 + T cells.
  • the T cell target sequence used in the present invention can effect the degradation of target cells or pathogens by CTL via this stimulation of CD8 + cells.
  • the genetic vaccine of the invention is administered by needleless injection.
  • Needleless injection is commonly used to introduce nucleic acid form factors into the body, and the present invention provides such painless and, therefore, little pain to the administered patient. Therefore, it is possible to provide an effect of improving the efficiency and providing a therapeutic effect or a preventive effect.
  • any technique known in the art can be used. Illustrative of such techniques are as performed elsewhere herein.
  • the genetic vaccine of the present invention further comprises particles.
  • the gene vaccine of the present invention is in a form more suitable for a gene gun by including particles as described above. More preferably, the particles used here are advantageously, but not limited to, gold particles. Gold particles are commonly used particles because of their high transformation efficiency. .
  • the present invention provides a method for producing the genetic vaccine or the genetic construct of the present invention, comprising: a nucleic acid sequence encoding ubiquitin; and a nucleic acid sequence encoding an antigen protein including a T cell target sequence. Operably linking the two.
  • the ubiquitin T cell target sequence or the antigen derived from the intracellular parasitic pathogen is as described above. Operably linking such two sequences can also be performed using techniques well known in the art.
  • the present invention provides a method for preventing or treating a disease caused by an antigen protein containing a T cell target sequence or an intracellular parasitic pathogen.
  • the method comprises the steps of: A) administering to a patient a genetic vaccine comprising a nucleic acid sequence encoding ubiquitin and a nucleic acid sequence encoding an antigen protein comprising a T cell target sequence or an antigen protein derived from an intracellular parasitic pathogen; Is included.
  • a genetic vaccine comprising a nucleic acid sequence encoding ubiquitin and a nucleic acid sequence encoding an antigen protein comprising a T cell target sequence or an antigen protein derived from an intracellular parasitic pathogen; Is included.
  • any form as described above can be used.
  • the genetic vaccine is administered by a gene gun (genegun).
  • a gene gun gene gun
  • Methods for performing a gene gun are known in the art and can be performed using any commercially available device.
  • the gene vaccine of the invention is also administered by needleless injection. Needleless injections are also known in the art and are commercially available Can be carried out using the Depeise.
  • the disease or disorder targeted by the treatment method and the prevention method of the present invention may be any disease or disorder as long as it is a disease or disorder related to a T cell target sequence or a cell-parasitic pathogen.
  • Bacteria, fungi, parasites protozoa, protozoa, leishmania, toxoplasma, etc.
  • neoplasms eg, cancer
  • protozoa causes include, but are not limited to.
  • the gene vaccine of the present invention is administered before, during or after the onset of the disease. If administered before the onset of the disease, in principle, the invention will be used for prophylactic purposes and after the onset of the disease will be, but not limited to, for therapeutic purposes.
  • one disease or disorder may be a treatment target, or a plurality of diseases or disorders may be a treatment target.
  • the gene vaccine of the present invention is administered without using a retrovirus.
  • a retrovirus By not using a retrovirus, side effects unique to the retrovirus (such as the occurrence of viral diseases) can be suppressed. This can be said to be one remarkable effect of the present invention, considering the situation where retroviruses were almost essential in conventional treatment methods.
  • the present invention provides a composition comprising a nucleic acid sequence encoding ubiquitin and a nucleic acid sequence encoding an antigen protein containing a T cell target sequence or an antigen protein derived from an intracellular parasitic pathogen as a gene vaccine.
  • the present invention includes a nucleic acid sequence encoding ubiquitin and a nucleic acid sequence encoding an antigen protein containing a T cell target sequence or an antigen protein derived from an intracellular pathogen for producing a genetic vaccine.
  • the use of the composition is provided.
  • the detailed description of ubiquitin, antigen protein, gene vaccine, etc. is as described above, and such a preferred embodiment is implemented by appropriately selecting any preferred embodiment according to actual use. can do.
  • Example 1
  • Plasmids encoding the SAG-1 gene from Toxoplasma gondii were constructed as follows: The SAG-1 gene was replaced with a sense primer (5, -ATGTT CACTCTCAAGTGCCCTAAAACA-3 '(SEQ ID NO: 59)) and an antisense primer ( 5, and amplified by PCR using -TTACTCCAGTTTCACGGTAC AGTGATGC-3 '(SEQ ID NO: 60). The PCR product of the SAG-1 gene was inserted into the pCDNA vector, pJW4303 vector. These plasmids were named pcDNA-SAGl (basic type) and pJW4304-SAG1 (secreted type), respectively (FIG. 1).
  • Escherichia coli DH5a was transformed with the expression plasmid containing the UB-SAG-1 gene, and cultured in a LB medium (50 Om1) at 37 ° C in a constant temperature bath with shaking ( ⁇ 16 hours).
  • an anion exchange resin column for nucleic acid purification (manufactured by MARL I GEN BI ⁇ SCIENCE I NC.) Equilibrated with 30 • ml of 60 OmM NaC 1 10 OmM sodium acetate (pH 5.0) was used. The supernatant was centrifuged at 12000 rpm for 10 minutes at room temperature, and the supernatant was taken as 1 oad, and the column was washed with 800 mM NaC1 / 100 mM potassium acetate solution. Elution was carried out with 10 OmMT ris-HC 1 (pH 8.5).
  • Cytotoxic T cell activity was calculated by measuring the amount of fragmented DNA in target cells using the JAM test.
  • target cells using a mouse R enca cells (H- 2 d) of SAG1 stable transformants, 10% containing FCS RPMI-1640 in 2/3 H- thymidine iC i / m 1 over ⁇ Labeled.
  • the target cells were harvested, washed and suspended in complete medium at a concentration of 2 X 1 0 5 cells Zm 1. This solution (100 ⁇ l) was then added to each well of a 96-well U-bottom plate.
  • FIGS. Figure 4 shows killer T cells with DNA vaccine 6 is a graph showing the activation of.
  • UB-SAG1 immunized mice (3 mice per group) were used and the average value was shown.
  • G1 and pcDNUB-SAG1 were transfected and cultured for ⁇ ( ⁇ 16 hours) for constant expression. Then, the proteasome inhibitor MG-132 was added to 0, 0.
  • the pcDNA-SAG1 and pcDNUBSAG1 prepared in Example 2 were transfected, and at the same time, the proteasome inhibitor epoxomycin ( ⁇ ⁇ ⁇ ) was added.
  • the cells were cultured at 37 ° C. in the presence of 5% CO 2 and dissolved after 24 hours.
  • Western plot was performed using an anti-SAG-1 antibody, and quantified by densitometric analysis.
  • HSP 90 was used as an internal control and indicated as a relative value.
  • mice were shot with pcDNA, pcDNA—SAG1, JW4304—SAG1 and pcDNAUB—SAG1 four times every two weeks and three times using a gene gun. Two weeks after the last vaccination, a lethal dose of T. gondii (RH) strain was infected (70 parasites / mouse).
  • RH T. gondii
  • Fig. 8 shows the results. More than 80% of the mice transfected with pcDNAUB-SAG1 survived 30 days after infection. p JW4304—Approximately 28% survived in mice transfected with SAG1. On the other hand, p cDNA, pc DNA—SAG In the group of mice into which 1 was introduced, all mice died 10 days after infection. Therefore, remarkable infection resistance was confirmed in mice vaccinated with pcDNAUB-SAGl. This demonstrated the effectiveness of the Ub-conjugated vaccine.
  • mice were immunized four times with pcDNA, pcDNA-SAG1 p JW4304-SAG1 and pcDNAUB—SAGl, and spleen cells were collected two weeks after the final immunization to produce SAG-1 specific cytokines.
  • the performance was analyzed using the ELISA method. The procedure is briefly described below.
  • BALBZc mice were shot with pcDNA, pcDNA-SAG1, pJW4304-SAG1 and pcDNAUB-SAG1 four times every two weeks and three times each using a gene gun.
  • splenocytes were harvested and sorted by MACS into CD8 + and CD4 + cells.
  • APC treated with infectious radiation was used as a stimulus
  • APC (3 OGy) treated with uninfected radiation was used as a control.
  • the interferon- ⁇ in the supernatant was detected by ELISA.
  • IFN- ⁇ mRN ⁇ in CD8 + cells was detected by the real-time PCR method. Relative evaluation was performed using b-actin as an internal control.
  • Figure 10 shows the results of the ELISA method.
  • pcDNAUB-SAGI produced nearly twice as much IFN- ⁇ as compared to the other groups.
  • Figure 11 shows the results of the real-time PCR method. IFN- ⁇ mRNA was expressed significantly higher in the ubiquitinated vector-administered group than in the other groups. These results indicated that CD8 + cells were activated.
  • T. gondii-specific IFN- ⁇ -producing cells are determined by ELIS POT.
  • Six to eight-week-old BALB / c mice are immunized with pcDNA, pcDNA-SAG1, pJW4304-SAG1 and pcDNAUB-SAG1 (2 / g each).
  • 96 Ueno Les plate Movable ltiscreen IPF iltrationlate MA I PS45 10, Mi llipore
  • mice were immunized with pcDNAUB-SAG1.
  • Anti-CD4 mAb (clone GK1.5), anti-CD8 mAb (clone 53--6.72), rat IgG (IgG binding column from normal rat serum) to deplete the corresponding T cell subset )
  • pcDNA was injected intraperitoneally into mice immunized with pcDN AUB-SAG1. They were then infected with T. gondii (day 0). Death of each T cell subset was confirmed by flow cytometry; more than 98% of the appropriate cell subset was killed.
  • mice treated with the anti-CD8 antibody pcDNA died approximately 10-12 days after infection (FIG. 13). About 18% of mice treated with anti-CD4 antibody survived 30 days after infection. In contrast, more than 80% of mice treated with rat IgG survived 30 days after infection.
  • a vaccine containing a gene in which a malaria parasite-derived MS P_1 gene and a ubiquitin gene were linked was prepared.
  • a plasmid encoding the MS P-1 gene from Malaria parasite was constructed as follows: The MS P-1 gene was replaced with a sense primer (5, -AATTGCTA
  • the gene encoding MS P-1 was further amplified by PCR using the same primer as a sense primer (5, one AATTGCT No. 62)) and an antisense primer (5, -GGCAGCCTTAAG T CCCATAAAGCTGGAAGA-3 '(SEQ ID NO: 63)). Amplified.
  • a gene encoding a mutant ubiquitin in which the Gly residue at the C-terminus was replaced with Ala was amplified by PCR using genomic DNA obtained from the liver of BALBZc mice. This mutant ubiquitin cDNA is ligated in-frame to the 5, side of the gene encoding MSP-1 and! ) Inserted into cDNA.
  • This plasmid was designated as pU (ubiquitin fusion type).
  • plasmids for the TPA leader sequence sequence or CTLA-4 were constructed. The following is a brief summary.
  • the TPA leader sequence sequence fusion type (secretion type: Tp) is integrated with the leader sequence of the mouse tissue plasminogen activator (SEQ ID NO: 64) in the pcDNA vector, and MSP is in-frame downstream of the leader sequence. Constructed by combining 1.
  • sense 5,- AG-3 As a primer for amplification of TP A 1 eader gene, sense 5,- AG-3, (SEQ ID NO: 66) and antisense 5, -AATGCCTCTAG ACGCACAAGAGCGTGAGCT-3, (SEQ ID NO: 67) were used.
  • a TLA-4 fusion type (secretion type: pC) was constructed by incorporating the extracellular domain of mouse CTLA-4 (CD152) into a pcDNA vector and binding MSP1 in-frame downstream thereof.
  • the CTLA-4 width has a
  • the expression plasmid containing the UB-MSP-1 gene was transformed into Escherichia coli DH5 ⁇ , and shaking culture was performed overnight ( ⁇ : 6 hours in a 37 ° C constant temperature bath in 500 ml of L : medium). went.
  • the pM (2 ⁇ g) prepared in Example 12 is shot three times at 578/6 mice. 48 Eight hours later, remove lymph nodes and perform RT-PCR. The expression of MS P_1 mRNA in the removed lymph nodes is detected. As a result, MSP-1 mRNA is detected, and MSP-1 is expressed in the local lymph node.
  • MSP-1 polyubiquitinated MSP-1 protein is synthesized in the cells.
  • Macaque kidney derived cells COS 7 cells contain pcDNA, pU, pT, pC and! )
  • Target cells are cells in which MSP-1 is constantly expressed in EL-4 cells derived from C57BL / 6 mice. 6-8 week old female. 5 78 Mr / / 6 Mausu, respectively is 2 / g of pc DNA (control), pM (basic type), p T (secreted), C (Secret type) and pU (Ubiquitin fusion type) are shot three times using a gene gun. Mouse splenocytes after immunization are used as effectors. Measure killer T cell activity by 3 H-thymidine labeling. The procedure is described below.
  • Cytotoxic T cell activity is calculated by measuring the amount of fragmented DNA in target cells using the JAM test.
  • target cells MSP-1 stable transformants of mouse EL-4 cells were used, and 2 of R'1 ⁇ 1-1640 containing 10% FCS. Label with 1/1111 3 H-thymidine.
  • Immunized splenocytes stimulated in vivo for 5 days with irradiated EL-4 cell stable transformants as effector cells were collected, washed, and suspended at 5 ⁇ 10 6 cells / ml in complete medium I do.
  • This effector-cell suspension (100 ⁇ l) ′ is added to each well of a 96-well U-bottom plate at various effector cell: target cell ratios.
  • the plate is centrifuged at 2000 g for 5 minutes and then incubated at 37 ° C. in 5% CO 2 for 4.5 hours. After incubation, the plates are collected on a 96-well harvester (Packard) onto a glassware finlet (Packard Instruments BV Chemical Operations, Groningen, the Netherlands), and The radioactivity captured on the filter is measured using a counter (Packard).
  • the percentage of DNA fragmentation is calculated by the following formula:
  • Example 14 it is examined using a proteasome inhibitor that the killer cell activating effect specific to MSP-1 is via a ubiquitin proteasome system. (Proteasome inhibitor MG-132)
  • PM and pU prepared in Example 11 are transfected into COS 7 cells, and cultured for a period of time (up to 16 hours) to allow steady expression. Then, add the proteasome inhibitor MG-132 to the culture medium at 0, 0.625, 1.25, 2.5, 5.0, 10.0 ⁇ at 37 ° C in the presence of 5% CO 2. Incubate for an additional 24-36 hours. After culturing, the cells are lysed, and the lysate is analyzed by Western blotting using an anti-MSP-1 antibody. As a result, it is confirmed that the amount of MSP1 detected by the anti-MSP1 antibody increases depending on the concentration of the proteasome inhibitor MG-132. (Proposome inhibitor epoxomycin)
  • Example 15 Transfect the pM and pU prepared in Example 11 into COS 7 cells, and simultaneously add the proteasome inhibitor epoxomycin (100 nM). Incubate at 37 ° C in the presence of 5% CO 2 and dissolve after 24 hours. Western plot using anti-MSP-1 antibody and quantify by densitometric analysis. Use HSP 90 as internal control and display as relative value. As a result, in pU, the addition of the proteasome inhibitor epoxomycin increases the amount of MSP-1. On the other hand, in pM, the amount of MSP-1 hardly changes in the presence or absence of epoxomycin.
  • epoxomycin 100 nM
  • 6- to 8-week-old female 057 6 // 6 mice (6 / group), 2 / ig pcDNA (control), pM, pT, pU and pC, 4 times every 2 weeks Make three shots each with a gun. Two weeks after the last vaccination, the Py17XL strain was infected (1 ⁇ 10 5 infected erythrocytes / mouse). At 5, 7, 10, and 12 days after the infection, the erythrocyte infection rate of each mouse was examined.
  • mice In the non-immunized group and!) CDNA group, 4 out of 6 mice died by 12 days later. Erythrocyte infection rate was lowest in mice immunized with pU ( Figure 14 one A to D) 0
  • mice 6-8 week old female.
  • P M the Pyl7XL strain was infected (1 ⁇ 10 5 infected erythrocytes / mouse).
  • untreated mice were infected with the bCSP strain (1 ⁇ 10 5 infected erythrocytes / mouse).
  • the survival rate of each group of mice was examined from 5 to 13 days after infection.
  • spleen cells were collected two weeks after the final immunization, and ELISA was performed for the ability to produce MSP-1-specific cytokines. Perform analysis using The procedure is briefly described below.
  • CD8 + T cells are sorted by MACS column.
  • the cells (5 XI 0 6 cells / Ueru), together with infectious radiation treated A PC 72 hours co-culture with RPMI 16 40 medium, recovering the culture supernatant.
  • 5 ⁇ 10 6 cells Zm1 lymphocytes are cultured for 3 days using recombinant-MSP1, and the supernatant is collected.
  • IFN- ⁇ a 96-well plate is overcoated with anti-cytokine capture Ab as the primary antibody. Wash plates with PBS containing 0.05% Tween-20, block with PBS containing 10% FCS, and add dilutions or standards of the supernatant.
  • a dilution of the culture supernatant is incubated overnight at 4 ° C, and after washing, the gel is incubated with the biotin-conjugated anti-cytokine detection mAb. After incubation for 2 hours, the plate is washed and streptavidin-conjugated al-rich phosphatase is added. After color development, measure the absorbance at 415 nm. Estimate the amount of cytokine in each supernatant from the standard curve.
  • the Ab pairs used are as follows (Supplementary antibody biotinylation detection Ab): IFN_y, R4-6 A2 / XMG1.2.
  • BALB / c mice are shot with pcDNA, pM, pT, pC and pU four times every two weeks and three times with a gene gun.
  • Splenocytes were harvested from the last vaccination after 2 weeks, divided into CD8 + cells and CD 4 + cells by MACS column You.
  • APC treated with infectious radiation (3 OGy) was used as a stimulus, and APC treated with uninfected radiation was used as a control. After culturing for 72 hours, the interferon y in the supernatant is detected by ELISA.
  • IFN- ⁇ mRNA in CD8 + cells is detected by real-time PCR. Relative evaluation using b-actin as an internal control. It is confirmed that IFN-Y mRNA is significantly higher expressed in the ubiquitinated vector administration group than in the other groups.
  • B ALBZc mice were immunized with pc DNA, pU, pT, pC and ⁇ , and after 2 immunizations and 4 immunizations were collected over time, ELISA was performed to determine the MSP-1 specific antibody titer in the serum. Analyze using the method. As a result, a high MSP-1-specific antibody titer could be induced in the serum after the fourth immunization, indicating that the immunization normally induced an immune response.
  • 6- to 8-week-old female C57BL / 6 mice receive 2 ⁇ g each of pcDNA, pU, pT, pC, and! M 4 times every 2 weeks and 3 times each using a gene gun. Shot and immunize.
  • 96 ⁇ El plate Mo I ti S creen IPF iltrationplate MA I PS 45 10, Mi llipore
  • a 50 / in 0. 1M NaHC_ ⁇ 3 zl
  • R4- 6A2 P a rMi ngen, S an Diego, CA
  • mice are immunized with pU.
  • the cells are infected with Pb CSP (day 0). Depletion of each T cell subset is confirmed by flow cytometry; more than 98% of the appropriate cell subset is depleted.
  • the protective ability provided by pU was canceled.
  • a vaccine containing a gene in which a gene such as Ag85B or HSP65 derived from Mycobacterium tuberculosis and a ubiquitin gene were linked was prepared.
  • Escherichia coli DH5 ⁇ is transformed with an expression plasmid containing the UB-Ag85B gene or the UB-HSP65 gene, etc., and placed in a constant temperature bath at 37 ° C in 500 ml of L ⁇ medium ( ⁇ : L 6 hours) Shaking culture was performed.
  • the neutralized solution was then applied to an anion exchange resin column for nucleic acid purification (MARL I GEN BIOS CIENCE INC.) Equilibrated with 30 OmM NaC 1/10 OmM sodium acetate (pH 5.0). After centrifugation at 12,000 rpm for 10 minutes at room temperature, the supernatant was centrifuged for 1 oad, and the column was washed with 800 mM NaC1 / 100 mM potassium acetate solution. Elution at 1 (pH 8.5). It is shown to be as effective as Toxoplasma and Malaria. As described above, the present invention has been exemplified using the preferred embodiments and examples of the present invention.
  • the present invention is useful in providing effective prevention, treatment methods, techniques, and medicaments for diseases that have been difficult to treat conventionally (particularly, diseases caused by intracellular parasitic pathogens including protozoa such as malaria and toxoplasma). Has the property.

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Abstract

La présente invention concerne un vaccin à ADN fusionné qui est préparé par construction d'un gène fusionné (ADN fusionné) d'un gène provenant d'un parasite protozoaire intracellulaire (par exemple du bacille tubercule, du parasite de la Malaria, du toxoplasme, etc.) avec un gène d'ubiquitine. L'utilisation de ce vaccin génique chez un hôte porteur de l'infection permet le traitement d'un antigène pathogène ubiquitiné (par ex. un produit génique) avec un protéasome et ainsi d'induire fortement les cellules CD8+T spécifiques de l'agent pathogène. L'invention a pour objet un vaccin génique qui se caractérise en ce qu'il contient un gène composé d'un gène provenant d'un parasite protozoaire intracellulaire et d'un gène d'ubiquitine lié à celui-ci.
PCT/JP2004/000975 2003-01-31 2004-01-30 Vaccin genique contre les parasites intracellulaires WO2004067040A1 (fr)

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WO2024064886A1 (fr) * 2022-09-23 2024-03-28 BioNTech SE Utilisation de dégrons n-terminaux pour améliorer l'immunogénicité d'un vaccin à lymphocytes t à arn
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WO2021076570A3 (fr) * 2019-10-17 2021-05-27 Baylor College Of Medicine Antigènes de vaccin de la maladie de chagas à stabilité améliorée et agrégation réduite
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