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• • C—CHEMISTRY; METALLURGY
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  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • 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
  • A61K2039/525—Virus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/20011—Papillomaviridae
  • C12N2710/20023—Virus like particles [VLP]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/20011—Papillomaviridae
  • C12N2710/20061—Methods of inactivation or attenuation

Definitions

• the invention relates to a papilloma pseudovirus capable of generating an immune response through an oral route and a preparation method thereof.
• This pseudovirus can be used in vaccines to prevent and treat mucosal pathogens or mucosal tumors.
• This pseudovirus can also be used in gene therapy.
• pathogenic microorganisms and human immune function defects are the root cause of many diseases.
• the human body is often attacked by pathogenic microorganisms and the destruction of hidden tumors inside the human body. Therefore, the human body also needs a kind of body resistance or immunity to resist the invasion and infection of pathogenic microorganisms and diseases inside the body.
• the current clinically used vaccine is injected subcutaneously or intramuscularly, which results in a specific immune response, leading to resistance to the disease and preventing the disease from occurring in the future.
• these vaccines are often injected subcutaneously or intramuscularly into the human body. After the vaccine is injected, it can only produce a systemic immune response, not a mucosal immune response. Such vaccines often fail to prevent and treat mucosal infections.
• many pathogenic bacteria are transmitted by the mucous membranes, such as the Aids virus.
• An object of the present invention is to develop a virus-like pseudovirus that does not cause disease.
• a gene or DNA vaccine that is used to treat and prevent disease is injected into the papilloma virus shell, orally or through other methods.
• the mucosa and systemic lymphoid tissues at the entrance of the mucosa induce the immune response of the human body and play a role in preventing and treating diseases.
• the papilloma pseudovirus in the present invention is a human papilloma virus-like particle (HPV-VLP) or a bovine (or other animal) papilloma virus-like particle (BPV-VLP) is dissociated and incorporated into plasmids (plasmids). Combination of adult or bovine (or other animal) papilloma pseudoviruses.
• HPV-VLP human papilloma virus-like particle
• BPV-VLP bovine (or other animal) papilloma virus-like particle
• a normal virus is made up of a shell and the DNA inside the shell.
• the papilloma pseudovirus, which is described in the invention has only a shell of the virus, but no virus DNA in the shell. If a DNA vaccine is placed in the shell of the virus, it can be delivered into the body by mouth.
• the DNA vaccine described in the present invention is only a piece of DNA fragment, not viral DNA. If this DAN fragment is injected subcutaneously or intramuscularly into a human body, a systemic immune response can be induced. Therefore, the viral shell containing the DNA vaccine is described in the present invention. Fake virus. This pseudovirus is prepared as follows ⁇
• Dissociation buffer ethylene glycol bis (e-aminoethyl ether) tetraacetic acid (EGTA) 20mM, dithiothreitol (DTT) 40mM, sodium chloride (NaCl) 300mM, tris (hydroxymethyl) ) Carbamate hydrochloride (Tris—HC1) (PH 8.0) 100 mM;
• stop buffer calcium chloride (CaCb) 25mM, dimethyl sulfoxide (DMSO) 20% (total volume of stop buffer);
• the dissociation buffer was: ethylene glycol di (em-based ether) tetraacetic acid (EGTA) 20 mM, dithiothreo Sugar alcohol (DTT) 40 mM, sodium chloride (NaCl) 300 mM, tris (hydroxymethyl) 'carbamidine hydrochloride (Tris-HC1) (PH 8.0) 100 mM, then add 1/10 volume of 0.5 g / 4l PCI-GLP-LCMV plasmid, and then gradually add an equal volume of stop buffer, the stop buffer is: calcium chloride (CaCl 2 ) 25mM, dimethyl sulfoxide (DMSO) 20% by volume, mix well The solution was left at 4 ° C overnight.
• EGTA ethylene glycol di (em-based ether) tetraacetic acid
• DTT dithiothreo Sugar alcohol
• Tris-HC1 tris (hydroxymethyl) 'carbamidine hydrochloride (Tris-HC1) (PH
• pseudovirus vaccine is more effective than the gene vaccine in inducing cellular immune response.
• the shell of bovine papilloma virus and dissociation buffer were mixed in a volume of 1: 1 and cultured for 60 minutes at room temperature.
• the dissociation buffer was: EGTA 20mM, DTT 40mM, NaCl 300mM, tris (hydroxymethyl) aminomethane salt.
• Tris—HC1) (PH 8.0) .00mM then add 1/10 volume of 1.0 ⁇ ⁇ / ⁇ 1 plasmid carrying the green fluorescent protein GLP gene, and then gradually add equal volume of stop buffer to stop buffer
• Stop buffer For: CaCh25mM, DMSO 20% by volume, put the mixed mixture at 4'C overnight. Use this fake disease.
• the mice were given orally (using a gastric tube) and then tested for GLP expression.
• GLP cannot be detected if the plasmid is taken orally.
• a pseudovirus containing the HPV 16E7 plasmid prepared by the same method as in Example 1 was orally used to immunize animals, and mucosal and systemic E7-specific cytotoxic cells were induced.
• the plasmid itself was used directly, and no immunity was induced after oral administration. reaction. This pseudovirus can therefore be used to induce mucosal and systemic immune responses.
• HPV and BPV pseudoviruses containing the HPV16E7 plasmid were prepared in the same way as in Examples 1 and 2.
• the mice were first orally immunized with the HPV pseudovirus and then infected with the BPV pseudovirus.
• the HPV pseudovirus was found to prevent BPV. Pseudovirus infection. This pseudovirus can be used to provide immune protection.
• Example 1 The method of Example 1 or Example 2 was used to prepare the interleukin-II-containing pseudovirus, and the intestinal mucosal lymphatic tissue and systemic lymphoid tissue were orally administered to improve the efficiency of mucosal or systemic immune induction.
• This pseudovirus is not pathogenic and can be used for gene therapy. That is, the gene plasmid to be expressed is incorporated into virus-like particles. This pseudovirus can orally deliver this gene into the intestinal mucosa and systemic lymphoid tissues, as well as mucosal epithelial tissues. More importantly, the pseudovirus can be used as a vaccine, and a plasmid expressing the antigen gene is incorporated to synthesize the pseudovirus, which can produce a protective immune response when taken orally. This differs from other vaccines in that many vaccines can only be injected subcutaneously or intramuscularly and cannot be taken orally. It can only produce a systemic immune response but not a mucosal immune response.
• pathogenic bacteria are transmitted through the mucous membranes, only this pseudovirus can produce an effective immune response to prevent or treat mucosal pathogenic infections.
• pathogenic bacteria include bacteria such as Salmonella.
• viruses such as HIV (human immunodeficiency virus).
• HIV human immunodeficiency virus
• the virus can also be used to induce anti-tumor immune responses to treat tumors, especially mucosal tumors, such as colon tumors.
• Pseudoviruses can induce a stronger immune response than genetic vaccines.

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• 2001
  • 2001-05-15 CN CN01118003A patent/CN1114690C/zh not_active Expired - Fee Related
  • 2001-10-19 JP JP2001321828A patent/JP4511090B2/ja not_active Expired - Fee Related
• 2002
  • 2002-03-22 WO PCT/CN2002/000187 patent/WO2002092796A1/zh not_active Ceased
  • 2002-11-14 US US10/294,087 patent/US6878541B2/en not_active Expired - Lifetime
• 2005
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• 2007
  • 2007-04-09 US US11/784,626 patent/US8129144B2/en not_active Expired - Fee Related
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