WO2004082712A1 - Vaccin divalent - Google Patents

Vaccin divalent Download PDF

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Publication number
WO2004082712A1
WO2004082712A1 PCT/CN2004/000232 CN2004000232W WO2004082712A1 WO 2004082712 A1 WO2004082712 A1 WO 2004082712A1 CN 2004000232 W CN2004000232 W CN 2004000232W WO 2004082712 A1 WO2004082712 A1 WO 2004082712A1
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japanese encephalitis
virus
encephalitis virus
recombinant
genome
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PCT/CN2004/000232
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English (en)
Chinese (zh)
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Xiaowu Pang
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Shanghai Tengen Biomedical Co., Ltd.
Tengen Biomedical Co.
Beijing Oriental Tengen Technology Development Co., Ltd.
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Publication of WO2004082712A1 publication Critical patent/WO2004082712A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • 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
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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 invention relates to the field of genetic recombination, and in particular to a recombinant virus vaccine constructed by using Japanese encephalitis virus as a vector. Background technique
  • Japanese encfephalitis and Hepatitis B are multiple infectious diseases in Asia.
  • Japanese encephalitis is caused by Japanese encephalitis virus (JEV) infection.
  • JEV Japanese encephalitis virus
  • the mortality rate is close to 40%, and 10% to 30% of survivors have severe permanent neurological sequelae.
  • the live attenuated JE vaccine is essentially a live attenuated JEV.
  • the selected strain in China is SA14-14_2, which was approved for marketing in China in 1988.
  • the protection period is more than 30 years, and its safety and effectiveness exceed the inactivated vaccine.
  • the immunity cost of live attenuated JE vaccine is much lower than that of inactivated vaccine, and the use of live attenuated vaccine can save 47% of the immune cost than inactivated vaccine (Ding Ding, et al. Cost-ef f ectivess of routine immunization to control Japanese encephalitis in Shanghai, China. Bulletin of the World Health Organization 2003, 81 (5): 334-342).
  • Some Asian countries have started clinical trials of live attenuated JE vaccines, such as Korea and Nepal.
  • the safe and effective live attenuated Japanese encephalitis vaccine shows its promising prospect as a gene therapy carrier.
  • the live attenuated Japanese encephalitis vaccine has gradually gained international recognition in recent years and the complexity of its RNA genome recombination technology To date, there have been no reports of any biological products based on live JE vaccines.
  • Hepatitis B is the most serious infectious disease that is widespread in Asia. At present, Hepatitis B virus (HBV) infection has become a worldwide health problem. About 300 million people worldwide are infected with HBV, and about 1 million people die each year from HBV-related diseases such as cirrhosis or liver cancer. China is a high-incidence area of hepatitis B, with about 100 million HBV infections. The infection rate in some areas can reach 14.29%. About 300,000 people die each year from cirrhosis or liver cancer. About 2.7 million cases of acute hepatitis are reported each year, of which 10 % ⁇ 30% are acute hepatitis B cases.
  • HBV Hepatitis B virus
  • Hepatitis B vaccine is an important means to prevent and control HBV infection.
  • the hepatitis B vaccines in the international market are basically genetically engineered HBV subunit vaccines. Their safety and effectiveness have been fully confirmed, but the production cost is high, and they need to be vaccinated 3 times (that is, 1 time after the first vaccination). Months and 6 months).
  • the effective protection rate of hepatitis B vaccine is 80-95%.
  • the concentration of protective antibodies in the body decreases year by year. About 7 to 50% of immunized people will not detect antibodies in the body after 7 years. Therefore, the hepatitis B vaccine should generally be Repeat immunization once every 5 ⁇ 7 years.
  • hepatitis B vaccines that are being developed internationally include synthetic peptide vaccines, DNA vaccines, and genetically engineered vaccines using Salmonella (53 ⁇ 4 ⁇ / 0 / 1 ⁇ 2 s) as carriers.
  • Salmonella 53 ⁇ 4 ⁇ / 0 / 1 ⁇ 2 s
  • these hepatitis B vaccines also have aspects of immune efficiency and production costs. Unsatisfactory.
  • the invention provides a novel vaccine based on attenuated Japanese encephalitis virus.
  • a recombinant Japanese encephalitis virus is provided.
  • the recombinant Japanese encephalitis virus is composed of Japanese encephalitis virus capsid and recombinant Japanese encephalitis virus genome, and the Japanese encephalitis virus genome is inserted with external Source nucleic acid sequence, and the recombined genome retains self-replicating function.
  • the exogenous nucleic acid contains a nucleic acid sequence encoding an antigen, an antigenic determinant, a cytokine, a growth factor, a polypeptide hormone, an enzyme, a receptor, an antibody, and / or a cancer-related protein.
  • the exogenous nucleic acid sequence contains a nucleic acid sequence of a hepatitis B virus antigen or an epitope.
  • the insertion site of the exogenous nucleic acid is selected from the following group:
  • the JE virus genome is partially deleted from the JE structural protein gene sequence. More preferably, the deleted structural protein gene sequence is selected from the group consisting of: protein C, prM protein, protein E, or a combination thereof.
  • the JE virus is attenuated, such as the strain SA14-14-2.
  • a second aspect of the present invention there is provided the use of the recombinant JE virus according to the present invention, which is used to prepare a therapeutic or preventive vaccine, especially to prepare a bivalent epidemic for the prevention and treatment of JE and hepatitis B .
  • a pharmaceutical composition which comprises the recombinant JE virus according to the present invention and a pharmaceutically acceptable carrier.
  • a method for preparing a recombinant Japanese encephalitis virus comprising the steps:
  • packaging cells are selected from the group:
  • step (a) a cell having a genome integrated with the structural protein gene deleted by the virus; (b) culturing the packaging cells of step (a);
  • the JE virus genome does not lack structural proteins, it can be directly introduced into host cells that can be infected by JE virus to produce recombinant JE virus.
  • host cells that can be infected by JE virus to produce recombinant JE virus.
  • Figure 1 is a schematic diagram of the RNA gene structure of Japanese encephalitis virus.
  • Figure 2 is a flow chart for the conversion of Japanese encephalitis virus RNA into four Japanese encephalitis virus cDNA fragments.
  • Figure 3 is a flowchart of the preparation of a full-length JE virus cDNA clone.
  • Figure 4 is a flow chart for the preparation of a bivalent vaccine with an insertion point between NS2B and NS3.
  • Figure 5 is a flow chart for the preparation of a bivalent vaccine with insertion points between E and NS1.
  • Figure 6 is a flow chart for the preparation of a bivalent vaccine with insertion points between C and prM.
  • Figure 7 is a flow chart of the preparation of a HBV-JE bivalent vaccine containing a JEV subgenome. Detailed ways
  • the invention provides a method for constructing and applying a recombinant Japanese encephalitis virus.
  • An exogenous non-JEV) nucleic acid sequence is inserted into the genome of the attenuated Japanese encephalitis virus, and the insertion of the exogenous nucleic acid sequence does not affect the self-replication function of the genome.
  • the recombinant Japanese encephalitis virus provided by the present invention not only has all the immunological activities of a live attenuated Japanese encephalitis vaccine, but also can express foreign nucleic acid in a user (patient, susceptible population, etc.) to produce a foreign polypeptide.
  • This recombinant JE virus can trigger the user's body's immune response to foreign peptides, and then achieve the role of preventing and / or treating diseases related to foreign peptides.
  • the Japanese encephalitis virus genome according to the present invention may be the whole genome or a subgenome in which a part of the nucleic acid sequence is deleted.
  • the JE virus genome is the whole genome.
  • the Japanese encephalitis virus genome is a subgenome in which a part of the structural protein gene sequence is deleted, wherein the deleted structural protein gene sequence is selected from the following genes: protein C, prM protein, protein E Or a combination.
  • the exogenous nucleic acid sequence described in the present invention may encode one or several polypeptides, and the types of encoded polypeptides include, but are not limited to, antigens or determinants thereof, cytokines, cancer-related proteins, growth factors, polypeptide hormones, receptors, Enzymes, antibodies, etc. If the exogenous nucleic acid contains a sequence encoding an antigen or epitope of a certain pathogen, the recombinant JE virus can stimulate the human body's immune response to the JEV and the pathogen, thereby preventing and / or treating JEV and the pathogen-related diseases at the same time. Role.
  • the invention provides a method for inserting a foreign nucleic acid sequence in a recombinant Japanese encephalitis virus.
  • said The insertion site of the exogenous nucleic acid sequence can be any suitable position in the JEV genome, but the insertion of the exogenous nucleic acid sequence cannot cause frameshift mutations in the JEV genome, cannot affect the function of JEV non-structural proteins, and cannot affect the self-replication of the recombinant genome Features.
  • the insertion position of the exogenous nucleic acid sequence is selected from the group consisting of between the NS2B and NS3 coding regions of the JEV genome; between the E and NS1 coding regions of the JEV genome; and the C of the JEV genome And prM coding region.
  • the invention provides a method for producing recombinant JE virus.
  • JEV containing the entire genome of JEV the existing production method of live attenuated JE vaccine can be used.
  • JEVs containing the JEV subgenome a special packaging cell is needed to produce the recombinant JE virus. the term
  • the "JE virus” described herein generally refers to attenuated JE virus, which cannot cause nervous system disease or other serious pathological changes after infection. It can be a live attenuated JE vaccine used in China.
  • SA14-14-2 can also be other attenuated Japanese encephalitis virus. Particularly preferred are attenuated Japanese encephalitis viruses that have been used clinically.
  • nucleic acid and “nucleic acid sequence” refer to nucleotides (ribonucleotides or deoxyribonucleotides) of any length in a polymerized form. It includes, but is not limited to, single- or double-stranded DNA or RNA, genomic DNA, and cDNA.
  • user refers to any individual, including humans and animals, who needs to be vaccinated or diagnosed and treated, especially those that are susceptible to JEV infection.
  • the "antigen” described herein refers to a substance that can induce an immune response in the immune system and can specifically react with antibodies or effector cells in vivo or in vitro. These include, but are not limited to, surface antigens of pathogens (pathogens such as bacteria and viruses), tumor antigens, and the like.
  • packaging cell refers to a cell that provides a JEV protein that cannot be expressed by the recombinant JEV genome.
  • the packaging cells contain the JEV nucleic acid sequence deleted from the JE virus subgenome and can express the protein encoded by the sequence. Therefore, when the recombinant JE virus subgenome is transferred into the packaging cells, all JEV proteins required for self-assembly can be obtained And then packaged into recombinant JE virus particles.
  • helper virus vector refers to a heterologous virus or a nucleic acid (including, but not limited to, DNA, RNA, and cDNA) containing a JEV nucleic acid sequence with a JEV subgenome deleted, and is transfected after being transfected into a JEV host cell
  • the cell line can become a packaging cell.
  • the invention provides a method for constructing and applying a recombinant Japanese encephalitis virus.
  • the recombinant JE virus can carry foreign nucleic acid into the human body, and is suitable for the prevention of infectious diseases and the gene therapy of some malignant diseases.
  • exogenous polypeptide encoded by the exogenous nucleic acid may be various functional proteins, and the HBV surface antigen is only one non-limiting example.
  • the following description of Japanese encephalitis virus with recombinant HBV surface antigen can also be generally used for other recombinant Japanese encephalitis Virus.
  • JEV is spherical
  • the diameter of the virus genome is a single-stranded positive-stranded RNA with a total length of 1 kb
  • the structural protein C prM precursor of M protein
  • E and non-structural protein NS1 NS5 are sequenced from the 5 'to 3' ends.
  • Virus RNA directly acts as mRNA in the cytoplasm and translates structural and non-structural proteins.
  • the length of the exogenous nucleic acid inserted into the recombinant JE virus is usually less than 5 kb, and it can encode a single polypeptide or multiple polypeptides. Due to its small genome, the optimal length of the exogenous nucleic acid is about 200 2500 kb.
  • a sequence encoding a proteolytic enzyme ie, a release element
  • the protease sequence is a foot-and-mouth disease virus 2A sequence.
  • Nucleotide sequences of Ruqian attenuated Japanese Encephalitis virus strains can be obtained from public databases, including, for example, GenBank.
  • GenBank The model strain is "SA14- 14-2".
  • the nucleotide sequence of the JEV SA14-14-2 genome can be obtained at GenBank accession number AF315119.
  • the production of attenuated Japanese encephalitis virus particles can be performed using production methods well known in the art.
  • Many attenuated JE virus strains are commercially available, for example from ATCC.
  • exogenous polypeptides of the present invention are located at different sites in the genome of the Japanese encephalitis virus.
  • a foreign nucleic acid sequence can be inserted into one or more of the following positions: (1) the N-terminus of a viral polypeptide; (2) between viral proteins C and prM; (3) viral proteins NS2A and NS2B (4) between the viral proteins NS2B and NS3; (5) between the viral proteins NS3 and NS4A; (5) between NS4A and NS4B; (6) between the E coding region and the NS1 coding region.
  • Exogenous nucleic acid can also be inserted into other sites of the Japanese encephalitis virus genome.
  • the insertion of the exogenous nucleic acid does not disrupt the function of the JE protein, and / or the proteolytic processing of the viral polypeptide, and / or the replication of the virus.
  • the recombinant JE virus of the present invention can be used as a preventive vaccine against hepatitis B.
  • the recombinant JE virus of the present invention will continue to multiply / replicate the recombinant JE virus genome in the host cell until the immune system is fully activated to stop the recombinant JE virus infection or clear the recombinant JE virus genome, so it can elicit sufficient strength of immunity Answer.
  • the present invention also provides various compositions including the aforementioned recombinant Japanese encephalitis virus, including pharmaceutical compositions, especially vaccine compositions.
  • compositions containing recombinant Japanese encephalitis virus may contain buffers
  • buffers well known to those skilled in the art, as well as a variety of materials suitable for a particular use, can be used in pharmaceutical compositions.
  • the composition may contain pharmaceutically acceptable excipients.
  • Various pharmaceutically acceptable excipients have been detailed in various publications, such as "Remington: Practice in Pharmacy and Pharmacology", 19th Edition (1995) Mack Publ ishing Co.
  • the pharmaceutical composition can be prepared into various dosage forms, including but not limited to the "Pharmacopoeia of the People's Republic of China" 2000 Various dosage forms described in the annual edition, such as injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays, suppositories, transdermal drugs (such as patches, etc.), ointments, lotions, etc.
  • organic or inorganic carriers and / or diluents suitable for oral or topical use can be used to formulate various compositions containing recombinant Japanese encephalitis virus. Diluents known in the art include aqueous media, vegetable and animal oils and fats.
  • stabilizers, wetting agents, and emulsifiers, salts that change osmotic pressure, various buffers and skin penetration enhancers that maintain different pH values can also be used as auxiliary materials.
  • the recombinant JE virus of the present invention can be formulated by various methods.
  • the vaccines of the present invention can be formulated using a variety of methods well known in the art using suitable pharmaceutical carriers and / or vehicles.
  • Sterile saline is a suitable vaccine medium.
  • Other aqueous and non-aqueous isotonic sterile injections, suspensions, etc., which are well known to those skilled in the art, can also be used for vaccine formulation.
  • the formulation of the vaccine composition of the present invention may further contain other ingredients well known to those skilled in the vaccine field, including, for example, adjuvants, stabilizers, pH adjusters, preservatives, and the like.
  • Adjuvants include (but are not limited to) aluminum salt adjuvants; saponin adjuvants; Ribi adjuvants (Ribi ImmunoChem Research In., Hamilton, MT); Montanide ISA adjuvants (Seppic, Paris, France); Hunter'sTiterMax adjuvant Agents (CytRx Corp., Norcross, GA); Gerbu adjuvant (Gerbu Biotechnik GmbH, Gaiberg, Germany) and the like.
  • other ingredients that modulate the immune response may be included in the formulation. How to use recombinant Japanese encephalitis virus
  • the invention provides a variety of methods for eliciting an immune response to a foreign polypeptide, including administering to a user the recombinant Japanese encephalitis virus or a composition thereof provided by the user, allowing the recombinant Japanese encephal virus to enter a cell, expressing and releasing the foreign polypeptide, and further Elicit an immune response to a foreign polypeptide.
  • the exogenous polypeptide is an antigenic / antigenic determinant of a pathogen.
  • Administration of this recombinant JE virus to a user can prevent or treat a disease caused by the pathogen.
  • This program is particularly suitable for the prevention and treatment of diseases caused by intracellular infection pathogens (viruses, bacteria, protozoa, etc.).
  • the recombinant JE virus can be used as a bivalent vaccine of the JE virus and the pathogen; when the exogenous polypeptide is the antigen of two or more pathogens, the recombinant JE virus can Multivalent vaccines for Japanese encephalitis virus and pathogens containing the above antigen / determinants.
  • the exogenous polypeptide is a tumor antigen.
  • This program is suitable for cancer patients or high-risk populations.
  • Tumor antigens are generally weak in immunogenicity.
  • Encephalitis virus as a tumor antigen carrier can enhance the immune response.
  • the exogenous polypeptide may also be a cytokine, a polypeptide hormone, an enzyme, an antibody, etc., which is suitable for a specific gene therapy scheme.
  • Mode of administration and dosage may also be a cytokine, a polypeptide hormone, an enzyme, an antibody, etc., which is suitable for a specific gene therapy scheme.
  • vaccine formulations contain In addition to Japanese encephalitis virus, it may also include a pharmaceutically acceptable carrier, and optionally an adjuvant, flavoring agent, or stabilizer.
  • the recombinant JE virus or its composition can be administered by various conventional and pharmaceutically acceptable routes, such as: intranasal, intramuscular, intratracheal, subcutaneous, intradermal, transvaginal, intrapulmonary, intravenous, Nasal, oral or other parenteral routes of administration.
  • the route of administration can be combined as required, or adjusted according to the type of antigen and disease characteristics.
  • Vaccine compositions can be administered in single or multiple dose booster doses to elicit and / or maintain immunity.
  • Recombinant JE virus or its composition should be administered in an "effective amount", that is, the amount of recombinant JE virus is sufficient to elicit an immune response in the chosen route of administration, which can effectively promote protection of the host against JE virus and contain foreign Antigen pathogen infection.
  • a single dose of recombinant Japanese encephalitis virus in a vaccine formulation is based on a dose that elicits an immunoprotective response without significant side effects.
  • an effective dose of the recombinant Japanese encephalitis virus vaccines typically range from about 102--108 plaque forming units (plaque forming units, Pfu), preferably from about 10 3 - 10 7 Pfu, best of 104 -10 6 Pfu.
  • plaque forming units plaque forming units
  • Pfu plaque forming units
  • the optimal amount of vaccine can be determined by standard methods in the art, such as antibody titer determination.
  • the recombinant JE virus provided by it is less or similar to existing live JE vaccines.
  • Another advantage of the present invention is that it is highly efficient.
  • the recombinant JE virus provided by the invention can self-replicate the JEV genome in a host cell, and continuously express foreign polypeptides for a period of time. Therefore, it can trigger an efficient immune response.
  • Another advantage of the invention is its low cost of use.
  • the cost of producing recombinant Japanese encephalitis virus according to the manufacturing method provided by the present invention is equivalent to or slightly higher than the existing live attenuated Japanese encephalitis vaccine.
  • the present invention also provides a recombinant JE virus containing an HBV antigen.
  • the exogenous nucleic acid encodes a HBV surface antigen / antigenic determinant, and the resulting recombinant JE virus can be used as a bivalent vaccine for preventing JE and Hepatitis B.
  • the bivalent vaccine provided by the present invention has the following advantages:
  • HBV surface antigen can be continuously expressed in cells, and it is more immunogenic than a hepatitis B subunit vaccine that cannot proliferate in vivo.
  • the application of the vaccine can achieve long-term protection without repeated immunizations, thus reducing the suffering of recipients, saving medical resources, and avoiding poor immune effects and failure due to missed seeds;
  • the vaccine is a recombinant Japanese encephalitis virus, and its effectiveness in preventing Japanese encephalitis is the same as that of the parental virus.
  • the vaccine is a recombinant Japanese encephalitis virus. All JEV functional proteins that make up the recombinant Japanese encephalitis virus are the same as the parental virus, a live attenuated Japanese encephalitis vaccine.
  • the live vaccine is the same, so its safety is similar to that of a live attenuated JE vaccine.
  • Recombinant viruses lacking part of the JEV structural protein gene, because they cannot self-proliferate in the body, will not cause secondary infection, so their safety is higher than that of live attenuated JE vaccines.
  • the production cost of this vaccine is equal to or slightly higher than that of live attenuated JE vaccine. Compared with the current hepatitis B vaccine and hepatitis B vaccine, it is equivalent to saving the production cost of hepatitis B vaccine, and the current selling price of hepatitis B vaccine is much higher than that of JE vaccine, so the vaccine has extremely high economic value.
  • the vaccine's low production cost and low vaccination cost help to increase the coverage of vaccination, improve people's health, and reduce encephalitis, neurological disorders, acute and chronic diseases caused by missed vaccines. Medical expenses for diseases such as hepatitis and liver cancer.
  • the present invention is further described below with reference to specific embodiments. These examples are only used to illustrate the present invention and not to limit the scope of the present invention.
  • the experimental methods without specific details in the following examples are generally based on conventional conditions such as those described in Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) by Satnbrook et al. Conditions recommended by the manufacturer.
  • Example 1 Preparation of full-length cDNA clone of Japanese encephalitis virus:
  • RT-PCR was used to convert Japanese encephalitis virus RNA into four Japanese encephalitis virus cDNA fragments (5 'end cDNA,
  • both ends of the intermediate fragment cDNA near the 3 'end include a portion of the same gene sequence as the 3' end of the intermediate fragment cDNA near the 5 'end and the 5' end of the 3 'end cDNA fragment.
  • sequence of the DNA primers used is as follows: Primer name Primer sequence (5 '-3') SEQ ID NO.
  • 4b AAATATGGTACCAGATCCTGTGTTCTTCCTCACCAC 8 Obtain 4 modified JE virus cDNA fragments with lengths of 2.9kb, 2.8kb, 3.8kb and 1.8kb respectively. b) The modified 5 'end fragment and 3' end fragment were ligated and cloned into the pRS424 plasmid by conventional methods.
  • Example 1 The full-length Japanese encephalitis virus cDNA clone obtained in Example 1 was cut with a BspE I endonuclease and a BamH I endonuclease to a length of about 2.1 kb. This fragment includes the Japanese encephalitis virus cDNA sequence 3445-5576. Base.
  • Example 2 Use the full-length Japanese encephalitis virus cDNA clone obtained in Example 1 as a template, and use GGCATAGTCTTGG
  • ACTTTGAT SEQ ID NO: 9
  • GACGTCGAGTCCAACCCTGGCCCCGGGGCT TTCGGTTATTGGCTCAC TTTA SEQ ID NO: 10
  • CCATGCCCTGTCTGGTATC TC SEQ ID NO: 11
  • GGGGCCAGGGTTGGACTNO CGGGTCGGACTCGCGGACTCGCGGACTCAC DNA fragments.
  • Fusion PCR Fusing PCR was used to ligate the two DNA fragments into a DNA fragment containing the 2A gene sequence of foot-and-mouth disease virus between the NS2B and NS3 genes (an Afl II restriction site was introduced in the 2A sequence)
  • the ends contain BspE and BamH I restriction site sequences, respectively.
  • Afl II endonuclease was used to linearize the modified JE virus cDNA generated in step 3.
  • DNA-dependent Sp6 RNA polymerase DNA-dependent Sp6 RNA polymerase was used to transcribe the linearized recombinant JE virus cDNA into RNA.
  • Example 1 The full-length Japanese encephalitis virus cDNA clone obtained in Example 1 was cut with a BsrG I endonuclease and a BspE I endonuclease to remove a fragment of about 1.6 kb. This fragment includes the JE virus cDNA sequence 1887-3445.
  • Example 2 Use the full-length Japanese encephalitis virus cDNA clone obtained in Example 1 as a template, and use CAGGCCACCTGAA ATGTAGGC (SEQ ID NO: 15) and GACGTCTCCCGCAAGCTTAAGAAGGTCAAA ATTCAACAGCTGTCCA GTGTCAGCATGCACATT (SEQ ID NO: 16) to : 17) and ATCATGC ATAACAGGTCTGAT (SEQ ID NO: 18) as primers, two DNA fragments were generated by PCR method.
  • the above two DMA fragments were ligated by fusion PCR to form a DNA fragment containing the 2A gene sequence between the E and NS1 genes (an AII II restriction site was introduced in the middle of the 2A sequence), and the ends of the DNA fragment contained BsrG I and BspE I restriction site sequence.
  • step 3 The 1.6-kb Japanese encephalitis virus cDNA fragment and the DNA fragment generated in step 2 were transformed into yeast (ATCC 76628) in step 1 to obtain a modified full-length Japanese encephalitis virus cDNA clone.
  • Example 4 Preparation of a bivalent vaccine with an insertion point between C and prM
  • Example 1 The full-length Japanese encephalitis virus cDNA clone obtained in Example 1 was cut with a Spe I endonuclease to remove a 612 bp fragment. This fragment includes JE virus cDNA sequences 175-785.
  • Example 2 Use the full-length Japanese encephalitis virus cDNA clone obtained in Example 1 as a template, and use GGTAAAAACCGGGCTA TCAAT (SEQ ID NO: 19) and AGGGTTGGACTCGACGTCTCCCGCAAGCTTAAGA AGGTCAAAATTCAAC CTGGGCTCCTGCACAAGCTATGAC (SEQ ID NO: 20), and GGAGAC GTCGATCGCGCGAGCCCGACCGAGCGATCCCAACC 21) and TCAGTTTTCAT GAGATATCGT (SEQ ID NO: 22) as primers, and two DNA fragments were generated by PCR.
  • the above two DNA fragments were ligated by fusion PCR to form a DNA fragment containing the 2A gene sequence between the C and prM genes (an Afl II restriction site was introduced in the middle of the 2A sequence), each of which contains Spe I enzyme Cleavage sequence.
  • Example 5 Neurotoxicity of Hepatitis B and Encephalitis Bivalent Vaccine
  • the recombinant JE virus particles obtained in Example 2, the recombinant JE virus particles obtained in Example 3, and the JEV attenuated strain SA14-14-2 virus particles were injected intraperitoneally at a dose of 10 4 PFU (0.5 ml), respectively.
  • Plaque reduction test for detection of Japanese encephalitis virus neutralizing antibodies The recombinant Japanese encephalitis virus particles (bivalent vaccine) and live attenuated Japanese encephalitis vaccine (SA14-14-2) obtained in Example 2 were intraperitoneally injected into two groups of 4 weeks of age at a dose of 10 e PFU (0.5 ml). ICR mice were exsanguinated after 7 days of immunization and serum was isolated. Mix the two groups of serum with diluted Japanese encephalitis virus P3 (about 200PFU / 0.4ml) in equal amounts, and dilute the diluted virus 1: 2 at the same time. As a virus control, place in a 37 ° C water bath for 90 minutes.
  • a cDNA clone from which the JEV structural protein C sequence is removed is prepared. 1.
  • the recombinant JE virus cDNA clone containing the HBsAg sequence obtained in Example 4 was digested with Apa I to make it linear.
  • the 72 bp at the 5 'end of this fragment includes the first 20 codons of structural protein C and its connected 5' non-coding region sequence, and 30b at the 3 'end.
  • P includes the 106th to 116th codons of the structural protein.
  • the linear recombinant JE virus cDNA and the 102 bp cDNA fragment were transformed into yeast (ATCC 76628) to obtain a recombinant JE virus cDNA clone ( ⁇ C-rJEV cDNA) from which the JEV structural protein C sequence was removed.
  • CMV cytomegalovirus, cytomegalovirus
  • CMV-JEV 5 end DNA fragment (referred to as CMV-JEV 5, end) was prepared by fusi ng PCR method.
  • the CMV-JEV 5 'end generated in step 1 and pRS424 plasmid were digested with Not I and Apa. The digestion products were purified using a Qiagen spin column (purchased from QIAGEN Inc.). The two DNA fragments were ligated with T4 ligase (purchased from New England Biolab) and transformed into E. coli. CMV-JEV 5 'end clone (pRS / CMV-JEV 5' end) was obtained by screening.
  • a 3' end fragment (referred to as JEV 3 'end) of the JEV cDNA was prepared by a PCR method.
  • a DNA fragment (referred to as HDVr) of hepatitis delta virus antigen blood ribozyme was prepared by fusion PCR method. .
  • pcDNA3 (purchased from Invi trogen) as a template and ⁇ and 3 ' ⁇ as primers, a DNA fragment of bovine growth hormone poly A (bovine growth hormone poly A (BGH pA)) (pA) was prepared by PCR. Using the above three fragments as templates and f / ⁇ T e ⁇ and 3, pA as primers, a DNA fragment of JEV 3, end- HDVr- pA was prepared by a fusion PCR method. SEQ ID Primer Name Primer Sequence (5 '-3')
  • the pRS / CMV- JEV5 'end generated in step 2 and the JEV3' end- HDVr- pA DNA fragment generated in step 3 were digested with Apa I and Sac II, and the digested product was purified with Qiagen spin column. The two DNA fragments were ligated with T4 ligase and transformed into E. coli. A clone of pRS / CMV- JEV5 'end-JEV3' end- HDVr- pA was obtained by screening.
  • the pRS / CMV-JEV5 'end- JEV3' end- HDVr- pA clone generated in step 4 above was digested with Apa I, and the full-length JE virus cDNA clone obtained in Example 1 was cloned with Notl and Kpn I Digestion.
  • the enzyme-digested product was purified, and the purified product was transformed into Saccharomyces cerevisiae.
  • the DNA was recombined with the same sequence in yeast to obtain a modified full-length JEVcDNA clone (referred to as plS / CMV / JEV).
  • the following recombinant Japanese encephalitis virus was constructed by a method similar to that of Examples 2 to 4, except that the foreign nucleic acid and the insertion site were different.
  • Examples 14-16 Various constructs of recombinant JE virus (containing JEV subgenome)
  • Example 17 Neutralization test of recombinant Japanese encephalitis virus vaccine

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Abstract

L'invention concerne un vaccin divalent pour assurer la prévention et le traitement de l'encéphalite japonaise B (également appelée encéphalite japonaise) et l'hépatite B. Ledit vaccin divalent est obtenu par introduction d'un gène d'antigène de virus de l'hépatite B dans le génome du virus de l'encéphalite B atténué. Il est possible d'utiliser le virus de l'encéphalite B atténué comme vecteur pour exprimer efficacement l'antigène du virus de l'hépatite B, le vaccin contre le virus de l'encéphalite B atténué conservant ses caractéristiques de fiabilité et d'immunogénéité. Ledit vaccin est par conséquent un vaccin divalent disponible pour prévenir et traiter à la fois l'encéphalite B et l'hépatite B.
PCT/CN2004/000232 2003-03-20 2004-03-19 Vaccin divalent WO2004082712A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2226031A (en) * 1988-12-09 1990-06-20 Nippon Zeon Co Expression of non-structural protein of Japanese encephalitis virus in insect cells
JPH06205672A (ja) * 1992-03-19 1994-07-26 Nippon Zeon Co Ltd 日本脳炎ウイルス及びｂ型肝炎ウイルスの表面抗原タンパク質の抗原部位を持つキメラタンパク質の製造法、およびそれに用いる組み換えバキュロウイルス
JP2001299355A (ja) * 2000-04-27 2001-10-30 National Institute Of Infectious Diseases 日本脳炎ウイルス遺伝子をコードするｃＤＮＡを含む発現ベクターおよびそれを用いたワクチン

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2226031A (en) * 1988-12-09 1990-06-20 Nippon Zeon Co Expression of non-structural protein of Japanese encephalitis virus in insect cells
JPH06205672A (ja) * 1992-03-19 1994-07-26 Nippon Zeon Co Ltd 日本脳炎ウイルス及びｂ型肝炎ウイルスの表面抗原タンパク質の抗原部位を持つキメラタンパク質の製造法、およびそれに用いる組み換えバキュロウイルス
JP2001299355A (ja) * 2000-04-27 2001-10-30 National Institute Of Infectious Diseases 日本脳炎ウイルス遺伝子をコードするｃＤＮＡを含む発現ベクターおよびそれを用いたワクチン

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