WO2021085650A1 - ワクチン - Google Patents

ワクチン Download PDF

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WO2021085650A1
WO2021085650A1 PCT/JP2020/041112 JP2020041112W WO2021085650A1 WO 2021085650 A1 WO2021085650 A1 WO 2021085650A1 JP 2020041112 W JP2020041112 W JP 2020041112W WO 2021085650 A1 WO2021085650 A1 WO 2021085650A1
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virus
protein
vaccine
domain
cells
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French (fr)
Japanese (ja)
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国昭 根路銘
繁夫 杉田
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Nerome Institute Of Biological Resources
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Nerome Institute Of Biological Resources
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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
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • the present invention relates to a novel vaccine.
  • influenza vaccines The quantitative transition of influenza vaccines is symbolically summarized as an increase in live attenuated vaccines or live low temperature adaptive vaccines. Their primary purpose is regional immunity, which can play an important role in the prevention of respiratory infections through mucosal immunity.
  • regional immunity which can play an important role in the prevention of respiratory infections through mucosal immunity.
  • influenza virus-like particle (VLP) vaccine containing the H5 type HA protein of influenza virus produced by Bombyx mori was developed (see Patent Document 1).
  • inactivated influenza vaccines have been developed using various techniques such as cell culture-derived vaccines; DNA vaccines; influenza virus-like particle (VLP) vaccines.
  • VLP influenza virus-like particle
  • the silkworm frogs for the H5 subtype VLP vaccine and the H7 subtype VLP vaccine of triinfluenza are the production levels of the H5 VLP vaccine or the H7HA VLP vaccine, and per frog.
  • We have developed a silkworm worm that exhibits a production level exceeding millions of HA titers see Patent Document 1).
  • These silk moth pupae can be used in chickens via partially purified products, but in the case of human vaccines, we must confirm their safety and efficacy in detail.
  • inactivated vaccines in recent years are required to provide high HI antibody titers through appropriate techniques.
  • interleukin 12 interleukin 12
  • the present inventors have conducted intensive studies, and interleukin 12, the M2 protein of influenza A virus associated with the interleukin 12, and the neurominidase.
  • a virus-like particle having an immunomodulator molecule (hereinafter, also referred to as M2 / N / IL12 molecule or IL12 / N / M2 molecule) containing a protein, and to apply the virus-like particle having this immunomodulator molecule to a vaccine.
  • the present inventors disclose the production and protective efficacy of HI antibody by a vaccine containing virus-like particles containing M2, N, and IL12 molecules.
  • M2 ⁇ N ⁇ IL12 molecule The design drawing of M2 ⁇ N ⁇ IL12 molecule is shown.
  • the M2, N, and IL12 molecules are Interleukin-12 (interleukin 12), NA Starks (NA Stark), NA Transmembrane (TM) Domein (NA transmembrane domain), NA Cytoplasmic Domein (NA cytoplasmic domain), and Flaglinker. (Flag linker), M2 Cytoplasmic Domain (M2 cytoplasmic domain), M2 Transmembrane Domain (TM) Domein (M2 cytoplasmic domain), and M2 Small Ectodomein (M2 small extracellular domain).
  • a schematic diagram of IL / N / M2 molecules is shown.
  • FIG. 1 indicates an H5HA protein
  • 2 indicates an M2 / N / IL12 molecule
  • 3 indicates an H7HA protein.
  • M2, N, and IL12 molecules are expressed in Sf9 cells.
  • FkH5 protein is expressed in Sf9 cell.
  • AnH7 protein is expressed in Sf9 cell.
  • PBS control
  • M2 ⁇ N ⁇ IL12 M2 ⁇ N ⁇ IL12 molecule
  • VLP containing M2 ⁇ IL12 and FkH5 protein VLP containing M2 ⁇ IL12 and AnH7 protein.
  • M2 ⁇ N ⁇ IL12 M2 ⁇ N ⁇ IL12 molecule
  • VLP containing M2 ⁇ IL12 and FkH5 protein VLP containing M2 ⁇ IL12 and AnH7 protein.
  • Interleukin-12 is a protein identified in 1989 as a factor that activates NK cells from EBV-transformed cells. It consists of subunits of p40 and p35, and p40 has a subunit in common with IL-23. IL is thought to coordinate various actions by combining subunits in this way.
  • the immune system of mammals includes acquired immunity by B cells and T cells and innate immunity centered on natural killer cells (Natural Ki11er Tce11, hereinafter abbreviated as NK cells).
  • axons have only innate immunity, and acquired immunity has been achieved since becoming a vertebrate, but acquired immunity has a very short history in evolution, and organisms other than vertebrates are innate immunized. Has been responsible for immunity.
  • NH cells have been discovered by Shigeo Koyasu's group as cells involved in the innate immunity of the lymphocyte system, and three types of innate immune cells, NK cells, lymphoid tissue inducer (LTi) cells, and natural helper (NH) cells, have been identified. It has become clear. In this way, the acquired immune system consists of Th1 cells corresponding to NK cells, Th2 cells corresponding to NH cells, and Th17 cells corresponding to Lti cells, and innate immunity is differentiated and developed to be acquired immunity. I have come to understand.
  • NKT cells mouse
  • MAIT cells human
  • innate immunity and acquired immunity are closely proliferating and responsible for immunity.
  • the research so far has focused on acquired immunity, and the research on innate immunity that supports them has been delayed due to the difficulty of research.
  • Speaking of immunity research on acquired immunity by T cells and B cells is the main focus. It has been considered that how to activate antibody production by B cells and how to activate cytotoxic T cells are the key to immunity.
  • there is a foundation of innate immunity and it is considered that activating acquired immunity as well as activating innate immunity is closer to the actual immune response.
  • NK cells are the key to innate immunity.
  • NK cells are known to be enhanced by 1FN- ⁇ / ⁇ , 1FN-1, 1L-2, 1L-4, lL-12, 1L-15, 1L-18, but the most NK cells among them.
  • APCs antigen-presenting cells
  • dendritic cells dendritic cells
  • macrophages / monomorphic cells and B cells
  • only dendritic cells turn naive T cells into activated T cells (effector T cells). And can show the strongest antigen presenting ability.
  • the vaccine containing M2, NA, and IL12 as one molecule aims at the vaccine effect by presenting the antigen of the M2 protein of influenza virus in dendritic cells. While the 23 amino acids in the N-terminal region of the M2 protein are exposed on the surface of the virus as antigens, they are well preserved and because they are the entrance to the ion channels of the virus, the antibody against this part has a neutralizing activity of the virus. It is believed that.
  • the 23 amino acids in the N-terminal region of the M2 protein are attracting attention as a universal vaccine that is effective against all influenza viruses regardless of subtype because of their good storage stability.
  • a 19-amino acid transmembrane region following the region exposed on the surface of the 23-amino acid lipid bilayer of M2 and a 54-amino acid lipid two
  • the IL-12 protein was placed behind the transmembrane region of the NA protein and the stalk region of the NA protein by binding to the inner region of the layer and the N-terminal region inside the lipid bilayer of the NA protein via the FLAG sequence. .. This allowed the IL-12 protein to float freely on top of the stalk and outside the lipid bilayer in a stalk-bound form.
  • the M2 protein of influenza virus and IL-12 are always in a close positional relationship, and NK cell phagocytosis of M2 protein and antigen presentation by dendritic cells can be expected.
  • membrane glycoproteins such as HA proteins
  • HA vaccines prepared by the same baculovirus expression system are mixed and mixed with the lipid double layer of baculovirus or an oil layer such as artificially added sesame oil by ultrasonic treatment or the like.
  • IL-12 and the antigen protein can be coordinated on the same artificial membrane, and the effects of both innate immunity and acquired immunity activated based on innate immunity are linked to all antigens. It can be used as a vaccine antigen.
  • the vaccine in the present invention is a vaccine characterized by containing an immune modulator molecule.
  • the immune modulator molecule comprises an IL-12 protein, an NA domain region derived from a neuraminidase (NA) protein, and an M2 protein domain region derived from an influenza virus.
  • the NA domain region includes an extracellular domain, a transmembrane domain, and an intracytoplasmic domain
  • the M2 protein domain region includes an extracellular domain, a transmembrane domain, and an intracytoplasmic domain.
  • the immunomodulator molecule the IL-12 protein is bound to the extracellular domain in the NA domain region, and the cytoplasmic domain in the M2 protein domain region is bound to the cytoplasmic domain in the NA domain region by a linker.
  • influenza virus-like particles containing a hemagglutinin (HA) protein derived from a virus classified into influenza virus H5 type and / or (ii) influenza virus H7 type. It may contain influenza virus-like particles containing hemagglutinin (HA) protein from the classified virus.
  • the influenza virus-like particles contained in the vaccine according to the present invention are classified into influenza virus H5 type hemagglutinin (HA) protein (hereinafter, also referred to as H5 protein) and / or influenza virus H7 type.
  • the vaccine of the present invention can provide a survival rate equivalent to that of an animal that has been vaccinated with an inactivated influenza virus H1N1 type and infected with the influenza virus H1N1 type.
  • at least one selected from virus-like particles containing immune modulator molecules, influenza virus-like particles (i), and influenza virus-like particles (ii) is produced by Sf9 cells or Eri silkworms by the method described later. It is preferable to let it. Further, it is preferable that the virus classified into influenza virus H5 type is H5N1 type virus and / or the virus classified into influenza virus H7 type is H7N9 type virus.
  • the virus-like particle of the present invention may have a virus-like particle structure containing only the chimeric cytokine M2, N, IL12, or may have a virus-like particle structure containing HA protein and M2, N, IL12. ..
  • the virus-like particles contain the HA protein of the virus, which does not contain RNA derived from influenza virus, and the structure of the virus-like particles is preferably particles having a diameter of 50 to 150 nm, more preferably 60 nm to 120 nm. It has a structure in which distinct spikes (eg, HA spikes) are densely arranged on the surface of the virus, and is morphologically very similar to a virus particle.
  • virus-like particles of the present invention are expressed by the silk moth, they may have lipids or sugar chain modifications derived from the silk moth.
  • the lipid include glyceroglycolipid, glycosphingolipid, cholesterol, phospholipid and the like.
  • glyceroglycolipids examples include sulfoxyribosylglyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride and the like.
  • glycosphingolipids include galactosyl cerebroside, lactosyl cerebroside, and ganglioside.
  • Examples of phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylic acid, phosphatidylglycerol, phosphatidylinositol, lysophosphatidylcholine, sphingomyelin and the like.
  • fatty acids and the like induced by hydrolysis and the like may be contained.
  • the fatty acid include myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linolenic acid and the like.
  • the vaccine containing virus-like particles of the present invention has a structure in which spikes of recombinant virus membrane proteins having immunogenicity are densely provided, so that an unprecedentedly high immunogenicity can be achieved without using an adjuvant. It becomes possible to show the sex. Since no adjuvant is used, side effects such as allergies and anaphylactic shock can be reduced.
  • the virus-like particles of the present invention containing the virus-like particles of the present invention have high immunogenicity. Therefore, even if a commonly used adjuvant is not used, an unprecedented high immunogenicity is obtained. It is possible to show sex. Moreover, since a commonly used adjuvant is not used, side effects such as allergies and anaphylactic shock can be reduced. Further, as a matter of course, since the nucleic acid derived from the virus does not exist inside the hollow of the spherical outer shell of the virus-like particle of the present invention, it is non-pathogenic.
  • the vaccine of the present invention can effectively prevent viral infections in animals including humans.
  • the vaccine of the present invention can significantly shorten the period required for production as compared with the conventional production method using chicken eggs. Therefore, in the event of an epidemic of viral infections such as influenza, vaccines can be procured in a short period of time, and it is possible to make a great contribution to protecting people from infectious diseases and maintaining their health. Furthermore, in the production of a vaccine using conventional chicken eggs, it is necessary to have a highly safe facility such as a P3 facility in order to handle the seed virus used for the vaccine, and it is necessary to produce a large amount of vaccine because the immunogenicity of the vaccine is low. While a huge manufacturing cost is required for some reason such as the above, the manufacturing method of the present invention can significantly reduce the manufacturing cost.
  • the invention is a method of inoculating an animal with a vaccine against viral infection, the effective amount of a vaccine comprising the polypeptide according to the invention or produced according to the production method according to the invention. Is a method comprising administering to the animal.
  • the animal is not particularly limited as long as it is a homeothermic animal, and may be a non-human animal such as a bird, a pig, a cow, a dog, or a cat.
  • the invention is a method of inducing an immune response against a virus to an animal, the efficacy of a vaccine comprising the polypeptide according to the invention or produced by the production method according to the invention. A method comprising administering an amount to the animal.
  • the invention in another aspect, relates to a vaccine composition.
  • the vaccine composition may contain additional components and a pharmaceutically acceptable carrier.
  • additional component include an adjuvant and a component that enhances the anti-influenza virus action such as an anti-influenza virus agent.
  • the adjuvant include aluminum hydroxide, aluminum phosphate, saponin, water-in-oil emulsion, oil-in-water emulsion, emulsion in water-in-oil, acrylic or methacrylic acid polymer, maleic anhydride, alkenyl derivative, carbomer, and block copolymer. ..
  • antiviral agent examples include neuromitase inhibitors such as zanamivir, oseltamivir, peramivir, and laninamivir, amantadine, rimantadine, and RNA polymerase inhibitors.
  • the pharmaceutically acceptable carriers contained in the vaccine composition of the present invention include solvents, dispersants, coating agents, stabilizers (albumin, ethylenediamine tetraacetate alkali salt), and diluents that are permitted to be used as pharmaceuticals.
  • the vaccine composition of the present invention can be in a dosage form suitable for parenteral administration or oral administration.
  • the composition for parenteral administration include injections, nasal drops and the like, and injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections and the like. Includes the dosage form of.
  • Such injections can be prepared according to known methods, for example, by dissolving, suspending or emulsifying the antigenic protein in a sterile aqueous or oily solution usually used for injections. The prepared injection solution is usually filled in a suitable ampoule, vial or syringe.
  • the parenteral pharmaceutical composition is prepared in a dosage form of a dosage unit suitable for the dose of the active ingredient.
  • An effective amount of a vaccine is an amount sufficient to achieve a biological effect such as inducing sufficient humoral or cell-mediated immunity against the virus.
  • administration methods include inhalation, intranasal, oral, parenteral (eg, intradermal, intramuscular, intravenous, intraperitoneal, and subcutaneous administration). The effective amount and method of administration may depend on the age, sex, condition and body weight of the person being administered.
  • influenza vaccine in general, a vaccine containing 15 ⁇ g or more of HA protein per strain in 1 ml, 0.25 ml subcutaneously for those aged 6 months or more and less than 3 years old, and those aged 3 to 13 years old 0.5 ml is injected subcutaneously twice at intervals of approximately 2-4 weeks. For persons 13 years and older, 0.5 ml is injected subcutaneously once or twice at intervals of approximately 1 to 4 weeks.
  • ⁇ Method for producing virus-like particles containing immunomodulator molecules> it is preferable to produce virus-like particles using a protein expression system using Sf9 cells or Eri silkworm.
  • the amino acid sequence of the protein of the encoded immunomodulator molecule does not change as compared with the corresponding mammalian amino acid sequence.
  • a step of modifying the codon of the DNA fragment to obtain a codon-optimized DNA fragment for expression a step of inserting the obtained codon-optimized DNA fragment into a vector, the obtained vector, and a baculovirus-derived DNA.
  • the step of co-transfecting Sf9 cells the step of obtaining a baculovirus recombinant containing a codon-optimized DNA fragment from the obtained Sf9 cells, and infecting the eri silkworm with the baculovirus recombinant to produce the eri silkworm. It is preferable to include a step of breeding and a step of isolating the immunomodulator molecule from the Eri silkworm.
  • an amino acid sequence corresponding to the NA domain region, IL-12 protein, and NA domain region used in the present invention is obtained from an amino acid sequence registered in Genbank or the like in a mouse, and is a basis for nucleic acid design. And.
  • the virus-like particle gene sequence is determined, and the virus-like particle gene sequence is modified in consideration of optimizing the codon usage frequency of the silk moth.
  • a codon-optimized DNA fragment for expression of Eri silkworm of M2, N, IL12 molecule is obtained.
  • the obtained codon-optimized DNA fragment is inserted into a known vector suitable for baculovirus by using any known method, and the obtained vector and the linearized baculovirus-derived DNA are obtained.
  • a step of co-transfecting Sf9 cells a step of obtaining a baculovirus recombinant obtained by recombining a codon-optimized DNA fragment from the obtained Sf9 cells, infecting Eri silkworm with the baculovirus recombinant to produce Eri silkworm.
  • Virus-like particles may be obtained through a step of breeding and a step of isolating virus-like particles from the Eri silkworm.
  • an optimized DNA fragment for expressing influenza virus-like particles containing hemagglutinin (HA) protein derived from a virus classified into influenza virus H5 type which is obtained by the same method as described above.
  • An optimized DNA fragment for expressing influenza virus-like particles containing a hemagglutinin (HA) protein derived from a virus classified into influenza virus H7 was prepared, and the same operation was performed to prepare the above-mentioned in Eri silkworm.
  • (I) and / or (ii) above may be expressed, or these optimized DNA fragments may be co-expressed together with a codon-optimized DNA fragment for the expression of Eri silkworms of M2, N, IL12 molecules. Good.
  • the time of introduction (inoculation) of the baculovirus recombinant into the silk moth is not particularly limited as long as it is in the silk moth stage, but it is preferably early in the silk moth stage, specifically, on the day of molting (molting 1).
  • the day) to the 4th day (5th day of molting) is preferable, and the next day (2nd day of molting) to the day after next (3rd day of molting) is more preferable.
  • these pupae are bred for a predetermined period, and the temperature conditions at that time are preferably 20 ° C. to 30 ° C., more preferably 22 ° C.
  • the period of pupal breeding is not particularly limited as long as it is the period from after the above inoculation to before the pupae metamorphose into adults (moths), but 2 to 6 days after the above inoculation is preferable, and 2 to 4 days is more preferable. It is preferable, and 3 days is more preferable. Any method well known to those skilled in the art can be used for the recovery of virus-like particles from Eri silkworm.
  • Eri silkworm can be homogenized in an isotonic buffer solution and then recovered using immobilized erythrocytes or a sialic acid column (fetin column). It is also possible to obtain a fraction contained in the form of virus-like particles by using a fractionation method such as a sucrose density gradient centrifugation method.
  • a fractionation method such as a sucrose density gradient centrifugation method.
  • Ac NPV recombinant (hereinafter, also referred to as Ac M2 / N / IL12 recombinant) in which a DNA sequence encoding M2 / N / IL12 molecule is incorporated into Autographa California nuclear polymorphic disease virus (Ac NPV), FkH5HA protein in Ac NPV.
  • Ac NPV recombinant (hereinafter, also referred to as Ac FkH5 recombinant) incorporating a DNA sequence encoding AnH7HA protein
  • Ac NPV recombinant (hereinafter, also referred to as Ac AnH7 recombinant) incorporating a DNA sequence encoding AnH7HA protein in Ac NPV.
  • a / PR / 8/34 (H1N1) strain (PR8H1) of influenza virus was cultured and PR8H1 was infected with mice in order to restore the virulence in the mice.
  • HKH5 RG-A / BarnSwallow / HongKong / 1161/2010-A / PR / 8/34 [R] (6 + 2) (H5N1)
  • AnH7 RG-A
  • Anhui / 1/2013-A / PR / 8/34 [R] (6 + 2) (H7N9) will be sold to Dr. RG Webster of St Jude Chicken's Research Hospital, Memphis, TN, USA. It was propagated in 10-day-old fertilized chicken eggs and MDCK cells.
  • the M2, N, and IL12 genes were also synthesized by Japan Genewith Co., Ltd.
  • the M2, N, and IL12 genes include glycine linker (L1), M2 small extracellular domain (23 amino acids), M2 transmembrane domain (19 amino acids), M2 cytoplasmic domain (54 amino acids), Flag linker (11 amino acids), and NA cytoplasm.
  • L1 glycine linker
  • M2 small extracellular domain 23 amino acids
  • M2 cytoplasmic domain 54 amino acids
  • Flag linker 11 amino acids
  • NA cytoplasm Designed to encode a membrane protein constructed by combining two subsystems of IL12 (p35 and p40) with a domain (6 amino acids), an NA transmembrane domain (31 amino acids), and NA Stoke (41 amino acids). It was.
  • the synthesized gene was inserted downstream of the pFast Bac1 (pFast Bact) polyhedrosis promoter, and the results and plasmid were used to introduce into DH10Bac.
  • pFast Bac1 pFast Bact
  • the results and plasmid were used to introduce into DH10Bac.
  • VLP vaccine Preparation of VLP Vaccine in Eli silk moth
  • the pupa of Eli silk moth was used to prepare the VLP vaccine.
  • tussah pupae were infected with the Ac NPV recombinant described above.
  • Inoculation of baculovirus into pupae and preparation of each VLP vaccine are performed by Maegawa K, Shibata T, Yamaguchi R, Hiroike K, Izzati U.Z, et al.
  • Overexpression of a virus-like particke influenza vaccine in Eri silkworm pupae using Autographa californica nuclear polyhedrosis virus and host-range expansion. Arch Virol 2018; 163 (10): 2787-2797.
  • Blood cell adsorption test Sf9 cells infected with the Ac NPV recombinant described above were washed once with PBS, and Maegawa K, Shibata T, Yamaguchi R, Hiroike K, Izzati U. Z, et al. Overexpression of a virus-like. particke influenza vaccine in Eri silkworm pupae, using Autographa californica nuclear polyhedrosis virus and host-range expansion. Arch Virol 2018; 163 (10): 2787-2797. A blood cell adsorption test was performed using 0.5% chicken erythrocytes.
  • the anti-Flag mouse monoclonal antibody M2 and the anti-DYKDDDDK tag mouse monoclonal antibody were purchased from Sigma Aldrich (St. Louis, MO, USA) and Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka, Japan), respectively.
  • Antisera against HKH5 virus, PRHA virus, and AnH7 virus were prepared in mice immunized with each purified virus (HA titer of 2,048-4,096) three times.
  • a series of mouse groups including 5 mice each, were subjected to VLP, FkH5 protein and M2 containing inactivated PRH1, M2, N, IL12 molecules in PBS.
  • mice were infected with the virus by antigen administration with challenge virus via the nasal route, and the health status and survival of the mice immunized with the above three vaccines were observed daily for 9 days. Mice that died during the course of the experiment were autopsied for lung removal and collection of whole blood. The viral load in the removed lung was measured by the plaque method for homogenates in all the removed lungs. For infection with antigen virus, undiluted virus stock solutions of pathogenic PRH1 virus and FkH5 virus were used.
  • VLPs containing M2 ⁇ N ⁇ IL12 molecules and FkH5 protein and / or M2 ⁇ N ⁇ IL12 molecules and AnH7 protein When VLPs were prepared, it was predicted that monovalent VLP vaccines and divalent VLP vaccines associated with M2, N, and IL12 molecules could be synthesized.
  • Sf9 cells were subjected to their Ac NPV recombinants. Double infected. This result is shown in the FA test of FIG. Expression of the M2, N, and IL12 molecules was confirmed in FIG. 3A (red) treated with the IL12 monoclonal antibody.
  • Sf9 cells infected with Ac M2 ⁇ N ⁇ IL12 were determined to be green labeled with anti-mouse IL12 antibody (FIG. 3B).
  • the purpose was to observe the neutral colors showing expression (FIG. 3D).
  • Figures 3E and 3F highlighted a much more diverse neutral color, indicating co-expression between VLP antigens containing M2, N, IL12 molecules and FkH5 and AnH7 proteins.
  • FIGS. 3A to 3F Hemagglutinin adsorption activity of double-infected cells
  • the present inventors were able to confirm that a large number of cells were capable of co-expression of the above three recombinants.
  • co-expression by the above three Ac NPV recombinants was confirmed by exposure on infected cells by a blood cell adsorption test of FkH5 VLP antigen or AnH7 VLP antigen.
  • FIG. 4A showed negative hemodsorption in cells infected with the Ac M2 ⁇ N ⁇ IL12 recombinant, and as a result, the expressed M2 ⁇ N ⁇ IL12 molecules contained hemagglutination molecules. It was shown not to.
  • mice M2, N, IL12 molecules (abbreviated as "M2, IL12” in FIG. 5) are possible immunodefensive M2 proteins of influenza A virus. Since it contains an M2 protein showing a very high degree of homology between influenza A viruses, the protective activity of a vaccine containing virus-like particles containing an immune modulator molecule was examined in mice. The vaccine showed 80% and 60% survival against infection with PRbH1 and HKH5 viruses, indicating protection against the above two viruses (FIG. 5A). Since the H7 virus did not show pathogenicity in mice, no effective mortality and survival data could be obtained from this experiment.
  • a vaccine containing a virus-like particle containing an immunomodulator molecule and an FkH5 protein or a virus-like particle containing an immunomodulator molecule and an H7 protein is effective against infection with a highly pathogenic PR8H1 virus. , 90-100%, which is extremely high protection (Fig. 5B).
  • immunization of mice with virus-like particles containing only immunomodulator molecules showed a 20% mortality rate, suggesting a weak but protective protection (Fig. 5B).
  • HkH5, AnH7, and PR8H1 challenge virus
  • mice treated with PBS showed 60% and 20% survival rates for HkH5 virus and PR8H1 virus, respectively, but 100% survival rates for AnH7 virus ().
  • FIG. 6C control mice treated with PBS
  • FIG. 6C control mice treated with PBS
  • FIG. 6D the protective activity of the simple inactivated vaccine (PR8 vaccine) against the PR8H1, HkH5, and AnH7 challenge viruses was 100%, 60%, and 100%, respectively.
  • mice immunized with VLP containing M2 ⁇ N ⁇ IL12 molecule Fig. 6E
  • mice immunized with VLP containing FkH5 protein and M2 ⁇ N ⁇ IL12 molecule Fig. 6F
  • AnH7 protein All mice immunized with VLP containing M2, N, and IL12 molecules (FIG. 6G) were shown to have improved survival rates as compared to FIGS. 6C and 6D.
  • Each experimental group consisted of 10 mice.
  • virus-like particles containing an immunomodulator molecule (M2, N, IL12 molecule) and FkH5 protein, and a virus containing an immunomodulator molecule (M2, N, IL12 molecule) and AnH7 protein As can be seen in Table 1, virus-like particles containing an immunomodulator molecule (M2, N, IL12 molecule) and FkH5 protein, and a virus containing an immunomodulator molecule (M2, N, IL12 molecule) and AnH7 protein.
  • the divalent H5 and H7 VLP vaccines inhibited mouse PR8H1 (H1 subtype), HKH5 (H5 subtype) and AnH7 (H7 subtype).
  • H1 subtype mouse PR8H1
  • H5 subtype H5 subtype
  • AnH7 H7 subtype
  • co-administration of VLP vaccines containing M2, N, IL12 molecules with H5 and H7 proteins could protect animals from completely different PR8H1 belonging to the HA subtype, and protected them from mutual infection.
  • the numbers in parentheses in the column of protective activity represent the mortality rate (%) due to challenge virus infection, and the molecule (left) is immunized with the vaccine and killed mice when infected with the virus. It shows the rate.
  • the denominator (right) shows the mouse mortality rate when immunized with a negative control PBS and infected with the virus.
  • mice vaccinated with PBS as a negative control of the vaccine were immunized with the M2 ⁇ N ⁇ IL12 + HKH5 VLP vaccine when the survival rate was 20%, followed by a significantly different subtype of PR8H1 virus. It was interesting to find that the survival rate of the mice fed was 90% (Fig. 5B). Not surprisingly, the same vaccine showed 100% protective activity against each of the AnH7 and HKH5 viruses.
  • Table 3 summarizes the cross-protective activity of the VLP vaccine according to the invention evaluated in mice immunized with various vaccines and infected with the challenge virus.
  • the present inventors succeeded in constructing the M2 peptide and the stalk peptide of the neuraminidase protein of IL-12, which is an immune reaction modulator, and the neuraminidase protein of influenza A virus, based on the molecular design of each structural gene. did.
  • the DNA encoding all the above proteins which is 2,130 nucleotides in length, is recombinant DNA (M2 ⁇ N) by the Autographa California nuclear polyhedrosis virus (Ac NPV). -The preparation of IL12) was carried out.
  • the chimeric cytokine (immune modulator molecule) system may be useful in the field of other infectious viruses, including the broad viral family.
  • the present inventors have already developed the DPT vaccine, which is a vaccine for measles, mumps and rubella virus, and the ZIKA and mad dog disease VLP vaccines in Kaiko, and these vaccines have also been developed as chimeric cytokines. We are promoting.
  • GGGGTGGGGSGGGGTGGGG (16) Amino acid sequence of p35 subunit (193 amino acids) of murine IL12 (SEQ ID NO: 16) RVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKM

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