WO2020179797A1 - インフルエンザｈａスプリットワクチンの製造方法 - Google Patents

インフルエンザｈａスプリットワクチンの製造方法 Download PDF

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WO2020179797A1
WO2020179797A1 PCT/JP2020/008974 JP2020008974W WO2020179797A1 WO 2020179797 A1 WO2020179797 A1 WO 2020179797A1 JP 2020008974 W JP2020008974 W JP 2020008974W WO 2020179797 A1 WO2020179797 A1 WO 2020179797A1
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Prior art keywords
influenza
split vaccine
vaccine
split
lah
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PCT/JP2020/008974
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English (en)
French (fr)
Japanese (ja)
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宜聖 高橋
悠 安達
学 阿戸
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公益財団法人ヒューマンサイエンス振興財団
大日本住友製薬株式会社
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Priority to EP20766488.9A priority Critical patent/EP3936147A4/en
Priority to KR1020217030986A priority patent/KR20210135261A/ko
Priority to SG11202109566X priority patent/SG11202109566XA/en
Priority to AU2020233456A priority patent/AU2020233456A1/en
Priority to CN202080033434.5A priority patent/CN114096273A/zh
Priority to CA3132578A priority patent/CA3132578A1/en
Priority to EA202192398A priority patent/EA202192398A1/ru
Priority to BR112021017310A priority patent/BR112021017310A8/pt
Application filed by 公益財団法人ヒューマンサイエンス振興財団, 大日本住友製薬株式会社 filed Critical 公益財団法人ヒューマンサイエンス振興財団
Priority to US17/435,590 priority patent/US20220152191A1/en
Priority to PH1/2021/552094A priority patent/PH12021552094A1/en
Priority to JP2021504119A priority patent/JP7545955B2/ja
Priority to MX2021010685A priority patent/MX2021010685A/es
Publication of WO2020179797A1 publication Critical patent/WO2020179797A1/ja
Priority to IL285984A priority patent/IL285984A/en

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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
    • 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
    • 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
    • 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
    • A61K2039/5252—Virus inactivated (killed)
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a method for producing an influenza HA split vaccine.
  • hemagglutinin The current influenza hemagglutinin (hereinafter, hemagglutinin may be referred to as "HA") vaccine exerts an infection protective effect by inducing HA antibody.
  • the HA antibody binds to a region exposed to the outside from the virus membrane, which is called the head region, and this region is the region with the most structural changes among virus strains. As a result, there are cases where the HA antibody cannot bind to the antigen-mutated virus infection different from the vaccine strain and the vaccine does not work.
  • Patent Document 1 In order to efficiently induce stem antibodies, HA stem proteins that have succeeded in stabilizing unstable stems by binding artificial mutations and linkers have been developed and are being tested in humans.
  • the present invention has been made in view of such problems, and an object of the present invention is to provide a method for producing an influenza HA split vaccine that produces an antibody that binds to the HA stem region of influenza, which is less likely to cause antigenic variation.
  • the method for producing an HA split vaccine according to the present invention also applies to an antigenic drift influenza virus that produces an antibody that binds to LAH (long alpha helix) in the HA stem region by subjecting the influenza HA split vaccine to acid treatment. It is an effective method for producing influenza HA split vaccine.
  • the present invention relates to the following.
  • [Item 1] A method for producing an influenza HA split vaccine, which produces an antibody that binds to LAH in the HA stem region by subjecting the influenza HA split vaccine to an acidic treatment.
  • [Item 1a] A method for producing an influenza HA split vaccine, which produces an antibody that binds to LAH in the HA stem region by subjecting an influenza HA split vaccine that has not been treated with formalin to an acid treatment.
  • [Item 1b] A method for producing an influenza HA split vaccine, which comprises a step of acidifying an influenza HA split vaccine and then a step of formalin treatment to produce an antibody that binds to LAH in the HA stem region.
  • [Item 1c] A method for producing an influenza HA split vaccine, which comprises a step of acidifying an influenza HA split vaccine that has not been treated with formalin and then a step of subjecting it to a formalin treatment to produce an antibody that binds to LAH in the HA stem region.
  • [Item 2] Item 2. The method according to Item 1, 1a to 1c, wherein the influenza HA split vaccine is also effective against an antigen-mutated influenza virus.
  • [Item 3] A method for producing an influenza HA split vaccine against an antigenic drift influenza virus, which produces an antibody that binds to LAH in the HA stem region by subjecting the influenza HA split vaccine to an acidic treatment.
  • [Item 4] The method for producing an influenza HA split vaccine according to any one of claims 1 to 3, 1a to 1c, wherein the acidic treatment is performed at pH 4.4 to 5.8.
  • [Item 5] The method for producing an influenza HA split vaccine according to any one of claims 1 to 4, 1a to 1c, wherein the influenza HA split vaccine is H3N2 type or H1N1 type.
  • [Item 5c] The method for producing an influenza HA split vaccine according to any one of claims 1 to 5, 1a to 1c, wherein the influenza HA split vaccine is H3N2 type or H1N1 type, except for the following production method.
  • 0.15M citrate buffer (pH 3.5) was added to the phosphate buffered saline suspension of the influenza HA split vaccine prepared from the H3N2 type X31 strain or the H1N1 type A / Puerto Rico / 8/34 strain. After adjusting to pH 5.0 and treating at room temperature for 30 minutes, add 1M Tris buffer (pH 8.0) to return the pH to 7.3 and centrifuge to obtain a membrane-fused HA split vaccine, and then 0.05 v / v.
  • a manufacturing method comprising the step of adding formalin at a final concentration of% and treating for several days. [Item 5a] Item 2.
  • the method for producing an influenza HA split vaccine according to any one of Items 1 to 5, 1a to 1c, 5c or 5d, wherein the influenza HA split vaccine is a single HA subtype influenza HA split vaccine. ..
  • Items 1-5, 1a-1c, 5c or 5d comprising the step of mixing two or more influenza HA split vaccine antigens produced by acidifying each single subtype of influenza HA split vaccine.
  • Item 7 Item 7. The influenza HA split vaccine according to Item 6, wherein the influenza HA split vaccine is also effective against an antigen variant influenza virus.
  • Item 8 Item 8. The influenza HA split vaccine according to Item 6 or 7, which has a shape in which the HA stem region is exposed to the outside. [Item 9] Since the HA stem region of the influenza HA split vaccine antigen is exposed to the outside, the antigenicity of LAH in the HA stem region is enhanced, and an antibody that binds to LAH in the HA stem region can be produced. Item 8. The influenza HA split vaccine according to any one of Items 6 to 8. [Item 10] An influenza HA split vaccine that produces antibodies that bind to LAH in the HA stem region, which is produced by subjecting an influenza HA split vaccine to acidic treatment.
  • An influenza HA split vaccine that produces an antibody that binds to LAH in the HA stem region, which is produced by subjecting an influenza HA split vaccine containing a single subtype to acidic treatment.
  • [Item 10e] Produces an antibody that binds to LAH in the HA stem region, which is a vaccine antigen that is a mixture of two or more influenza HA split vaccine antigens produced by acidifying an influenza HA split vaccine containing each single subtype. Influenza HA split vaccine.
  • Item 10f Item 10. The influenza HA split vaccine according to any one of Items 10 to 10e, wherein the influenza HA split vaccine is also effective against an antigen-mutated influenza virus.
  • [Item 10g] Item 10.
  • the influenza HA split vaccine according to any one of Items 10 to 10f, which is produced by the production method according to 5c or 5d.
  • An influenza HA split vaccine that is produced by acidifying an influenza HA split vaccine and produces an antibody that binds to LAH in the HA stem region, and is also effective against antigenic drift influenza virus.
  • a method for producing the influenza HA split vaccine according to any one of the above items 1 to 5, 1a to 1c, 5a, and 5b, except for the following production method A, is also included as one embodiment of the present application.
  • Manufacturing method A Polyoxyethylene sorbitan monooleate (eg, H3N2 type X31 strain or H1N1 type A / Puerto Rico / 8/34 strain suspended in phosphate buffered saline so that the final concentration is 0.1v / v%.
  • Tween80 to suspend, add diethyl ether and suspend further, leave still until the aqueous layer and the diethyl ether layer are completely separated, remove the diethyl ether layer, and remove the residual diethyl ether in the recovered aqueous layer.
  • 0.15M citrate buffer (pH3.5) to the phosphate-buffered saline suspension of the HA split vaccine obtained by evaporation under pressure, adjust the pH to 5.0 and treat at room temperature for 30 minutes
  • 1M Tris buffer (pH8.0) was added to adjust the pH to 7.3, centrifugation was performed, and the process of adding formalin to the obtained membrane fusion HA split vaccine at a final concentration of 0.05 v/v% was included. ..
  • An influenza HA split vaccine produced by the method for producing an influenza HA split vaccine according to any one of the above items 1 to 5, 1a to 1c, 5a, and 5b, excluding the production method A, is also mentioned as one aspect of the present application. ..
  • influenza HA split vaccine according to any one of Items 6 to 11 and 10a to 10f, excluding the influenza HA split vaccine produced by the production method A, is also mentioned as one aspect of the present application.
  • the method for producing an influenza HA split vaccine according to any one of the above items 1 to 5, 1a to 1c, 5a, and 5b, excluding the following production method B, is also mentioned as one aspect of the present application.
  • Manufacturing method B To the H3N2 type influenza virus particles or H1N1 type influenza virus particles suspended in phosphate buffered saline, polyoxyethylene sorbitan monooleate (for example, Tween80) was added and suspended so that the final concentration was 0.1 v / v%.
  • An influenza HA split vaccine produced by the method for producing an influenza HA split vaccine according to any one of the above items 1 to 5, 1a to 1c, 5a, and 5b excluding the production method B is also mentioned as one aspect of the present application. ..
  • influenza HA split vaccine according to any one of Items 6 to 11 and 10a to 10f, excluding the influenza HA split vaccine produced by the production method B, is also mentioned as one aspect of the present application.
  • the method for producing the influenza HA split vaccine according to any one of the above items 1 to 5, 1a to 1c, 5a, and 5b, except for the following production method C, is also an aspect of the present application.
  • Manufacturing method C 0.15M citrate buffer (pH 3.5) was added to the phosphate buffered saline suspension of the influenza HA split vaccine prepared from the H3N2 type X31 strain or the H1N1 type A / Puerto Rico / 8/34 strain. After adjusting the pH to 5.0 and treating at room temperature for 30 minutes, add 1M Tris buffer (pH8.0) to adjust the pH to 7.3 and centrifuge to obtain 0.05 v/v% of the obtained membrane fusion type HA split vaccine.
  • a manufacturing method comprising the step of adding formalin at the final concentration of.
  • An influenza HA split vaccine produced by the method for producing an influenza HA split vaccine according to any one of the above items 1 to 5, 1a to 1c, 5a, and 5b, excluding the production method C, is also mentioned as one aspect of the present application. ..
  • influenza HA split vaccine according to any one of Items 6 to 11 and 10a to 10f, excluding the influenza HA split vaccine produced by the production method C, is also mentioned as one aspect of the present application.
  • the method for producing the influenza HA split vaccine according to any one of the above items 1 to 5, 1a to 1c, 5a, and 5b, except for the following production method D, is also an aspect of the present invention.
  • Manufacturing method D 0.15M citrate buffer (pH3.5) was added to a phosphate buffered saline suspension of H3N2 or H1N1 influenza HA split vaccine to pH 5.0 and treated at room temperature for 30 minutes.
  • a method for producing comprising the steps of adding Tris buffer (pH 8.0) to adjust the pH to 7.3, centrifuging the mixture, and adding formalin to the obtained membrane-fused HA split vaccine at a final concentration of 0.05 v/v %.
  • An influenza HA split vaccine produced by the method for producing an influenza HA split vaccine according to any one of the above items 1 to 5, 1a to 1c, 5a, and 5b, excluding the production method D, is also mentioned as one aspect of the present application. ..
  • influenza HA split vaccine according to any one of Items 6 to 11 and 10a to 10f, excluding the influenza HA split vaccine produced by the production method D, is also mentioned as one aspect of the present application.
  • an influenza HA split vaccine that produces an antibody that binds to the HA stem region of influenza, which is unlikely to cause antigenic variation, can be obtained by a simple method. Therefore, an influenza HA split vaccine that is also effective against antigenic drift influenza virus can be obtained.
  • FIG. 3 is a graph showing an increase in LAH antibody titer in serum of mice inoculated with H3N2-type membrane fusion HA split vaccine ((pre-fix) and (post-fix)).
  • the method for producing an influenza HA split vaccine according to the present embodiment includes a step of acidifying the influenza HA split vaccine.
  • the influenza HA split vaccine is obtained by treating the whole particle vaccine with ether to remove the lipid component that becomes a pyrogen, and recovering the HA protein on the surface of the virus particles necessary for immunity by the density gradient translocation method.
  • HA protein is the main component for production.
  • Influenza A virus has two types of spike proteins, HA and NA (neuraminidase), which play a role in causing infection.
  • HA binds to the cells that are trying to infect and is responsible for taking the virus into the cells.
  • HA frequently causes antigenic variation.
  • NA has the role of breaking the bond between infected cells and HA and releasing the replicated virus from the cells.
  • HA of influenza A virus is divided into two regions, the spherical region and the stem region (Fig. 1).
  • the spherical region contains a receptor binding site for the virus to bind to the target cell.
  • the stem region also contains a fusion peptide sequence required for membrane fusion between the viral membrane and the cell membrane of the target cell.
  • the HA protein changes to a structure called a membrane fusion type.
  • the stem region is exposed to the outside from the virus membrane instead of the spherical region, with a large structural change in the three-dimensional structure of the antigen stem.
  • the present inventor has found as a new finding in vivo that when membrane-fused HA is used as a vaccine, an antibody that binds to LAH in the stem region is induced, and this antibody has a protective effect against antigenic drift virus strains. , The present invention was completed based on such a fact.
  • the acid treatment is not particularly limited, but is, for example, pH 2.0 to 6.5, preferably 3.0 to 6.5, more preferably 4.0 to 6.0, and further preferably 4.4 to 5.8. Specifically, for example, pH 2.0 to 2.9, 2.0 to 4.0, 2.0 to 5.0, 2.0 to 6.0, 3.0 to 4.0, 3.0 to 5.0, 3.0 to 6.0, 4.0 to 5.8, 4.0 to 6.5, 5.0 to 6.5, or 6.0 to 6.5 can be mentioned.
  • the acid used for the acid treatment is not particularly limited, but for example, phosphoric acid, citric acid, maleic acid, hydrochloric acid and the like can be used.
  • Examples of the temperature for the acid treatment include 0 ° C. to 75 ° C., preferably 10 ° C. to 60 ° C., more preferably 20 ° C. to 45 ° C., and further preferably 25 ° C. to 42 ° C. Specifically, for example, 0 ° C to 20 ° C, 5 ° C to 25 ° C, 10 ° C to 30 ° C, 15 ° C to 35 ° C, 20 ° C to 37 ° C, 25 ° C to 37 ° C, 30 ° C to 50 ° C, 38.
  • the treatment time is, for example, 5 minutes to 120 minutes, preferably 15 minutes to 60 minutes, and more preferably 20 minutes to 45 minutes. Specifically, for example, 5 to 60 minutes, 20 to 60 minutes, 15 to 120 minutes, 15 to 45 minutes, 20 to 60 minutes, 20 to 120 minutes, 45 minutes to 120 minutes, or 60. Minutes to 120 minutes can be raised.
  • HA of influenza A virus can be divided into 18 subtypes (H1 to H18) and NA into 9 subtypes (N1 to N9). It is possible to target subtypes. Further, in the method for producing an influenza HA split vaccine according to the present invention, not only type A vaccine but also type B vaccine having HA can be produced.
  • influenza HA split vaccine obtained by the production method according to the present invention produces an antibody that binds to LAH with few mutations, it can be used as an influenza virus known as an antigenic variant within the same HA subtype. Against this, cross defense may be possible. In addition, cross-reactivity can be exhibited between HA subtypes with similar LAH amino acid sequences (eg, H3 and H7 types).
  • the "influenza HA split vaccine which is a single HA subtype” refers to 18 subtypes of influenza A virus (H1 to H18) or one type of HA subtype selected from influenza B virus. Means the influenza HA split vaccine. If it is a single HA subtype, the NA subtype may be the same or different. Preferred HA subtypes include H1, H3 and B types.
  • influenza HA split vaccine consisting of each single HA subtype is subjected to acid treatment, and the obtained influenza HA split vaccine is used in multiple types (two or more). (Above) Can be mixed and manufactured.
  • influenza HA split vaccine in which two or more types of HA subtypes are mixed can be first subjected to acidic treatment to be produced.
  • inoculated as a vaccine containing a plurality of subtypes it is preferable that 1 to 3 subtypes selected from the group consisting of H1, H3 and B types are contained.
  • the influenza HA split vaccine obtained by the production method according to the present invention preferably binds to the LAH-binding monoclonal antibody more strongly than the current HA split vaccine.
  • the LAH-binding monoclonal antibody binds 1.05 times or more, preferably 1.1 times or more, more preferably 1.5 times or more, still more preferably 2 times or more stronger than the current HA split vaccine.
  • binding 1.05 times, 1.1 times, 1.5 times, or 2 times or more stronger than the current HA split vaccine means that, for example, the reciprocal of the antibody concentration when the absorbance obtained by regression is 0.7 is the current HA split.
  • the reciprocal of the antibody concentration of the vaccine is 1.05 times, 1.1 times, 1.5 times, or 2 times or more, respectively.
  • the influenza HA split vaccine of the present invention has higher binding to the LAH-binding monoclonal antibody as compared with the current HA split vaccine, and the upper limit is not particularly limited, but for example, 1.05-200 times, 1.1-150 times. , 1.5 to 100 times, 2 to 50 times range is exemplified.
  • the range of binding of the influenza HA split vaccine of the present invention to the LAH-binding monoclonal antibody compared to the current HA split vaccine is 200 with a lower limit selected from 1.05, 1.1, 1.5, 2, 3, 4, and 5.
  • the method for measuring the binding property of the influenza HA split vaccine to the LAH-binding monoclonal antibody is not particularly limited and can be measured by a general method known to those skilled in the art, but can be measured, for example, according to the method of the examples of the present application. ..
  • the LAH-binding monoclonal antibody means a monoclonal antibody that binds to LAH, and the production method thereof is not particularly limited, and can be produced by a general method known to those skilled in the art.
  • the LAH-binding monoclonal antibody can bind to a peptide corresponding to at least a portion of the LAH of the influenza virus from which the influenza HA split vaccine is derived. It means that it is a thing.
  • the current HA split vaccine means a vaccine obtained by treating a whole particle vaccine with ether to remove a lipid component which becomes a pyrogen, and can be produced, for example, by the method of Example 1 of the present application.
  • the current HA split vaccine may be an influenza HA split vaccine produced without acid treatment with respect to the influenza HA split vaccine of the present invention produced by a method having the following steps of acid treatment.
  • Formalin treatment may be performed in the production of the influenza HA split vaccine of the present invention.
  • the timing of acid treatment of influenza HA split vaccine is preferably before formalin treatment.
  • the influenza HA split vaccine antigen influenza HA split vaccine antigen capable of producing an antibody that binds to LAH in the HA stem region
  • the HA fraction used in the current influenza HA split vaccine is subjected to acidic treatment.
  • an influenza HA split vaccine antigen that has a strong effect of producing a cross-reactive antibody and is therefore more preferable as a universal influenza vaccine antigen can be obtained. That is, in the present application, it is preferable that the virus particles are treated with ether or the like, and the HA fraction excluding the fat solvent is subjected to an acid treatment and then a formalin treatment.
  • influenza HA split vaccine before the acid treatment is preferably a split vaccine not treated with formalin.
  • the commercially available influenza HA vaccine (brand name) is described in the Biologics Standards (March 30, 2004, Ministry of Health, Labor and Welfare Notification No. 155, Final Amendment: November 30, 2018, Ministry of Health, Labor and Welfare Notification No. 409). As shown above, a step of decomposing the virus with ether or the like, removing the fat solvent, and then treating with formaldehyde or a substance having an action equivalent thereto has already been performed.
  • the commercially available influenza HA vaccine (trade name) is an influenza HA split vaccine, it is preferably not used in the production of the influenza HA split vaccine of the present invention because it has already been treated with formaldehyde or the like.
  • the concentration of formalin in the formalin-treated solution when formalin is applied to the acid-treated influenza HA split vaccine is, for example, 0.0005v / v% to 10v / v%, preferably 0.001v / v% to 1v / v%. More preferably, 0.003v / v% to 0.5v / v%, and even more preferably 0.005v / v% to 0.1v / v%.
  • the time for formalin treatment is, for example, 1 hour to 10 days, preferably 2 hours to 5 days, and more preferably 12 hours to 3 days.
  • Examples of the formalin treatment temperature include 0 ° C. to 75 ° C., preferably 1 ° C. to 37 ° C., and more preferably 1 ° C. to 30 ° C.
  • the grade of formalin used is preferably a grade that can be used for medical purposes.
  • the method for producing an influenza HA split vaccine according to the present invention can have a step of including an adjuvant.
  • the adjuvant is not particularly limited, but for example, aluminum salts such as aluminum hydroxide and aluminum phosphate, chitosan, oligodeoxynucleotides, oil-in-water emulsions and the like can be used.
  • Aluminum hydroxide is preferable, and the immunogenicity can be enhanced by using aluminum hydroxide as an adjuvant.
  • the influenza HA split vaccine obtained by the production method according to the present invention can be used, for example, for additional inoculation after the lapse of a predetermined period after the initial inoculation.
  • the period from the initial vaccination to the booster vaccination is not particularly limited, but is, for example, 20 days to 3 years, preferably March to 2 years, and more preferably June to 1 year. Is.
  • the amount of the initial and booster influenza HA split vaccines is not particularly limited, but is, for example, 1 ⁇ g to 200 ⁇ g, preferably 10 ⁇ g to 30 ⁇ g, and more preferably 15 ⁇ g per dose.
  • One dose is, for example, 0.5 mL.
  • the administration method is not particularly limited in the initial vaccination and the booster vaccination, but for example, nasal, subcutaneous, intradermal, transdermal, intraocular, mucosal, or oral administration is preferable, and intramuscular administration is preferable. is there.
  • the influenza HA split vaccine obtained by the production method according to the present invention has a protective effect against antigenic drift virus strains.
  • the current HA split vaccine is prepared from H3N2 influenza virus particles (A / Fujian / 411/02 (H3N2)) and subjected to acid treatment, not only A / Fujian / 411/02 (H3N2) but also, for example.
  • H1N1 influenza virus particles A / Puerto Rico / 8/34 (H1N1)
  • H1N1 influenza virus particles A / Puerto Rico / 8/34 (H1N1)
  • A/Narita/1/09 H1N1
  • A/Beijing/262/95 H1N1
  • A/Brazil/11/78 H1N1
  • A/Chile/1/83 H1N1
  • A/New Jersey/8/76 H1N1
  • H1N1N1 A/Taiwan/1/86
  • H1N1N1 A/Yamagata/32/89 (H1N1)
  • A/NewCaledonia/20/99 H1N1)
  • A/Solomon Islands/3/ 2006 H1N1
  • A/Brisbane/59/2007 H1N1)
  • A/Mexico/4108/2009 H1N1N1
  • etc. may have an infection protective effect
  • H3N2 influenza virus particles X31 strain
  • H1N1 influenza virus particles A / Puerto Rico / 8/34 strain
  • Tween 80 was added and suspended so as to be / v%.
  • Diethyl ether was added and further suspended, and the mixture was allowed to stand until the aqueous layer and the diethyl ether layer were completely separated, and then the diethyl ether layer was removed. After repeating this ether extraction, diethyl ether remaining in the recovered aqueous layer was distilled off at normal pressure to prepare an HA split vaccine.
  • a synthetic peptide (H3; Ac-RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENADYKDDDDKC) (SEQ ID NO: 1) corresponding to a part of the stem portion (long alpha helix) was dissolved in phosphate buffered saline (pH 7.3) at 10 ⁇ g / ml. 100 ⁇ l was added to each 96-well plate. After allowing to stand overnight at 4 ° C., each well was washed 3 times with phosphate buffered saline, and 150 ⁇ l of phosphate buffered saline containing 1v / v% bovine serum albumin was added.
  • each well was washed 3 times with phosphate buffered saline, and Tween 20 was serially diluted with phosphate buffer containing 0.05 v/v% and 1 v/v% bovine serum albumin.
  • Mouse serum and a standard monoclonal antibody H3; clone name V15-5 of known concentration were added to each well in an amount of 100 ⁇ l.
  • each well was washed 3 times with phosphate buffered saline (containing 0.05% v/v% of Tween20) and contained 0.05v/v% Tween20 and 1v/v% bovine serum albumin.
  • the LAH antibody titer in the serum of BALB / c mice intraperitoneally vaccinated with the membrane-fused HA split vaccine is the LAH antibody titer in the serum of BALB / c mice intraperitoneally vaccinated with the current HA split vaccine. It was significantly higher than the antibody titer.
  • mice In the H3N2 virus infection protection experiment, sera collected from non-vaccinated mice or mice after vaccination with the current H3N2-type HA split vaccine or membrane fusion HA split vaccination were treated with BALB/c. 200 ⁇ l of each was intraperitoneally administered to mice ( ⁇ , 6-12 weeks old).
  • H3N2 influenza virus (A / Guizhou / 54/89), which has a different antigenicity from the vaccine strain, was transmitted to 5 mice lethal dose50 (5% of the virus amount that causes lethal infection in 50% of mice). The virus was infected by nasal administration under anesthesia.
  • mice were weighed and observed daily for 21 days after virus infection, and the changes in body weight and survival rate were examined. Mice with 25% weight loss were euthanized.
  • BALB / c mice inoculated with the membrane-fused HA split vaccine were able to significantly suppress the decrease in survival rate from 9 days after infection with another H3N2 influenza virus having different antigenicity.
  • H1N1 influenza virus particle C57BL / 6 mice ( ⁇ , 6-12 weeks old) with H1N1 current HA split vaccine or membrane fusion HA split vaccine (10)
  • the ⁇ g vaccine + 10 ⁇ g CpG-ODN1760 was suspended in phosphate buffered physiological saline and mixed with an equal volume of Freund's incomplete adjuvant (ROCKLAND) to make a liquid volume of 200 ⁇ l).
  • ROCKLAND Freund's incomplete adjuvant
  • a membrane-fused HA split vaccine similar to the initial vaccination, 10 ⁇ g vaccine + 10 ⁇ g CpG-ODN suspended in phosphate-buffered physiological saline and an equal amount of Freund's incomplete adjuvant (ROCKLAND)) (Mixed with 200 ⁇ l) was inoculated intraperitoneally. After 14 days after the booster vaccination, blood was collected from the vaccinated mice and the serum was collected.
  • a synthetic peptide (H1; Ac-RIENLNKK VDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNADYKDDDDKC) (SEQ ID NO: 2) corresponding to a part of the stem part (long alpha helix) was used, except that a standard monoclonal antibody (H1; clone name F2) having a known concentration was used. The same method as above was used.
  • the LAH antibody titer in the serum of C57BL/6 mice intraperitoneally inoculated with the membrane fusion type HA split vaccine was the same as that of LAH in the serum of C57BL/6 mice intraperitoneally inoculated with the current HA split vaccine. It was significantly higher than the antibody titer.
  • H1N1 influenza virus (A / Narita / 1/09) with a different antigenicity from the vaccine strain was transmitted to 5 mice lethal dose50 (5 of the amount of virus that causes lethal infection in 50% of mice). The virus was infected by nasal administration under anesthesia.
  • mice were observed daily for 20 days after virus infection to examine the survival rate. As shown in FIG. 5, in the C57BL/6 mice vaccinated with the membrane fusion type HA split vaccine, the decrease in survival rate could be significantly suppressed from the 9th day after infection with another H1N1 influenza virus having different antigenicity.
  • LAH-binding monoclonal antibody (#1 to #5 in Fig. 6) prepared from mouse or human peripheral blood infected with X31 strain by an antibody-binding ELISA method against the LAH epitope (Enzyme-Linked Immuno Sorbent Assay) , Binding to current HA split vaccine or membrane fusion HA split vaccine was measured.
  • the current HA split vaccine or membrane fusion type HA split vaccine of H3N2 influenza virus (X31 strain) was dissolved in phosphate buffered saline (pH 7.3), and 50 ⁇ l of each was added to a 96-well plate.
  • each well was washed 3 times with phosphate buffered saline, and 150 ⁇ l of phosphate buffered saline containing 1v / v% bovine serum albumin was added. After standing at room temperature for 2 hours, each well is washed 3 times with phosphate buffered saline (containing 0.05v / v% Tween 20) and stepped with phosphate buffer containing 1v / v% bovine serum albumin. 50 ⁇ l of diluted LAH-binding monoclonal antibody was added.
  • each well was washed 3 times with phosphate buffered saline (containing 0.05v / v% Tween20), 0.05v / v% Tween20 and 1v / v% bovine serum albumin.
  • Peroxidase-labeled anti-mouse IgG antibody (Southern Biotech) diluted with phosphate buffered saline containing 100 ⁇ l was added to each well.
  • the LAH-binding monoclonal antibody bound 1.05 to 21 times stronger to the membrane-fused HA split vaccine than the current HA split vaccine.
  • H3N2 influenza vaccine BALB / c mice ( ⁇ , 6-12 weeks old) with current H3N2 HA split vaccine or membrane-fused HA split vaccine (10 ⁇ g vaccine + 10 v / v% AddaVax adjuvant (InvivoGen)) in phosphate buffered saline Dissolve in water to make a liquid volume of 200 ⁇ l) was inoculated intraperitoneally. Twelve days after inoculation, blood was collected from the vaccinated mice and serum was collected.
  • the vaccine (Post-fix) subjected to the formalin treatment after the acidic treatment in the preparation step of the membrane fusion HA split vaccine is compared with the vaccine (Pre-fix) given before the acidic treatment.
  • the LAH-binding monoclonal antibody bound more strongly.
  • the LAH antibody titer in the serum of BALB / c mice intraperitoneally inoculated with the membrane-fused HA split vaccine is the LAH in the serum of BALB / c mice intraperitoneally inoculated with the current HA split vaccine. It was higher than the antibody titer. Furthermore, the membrane fusion type HA split vaccine (post-fix) had a higher LAH antibody titer than the membrane fusion type split vaccine (Pre-fix).
  • HA split vaccine Using H3N2 influenza virus particles (X31 strain), a HA split vaccine is prepared by the method described in 1. above.
  • Acid treatment After suspending the HA split vaccine in phosphate buffered saline, add 0.15M citrate buffer (pH 3.5) or dilute hydrochloric acid as acid treatment, and adjust the pH to 5 conditions (2.0, 3.0, 4.0, 5.0). , Or 6.0). After standing at 5 conditions (10°C, 25°C, 35°C, 45°C or 55°C) under 3 conditions (10 minutes, 30 minutes or 1 hour), add 1M Tris buffer (pH8.0). And return the pH to 7.3. Then, centrifuge to obtain a membrane-fused HA split vaccine. Formalin is added to the membrane-fused HA split vaccine to a final concentration of 0.05 v / v%, and the vaccine is allowed to stand for several days.
  • SEQ ID NO: 1, 2 Synthetic peptide

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EA202192398A EA202192398A1 (ru) 2019-03-04 2020-03-03 Способ приготовления сплит-вакцины против гриппа на основе гемагглютинина
SG11202109566X SG11202109566XA (en) 2019-03-04 2020-03-03 Method for preparing influenza ha split vaccine
AU2020233456A AU2020233456A1 (en) 2019-03-04 2020-03-03 Method for preparing influenza HA split vaccine
CN202080033434.5A CN114096273A (zh) 2019-03-04 2020-03-03 用于生产流感ha裂解疫苗的方法
CA3132578A CA3132578A1 (en) 2019-03-04 2020-03-03 Method for producing influenza ha split vaccine
EP20766488.9A EP3936147A4 (en) 2019-03-04 2020-03-03 METHOD FOR PREPARING A SPLIT VACCINE AGAINST INFLUENZA HEMAGGLUTININ (HA)
PH1/2021/552094A PH12021552094A1 (en) 2019-03-04 2020-03-03 Method for preparing influenza ha split vaccine
BR112021017310A BR112021017310A8 (pt) 2019-03-04 2020-03-03 Método para produzir vacina fracionada de ha de influenza
US17/435,590 US20220152191A1 (en) 2019-03-04 2020-03-03 Method for preparing influenza ha split vaccine
KR1020217030986A KR20210135261A (ko) 2019-03-04 2020-03-03 인플루엔자 ha 스플릿 백신의 제조 방법
JP2021504119A JP7545955B2 (ja) 2019-03-04 2020-03-03 インフルエンザｈａスプリットワクチンの製造方法
MX2021010685A MX2021010685A (es) 2019-03-04 2020-03-03 Método para producir vacuna fraccionada de hemaglutinina (ha) contra la influenza.
IL285984A IL285984A (en) 2019-03-04 2021-08-31 A method for producing a split influenza component

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