WO2008041710A1 - Vaccin contre la grippe précipité et inactivé et son procédé de production - Google Patents

Vaccin contre la grippe précipité et inactivé et son procédé de production Download PDF

Info

Publication number
WO2008041710A1
WO2008041710A1 PCT/JP2007/069307 JP2007069307W WO2008041710A1 WO 2008041710 A1 WO2008041710 A1 WO 2008041710A1 JP 2007069307 W JP2007069307 W JP 2007069307W WO 2008041710 A1 WO2008041710 A1 WO 2008041710A1
Authority
WO
WIPO (PCT)
Prior art keywords
virus
influenza
stock solution
influenza virus
aluminum hydroxide
Prior art date
Application number
PCT/JP2007/069307
Other languages
English (en)
Japanese (ja)
Inventor
Shuro Goto
Yo-Ichiro Kino
Toru Gotanda
Setsuo Arai
Kazuo Hosoi
Kazuyuki Takizawa
Isao Fuke
Yoshikazu Tada
Original Assignee
Juridical Foundation The Chemo-Sero-Therapeutic Research Institute
The Kitasato Institute
Denka Seiken Co., Ltd.
The Research Foundation For Microbial Diseases Of Osaka University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Juridical Foundation The Chemo-Sero-Therapeutic Research Institute, The Kitasato Institute, Denka Seiken Co., Ltd., The Research Foundation For Microbial Diseases Of Osaka University filed Critical Juridical Foundation The Chemo-Sero-Therapeutic Research Institute
Priority to KR1020097009143A priority Critical patent/KR101108913B1/ko
Priority to JP2008537539A priority patent/JP4642114B2/ja
Priority to CN200780044427.XA priority patent/CN101594881B/zh
Publication of WO2008041710A1 publication Critical patent/WO2008041710A1/fr

Links

Classifications

    • 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
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • 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
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • 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 sediment-inactivated whole-particle influenza vaccine and a method for producing the same.
  • Influenza viruses are RNA enveloped viruses having a particle size of about lOOnm in diameter belonging to the Orthomyxoviridae family, and are classified into A, B, and C types based on the antigenicity of internal proteins.
  • Influenza viruses consist of a core of ribonucleic acid (RNA) associated with an internal nucleocapsid or nucleoprotein surrounded by a viral envelope with a lipid bilayer structure and an external glycoprotein.
  • the inner layer of the virus envelope is mainly composed of matrix proteins, and the outer layer is mostly composed of host-derived lipid substances.
  • Influenza virus RNA has a segmented structure.
  • the influenza pandemic around the world is caused by influenza A virus.
  • Type A has two types of envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA), and is differentiated into 16 subtypes in HA and 9 subtypes in NA by the difference in antigenicity.
  • HA hemagglutinin
  • NA
  • influenza A viruses are distributed among animals such as birds, pigs, and horses in addition to humans, gene recombination occurs between human and animal viruses, resulting in a new antigenicity. It has been elucidated that the emergence of type I virus (new type virus) and that this new type of virus will cause a pandemic in non-immune populations. Up to now, the epidemic of Spanish funnoleza (HswlNl), Asia Infnoleenza (H2N2) and Hong Kong Infnoleenza (H3N2) is known. In recent years, outbreaks and epidemics of H5N1 subtype highly pathogenic avian influenza have been observed mainly in Southeast Asia, and more than 150 million poultry have been killed or killed so far.
  • Influenza vaccines are roughly divided into two types. For one thing, currently available influenza vaccines are split vaccines that contain subvirions that have been broken down with organic solvents / surfactants or other reagents to solubilize the purified viral particles into lipid envelopes. vaccine), or a subunit vaccine containing purified HA and NA, administered mainly intramuscularly or subcutaneously.
  • the other is a vaccine in which purified virus particles are chemically inactivated with drugs such as formalin, and is called a whole-particle vaccine. In this case, since the virus gene or virus protein is only chemically modified, the infectivity only disappears, so that the virus remains in the particle form and becomes a vaccine antigen.
  • Whole particle vaccines are considered to be more immunogenic compared to split vaccines, especially those that have not yet experienced infections! /I.e. No immune memory! Good immunity against na ⁇ ve individuals A response can be elicited. Because humans have immune memory against the new influenza, this feature of whole-particle vaccines is preferred from an efficacy perspective.
  • the highly immunogenic properties of all-particle vaccines mean that their biological activity is high and are closely related to side reactions such as local swelling, redness, or fever when inoculated. In other words, it was thought that an effective and highly safe vaccine could be developed if some method could be established that could reduce the side-reaction while maintaining the high immunogenicity of the all-particle vaccine.
  • a whole-particle vaccine having high immunogenicity has been expected as a dosage form for vaccine against new influenza.
  • H5N1 subtype it has been reported that even this whole particle vaccine cannot elicit a good immune response in humans. Measures to improve the performance were necessary.
  • Adjuvants are immunopotentiators added to vaccines to increase the immunogenicity of antigens, and many substances have been studied so far. For example, in order to increase the effectiveness of influenza vaccines, the use of an aluminum adjuvant has also been studied (Patent Document 7). It is also known to use a nonionic surfactant such as polyoxyethylene ether (Tween (registered trademark)) as an adjuvant for a vaccine for mucosal administration (Patent Document 8).
  • a nonionic surfactant such as polyoxyethylene ether (Tween (registered trademark)
  • Aluminum adjuvants which are added to DPT vaccines and hepatitis B vaccines and have been widely recognized for safety over the years, are most widely studied.
  • Aluminum adjuvant has an immunostimulatory effect and has a so-called deposit effect that keeps a small amount of antigen locally by adsorbing the antigen, and therefore has an excellent characteristic that the antigen amount of the vaccine can be reduced.
  • a pandemic vaccine that is intended for all humans, how many vaccines can be supplied is a major proposition, and this characteristic is a major advantage in vaccine design. Therefore, if aluminum adjuvant is used as a vaccine, the adsorptivity of the antigen to the aluminum gel is a very important factor.
  • Patent Document 1 WO91 / 03552
  • Patent Document 2 WO01 / 004333
  • Patent Document 3 WOOO / 060050
  • Patent Document 4 WO2001 / 083794
  • Patent Document 5 WO2002 / 067983
  • Patent Document 6 WO2002 / 074336
  • Patent Document 7 WOOO / 015251
  • Patent Document 8 WO 99/52549
  • the object of the present invention is to reduce the amount of antigen, the amount of antigen and the amount of side effects that are effective against a new influenza virus, particularly immunogenicity is low! It is to provide a fluenza vaccine and a method for producing the same. Means for solving the problem
  • the inventors of the present invention have made extensive studies in view of the above problems, and as a result, combined with an inactivated virus whole particle an adjuvant that is highly immunogenic to humans and has few side effects. It has been found that it can be achieved, and the present invention has been completed. That is, the present invention is as follows.
  • a sedimentation-inactivated infnorenza vaccine comprising, as active ingredients, an aluminum hydroxide gel prepared from sodium carbonate and potassium aluminum sulfate and influenza virus whole particles.
  • Adsorption rate of all influenza virus particles on aluminum hydroxide gel is 90% or more
  • the hemagglutinin activity of all influenza virus particles with respect to the total hemagglutinin activity is 80% or more.
  • the aluminum concentration is 0 ⁇ lmg / mL or more and less than 0 ⁇ 8mg / mL, and the concentration of all particles of influenza virus is 3 ⁇ 4 ⁇ ;! OO ⁇ g HA / mL, [1] to [3]
  • the vaccine according to any one of the above.
  • influenza virus in the virus stock solution before mixing with the aluminum hydroxide gel has the following properties:
  • Influenza prophylaxis including administering to a subject an effective amount of precipitated inactivated influenza vaccine containing aluminum hydroxide gel prepared from sodium carbonate and potassium aluminum sulfate and all influenza virus particles as active ingredients Or a mitigation method.
  • Adsorption rate of all influenza virus particles on aluminum hydroxide gel is 90% or more
  • Total hemagglutinin activity is 80% or more.
  • the aluminum concentration is 0.1 mg / mL or more and less than 0.8 mg / mL, and the concentration of all influenza virus particles is 3.4 to;! OO ⁇ g HA / mL, [11] to [13 What The method according to any one of the above.
  • influenza virus in the virus stock solution before mixing with the aluminum hydroxide gel has the following properties:
  • Adsorption rate of all influenza virus particles on aluminum hydroxide gel is 90% or more
  • Total hemagglutinin activity is 80% or more.
  • the aluminum concentration is 0.1 mg / mL or more and less than 0.8 mg / mL, and the concentration of all influenza virus particles is 3.4 to;! OO ⁇ g HA / mL, [18] to [20 Use of any one of the above.
  • influenza virus in the virus stock solution before mixing with the aluminum hydroxide gel has the following properties: (i) When the virus stock solution is analyzed by analytical sucrose density gradient centrifugation, more than 80% of the total hemagglutinin activity accumulates in the virus particle fraction, and
  • a novel influenza vaccine or a sedimentation-inactivated whole-particle influenza vaccine that uses whole influenza particles as an active ingredient, induces an effective immune response with a small amount of antigen to humans, and has few side reactions. Will be able to respond to the global epidemic of the new Inflenenza. Since the vaccine of the present invention contains an aluminum hydroxide gel (salt) prepared from sodium carbonate and potassium aluminum sulfate as an adjuvant, it can induce an effective immune response against humans with a small amount of antigen. Sufficient supply of influenza vaccine can be secured. According to the method for producing a precipitation-inactivated whole-particle influenza vaccine of the present invention, a novel influenza vaccine that induces an effective immune response and has few side reactions is provided by maintaining the antigenicity of the virus particles well. be able to.
  • FIG. 1 shows a schematic diagram of the production process of the virus stock solution and vaccine of the present invention.
  • FIG. 2 shows a schematic diagram of a production process of a virus stock solution and a vaccine of a comparative example.
  • FIG. 3 is a graph showing the effect of vaccines prepared using the gels of Example 2 and Comparative Example 2 on animals.
  • Fig. 4 is a graph showing the results of a vaginal test conducted on healthy adult men aged 20 to 40 years old using a precipitated new influenza vaccine produced by Method B.
  • FIG. 5 is an electron micrograph of virus stock solutions produced in Example 1 (Method A) and Comparative Example 1 (Method B), respectively.
  • FIG. 6 is a graph showing the analytical results of analytical sucrose density gradient centrifugation for the virus stock solutions produced in Example 1 (Method A) and Comparative Example 1 (Method B), respectively.
  • FIGS. 7A and B are graphs showing the results of gel filtration HPLC analysis of the virus stock solutions produced in Example 1 (Method A) and Comparative Example 1 (Method B), respectively.
  • FIG. 8 is a graph showing neutralizing antibody titers after vaccination in phase I clinical trials.
  • FIG. 9 is a graph showing HI antibody production in vaccinated mice.
  • FIG. 10 is a graph showing the results of an abnormal toxicity negative test in guinea pigs.
  • FIG. 11 is a graph showing the virus adsorption rate in vaccines prepared using the gels of Example 2 and Comparative Example 2.
  • the present invention provides a precipitated inactivated influenza vaccine comprising, as active ingredients, an aluminum hydroxide gel prepared from sodium carbonate and potassium aluminum sulfate and all influenza virus particles.
  • the vaccine of the present invention is characterized by containing an aluminum hydroxide genore prepared as an adjuvant by using sodium carbonate and aluminum sulfate.
  • an adjuvant raw material sodium carbonate and potassium ammonium sulfate can be used without limitation the reagents usually used for preparing aluminum hydroxide gel.
  • aluminum potassium sulfate aluminum potassium sulfate decahydrate AlK (SO 4) -12H 2 O, which is also referred to as potash ivan, is preferably used.
  • influenza viruses include all currently known subtypes as well as subtypes that will be isolated and identified in the future. No influenza epidemic has been observed so far, and from the standpoint of effectively preventing human infection in the future, influenza viruses have been excluding HI and H3. H;! ⁇ 16 (ie, H2 and H4 ⁇ A subtype consisting of a combination of a type selected from 16) and a type selected from N1-9 is preferred. These subtypes are also called new influenza viruses.
  • the subtype is more preferably a combination of a type selected from H5, 7 and 9 and a type selected from Nl to 9 forces.
  • Influenza viruses can be one strain belonging to the same subtype or two or more strains belonging to different subtypes, or two or more strains belonging to the same subtype.
  • influenza virus total particle refers to a virus particle purified from a virus suspension obtained by culturing influenza virus while retaining the form of the virus. Therefore, the influenza vaccine of the present invention means a vaccine excluding a split vaccine containing a subvirion and a subunit vaccine containing purified HA or NA, and is also called a whole particle vaccine.
  • the present inventors can obtain an effective vaccine with a small amount of antigen and less side-effect reaction.
  • the present inventors can obtain an effective vaccine with a small amount of antigen and less side-effect reaction.
  • influenza virus whole particles are preferably purified from a virus suspension in the absence of a surfactant and ethers.
  • a virus solution containing whole influenza virus particles purified or concentrated for adsorption with aluminum hydroxide gel is referred to as a “virus stock solution”. The method for purifying or concentrating influenza virus will be described later.
  • the vaccine of the present invention preferably has the following properties (1) and (2) in order to make the vaccine effective with a small amount of antigen and with few side reactions. .
  • Adsorption rate of all influenza virus particles on aluminum hydroxide gel is 90 % Or more. If this adsorption rate exceeds 90%, the synergistic effect with the whole virus particles will increase.
  • the adsorption rate refers to aluminum hydroxide gel and all influenza virus particles mixed in physiological saline at 4-8 ° C, and then the supernatant is recovered and the hemagglutinin (HA) titer of the supernatant is recovered. Or the value obtained by measuring the force to measure the protein mass, or the protein mass adsorbed on the gel. A detailed method for measuring the adsorption rate is described in the examples.
  • the adsorption rate is more preferably 95% or more, and more preferably 97% or more.
  • the adsorption rate can be less than 90% when the virus is mixed under the condition that all the virus particles are supersaturated with respect to the gel. Therefore, in the present invention, when the adsorption rate is less than 90%, What is necessary is just to reduce an antigen amount suitably so that it may become 90% or more.
  • the hemagglutinin activity of all influenza virus particles with respect to the total hemagglutinin activity is 80% or more. More preferably, it is 85% or more, and still more preferably 90% or more. This ratio is measured by the following operation.
  • the inactivated virus solution is fractionated by sucrose density gradient centrifugation before adsorbing to the gel. Usually, viral whole particles accumulate at a sucrose concentration of around 40%, and can be obtained by dividing the amount of hemagglutinin in this fraction by hemagglutinin in all fractions.
  • the amount of hemagglutinin can be measured as an HA value using hemagglutination ability as an index.
  • the presence of over 80% of total hemagglutinin on all influenza virus particles is an indicator that the virus particle morphology in the vaccine is almost completely maintained. Such good whole particle property is important for obtaining the synergistic effect in the vaccine of the present invention.
  • virus particles can also be confirmed by the following method.
  • the absorption spectrum at 280 nm has no shoulder! / is a single peak. Whether or not the absorption spectrum is a single peak without a shoulder can be confirmed by observing the chart obtained by the high performance liquid chromatography with the naked eye and using the usual judgment criteria in the field.
  • the vaccine of the present invention preferably has an aluminum concentration of 0.1 mg / mL or more and less than 0.8 mg / mL, more preferably 0.2 mg / mL or more and less than 0.8 mg / mL.
  • concentration is the value calculated from the usage-amount of the potash iban as a raw material.
  • the aluminum concentration can be determined by atomic absorption analysis.
  • the concentration of all particles of influenza virus is preferably 3.4 to 100 g HA / mL, more preferably 10 to 30; ⁇ HA / mL.
  • the concentration is a value obtained by measuring the HA concentration of the whole protein of wineless. The method for measuring protein is described in the examples.
  • the vaccine of the present invention may further contain a pharmaceutically acceptable carrier in addition to influenza virus whole particles and aluminum hydroxide gel.
  • a pharmaceutically acceptable carrier in addition to influenza virus whole particles and aluminum hydroxide gel.
  • carriers commonly used in vaccine production can be used. Specific examples include saline, buffered saline, dextrose, water, glycerol, isotonic aqueous buffers, and combinations thereof. It is done. Further, a preservative (eg, thimerosal), an isotonic agent, a pH adjuster, an inactivating agent (eg, formalin) and the like are appropriately blended therein.
  • the vaccine of the present invention may be administered systemically or locally.
  • systemic administration it is injected intramuscularly, subcutaneously, or intradermally.
  • topical administration administration to the nasal cavity and the pharynx is exemplified, and examples of the administration method include spraying and application.
  • the administration target of the vaccine of the present invention is not particularly limited, and examples thereof include mammals including humans and birds.
  • the vaccine of the present invention can be used to prevent or reduce the symptoms of influenza.
  • the present invention provides a method for preventing or reducing influenza comprising the step of administering an effective immunization amount of the vaccine of the present invention to a subject.
  • the method for administering the vaccine is as described above.
  • the dose is determined in consideration of the age, sex, weight, etc. of the subject, but as an antigen, it is usually 1.7-50 ⁇ HA, preferably 5 to 15; ⁇ HA is administered once or twice or more can do.
  • the administration is performed multiple times, and in this case, administration is preferably performed with an interval of 1 to 4 weeks.
  • the present invention also provides a method for producing the sediment-inactivated influenza vaccine.
  • the production method of the present invention comprises preparing influenza virus whole particles as an antigen, In the entire process of adsorption onto the aluminum hydroxide gel, a solvent that does not contain a surfactant and ethers is used.
  • surfactant refers to any surfactant that is usually used in the field of production of cutin, and examples include an anionic surfactant, a cationic surfactant, and a nonionic surfactant. It is done.
  • a nonionic surfactant is often used so as not to lower the immunogenicity of the cutin, and for example, polyoxyethylene ether (Tween (registered trademark)) is widely used. These surfactants are not used in the production method of the present invention.
  • the ethers mean all ethers usually used in the field of vaccine production, and examples thereof include dimethyl ether. These ethers are not used in the production method of the present invention.
  • Influenza virus can be a strain isolated from an infected animal or patient, or a recombinant virus established by genetic engineering in cultured cells! /. Influenza virus may be cultured by infecting a culture cell that may be cultured by inoculating a virus in the allantoic cavity of a chicken egg. The culture conditions are 30 to 37 ° C for chicken eggs;! To 7 days, and in the case of cultured cells, the medium, temperature, and culture time are set according to the cells used. After completion of the culture, the virus suspension is collected by a conventional method.
  • the resulting virus suspension is centrifuged or filtered for clarification and, if necessary, ultrafiltered for concentration. Then, for virus purification, ultracentrifugation such as sucrose density gradient centrifugation is performed! / ⁇ Purified virus solution is obtained.
  • the purified virus solution is inactivated.
  • a formalin treatment As a method for inactivating the virus, a formalin treatment, an ultraviolet ray irradiation, a force S such as / 3_propiolatatone, and a formalin treatment are preferable.
  • the formalin treatment conditions can be appropriately set.
  • the formalin treatment can be performed by incubating at a low temperature for several weeks using a 0.02 v / v% formalin solution.
  • the ability to prepare virus stock solution by filtering virus solution The purpose is to concentrate the virus simultaneously with the filtration.
  • Examples of such filtration include ultrafiltration. Ultrafiltration can be performed by conventional methods.
  • Adsorption of influenza virus and aluminum hydroxide gel can be achieved by using a virus stock solution adjusted to a predetermined protein concentration and an aluminum hydroxide gel suspension for several hours at a low temperature of about 4-8 ° C. ⁇ Mix it! / Known aluminum hydroxide gels can be used. As described above, it is preferable to use sodium carbonate and potassium aluminum sulfate.
  • Example 1 and FIG. 1 a virus stock solution and a prototype vaccine were produced in the same manner as in Example 1 except that Tween (registered trademark) 80 was used in the purification process of the virus suspension.
  • Figure 2 shows the outline.
  • 0. 21M salt aqueous solution (pH 2.95) 50. While maintaining OmL, 0.50M NaOH aqueous solution 63.lmL was gradually added dropwise at room temperature and added for 55 minutes. P at completion of addition H was 9.00. The solution was stirred at room temperature for an additional 30 minutes. The pH at this time was 9.00. The stirred solution was allowed to stand at 4 ° C. for 21.5 hours to obtain 113 mL of aluminum hydroxide gel (pH 8.4). The obtained aluminum hydroxide gel was subjected to centrifugal separation at 4 ° C. and 2000 rpm for 10 minutes, and the precipitate was collected.
  • Example 2 The virus stock solution obtained in Example 1 and the aluminum hydroxide gel obtained in Example 2 were mixed to obtain a sedimentation-inactivated whole particle vaccine.
  • the virus stock solution obtained in Comparative Example 1 and the aluminum hydroxide gel obtained in Comparative Example 2 were mixed to obtain a sedimentation-inactivated whole particle vaccine.
  • NIBRG 14 strain influenza inactivated whole particle vaccine stock solution (FPBM Z0401, real (Prepared by the method described in Example 1)
  • the aluminum gel and the virus suspension were mixed, and the supernatant was collected.
  • the tantalum content and HA value of the supernatant were measured, and the respective adsorption rates were calculated.
  • the mixture was centrifuged (Tomy micro high-speed centrifuge MC-150, 1000 rpm, 5 minutes), and the supernatant was collected.
  • the collected supernatant was used as a protein content measurement material or a HA value measurement material.
  • the protein content of the supernatant was measured using Micro BCA Protein Assay Kit (Pierce).
  • the standard substance used was the BSA solution supplied with the kit.
  • the HA value of the supernatant was measured according to the section “Measurement of influenza HA titer” in the “Pathogen Detection Manual” of the National Institute of Infectious Diseases.
  • As the blood cell suspension 0.5% chicken erythrocyte suspension was used.
  • the adsorption rate (%) was calculated by dividing the antigen content in the supernatant of each specimen by the antigen content in the supernatant of a control specimen not containing gel.
  • a / Wyoming / 3/2003 has good adsorption ability to any hydroxide hydroxide gel.
  • NIBRG-14 has poor adsorption to aluminum chloride aluminum hydroxide gel, only to potassium aluminum hydroxide gel. It showed good adsorption. This indicates that when the H5N1 subtype strain is the antigen, it is better to use potassium aluminum hydroxide gel manufactured by Calimiyoban.
  • Aluminum hydroxide gel prepared in Comparative Example 2
  • aluminum hydroxide gel prepared in Example 2
  • Example 2 which is made from potash ivan
  • An inactivated virus antigen supplemented with was prepared. These antigens were immunized to ddY mice twice at 3-week intervals so that each mouse had a dose of 0.5, 0.05 (protein mass). Secondary immunization Blood was collected 2 weeks later, and the resulting serum was The HI antibody titer was measured using this (Fig. 3).
  • a phase I study was conducted on healthy adult males aged 20 to 40 years using the precipitating new influenza vaccine investigational product produced in Comparative Example 3 (Method B).
  • the vaccine was given twice subcutaneously at 3 week intervals.
  • the amount of antigen per inoculation was compared in three types: 1. HA (low dose), 5 g HA (medium dose) and 15; ⁇ HA (high dose).
  • the neutralizing antibody titer that strongly reflects the protective ability against infection with the virus was compared before and after vaccination and used as an index.
  • FIG. 4 even after the 15 ⁇ g HA vaccine was administered twice, the seroconversion rate of antibody titer of 1:40 or more was not satisfactory at 40% or less.
  • Example 3 The immunogenicity in mice and rabbits of the new influenza vaccines produced in Example 3 (Method A) and Comparative Example 3 (Method B) was evaluated using the HI antibody titer as an index.
  • each group of 8 to 10 BALB / c mice was treated with an aluminum hydroxide gel (alum) adjuvant vaccine containing inactivated virus antigens to achieve 0 ⁇ 04 to 3 g HA / animal. Inoculated twice into the lower limb muscles at 3-week intervals. The HI antibody titer of the mouse serum 14 days after the secondary immunization was measured, and the average HI antibody titer of 10 mice in each group is shown in the table.
  • alum aluminum hydroxide gel
  • a 15 mL to 60% sucrose density gradient was overlaid with a 1 mL stock solution with a protein concentration of 180 g / mL and centrifuged at 4 ° C and 18000 rpm for about 16 hours. About 0.5 mL fraction was fractionated per fraction, and sucrose concentration, HA activity and protein concentration of each fraction were measured. The HA value was measured three times using chicken erythrocytes, and the geometric mean value was graphed. The protein concentration was analyzed by densitometry after 50 L of each fraction was transferred to a PVDF membrane, stained with Coomassie brilliant blue (Figure 6).
  • HA activity and protein peaks were observed in the fraction with a sucrose concentration of about 40%.
  • method A more than 80% of the applied HA activity was accumulated at the virus peak, but in method B, the accumulation rate remained below 50%.
  • Method B the protein peak and the HA activity were not observed even in the fraction with a sucrose concentration of about 17%.
  • UV absorptiometer (measurement wavelength: 210, 280nm)
  • FIG. 7A Shown are gel filtration chromatograms of the stock solution (FIGS. 7A and B, top: 210 nm, bottom: 280 ⁇ m). From FIG. 7, a peak expected to be a virus particle was observed at a retention time of 9.9 minutes. In Method B, a shoulder was also observed at a retention time of 10.9 minutes.
  • a new type of precipitated influenza vaginal cutin drug containing the aluminum hydroxide gel prepared in Example 1 was manufactured, and the vaginal test was conducted on healthy adult men aged 20 to 40 years (FIG. 8).
  • This vaccine was given twice subcutaneously or intramuscularly at 3-week intervals.
  • the amount of antigen per inoculation was compared in three types: 1.7 g HA (low dose), 5 g HA (medium dose), and 15 g HA (high dose).
  • the neutralizing antibody titer that strongly reflects the ability to protect against viruses was compared before and after vaccination. In the high-dose group, the antibody titer rose 73% in the subcutaneous group, and in all cases in the intramuscular group, the antibody titer increased more than 4-fold! /.
  • the antibody titer increased in 58% in the subcutaneous inoculation group and 75% in the intramuscular group. From this, it was confirmed that the precipitated influenza vaccine to which aluminum hydroxide gel was added often elicited an immune response against humans.
  • mice of the new influenza vaccine supplemented with the aluminum hydroxide gel (Alham) prepared in Example 1 were evaluated using the HI antibody titer as an index.
  • Ten mice in each group were inoculated subcutaneously or intramuscularly twice at intervals of 3 weeks with a product with or without alum containing 0.04 to 3 g HA / inactivated virus antigen.
  • the HI antibody titer of mouse serum 14 days after secondary immunization was measured, and the average HI antibody titer of 10 mice in each group is shown in FIG. From Fig. 9, the HI antibody titer of the group with alamu was higher in both the subcutaneous and intramuscular administration groups compared to the formulation without alamu. From this, it was considered that the addition of alum induces a higher antibody titer than the non-added group.
  • Experimental Example 8 Vaccine safety

Abstract

La présente invention concerne un vaccin contre le virus entier de la grippe, précipité et inactivé, qui peut agir efficacement sur un nouveau type de virus de la grippe, particulièrement un nouveau type de virus doté d'une faible immunogénicité, ne contient qu'une faible quantité d'antigène, et ne produit que très peu d'effets secondaires indésirables. L'invention porte également sur un procédé permettant de produire le vaccin. Elle concerne spécifiquement un vaccin contre la grippe, précipité et inactivé, contenant un gel d'hydroxyde d'aluminium préparé à partir de carbonate de sodium et de sulfate d'aluminium et de potassium, ainsi que des particules virales de la grippe, entières et isolées, en tant que principes actifs. L'invention concerne en outre un procédé permettant de produire le vaccin contre la grippe, précipité et inactivé, le procédé étant caractérisé par la mise en œuvre de chaque étape au moyen d'un solvant ne contenant aucun agent tensioactif ou aucun éther.
PCT/JP2007/069307 2006-10-02 2007-10-02 Vaccin contre la grippe précipité et inactivé et son procédé de production WO2008041710A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
KR1020097009143A KR101108913B1 (ko) 2006-10-02 2007-10-02 침강/비활성화 인플루엔자 백신 및 그의 제조 방법
JP2008537539A JP4642114B2 (ja) 2006-10-02 2007-10-02 沈降不活化インフルエンザワクチンおよびその製造方法
CN200780044427.XA CN101594881B (zh) 2006-10-02 2007-10-02 吸附灭活流感疫苗及其制备方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2006271295 2006-10-02
JP2006-271295 2006-10-02

Publications (1)

Publication Number Publication Date
WO2008041710A1 true WO2008041710A1 (fr) 2008-04-10

Family

ID=39268564

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2007/069307 WO2008041710A1 (fr) 2006-10-02 2007-10-02 Vaccin contre la grippe précipité et inactivé et son procédé de production

Country Status (4)

Country Link
JP (1) JP4642114B2 (fr)
KR (1) KR101108913B1 (fr)
CN (1) CN101594881B (fr)
WO (1) WO2008041710A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09124493A (ja) * 1995-11-02 1997-05-13 Fuji Chem Ind Co Ltd 制酸剤及びその製法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09124493A (ja) * 1995-11-02 1997-05-13 Fuji Chem Ind Co Ltd 制酸剤及びその製法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LU X. ET AL.: "A Mouse Model for the Evaluation of Pathogenesis and Immunity to Influenza A (H5N1) Viruses Isolated from Humans", JOURNAL OF VIROLOGY, vol. 73, no. 7, 1999, pages 5903 - 5911, XP002162397 *
LU X. ET AL.: "Immunity to Influenza A H9N2 Viruses Induced by Infection and Vaccination", JOURNAL OF VIROLOGY, vol. 75, no. 10, 2001, pages 4896 - 4901, XP003022014 *
LUKE C.J. ET AL.: "Vaccines for Pandemic Influenza", EMERGING INFECTIOUS DISEASES, vol. 12, no. 1, 2006, pages 66 - 72, XP003022015 *
WADMAN M.: "Race is on for flu vaccine", NATURE, vol. 438, no. 7064, 3 November 2005 (2005-11-03), pages 23, XP003022016 *

Also Published As

Publication number Publication date
CN101594881A (zh) 2009-12-02
KR20090087878A (ko) 2009-08-18
CN101594881B (zh) 2017-07-21
JPWO2008041710A1 (ja) 2010-02-04
KR101108913B1 (ko) 2012-02-09
JP4642114B2 (ja) 2011-03-02

Similar Documents

Publication Publication Date Title
JP6949027B2 (ja) ジカウイルスワクチン
AU646257B2 (en) Oral vaccine comprising antigen surface-associated with red blood cells
JP3980884B2 (ja) プロテオソーム・インフルエンザ・ワクチン
US11684669B2 (en) CpG-adjuvanted SARS-CoV-2 virus vaccine
CN111603556B (zh) 一种新型冠状病毒亚单位纳米疫苗的制备和应用
CN113730566B (zh) 一种流感新冠联合疫苗及其制备方法
TW202203967A (zh) 不活化ＳＡＲＳ—ＣｏＶ—２病毒疫苗
CN112043825A (zh) 一种基于新型冠状病毒突刺蛋白s1区域预防新型冠状病毒感染的亚单位疫苗
CN108697786B (zh) 含有固定化病毒粒子的疫苗
CN112569348B (zh) 一种带状疱疹疫苗
JP2002532435A (ja) Ｒｓｖサブユニット成分およびインフルエンザウイルス調製物を含む多価免疫原性組成物
JP5918870B2 (ja) インフルエンザに対する改善されたワクチン接種
JP2011524372A (ja) インフルエンザワクチン接種のための新規ペプチドアジュバント
JP6985384B2 (ja) 弱毒変異型ジカウイルスを含むワクチン組成物
US20120107354A1 (en) Viral vaccine and process for preparing the same
Ewasyshyn et al. Comparative analysis of the immunostimulatory properties of different adjuvants on the immunogenicity of a prototype parainfluenza virus type 3 subunit vaccine
WO2016010081A1 (fr) Vaccin contenant des particules pseudo-virales
CN107050446B (zh) 经修饰的季节流感-rsv联合疫苗及其制备方法
JP7288270B2 (ja) 不活化全粒子インフルエンザワクチン及びその調製法
JP4642114B2 (ja) 沈降不活化インフルエンザワクチンおよびその製造方法
Noori et al. Construction of Influenza A/H1N1 virosomal nanobioparticles

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200780044427.X

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07829047

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2008537539

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 1020097009143

Country of ref document: KR

122 Ep: pct application non-entry in european phase

Ref document number: 07829047

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)