JP2009149583A - Flavivirus infectious disease vaccine and adjuvant for flavivirus infectious disease vaccine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an adjuvant for a flavivirus infectious disease vaccine and an effective and safe flavivirus infectious disease vaccine for single inoculation using the same. <P>SOLUTION: The flavivirus infectious disease vaccine comprises a safe flavivirus antigen, and a biodegradable nanoparticle mainly composed of a polyamino acid or an aluminum hydroxide gel as an effective adjuvant. The adjuvant for the flavivirus infectious disease vaccine comprises a biodegradable nanoparticle mainly composed of a polyamino acid. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a flavivirus infectious disease vaccine for single inoculation containing a flavivirus infectious disease vaccine and an immunological adjuvant (adjuvant) for flavivirus infectious disease vaccine. In detail, biodegradable γ-PGA nanoparticles (γ-PGA-NP) or aluminum hydroxide gel is used as an adjuvant to enhance the protective immune immunity. The present invention relates to a flavivirus infection vaccine for preventing flavivirus infection.

  The flavivirus is an RNA virus having a single-stranded plus-strand RNA as a genome (about 11 kb). This virus includes Japanese encephalitis virus, West Nile virus, yellow fever virus, dengue virus, tick-borne encephalitis virus, and St. Louis encephalitis virus. Flaviviruses are widespread in vertebrates, many of which are transmitted through mosquitoes and ticks. Many vertebrate infections are subclinical, but rarely can cause serious illnesses such as those of the central nervous system characterized by encephalitis. A large number of clinical cases and death cases are reported every year worldwide. In recent years, the number of flavivirus infections has decreased in Japan, but there are strong concerns about landing in Japan from overseas infected areas. Therefore, there is a strong demand for measures to prevent the spread of infection.

There is an urgent need to develop an effective vaccine to prevent flavivirus infection.
Taking Japanese encephalitis (hereinafter also referred to as JE) as an example, the Foundation for Microbial Diseases of Osaka University received a formalin-inactivated mouse brain-derived JE vaccine (hereinafter also referred to as JE vaccine or JE vaccine Biken) from Beijing 1 strain. Manufactured and distributed mainly in Japan. The study group also produced another mouse brain-derived JE vaccine (JE-VAX) from Nakayama strain of Japanese encephalitis virus (hereinafter also referred to as JEV). JE-VAX is distributed commercially and widely available internationally (Non-Patent Document 1).
Internationally, the recommended dosing schedule for JE-VAX vaccine is a total of 3 vaccinations on days 0, 7, and 30 respectively, and additional booster vaccinations are required after the first series However, the optimal time has not been solved sufficiently.
In Japan, child JE vaccine is required by law. The first series of immunizations requires 3 vaccinations by 90 months of age, as well as a single booster between 9 and 12 years of age. The Japanese government recommends additional regular boosters to maintain immunity against JEV. As described above, the problem of the current Japanese encephalitis vaccine is that it is inconvenient and uncomfortable to require multiple basic immunizations and further immunizations. It is to give little JE prevention in a short period of time in emergency situations such as travel. Therefore, there is a great need for the development of an effective single vaccination vaccine against JEV.
However, useful adjuvants for these flavivirus infection vaccines have not yet been developed.

A number of adjuvants have been evaluated for use in vaccines. However, the only adjuvant commonly used clinically in Japan with an unacceptable level of toxicity is an aluminum-based mineral salt (aluminum adjuvant; alum).
γ-PGA is a natural component of natto, a traditional Japanese food, and is a bacterial exopolymer produced by a specific strain of Bacillus natto (Non-Patent Documents 2 to 5). We have created biodegradable (γ-PGA) -graft-L-phenylalanine (L-PAE) copolymers (γ-PGA nanoparticles [γ-PGA-NP]), where γ-PGA-NP is Because of its natural origin, water solubility, biodegradability and safety, it is well suited for medical applications, and has been proposed for use in drug delivery, tissue engineering, and as a thermosensitive polymer.
Kurane I.I. Et al., Vaccine, 2000; 18 Suppl 2: p33-35. Akagi T. et al. Et al., Macromol Biosci, 2005; 5 (7): p598-602. Akagi T. et al. Et al., J Control Release, 2005; 108 (2-3): p226-236. Akagi T. et al. Et al., J Biometer Sci Poly Ed, 2006; 17 (8): p875-92. Akagi T. et al. Et al., Biomaterials, 2007; 28 (23): p3427-3436.

  The object of the present invention is to provide a flavivirus infectious disease vaccine capable of protecting against infection by a single inoculation containing a safe and effective adjuvant for flavivirus infectious disease vaccine.

  The present inventors have developed a single inoculation method that can obtain an immunoprotective ability equivalent to several inoculations with a single inoculation for a flavivirus vaccine that requires multiple inoculations such as Japanese encephalitis vaccine. want to be. It is also necessary to develop a safe next-generation vaccine that does not use Japanese encephalitis virus in order to improve the safety of vaccine production. Therefore, a vaccine for single inoculation using an adjuvant in combination with an existing Japanese encephalitis vaccine or a Japanese encephalitis hollow particle (JE-VLP) or West Nile hollow particle that can be produced without using Japanese encephalitis virus and has a vaccine effect. Started development. As an adjuvant, γ-PGA-NP prepared by partially hydrophobizing aluminum hydroxide gel and γ-PGA derived from Bacillus natto with L-ethylalanine ethyl ester to make amphiphile was used. By adding the nanoparticles as an adjuvant to the flavivirus vaccine, it was found that a single inoculation induces sufficient protective immunity, and the present invention has been completed. That is, the present invention is as follows.

[1] A flavivirus infection vaccine containing a flavivirus antigen for preventing flavivirus infection and an adjuvant.
[2] The vaccine according to [1] above, wherein the adjuvant is biodegradable nanoparticles mainly composed of polyamino acids.
[3] The vaccine according to [1] above, wherein the adjuvant is aluminum hydroxide gel.
[4] The vaccine according to [3] above, wherein the aluminum hydroxide gel is prepared from sodium carbonate and potassium aluminum sulfate.
[5] The vaccine according to any one of [1] to [4] above, which is a vaccine for preventing flavivirus infection by inoculation once.
[6] The vaccine according to any one of [1] to [5] above, wherein the flavivirus is Japanese encephalitis virus and / or West Nile virus.
[7] The vaccine according to any one of [1] to [6] above, wherein the flavivirus antigen is flavivirus-like hollow particles.
[8] The vaccine according to any one of [1] to [6] above, wherein the flavivirus antigen is whole virus particles.
[9] The polyamino acid is poly (γ-glutamic acid), poly (α-aspartic acid), poly (ε-lysine), poly (α-glutamic acid), poly (α-lysine), polyasparagine, and modified products thereof The vaccine according to any one of [2], [5] to [8] above, which is selected from the group consisting of derivatives thereof and mixtures thereof.
[10] Any of the above-mentioned [2], [5] to [9], wherein the polyamino acid is selected from the group consisting of poly (γ-glutamic acid), a modified form thereof, a derivative thereof and a mixture thereof The vaccine according to 1.
[11] The vaccine according to any one of [2], [5] to [10] above, wherein the polyamino acid is amphiphilized.
[12] The vaccine according to any one of [2], [5] to [11] above, wherein the polyamino acid is a graft copolymer of poly (γ-glutamic acid) and phenylalanine ethyl ester.
[13] An adjuvant for a flavivirus infection vaccine comprising biodegradable nanoparticles mainly composed of a polyamino acid.

  The adjuvant of the present invention is an effective and safe adjuvant for flavivirus infection vaccines. The flavivirus infectious disease vaccine of the present invention containing a flavivirus antigen with γ-PGA-NP or aluminum hydroxide gel as an adjuvant exhibits sufficient protective immunity equivalent to that obtained by inoculating a conventional plain vaccine multiple times. It is possible to induce by inoculating once.

  The present invention provides a flavivirus infection vaccine containing a flavivirus antigen for preventing flavivirus infection and an adjuvant.

  Specifically, the present invention provides a flavivirus infection vaccine containing flavivirus antigens and biodegradable nanoparticles based on polyamino acids. Since the vaccine has a sufficient effect by a single inoculation, the use is preferably a single inoculation.

  The present invention also provides a flavivirus infection vaccine comprising a flavivirus antigen and an aluminum hydroxide gel. Since the vaccine has a sufficient effect by a single inoculation, the use is preferably a single inoculation.

  In the present invention, flavivirus is a virus belonging to the genus Flavivirus in the Flaviviridae family, such as Japanese encephalitis virus, West Nile virus, yellow fever virus, dengue virus, tick-borne encephalitis virus, and St. Louis encephalitis virus. It is done. Particularly preferred is Japanese encephalitis virus or West Nile virus. The subtype of flavivirus is not particularly limited, and may be a subtype isolated so far or a subtype isolated in the future.

  The flavivirus antigen contained in the vaccine of the present invention is not particularly limited as long as it is at least a part of various components constituting the flavivirus, and may be a virus subunit including M protein and E protein. Well, it can be whole virus particles. Alternatively, the flavivirus antigen may be a flavivirus-like hollow particle (hereinafter also referred to as VLP) that can be produced without using a flavivirus and has been reported to have a vaccine effect. From the viewpoint of immunogenicity, VLP or whole virus particles are preferred. Regarding the whole virus particles, all virus particles inactivated by formalin or the like are more preferable from the viewpoint of safety. One or more flavivirus antigens contained in the flavivirus infection vaccine of the present invention can be appropriately selected and used. One kind of antigen derived from a plurality of subtypes may be used.

When the flavivirus antigen is a Japanese encephalitis virus antigen, the production method is as follows.
When the antigen is whole virus particles, for example, the brain emulsion of a mouse infected with Japanese encephalitis virus is treated with protamine sulfate to isolate and purify the virus, and inactivate it with formalin, as in the method for producing the Japanese encephalitis vaccine Biken. Then, it may be purified by ultracentrifugation and diluted with TCM-199. Alternatively, as disclosed in, for example, International Publication No. WO00 / 20565, a method of culturing Japanese encephalitis virus by infecting a cell line may be used.
When the antigen is VLP, for example, according to the method disclosed in Japanese Patent Application Laid-Open No. 2004-65118, cDNA prepared from Japanese encephalitis virus genomic RNA is incorporated into an expression vector to transform animal cells, and the transformant is cultured. Thus, VLP can be obtained.

West Nile virus antigen can also be produced according to the above Japanese encephalitis virus antigen. That is, when the antigen is whole virus particles, for example, the brain emulsion of a mouse infected with West Nile virus is treated with protamine sulfate, the virus is separated and purified, and this is inactivated with formalin and then purified by ultracentrifugation. Then, it may be diluted with TCM-199. Alternatively, according to the method disclosed in International Publication No. WO00 / 20565, a method of culturing by infecting a cell line with West Nile virus may be used.
In addition, when the antigen is West Nile virus VLP, West Nile virus prM protein and E protein are based on the amino acid sequence of West Nile virus (eg, NY99-flamingo 382-99 strain: GenBank Accession No. AF196835). By introducing an expression vector containing the encoded DNA fragment into an animal cell and culturing the resulting transformed cell, West Nile virus VLP can be secreted into the medium. Examples of mutant West Nile virus strains other than NY99-flamingo382-99 strain include, for example, “Table I” of Ebel, GD et al., Am. J. Trop. Med. Hyg., 71 (4): 493-500, 2004. And the like, and each Accession No. shown in the table. The VLP of the mutant West Nile virus strain can be easily obtained by obtaining the sequence of and determining the corresponding region.
Flavivirus antigens other than Japanese encephalitis virus and West Nile virus can also be produced according to the above method.

  In the present invention, an adjuvant, when used together with an antigen, plays a role of non-specifically enhancing an immune response to the antigen, enhancing cellular immunity against the antigen and antibody production, Is an oil emulsion (for example, Freund's incomplete adjuvant, Freund's completeness) that wraps an inorganic solution (such as aluminum hydroxide, aluminum phosphate, alum, etc.) or an aqueous antigen solution into an emulsion. Adjuvant etc.). The adjuvant used in the present invention is not limited as long as the above-mentioned object can be achieved. However, when used together with the flavivirus antigen, a single inoculation provides a sufficient protective immunity induction effect. Degradable nanoparticles and aluminum hydroxide gel are preferred.

  In one embodiment, the vaccine of the present invention contains biodegradable nanoparticles based on polyamino acids in addition to the flavivirus antigen.

  The “biodegradable nanoparticle” is mainly composed of polyamino acid, is subjected to the action of an enzyme or the like in a living body, and after a predetermined time elapses, the particle can be disintegrated and the polyamino acid skeleton can be decomposed into an amino acid monomer or oligomer. Refers to particles.

  The “main component” of the biodegradable nanoparticle in the present invention refers to a skeleton constituting the nanoparticle.

  The shape of the biodegradable nanoparticles in the present invention is not particularly limited, but is generally spherical. The size of the spherical particles is usually about 10 nm to 100 μm, preferably 10 nm to 500 nm. By using such a size, the amount of immobilized flavivirus antigen increases with the increase in particle surface area per unit weight, the uptake of antigen into antigen-presenting cells, the accompanying activation of CTL, the induction of antibody production, etc. The effect of.

  The shape of the particles can be confirmed by observing with a microscope. The size of the particles refers to an average particle diameter obtained by measuring an aqueous dispersion of nanoparticles by dynamic light scattering. In addition, the size of the nanoparticles having a shape other than the spherical shape can be determined according to the spherical nanoparticles.

  The polyamino acid serving as the skeleton of the biodegradable nanoparticles may be a naturally occurring amino acid or a polymer of artificial amino acids. From the viewpoint of safety or toxicity, polyamino acids consisting of naturally occurring amino acids are preferred, but their origin may be natural products or synthetic products. The naturally occurring amino acid is preferably glutamic acid, aspartic acid, lysine or asparagine. Further, the amino acid may be L-form or D-form. The polyamino acid may contain L-form and D-form at a predetermined ratio.

  Examples of polyamino acids consisting of naturally occurring amino acids include poly (γ-glutamic acid), poly (α-aspartic acid), poly (ε-lysine), poly (α-glutamic acid), poly (α-lysine) or polyasparagine. Is preferred. In order to achieve the intended purpose and impart various properties to the nanoparticles, modified polyamino acids, derivatives thereof or mixtures thereof are also included in the preferred polyamino acids of the present invention. Particularly preferred polyamino acids are poly (γ-glutamic acid), a modified form thereof, a derivative thereof or a mixture thereof.

  The terms “modified amino acid”, “amino acid derivative”, “modified form” and “derivative” of a polyamino acid shall have the meanings commonly used in the art. Examples of “modified products” and “derivatives” of polyamino acids include those in which a part of the constituent amino acids is changed to another amino acid, and those that have been modified using an available functional group of the constituent amino acids. Specifically, a poly (γ-glutamic acid) peptide chain into which one or more other amino acids, modified products or derivatives thereof are introduced, a graft copolymer, or poly (ε -Lysine) The amino acid lysine, which is partially methylated at the available α-position, can be mentioned.

  The “mixture” of polyamino acids contains two or more amino acids as constituent components, and it is preferable to select two or more of the amino acids constituting the polyamino acids exemplified above, modified products thereof and derivatives thereof. .

  Examples of preferred “modified products” or “derivatives” include those in which polyamino acids are modified or derived so that the polyamino acids are amphiphilized. For example, a hydrophobic amino acid can be introduced into the side chain of a hydrophilic polyamino acid to achieve a desired hydrophilic-hydrophobic balance. A specific example is a polyamino acid which is a graft copolymer of poly (γ-glutamic acid) and phenylalanine ethyl ester. Such polyamino acids are also preferable because they can be easily formed into nanoparticles by association of hydrophobic groups within and between molecules.

  The polyamino acid may contain components other than amino acids, such as carbohydrates and lipids. A preferable polyamino acid has 50% by weight or more of the constituent components as amino acids.

  The bonds between all constituent amino acids in the polyamino acid may be of the same type or different types. For example, all constituent amino acids may be linked by peptide bonds, or partially or wholly bound by amino acids other than peptide bonds or by linkers. Examples of bonds other than peptide bonds include ester bonds and ether bonds, and examples of linkers include, but are not limited to, glutaraldehyde and diisocyanate. Furthermore, it may be cross-linked between the functional groups of the polyamino acid. By crosslinking, the physical properties of the polyamino acid can be changed to obtain desired adjuvant properties. Examples of the crosslinking agent include carbodiimide and diglycidyl ester, but are not limited thereto.

  The molecular weight of the polyamino acid is not particularly limited, but can be changed according to the desired viscosity and solubility. The molecular weight is usually in the range of 1000 to 5 million. Preferably it is the range of 5000-2 million. The molecular weight is a value measured by gel filtration chromatography in an aqueous solution or an organic solvent.

  A polyamino acid can be produced by appropriately selecting a known method such as a chemical synthesis method or a fermentation method. The formation of nanoparticles of polyamino acid can be performed by a known method. For example, a submerged drying method, a spray drying method, a spherical crystallization method, a solvent substitution method (precipitation / dialysis method), or a direct ultrasonic dispersion method can be used. For example, biodegradable nanoparticles composed of poly (γ-glutamic acid) or poly (ε-lysine) can be produced by a solvent substitution method. By appropriately selecting or combining such methods, the material, constituent components, molecular weight, size, charge, and other parameters of the biodegradable nanoparticles can be made suitable for the purpose.

  In another embodiment, the vaccine of the present invention contains aluminum hydroxide gel in addition to the flavivirus antigen.

  The aluminum hydroxide gel used in the vaccine of the present invention may be any aluminum hydroxide gel as long as it has an action of enhancing the immunogenicity of an antigen when added to the vaccine. For example, aluminum hydroxide gel can be produced by a method known per se. Aluminum hydroxide gel “Aluminum Hydroxide Gel (A-8222)” (manufactured by SIGMA), “Alhydrogel (2%)) (Brenntag Biosector (Commercially available) may be used.

The aluminum hydroxide gel is preferably prepared using sodium carbonate and potassium aluminum sulfate as raw materials. As the raw materials, sodium carbonate and aluminum potassium sulfate, any reagent usually used for preparing an aluminum hydroxide gel can be used without limitation. As the aluminum potassium sulfate, aluminum potassium sulfate dodecahydrate AlK (SO ₄ ) ₂ · 12H ₂ O, which is also referred to as potassium alum, is preferably used.

  For example, the aluminum hydroxide gel used in the present invention is prepared as follows using sodium carbonate and potassium aluminum sulfate as raw materials. Sodium carbonate (143.1 g) is dissolved in 9 L saline and filtered to prepare a sodium carbonate solution. Potassium aluminum sulfate dodecahydrate (Kari Alum) (316.0 g) is dissolved in 9 L of physiological saline and filtered to prepare a potassium aluminum sulfate solution. The obtained sodium carbonate solution and aluminum potassium sulfate solution are mixed and stirred to obtain an aluminum hydroxide gel, which can be used as an adjuvant (aluminum hydroxide adjuvant) of the present invention by washing.

  In the flavivirus infectious disease vaccine of the present invention, the flavivirus antigen may be included in the biodegradable nanoparticles, or the flavivirus antigen may be immobilized on the surface of the biodegradable nanoparticles.

  The ratio between the flavivirus antigen and the biodegradable nanoparticles contained in the vaccine of the present invention cannot be defined unconditionally depending on the type of antigen and the characteristics of the nanoparticles. For example, the flavivirus antigen is VLP and biodegradable nanoparticle. When poly (γ-glutamic acid) is used as the particles, the weight ratio is 1: 1 to 1: 1000, preferably 1:10 to 1: 200.

  Moreover, the flavivirus infectious disease vaccine of this invention can be manufactured by mixing the said flavivirus antigen and the aluminum hydroxide gel as said adjuvant.

  Although the ratio of flavivirus antigen and aluminum hydroxide gel contained in the vaccine of the present invention cannot be defined unconditionally depending on the type of antigen, for example, when VLP is used as the flavivirus antigen, the ratio by weight is 1:10 to 10. 1: 500 is exemplified, and preferably 1:30 to 1: 200.

  Furthermore, the vaccine of the present invention may contain a pharmaceutically acceptable carrier. As the pharmaceutically acceptable carrier, carriers commonly used for the production of vaccines can be used without limitation, and specifically, saline, buffered saline, dextrose, water, glycerol, isotonic aqueous buffer. Liquids and combinations thereof. Moreover, an emulsifier, 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. In the case of systemic administration, intramuscular, subcutaneous, intradermal, intravenous, intraperitoneal and the like can be mentioned, and the administration method includes injection or infusion.

  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 preferably has a form suitable for the mode of administration of the vaccine. Examples of the injectable form include a solution, a suspension or an emulsion. Or solid forms, such as a lyophilized formulation, are mentioned as a form used for a liquid solution, suspension, or an emulsion.

  The vaccine of the present invention can be used to prevent or reduce the symptoms of flavivirus infection. The present invention provides a method for preventing or reducing flavivirus infection, which comprises 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 when VLP is used as an antigen, 5 μg to 100 μg can usually be administered once or twice or more. The vaccine of the present invention is recommended to be inoculated once because it has a sufficient infection-protecting effect. When administering several times, it is preferable to administer at intervals of 1 to 2 weeks. When virus whole particles are used as an antigen, the dose may be set in terms of VLP. The weight of the VLP is a value obtained by protein quantification by a protein nitrogen quantification method.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail by a test example, this invention is not limited at all by these test examples.

The following test examples were performed as follows.
As the mouse, a 4-week-old female BALB / c mouse (Japan SLC, Hamamatsu, Japan) was used.
JEV Beijing 1 strain was used for virus neutralizing antibody titer assay, and JaTH-160 strain was used for mouse challenge experiments.
As JE vaccine Viken (JE vaccine) and aluminum hydroxide adjuvant (Lot No .: MAH0601), those prepared by Osaka University Microbial Disease Research Society, Kannonji Institute (BIKEN, Kannonji, Japan) were used.
JE-VLP was purified from the culture supernatant of the JEV cDNA highly expressing J12 # 26 cell line (Japanese Patent Laid-Open No. 2004-65118).

  γ-PGA-NP uses γ-PGA (average molecular weight Mn = 380,000, Meiji Seika Co., Ltd., Tokyo, Japan) in accordance with previous reports to form a nanoparticle composed of a γ-PGA hydrophobic derivative at Osaka University. (Akagi T. et al., Macromol Biosci, 2005; 5 (7): p598-602, Kaneko T. et al., Chem Mater, 2005; 17: p2484-86, and Matsusaki M. et al., Chem Lett, 2004; 33: see p398-99). Nanoparticles consisting of γ-PGA-graft-L-Phe were prepared by precipitation and dialysis. First, γ-PGA-graft-L-Phe (10 mg) was dissolved in 1 ml of dimethyl sulfoxide (DMSO), and the same amount of physiological saline was added to obtain a translucent solution. Thereafter, the solution was dialyzed against distilled water with a cellulose membrane tube (cutoff having a molecular weight of 50,000) to remove the organic solvent. The dialyzed solution was lyophilized. In the following test examples, 53% grafted γ-PGA-graft-L-Phe was used.

Regarding vaccination, the dose to mice was 1 μg each for JE vaccine and JE-VLP, and 100 μg each for aluminum hydroxide adjuvant and γ-PGA-NP as adjuvant, except for Test Example 4, as the antigen amount. The method was intraperitoneal injection.
As a viral challenge, immunized mice groups were inoculated 15 days after the first vaccination with an intraperitoneal injection of 3 × 10 ⁵ PFU (20 × LD ₅₀ ) of JEV's JaTH-160 strain. Concurrent with the JEV challenge, mice were inoculated intracerebrally with 10 μl PBS to disrupt the blood brain barrier (Srivastava AK. Et al., Vaccine, 2001; 19 (31): p4557-4565, and Kojima A. et al.) As previously reported. , J Virol, 2003; 77 (16): p8745-8755).

The test for neutralizing antibodies in serum samples was performed as follows.
Serum from mice in a single inoculation group was collected 15 days after the first immunization and tested for virus neutralizing antibodies according to previous reports (Srivastava AK. Et al., Vaccine, 2001; 19 (31): p4557-4565, And Kojima A et al., J Virol, 2003; 77 (16): p8745-8755). Neutralizing antibody titer is expressed as the reciprocal of diluted serum that reduces the average number of plaques per well by 50% relative to control wells.

  Fisher's exact test was performed using StatView software (SAS Institute, Cary, NC) to assess differences between groups in the virus challenge test. A parametric Welch t test was used to analyze data from other tests. A p value of <0.05 was considered significant.

Test Example 1 Challenge test with lethal JEV after inoculation with JE vaccine once or twice and neutralizing antibody titer (1) Protection of mice in lethal JEV challenge test with inoculation once or twice with JE vaccine (Fig. 1)
(A) PBS as a JE vaccine or control group was inoculated twice into the abdominal cavity of mice on day 0 and day 7. On day 15, mice were infected with JEV. Survival rate transition until 20 days after virus challenge was examined (FIG. 1A).
(B) Mice were inoculated once per day on day 0 with JE vaccine or PBS. On day 15, mice were infected with JEV. The survival rate transition was examined every day until the 20th day after infection (FIG. 1B).
All mice in the two immunization group survived after JEV challenge (FIG. 1A), while only 50% of the single inoculation group survived (FIG. 1B).
(2) Anti-JEV neutralizing antibody titer in the serum of mice immunized once or twice with JE vaccine (FIG. 2)
Mice were immunized by intraperitoneal injection with PBS or PBS solution containing JE vaccine once or twice as described in (1). Fifteen days after the first immunization, mouse serum was collected and examined for anti-JEV neutralizing antibody titer.
As shown in FIG. 2, the neutralizing antibody titer in the sera of mice immunized once with the JE vaccine was significantly lower than that in the sera of mice immunized twice.

Test Example 2 Challenge test and neutralizing antibody titer when mice were inoculated once with JE vaccine supplemented with γ-PGA-NP or aluminum hydroxide adjuvant as an adjuvant (FIGS. 3 and 4)
(A) JE vaccine + aluminum hydroxide adjuvant, JE vaccine + γ-PGA-NP as a group containing an adjuvant, and JE vaccine, PBS, aluminum hydroxide adjuvant, γ-PGA-NP (100 μg) as a control group intraperitoneally Was inoculated once. Mice were infected with JEV 15 days after immunization. The change in survival rate until 20 days after infection was examined. As shown in the results of the virus challenge test in FIG. 3, 20 days after the JEV challenge, the survival rate of the mice was 100% in both the JE vaccine + aluminum hydroxide adjuvant and JE vaccine + γ-PGA-NP groups. Compared with the JE vaccine, it was found that the single inoculation of the JE vaccine + aluminum hydroxide adjuvant, JE vaccine + γ-PGA-NP adjuvanted group significantly increased the survival rate.
(B) JE vaccine + aluminum hydroxide adjuvant, JE vaccine + γ-PGA-NP as a group containing an adjuvant, and JE vaccine, PBS, aluminum hydroxide adjuvant, γ-PGA-NP (100 μg) as a control group intraperitoneally Was inoculated once. Fifteen days after immunization, mouse serum was collected and examined for anti-JEV neutralizing antibody titer.
As shown in FIG. 4, the neutralizing antibody titers of the JE vaccine, JE vaccine + aluminum hydroxide adjuvant, JE vaccine + γ-PGA-NP group were 1.765, 3.155, and 3.163, respectively. The neutralizing antibody titer of the JE vaccine + γ-PGA-NP group was significantly higher than that of the JE vaccine.

Test Example 3 Challenge test and neutralizing antibody titer when mice were inoculated once with JE-VLP supplemented with γ-PGA-NP or aluminum hydroxide adjuvant as an adjuvant (FIGS. 5 and 6)
(A) JE-VLP + aluminum hydroxide adjuvant, JE-VLP + γ-PGA-NP, JE-VLP as the group containing adjuvant, PBS, aluminum hydroxide adjuvant, γ-PGA-NP (100 μg) as the control group Inoculated once. 15 days after immunization, mice were infected with JEV, and the survival rate transition until 20 days was examined.
As shown in FIG. 5, the survival rate of the JE-VLP group was 40%, and the survival rates of both the JE-VLP + aluminum hydroxide adjuvant supplemented with adjuvant and JE-VLP + γ-PGA-NP were 94%. there were.
(B) As a group containing adjuvant, JE-VLP + aluminum hydroxide adjuvant, JE-VLP + γ-PGA-NP, JE-VLP, and PBS, aluminum hydroxide adjuvant, γ-PGA-NP as the control group A second inoculation was performed. Fifteen days after immunization, mouse serum was collected and examined for anti-JEV neutralizing antibody titer.
As shown in FIG. 6, the neutralizing antibody titers of both the JE-VLP + aluminum hydroxide adjuvant and JE-VLP + γ-PGA-NP groups after the single inoculation were determined by the double inoculation of the JE vaccine without adjuvant (FIG. 2). The neutralizing antibody titer was similar to that of JE vaccine and JE-VLP was confirmed to induce sufficient infection protective ability by single inoculation by adding aluminum hydroxide adjuvant to JE vaccine and JE-VLP, respectively.
The neutralizing antibody titers of both groups after the first inoculation with the adjuvant were the neutralizing antibody titer levels after the second inoculation without the adjuvant, and the survival rate was 94% or more. By adding an aluminum adjuvant to JE-VLP, it was confirmed that a single inoculation induced sufficient protective immunity, and the same effect was obtained even when γ-PGA-NP was used as an adjuvant.
Therefore, it was suggested that a single inoculation method of JE vaccine could be constructed by combining JE-VLP with γ-PGA-NP or aluminum hydroxide adjuvant.

Test Example 4 Challenge test and neutralizing antibody titer when mice were inoculated twice with JE-VLP supplemented with γ-PGA-NP or aluminum hydroxide adjuvant as an adjuvant (FIG. 7-A, FIG. 7-B, FIG. 8) )
JE-VLP (0.1 μg, 1 μg) was inoculated intraperitoneally into mice twice a week each, either alone or suspended in either aluminum adjuvant or γ-PGA-NP, 15 days after the initial inoculation Later, 30 x LD ₅₀ doses of JEV were intraperitoneally infected, the mice were infected with JEV, and the survival rate changes up to 20 days later were examined. FIG. 7-A and FIG. 7-B show changes in survival rate (survival rate) after the second inoculation.
JE-VLP (0.1 μg, 0.3 μg, 1 μg) alone or suspended in either aluminum adjuvant or γ-PGA-NP was used to determine the neutralizing antibody titer in the serum 2 weeks after the initial inoculation. It was measured. FIG. 8 shows the neutralizing antibody titer after each inoculation twice.
As for the combined vaccine with JE-VLP + aluminum hydroxide adjuvant, the neutralizing antibody titer when the inoculation amount of JE-VLP was 1 μg was 2.32, compared with 1.69 at 0.3 μg, 0.8. It was 1.69 at 1 μg.
On the other hand, for the combination vaccine with JE-VLP + γ-PGA-NP, the inoculum of JE-VLP is 3.05 at 0.3 μg and 2.15 at 0.1 μg, compared with the neutralizing antibody titer of 2.32 at 1 μg. 91.

Further, compared with the case of only 1 μg of JE vaccine, 0.1 μg of JE-VLP + aluminum hydroxide adjuvant has neutralizing antibody titers of 3.63 and 3.56, and the antigen amount is 1/10. It was the same level. The neutralizing antibody titer of 0.1 μg of JE-VLP + γ-PGA-NP was almost the same level as 2.91.
From this, it was found that the vaccine for single inoculation of the present invention can provide a sufficient vaccine effect even if the amount of antigen is reduced.

It is a figure which shows the survival rate of the mouse | mouth at the time of lethal JEV challenge after 1 time or 2 times inoculation of JE vaccine. Mouse (n = 10), ○: 400 μl of PBS solution containing 1 μg of JE vaccine, ●: 400 μl of PBS solution It is a figure which shows the anti- JEV neutralizing antibody titer in the serum of the mouse | mouth after inoculating once or twice with a JE vaccine. Mice (n = 5), mean, ^* : p <0.05, N.I. D. : Not detectable It shows the survival rate transition at the time of lethal JEV challenge in mice after a single inoculation with JE vaccine supplemented with aluminum hydroxide adjuvant or γ-PGA-NP as an adjuvant. ●: PBS, n = 31, ■: alum = aluminum hydroxide adjuvant, n = 35, ◆: PGA-NPs = γ-PGA-NP, n = 35, ○: JE vaccine = JE vaccine, n = 31, □ : JE vaccine + alum = JE vaccine + aluminum hydroxide adjuvant, n = 20, ◇: JE vaccine + γ-PGA-NPs = JE vaccine + γ-PGA-NP, n = 33, ^* : p <0.05 It is a neutralizing antibody titer when a mouse is inoculated once with JE vaccine supplemented with γ-PGA-NP or an aluminum hydroxide adjuvant as an adjuvant. Average value, ^* : p <0.05. N. D. : Not detectable The change in survival rate at the time of lethal JEV challenge in mice after inoculating mice once with JE-VLP supplemented with γ-PGA-NP or aluminum hydroxide adjuvant as an adjuvant is shown. It is a figure which shows the neutralizing antibody titer when a mouse | mouth is inoculated once with JE-VLP which added (gamma) -PGA-NP or aluminum hydroxide adjuvant as an adjuvant. The change in the survival rate at the time of lethal JEV challenge in mice is shown when JE-VLP (1 μg) supplemented with γ-PGA-NP or aluminum hydroxide adjuvant as an adjuvant is inoculated twice. It shows the survival rate change at the time of lethal JEV challenge in mice when the mice were inoculated twice with JE-VLP (0.1 μg) supplemented with γ-PGA-NP or aluminum hydroxide adjuvant as an adjuvant. The neutralizing antibody titer when mice were inoculated twice with JE-VLP (0.1 μg, 0.3 μg, 1 μg) supplemented with γ-PGA-NP or aluminum hydroxide adjuvant as an adjuvant is shown.

Claims (13)

  A flavivirus infection vaccine comprising a flavivirus antigen for preventing flavivirus infection and an adjuvant.   The vaccine according to claim 1, wherein the adjuvant is a biodegradable nanoparticle mainly composed of a polyamino acid.   The vaccine of claim 1, wherein the adjuvant is aluminum hydroxide gel.   The vaccine according to claim 3, wherein the aluminum hydroxide gel is prepared from sodium carbonate and potassium aluminum sulfate.   The vaccine according to any one of claims 1 to 4, which is a vaccine for preventing flavivirus infection by inoculation once.   The vaccine according to any one of claims 1 to 5, wherein the flavivirus is Japanese encephalitis virus and / or West Nile virus.   The vaccine according to any one of claims 1 to 6, wherein the flavivirus antigen is a flavivirus-like hollow particle.   The vaccine according to any one of claims 1 to 6, wherein the flavivirus antigen is whole virus particles.   Polyamino acid is poly (γ-glutamic acid), poly (α-aspartic acid), poly (ε-lysine), poly (α-glutamic acid), poly (α-lysine), polyasparagine, modified products thereof, The vaccine according to any one of claims 2, 5 to 8, which is selected from the group consisting of derivatives and mixtures thereof.   The vaccine according to any one of claims 2 and 5 to 9, wherein the polyamino acid is selected from the group consisting of poly (γ-glutamic acid), a modified form thereof, a derivative thereof and a mixture thereof.   The vaccine according to any one of claims 2, 5 to 10, wherein the polyamino acid is amphiphilized.   The vaccine according to claim 2, wherein the polyamino acid is a graft copolymer of poly (γ-glutamic acid) and phenylalanine ethyl ester.   An adjuvant for flavivirus infection vaccine comprising biodegradable nanoparticles mainly composed of polyamino acids.