WO2024048570A1 - Hepatitis b vaccine composition for nasal administration and nasal administration system thereof - Google Patents

Abstract

The present invention addresses the problem of providing a vaccine composition for nasal administration that is usable for preventing and treating hepatitis B, and a nasal administration system of the vaccine.　Provided is a hepatitis B vaccine composition that comprises: (i) virus-like particles containing hepatitis B surface L antigen proteins (HBs-L antigen proteins) of two or more genotypes selected from the group consisting of types A, B, C and D, and a hepatitis B nucleocapsid antigen (HBc antigen) protein; and (ii) a base material containing a carboxyvinyl polymer having been treated by externally applying a shear force. Also provided is a nasal administration system of the hepatitis B vaccine, said system comprising the composition filled into a sprayable device equipped with a nasal spray nozzle.

Description

Hepatitis B vaccine composition for nasal administration and nasal administration system for the vaccine composition

The present invention relates to a vaccine composition for nasal administration that can be used for the prevention and treatment of hepatitis B, and a nasal administration system for the vaccine.

Hepatitis B is hepatitis caused by infection with hepatitis B virus (HBV), and HBV is transmitted through blood and body fluids. Persistent infection of hepatocytes with HBV is a serious infectious disease that causes chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, but there is no treatment that can eliminate the virus.

Currently, pegylated interferon preparations (PEG-IFN) and nucleic acid analog preparations (NA) are mainly used for the treatment of chronic hepatitis B (CHB), but PEG-IFN has immunostimulatory and antiviral effects. Although there are some effective examples of HBV treatment that strengthens immunity and effectively sustains suppression of virus proliferation, the HBV elimination rate is generally low and the high frequency and variety of side effects are major problems. On the other hand, although NA shows a high HBV DNA negative conversion rate of approximately 95% by inhibiting viral replication, it cannot be eliminated and its efficacy quickly disappears upon discontinuation of administration. Therefore, lifelong use is required, which poses major problems in terms of compliance and medical economics, and it has also been reported that there is a possibility of the emergence of resistant viruses with long-term use.

On the other hand, in order to prevent HBV infection, in Japan, preventive vaccines are administered to those at high risk of infection (HBV carrier families, medical workers), and this has achieved some results in significantly reducing the number of HBV carriers. As a treatment for CHB, trials of immunotherapy using an HBV vaccine are being conducted.

Some of the present inventors have investigated HBs antigens for the purpose of inducing neutralizing antibodies against HBs antigen (hepatitis B virus surface antigen) and inducing acquired immunity against HBc antigen (hepatitis B nucleocapsid antigen) in the treatment of HBV. We improved a formulation containing two types of antigens, HBs-S antigen and HBc antigen, and succeeded in developing a composition for nasal administration containing carboxyvinyl polymer (CVP) (Patent Documents 3 and 4). Combining HBs-S antigen and HBc antigen strongly activates cell-mediated immunity, and further combining CVP delays the clearance of the vaccine in the nasal mucosa and enhances the immune response in the nasal mucosa. As a result, we were able to obtain a vaccine composition that exhibits strong activity in both inducing anti-HBs neutralizing antibodies and activating cell-mediated immunity.

Clinical trials are underway for immunotherapy in which such a composition is filled into a dedicated device and administered nasally. The phase 1 clinical trial included 29 patients with chronic hepatitis B (CHB) on treatment with nucleic acid analogues (NA) and 42 untreated hepatitis B virus (HBV) carriers, who received once every two weeks. The composition was intranasally administered 10 times in total. Anti-HBs antibodies were induced in about half of the cases, and the amount of HBs antigen decreased over time. In some cases, HBs antigen disappeared and functional cure was achieved. Moreover, adverse events were minor (Non-Patent Document 1).

HBV is a particle composed of DNA at its center and a capsid surrounding it, and further has an envelope structure containing a large number of proteins in the lipid membrane on its surface. The envelope is the outermost structure of the virus and is used for immunological detection of HBV, so it is also called HBV surface antigen (HBs).

The full-length protein that forms the surface antigen of HBV is called L protein, and there are three regions, Pre-S1 region, Pre-S2 region, and S region, in order from the N-terminus presented on the outermost part of the particle.

The full-length HBs-L antigen protein, called the HBs-L antigen protein, lacks the Pre-S1 region, the protein consisting of the Pre-S2 region and the S region is called the M protein, and the HBs-L antigen protein lacks the Pre-S1 and Pre-S2 regions. , a protein consisting only of the S region is called an S protein.

The various HBV antigen proteins mentioned above can be produced using an expression system using eukaryotic cells such as yeast or animal cells into which genes encoding them have been introduced, and these genetically recombinant surface antigens is widely used as a preventive vaccine against HBV. Among these, the use of S antigen produced in yeast is the mainstream in the current market. The HBs antigen used in the preparation containing HBc antigen, HBs antigen, and CVP used in the above clinical trial is S protein (HBs-S antigen protein).

On the other hand, the full-length L protein has, in addition to the S region and Pre-S2 region contained in the M protein, a Pre-S1 region that is the HBV sensor region. Therefore, when the L protein is used as a vaccine, antibodies against the outermost Pre-S1 region are also produced, making it an excellent prophylactic vaccine. Some of the present inventors have confirmed that a vaccine composition that combines HBs-L antigen and HBc antigen exhibits a high ability to activate cellular immunity (Patent Documents 2 and 3).

It is known that there are multiple HBV genotypes, A to J, but A, B, C, and D are the main HBV genotypes. Regarding the S region and Pre-S2 region, the relationship between each genotype and phenotype has been clarified. For example, in the S region, the 124th to 147th amino acid sequence is generally known as the highly immunogenic antigenic determinant "a". It is known that in the Pre-S2 region, the antigenic determinant "b" is present in common in all genotypes (Non-Patent Document 2). Therefore, with respect to these regions, even if the genotypes are different, it is thought that common antibodies will be produced for at least some of the genotypes.

On the other hand, regarding the Pre-S1 region, analysis of each genotype and phenotype has not been sufficiently conducted, and the amino acid sequence homology between each genotype of HBV is low. Therefore, even if a vaccine containing the HBs-L antigen protein containing the Pre-S1 region derived from one type of genotype is produced as an active ingredient, the Pre-S1 region is It is not always possible to elicit an immune response through

Some of the present inventors have developed an HBs-L antigen protein whose genotype is any of A, B, C, D or variants of each of these genotypes as a vaccine for different genotypes. have proposed virus-like particles containing one or more types of (Patent Documents 1 and 2). Using a combination of virus-like particles having HBs-L antigen protein of one of genotypes A, B, C, or D in one virus-like particle, or using two virus-like particles in one virus-like particle. It has been confirmed that by using virus-like particles containing HBs-L antigen proteins of the above genotypes, it is possible to induce HBs-neutralizing antibodies against multiple types of genotypes. For example, antibodies induced by virus-like particles containing type C HBs-L antigen protein and type D HBs-L antigen protein are high against all HBs antigens of genotypes A, B, C, and D. It has been confirmed that it has neutralizing activity (Patent Document 1).

Patent No. 6613399 WO2019/013361 WO2019/070019 WO2019/235466

History of Medicine Vol. 281 No. 3, 265-268 (2022) Usuda S et al. (1999) J. Virol Methods. 80(1), 97-112

The purpose of this application is to provide a stable vaccine composition for nasal administration and a nasal administration system for the vaccine.

The present application provides: (i) a virus-like particle containing hepatitis B surface L antigen protein (HBs-L antigen protein) of two or more genotypes selected from the group consisting of type A, type B, type C, and type D; Provided is a hepatitis B vaccine composition for nasal administration, comprising a base containing a hepatitis B nucleocapsid antigen (HBc antigen) protein, and (ii) a carboxyvinyl polymer treated with external shear force.

The present application also provides a hepatitis B vaccine nasal administration system, which comprises filling the hepatitis B vaccine composition for nasal administration of the present application into a syringe-type ejector having a nasal spray nozzle.

In another aspect, the present application provides a method for treating hepatitis B, comprising nasally administering an effective amount of the nasally administered hepatitis B vaccine composition of the present application to a subject in need of treatment for hepatitis B. provide. In the methods of the present application, the hepatitis B vaccine composition for nasal administration may be administered using a syringe-type ejector having a nasal spray nozzle.

In yet another aspect, the present application provides (i) hepatitis B surface L antigen protein (HBs-L antigen protein) of two or more genotypes selected from the group consisting of type A, type B, type C, and type D. B for nasal administration of a composition comprising a base containing virus-like particles containing virus-like particles, hepatitis B nucleocapsid antigen (HBc antigen) protein, and (ii) a carboxyvinyl polymer treated by applying an external shear force. Provides for use in the manufacture of hepatitis vaccines. The composition of the present application may be filled into a syringe-type squirt device having a nasal spray nozzle.

Virus-like particles containing HBs-L antigen protein have poor stability, and especially when used simultaneously with hepatitis B nucleocapsid antigen (HBc antigen), the stability further decreases depending on the amount of HBc antigen. At the same time, the present inventors' research revealed that the HBc antigen also becomes unstable. Although the hepatitis B vaccine composition of the present application contains virus-like particles containing HBs-L antigen protein and HBc antigen protein, it is a stable vaccine in terms of formulation, and can be used as a preventive and therapeutic vaccine for hepatitis B. Suitably used.

Schematic diagram of HBs-L hybrid antigen (HBs-Lh, including genotype C type and D type HBs-L antigen proteins) used in Examples. FIG. 1 is a partially cutaway side view showing a schematic configuration of the entire medical syringe (prefilled device) equipped with a nasal spray nozzle used in Examples. (a) and (b) are exploded perspective views showing the schematic structure of the nasal spray nozzle used in the examples, and show the states before and after the filling rod is inserted into the nozzle body. . (a) is a vertical cross-sectional view of the nasal spray nozzle shown in FIG. 3(b) when viewed from a vertical plane, and (b) to (d) are taken along the line BB in FIG. 4(a). , CC line, and DD line. In Example 3, antibody titers against HBs-LH antigen in mouse serum were examined by ELISA two weeks after nasal administration of formulations 1 to 6 (examples) and formulations 7 and 8 (comparative examples) to mice. Show the results. In Example 3, the results of examining the antibody titer against HBc antigen in mouse serum by ELISA two weeks after nasally administering formulations 1 to 6 (example) and formulations 7 and 8 (comparative example) to mice, respectively. show.

In this specification, when a numerical value is accompanied by the term "about," it is intended to include a range of ±10% of that value. For example, "about 20" includes "18 to 22". The range of numbers includes all numbers between and including the endpoints. "About" in relation to a range applies to both endpoints of the range. Therefore, for example, "about 20-30" includes "18-33".

Virus-like particle containing HBs-L antigen protein The virus-like particle containing HBs-L antigen protein contained in the hepatitis B vaccine composition of the present application is a protein having one or more transmembrane domains. These particles are mainly composed of a certain HBs-L antigen protein and a lipid bilayer membrane, and do not contain nucleic acids such as DNA or RNA that control genetic information inside the particles.

The genotype of the HBs-L antigen protein contained in the virus-like particle is selected from A, B, C and D. Furthermore, these genotype variants can also be used as the HBs-L antigen protein contained in the virus-like particles used in the present application. Note that the variants defined herein are not limited to variants due to mutation, but also include variants obtained by artificially introducing mutations.

The specific amino acid sequence of the HBs-L antigen protein of each genotype may be any known one and is not particularly limited. For example, the amino acid sequence shown in any one of SEQ ID NOs: 1 to 9 as genotype A, the amino acid sequence shown in any one of SEQ ID NOs 10 to 18 as genotype B, and the amino acid sequence shown in any one of SEQ ID NOs 29 to 38 as genotype D. Amino acid sequences can be mentioned.

The amino acid sequences that specify the HBs-L antigen protein of each genotype described above are the amino acid sequences shown in SEQ ID NOs: 1 to 38 that specify the HBs-L antigen protein of each genotype, to the extent that the effects of the present invention are not significantly impaired. It can also be a variant of. Such mutations are not particularly limited, and may include, for example, substitutions, insertions, or deletions.

The degree of mutation in the amino acid sequence of each genotype described above is not particularly limited. For example, introducing mutations of about 33 amino acids into the amino acid sequence shown in any one of SEQ ID NOs: 1 to 9 (genotype A), or introducing mutations into the amino acid sequence shown in any one of SEQ ID NOs: 10 to 18 (genotype B). Introduction of mutations of about 26 amino acids, introduction of mutations of about 28 amino acids into the amino acid sequence (genotype C) shown in any of SEQ ID NOS: 19 to 28, and introduction of mutations of about 28 amino acids to the amino acid sequence shown in any of SEQ ID NOS: 29 to 38 ( For genotype D), mutations of about 38 amino acids can be introduced.

The degree of mutation introduction into the amino acid sequence of each genotype defined in the present invention is as described in Patent Document 1. As a specific example of a variant, if the number of amino acids is 400 among SEQ ID NOs: 1 to 38, a region of 11 amino acids from the N-terminus and/or a region of 6 amino acids from positions 163 to 168 is deleted. Examples include mutants that have Furthermore, among SEQ ID NOs: 1 to 38, when the number of amino acids is not 400, examples include mutants in which the amino acid sequences in the regions corresponding to these are deleted.

As an artificially mutated variant, for example, the three regions of the HBs-L antigen protein, the Pre-S1 region, the Pre-S2 region, and the S region, are replaced with regions derived from different genotypes. Or mutants with deletion can be mentioned. Specifically, the Pre-S1 region of one genotype is replaced with a Pre-S1 region derived from another genotype, and the Pre-S1 region and Pre-S2 region of one genotype are replaced with another genotype. Substitutions to the derived Pre-S1 and Pre-S2 regions, and further examples include mutants in which the Pre-S2 region of one genotype is replaced with Pre-S2 derived from another genotype. It is also possible to replace the Pre-S2 region with a Pre-S1 region derived from another genotype, and also include mutants in which the Pre-S2 region is deleted. In addition, in the case of these artificial substitutions, it is possible to introduce mutations exceeding the number of mutations introduced into the amino acid sequence of each genotype described above. In the specification and claims of the present application, "genotype A," "genotype B," "genotype C," and "genotype D" refer to the respective genes of the HBs-L antigen protein unless otherwise specified. shall mean either the type or a variant thereof.

"Virus-like particles containing hepatitis B surface L antigen protein (HBs-L antigen protein) of two or more genotypes selected from the group consisting of type A, type B, type C, and type D" used in this application may be a combination of particles containing one type of HBs-L antigen protein in each particle, or a combination of particles containing two types of HBs-L antigen protein in one particle. . Hereinafter, a virus-like particle containing one genotype of HBs-L antigen protein per particle will be referred to as "HBs-L antigen". Virus-like particles containing two types of HBs-L antigen proteins in one particle are called "HBs-L hybrid antigen" or "HBs-Lh antigen." The hepatitis B vaccine composition of the present application contains HBs-L antigen proteins of two or more genotypes selected from A, B, C, and D, by appropriately combining HBs-L antigen and/or HBs-Lh antigen. Preferably.

Combinations of HBs-L antigen proteins include [type A HBs-L antigen protein and type B HBs-L antigen protein], [type A HBs-L antigen and type D HBs-L antigen protein], [ HBs-L antigen protein of type B and HBs-L antigen protein of type C], [HBs-L antigen protein of type B and HBs-L antigen protein of type D], [HBs-L antigen protein of type C and D HBs-L antigen protein of type B], and HBs-L antigen protein of type B, HBs-L antigen protein of type C, and HBs-L antigen protein of type D]. Preferably, it is a combination of an HBs-L antigen protein of type C genotype and an antigen protein of type D genotype. HBs-Lh antigen, which is a virus-like particle containing two types of antigen proteins, an HBs-L antigen protein with genotype C type and an antigen protein with genotype D type, is particularly preferably used.

HBs-L antigen and HBs-Lh antigen, including HBs-L antigen proteins of each genotype, can be produced using genetically modified yeast (Patent Documents 1 and 2). HBs-L antigen protein has self-assembly ability and can present antigen by assembling on a lipid membrane to form particles. The HBs-L antigen protein has an S region with high lipid affinity, and regardless of the genotype, when produced using biological cells, it sticks into lipid membranes and exists. As a result, the protein takes the form of a virus-like particle, which is a stable antigenic particle structure, and has high immunogenicity due to this particle structure.

Hepatitis B nucleocapsid antigen (HBc antigen)
The hepatitis B nucleocapsid antigen (HBc antigen) included in the hepatitis B vaccine composition of the present application is also known as hepatitis B core antigen. Any protein known as an HBc antigen may be used, and is not particularly limited. HBc antigen can be produced using previously reported methods, such as Rolland et al. J Chromatogr B Biomed Sci Appl. 2001 25;753(1):51-65). For example, an HBc antigen expression vector can be obtained by inserting the full-length HBc antigen DNA (ACC# X01587) into a pET-19b vector from which sequences such as His-tag have been removed. The obtained expression vector is introduced into E. coli to obtain an expression strain, the E. coli strain is cultured to obtain bacterial cells, and the HBc antigen can be obtained by purifying the obtained bacterial cells. It is known that the capsid proteins of the HBc antigen bind to each other to form particles, and the HBc antigen produced by Escherichia coli also self-assembles to form particles.

In the hepatitis B vaccine composition of the present application, the blending ratio of HBs-L antigen and HBc antigen is not limited, and the HBc antigen with respect to the total weight of HBs-L antigen and/or HBs-Lh antigen as virus-like particles The ratio of protein amounts is preferably from 1:0.1 to 1:10, for example, 1: about 0.25, 1: about 0.5, 1: about 1, 1: about 1.25, 1: Examples include cases where the ratio is about 1.5 and 1:about 2.

Base containing a carboxyvinyl polymer treated by applying an external shearing force The base containing a carboxyvinyl polymer treated by applying an external shearing force included in the hepatitis B vaccine composition of the present application includes, for example: An example is "gel base containing skin/mucosal adhesive" disclosed in WO2007/123193.

A carboxyvinyl polymer is a polymer of acrylic acid, and the carboxyl group content of a carboxyvinyl polymer means the ratio (% by mass) of carboxyl groups contained in the polymer to the total amount of the polymer. The method for quantifying the carboxyl group content is not particularly limited, but may be determined using, for example, the method for quantifying carboxyvinyl polymers listed in the Pharmaceutical Excipient Standards. CVP used in the vaccine composition of the present application preferably has a carboxyl group content of 60.0 to 62.0% by mass.

The polymer particle diameter of CVP used in the composition of the present invention is such that the mode diameter based on the number of particles is adjusted to 0.05 to 10 μm (preferably 0.1 to 2.0 μm) by applying external shear force. is preferably used. Hereinafter, unless otherwise specified, the term "mode diameter" means the mode diameter based on the number standard.

The polymer particle size [particle size distribution] is measured using a number-based particle size distribution that can be measured with a laser diffraction type particle size distribution measuring device, a dynamic light scattering type particle size distribution measuring device, etc. It is preferable that there is no large difference from the distribution results. If the analysis results differ depending on the device, if the mode diameter of the polymer particles is within a predetermined range in at least one device, the mode diameter of the polymer particles can be considered to be within the predetermined range. The mode diameter may be determined by checking the state of the polymer particles using a phase contrast microscope, an opt-SEM (Optical Shadow Effect Mode microscope), or the like. Further, the polymer particle size may be determined by a method using a laser diffraction particle size distribution measuring device according to the method described in the test examples of the present application.

The mode diameter of the CVP is adjusted by applying an external mechanical shearing force to the CVP. For example, by applying an external shearing force to commercially available CVP, the mode diameter of the polymer particles can be adjusted to a desired range. The operation of applying shear force is performed by a method known to those skilled in the art. For example, as a device for applying mechanical shearing force, a high-speed rotation emulsifier, a colloid mill emulsifier, a high-pressure emulsifier, a roll mill emulsifier, an ultrasonic emulsifier, and a membrane emulsifier can be used. Particularly preferred are homomixer type, comb type, and intermittent jet stream generation type high-speed rotating emulsifiers.

The hepatitis B vaccine composition of the present application contains a gel base containing CVP that has been treated by applying shearing force from the outside. CVP may be neutralized. Examples of the neutralizing agent added to the gel base containing CVP include sodium hydroxide, potassium hydroxide, and L-arginine. By neutralizing CVP, which is a produced substance, CVP is ionized and increases sterically, thereby increasing the viscosity of the gel base. For example, if L-arginine is added, its weight ratio to CVP may range from, for example, 1: about 0.5 to 1: about 3, preferably from 1: about 1 to 1: about 2.5, such as 1: about 2. This is an example of what you can do.

The hepatitis B vaccine composition of the present application may further contain a pharmaceutically acceptable carrier. As the carrier, carriers commonly used in the manufacture of vaccines and intranasal preparations can be used, such as saline, buffered saline, dextrose, water, glycerin, isotonic aqueous buffers, etc. and combinations thereof. In addition, preservatives (e.g., thimerosal), isotonic agents, pH adjusters, surfactants, stabilizers (e.g., sodium edetate hydrate), inactivating agents (e.g., formalin), etc. are added as appropriate. May be blended.

It is particularly preferred to add at least one cationic surfactant selected from the group consisting of cetylpyridinium chloride, benzalkonium chloride and benzethonium chloride, particularly preferably cetylpyridinium chloride. Add chloride or cetylpyridinium chloride hydrate. When cetylpyridinium chloride hydrate is added, the amount may be determined as appropriate, but for example, the weight ratio with CVP is 1:about 0.01 to 0.03, more preferably 1:about 0.018wt. For example, it may be a section.

The concentration of CVP in the hepatitis B vaccine composition of the present application is preferably from 0.1 to 2.0 w/v%, more preferably from 0.3 to 1.0 w/v%, for example about 0.55 w/v%. It is. As an example, as a base material containing a carboxyvinyl polymer treated by applying an external shearing force of the present application, the concentration of the carboxyvinyl polymer treated by applying an external shearing force is about 0.55 w / v %, An example is a base containing about 2 parts by weight of L-arginine per 1 part by weight of CVP. A base further containing about 0.018 parts by weight of cetylpyridinium chloride hydrate per 1 part by weight of CVP is also exemplified.

The concentration of each antigen in the hepatitis B vaccine composition of the present application is determined by the total amount of HBs-L antigen (total amount of HBs-L antigen and/or HBs-Lh antigen) and the amount of HBs antigen from 0.01 to 0.01, respectively. Examples include 10 mg/mL, preferably within the range of 0.05 to 5 mg/mL, for example about 0.1 mg/mL each.

The hepatitis B vaccine composition of the present application contains a carboxyvinyl polymer obtained by mixing and stirring stock solutions in which HBs-L antigen and HBc antigen are each suspended separately, and treating the mixed antigen solution by applying a shearing force from the outside. It can be manufactured by mixing with a base. There is no need to add adjuvants other than these ingredients to the compositions of the present application. This mixing and stirring can be achieved in a short time by gentle mixing and stirring without applying stress such as heat or pressure to each antigen.

The vaccine composition of the present application is administered by spray into the nasal cavity for the prevention or treatment of hepatitis B. Preferably, the average particle size of the sprayed preparation is in the range of 50 μm to 120 μm (preferably in the range of 50 μm to 80 μm), and the particle size distribution of the sprayed preparation is in the range of 10 μm to 100 μm (preferably 50% or more, preferably in the range of 10 μm to 100 μm). 70% or more), the spray angle from the device is in the range of 30° to 70° (preferably in the range of 40° to 60°), and the spray is administered evenly into the nasal cavity with a full cone spray density. "Full cone", which means a uniform spray density with no bias, is one of the spray pattern shapes and means a homogeneous circular area, while the opposite term refers to a donut-shaped area that is localized only on the periphery. It is a "Holocone".

The vaccine composition of the present application is administered by spraying into one or both nostrils intranasally once or multiple times. The dose is determined taking into consideration the subject's age, sex, body weight, etc., but the amount of antigen to be administered is 0.01 to 5 mg of HBc antigen and the total amount of HBs-L antigen (HBs-L antigen). The total amount of antigen and/or HBs-Lh antigen) is preferably in the range of 0.01 to 5 mg, more preferably in the range of 0.05 to 2 mg, respectively. Further, the amount of the formulation sprayed per nostril is preferably 50 μL to 1000 μL, more preferably 250 μL to 1000 μL.

The frequency and period of administration may be determined by taking into consideration the subject's age, sex, weight, etc., and the severity of the disease. For example, administration may be performed multiple times, for example, 3 to 20 times, 5 to 15 times, about 10 times, at intervals of 3 to 21 days, 7 to 17 days, or about 14 days.

The hepatitis B vaccine composition of the present application may be administered by being filled into a sprayable device having a nasal spray nozzle. The sprayable device having a nasal spray nozzle is not limited to any commonly used nasal spraying device, and may be a sprayable device without a pump function. By using an upward pressure airless spray container as a multi-dose spray container, as described for example in WO 2007/123193 and WO 2007/123207, it is possible to administer the spray container at any angle or range of angles without reducing the amount remaining in the container. It can be used without. As a disposable type device limited to use for one person, there is a medical syringe equipped with a nasal spray nozzle disclosed in WO2015/199130, and also described in WO2021/066195 and Japanese Patent Application No. 2021-126673. A device having a nasal spray/injection nozzle such as the above can be used.

The present invention will be explained below with reference to Examples, but the present invention is not limited to these Examples in any way.

A CVP base, an HBs-Lh antigen stock solution, and an HBc antigen stock solution were prepared by the method shown below and mixed as follows to prepare a nasal hepatitis B vaccine composition.

[Production of carboxyvinyl polymer (CVP) base]

[Preparation of HBs-Lh antigen stock solution]

A schematic diagram of the HBs-Lh antigen produced in this example is shown in FIG. HBs-Lh antigen is produced by feeding yeast transformed to express two HBs-L antigen proteins with genotypes C and D, crushing the resulting bacterial cells, heat treatment, and affinity treatment. A cryopreserved product of virus-like particles produced by gel filtration was thawed and used. The molecular weight of L protein is approximately 45KDa, the purity is over 99%, and the particle size is 60 to 70 nm.
The HBs-L antigen stock solution was obtained by suspending the obtained virus-like particles in purified water to which the respective components were added in the amounts listed in Table 2.

[Preparation of HBc antigen stock solution]

HBc antigen was obtained by feeding-batch culture of Escherichia coli transformed to express HBc antigen protein (full length), cell disruption, ammonium sulfate precipitation, gel filtration, and endotoxin removal. The molecular weight was approximately 21 KDa, the purity was 99% or more, the particle size was 30 to 35 nm, and the endotoxin concentration was <50 EU/mg protein.

[Mixing carboxyvinyl polymer (CVP) base and virus stock solution]
After mixing the above HBs-Lh antigen stock solution with HBc antigen stock solution [ratio is 1:1], equal amounts of the antigen mixture and CVP base were mixed, stirred until homogeneous, and then administered by nasal hepatitis B. A vaccine composition was obtained (Example 1). This mixing and agitation can be achieved in a short time by gentle mixing and agitation without applying stress such as heat or pressure to the hepatitis B vaccine antigen.

The obtained nasal hepatitis B vaccine composition was filled into a device for nasal administration, and the droplet size distribution, spray homogeneity, and spray angle were measured. The nasal administration device used was a medical syringe (metered syringe type ejector) equipped with a nasal spray nozzle described in WO2015/199130. The devices used are shown in Figures 2 to 4. The diameter of the ejection port 21 at the tip of the nozzle is 0.26 mm. (1) In the formulation droplet size distribution, the average formulation droplet size is in the range of 30 μm to 80 μm [63.6 μm], and 80% or more of the formulation droplet size distribution is in the range of 10 μm to 100 μm [85.3%], (2) The spray density is uniform and full cone, and (3) the injection angle is controlled within the range of 30° to 70° [51°], making it an extremely good nasal administration system for hepatitis B vaccine. confirmed.
The hepatitis B vaccine nasal administration system of Example 1 is summarized below.

Mixing ratio of HBs-Lh antigen and HBc antigen stability and effect of CVP HBs-Lh antigen and HBc antigen used in Example 1 were each suspended in PBS, and an HBs-Lh antigen solution (0.1 μg/mL PBS solution) was prepared. , an HBc antigen solution (0.1 μg/mL PBS solution) was obtained. The following formulations were prepared using each antigen solution and the CVP base used in the production of Example 1 using PBS (-) containing no antigen.

これを各検体に含まれる分量で示すと下記となる

This is shown below in terms of the amount contained in each sample.

　すなわち、ＨＢｓ－Ｌｈ抗原（２５ｎｇ）とＨＢｃ抗原０ｎｇ（ＨＢｓ－Ｌｈ抗原単独）、０．６２５ｎｇ（４：１）、１２．５ｎｇ（２：１）、２５ｎｇ（１：１）を混合後、実施例１の製造に使用したＣＶＰ基剤を等量混合もしくはＰＢＳ（－）を等量混合し、４℃で１週間静置した。（検体０９は－３０℃で保存した）その後、ウエスタンプロット法によりＨＢｓ－Ｌｈ抗原およびＨＢｃ抗原を検出した。（図１）タンパク質の検出と定量には、Fusion Solo S(M&S Instrument Inc)を用いて行った。結果を表７に示す。

That is, after mixing HBs-Lh antigen (25 ng) and HBc antigen 0 ng (HBs-Lh antigen alone), 0.625 ng (4:1), 12.5 ng (2:1), and 25 ng (1:1), Equal amounts of the CVP base used in the production of Example 1 or PBS (-) were mixed together, and the mixture was allowed to stand at 4° C. for one week. (Sample 09 was stored at -30°C) Thereafter, HBs-Lh antigen and HBc antigen were detected by Western blotting. (Figure 1) Protein detection and quantification were performed using Fusion Solo S (M&S Instrument Inc). The results are shown in Table 7.

括弧の中の数値はＣＶＰ＋（ＣＶＰ配合有）に対するＣＶＰ-（ＣＶＰ配合無）の比率を示す。 Quantification results of HBs-Lh antigen and HBc antigen protein

The numbers in parentheses indicate the ratio of CVP- (without CVP) to CVP+ (with CVP).

Hepatitis B surface hybrid antigen (HBs-Lh antigen) has poor stability, and especially when used simultaneously with hepatitis B nucleocapsid antigen (HBc antigen), the stability may further decrease depending on the amount of HBc antigen. At the same time, the stability of HBc antigen also decreased. It was confirmed and discovered that the decrease in stability of both antigens can be greatly improved by incorporating carboxyvinyl polymer (CVP).

Antibody production induction ability test in mice (nasal mucosal inoculation)
1. Preparation of vaccine formulation (1) Preparation of antigen stock solution: Preparation of HBs-Lh/HBc antigen stock solution As the HBs-Lh antigen (type C, type D), the virus particles produced in Example 1 were used. An HBs-Lh antigen stock solution was obtained by suspending 2.857 mg of the obtained virus-like particles in phosphate buffered saline (Gibco PBS, pH 7.4).
As the HBc antigen, the antigen produced in Example 1 was used. The HBc antigen stock solution was obtained by suspending 2.857 mg of HBc antigen in phosphate buffered saline (Gibco PBS, pH 7.4).

表中の「－」は「不含」を意味する。 (2) Production of carboxyvinyl polymer (CVP) base A CVP base was produced using the components listed in Table 8 and subjected to shearing force in the same manner as in Example 1. Cetylpyridinium chloride hydrate, polysorbate 80, macrogol 4000, and concentrated glycerin were all added to the CVP base of Example 1 in the amounts shown in the table after obtaining the CVP base.

"-" in the table means "not included".

Table 9 shows the pH and viscosity of the mixture obtained by mixing each of Bases 1 to 5 with phosphate buffered saline (Gibco PBS, pH 7.4) at a ratio of 1:1 (volume ratio).

(3) Preparation of vaccine formulations (3)-1 Vaccine formulations for nasal vaccination 1 to 5
Mix HBs-Lh antigen stock solution (antigen concentration 2.857 mg/mL) and HBc antigen stock solution (antigen concentration 2.857 mg/mL) at a 1:1 (volume ratio) to obtain each antigen concentration of 1.429 mg/mL (20 μg/14 μL). And so. Vaccine formulations 1 to 5 with each antigen concentration of 0.715 mg/mL (10 μg/14 μL) were obtained by mixing with Bases 1 to 5 at a ratio of 1:1 (volume ratio).

(3)-2 Vaccine formulation for nasal inoculation 6
HBs-Lh antigen stock solution (antigen concentration 2.857 mg/mL), HBc antigen stock solution (antigen concentration 2.857 mg/mL), and phosphate buffered saline (Gibco PBS, pH 7.4) at a ratio of 1:0.25:0.75 (volume ratio). Mix and mix with Example Base 1 in a 1:1 (volume ratio) to give an HBs-Lh antigen concentration of 1.429 mg/mL (20 μg/14 μL) and an HBc antigen concentration of 0.357 mg/mL (5 μg/14 μL). , [HBs-Lh antigen concentration 0.715 mg/mL (10 μg/14 μL), HBc antigen concentration 0.179 mg/mL (2.5 μg/14 μL)] was obtained.

(3)-3 Preparation of antigen-only vaccine formulation for nasal inoculation HBs-Lh antigen stock solution (antigen concentration 2.857 mg/mL), HBc antigen stock solution (antigen concentration 2.857 mg/mL) and phosphate buffered saline (Gibco PBS) , pH 7.4) at a ratio of 1:1:2 (volume ratio) to prepare a vaccine preparation composition [HBs-Lh antigen concentration 0.715 mg/mL (10 μg/14 μL), HBc antigen concentration 0.715 mg/mL (10 μg/14 μL). )], vaccine formulation 7 containing no CVP base was obtained.
HBs-Lh antigen stock solution (antigen concentration 2.857 mg/mL), HBc antigen stock solution (antigen concentration 2.857 mg/mL), and phosphate buffered saline (Gibco PBS, pH 7.4) at a ratio of 1:0.25:2.75 (volume ratio). By mixing, vaccine formulation composition [HBs-Lh antigen concentration 0.715 mg/mL (10 μg/14 μL), HBc antigen concentration 0.179 mg/mL (2.5 μg/14 μL)], vaccine formulation 8 containing no CVP base was obtained. Ta. The ingredients of each formulation are shown in Table 10.

表中の「－」は「不含」を意味する。

"-" in the table means "not included".

2. Antibody production induction ability test in mice (nasal mucosal inoculation)
(Method)
Vaccine formulations 1 to 8 were injected into both nostrils of BALB/c mice (female, 6 weeks old, 4 mice/group) in 7 μL doses (total 14 μL: HBs-Lh antigen amount 10 μg, HBc antigen amount 10 μg (Formulations 1 to 5). , 7) or 10 μg of HBs-Lh antigen and 2.5 μg of HBc antigen (Preparations 6 and 8).Blood was collected after 2 weeks, and antibody production was induced by measuring the antigen-specific antibody titer in the serum. The results are shown in Table 11 and Figures 5-6.

Vaccine formulations 1 and 6 using a CVP base provided in the present application have stronger antibody induction against both HBs-Lh and HBc compared to vaccine formulations 7 and 8 (comparative examples) that do not contain a CVP base. showed ability. Antibody induction ability was enhanced by further adding cetylpyridinium chloride hydrate to the CVP base.

Examples of formulations provided in this application are shown below. In the table, carboxyvinyl polymer is the carboxyvinyl polymer produced in Example 1 or 2. HBs-Lh antigen and HBc antigen were produced in Example 1.

DESCRIPTION OF SYMBOLS 1...Medical syringe, 2...Preparation, 3...Syringe barrel, 4...Syringe body, 5...Plunger rod, 5a...Fixing part, 6...Tip opening, 7...Piston, 8...Finger hook part, 9...Plunger operation part , 10... Nasal spray nozzle, 20... Nozzle main body, 21... Nozzle ejection hole, 22... Tip, 23... Inner wall of nozzle main body, 23a... Projection, 24... Internal space, 25... Nozzle small diameter part, 26... Nozzle large diameter part, 27... Nozzle shoulder part, 30... Filling rod (filling rod), 33... Outer wall of filling rod, 33a... Recessed part, 35... Rod small diameter part, 36... Rod large diameter part, 37... Rod shoulder Parts, 38, 39... Groove portion, 40... Gap, 42... Nozzle chamber, 44... Eddy flow forming part, 50... Protective cap.

Claims (8)

(i) Virus-like particles containing hepatitis B surface L antigen protein (HBs-L antigen protein) of two or more genotypes selected from the group consisting of type A, type B, type C, and type D; A hepatitis B vaccine composition comprising a base comprising a hepatitis nucleocapsid antigen (HBc antigen) protein, and (ii) an externally sheared carboxyvinyl polymer. 2. The virus-like particle containing HBs-L antigen protein comprises an HBs-L hybrid antigen (HBs-Lh) containing two genotypes of HBs-L antigen protein in one virus-like particle. Hepatitis B vaccine composition. The hepatitis B vaccine composition according to claim 2, wherein the HBs-L hybrid antigen has HBs-L antigen proteins of genotype C and genotype D. Claims 1 to 3, wherein the carboxyvinyl polymer treated by applying external shearing force has a carboxyl group content of 60.0 to 62.0% by mass and a number-based mode diameter of 0.05 to 10 μm. Hepatitis B vaccine composition according to any one of the above. The hepatitis B vaccine composition according to claim 4, further comprising a cationic surfactant. The hepatitis B vaccine composition according to claim 5, wherein the cationic surfactant is at least one selected from the group consisting of cetylpyridinium chloride, benzalkonium chloride, and benzethonium chloride. 7. The hepatitis B vaccine composition according to claim 6, wherein the cationic surfactant is cetylpyridinium chloride. A hepatitis B vaccine nasal administration system, which comprises filling the hepatitis B vaccine composition according to any one of claims 1 to 7 into a sprayable device having a nasal spray nozzle.

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