Hepatitis b vaccine composition for nasal administration and nasal administration system thereof

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 said vaccine composition

[0001] 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 said vaccine.

[0002] Hepatitis B is a liver disease caused by infection with the hepatitis B virus (HBV), which is transmitted through blood and bodily fluids. Persistent infection of liver cells with HBV is a serious infectious disease that can cause chronic hepatitis, cirrhosis, and hepatocellular carcinoma, but there is no treatment that can eliminate the virus.

[0003] Currently, pegylated interferon (PEG-IFN) and nucleoside analogs (NA) are the primary treatments for chronic hepatitis B (CHB). While PEG-IFN possesses immunostimulatory and antiviral properties, and has been shown to be effective in some cases in strengthening the immune system and sustaining viral replication suppression, it generally has low HBV elimination rates and frequent and diverse side effects. NA, on the other hand, inhibits viral replication, demonstrating a high HBV DNA negativity rate of approximately 95%, but does not eliminate the virus, and its efficacy rapidly disappears upon discontinuation of administration. This requires lifelong administration, posing significant compliance and medical economic challenges. Furthermore, there are reports of the potential for the emergence of resistant viruses with long-term use.

[0004] Meanwhile, in Japan, to prevent HBV infection, preventive vaccines are administered to those at high risk of infection (family members of HBV carriers, medical workers), and some success has been achieved in significantly reducing the number of HBV carriers. Immunotherapy using HBV vaccines is also being tested as a treatment for CHB.

[0005] Some of the present inventors have improved a formulation containing two antigens, HBs antigen (HBs-S antigen) and HBc antigen, and succeeded in developing a composition for nasal administration containing carboxyvinyl polymer (CVP) with the aim of inducing neutralizing antibodies against HBs antigen (hepatitis B virus surface antigen) and adaptive immunity against HBc antigen (hepatitis B nucleocapsid antigen) in HBV treatment (Patent Documents 3 and 4). The combination of HBs-S antigen and HBc antigen strongly activates cellular immunity, and the addition of CVP delays clearance of the vaccine in the nasal mucosa and enhances the immune response in the nasal mucosa, resulting in a vaccine composition that exhibits strong activity in both inducing anti-HBs neutralizing antibodies and activating cellular immunity.

[0006] Clinical trials of immunotherapy using this composition loaded into a dedicated device and administered intranasally are currently underway. The Phase 1 clinical trial involved 29 patients with chronic hepatitis B (CHB) undergoing treatment with nucleic acid analogs (NAs) and 42 untreated hepatitis B virus (HBV) carriers. The composition was administered intranasally once every two weeks for a total of 10 doses. Anti-HBs antibodies were induced in approximately half of the cases, and HBs antigen levels decreased over time. In some cases, HBs antigen disappeared, achieving a functional cure. Furthermore, adverse events were mild (Non-Patent Document 1).

[0007] HBV is a particle composed of DNA at its center and a capsid surrounding it, and further has an envelope structure containing many proteins in a 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 sometimes called the HBV surface antigen (HBs).

[0008] The full-length protein that forms the surface antigen of HBV is called the L protein, which has three regions, Pre-S1 region, Pre-S2 region, and S region, displayed on the outermost surface of the particle, in this order from the N-terminus.

[0009] A protein that lacks the Pre-S1 region from the HBs-L antigen protein called the full-length L protein and consists of the Pre-S2 region and S region is called an M protein, and a protein that lacks the Pre-S1 and Pre-S2 regions from the HBs-L antigen protein and consists of only the S region is called an S protein.

[0010] The various HBV antigen proteins described above can be produced in an expression system using eukaryotic host cells, such as yeast or animal cells, into which genes encoding them have been introduced, and these recombinant surface antigens are widely used as preventive vaccines against HBV. Among these, the use of S antigen produced in yeast is currently mainstream on the market. The HBs antigen used in the formulation containing HBc antigen, HBs antigen, and CVP used in the clinical trials described above is the S protein (HBs-S antigen protein).

[0011] On the other hand, the full-length L protein contains the S region and Pre-S2 region contained in the M protein, as well as the Pre-S1 region, which is the HBV sensor region. Therefore, when the L protein is used as a vaccine, antibodies against the Pre-S1 region displayed on the outermost surface are also produced, making it an excellent preventive vaccine. Some of the present inventors have confirmed that a vaccine composition combining HBs-L antigen and HBc antigen exhibits high cellular immunity activation capacity (Patent Documents 2 and 3).

[0012] It is known that there are multiple HBV genotypes, ranging from A to J, with types A, B, C, and D being the major HBV genotypes. The relationship between each genotype and phenotype has been clarified for the S region and pre-S2 region. For example, in the S region, the amino acid sequence from positions 124 to 147 is generally known as the highly immunogenic antigenic determinant "a." In the pre-S2 region, it is known that the antigenic determinant "b" is commonly present in each genotype (Non-Patent Document 2). Therefore, with regard to these regions, it is believed that common antibodies will be produced against at least some genotypes, even if the genotypes differ.

[0013] On the other hand, the Pre-S1 region has not been sufficiently analyzed with respect to each genotype and phenotype, and there is low homology in the amino acid sequence between each HBV genotype. Therefore, even if a vaccine is produced that contains an HBs-L antigen protein containing a Pre-S1 region derived from one genotype as an active ingredient, it does not necessarily elicit an immune response via the Pre-S1 region against HBV of other genotypes.

[0014] Some of the present inventors have proposed virus-like particles containing one or more HBs-L antigen proteins of genotype A, B, C, or D, or variants of any of these genotypes, as vaccines that address differences in genotype (Patent Documents 1 and 2). It has been confirmed that HBs-neutralizing antibodies against multiple genotypes can be induced by using a single virus-like particle containing HBs-L antigen proteins of one of the genotypes A, B, C, or D, in combination with multiple genotypes, or by using a single virus-like particle containing HBs-L antigen proteins of two or more genotypes. For example, it has been confirmed that antibodies induced by virus-like particles containing HBs-L antigen proteins of type C and type D have high neutralizing activity against all HBs antigens of type A, B, C, and D (Patent Document 1).

[0015] Patent No. 6613399 WO2019 / 013361 WO2019 / 070019 WO2019 / 235466

[0016] Medical Progress Vol. 281 No. 3, 265-268 (2022) Usuda S et al. (1999) J. Virol Methods. 80(1), 97-112

[0017] The present application aims to provide a stable vaccine composition for nasal administration and a nasal administration system for said vaccine.

[0018] The present application provides a hepatitis B vaccine composition for nasal administration, comprising: (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 types A, B, C, and D, and a hepatitis B nucleocapsid antigen (HBc antigen) protein; and (ii) a base containing a carboxyvinyl polymer treated by applying external shear force.

[0019] The present application also provides a nasal hepatitis B vaccine administration system, which comprises a syringe-type sprayer having a nasal spray nozzle filled with the nasal hepatitis B vaccine composition of the present application.

[0020] In another aspect, the present application provides a method for treating hepatitis B, comprising nasally administering an effective amount of the intranasal hepatitis B vaccine composition of the present application to a subject in need of treatment for hepatitis B. In the method of the present application, the intranasal hepatitis B vaccine composition may be administered using a syringe-type spray device having a nasal spray nozzle.

[0021] In yet another aspect, the present application provides use of a composition comprising: (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 A, B, C, and D, and hepatitis B nucleocapsid antigen (HBc antigen) protein, and (ii) a base containing a carboxyvinyl polymer treated by applying external shear force, for the production of a hepatitis B vaccine for nasal administration. The composition of the present application may be filled in a syringe-type spray device having a nasal spray nozzle.

[0022] Research by the present inventors has revealed that virus-like particles containing HBs-L antigen protein have poor stability, and that when used simultaneously with hepatitis B nucleocapsid antigen (HBc antigen), in particular, their stability further decreases depending on the amount of HBc antigen incorporated, and that the HBc antigen also becomes unstable at the same time. The hepatitis B vaccine composition of the present application is a stable vaccine formulation, despite containing virus-like particles containing HBs-L antigen protein and HBc antigen protein, and is suitable for use as a vaccine for the prevention and treatment of hepatitis B.

[0023] 3(a) and 3(b) are exploded perspective views showing the schematic configuration of the nasal spray nozzle used in the Examples, showing the state before and after the filling rod is inserted into the nozzle body. 3(a) is a vertical cross-sectional view of the nasal spray nozzle shown in FIG. 3(b), and (b) to (d) are horizontal cross-sectional views taken along lines B-B, C-C, and D-D in FIG. 4(a). 3(b) shows the results of ELISA analysis of antibody titers against HBs-LH antigen in mouse serum two weeks after nasal administration of Formulations 1 to 6 (Examples) and Formulations 7 and 8 (Comparative Examples). In Example 3, the antibody titer against HBc antigen in the serum of mice was examined by ELISA two weeks after nasal administration of Preparations 1 to 6 (Examples) and Preparations 7 and 8 (Comparative Examples) to mice.

[0024] As used herein, when a numerical value is accompanied by the term "about," it is intended to encompass a range of ±10% of that value. For example, "about 20" is intended to include "18 to 22." A range of numerical values ​​includes all values ​​between and at the endpoints. "About" in reference to a range applies to both endpoints of the range. Thus, for example, "about 20 to 30" is intended to include "18 to 33."

[0025] Virus-like particles containing HBs-L antigen protein The virus-like particles containing HBs-L antigen protein contained in the hepatitis B vaccine composition of the present application are particles whose main components are HBs-L antigen protein, which is a protein having one or more transmembrane domains, and a lipid bilayer membrane, and do not contain nucleic acids such as DNA or RNA that control genetic information inside the particles.

[0026] The genotype of the HBs-L antigen protein contained in the virus-like particles is selected from A, B, C, and D. Mutants of these genotypes are also used as the HBs-L antigen protein contained in the virus-like particles used in the present application. Note that the mutants defined herein are not limited to mutants obtained by mutation, but also include mutants obtained by artificially introducing mutations.

[0027] The amino acid sequence of the HBs-L antigen protein of each genotype may be any known sequence, and is not particularly limited. Examples include the amino acid sequence shown in any of SEQ ID NOS: 1 to 9 for genotype A, the amino acid sequence shown in any of SEQ ID NOS: 10 to 18 for genotype B, and the amino acid sequence shown in any of SEQ ID NOS: 29 to 38 for genotype D.

[0028] The amino acid sequences specifying the HBs-L antigen proteins of each genotype described above can also be variants of the amino acid sequences specified by SEQ ID NOS: 1 to 38, which specify the HBs-L antigen proteins of each genotype, as long as the effects of the present invention are not significantly impaired. Such variants are not particularly limited, and examples include substitutions, insertions, deletions, and the like.

[0029] The degree of mutation in the amino acid sequence of each of the above genotypes is not particularly limited. Examples include introduction of about 33 amino acid mutations into the amino acid sequence shown in any one of SEQ ID NOS: 1 to 9 (genotype A), introduction of about 26 amino acid mutations into the amino acid sequence shown in any one of SEQ ID NOS: 10 to 18 (genotype B), introduction of about 28 amino acid mutations into the amino acid sequence shown in any one of SEQ ID NOS: 19 to 28 (genotype C), and introduction of about 38 amino acid mutations into the amino acid sequence shown in any one of SEQ ID NOS: 29 to 38 (genotype D).

[0030] The degree of mutation introduced into the amino acid sequence of each genotype defined in the present invention is as described in Patent Document 1. Specific examples of mutants include those in which, if the sequence of SEQ ID NOs: 1 to 38 consists of 400 amino acids, a region of 11 amino acids from the N-terminus and / or a region of 6 amino acids from positions 163 to 168 are deleted. Furthermore, if the sequence of SEQ ID NOs: 1 to 38 does not consist of 400 amino acids, mutants in which the amino acid sequence of the corresponding region is deleted can be mentioned.

[0031] Examples of artificially mutated mutants include mutants in which the three regions of the HBs-L antigen protein, the Pre-S1 region, the Pre-S2 region, and the S region, are replaced with or deleted from regions derived from different genotypes. Specifically, examples include mutants in which the Pre-S1 region of one genotype is replaced with the Pre-S1 region of another genotype, mutants in which the Pre-S1 region and the Pre-S2 region of one genotype are replaced with the Pre-S1 region and the Pre-S2 region of another genotype, and mutants in which the Pre-S2 region of one genotype is replaced with the Pre-S2 region of another genotype. It is also possible to replace the Pre-S2 region with the Pre-S1 region of another genotype, and further mutants in which the Pre-S2 region is deleted. In these artificial substitutions, it is possible to introduce mutations in excess of the number of mutations introduced into the amino acid sequence of each genotype. In the present specification and claims, unless otherwise specified, the terms "genotype A," "genotype B," "genotype C," and "genotype D" refer to the respective genotypes of HBs-L antigen protein or any of their mutants.

[0032] As used herein, "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 A, B, C, and D" may refer to a combination of particles containing HBs-L antigen proteins of one genotype each, or to particles containing HBs-L antigen proteins of two genotypes each. Hereinafter, virus-like particles containing HBs-L antigen proteins of one genotype each will be referred to as "HBs-L antigen." Virus-like particles containing HBs-L antigen proteins of two genotypes each will be referred to as "HBs-L hybrid antigen" or "HBs-Lh antigen." The hepatitis B vaccine composition of the present application preferably contains HBs-L antigen proteins of two or more genotypes selected from A, B, C, and D, in appropriate combinations of HBs-L antigen and / or HBs-Lh antigen.

[0033] Examples of 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 protein and type D HBs-L antigen protein], [type B HBs-L antigen protein and type C HBs-L antigen protein], [type B HBs-L antigen protein and type D HBs-L antigen protein], [type C HBs-L antigen protein and type D HBs-L antigen protein], and [type B HBs-L antigen protein, type C HBs-L antigen protein and type D HBs-L antigen protein]. Preferably, the combination is an HBs-L antigen protein of genotype C and an antigen protein of genotype D. Particularly preferably used is an HBs-Lh antigen, which is a virus-like particle containing two types of antigen proteins, an HBs-L antigen protein of genotype C and an antigen protein of genotype D.

[0034] HBs-L antigen and HBs-Lh antigen, including HBs-L antigen proteins of each genotype, can be produced using genetically engineered yeast (Patent Documents 1 and 2). HBs-L antigen proteins have the ability to self-assemble, and can be presented as antigens by assembling on lipid membranes to form particles. HBs-L antigen proteins have an S region with high lipid affinity, and regardless of the genotype, when produced using biological cells, they are embedded in lipid membranes. This allows the protein to take the form of a virus-like particle, which is a stable antigen particle structure, and this particle structure gives the protein high immunogenicity.

[0035] Hepatitis B Nucleocapsid Antigen (HBc Antigen) The hepatitis B nucleocapsid antigen (HBc antigen) contained in the hepatitis B vaccine composition of the present application is also known as hepatitis B core antigen. Any known protein may be used as an HBc antigen, and there is no particular limitation. 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, a full-length HBc antigen DNA (ACC# X01587) can be inserted into a pET-19b vector from which sequences such as His-tag have been removed to obtain an expression vector for the HBc antigen. The resulting expression vector can be introduced into Escherichia coli (E. coli) to obtain an expression strain, which is then cultured to obtain bacterial cells, from which the HBc antigen can be purified. It is known that HBc antigens form particles by mutual binding of individual capsid proteins, and HBc antigens produced by E. coli also self-assemble to form particles.

[0036] In the hepatitis B vaccine composition of the present application, the blending ratio of HBs-L antigen to HBc antigen is not limited, and the ratio of the amount of HBc antigen protein to the total weight of HBs-L antigen and / or HBs-Lh antigen as virus-like particles is preferably 1:0.1 to 1:10, and examples thereof include 1:about 0.25, 1:about 0.5, 1:about 1, 1:about 1.25, 1:about 1.5, and 1:about 2.

[0037] Base containing a carboxyvinyl polymer treated by applying external shear force Examples of the base contained in the hepatitis B vaccine composition of the present application and containing a carboxyvinyl polymer treated by applying external shear force include the "gel base containing a skin / mucosa adhesive agent" disclosed in WO2007 / 123193.

[0038] Carboxyvinyl polymers are polymers of acrylic acid, and the carboxyl group content of a carboxyvinyl polymer refers to the proportion (% by mass) of carboxyl groups contained in the polymer relative to the total amount of the polymer. The method for quantifying the carboxyl group content is not particularly limited, and may be determined, for example, using the method for quantifying carboxyvinyl polymers listed in the Pharmaceutical Excipients Standards. The CVP used in the vaccine composition of the present application preferably has a carboxyl group content of 60.0 to 62.0% by mass.

[0039] The polymer particle diameter of the CVP used in the composition of the present invention is preferably adjusted to a number-based mode diameter of 0.05 to 10 μm (preferably 0.1 to 2.0 μm) by applying an external shear force. Hereinafter, unless otherwise specified, the term "mode diameter" refers to the number-based mode diameter.

[0040] The polymer particle size (particle size distribution) is measured as a number-based particle size distribution that can be measured using a laser diffraction particle size distribution analyzer, a dynamic light scattering particle size distribution analyzer, or the like, but it is preferable that the result does not differ significantly from the volume-based particle size distribution result. Even if the analysis results differ depending on the analyzer, if the mode diameter of the polymer particles is within a predetermined range using at least one analyzer, the mode diameter of the polymer particles can be considered to be within the predetermined range. The mode diameter may also be determined by observing the state of the polymer particles using a phase-contrast microscope, Opt-SEM (Optical Shadow Effect Mode Microscope), or the like. Alternatively, the polymer particle size may be measured using a laser diffraction particle size distribution analyzer according to the method described in the test examples of this application.

[0041] The mode diameter of the CVP is adjusted by applying an external mechanical shear force to the CVP. For example, the mode diameter of the polymer particles can be adjusted to a desired range by applying an external shear force to a commercially available CVP. The shear force can be applied by a method known to those skilled in the art. For example, devices that can apply mechanical shear force include a high-speed rotary emulsifier, a colloid mill emulsifier, a high-pressure emulsifier, a roll mill emulsifier, an ultrasonic emulsifier, and a membrane emulsifier. High-speed rotary emulsifiers such as a homomixer, a comb-tooth type, and an intermittent jet flow generating type are particularly preferred.

[0042] The hepatitis B vaccine composition of the present application contains a gel base containing CVP treated by external shear force. The CVP may be neutralized. Examples of neutralizing agents added to the CVP-containing gel base include sodium hydroxide, potassium hydroxide, and L-arginine. Neutralizing the CVP, which is the product, ionizes the CVP and sterically expands it, thereby thickening the gel base. For example, when L-arginine is added, the weight ratio of L-arginine to CVP is, for example, in the range of about 1:0.5 to about 1:3, preferably about 1:1 to about 1:2.5, e.g., about 1:2.

[0043] The hepatitis B vaccine composition of the present application may further contain a pharmaceutically acceptable carrier. Examples of the carrier include those commonly used in the production of vaccines and intranasal preparations, such as saline, buffered saline, dextrose, water, glycerin, isotonic aqueous buffer solutions, and combinations thereof. Preservatives (e.g., thimerosal), isotonicity agents, pH adjusters, surfactants, stabilizers (e.g., edetate sodium hydrate), and inactivators (e.g., formalin) may also be added as appropriate.

[0044] In particular, it is preferable to add at least one cationic surfactant selected from the group consisting of cetylpyridinium chloride, benzalkonium chloride, and benzethonium chloride, and it is particularly preferable to add cetylpyridinium chloride or cetylpyridinium chloride hydrate. When cetylpyridinium chloride hydrate is added, the amount may be determined appropriately, and for example, the weight ratio to CVP is 1: about 0.01 to 0.03, more preferably 1: about 0.018 parts by weight.

[0045] The concentration of CVP in the hepatitis B vaccine composition of the present application is preferably 0.1 to 2.0 w / v%, more preferably 0.3 to 1.0 w / v%, for example, about 0.55 w / v%. One example of a base containing a carboxyvinyl polymer treated by externally applying shear force of the present application is a base containing a carboxyvinyl polymer treated by externally applying shear force at a concentration of about 0.55 w / v% and about 2 parts by weight of L-arginine per 1 part by weight of CVP. Another example is a base further containing about 0.018 part by weight of cetylpyridinium chloride hydrate per 1 part by weight of CVP.

[0046] The concentrations of each antigen in the hepatitis B vaccine composition of the present application are, for example, within the range of 0.01 to 10 mg / mL, preferably 0.05 to 5 mg / mL, for the total amount of HBs-L antigen (total amount of HBs-L antigen and / or HBs-Lh antigen) and the amount of HBs antigen, for example, each is approximately 0.1 mg / mL.

[0047] The hepatitis B vaccine composition of the present application can be produced by mixing and stirring stock solutions in which HBs-L antigen and HBc antigen are separately suspended, and then mixing the mixed antigen solution with a base containing a carboxyvinyl polymer treated by external shear force. No adjuvants other than these components need to be added to the composition of the present application. This mixing and stirring can be achieved in a short time by gentle mixing and stirring without subjecting each antigen to stress such as heat or pressure.

[0048] 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 sprayed formulation has an average particle size in the range of 50 μm to 120 μm (preferably, 50 μm to 80 μm), a particle size distribution of 50% or more in the range of 10 μm to 100 μm (preferably, 70% or more in the range of 10 μm to 100 μm), a spray angle from the device in the range of 30° to 70° (preferably, 40° to 60°), and a spray density of a full cone that is uniform and even. Note that a "full cone," which refers to a uniform spray density without any bias, is one type of spray pattern shape and refers to a uniform, circular area; the opposite term is a "hollow cone," which is a donut-shaped spray with the spray density localized only to the periphery.

[0049] The vaccine composition of the present application is administered intranasally by spraying into one or both nostrils one or more times. The dose is determined taking into consideration the age, sex, weight, etc. of the subject, but the amount of antigen administered is preferably 0.01 to 5 mg of HBc antigen and 0.01 to 5 mg of the total amount of HBs-L antigen (total amount of HBs-L antigen and / or HBs-Lh antigen), more preferably 0.05 to 2 mg each. The amount of formulation sprayed per nostril is preferably 50 μL to 1000 μL, more preferably 250 μL to 1000 μL.

[0050] The frequency and duration of administration may be determined taking into consideration the age, sex, weight, etc. of the subject, the severity of the disease, etc. For example, the drug may be administered multiple times, for example, 3 to 20 times, 5 to 15 times, or about 10 times, at intervals of 3 to 21 days, 7 to 17 days, or about 14 days.

[0051] The hepatitis B vaccine composition of the present application may be administered by filling it into a sprayable device having a nasal spray nozzle. The sprayable device having a nasal spray nozzle is not limited to commonly used nasal devices, and may also be a sprayable device without a pump function. For example, by using an upward pressure airless spray container as a multi-dose spray container, as described in WO 2007 / 123193 and WO 2007 / 123207, the spray container can be used without remaining in the container regardless of the angle or range of angles at which the spray container is administered. Examples of disposable devices limited to use by a single vaccinee include a medical syringe equipped with a nasal spray nozzle as disclosed in WO 2015 / 199130, and a device with a nasal spray / spray nozzle as described in WO 2021 / 066195 and Japanese Patent Application No. 2021-126673.

[0052] The present invention will be described below with reference to examples, but the present invention is not limited to these examples in any way.

[0053] A CVP base, a stock solution of HBs-Lh antigen, and a stock solution of HBc antigen were prepared by the methods described below, and mixed as follows to prepare a nasal hepatitis B vaccine composition.

[0054] Preparation of Carboxyvinyl Polymer (CVP) Base

[0055] [Preparation of HBs-Lh antigen stock solution]

[0056] A schematic diagram of the HBs-Lh antigen produced in this example is shown in Figure 1. The HBs-Lh antigen was prepared by feeding-batch culturing yeast transformed to express two HBs-L antigen proteins, genotypes C and D. The resulting cells were disrupted, heat-treated, and subjected to affinity gel filtration to generate frozen virus-like particles. The L protein had a molecular weight of approximately 45 kDa, a purity of 99% or greater, and a particle diameter of 60-70 nm. The HBs-L antigen stock solution was prepared by suspending the resulting virus-like particles in purified water containing the respective components in the amounts listed in Table 2.

[0057] [Preparation of HBc antigen stock solution]

[0058] HBc antigen was obtained by feeding culture of Escherichia coli transformed to express the full-length HBc antigen protein, followed by cell disruption, ammonium sulfate precipitation, gel filtration, and endotoxin removal. The HBc antigen had a molecular weight of approximately 21 kDa, a purity of 99% or more, a particle size of 30-35 nm, and an endotoxin concentration of <50 EU / mg protein.

[0059] [Mixing of carboxyvinyl polymer (CVP) base and virus stock solution] The above HBs-Lh antigen stock solution was mixed with HBc antigen stock solution (ratio 1:1), and then the antigen mixture was mixed with equal amounts of CVP base and stirred until homogeneous, to obtain a nasal hepatitis B vaccine composition (Example 1). This mixing and stirring can be achieved in a short time by gentle mixing and stirring without subjecting the hepatitis B vaccine antigen to stress such as heat or pressure.

[0060] The resulting nasal hepatitis B vaccine composition was loaded into a nasal administration device, and the droplet size distribution, spray uniformity, and spray angle of the formulation were measured. The nasal administration device used was a nasal spray nozzle described in WO 2015 / 199130 attached to a medical syringe (metered-dose syringe-type sprayer). Figures 2 to 4 show the device used. The nozzle 21 at the tip of the nozzle had a hole diameter of 0.26 mm. The results confirmed that the nasal hepatitis B vaccine administration system was extremely effective, with (1) a mean droplet size distribution of the formulation ranging from 30 μm to 80 μm (63.6 μm), with 80% or more of the droplet size distribution falling within the range of 10 μm to 100 μm (85.3%), (2) a uniform full cone spray density, and (3) a spray angle controlled within the range of 30° to 70° (51°). The hepatitis B vaccine intranasal administration system of Example 1 is summarized below.

[0061]

[0062] Effect of Mixing Ratio and CVP on HBs-Lh Antigen and HBc Antigen Stability The HBs-Lh antigen and HBc antigen used in Example 1 were each suspended in PBS to obtain an HBs-Lh antigen solution (0.1 μg / mL PBS solution) and an HBc antigen solution (0.1 μg / mL PBS solution). The following formulations were prepared using each antigen solution and PBS (-) containing no antigen, as well as the CVP base used in the production of Example 1.

[0063] The amounts contained in each sample are as follows:

[0064] Specifically, HBs-Lh antigen (25 ng) was mixed with HBc antigen (0 ng (HBs-Lh antigen alone), 0.625 ng (4:1), 12.5 ng (2:1), or 25 ng (1:1), followed by an equal volume of the CVP base used in Example 1 or an equal volume of PBS (-) and allowed to stand at 4°C for one week. (Sample 09 was stored at -30°C.) HBs-Lh antigen and HBc antigen were then detected by Western blot analysis (Figure 1). Protein detection and quantification were performed using a Fusion Solo S (M&S Instrument Inc.). The results are shown in Table 7.

[0065] Quantitative results of HBs-Lh antigen and HBc antigen protein The numbers in parentheses indicate the ratio of CVP- (without CVP) to CVP+ (with CVP).

[0066] Hepatitis B surface hybrid antigen (HBs-Lh antigen) has poor stability, and when used simultaneously with hepatitis B nucleocapsid antigen (HBc antigen), its stability further decreased depending on the amount of HBc antigen added, and the stability of HBc antigen also decreased. It was confirmed and discovered that the decreased stability of both antigens can be significantly improved by adding carboxyvinyl polymer (CVP).

[0067] Antibody Production Induction Ability Test in Mice (Nasal Mucosal Inoculation) 1. Preparation of Vaccine Preparation (1) Preparation of Antigen Stock Solution: Preparation of HBs-Lh / HBc Antigen Stock Solution HBs-Lh antigen (types C and D) was prepared using the virus particles produced in Example 1. The HBs-Lh antigen stock solution was obtained by suspending 2.857 mg of the resulting virus-like particles in phosphate-buffered saline (Gibco PBS, pH 7.4). The HBc antigen was prepared using the antigen produced in Example 1. The HBc antigen stock solution was obtained by suspending 2.857 mg of HBc antigen in phosphate-buffered saline (Gibco PBS, pH 7.4).

[0068] (2) Preparation of Carboxyvinyl Polymer (CVP) Base A CVP base was prepared by applying shear force using the components listed in Table 8 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. In the table, "-" means "not included."

[0069] Table 9 shows the pH and viscosity of the mixtures obtained by mixing each of Bases 1 to 5 with phosphate buffered saline (Gibco PBS, pH 7.4) in a 1:1 (volume ratio).

[0070] (3) Preparation of vaccine preparations (3)-1 Vaccine preparations 1 to 5 for nasal administration HBs-Lh antigen stock solution (antigen concentration 2.857 mg / mL) and HBc antigen stock solution (antigen concentration 2.857 mg / mL) were mixed at a 1:1 (volume ratio) to give an antigen concentration of 1.429 mg / mL (20 μg / 14 μL). These were mixed at a 1:1 (volume ratio) with bases 1 to 5 to give vaccine preparations 1 to 5 with antigen concentrations of 0.715 mg / mL (10 μg / 14 μL).

[0071] (3)-2 Vaccine Preparation 6 for Nasal Inoculation An HBs-Lh antigen stock solution (antigen concentration 2.857 mg / mL), an HBc antigen stock solution (antigen concentration 2.857 mg / mL), and phosphate-buffered saline (Gibco PBS, pH 7.4) were mixed in a volume ratio of 1:0.25:0.75 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). This was mixed with Example Base 1 in a volume ratio of 1:1 to give Vaccine Preparation 6 (HBs-Lh antigen concentration 0.715 mg / mL (10 μg / 14 μL) and an HBc antigen concentration 0.179 mg / mL (2.5 μg / 14 μL)).

[0072] (3)-3 Preparation of antigen-only vaccine formulation for nasal inoculation An HBs-Lh antigen stock solution (antigen concentration 2.857 mg / mL), an HBc antigen stock solution (antigen concentration 2.857 mg / mL), and phosphate-buffered saline (Gibco PBS, pH 7.4) were mixed in a 1:1:2 (volume ratio) to obtain vaccine formulation composition [HBs-Lh antigen concentration 0.715 mg / mL (10 μg / 14 μL), HBc antigen concentration 0.715 mg / mL (10 μg / 14 μL)] and vaccine formulation 7, which did not contain a CVP base. 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) were mixed in a volume ratio of 1:0.25:2.75 to obtain a 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)] and vaccine formulation 8, which does not contain a CVP base. The components of each formulation are shown in Table 10.

[0073] In the table, "-" means "not included."

[0074] 2. Antibody production induction test in mice (nasal mucosal inoculation) (Method) BALB / c mice (female, 6 weeks old, 4 mice / group) were inoculated with 7 μL of each of vaccine preparations 1 to 8 into both nostrils (total 14 μL: 10 μg HBs-Lh antigen and 10 μg HBc antigen (preparations 1 to 5, 7) or 10 μg HBs-Lh antigen and 2.5 μg HBc antigen (preparations 6 and 8). Blood was collected two weeks later, and antibody production induction was analyzed by measuring the antigen-specific antibody titer in the serum. The results are shown in Table 11 and Figures 5-6.

[0075] Vaccine preparations 1 and 6, which use the CVP base provided herein, exhibited stronger antibody induction capabilities against both HBs-Lh and HBc than vaccine preparations 7 and 8 (comparative examples) which do not contain a CVP base. The antibody induction capabilities were enhanced by further adding cetylpyridinium chloride hydrate to the CVP base.

[0076] Examples of formulations provided in the present application are shown below. In the table, the carboxyvinyl polymer is the carboxyvinyl polymer produced in Example 1 or 2. The HBs-Lh antigen and HBc antigen are those produced in Example 1.

[0077] 1...medical syringe, 2...preparation, 3...syringe barrel, 4...syringe body, 5...plunger rod, 5a...fixing portion, 6...tip opening, 7...piston, 8...finger hook portion, 9...plunger operation portion, 10...nasal spray nozzle, 20...nozzle body portion, 21...nozzle ejection hole, 22...tip portion, 23...inner wall of nozzle body portion, 23a...projection portion, 24...internal space, 25...nozzle small diameter portion, 26...nozzle large diameter portion, 27...nozzle shoulder portion, 30...filling rod (filling rod), 33...outer wall of filling rod, 33a...recess, 35...rod small diameter portion, 36...rod large diameter portion, 37...rod shoulder portion, 38, 39...groove portion, 40...gap, 42...nozzle chamber, 44...vortex forming portion, 50...protective cap

Claims

1. (i) Virus-like particles containing two or more genotypes of hepatitis B surface L antigen protein (HBs-L antigen protein) selected from the group consisting of types A, B, C, and D, and hepatitis B nucleocapsid antigen (HBc antigen) protein, (ii) A hepatitis B vaccine composition comprising a base containing a carboxyvinyl polymer that has been treated by applying external shear force.

2. The hepatitis B vaccine composition according to claim 1, wherein the virus-like particles containing HBs-L antigen protein contain an HBs-L hybrid antigen (HBs-Lh) that contains two genotypes of HBs-L antigen protein in a single virus-like particle.

3. 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.

4. The hepatitis B vaccine composition according to claim 1, wherein the carboxyvinyl polymer, which has been treated by applying an external shear force, has a carboxyl group content of 60.0 to 62.0% by mass and a modal diameter based on the number of molecules of 0.05 to 10 μm.

5. The hepatitis B vaccine composition according to claim 4, further comprising a cationic surfactant.

6. 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.

8. A hepatitis B vaccine nasal administration system comprising filling a sprayable device having a nasal spray nozzle with the hepatitis B vaccine composition described in any one of claims 1 to 7.