Nasal hepatitis b vaccine composition and method for producing same

Abstract

The present invention relates to a hepatitis B vaccine composition for spray administration to the nasal mucosa, characterized by comprising a hepatitis B surface antigen (HBs antigen), a hepatitis B nucleocapsid antigen (HBc antigen) and a gel base containing a carboxyl vinyl polymer, and by being applicable to hepatitis B prevention and treatment. The vaccine composition produces an improved immune response.

Description

Intranasal hepatitis B vaccine composition and method for producing same

[0001] The present invention relates to a hepatitis B vaccine composition for nasal mucosal spray administration used for the prevention and treatment of hepatitis B, and a method for producing the same.

[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 hepatocytes with HBV can lead to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma.

[0003] Currently, interferon (IFN) and nucleic acid analogs (NA) are the primary treatments for chronic hepatitis B (CHB). While IFN has been shown to be effective in some cases, strengthening immunity and sustaining viral replication suppression, it generally suffers from low HBV elimination rates and severe side effects. Meanwhile, NA has a high HBV elimination rate of approximately 95%, but does not provide a cure and requires lifelong administration, posing significant compliance and medical economic challenges. Furthermore, there have been reports of the possibility of the virus developing resistance with long-term use. Therefore, the development of a new treatment for CHB was needed.

[0004] On the other hand, 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.On the other hand, immunotherapy using HBV vaccines has also been tried as a treatment for CHB, but sufficient therapeutic effects have not yet been achieved.

[0005] HBV contains multiple antigens, and the present inventors have attempted immunotherapy, focusing on the HBs antigen (hepatitis B surface antigen), which induces neutralizing antibodies. Furthermore, recent advances in research have revealed that acquired immunity against the HBc antigen (hepatitis B nucleocapsid antigen) contributes to the inhibition and elimination of HBV proliferation. In this context, the Cuban Institute of Biomedical Engineering (CECMED) developed a nasal therapeutic vaccine containing two antigens, HBs antigen and HBc antigen. Clinical trials were conducted in Bangladesh, and the vaccine was successfully commercialized as HeberNasvac (registered trademark, Non-Patent Document 1). However, because nasal administration alone does not generate an adequate immune response, two cycles of vaccination, including subcutaneous administration, are required, and the vaccine is not a fully mucosal nasal vaccine.

[0006] As described above, a versatile intranasal vaccine formulation for therapeutic and preventive HBV vaccines has been eagerly awaited as a next-generation hepatitis B vaccine, but in reality, it has not been possible to fully realize this vaccine because a sufficient immune response has not been obtained.

[0007] WO2007 / 123193

[0008] HeberNasvac package insert

[0009] The object of the present invention is to provide a hepatitis B vaccine composition for simpler administration by nasal mucosal spray, which does not require concomitant use by other administration routes such as subcutaneous administration and does not contain auxiliary components such as adjuvants that may be toxic, a method for producing the same, and a method for treating and preventing hepatitis B.

[0010] In view of the above problems, the present inventors have conducted extensive research and have found that a gel base for nasal mucosal spray administration, which is made of a carboxyvinyl polymer that has been treated with external shear force to enhance spraying performance, can be administered via nasal administration with two antigens, HBs antigen and HBc antigen, without the use of an adjuvant, thereby sufficiently enhancing the immune-inducing ability in humans, and have thus completed the present invention.

[0011] [1] A hepatitis B vaccine composition for nasal mucosal spray administration, comprising: (i) hepatitis B surface antigen (HBs antigen) and / or hepatitis B nucleocapsid antigen (HBc antigen), and (ii) a gel base containing a carboxyvinyl polymer that has been treated by applying external shear force to impart sprayability.

[0012] [2] The hepatitis B vaccine composition for nasal mucosal spray administration according to [1], wherein the hepatitis B vaccine (i) is 0.01 to 10 mg / mL per antigen.

[0013] [3] The hepatitis B vaccine composition for nasal mucosal spray administration described in [1] or [2], which contains 0.1 w / v% to 1.0 w / v% carboxyvinyl polymer.

[0014] [4] A hepatitis B vaccine composition for nasal mucosal spray administration according to any one of [1] to [3], which uses a gel base for spray administration containing a carboxyvinyl polymer that has been treated by applying external shear force to add spray performance such as (1) a range of particle size distribution of the formulation, (2) uniformity of spray density, and / or (3) spray angle control.

[0015] [5] A hepatitis B vaccine composition for nasal mucosal spray administration according to any one of [1] to [4], which is obtained by using a gel base containing 0.5 w / v % to 2.0 w / v % of a carboxyvinyl polymer, and obtaining a gel base for spray administration containing a carboxyvinyl polymer that has been treated by applying external shear force to add spray performance such as (1) a range of formulation particle size distribution, (2) uniformity of spray density, and / or (3) spray angle control, and then uniformly mixing the gel base with a virus stock solution containing HBs antigen and HBc antigen in a short period of time without applying stress.

[0016] [6] The hepatitis B vaccine composition for nasal mucosal spray administration according to any one of [1] to [5], which is produced by applying external shear force to a base containing a carboxyvinyl polymer, using a gel base for spray administration to which (1) the average particle size of the formulation is in the range of 50 μm to 120 μm, with 50% or more of the particle size distribution being in the range of 10 μm to 100 μm, (2) the spray density becomes a uniform full cone without bias, and (3) spray performance is added, with the spray angle controlled to the range of 30° to 70°.

[0017] [7] The hepatitis B vaccine composition for nasal mucosal spray administration according to any one of [1] to [5], which is produced by applying external shear force to a base containing a carboxyvinyl polymer, using a gel base for spray administration to which (1) the average particle size of the formulation is in the range of 70 μm to 100 μm, and 60% or more of the particle size distribution is in the range of 10 μm to 100 μm, (2) the spray density becomes a uniform full cone without bias, and (3) spray performance is added, with the spray angle controlled to the range of 40° to 60°.

[0018] [8] A hepatitis B vaccine composition for nasal mucosal spray administration according to any one of [1] to [7], which uses a gel base for spray administration containing a carboxyvinyl polymer that has been treated by applying external shear force to add (1) a formulation particle size distribution range, (2) spray density uniformity, and (3) spray angle control, so as to enable spray administration using a sprayable device without a pump function.

[0019] [9] A method for preventing and / or treating hepatitis B, comprising administering the hepatitis B vaccine composition for nasal mucosal spray administration described in any one of [1] to [8] to a subject in need thereof using a device that can spray a viscous formulation onto the intranasal mucosa from each nostril.

[0020]

[10] Use of the hepatitis B vaccine composition for nasal mucosal spray administration according to any one of [1] to [8] for treating and / or preventing hepatitis B.

[0021] According to the present invention, a vaccine composition containing HBs antigen and HBc antigen as active ingredients can induce an effective immune response with a small amount of antigen, making it possible to provide a hepatitis B vaccine composition for nasal mucosal spray administration that does not require concomitant administration via other administration routes such as subcutaneous administration and does not require the use of an adjuvant. Furthermore, as a formulation that is easy to administer and has few side effects, it can not only prevent hepatitis B but also induces a sufficient immune response, making it applicable to the treatment of chronic hepatitis B, and its strong activity is expected to lead to a complete cure of CHB with continuous administration.

[0022] This graph shows the results of an immune response analysis test, showing the results of neutralizing antibody titers measured one week after the final vaccination. In the graph, "sc" means subcutaneous vaccination, and "in" means nasal vaccination. The vehicle refers to CVP treated with external shearing force; (-) means vehicle that does not contain CVP, and (+) means vehicle that contains CVP. This graph shows the results of an immune response analysis test, showing the results of IgA antibody measurements measured one week after the final vaccination. In the graph, "sc" means subcutaneous vaccination, and "in" means nasal vaccination. The vehicle refers to CVP treated with external shearing force; (-) means vehicle that does not contain CVP, and (+) means vehicle that contains CVP.

[0023] The present invention provides a hepatitis B vaccine composition for nasal mucosal spray administration, which contains a gel base for nasal mucosal spray administration consisting of a carboxyvinyl polymer treated by applying external shear force to impart spraying properties, and a hepatitis B antigen.

[0024] The term "gel base containing a carboxyvinyl polymer treated by external application of shear force to impart sprayability" as used herein refers to, for example, the "gel base containing a skin / mucosa adhesive" disclosed in WO 2007 / 123193, which is a base containing a carboxyvinyl polymer and optionally gellan gum, and whose viscosity has been adjusted by external application of shear force. The specific external shear force described in WO 2007 / 123193 is not simply stirring or shaking, but is applied using a shear force-applying device known to those skilled in the art. Specifically, a high-speed rotary emulsifier, colloid mill emulsifier, high-pressure emulsifier, roll mill emulsifier, ultrasonic emulsifier, or membrane emulsifier can be used. High-speed rotary emulsifiers, such as homomixer, comb-tooth, and intermittent jet flow-generating types, are particularly preferred. Such bases are characterized by the ability to adjust viscosity to various levels by external application of shear force, and the spray angle and spray density from the spray container can be controlled to suit the intended purpose.

[0025] The device used for spray administration is not limited to any commonly used nasal instillation device, and the composition of the present invention can be used with a spray-capable 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 (see Figures 1 and 2) and WO 2007 / 123207 (see Figures 1 to 11), the spray container can be used without residual amount in the container at any angle or range of angles, making it suitable for vaccinating large numbers of people in developing countries. Furthermore, as a disposable device limited to use by a single vaccinee, the nasal spray nozzle disclosed in WO 2015 / 199130 (see Figures 1 to 4) can be used. In the present invention, the hepatitis B virus antigen administered using such a spray administration device is spread over a wide area and for a long period of time on the nasal mucosa, thereby enhancing the immunogenicity of the vaccine.

[0026] The carboxyvinyl polymer used as the raw material for the base for nasal mucosal spray administration is a hydrophilic polymer obtained by polymerizing acrylic acid as the main component, and pharmaceutical additives commonly used for preparing aqueous gels can be used without limitation. The content of the gel base containing the carboxyvinyl polymer treated by applying external shear force to impart sprayability is 0.1 to 1.0% w / v, preferably 0.3 to 0.7% w / v, calculated as the carboxyvinyl polymer content.

[0027] The vaccine of the present invention is characterized by containing both or either of the surface type (HBs antigen) and core type (HBc antigen) of hepatitis B virus antigen as antigens. The hepatitis B virus antigen used in the present invention refers to hepatitis B virus surface antigen and hepatitis B virus nucleocapsid antigen produced by yeast using recombinant DNA technology. The hepatitis B virus antigen is prepared using a purified or concentrated virus stock solution for mixing with a gel base for nasal mucosal spray administration. The vaccine of the present invention preferably has a hepatitis B virus antigen concentration of 0.01 to 10 mg / mL per vaccine antigen, more preferably 0.05 to 5 mg / mL. Hepatitis B virus surface antigen (HBsAg) is in the form of particles (approximately 50 to 60 nm in diameter) with many antigenic proteins displayed on a lipid membrane. The antigen protein displayed is originally composed of three domains (S, Pre-S1, Pre-S2). Antigens that possess all three domains are called L-type antigens (HBsAg L-protein), those that lack Pre-S1 are called M-type antigens, and those that lack both Pre-S1 and Pre-S2 are called S-type antigens (HBsAg S-protein). All antigens are produced using genetically modified yeast.

[0028] Adjuvants are a general term for substances that have regulatory activity, such as enhancing or suppressing immune responses. They are immune-enhancing agents added to vaccines to increase the immunogenicity of antigens, and many such substances have been investigated. While the use of adjuvants improves the immune effect of vaccines, they also have the disadvantage of potentially causing side effects such as inflammation. Naturally, several adjuvants have been proposed as candidates for nasal vaccines, but because there are no adjuvants whose safety has been widely recognized, no nasal vaccines containing adjuvants with established efficacy and safety have yet been approved.

[0029] In the present invention, in the method of mixing the spray-administered gel base with the virus stock solution, "without applying stress" means that heat, pressure, etc. are not applied and high-speed stirring is not performed.

[0030] The present inventors have discovered a nasal mucosal spray hepatitis B vaccine composition that uses a gel base for nasal mucosal spray administration, which has excellent spraying performance and a high rate of nasal mucosa spread, for the hepatitis B vaccine, thereby making it possible to obtain an effective vaccine with fewer side effects and a smaller amount of antigen without the need for concomitant use with other administration routes such as subcutaneous administration and without the use of adjuvants. By combining this with a device that can spray even highly viscous gel bases, the average particle size of the sprayed formulation is in an appropriate range of 50 to 120 μm (preferably, 70 to 100 μm), the formulation particle size distribution is 50% or more in the range of 10 to 100 μm (preferably, 60% in the range of 10 to 100 μm), the spray angle from the device is in the range of 30 to 70° (preferably, 40 to 60°) so that the vaccine can be administered to the required site in the nasal cavity, and the spray density is uniformly distributed over the full cone. These discoveries led to the present invention.

[0031] In the present invention, a "full cone" which represents a uniform spray density without any bias in the spray density is one type of spray pattern shape and means a uniform circular area, while the opposite term is a donut-shaped "hollow cone" which is localized only to the periphery.

[0032] The vaccine of the present invention may further contain a pharmaceutically acceptable carrier in addition to the hepatitis B virus antigen and the gel base for nasal mucosal spray administration. The carrier may be any of those commonly used in the production of vaccines and intranasal administration formulations, including 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.

[0033] The dosage is determined taking into consideration the age, sex, weight, etc. of the subject, but typically 0.1 to 100 μg, preferably 0.5 to 10 μg per antigen can be administered once or twice or more times. Multiple administrations are preferred, and in this case, administration is preferably performed at intervals of 1 to 4 weeks.

[0034] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0035] A gel base and a stock solution of hepatitis B virus were prepared by the methods described below, and the two were mixed as follows to prepare a hepatitis B vaccine composition. [Preparation of Gel Base] Gel Base Example 1

[0036] [Virus stock solution containing hepatitis B virus antigen] Virus stock solution example 1

[0037] Virus stock solution example 2

[0038] [Mixing of Gel Base and Virus Stock Solution] The above-mentioned Virus Stock Solution Example 1 and Virus Stock Solution Example 2 (ratio 1:1) and Gel Base Example 1 were mixed and stirred to obtain a homogeneous intranasal hepatitis B vaccine composition (Examples 1 and 2). This mixing and stirring can be achieved by gentle mixing and stirring in a short time without subjecting the hepatitis B vaccine antigen to stress such as heat or pressure. The component amounts and physical properties of the obtained influenza vaccine composition, as well as the added spray performance when sprayed using an appropriate device, are shown below.

[0039] Example 1

[0040] Example 2

[0041] A nasal hepatitis B vaccine composition not containing a gel base was prepared with the following composition. Comparative Example 1

[0042] Comparative Example 2

[0043] Immune Response Analysis Test: Tree shrews were used as experimental animals to evaluate the antibody induction ability of the nasal hepatitis B virus vaccine compositions prepared in Examples 1 and 2, and Comparative Examples 1 and 2, as follows. (Antibody Induction in Samples) Tree shrews (Tupaia belangeri, purchased from the Kunming Institute of Zoology, Chinese Academy of Sciences) were used as samples. They were randomly divided into four groups, two groups of four or three animals each. Antibody induction was performed via nasal administration in the four-animal group using Examples 1 and 2, and in the three-animal group using Comparative Examples 1 and 2. Nasal administration was performed by spraying 0.05 mL into each nostril using a high-pressure syringe and a liquid spray device (10 μg antigen in total for both nostrils). After the initial vaccination, a total of five vaccinations were administered at two-week intervals, followed by another vaccination four weeks later. Blood samples were collected at each vaccination and one week after the final vaccination, and neutralizing antibody titers were measured and evaluated. Separately, two groups of three treeshrews were prepared, and 0.1 mL (10 μg of each antigen) of Comparative Example 1 and Comparative Example 2 were subcutaneously administered to the backs of the treeshrews. The administration was continued in the same manner as in the case of intranasal administration described above, and blood samples were taken.

[0044] (Measurement of neutralizing antibody titer and serum IgA antibody) The virus solution used to measure neutralizing antibody titer was prepared by infecting and culturing primary human hepatocytes (PXB cells, manufactured by Phoenix Bio) with genotype C (C_JPNAT) hepatitis B virus. HepG2-NTCP30 cells were used for the neutralization test, and neutralizing antibody titers were measured according to standard methods. In addition, serum IgA antibodies against various antigens were detected and measured by ELISA.

[0045] (Results) The results of measuring the neutralizing antibody titer and IgA antibody one week after the final inoculation are shown in Table 1 and Figures 1 and 2. Nasal inoculation had a higher antibody induction ability than subcutaneous inoculation, while in nasal inoculation, Examples 1 and 2, which used carboxyvinyl polymer (CVP) treated by external shearing force as the base, had significantly higher antibody induction ability than Comparative Examples 1 and 2, which did not use CVP. [Table 1]

Claims

1. A hepatitis B vaccine composition for nasal mucosal spray administration, comprising: (i) hepatitis B surface antigen (HBs antigen) and / or hepatitis B nucleocapsid antigen (HBc antigen), and (ii) a gel base containing a carboxyvinyl polymer that has been treated by applying external shear force to impart sprayability.

2. A hepatitis B vaccine composition for administration via a nasal mucosal spray according to claim 1, wherein the hepatitis B vaccine (i) is 0.01 to 10 mg / mL per antigen.

3. A hepatitis B vaccine composition for nasal mucosal spray administration according to claim 1 or 2, containing 0.1 w / v% to 1.0 w / v% of a carboxyvinyl polymer.

4. A hepatitis B vaccine composition for nasal mucosal spray administration according to any one of claims 1 to 3, which uses a spray gel base containing a carboxyvinyl polymer that has been treated by applying external shear force to add spray performance such as (1) a range of particle size distribution of the formulation, (2) uniformity of spray density, and / or (3) spray angle control.

5. A hepatitis B vaccine composition for nasal mucosal spray administration according to any one of claims 1 to 4, which is obtained by using a gel base containing 0.5 w / v % to 2.0 w / v % carboxyvinyl polymer, and obtaining a gel base for spray administration containing carboxyvinyl polymer that has been treated by applying external shear force to add spray performance such as (1) a range of formulation particle size distribution, (2) uniformity of spray density, and / or (3) spray angle control, and then uniformly mixing the gel base with a virus concentrate containing HBs antigen and HBc antigen in a short period of time without applying stress.

6. A hepatitis B vaccine composition for nasal mucosal spray administration according to any one of claims 1 to 5, which is produced using a spray gel base that is imparted with spray performance by applying external shear force to a base containing a carboxyvinyl polymer, such that (1) the average particle size of the formulation is in the range of 50 μm to 120 μm, with 50% or more of the particle size distribution being in the range of 10 μm to 100 μm, (2) the spray density is uniform and the cone is full, and (3) the spray angle is controlled within the range of 30° to 70°.

7. A hepatitis B vaccine composition for nasal mucosal spray administration according to any one of claims 1 to 5, which is produced using a spray gel base that is imparted with spray performance by applying external shear force to a base containing a carboxyvinyl polymer, such that (1) the average particle size of the formulation is in the range of 70 μm to 100 μm and 60% or more of the particle size distribution is in the range of 10 μm to 100 μm, (2) the spray density becomes a uniform full cone without bias, and (3) the spray angle is controlled within the range of 40° to 60°.

8. A hepatitis B vaccine composition for nasal mucosal spray administration according to any one of claims 1 to 7, which uses a spray administration gel base containing a carboxyvinyl polymer that has been treated by applying external shear force to add (1) a range of formulation particle size distribution, (2) uniformity of spray density, and (3) spray angle control, so as to enable spray administration using a sprayable device without a pump function.

9. A method for preventing and / or treating hepatitis B, comprising administering the hepatitis B vaccine composition for nasal mucosal spray administration described in any one of claims 1 to 8 to a subject in need thereof using a device that can spray a viscous formulation onto the intranasal mucosa from each nostril.

10. Use of the hepatitis B vaccine composition for nasal mucosal spray administration according to any one of claims 1 to 8 for treating and / or preventing hepatitis B.

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