KR101108913B1 - Precipitated/inactivated influenza vaccine and method for production thereof - Google Patents

Abstract

New influenza viruses, particularly sedimentation / inactivating whole virus influenza vaccines that can effectively function in new viruses with low immunogenicity with low content of antigens and have less adverse side effects; And a method for preparing the vaccine is disclosed. Specifically, sedimentation / inactivating influenza vaccines comprising aluminum hydroxide gels prepared from sodium carbonate and potassium aluminum sulfate and total influenza virus particles isolated as active ingredients; And a method for preparing said settling / inactivating influenza vaccine, characterized in that each step is carried out using a solvent free of surfactant or free of ether.

Adsorbed Inactivated Influenza Vaccine

Description

Sedimentation / Inactivating Influenza Vaccines and Methods for Making the Same {PRECIPITATED / INACTIVATED INFLUENZA VACCINE AND METHOD FOR PRODUCTION THEREOF}

The present invention relates to an adsorbed inactivated whole virion influenza vaccine and a method of making the same.

Influenza viruses are RNA envelope viruses with a virion size of about 100 nm, belonging to the family orthomyxoviridae. It is divided into types A, B and C, based on the antigenicity of internal proteins. Influenza viruses consist of a core and external glycoproteins of ribonucleic acid (RNA) associated with an internal nucleocapsid or nucleoprotein surrounded by a viral envelope having a lipid bilayer structure. The inner layer of the viral envelope consists mainly of matrix proteins, and the outer layer consists mostly of host-derived fat material. The RNA of influenza virus has a segment structure. The pandemic influenza is caused by the influenza A virus. Type A has two types of envelope glycoproteins of hemagglutinin (HA) and neuramidinase (NA), and HA is divided into 16 subtypes based on the difference in antigenicity, and NA is 9 subtypes. Divided into

Influenza A viruses are distributed among animals such as birds, pigs and horses in addition to humans. It has been clearly demonstrated that the recombination of genes between human and animal viruses results in the appearance of subtype viruses with new antigenicity (new viruses), and the new viruses are prevalent in a population of nonimmunized humans. To date, it is known that there have been epidemics of Spanish influenza (Hsw1N1), Asian influenza (H2N2) and Hong Kong influenza (H3N2). Recently, the incidence and epidemic of the highly pathogenic avian influenza of the H5N1 subtype has been observed in Southeast Asia, so that more than 150 million birds have been killed or discarded so far. In addition, human infections with the virus have been reported, confirming 246 infections in September 2006. Threatening was the mortality rate, of which 144 of 246 people died. In general, avian influenza viruses are believed not to easily infect humans, and their properties change fundamentally. However, if the virus easily infects and pandemics humans by mutation, it will be an unprecedented tragedy. To prepare for the pandemic, the development of new influenza vaccines is urgently needed. However, since the first human infection was reported in Hong Kong in 1997, the urgency of vaccine development has not been easily advanced, even though it has been perceived. The main cause of this is the low immunogenicity of the H5N1 subtype avian influenza virus, which prevents conventional influenza vaccines from giving the vaccinees sufficient vaccine effect. Therefore, some measures to increase immunogenicity are required.

Influenza vaccines are largely divided into two. One is the currently available influenza vaccine, which is a split vaccine comprising subvirions obtained by digesting purified viral virions with organic solvents / surfactants or another reagent to solubilize the lipid envelope. vaccines, or subunit vaccines containing purified HA and NA, and are administered primarily intramuscularly or subcutaneously. The other is a vaccine obtained by chemically inactivating purified virus virions with a drug such as formalin, and is called a whole virion vaccine. In this case, since the viral gene or viral protein is chemically modified to lose infectivity alone, the virus becomes a vaccine antigen while maintaining the virion form. The whole virion vaccine is considered to have higher immunogenicity compared to the split vaccine and can induce a good immune response especially for naive individuals who have never experienced infection and therefore do not have immune memory. Since humans do not have immune memory for new influenza, this feature of the entire virion vaccine is desirable in terms of effectiveness. However, the high immunogenicity of the entire virion vaccine means high biological activity, which is also closely related to side reactions such as local swelling, redness, or fever upon inoculation. Therefore, if some method can be established that can utilize the high immunogenicity of the entire virion vaccine and reduce its side reactions, then an effective and safe vaccine will be developed.

The whole virion vaccine was expected because of its high immunogenicity as a formulation of the vaccine against new influenza. However, even with the entire virion vaccine, a record that can not cause a good immune response to the H5N1 subtype in humans has been reported, and another method for improving immunogenicity is needed.

Adjuvant is an immunoadjuvant added to the vaccine in order to increase the immunogenicity of the antigen, and many substances have been studied so far. For example, in order to raise the effectiveness of an influenza vaccine, the combination of aluminum adjuvant is also considered (patent document 7). In addition, it is also known to use a nonionic surfactant such as polyoxyethylene ether (Tween®) as an adjuvant for a mucosal vaccine (Patent Document 8).

There are several adjuvant candidates and the most extensively reviewed aluminum adjuvant added to DPT vaccines and hepatitis B vaccines has been extensively identified over the years. The aluminum adjuvant, together with the immune stimulating action, has a so-called deposit effect (locally retains a small amount of antigen by adsorption of the antigen), thus providing excellent properties of reducing the antigen content of the vaccine. For pandemic vaccines that are given to humans, the question is how many people can get the vaccine. This property of the aluminum adjuvant is very advantageous in the design of the vaccine. Therefore, when the aluminum adjuvant is used as a vaccine, the adsorption of the antigen to the aluminum gel is a very important factor.

Patent Document 1: WO 91/03552

Patent Document 2: WO 01/004333

Patent Document 3: WO 00/060050

Patent Document 4: WO 2001/083794

Patent Document 5: WO 2002/067983

Patent Document 6: WO 2002/074336

Patent Document 7: WO 00/015251

Patent Document 8: WO 99/52549

DISCLOSURE OF INVENTION

Problems to be Solved by the Invention

It is an object of the present invention to provide an inactivated inactivated whole virion influenza vaccine with a low antigenic amount and a low side effect response against a new influenza virus, especially a new virus with low immunogenicity, and a method for producing the same.

Means to solve the problem

In view of the above problems, the present inventors conducted a intensive study and found that the above object can be achieved by combining an adjuvant with whole inactivation virus with an adjuvant having elevated immunogenicity for humans and low side effects, It has led to the completion of the present invention. Accordingly, the present invention provides the following.

[1] Adsorbed inactivated influenza vaccine comprising as an active ingredient an aluminum hydroxide gel prepared from sodium carbonate and potassium aluminum sulfate and an influenza virus whole virion.

[2] The vaccine of [1], wherein the influenza virus whole virion is purified from the virus suspension in the absence of a surfactant and an ether.

[3] The vaccine according to [1] or [2], having the following characteristics:

(1) the adsorption rate of influenza virus total virions to the aluminum hydroxide gel is at least 90%, and

(2) The hemagglutinin activity of influenza virus total virions on total hemagglutinin activity is 80% or more.

[4] The vaccine according to any one of [1] to [3], wherein the aluminum concentration is 0.1 mg / mL or more and less than 0.8 mg / mL, and the concentration of influenza virus total virion is 3.4 to 100 μg HA / mL.

[5] The vaccine according to any one of [1] to [4], wherein the influenza virus in the virus stock solution before mixing with the aluminum hydroxide gel has the following characteristics:

(i) when the viral stock is analyzed by an analytical sucrose density gradient centrifugation method, at least 80% of the total hemagglutinin activity accumulates in the viral virion fraction, and

(ii) When the virus stock was subjected to gel filtration high performance liquid chromatography, the absorbance spectrum at 280 nm had a single shoulderless peak.

[6] The method of any one of [1] to [5], wherein 80% or more of influenza viruses in the virus stock solution before mixing with the aluminum hydroxide gel observe the virus stock solution under an electron microscope at 20000 to 30000 times magnification. , A vaccine that maintains a complete virion form.

[7] The vaccine according to any one of [1] to [6], wherein the influenza virus is a subtype comprising a combination of a form selected from H2 and H4-16 and a form selected from N1-9.

[8] A method for preparing an adsorbed inactivated influenza vaccine comprising as an active ingredient total influenza virus virions, the method comprising performing the following steps (1) to (5) in a solvent free of a surfactant and an ether :

(1) culturing the influenza virus to provide a virus suspension,

(2) purifying the virus suspension by filtration or centrifugation,

(3) inactivating the purified virus solution with formalin,

(4) filtering the inactivated virus solution to produce a virus stock solution, and

(5) adsorbing influenza virus in the virus stock solution to an aluminum hydroxide gel.

[9] The production method according to [8], wherein the aluminum hydroxide gel is prepared from sodium carbonate and potassium aluminum sulfate.

[10] The production method according to [8] or [9], wherein the influenza virus is a subtype comprising a combination of a form selected from H2 and H4-16 and a form selected from N1-9.

[11] A method for preventing or alleviating influenza, comprising administering to a subject an effective amount of an inactivated influenza vaccine comprising an aluminum hydroxide gel prepared from sodium carbonate and potassium aluminum sulfate and an influenza virus whole virion as an active ingredient .

[12] The method of [11], wherein the influenza virus whole virion is purified from the virus suspension in the absence of a surfactant and an ether.

[13] The method of [11] or [12], wherein the vaccine has the following characteristics:

(1) the adsorption rate of influenza virus total virions to the aluminum hydroxide gel is at least 90%, and

(2) Total hemagglutinin activity is 80% or more.

[14] The method according to any one of [11] to [13], wherein the aluminum concentration is 0.1 mg / mL or more and less than 0.8 mg / mL, and the concentration of influenza virus total virion is 3.4 to 100 µg HA / mL.

[15] The method according to any one of [11] to [14], wherein the influenza virus in the viral stock before mixing with the aluminum hydroxide gel has the following characteristics:

(i) when the viral stock is analyzed by an analytical sucrose density gradient centrifugation method, at least 80% of the total hemagglutinin activity accumulates in the viral virion fraction, and

(ii) Gel Filtration High Performance Liquid Chromatography of Virus Stocks with Absorption Spectrum at 280 nm with Single Peak Without Shoulder.

[16] The method of any one of [11] to [15], wherein when the virus stock is observed under an electron microscope at 20000 to 30000 times magnification, at least 80% of the influenza virus in the virus stock before mixing with the aluminum hydroxide gel is incomplete. How to maintain virion form.

[17] The method according to any one of [11] to [16], wherein the influenza virus is a subtype comprising a combination of a form selected from H2 and H4-16 and a form selected from N1-9.

[18] Use of aluminum hydroxide gel and influenza virus whole virion prepared from sodium carbonate and potassium aluminum sulfate for the production of adsorbed inactivated influenza vaccines.

[19] The use of [18], wherein the influenza virus whole virion is purified from the virus suspension in the absence of a surfactant and an ether.

[20] The use of [18] or [19], wherein the vaccine has the following properties:

(1) the adsorption rate of influenza virus total virions to the aluminum hydroxide gel is at least 90%, and

(2) Total hemagglutinin activity is 80% or more.

[21] The use according to any one of [18] to [20], wherein the aluminum concentration is 0.1 mg / mL or more and less than 0.8 mg / mL, and the concentration of influenza virus total virion is 3.4 to 100 μg HA / mL. .

[22] The use according to any one of [18] to [21], wherein the influenza virus in the viral stock before mixing with the aluminum hydroxide gel has the following characteristics:

(i) when the viral stock is analyzed by an analytical sucrose density gradient centrifugation method, at least 80% of the total hemagglutinin activity accumulates in the viral virion fraction, and

(ii) When gel stocks were subjected to gel filtration high performance liquid chromatography, the absorbance spectrum at 280 nm had a single shoulderless peak.

[23] The method of any one of [18] to [22], wherein when the virus stock solution is observed under an electron microscope at 20000 to 30000 times magnification, at least 80% of the influenza virus in the virus stock solution before mixing with the aluminum hydroxide gel. For maintaining complete virion form.

[24] The use according to any one of [18] to [23], wherein the influenza virus is a subtype comprising a combination of a form selected from H2 and H4-16 and a form selected from N1-9.

[25] A commercial package comprising the adsorbed inactivated influenza vaccine according to any one of [1] to [7] above, and documents stating that the vaccine can be used or should be used for the prevention or reduction of influenza.

According to the present invention, a novel influenza vaccine, an adsorbed inactivated whole virion influenza vaccine, which contains influenza total virion as an active ingredient, induces an effective immune response with a low antigenic amount to humans, and has a low side reaction, can be provided, This makes it possible to control the new flu pandemic. Since the vaccine of the present invention contains an aluminum hydroxide gel (salt) prepared from sodium carbonate and potassium potassium sulfate as an adjuvant, it is possible to induce an effective immune response against humans with a low antigen content and to secure a sufficient supply amount of the influenza vaccine. Can be. According to the method for preparing the adsorbed inactivated whole virion influenza vaccine of the present invention, a new influenza vaccine can be provided which maintains high antigenicity of the viral virion, induces an effective immune response, and has low side reactions.

1 shows a schematic of the preparation of the viral stock and vaccine of the invention.

2 shows a schematic of the preparation of the viral stock and vaccine of the comparative example.

3 is a graph showing immunogenicity in animals of vaccines prepared using the gels of Example 2 and Comparative Example 2. FIG.

Figure 4 is a graph showing the results of Phase I studies in healthy adult males aged 20 to 40 years using the new adsorptive influenza vaccine prepared by the B method.

5 shows electron micrographs of virus stocks prepared in Example 1 (A method) and Comparative Example 1 (B method).

6 is a graph showing the results of analytical sucrose density gradient centrifugation on virus stocks prepared in Example 1 (A method) and Comparative Example 1 (B method).

7 A and 7B are graphs showing the results of gel filtration HPLC analysis using the virus stock solutions prepared in Example 1 (A method) and Comparative Example 1 (B method).

8 is a graph showing post-vaccination neutralizing antibody titers in phase I studies.

9 is a graph showing HI antibody production in vaccinated mice.

10 is a graph showing the results of abnormal toxicity tests in guinea pigs.

FIG. 11 is a graph showing the adsorption rate of viruses in vaccines prepared using the gels of Example 2 and Comparative Example 2. FIG.

Best Mode for Carrying Out the Invention

The present invention provides an adsorbed inactivated influenza vaccine containing as an active ingredient an aluminum hydroxide gel and influenza virus whole virion prepared from sodium carbonate and potassium aluminum sulfate.

The vaccine of the present invention is characterized by containing aluminum hydroxide gel prepared from sodium carbonate and potassium aluminum sulfate as an adjuvant. By using the gel, the adsorption rate of the whole viral virion can be significantly improved, and the immunogenicity of the vaccine against humans can be improved. As sodium carbonate and potassium aluminum sulfate, which are raw materials for the auxiliary, reagents commonly used to prepare aluminum hydroxide gel can be used without limitation. As potassium aluminum sulfate, aluminum potassium potassium sulfate hydrate AlK (SO ₄ ) _{2 to} 12H ₂ O, also referred to as potassium alum, is preferably used. The manufacturing method of aluminum hydroxide gel is mentioned later.

The vaccines of the invention characteristically contain influenza virus whole virions as antigens. In the present invention, influenza viruses include any subtypes currently known, and subtypes that are isolated and identified in the future. Influenza viruses are selected from H1 to H16 (that is, H2 and H4 to 16) excluding H1 and H3 because no epidemic in humans has been observed so far, and it is necessary to effectively prevent infection in humans in the future. The subtype which consists of a combination of a form and the form chosen from N1-N9 is preferable. The subtype is also referred to as new influenza virus. The subtype is more preferably a subtype consisting of a combination of a form selected from H5, H7 and H9 and a form selected from N1 to N9. Influenza viruses may be one strain belonging to the same subtype, or two or more strains belonging to the same subtype, or two or more strains belonging to a different subtype.

In the present invention, the influenza virus whole virion refers to a virus virion purified while maintaining the form of the virus from a virus suspension obtained by culturing the influenza virus. Thus, the influenza vaccine of the present invention refers to a vaccine excluding a split vaccine including subvirions, and a subunit vaccine including purified HA or NA, and is also referred to as a whole virion vaccine.

The present inventors can use the above-mentioned adsorbent having a high adsorption rate for the entire virion vaccine to obtain a vaccine having a low antigen content and a low side effect reaction, and especially when compared with other vaccines, mammals other than humans. Among them, even when the immunogenicity is not different, it was found that a large difference occurs in the immune response to humans, and the present invention was reached.

The influenza virus whole virion is purified from the virus suspension, preferably in the absence of surfactants and ethers. The virus solution containing the entire influenza virus virion purified or concentrated for adsorption with aluminum hydroxide gel is referred to as "viral stock solution". Methods for purifying or concentrating influenza viruses are described below.

About the vaccine of this invention which becomes an effective vaccine with a small amount of antigen, and has a small side reaction, Preferably, the characteristic of following (1) and (2) is shown.

(1) The adsorption rate of the entire influenza virus virion to the aluminum hydroxide gel is 90% or more. If the adsorption rate is greater than 90%, the synergistic effect with the viral virion is increased. Here, the adsorption rate is a mixture of aluminum hydroxide gel and influenza virus whole virion overnight in physiological saline at 4 ~ 8 ℃, the supernatant is recovered, the hemagglutinin (HA) value or the protein mass of the supernatant or gel The value determined by measuring the mass of protein adsorbed on the The detailed measuring method of the said adsorption rate is described in the Example. The adsorption rate is more preferably 95% or more, even more preferably 97% or more. The adsorption rate can be at least 90% when the viral virion is mixed with the gel under supersaturation conditions. When the adsorption rate is less than 90% in the present invention, the amount of antigen can be appropriately reduced so as not to be 90% or more.

(2) The hemagglutinin activity of influenza virus total virions on total hemagglutinin activity is at least 80%. More preferably, it is 85% or more, More preferably, it is 90% or more. The ratio is measured by the following operation. The inactivated virus solution is dispensed by sucrose density gradient centrifugation or the like before adsorption on the gel. Generally, virus total virions are accumulated at sucrose concentrations near 40%. Accordingly, the ratio is determined by dividing the amount of hemagglutinin in the fraction by the amount of hemagglutinin in the total fraction. Hemagglutinin amount can be measured as an HA value along with erythrocyte clotting ability as an indicator. The presence of at least 80% of the total hemagglutinin relative to the influenza virus total virions is indicative of near complete maintenance of the form of the viral virions in the vaccine. Such good total virions are important for the vaccine of the present invention to achieve the synergistic effect.

Morphology of viral virions can also be identified by the following method.

i) The adsorption spectrum at 280 nm has a single peak without shoulder when the virus stock is subjected to gel filtration high performance liquid chromatography. Whether the adsorption spectrum has a single shoulderless peak can be confirmed by visual observation of the plot obtained by high performance liquid chromatography and its judgment in accordance with conventional criteria used in the relevant art.

ii) When viral stock is observed under an electron microscope at 20000 to 30000 magnification, at least 80% of the virus exhibits a complete virion form. Regarding the complete virion form of the virus, about 20-30 viruses are observed with the presence of HA and NA spikes observed around the virion as an indicator, and the proportion of virus with a complete virion form is determined. .

The vaccine of the present invention preferably has an aluminum concentration of at least 0.1 mg / mL and less than 0.8 mg / mL, more preferably at least 0.2 mg / mL and less than 0.8 mg / mL. The said concentration is calculated from the usage-amount of potassium alum as a raw material. Alternatively, aluminum concentration can also be determined by atomic absorption analysis.

The vaccine of the present invention preferably comprises influenza virus total virion at a concentration of 3.4-100 μg HA / mL, more preferably 10-30 μg HA / mL. This concentration can be obtained by measuring the HA concentration of the viral whole virion protein. The measuring method of a protein is described in the Example.

The vaccine of the present invention may include a pharmaceutically acceptable carrier in addition to the influenza virus whole virion and the aluminum hydroxide gel. As said carrier, the carrier normally used for manufacture of a vaccine can be used, Specifically, saline, buffered saline, dextrose, water, glycerol, isotonic aqueous buffer, and combinations thereof can be mentioned. Moreover, a preservative (for example, thimerosal), an isotonicity agent, a pH adjuster, an inactivating agent (for example, formalin), etc. can be mix | blended suitably.

The vaccine of the present invention can be administered systemically or topically. In systemic administration, it is injected intramuscularly, subcutaneously, intradermally, or the like. In the case of topical administration, mention may be made of intranasal administration and administration to the pharynx, the method of administration being spraying, application and the like.

The administration target of the vaccine of the present invention is not particularly limited, and examples thereof include mammals, birds, and the like including humans.

Using the vaccine of the present invention, influenza can be prevented or its symptoms can be alleviated. The present invention provides a method for the prevention or alleviation of influenza comprising administering to a subject an effective immunosensitizing dose of a vaccine of the present invention.

The method of administering the vaccine is exemplified above. The dosage is determined in consideration of the age, sex, weight, and the like of the subject, but as the antigen, 1.7 to 50 μg HA, and preferably 5 to 15 μg HA, may be administered once or twice or more. Preferably, it is administered a plurality of times, and in this case, it is preferable to administer at intervals of 1 to 4 weeks.

The present invention provides a method for producing the adsorbed inactivated influenza vaccine. The production method of the present invention is characterized in that a solvent containing no surfactant and an ether is used in the entire step of preparing an influenza virus whole virion as an antigen and adsorbing the virion to an auxiliary agent and an aluminum hydroxide gel. . Surfactant means any surfactant commonly used in the field of vaccine manufacture, and includes anionic surfactants, cationic surfactants, nonionic surfactants, and the like. In order to prevent the immunogenicity of the vaccine from lowering, nonionic surfactants are often used, for example, polyoxyethylene ether (Tween®) and the like are widely used. In the manufacturing method of this invention, these surfactant is not used. By ether is meant any ether commonly used in the field of vaccine manufacture, including diethyl ether and the like. In the production method of the present invention, these ethers are not used.

(1) culturing the influenza virus to provide a virus suspension

Influenza viruses may be recombinant viruses established genetically in cultured cells or in strains isolated from infected animals or patients. As a method of culturing influenza viruses, a virus can be inoculated and cultured in the ureter cavity of an egg, or cultured cells can be infected with a virus, and the cells can be cultured. Culture conditions for eggs is 30 ~ 37 ℃ for about 1-7 days, in the case of cultured cells, the medium, temperature and incubation time is determined according to the cells used. After incubation, the virus suspension is recovered by conventional methods.

(2) purifying the virus suspension by filtration or centrifugation

The resulting virus suspension is centrifuged or filtered to provide a clear solution. If necessary, ultrafiltration is carried out for concentration. Thereafter, for virus purification, ultracentrifugation such as sucrose density gradient centrifugation is performed to obtain purified virus liquid.

(3) inactivating the purified virus solution with formalin

Purified virus liquid performs an inactivation process. The virus inactivation method includes formalin treatment, ultraviolet irradiation, β-propiolactone treatment, and the like, and formalin treatment is preferable. The conditions of formalin treatment can be appropriately determined. For example, cells are incubated for several weeks at low temperature using a formalin solution of 0.02 v / v%.

(4) filtering the inactivated virus solution to produce a virus stock solution

In this step, the virus solution is filtered to prepare a virus stock solution. Concentrate the virus simultaneously with filtration as desired. Filtration includes ultrafiltration and the like. Ultrafiltration may be carried out by conventional methods.

(5) adsorbing influenza virus in the virus stock solution to an aluminum hydroxide gel

The suspension of the virus stock and the aluminum hydroxide gel adjusted to the predetermined protein concentration are mixed at a low temperature of about 4 to 8 ° C. for several hours to overnight, so that the influenza virus is adsorbed with the aluminum hydroxide gel. As the aluminum hydroxide gel, known ones can be used. As mentioned above, those prepared from sodium carbonate and potassium aluminum sulfate are preferred.

The present invention is described in detail by the following examples, but the present invention is not limited to these examples.

Example 1 Preparation of Viral Stock and Preparation of Preproduction Vaccine (Step A Method)

Viral stock solutions and prototype vaccines were prepared according to a common method of SAIKIN SEIZAI KYOUKAI. The outline is shown in FIG.

Comparative Example 1 Preparation of Viral Stock Solution and Preparation of Prototype Vaccine (Step: Method B)

Viral stock solutions and starting vaccines were prepared in the same manner as in Example 1, except that Tween 80 was used for the purification of the virus suspension in Example 1 and FIG. 1. The outline is shown in FIG.

Example 2 Preparation of Aluminum Hydroxide Gel

Sodium carbonate (143.1 g) was dissolved in physiological saline (9 L) and the mixture was filtered to give a sodium carbonate solution. Aluminum potassium potassium sulfate 12 hydrate (potassium alum, 316.0 g) was dissolved in physiological saline (9 L), and the mixture was filtered to provide an aluminum potassium sulfate solution. The obtained sodium carbonate solution and aluminum potassium sulfate solution were mixed, and the mixture was stirred. The obtained aluminum hydroxide gel was washed and used as an adjuvant of the present invention.

Comparative Example 2 Preparation of Aluminum Hydroxide Gel

To a 0.21 M aqueous aluminum chloride solution (pH 2.95, 50.0 mL) was added dropwise 0.50 M aqueous NaOH solution (63.1 mL) over 55 minutes with stirring at room temperature. The pH at the completion of addition was also 9.00. The solution was further stirred for 30 minutes at room temperature. The pH at that time was also 9.00. The solution after stirring was left at 4 ° C. for 21.5 hours to obtain an aluminum hydroxide gel (113 mL, pH 8.4). The obtained aluminum hydroxide gel was centrifuged at 4 ° C. at 2000 rpm for 10 minutes and the precipitate was collected by filtration. 0.01 M PBS (pH 7.1, 29.25 mL) was added to the precipitate (12.5 mL), and after confirming that the pH had risen to 9.4, the mixture was autoclaved at 121 ° C. for 20 minutes. The pH of the aluminum hydroxide gel after autoclaving was 8.4.

Example 3 Preparation of Adsorbed Inactivated Whole Virion Vaccine

The virus stock obtained in Example 1 and the aluminum hydroxide gel obtained in Example 2 were mixed to obtain an adsorbed inactivated whole virion vaccine.

Comparative Example 3 Preparation of Adsorbed Inactivated Whole Virion Vaccine

The virus stock solution obtained in Comparative Example 1 and the aluminum hydroxide gel obtained in Comparative Example 2 were mixed to obtain an adsorbed inactivated whole virion vaccine.

Experimental Example 1 Adsorption Rate for Gels of Viruses

Then, using the inactivated total virions of the new influenza virus (NIBRG-14 strain), antigen adsorption rates for various aluminum gels were studied. In addition to aluminum hydroxide gels prepared from potassium alum (Example 2) and aluminum hydroxide gels prepared from aluminum chloride (Comparative Example 2), commercially available aluminum hydroxide gels and aluminum phosphate gels shown in the following list were also subjected to measurements. Antigen adsorption rates were compared.

(Experimental material)

(1) aluminum gel:

1) Aluminum Hydroxide Gel "Aluminum Hydroxide Gel (A-8222)" (manufactured by SIGMA)

13 mg-Al / ml

2) Aluminum Hydroxide Gel "Alhydrogel (2%)" (Brenntag Biosector)

10.8 mg-Al / ml

3) Aluminum Phosphate Gel "Adju-Phos" (manufactured by Superfos Biosector)

4.4 mg-Al / ml

(2) virus suspension:

NIBRG-14 week influenza inactivated whole virion vaccine stock (FPBM Z0401, prepared by the method described in Example 1)

(3) Diluent:

1) 0.01M PBS (-)

2) saline solution

(Experimental method)

The aluminum gel and the virus suspension were mixed and the supernatant was recovered. The protein content and the HA value of the supernatant were measured to calculate the respective adsorption rates.

(Measurement Method of Adsorption Rate for Aluminum Gel of Antigen)

(1) gel and antigen mixing

To the aluminum gel suspension (equivalent to 0.3 mg-Al) and the virus suspension (11.7, 33.3, or 100 μg-protein equivalent) the diluent was added to a total volume of 1 ml and the mixture was stirred overnight in a cold room (4-8 ° C.). .

(2) Collection of supernatant

The mixed solution was centrifuged (TOMY trace high speed centrifuge MC-150, 1000 rpm, 5 minutes) to recover the supernatant. The recovered supernatant was used as a material for measuring the protein content or a material for measuring the HA value.

(3) determination of protein content

Protein content of the supernatants was measured using a Micro BCA Protein Assay Kit (Pierce Biotechnology Inc). As standard material, the BSA solution attached to the kit was used.

(4) HA value measurement

The HA value of the supernatant was measured according to the "Pathogen Detection Manual", "Measurement of Influenza HA Value" of the National Institute of Infectious Diseases. The blood cell suspension used a 0.5% chicken erythrocyte suspension.

(5) adsorption rate calculation

The adsorption rate (%) was calculated by dividing the antigen content in the supernatant of each test subject gel by the antigen content in the supernatant of a control test subject not containing the gel.

(result)

Adsorption rates for various commercially available aluminum gels calculated from protein content and HA content when using 0.01 M-PBS in the diluents are shown in Table 1.

Type of aluminum gel
Adsorption rate (%)
Amount of antigen
11.7 μg 33.3 μg 100.0 μg Aluminum hydroxide gel, A-8222 (manufactured by Sigma) 62.6 42.1 Aluminum hydroxide gel, Alhydrogel (manufactured by Brunntag Biosector) 67.0 62.0 48.4 Aluminum phosphate gel, Ajyu-Phos (manufactured by Superfos Biosector) 0.0 25.9 7.9 None 0.0 0.0 0.0

Aluminum hydroxide (made of aluminum chloride) gel (manufactured in Comparative Example 2) and aluminum hydroxide (potassium alum) gel of inactive NIBRG-14 (H5N1 subtype) or A / Wyoming / 3/2003 (H3N2 subtype) Adsorption rate) (compared to Example 2). These were combined so that the final protein concentration of both inactivated viruses was 100 μg / mL and the final concentration of aluminum was 0.3 mg / mL, and the mixture was incubated at room temperature for 15 minutes. The gel fraction and the supernatant were separated by centrifugation, the protein mass remaining in the supernatant was measured, and the adsorption rate was calculated based on this. The result is shown in FIG. A / Wyoming / 3/2003 adsorbed well on all aluminum hydroxide gels, while NIBRG-14 exhibited poor adsorption on aluminum hydroxide (made of aluminum chloride) gels and only good on aluminum hydroxide (potassium alum) gels. Adsorption was shown. These results suggest that if the antigen is H5N1 subtype host, it is preferable to use an aluminum hydroxide (potassium alum) gel.

Experimental Example 2 Immunogenicity of Vaccines in Animals

To compare and evaluate the adjuvant effect of aluminum hydroxide salts prepared by different preparation methods, 2 of aluminum hydroxide gel (prepared in Comparative Example 2) and aluminum hydroxide gel (prepared in Example 2) using potassium alum as raw materials Inactivated viral antigens containing a kind of aluminum gel were prepared. The antigen (0.5 mass of protein per 0.05 μg) was immunized twice in ddY mice at 3 week intervals. Blood collection was performed two weeks after the second immunization, and the HI antibody value was measured using the obtained serum (FIG. 3).

From FIG. 3, when the aluminum hydroxide (potassium alum) gel was used as an adjuvant, high antibody response was observed also in any administration group.

Reference Example 1 Effectiveness of the vaccine in humans

A Phase I study was conducted in healthy adult males aged 20-40 years using the new adsorbed influenza vaccine mock test drug prepared in Comparative Example 3 (method B). The vaccine was inoculated twice subcutaneously at three week intervals. Antigen amount was compared with three types of 1.7 micrograms HA (low dose), 5 micrograms HA (medium dose), and 15 micrograms HA (high dose) per dose inoculation. Neutralizing antibody values strongly reflecting viral infection defense responses were compared before and after vaccination and used as indicators. As a result, as shown in FIG. 4, even after administering 15 micrograms HA vaccine twice, the positive conversion rate in antibody value 1:40 or more was 40% or less, and was not satisfactory.

Reference Example 2 Effectiveness of Vaccine in Animals

The immunogenicity of mice and rabbits of the new influenza vaccine prepared in Example 3 (method A) and Comparative Example 3 (method B) was evaluated by HI antibody values as an index. In experiments using mice, an aluminum hydroxide gel (ALUM) adjuvant vaccine containing an inactivated viral antigen was injected intramuscularly twice a week at 0.04-3 μg HA / mouse in the lower extremities of 8-10 BALB / c mice in each group. Inoculated. HI antibody values of mouse serum 14 days after the second immunization were measured, and the average HI antibody values of 10 mice per group are shown in the table below. In experiments with rabbits, each vaccine (15 μg HA) was intramuscularly administered to white rabbits (3 per group). As a result, as shown in Table 2, differences in immunogenicity could not be distinguished between vaccines prepared by the A method and the B method in experimental animals.

animal
Method A
Method B Experiment 1 Experiment 2 Experiment 3 Experiment 4 mouse

3 μg
410 times 3 μg
150 times 3 μg
57 times 3μg no implementation 0.6 μg
240 times 1 μg
60 times 0.6 μg
61 times 1 μg
23 times 0.12 μg
50 times 0.2 μg
20 times 0.12 μg
12 times 0.2 μg
52 times rabbit 254 times There is no conduct 101 times 160 times

Experimental Example 3 Observation by Electron Microscope

The stock solutions prepared in Example 1 (Method A) and Comparative Example 1 (Method B) were placed in a grid for electron microscopic observation with a formvar film, respectively, and 2% phosphotungstic acid. And stained with an acceleration voltage of 120 KV (FIG. 5). As a result, when the virions in which HA and NA spikes were observed around the virions were completely virions, 80% or more of the virions in the stock solution prepared by the method A were completely virions, whereas the stock solution prepared by the method B was used. Flawless virions were rarely observed.

Experimental Example 4 Sucrose Density Gradient Centrifugation

Analytical sucrose density gradient centrifugation was performed on the stock solutions prepared in Example 1 (A method) and Comparative Example 1 (B method).

A stock solution (1 mL) with a protein concentration of 180 μg / mL was stratified on a sucrose density gradient of 15 to 60%, and centrifuged at 4 ° C. and 18000 rpm for about 16 hours. About 0.5 mL per fraction was fractionated and the sucrose concentration, HA activity and protein concentration of each fraction were measured. HA values were measured three times using chicken erythrocytes, and their geometric mean values were shown graphically. For protein concentration, 50 μL of each fraction was transferred to PVDF membrane, stained with Coomassie Brilliant Blue, and analyzed by density meter (FIG. 6).

For each stock, peaks of HA activity and protein were observed in fractions of about 40% sucrose concentration. In method A, more than 80% of the applied HA activity accumulated at the virus peak, while in method B, the accumulation ratio remained below 50%. In addition, in the method B, a peak of the protein was observed even in the fraction having a sucrose concentration of about 17%, but no HA activity was observed.

Experimental Example 5 Gel Filtration HPLC

Gel filtration HPLC was performed on the stock solutions prepared in Example 1 (A method) and Comparative Example (B method), respectively. The stock solution was diluted with mobile phase (PBS) so that the protein concentration was 100 μg / mL and used as a sample. The stock solutions prepared by the methods A and B were analyzed by liquid chromatography under the following conditions, respectively.

Detector: UV absorbance photometer (wavelength: 210, 280 nm)

Column: HPLC analysis column TSKgel G3000PWXL from Tosoh corporation

Column temperature: constant temperature around 25 ° C

Mobile phase: 0.01 mol / L sodium phosphate buffer (Note 1) PBS (pH 7.4)

Flow rate: approx. 0.5 mL per minute

(Note 1) 0.01 mol / L sodium phosphate buffer solution:

Potassium dihydrogen phosphate (0.238 g) and disodium hydrogen orthophosphate anhydrous 12 hydrate salt (2.955 g) were dissolved in water to make 1000 mL.

Gel filtration chromatogram of the stock solution is shown (FIGS. 7A and 7B, top: 210 nm, bottom: 280 nm). From Fig. 7, a peak expected to be viral virion was observed at the retention time of 9.9 minutes. In addition, in the method B, a shoulder was observed even at 10.9 minutes of holding time.

Experimental Example 6 Immunogenicity in Clinical Trials

A new adsorbed influenza vaccine mock test drug was prepared by adding aluminum hydroxide gel prepared in Example 1, and a phase I study was conducted using healthy adult males aged 20-40 years (FIG. 8). The vaccine was inoculated twice subcutaneously or intramuscularly at three week intervals. Antigen amount was compared with three types of 1.7 micrograms HA (low dose), 5 micrograms HA (medium dose), and 15 micrograms HA (high dose) per dose, and the effectiveness was compared. Neutralizing antibody values strongly reflecting viral infection defense responses were compared before and after vaccination and used as indicators. In the high dose inoculation group, an increase of 73% was observed in the subcutaneous inoculation group, and in the intramuscular inoculation group, the antibody value increased more than four times in all cases. In the medium dose group, an increase of 58% in the subcutaneous group and 75% in the intramuscular inoculation group was observed. From this it was confirmed that the adsorbed influenza vaccine with the addition of aluminum hydroxide gel caused an immune response in many cases for humans as well.

Experimental Example 7 Mouse HI Antibody Production

Immunogenicity in mice of the new influenza vaccine containing aluminum hydroxide gel (ALUM) prepared in Example 1 was evaluated using HI antibody values as an indicator. Formulations with or without ALUM containing 0.04-3 μg HA / marie inactivated viral antigen were inoculated subcutaneously or intramuscularly twice into the lower extremities of 10 mice in each group at three week intervals. HI antibody value of the mouse serum after 14 days of secondary immunity was measured, and the average HI antibody value of 10 mice of each group is shown in FIG. From FIG. 9, in both the subcutaneous and intramuscular administration groups, the HI antibody value of the ALUM preparation group was higher than the HI antibody value of the ALUM free formulation group. From this, it can be seen that by adding ALUM, higher antibody values are caused than in the non-added group.

Experimental Example 8 Safety of Vaccine

Abnormal Toxicity Test

The abnormal toxicity test in the guinea pigs of the new influenza vaccine which added the aluminum hydroxide gel (ALUM) manufactured in Example 1 as an adjuvant was performed. The test subjects were inactivated virus antigen 30 μg HA / mL + ALUM 0.3 mg / mL, inactivated virus antigen 30 μg HA / mL, ALUM only 0.3 mg / mL and saline. Each test subject (5 mL) was inoculated into three guinea pigs and the change in body weight was observed. The average of the weight change of three guinea pigs of each group is shown in FIG. At 1 day after inoculation, both the ALUM preparation group and the ALUM-free formulation group showed weight loss, but the ALUM formulation gained weight from day 2 and exceeded the physiological saline group on day 7. The ALUM-free formulation inoculated group did not exceed the saline inoculated group until day 7. From this, it can be seen that the side reaction is suppressed by the antigen binding to ALUM.

According to the present invention, a novel influenza vaccine which induces an effective immune response with a low antigenic amount to humans and a low side reaction can be provided, thereby enabling a proper control for pandemic of the new influenza.

This application is based on a patent application 2006-271295 (claim date: October 2, 2006) claimed in Japan, which is hereby incorporated by reference in its entirety.

Claims (25)

Adsorbed inactivated influenza vaccine comprising as an active ingredient an influenza virus whole virion and an aluminum hydroxide gel prepared from sodium carbonate and potassium aluminum sulfate, purified from a virus suspension in the absence of surfactants and ethers. The vaccine of claim 1 having the following characteristics: (1) the adsorption rate of influenza virus total virions to the aluminum hydroxide gel is at least 90%, and (2) The hemagglutinin activity of influenza virus total virions on total hemagglutinin activity is 80% or more. The vaccine according to claim 1, wherein the aluminum concentration is at least 0.1 mg / mL and less than 0.8 mg / mL, and the influenza virus total virion has a concentration of 3.4 to 100 μg HA / mL. The vaccine of claim 1 wherein the influenza virus in the viral stock solution prior to mixing with the aluminum hydroxide gel has the following characteristics: (i) when the viral stock is analyzed by an analytical sucrose density gradient centrifugation method, at least 80% of the total hemagglutinin activity accumulates in the viral virion fraction, and (ii) When the virus stock was subjected to gel filtration high performance liquid chromatography, the absorbance spectrum at 280 nm had a single shoulderless peak. The vaccine of claim 1, wherein at least 80% of the influenza virus in the viral stock prior to mixing with the aluminum hydroxide gel maintains the complete virion form when the viral stock is observed under an electron microscope at 20000 to 30000x magnification. The vaccine according to claim 1, wherein the influenza virus is a subtype consisting of a combination of a form selected from H2 and H4-16 and a form selected from N1-9. A process for the preparation of an adsorbed inactivated influenza vaccine comprising influenza virus whole virions as an active ingredient, the method comprising performing the following steps (1) to (5) in a solvent free of surfactant and ether: (1) culturing the influenza virus to obtain a virus suspension, (2) purifying the virus suspension by filtration or centrifugation, (3) inactivating the purified virus solution with formalin, (4) filtering the inactivated virus solution to produce a virus stock solution, and (5) adsorbing influenza virus in the virus stock solution to an aluminum hydroxide gel prepared from sodium carbonate and potassium aluminum sulfate. The production method according to claim 7, wherein the influenza virus is a subtype comprising a combination of a form selected from H2 and H4-16 and a form selected from N1-9. delete delete delete delete delete delete A commercial package comprising an adsorbed inactivated influenza vaccine according to any one of claims 1 to 6 and documents which can or should be used for the prevention or alleviation of influenza. delete delete delete delete delete delete delete delete delete delete

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