Method for producing an influenza ha split vaccine

Abstract

Provided is a method for producing an influenza HA split vaccine, which generates antibodies that bind to the stem region of the HA of influenza, wherein the stem region of the HA is difficult to generate antigenic variation. The influenza HA split vaccine is subjected to acidic treatment. By performing the acidic treatment, an influenza HA split vaccine is obtained, which generates antibodies that bind to the stem region of the HA. This influenza HA split vaccine has good protection against infection by other influenza viruses having different antigenicity.

Description

Technical Field

[0001] This invention relates to a method for producing an influenza HA split vaccine. Background Technology

[0002] Current influenza hemagglutinin (HA) vaccines induce anti-HA antibodies, thereby providing protection against infection. Anti-HA antibodies bind to a portion of the virus exposed to the outside of the viral membrane, called the "head region." This region undergoes the most frequent structural changes in viral strains. Therefore, in some cases, anti-HA antibodies may fail to bind to viruses that have undergone antigenic variation and are different from the vaccine strain, and the vaccine may not provide protection against infection.

[0003] In recent years, it has been revealed that antibodies binding to stem regions unlikely to cause antigenic variation include protective antibodies against infection (Patent Document 1). To effectively induce antibodies to bind to stem regions, HA stem proteins with stabilized stem portions have been developed, and clinical trials have been conducted in humans: initially unstable stem portions have been stabilized through artificial mutation or linker binding.

[0004] However, issues regarding production for practical applications remain to be resolved, and the development of HA vaccine antigens that can more easily induce anti-stem antibodies is anticipated.

[0005] Citation List

[0006] Patent documents

[0007] Patent Document 1: Japanese Unexamined Patent Publication (Translation of PCT International Application) No. 2016-516090. Summary of the Invention

[0008] Technical issues

[0009] In view of the foregoing, one object of the present invention is to provide a method for producing an influenza HA split vaccine, the vaccine generating antibodies that bind to the HA stem region of influenza, the HA stem region being unlikely to cause antigenic variation.

[0010] Solution to the problem

[0011] The method for producing an HA split vaccine according to the present invention includes acidifying an influenza HA split vaccine to produce an influenza HA split vaccine, said vaccine producing antibodies that bind to the long α-helix (LAH) of the HA stem region and are effective against influenza viruses that cause antigenic variation.

[0012] Specifically, the present invention relates to the following.

[0013] [Project 1]

[0014] A method for producing an influenza HA split vaccine, the vaccine being capable of generating antibodies that bind to the LAH region of the HA stem, the method comprising: acidifying the influenza HA split vaccine.

[0015] [Project 1a]

[0016] A method for producing an influenza HA split vaccine, the vaccine being capable of generating antibodies that bind to the LAH region of the HA stem, the method comprising: acidifying the influenza HA split vaccine that has not yet undergone formalin treatment.

[0017] [Project 1b]

[0018] A method for producing an influenza HA split vaccine, the vaccine being capable of generating antibodies that bind to the LAH region of the HA stem, the method comprising: acidifying the influenza HA split vaccine; and subsequently treating the influenza HA split vaccine with formalin.

[0019] [Project 1c]

[0020] A method for producing an influenza HA split vaccine, the vaccine being capable of generating antibodies that bind to the LAH region of the HA stem, the method comprising: acidifying an influenza HA split vaccine that has not yet undergone formalin treatment; and subsequently formalinizing the influenza HA split vaccine.

[0021] [Project 2]

[0022] According to the production methods of Projects 1, 1a to 1c, the influenza HA split vaccine is also effective against influenza viruses that cause antigenic mutations.

[0023] [Project 3]

[0024] A method for producing an influenza HA split vaccine, the vaccine being capable of generating antibodies that bind to the LAH region of the HA stem and are effective against influenza viruses that cause antigenic variation, the method comprising: acidifying the influenza HA split vaccine.

[0025] [Project 4]

[0026] The method of any one of items 1-3, 1a to 1c, wherein the acidic treatment is carried out at a pH of 4.4-5.8.

[0027] [Project 5]

[0028] The method of any one of items 1-4, 1a to 1c, wherein the influenza HA split vaccine belongs to the H3N2 or H1N1 type.

[0029] [Project 5c]

[0030] The method of any one of items 1-5 and 1a to 1c, wherein the influenza HA split vaccine is of type H3N2 or H1N1, except that the method includes the following steps: treating the H3N2 or H1N1 influenza HA split vaccine suspended in phosphate-buffered saline by adding 0.15 M citrate buffer (pH 3.5) to bring the pH to 5.0; after 30 minutes at room temperature, adding 1 M Tris buffer (pH 8.0) to restore the pH to 7.3; centrifuging to obtain a membrane fusion HA split vaccine; and then adding formalin to the membrane fusion HA split vaccine to a final concentration of 0.05 v / v%, and allowing it to stand for several days.

[0031] [Project 5D]

[0032] The method of any one of items 1-5 and 1a to 1c, wherein the influenza HA split vaccine is of type H3N2 or H1N1, except that the method includes the following steps: treating the influenza HA split vaccine prepared from strain X31 of type H3N2 or strain A / Puerto Rico / 8 / 34 of type H1N1 suspended in phosphate-buffered saline to bring the pH to 5.0 by adding 0.15 M citrate buffer (pH 3.5) to the influenza HA split vaccine suspended in phosphate-buffered saline; after 30 minutes at room temperature, adding 1 M Tris buffer (pH 8.0) to restore the pH to 7.3; centrifuging to obtain a membrane fusion HA split vaccine; and then adding formalin to the membrane fusion HA split vaccine to a final concentration of 0.05 v / v%, and allowing it to stand for several days.

[0033] [Project 5a]

[0034] According to the method of any one of items 1-5, 1a to 1c, 5c and 5d, the influenza HA split vaccine is an influenza HA split vaccine of a single HA subtype.

[0035] [Project 5b]

[0036] The method according to any one of items 1-5, 1a to 1c, 5c and 5d, the method comprising: mixing two or more influenza HA split vaccine antigens, each of which is produced by acidifying a single subtype of influenza HA split vaccine.

[0037] [Project 6]

[0038] An influenza HA split vaccine that produces antibodies that bind to the LAH in the HA stem region.

[0039] [Project 7]

[0040] The influenza HA split vaccine in Project 6 is also effective against influenza viruses that cause antigenic mutations.

[0041] [Project 8]

[0042] The influenza HA split vaccine of Project 6 or 7 has an HA stem region exposed to the outside.

[0043] [Project 9]

[0044] The influenza HA split vaccine of any of items 6-8, wherein the HA stem region of the influenza HA split vaccine antigen exposed to the outside enhances the antigenicity of the LAH in the HA stem region, and the influenza HA split vaccine is capable of producing antibodies that bind to the LAH in the HA stem region.

[0045] [Project 10]

[0046] An influenza HA split vaccine capable of producing antibodies that bind to the LAH in the HA stem region, said vaccine being produced by acidifying an influenza HA split vaccine.

[0047] [Project 10a]

[0048] An influenza HA split vaccine capable of producing antibodies that bind to the LAH in the HA stem region, said vaccine being produced by acid treatment of an influenza HA split vaccine that has not yet undergone formalin treatment.

[0049] [Project 10b]

[0050] An influenza HA split vaccine capable of producing antibodies that bind to the LAH in the HA stem region, said vaccine being produced by a production method comprising the following steps: acid treatment of the influenza HA split vaccine; and thereafter, formalin treatment of the influenza HA split vaccine.

[0051] [Project 10c]

[0052] An influenza HA split vaccine capable of producing antibodies that bind to the LAH of the HA stem region, said vaccine being produced by a production method comprising the following steps: acid treatment of an influenza HA split vaccine that has not yet undergone formalin treatment; and thereafter, formalin treatment of the influenza HA split vaccine.

[0053] [Project 10d]

[0054] An influenza HA split vaccine capable of producing antibodies that bind to the LAH in the HA stem region, said vaccine being produced by acidic treatment of an influenza HA split vaccine comprising a single subtype.

[0055] [Project 10e]

[0056] An influenza HA split vaccine capable of producing antibodies that bind to the LAH of the HA stem region, said vaccine being produced by mixing two or more of the influenza HA split vaccine antigens, each of which is produced by acidifying an influenza HA split vaccine comprising a single subtype.

[0057] [Project 10f]

[0058] The influenza HA split vaccine from any of the items 10-10e is also effective against influenza viruses that cause antigenic mutations.

[0059] [Item 10g]

[0060] The influenza HA split vaccine of any of items 10-10f, which is produced by the method of item 5c or 5d.

[0061] [Project 11]

[0062] An influenza HA split vaccine capable of producing antibodies that bind to the LAH in the HA stem region and is also effective against influenza viruses that cause antigenic variation, said vaccine being produced by acidifying an influenza HA split vaccine.

[0063] One embodiment of this application includes a method for producing an influenza HA split vaccine according to any one of items 1-5, 1a to 1c, 5a and 5b, other than production method A described below.

[0064] Production method A includes the following steps: adding polyoxyethylene dehydrated sorbitan monooleate (e.g., Tween 80) to a final concentration of 0.1 v / v% for strain X31 of type H3N2 or strain A / Puerto Rico / 8 / 34 of type H1N1 suspended in phosphate-buffered saline; adding and suspending diethyl ether, and allowing the suspension to stand until the aqueous layer and the diethyl ether layer are completely separated, then removing the diethyl ether layer; distilling off the remaining diethyl ether in the recovered aqueous layer at atmospheric pressure to obtain the HA split vaccine; then treating the HA split vaccine suspended in phosphate-buffered saline with 0.15 M citrate buffer (pH 3.5) to bring the pH to 5.0; after 30 minutes at room temperature, adding 1 M Tris buffer (pH 8.0) to restore the pH to 7.3; centrifuging to obtain a membrane fusion HA split vaccine; and then adding formalin to the membrane fusion HA split vaccine to a final concentration of 0.05 v / v%.

[0065] One embodiment of this application includes an influenza HA split vaccine, which is produced by a method other than production method A, according to any one of items 1-5, 1a to 1c, 5a and 5b.

[0066] One embodiment of this application includes an influenza HA split vaccine according to any one of items 6-11 and 10a to 10f, other than an influenza HA split vaccine produced by production method A.

[0067] One embodiment of this application includes a method for producing an influenza HA split vaccine according to any one of items 1-5, 1a to 1c, 5a and 5b, other than production method B described below.

[0068] Production method B includes the following steps: adding polyoxyethylene dehydrated sorbitan monooleate (e.g., Tween 80) to H3N2 or H1N1 influenza virus particles suspended in phosphate-buffered saline to a final concentration of 0.1 v / v%, and suspending therein; adding and suspending ether, and allowing the suspension to stand until the aqueous and ether layers are completely separated, then removing the ether layer; distilling off the ether remaining in the recovered aqueous layer at atmospheric pressure to obtain an HA split vaccine; then treating the HA split vaccine suspended in phosphate-buffered saline with 0.15 M citrate buffer (pH 3.5) to bring the pH to 5.0; after 30 minutes at room temperature, adding 1 M Tris buffer (pH 8.0) to restore the pH to 7.3; centrifuging to obtain a membrane fusion HA split vaccine; and then adding formalin to the membrane fusion HA split vaccine to a final concentration of 0.05 v / v%.

[0069] One embodiment of this application includes an influenza HA split vaccine, which is produced by a method other than production method B, according to any one of items 1-5, 1a to 1c, 5a and 5b.

[0070] One embodiment of this application includes an influenza HA split vaccine according to any one of items 6-11 and 10a to 10f, other than an influenza HA split vaccine produced by production method B.

[0071] One embodiment of this application includes a method for producing an influenza HA split vaccine according to any one of items 1-5, 1a to 1c, 5a and 5b, other than production method C described below.

[0072] Production method C includes the following steps: treating the influenza HA split vaccine prepared from strain X31 of type H3N2 or strain A / Puerto Rico / 8 / 34 of type H1N1 with 0.15 M citrate buffer (pH 3.5) suspended in phosphate buffered saline to bring the pH to 5.0; after 30 minutes at room temperature, adding 1 M Tris buffer (pH 8.0) to restore the pH to 7.3; centrifuging to obtain the membrane fusion HA split vaccine; and then adding formalin to the membrane fusion HA split vaccine to a final concentration of 0.05 v / v%.

[0073] One embodiment of this application includes an influenza HA split vaccine, which is produced by a method other than production method C, according to any one of items 1-5, 1a to 1c, 5a and 5b.

[0074] One embodiment of this application includes an influenza HA split vaccine according to any one of items 6-11 and 10a to 10f, other than an influenza HA split vaccine produced by production method C.

[0075] One embodiment of this application includes a method for producing an influenza HA split vaccine according to any one of items 1-5, 1a to 1c, 5a and 5b, other than production method D described below.

[0076] Production method D includes the following steps: treating the H3N2 or H1N1 influenza HA split vaccine by adding 0.15 M citrate buffer (pH 3.5) to a phosphate-buffered saline solution to bring the pH to 5.0; after 30 minutes at room temperature, adding 1 M Tris buffer (pH 8.0) to restore the pH to 7.3; centrifuging to obtain a membrane fusion HA split vaccine; and then adding formalin to the membrane fusion HA split vaccine to a final concentration of 0.05 v / v%.

[0077] One embodiment of this application includes an influenza HA split vaccine, which is produced by a method other than production method D, according to any one of items 1-5, 1a to 1c, 5a and 5b.

[0078] One embodiment of this application includes an influenza HA split vaccine according to any one of items 6-11 and 10a to 10f, other than an influenza HA split vaccine produced by production method D.

[0079] Advantages of the invention

[0080] According to the present invention, an influenza HA split vaccine is obtained by a simple technique, the vaccine producing antibodies that bind to the HA stem region of influenza, which is unlikely to cause antigenic variation. Therefore, an influenza HA split vaccine that is also effective against influenza viruses that cause antigenic variation is obtained. Attached Figure Description

[0081] [ Figure 1 ] Figure 1 This is a diagram illustrating the influenza virus.

[0082] [ Figure 2 ] Figure 2 The figure shows an increase in the titer of anti-LAH antibodies in the serum of mice vaccinated with the membrane fusion H3N2 HA split vaccine.

[0083] [ Figure 3 ] Figure 3 The figure shows the improved cross-protective ability against antigen variants in mice vaccinated with the membrane fusion H3N2 HA split vaccine.

[0084] [ Figure 4 ] Figure 4 The figure shows an increase in the titer of anti-LAH antibodies in the serum of mice vaccinated with the membrane fusion H1N1 HA split vaccine.

[0085] [ Figure 5 ] Figure 5 The figure shows the improved cross-protective ability against antigen variants in mice vaccinated with the membrane fusion H1N1 HA split vaccine.

[0086] [ Figure 6 ] Figure 6 The figures shown each indicate that monoclonal antibodies binding to LAH bind more strongly to membrane-fusion HA split vaccines than current HA split vaccines.

[0087] [ Figure 7 ] Figure 7 The figures shown each indicate that the monoclonal antibody binding to LAH strongly binds to the membrane fusion-type HA split vaccine, which has been formalin-treated after acid treatment.

[0088] [ Figure 8 ] Figure 8 The figure shows an increase in the titer of anti-LAH antibodies in the serum of mice vaccinated with the membrane fusion H3N2 HA split vaccine (pre-fixed and post-fixed). Detailed Implementation

[0089] Embodiments of the present invention will now be described in detail with reference to the accompanying drawings. However, these embodiments are intended to facilitate an understanding of the principles of the invention, and the scope of the invention is not limited to the embodiments described below. Other embodiments are also covered within the scope of the invention, wherein the configurations of the embodiments described below have been appropriately substituted by those skilled in the art.

[0090] The method for producing an influenza HA split vaccine according to this implementation plan includes the step of acidifying the influenza HA split vaccine.

[0091] Influenza HA split vaccines are prepared by treating intact viral vaccines with ether to remove lipid components that become pyrogens. Influenza HA split vaccines have HA protein as a major component because they are produced as follows: the HA protein required for immunization is collected from the surface of viral particles by density gradient centrifugation.

[0092] A glycoprotein called the "spike protein" extends from the surface of the influenza virus. Figure 1 Influenza A virus has two types of spike proteins, namely HA and NA (neuraminidase), which help the virus cause infection. HA binds to the cell to be infected and helps the virus enter the cell. HA often causes antigenic variation. NA causes the infected cell to detach from HA and is used to release the replicating virus from the cell.

[0093] The HA of influenza A virus is divided into two regions: the head region and the stem region. Figure 1 The head region contains the receptor binding site at which the virus binds to the target cell. The stem region contains the fusion peptide sequence required for membrane fusion between the viral membrane and the target cell membrane.

[0094] Acidic treatment of influenza HA split vaccines alters the structure of the HA protein to a structure known as a membrane fusion type. In the membrane fusion type HA protein, the stem region replaces the head region, which is exposed externally from the viral membrane, exhibiting a significant structural change in the conformation of the antigen stem. The inventors of this invention have discovered in vivo that when the membrane fusion type HA protein is used as a vaccine, it induces antibodies that bind to the LAH in the stem region, and these antibodies provide protection against viral strains that cause antigenic variation. Based on this fact, this invention has been completed.

[0095] The acid treatment is not particularly limited and can be carried out at pH values ​​of, for example, 2.0-6.5, preferably 3.0-6.5, more preferably 4.0-6.0, and even more preferably 4.4-5.8. Specific examples include pH values ​​of 2.0-2.9, 2.0-4.0, 2.0-5.0, 2.0-6.0, 3.0-4.0, 3.0-5.0, 3.0-6.0, 4.0-5.8, 4.0-6.5, 5.0-6.5, and 6.0-6.5. The acid used for the acid treatment is not particularly limited and can be, for example, phosphoric acid, citric acid, maleic acid, hydrochloric acid, or any other suitable acid.

[0096] The temperature for acid treatment is, for example, 0°C to 75°C, preferably 10°C to 60°C, more preferably 20°C to 45°C, and even more preferably 25°C to 42°C. Specific examples include 0°C to 20°C, 5°C to 25°C, 10°C to 30°C, 15°C to 35°C, 20°C to 37°C, 25°C to 37°C, 30°C to 50°C, 38°C to 55°C, 38°C to 60°C, 38°C to 65°C, 38°C to 70°C, 38°C to 75°C, 40°C to 55°C, 40°C to 60°C, 40°C to 65°C, 40°C to 70°C, 40°C to 75°C, 42°C to 55°C, 42°C to 60°C, 42°C to 65°C, 42°C to 70°C, 42°C to 75°C, 45°C to 55°C, 45°C to 60°C, 45°C to 65°C, 45°C to 70°C, and 45°C to 75°C. The processing time is, for example, 5 minutes to 120 minutes, preferably 15 minutes to 60 minutes, more preferably 20 minutes to 45 minutes. Specific examples include 5 minutes to 60 minutes, 20 minutes to 60 minutes, 15 minutes to 120 minutes, 15 minutes to 45 minutes, 20 minutes to 60 minutes, 20 minutes to 120 minutes, 45 minutes to 120 minutes, and 60 minutes to 120 minutes.

[0097] Based on antigenic differences, influenza A virus HA is classified into 18 subtypes (H1 to H18), and NA is classified into 9 subtypes (N1 to N9). The influenza HA split vaccine of the present invention is applicable to all these subtypes. In addition, the method for producing influenza HA split vaccine according to the present invention can produce a vaccine that is effective not only against influenza A virus but also against influenza B virus with HA.

[0098] The influenza split HA vaccine obtained by the production method according to the invention produces antibodies whose binding is unlikely to cause mutated LAH. Therefore, the vaccine can provide cross-protection against influenza viruses known as antigenic variants, provided that the viruses have the same HA subtype. Furthermore, the influenza split HA vaccine obtained by the production method according to the invention can exhibit cross-reactivity between HA subtypes (e.g., H3 and H7) with similar amino acid sequences of LAH.

[0099] In this application, "influenza HA split vaccine with a single HA subtype" refers to an influenza HA split vaccine selected from the 18 subtypes (H1 to H18) of influenza A virus or a single HA subtype of influenza B virus. As long as it has a single HA subtype, the NA subtype can be the same or different. Preferred HA subtypes include H1, H3, and B.

[0100] To produce a mixed vaccine containing two or more HA subtypes, each of the influenza HA split vaccines belonging to a single HA subtype is acidified, and multiple (two or more) influenza HA split vaccines thus obtained can be mixed together. Alternatively, the mixed vaccine can be produced by acidifying an influenza HA split vaccine previously prepared by mixing two or more HA subtypes. For administration as a vaccine comprising two or more subtypes, the vaccine preferably comprises 1-3 subtypes selected from H1, H3, and B.

[0101] In a preferred embodiment, the influenza HA split vaccine obtained by the production method of the present invention binds to LAH-binding monoclonal antibodies more strongly than current HA split vaccines. For example, the influenza HA split vaccine binds to LAH-binding monoclonal antibodies at least 1.05 times, preferably at least 1.1 times, more preferably at least 1.5 times, and even more preferably at least 2 times more strongly than current HA split vaccines. In this context, "an influenza HA split vaccine that binds more strongly than current HA split vaccines by at least 1.05 times, at least 1.1 times, at least 1.5 times, or at least 2 times" means, for example, that when the absorbance determined by regression is 0.7, the reciprocal of the antibody concentration is at least 1.05 times, at least 1.1 times, at least 1.5 times, or at least 2 times the reciprocal of the antibody concentration of current HA split vaccines. In a preferred embodiment, the influenza HA split vaccine of the present invention has a higher binding capacity to LAH-binding monoclonal antibodies than current HA split vaccines. Although there is no particular upper limit, the binding ability can be in the range of, for example, 1.05-200 times, 1.1-150 times, 1.5-100 times, or 2-50 times. Alternatively, the range of binding ability of the influenza HA split vaccine of the present invention to monoclonal antibodies binding to LAH compared to the binding ability of current HA split vaccines can be indicated by a combination of lower limits selected from 1.05, 1.1, 1.5, 2, 3, 4, and 5 and upper limits selected from 200, 150, 100, 50, 30, and 20. To measure the binding ability of the influenza HA split vaccine to monoclonal antibodies binding to LAH, any method can be used without particular limitation, and common methods known to those skilled in the art can be employed. For example, binding ability can be measured by the methods described in the embodiments of this application.

[0102] In this application, "LAH-binding monoclonal antibody" refers to a monoclonal antibody that binds to LAH. Any method can be used to produce the monoclonal antibody without particular limitation, and common methods known to those skilled in the art can be employed. When measuring the binding ability of an influenza HA split vaccine to an LAH-binding monoclonal antibody, it is assumed that the LAH-binding monoclonal antibody is capable of binding at least a portion of the peptide corresponding to the LAH of the influenza virus from which the influenza HA split vaccine is derived.

[0103] In this application, "current HA split vaccine" refers to a vaccine from which lipid components that become pyrogens have been removed by treating an intact viral vaccine with ether, and which can be produced by, for example, the method described in Example 1 of this application. The current HA split vaccine can also be an influenza HA split vaccine produced without acid treatment, which differs from the influenza HA split vaccine of the present invention prepared by a method including the acid treatment described below.

[0104] The production of the influenza HA split vaccine of the present invention may include formalin treatment. In a preferred embodiment, the influenza HA split vaccine is acid-treated prior to formalin treatment. In preparing the influenza HA split vaccine antigen of the present invention (an influenza HA split vaccine antigen capable of producing antibodies binding to the LAH of the HA stem region), the HA fraction used for the current influenza HA split vaccine is acid-treated and then formalin-treated. This makes it possible to obtain the influenza HA split vaccine antigen, which more effectively generates cross-reactive antibodies, and is therefore more preferred as a universal influenza vaccine antigen. That is, in a preferred embodiment of this application, the HA fraction from which the fatty solvent is removed by treating the virus particles with ether or any other suitable reagent is acid-treated and then formalin-treated.

[0105] In a preferred embodiment of this application, the influenza HA split vaccine prior to acid treatment is a split vaccine that has not yet undergone formalin treatment.

[0106] Commercially available Influenza HA Vaccine (trade name) has undergone treatment with formaldehyde or an equivalent substance after the virus has been decomposed with ether or any other suitable reagent and the fatty solvent has been removed, as described in Biological Products Standards (Ministerial Notification No. 155 of the Ministry of Health, Labour and Welfare, March 30, 2004; latest revised, Ministerial Notification No. 409, November 30, 2018). Preferably, the influenza HA split vaccine of the present invention is produced without using commercially available Influenza HA Vaccine (trade name), which is one of the influenza HA split vaccines, because it has been treated with formaldehyde or any other suitable reagent.

[0107] The formalin concentration in the formalin-treated solution used for influenza HA split vaccine after acid treatment is, for example, 0.0005 v / v% to 10 v / v%, preferably 0.001 v / v% to 1 v / v%, more preferably 0.003 v / v% to 0.5 v / v%, and even more preferably 0.005 v / v% to 0.1 v / v%. The formalin treatment time is, for example, 1 hour to 10 days, preferably 2 hours to 5 days, more preferably 12 hours to 3 days. The formalin treatment temperature is, for example, 0°C to 75°C, preferably 1°C to 37°C, more preferably 1°C to 30°C.

[0108] Preferably, medical-grade formalin is used.

[0109] A method for producing the influenza HA split vaccine of the present invention may include the step of adding an adjuvant. Examples of adjuvants include, but are not limited to, aluminum salts such as aluminum hydroxide and aluminum phosphate, chitosan, oligodeoxynucleotides, and oil-in-water emulsions. Among these, aluminum hydroxide is preferred, and the application of aluminum hydroxide as an adjuvant can enhance immunogenicity.

[0110] The influenza HA split vaccine obtained by the production method of the present invention can be used, for example, for subsequent vaccinations after a predetermined period following the initial vaccination. There is no particular limitation on the period between the initial vaccination and the subsequent vaccination, but it can be, for example, 20 days to 3 years, preferably 3 months to 2 years, more preferably 6 months to 1 year. There is no particular limitation on the amount of influenza HA split vaccine used for the initial and subsequent vaccinations, but each dose can be, for example, 1 μg to 200 μg, preferably 10 μg to 30 μg, more preferably 15 μg. A single dose is, for example, 0.5 mL. Any method of administration can be used for the initial and subsequent vaccinations without particular limitation, and can be administered, for example, nasally, subcutaneously, intradermally, transdermally, intraocularly, through mucous membranes, or orally. Intramuscular administration is preferred.

[0111] The influenza HA split vaccine obtained by the production method of the present invention has a protective effect against viral strains that cause antigenic variation. For example, if a current HA split vaccine is prepared from H3N2 influenza virus particles (A / Fujian / 411 / 02 (H3N2)) and subjected to acid treatment, the vaccine may be effective against not only A / Fujian / 411 / 02 (H3N2), but also, for example, A / Guizhou / 54 / 89 (H3N2), A / OMS / 5389 / 88 (H3N2), A / Beijing / 32 / 92 (H3N2), A / England / 427 / 88 (H3N2), A / Johannesburg / 33 / 94 (H3N2), A / Leningrad / 360 / 86 (H3N2), A / Mississippi / 1 / 85 (H3N2), A / Philippines / 2 / 82 (H3N2), A / Shandong / 9 / 93 (H3N2), A / Shanghai / 16 / 89 (H3N2), and A / Shanghai / 24 / 90 Protective effect against infection with (H3N2), A / Sichuan / 2 / 87 (H3N2), A / Kitakyushu / 159 / 93 (H3N2), A / Akita / 1 / 94 (H3N2), A / Panama / 2007 / 99 (H3N2), A / Wyoming / 03 / 03 (H3N2), A / New York / 55 / 2004 (H3N2) or A / Hiroshima / 52 / 2005 (H3N2). Furthermore, for example, if a current HA split vaccine is prepared from H1N1 influenza virus particles (A / Puerto Rico / 8 / 34(H1N1)) and subjected to acid treatment, the vaccine may also be effective against not only A / Puerto Rico / 8 / 34 (H1N1), but also against, for example, A / Narita / 1 / 09 (H1N1), A / Beijing / 262 / 95 (H1N1), A / Brazil / 11 / 78 (H1N1), A / Chile / 1 / 83 (H1N1), A / New Jersey / 8 / 76 (H1N1), A / Taiwan / 1 / 86 (H1N1), A / Yamagata / 32 / 89 (H1N1), A / New Caledonia / 20 / 99 (H1N1), A / Solomon Islands / 3 / 2006 (H1N1), and A / Blisban / 59 / 2007. Protective effect against infection with (H1N1) or A / Mexico / 4108 / 2009 (H1N1). Example

[0112] 1. Preparation of HA split vaccine

[0113] Tween 80 was added to H3N2 influenza virus particles (X31 strain) or H1N1 influenza virus particles (A / Puerto Rico / 8 / 34 strain) suspended in phosphate-buffered saline to a final concentration of 0.1 v / v%, and the mixture was suspended therein. Ether was added and the mixture was suspended, and the suspension was allowed to stand until the aqueous and ether layers were completely separated, then the ether layer was removed. This ether extraction was repeated, and the ether remaining in the recovered aqueous layer was distilled off at atmospheric pressure to obtain the HA split vaccine.

[0114] 2. Acid treatment

[0115] The HA split vaccine was suspended in phosphate-buffered saline and then acidified by adding 0.15 M citrate buffer (pH 3.5) to bring the pH to 5.0. After standing at room temperature for 30 minutes, 1 M Tris buffer (pH 8.0) was added to restore the pH to 7.3. Centrifugation was then performed to obtain the membrane fusion HA split vaccine. Formalin was added to the membrane fusion HA split vaccine prepared in this way to a final concentration of 0.05 v / v%, and the mixture was allowed to stand for several days.

[0116] The current HA split vaccine is prepared in the same manner as described in section 1 above, except that no acid treatment is provided.

[0117] 3. Measure the titer of anti-LAH antibody using ELISA.

[0118] 3-1. H3N2 influenza vaccination

[0119] BALB / c mice (female, 6–12 weeks old) were intraperitoneally vaccinated with either the current H3N2 HA split vaccine or the membrane fusion HA split vaccine (10 μg vaccine + 10 v / v% AddaVax adjuvant (InvivoGen), dissolved in phosphate-buffered saline to a volume of 200 μl). Twenty-eight days after the initial vaccination, the mice were intraperitoneally vaccinated with the membrane fusion HA vaccine (10 μg of the vaccine alone dissolved in phosphate-buffered saline to a volume of 200 μl). Blood was collected from the vaccinated mice at least 14 days after the additional vaccination, and serum was collected from them.

[0120] 3-2. Measurement by ELISA

[0121] The concentration of anti-LAH antibodies in the serum of BALB / c mice that were intraperitoneally inoculated with the current H3N2 HA split vaccine or membrane fusion HA split vaccine was measured by ELISA (enzyme-linked immunosorbent assay) as follows.

[0122] Specifically, the synthetic peptide (H3; Ac-RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENADYKDDDDKC) (SEQ ID NO: 1) corresponding to a portion of the stem (long α-helix) was dissolved at 10 μg / ml in phosphate-buffered saline (pH 7.3), and 100 μl was added to each well of a 96-well plate. After standing overnight at 4°C, each well was washed three times with phosphate-buffered saline, and 150 μl of phosphate-buffered saline containing 1 v / v% bovine serum albumin was added. After standing for 2 hours at room temperature, each well was washed three times with phosphate-buffered saline. Then, 100 μl of mouse serum (serially diluted with phosphate-buffered saline containing 0.05 v / v% Tween 20 and 1 v / v% bovine serum albumin) and 100 μl of a standard monoclonal antibody of known concentration (H3; clone name V15-5) were added to each well. After standing at room temperature for 2 hours, each well was washed three times with phosphate-buffered saline (containing 0.05 v / v% Tween 20), and 100 μl of peroxidase-labeled anti-mouse IgG antibody (Southern Biotech) (diluted with phosphate-buffered saline containing 0.05 v / v% Tween 20 and 1 v / v% bovine serum albumin) was added to each well. After standing at room temperature for 2 hours, each well was washed three times with phosphate-buffered saline (containing 0.05 v / v% Tween 20). Then, 30 mg o-phenylenediamine tablets (Sigma) and 24 μl of 30% hydrogen peroxide solution (30% w / w; Sigma) were added to 60 ml of citrate buffer (pH 5.0) as substrate, and 100 μl of the resulting mixture was added to each well. After color development, 50 μl of 1 mol / L sulfuric acid (Wako Pure Chemical Industries, Ltd.) was added to stop the reaction, and the absorbance at 490 nm was measured using a Microplate Reader 450 (Biorad).

[0123] As in Figure 2 As shown, the serum anti-LAH antibody titer in BALB / c mice that were intraperitoneally inoculated with the membrane fusion HA split vaccine was significantly higher than that in BALB / c mice that were intraperitoneally inoculated with the current HA split vaccine.

[0124] 4. Cross-protection against antigen variants

[0125] In the protective experiment against H3N2 virus infection, 200 μl of serum collected from unvaccinated mice, 200 μl of serum collected from mice vaccinated with the current H3N2 HA split vaccine, or 200 μl of serum collected from mice vaccinated with the membrane fusion HA split vaccine were administered intraperitoneally to BALB / c mice (female, 6-12 weeks old).

[0126] Three hours after serum administration, another H3N2 influenza virus (A / Guizhou / 54 / 89) with antigenicity different from the vaccine strain was administered intranasally under anesthesia at a lethal dose of 50 for mice (5 times the viral load that would kill 50% of mice).

[0127] Mice were weighed and observed daily for 21 days from the start of viral infection to study changes in body weight and survival rate. The humanitarian endpoint was set at 25% body weight loss.

[0128] As in Figure 3 As shown, in BALB / c mice vaccinated with the membrane fusion HA split vaccine, the decline in survival was significantly suppressed after the ninth day following infection with other H3N2 influenza viruses of different antigenicities.

[0129] 5. Measure the titer of anti-LAH antibody using ELISA.

[0130] 5-1. H1N1 influenza virus particles

[0131] Female C57BL / 6 mice, 6–12 weeks old, were intraperitoneally vaccinated with either the current H1N1 HA split vaccine or a membrane fusion HA split vaccine (10 μg vaccine + 10 μg CpG-ODN 1760, suspended in phosphate-buffered saline and mixed with an equal volume of Freund's incomplete adjuvant (ROCKLAND) to a liquid volume of 200 μl). Twenty-eight days after the initial vaccination, mice were intraperitoneally vaccinated with the membrane fusion HA split vaccine (10 μg vaccine + 10 μg CpG-ODN, suspended in phosphate-buffered saline and mixed with an equal volume of Freund's incomplete adjuvant (ROCKLAND) to a liquid volume of 200 μl, in the same manner as the initial vaccination). At least 14 days after the additional vaccination, blood was collected from the vaccinated mice, and serum was collected from them.

[0132] 5-2. Measurement by ELISA

[0133] The concentration of anti-LAH antibodies in the serum of C57BL / 6 mice that were intraperitoneally inoculated with the current H1N1 HA split vaccine or membrane fusion HA split vaccine was measured by ELISA in the following manner.

[0134] The measurements were performed in the same manner as described above, except that a synthetic peptide (H1; Ac-RIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNADYKDDDDKC) (SEQ ID NO: 2) corresponding to a portion of the stem (long α-helix) was used and a standard monoclonal antibody (H1; clone name F2) of known concentration was used.

[0135] As in Figure 4 As shown, the anti-LAH antibody titer in the serum of C57BL / 6 mice that were intraperitoneally vaccinated with a membrane fusion HA split vaccine was significantly higher than that in the serum of C57BL / 6 mice that were intraperitoneally vaccinated with the current HA split vaccine.

[0136] 6. Cross-protection against antigen variants

[0137] In the protective experiment against H1N1 virus infection, 200 μl of serum collected from unvaccinated mice, 200 μl of serum collected from mice vaccinated with the current H1N1 HA split vaccine, or 200 μl of serum collected from mice vaccinated with the membrane fusion HA split vaccine were administered intraperitoneally to C57BL / 6 mice (female, 6-12 weeks old).

[0138] Three hours after serum administration, another H1N1 influenza virus (A / Narita / 1 / 09) with antigenicity different from the vaccine strain was administered intranasally under anesthesia at a lethal dose of 50 to mice (5 times the viral load that would kill 50% of mice).

[0139] Mice were observed daily for 20 days from the onset of viral infection to study survival rates. (For example, in...) Figure 5 As shown, in C57BL / 6 mice vaccinated with a membrane fusion HA split vaccine, the decline in survival was significantly suppressed after the ninth day following infection with other H1N1 influenza viruses of different antigenicities.

[0140] 7. Antibody binding ability to LAH epitopes

[0141] Anti-LAH monoclonal antibodies prepared from peripheral blood of mice or humans infected with strain X31 were measured by ELISA (enzyme-linked immunosorbent assay). Figure 6(#1 to #5) Binding to the current HA split vaccine or membrane fusion HA split vaccine. The current HA split vaccine or membrane fusion HA split vaccine of H3N2 influenza virus (X31 strain) was dissolved in phosphate-buffered saline (pH 7.3), and 50 μl of each was added to a 96-well plate. After standing overnight at 4°C, each well was washed three times with phosphate-buffered saline, and 150 μl of phosphate-buffered saline containing 1 v / v% bovine serum albumin was added. After standing for 2 hours at room temperature, each well was washed three times with phosphate-buffered saline (containing 0.05 v / v% Tween 20), and 50 μl of monoclonal antibody binding to LAH, serially diluted with phosphate-buffered saline containing 1 v / v% bovine serum albumin, was added. After standing overnight at 4°C, each well was washed three times with phosphate-buffered saline (containing 0.05 v / v% Tween 20), and 100 μl of peroxidase-labeled anti-mouse IgG antibody (Southern Biotech) diluted with phosphate-buffered saline containing 0.05 v / v% Tween 20 and 1 v / v% bovine serum albumin was added to each well. After standing for 2 hours at room temperature, each well was washed three times with phosphate-buffered saline (containing 0.05 v / v% Tween 20). Then, 30 mg o-phenylenediamine tablets (Sigma) and 24 µl of 30% hydrogen peroxide solution (30% w / w; Sigma) were added to 60 ml of citrate buffer (pH 5.0) as substrate, and 50 µl of the resulting mixture was added to each well. After color development, 25 µL of 1 mol / L sulfuric acid (Wako Pure Chemical Industries, Ltd.) was added to stop the reaction, and the absorbance at 490 nm was measured using a Microplate Reader 450 (Biorad). The change in binding capacity was calculated from the absorbance values ​​measured for current HA split vaccines or membrane fusion HA split vaccines.

[0142] As in Figure 6 As shown, the monoclonal antibody binding to LAH exhibits a binding affinity for membrane-fusion HA split vaccines that is 1.05–21 times greater than that for current HA split vaccines. The results indicate that acidic treatment of the HA split vaccine enhances the binding affinity of the antibody to the LAH epitope.

[0143] 8. Effects of formalin treatment sequence on antibody binding and antibody induction abilities.

[0144] 8-1. Preparation of HA split vaccine pretreated with formalin

[0145] Add Tween 80 to H3N2 influenza virus particles (X31 strain) suspended in phosphate-buffered saline to a final concentration of 0.1 v / v%. Add ether and suspend, and allow the suspension to stand until the aqueous and ether layers are completely separated, then remove the ether layer.

[0146] After repeating the ether extraction, the ether remaining in the recovered aqueous layer is distilled off under normal pressure. Further, formalin is added to a final concentration of 0.05 v / v%, and the mixture is allowed to stand for several days to obtain a formalin-pretreated HA split vaccine.

[0147] 8-2. Acidic treatment of formalin-pretreated HA split vaccine

[0148] The formalin-pretreated HA split vaccine was resuspended in phosphate-buffered saline, and then acidified by adding 0.15 M citrate buffer (pH 3.5) to bring the pH to 5.0. After standing at room temperature for 30 minutes, 1 M Tris buffer (pH 8.0) was added to restore the pH to 7.3. Centrifugation was then performed.

[0149] 9. Antibody binding ability to LAH epitopes

[0150] The binding affinity of the antibody to the LAH epitope was measured in the same manner as described in section 7 above, and the change in binding affinity was calculated. Here, it will be compared with... Figure 6 The same antibodies shown in #2, #4 and #5 were used as monoclonal antibodies, and monoclonal antibody #6, which binds to the HA head region, was used as a control.

[0151] 10. Measure the titer of anti-LAH antibody by ELISA.

[0152] 10-1. H3N2 Influenza Vaccination

[0153] Female BALB / c mice (6–12 weeks old) were intraperitoneally inoculated with the current H3N2 HA split vaccine or membrane fusion HA split vaccine (10 μg vaccine + 10 v / v% AddaVax adjuvant (InvivoGen), dissolved in phosphate-buffered saline to a volume of 200 μl). Blood and serum were collected from the vaccinated mice at least 12 days post-inoculation.

[0154] 10-2. Measurement by ELISA

[0155] The titer of the anti-LAH antibody was measured in the same manner as described in 3-2 above.

[0156] As in Figure 7As shown, monoclonal antibodies binding to LAH bind more strongly to vaccines that have undergone formalin treatment after acid treatment during the preparation of membrane fusion HA split vaccines (post-fixation) compared to vaccines that have undergone formalin treatment after acid treatment (pre-fixation) before acid treatment. The results indicate that the timing of formalin treatment affects the enhancement of antibody binding to the LAH epitope (obtained through acid treatment of the HA split vaccine), and that formalin treatment is ideally performed after acid treatment. Figure 7 and 8 In this context, the vaccine obtained by subjecting the HA split vaccine to acid treatment after formalin treatment in the same manner as described in Example 8 above is referred to as a "membrane fusion split vaccine (pre-fixation)". Further, the vaccine obtained by subjecting the HA split vaccine to formalin treatment after acid treatment in the same manner as described in Example 1 according to the procedure of Example 1 is referred to as a "membrane fusion split vaccine (post-fixation)".

[0157] As in Figure 8 As shown, the serum anti-LAH antibody titers in BALB / c mice intraperitoneally vaccinated with the membrane fusion-type HA split vaccine were higher than those in BALB / c mice intraperitoneally vaccinated with the current HA split vaccine. Furthermore, the membrane fusion-type HA split vaccine (post-fixation) exhibited higher anti-LAH antibody titers than the membrane fusion-type split vaccine (pre-fixation).

[0158] 11. Preparation of HA split vaccine

[0159] An HA split vaccine was prepared using H3N2 influenza virus particles (X31 strain) via the method described in section 1 above.

[0160] 12. Preliminary check of acid treatment conditions using a mixing tank.

[0161] During the acidification process, the pH was adjusted by adding 0.15M citrate buffer (pH 3.5) or dilute hydrochloric acid to the phosphate buffer solution. Homogenization conditions after adding citrate buffer were examined by observing the stirring rate and color change over time in a mixing vessel with a pH indicator. Methyl red pH indicator was used in 100 mL and 20 L mixing vessels, and the color change was observed and recorded on video after 10 minutes following addition at stirring speeds of 100 rpm, 200 rpm, 300 rpm, 400 rpm, and 500 rpm.

[0162] 13. Acid treatment

[0163] The HA split vaccine was suspended in phosphate-buffered saline and then acidified by adding 0.15 M citrate buffer (pH 3.5) or dilute hydrochloric acid to achieve five pH conditions (2.0, 3.0, 4.0, 5.0, or 6.0). After standing at five temperature conditions (10°C, 25°C, 35°C, 45°C, or 55°C) and three time conditions (10 min, 30 min, or 1 h), 1 M Tris buffer (pH 8.0) was added to restore the pH to 7.3. Centrifugation was then performed to obtain the membrane fusion HA split vaccine. Formalin was added to the membrane fusion HA split vaccine prepared in this way to a final concentration of 0.05 v / v%, and the mixture was allowed to stand for several days.

[0164] The current HA split vaccine is prepared in the same manner as described in 11 above, except that no acid treatment is provided.

[0165] 14. The binding affinity of antibodies to LAH epitopes

[0166] Using the X31 strain of H3N2 influenza virus, anti-LAH monoclonal antibodies prepared from peripheral blood of mice or humans infected with the X31 strain were measured by ELISA in a manner similar to that described in section 7 above. Figure 6 (#1 to #5) are combined with current HA split vaccines or membrane fusion HA split vaccines.

[0167] Industrial application

[0168] This invention can be used to produce influenza vaccines.

[0169] [Sequence List Free Text]

[0170] SEQ ID NO: 1, 2: Synthetic peptides.

Claims

1. A method for producing a membrane fusion type influenza HA split vaccine capable of inducing the production of antibodies binding LAH of the HA stem region, the method comprising: subjecting an influenza HA split vaccine not yet subjected to formalin treatment to an acidic treatment, and thereafter subjecting the influenza HA split vaccine to a formalin treatment, wherein the influenza HA split vaccine not yet subjected to formalin treatment is obtained by treating a whole virus vaccine with an ether, the acidic treatment is performed at a pH of 4.0-6.0, the acidic treatment is performed at 10°C to 60°C, the acidic treatment is performed for a time of 15 minutes to 60 minutes, the formalin is added to a final concentration of 0.005 v / v% to 0.1 v / v%, and the influenza HA split vaccine is of the H3N2 type or of the H1N1 type.

2. The method according to claim 1, wherein the acidic treatment is performed at a pH of 4.4-5.

8.

3. The method according to claim 1 or 2, wherein the influenza HA split vaccine is a single HA subtype influenza HA split vaccine.

4. The method of claim 1 or 2, comprising: two influenza HA split vaccine antigens are mixed, each of which is produced by subjecting a single subtype influenza HA split vaccine to an acidic treatment.

5. A membrane fusion type influenza HA split vaccine capable of inducing the production of antibodies binding LAH of the HA stem region, the vaccine being produced by a production method comprising the steps of subjecting an influenza HA split vaccine not yet subjected to formalin treatment to an acidic treatment; and thereafter subjecting the influenza HA split vaccine to a formalin treatment, wherein the influenza HA split vaccine not yet subjected to formalin treatment is obtained by treating a whole virus vaccine with an ether, the acidic treatment is performed at a pH of 4.0-6.0, the acidic treatment is performed at 10°C to 60°C, the acidic treatment is performed for a time of 15 minutes to 60 minutes, the formalin is added to a final concentration of 0.005 v / v% to 0.1 v / v%, and the influenza HA split vaccine is of the H3N2 type or of the H1N1 type.

6. Use of the influenza HA split vaccine according to claim 5 for the manufacture of a vaccine for protection against influenza infection of the H3N2 type or of the H1N1 type.

7. Use of the influenza HA split vaccine according to claim 5 for the manufacture of a vaccine for protection against influenza infection of the H3N2 type, wherein the influenza HA split vaccine is of the H3N2 type.

8. Use of the influenza HA split vaccine according to claim 5 for the manufacture of a vaccine for protection against influenza infection of the H1N1 type, wherein the influenza HA split vaccine is of the H1N1 type.

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