Methods for producing influenza HA split vaccines

Abstract

A method for producing an influenza HA split vaccine is provided, wherein the vaccine produces antibodies that bind to the HA stem region of influenza, which is less likely to cause antigenic variation. The influenza HA split vaccine is subjected to an acidic treatment. By the acidic treatment, an influenza HA split vaccine is obtained, wherein the vaccine produces antibodies that bind to the LAH of the HA stem region. This influenza HA split vaccine has excellent resistance to infection by other influenza viruses with different antigenicity.

Description

Technical Field

[0001] The present invention relates to a method for producing an influenza HA split vaccine. Background Art

[0002] Existing influenza hemagglutinin (hereinafter also abbreviated as "HA") vaccines induce anti-HA antibodies, thereby exerting a protective effect against infection. Anti-HA antibodies bind to a portion of the virus called the "head region" that is exposed from the outside of the viral membrane. This region most often undergoes structural changes in virus strains. Therefore, in some cases, anti-HA antibodies may not be able to bind to viruses that cause antigenic variation and are different from the vaccine strain, and the vaccine cannot exert a protective effect against infection.

[0003] Recently, it has been revealed that antibodies that bind to the stem region, which is less likely to cause antigenic variation, include protective antibodies against infection (Patent Document 1). In order to effectively induce antibodies that bind to the stem region, HA stem proteins with stabilized stem portions have been developed, and clinical trials thereof have been conducted in humans: the initially unstable stem portion has been stabilized by artificial variation or linker binding.

[0004] However, issues regarding production for practical use remain to be resolved, and development of HA vaccine antigens that can more easily induce anti-stem antibodies has been desired.

[0005] Citation List

[0006] Patent Literature

[0007] Patent Document 1: Japanese Unexamined Patent Publication No. 2016-516090 (Translation of PCT International Application) SUMMARY OF THE INVENTION

[0009] Technical issues

[0010] In view of the foregoing, it is an object of the present invention to provide a method for producing an influenza HA split vaccine that produces antibodies that bind to the HA stem region of influenza that is less likely to cause antigenic variation.

[0011] Solution to the problem

[0012] The method for producing an HA split vaccine of the present invention comprises subjecting an influenza HA split vaccine to acidic treatment, thereby producing an influenza HA split vaccine, which produces antibodies that bind to the long alpha helix (LAH) of the HA stem region and is effective against influenza viruses that cause antigenic variation.

[0013] Specifically, the present invention relates to the following contents.

[0014] [Project 1]

[0015] A method for producing an influenza HA split vaccine, wherein the vaccine produces antibodies that bind to LAH in the HA stem region, the method comprising: subjecting the influenza HA split vaccine to acidic treatment.

[0016] [Project 2]

[0017] The production method of item 1, wherein the influenza HA split vaccine is also effective against influenza viruses that cause antigenic mutations.

[0018] [Item 3]

[0019] A method for producing an influenza HA split vaccine, wherein the vaccine produces antibodies that bind to LAH in the HA stem region and is effective against influenza viruses that cause antigenic variation, the method comprising: subjecting the influenza HA split vaccine to acidic treatment.

[0020] [Item 4]

[0021] The method of any one of items 1-3, wherein the acid treatment is carried out at a pH of 4.4-5.8.

[0022] [Item 5]

[0023] The method of any one of items 1-4, wherein the influenza HA split vaccine has an H3N2 type or an H1N1 type.

[0024] [Item 6]

[0025] An influenza HA split vaccine that generates antibodies that bind to LAH in the HA stem region.

[0026] [Item 7]

[0027] The influenza HA split vaccine of Item 6 is also effective against influenza viruses that cause antigenic mutations.

[0028] [Item 8]

[0029] The influenza HA split vaccine of item 6 or 7, which has an HA stem region exposed to the outside.

[0030] [Item 9]

[0031] The influenza HA split vaccine of any one of items 6-8, wherein the HA stem region of the influenza HA split vaccine antigen exposed to the outside enhances the antigenicity of LAH in the HA stem region, and the influenza HA split vaccine is capable of producing antibodies that bind to LAH in the HA stem region.

[0032] [Item 10]

[0033] An influenza HA split vaccine that is effective against influenza viruses that cause antigenic variation and produce antibodies that bind to LAH in the HA stem region, the vaccine being produced by subjecting the influenza HA split vaccine to acidic treatment.

[0034] [Item 11]

[0035] An influenza HA split vaccine, which is produced by subjecting an influenza HA split vaccine to acidic treatment, produces antibodies that bind to LAH in the HA stem region and is also effective against influenza viruses that cause antigenic variation.

[0036] Advantages of the invention

[0037] According to the present invention, an influenza HA split vaccine that produces antibodies that bind to the HA stem region of influenza, which is unlikely to cause antigenic variation, is obtained by a simple technique. Therefore, an influenza HA split vaccine that is also effective against influenza viruses that cause antigenic variation is obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] [ Figure 1 ] Figure 1 A schematic diagram illustrating the influenza virus.

[0040] [ Figure 2 ] Figure 2 The graph shows the increase in the titer of anti-LAH antibodies in the sera of mice vaccinated with the membrane fusogenic H3N2 HA split vaccine.

[0041] [ Figure 3 ] Figure 3 The graph shows the improvement of cross-protection ability of mice vaccinated with membrane fusogenic H3N2 HA split vaccine against antigenic variants.

[0042] [ Figure 4 ] Figure 4 The graph shows the increase in the titer of anti-LAH antibodies in the sera of mice vaccinated with the membrane fusogenic H1N1 HA split vaccine.

[0043] [ Figure 5 ] Figure 5 The graph shows the improvement of the cross-protection ability of mice vaccinated with membrane fusogenic H1N1 HA split vaccine against antigen variants.

[0044] [ Figure 6 ] Figure 6 Shown are graphs each indicating that LAH-binding monoclonal antibodies bind more strongly to membrane fusion-type HA split vaccines than to existing HA split vaccines.

[0045] Description of the implementation plan

[0046] Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. However, the embodiments are intended to promote an understanding of the principles of the present invention, and the scope of the present invention is not limited to the following embodiments. Other embodiments in which the configuration of the following embodiments has been appropriately replaced by those skilled in the art are also included in the scope of the present invention.

[0047] The method for producing an influenza HA split vaccine of this embodiment includes the step of subjecting the influenza HA split vaccine to acidic treatment.

[0048] The influenza HA split vaccine is prepared by treating the whole virus vaccine with ether to remove lipid components that become pyrogen. The influenza HA split vaccine has HA protein as the main component because the influenza HA split vaccine is produced by collecting the HA protein required for immunity from the surface of virus particles through density gradient centrifugation.

[0049] A glycoprotein called the "spike protein" protrudes from the surface of the influenza virus ( Figure 1 ). Influenza A virus has two types of spike proteins, 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 debinds the infected cell from HA and is used to release the replicated virus from the cell.

[0050] 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 for the virus to bind to the target cell. The stem region contains the fusion peptide sequence required for membrane fusion between the viral membrane and the cell membrane of the target cell.

[0051] The acid treatment of the influenza HA split vaccine changes the structure of the HA protein to a structure called a membrane fusion type. In the membrane fusion type HA protein, the stem region is exposed to the outside of the viral membrane instead of the head region, and the conformation of the antigen stem has a large structural change. The present inventors found in vivo that when the membrane fusion type HA protein is used as a vaccine, antibodies that bind to the LAH in the stem region are induced, and the antibodies have a protective effect against virus strains that cause antigenic mutations. The present invention was completed based on this fact.

[0052] The acid treatment is not particularly limited and may be performed at a pH of, for example, 3.0-6.5, preferably 4.0-6.0, and more preferably 4.4-5.8. The acid used for the acid treatment is not particularly limited and may be, for example, phosphoric acid, citric acid, maleic acid, or any other suitable acid.

[0053] Based on the difference in antigenicity, the HA of influenza A virus is classified into 18 subtypes (H1-H18), and NA is classified into 9 subtypes (N1-N9). The influenza HA split vaccine of the present invention is applicable to all these subtypes. In addition, the method for producing the influenza HA split vaccine of the present invention can produce a vaccine that is effective not only for influenza A virus but also for influenza B virus having HA.

[0054] The influenza HA split vaccine obtained by the production method of the present invention produces antibodies that bind to LAH that is less likely to cause mutations. Therefore, the vaccine can have a cross-protective effect on influenza viruses called antigenic variants, as long as the viruses have the same HA subtype. In addition, the influenza HA split vaccine obtained by the production method of the present invention can have cross-reactivity between HA subtypes (e.g., H3 and H7) of similar amino acid sequences of LAH.

[0055] In a preferred embodiment, the influenza HA split vaccine obtained by the production method of the present invention binds to the LAH-binding monoclonal antibody more strongly than the existing HA split vaccine. For example, the binding strength of the influenza HA split vaccine to the LAH-binding monoclonal antibody is at least 1.05 times that of the existing HA split vaccine, preferably at least 1.1 times, more preferably at least 1.5 times, and even more preferably at least 2 times. In this context, "the binding strength of the influenza HA split vaccine is at least 1.05 times, at least 1.1 times, at least 1.5 times or at least 2 times that of the existing HA split vaccine" 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 the existing HA split vaccine. In a preferred embodiment, the binding ability of the influenza HA split vaccine of the present invention to the LAH-binding monoclonal antibody is higher than the binding ability of the existing HA split vaccine. Although the upper limit is not particularly limited, the binding capacity may be, for example, in the range of 1.05-200 times, 1.1-150 times, 1.5-100 times, or 2-50 times. Alternatively, the range of the binding capacity of the influenza HA split vaccine of the present invention to the LAH-binding monoclonal antibody can be compared with the range of the existing HA split vaccine by a combination of a lower limit selected from 1.05, 1.1, 1.5, 2, 3, 4, and 5 and an upper limit selected from 200, 150, 100, 50, 30, and 20. In order to measure the binding capacity of the influenza HA split vaccine to the LAH-binding monoclonal antibody, any method can be used without particular limitation, and a common method known to those skilled in the art can be used. For example, the binding capacity can be measured by the method described in the embodiments of the present application.

[0056] In the present application, "LAH-binding monoclonal antibody" means a monoclonal antibody that binds to LAH. To produce the monoclonal antibody, any method may be used without particular limitation, and a common method known to those skilled in the art may be employed. When measuring the binding ability of the influenza HA split vaccine to the LAH-binding monoclonal antibody, it is assumed that the LAH-binding monoclonal antibody is able to bind to a peptide corresponding to at least a portion of the influenza virus LAH from which the influenza HA split vaccine is derived.

[0057] In the present application, "existing HA split vaccine" means a vaccine from which lipid components that become pyrogens are removed by treating a whole virus vaccine with ether, and can be produced, for example, by the method described in Example 1 of the present application. The existing HA split vaccine may also be an influenza HA split vaccine produced without acid treatment, compared to the influenza HA split vaccine of the present invention prepared by the method comprising the following acid treatment.

[0058] The 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 water-in-oil emulsions. Among them, aluminum hydroxide is preferred, and the use of aluminum hydroxide as an adjuvant can enhance immunogenicity.

[0059] The influenza HA split vaccine obtained by the production method of the present invention can be used, for example, for additional vaccination after a predetermined time after the initial vaccination. The time after the initial vaccination and before the additional vaccination is not particularly limited, but may be, for example, 20 days to 3 years, preferably 3 months to 2 years, and more preferably 6 months to 1 year. The amount of the influenza HA split vaccine used for the initial and additional vaccinations is not particularly limited, but may be, for example, 1 μg-200 μg per dose, preferably 10 μg-30 μg, and more preferably 15 μg. A single dose is, for example, 0.5 mL. Any administration method can be used for the initial and additional vaccinations without particular limitation, and, for example, nasal, subcutaneous, intradermal, transdermal, intraocular, mucosal or oral administration can be adopted. Intramuscular administration is preferred.

[0060] The influenza HA split vaccine obtained by the production method of the present invention has a protective effect against virus strains that cause antigenic variation. For example, if an existing HA split vaccine is prepared from particles of H3N2 influenza virus (A / Fujian / 411 / 02 (H3N2)) and subjected to acid treatment, the vaccine can be effective not only against infection with A / Fujian / 411 / 02 (H3N2), but also against infection with, 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 / Shanghai / 16 / 89 ... (H3N2), A / Shanghai / 24 / 90 (H3N2), A / Sichuan / 2 / 87 (H3N2), A / Kitakyushyu / 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). Similarly, for example, if an existing HA split vaccine is prepared from particles of an H1N1 influenza virus (A / Puerto Rico / 8 / 34 (H1N1)) and subjected to acid treatment, the vaccine can also be effective not only against infection with A / Puerto Rico / 8 / 34 (H1N1), but also against infection with, 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), A / Brisbane / 59 / 2007 (H1N1), etc. (H1N1) or A / Mexico / 4108 / 2009 (H1N1) infection.

[0061] [Example]

[0062] 1. Preparation of HA Split Vaccine

[0063] Tween 80 was added to particles of H3N2 influenza virus (strain X31) or particles of H1N1 influenza virus (A / Puerto Rico / 8 / 34 strain) suspended in phosphate buffered saline to a final concentration of 0.2% and suspended therein. Ether was added and suspended, and the suspension was allowed to stand until the water layer and the ether layer were completely separated, and then the ether layer was removed. After repeating the ether extraction, the ether remaining in the recovered water layer was distilled off at normal pressure to obtain the HA split vaccine.

[0064] 2. Acid treatment

[0065] The HA split vaccine was suspended in phosphate buffered saline and then acidified by adding 0.15 M citrate buffer (pH 3.5) to a pH of 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. Afterwards, centrifugation was performed to obtain a membrane fusion type HA split vaccine. Formalin was added to the thus prepared membrane fusion type HA split vaccine to a final concentration of 0.05%, and allowed to stand for several days.

[0066] The existing HA split vaccine was prepared in the same manner as described in 1 above, except that the acid treatment was not performed.

[0067] 3. Measurement of anti-LAH antibody titers by ELISA

[0068] 3-1. Vaccination of H3N2 influenza

[0069] BALB / c mice (female, 6-12 weeks old) were intraperitoneally vaccinated with the existing H3N2 HA split vaccine or the membrane-fused HA split vaccine (10 μg vaccine + 20 μg AddaVax adjuvant (InvivoGen) dissolved in phosphate-buffered saline to a liquid volume of 200 μl). 28 days after the initial vaccination, the mice were intraperitoneally vaccinated with the membrane-fused HA vaccine (only 10 μg vaccine dissolved in phosphate-buffered saline to a liquid volume of 200 μl). At least 14 days after the additional vaccination, blood was collected from the vaccinated mice, and serum was collected therefrom.

[0070] 3-2. Measurement by ELISA

[0071] The concentration of anti-LAH antibodies in the sera of BALB / c mice intraperitoneally vaccinated with the existing H3N2 HA split vaccine or the membrane fusion type HA split vaccine was measured by ELISA (enzyme-linked immunosorbent assay) in the following manner.

[0072] Specifically, a synthetic peptide corresponding to a portion of the stem portion (long α helix) (H3; Ac-RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENADYKDDDDKC) (SEQ ID NO: 1) was dissolved in phosphate buffered saline (pH 7.3) at 10 μg / ml and added to a 96-well plate at 100 μl each. After standing overnight at 4°C, each well was washed 3 times with phosphate buffered saline, and 150 μl of phosphate buffered saline containing 1% bovine serum albumin was added. After standing at room temperature for 2 hours, each well was washed 3 times with phosphate buffered saline. Then, 100 μl of mouse serum serially diluted with phosphate buffer containing 0.05% Tween 20 and 1% bovine serum albumin and 100 μl of a standard monoclonal antibody (H3; clone name V15-5) of known concentration were added to each well. After standing at room temperature for 2 hours, each well was washed 3 times with phosphate buffered saline (containing 0.05% Tween 20), and 100 μl of peroxidase-labeled anti-mouse IgG antibody (SouthernBiotech) diluted with phosphate buffered saline containing 0.05% Tween 20 and 1% bovine serum albumin was added to each well. After standing at room temperature for 2 hours, each well was washed 3 times with phosphate buffered saline (containing 0.05% Tween 20). Then, 30 mg of 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 substrates, and 100 μl of the resultant was added to each well. After color development, 50 μl of 2N sulfuric acid (Wako Pure Chemical Industries, Ltd.) was added to terminate the reaction, and the absorbance value at 490 nm was measured using Microplate Reader 450 (Biorad).

[0073] like Figure 2 As shown, the titer of anti-LAH antibodies in the serum of BALB / c mice intraperitoneally vaccinated with membrane fusion HA split vaccine was significantly higher than the titer of anti-LAH antibodies in the serum of BALB / c mice intraperitoneally vaccinated with existing HA split vaccine.

[0074] 4. Cross-protection against antigenic variants

[0075] In experiments on protection against H3N2 virus infection, 200 μl of serum collected from unvaccinated mice, 200 μl of serum collected from mice vaccinated with an existing H3N2 HA split vaccine, or 200 μl of serum collected from mice vaccinated with a membrane fusogenic HA split vaccine were administered intraperitoneally to BALB / c mice (female, 6-12 weeks old).

[0076] Three hours after the serum administration, another H3N2 influenza virus (A / Guizhou / 54 / 89) having different antigenicity from the vaccine strain was intranasally administered at 5 times the median lethal dose for mice (5 times the amount of virus that kills 50% of mice) under anesthesia.

[0077] The mice were weighed daily and observed for 21 days from the start of viral infection to study changes in body weight and survival rate. Mice that lost 25% of their body weight were sacrificed.

[0078] like Figure 3 As shown, for BALB / c mice vaccinated with membrane fusion HA split vaccine, the decline in survival rate was significantly inhibited on and after day 9 after infection with other H3N2 influenza viruses with different antigenicity.

[0079] 5. Measurement of anti-LAH antibody titers by ELISA

[0080] 5-1. H1N1 influenza virus particles

[0081] C57BL / 6 mice (female, 6-12 weeks old) were intraperitoneally vaccinated with the existing H1N1 HA split vaccine or the membrane-fused HA split vaccine (10 μg vaccine + 10 μg CpG-ODN 1760, which was suspended in phosphate-buffered saline and mixed with an equal volume of Freund's incomplete adjuvant (ROCKLAND) to a liquid volume of 200 μl). 28 days after the primary vaccination, the mice were intraperitoneally vaccinated with the membrane-fused HA split vaccine (10 μg vaccine + 10 μg CpG-ODN, which was suspended in phosphate-buffered saline in the same manner as the primary vaccination and mixed with an equal volume of Freund's incomplete adjuvant (ROCKLAND) to a liquid volume of 200 μl). At least 14 days after the additional vaccination, blood was collected from the vaccinated mice, and serum was collected therefrom.

[0082] 5-2. Measurement by ELISA

[0083] The concentration of anti-LAH antibodies in the sera of C57BL / 6 mice intraperitoneally vaccinated with the existing H1N1 HA split vaccine or the membrane fusion type HA split vaccine was measured by ELISA in the following manner.

[0084] The measurement was performed in the same manner as above, except that a synthetic peptide corresponding to a portion of the stem portion (long α helix) (H1; Ac-RIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNADYKDDDDKC) (SEQ ID NO: 2) and a standard monoclonal antibody (H1; clone name F2) of known concentration were used.

[0085] like Figure 4 As shown, the titer of anti-LAH antibodies in the serum of C57BL / 6 mice intraperitoneally vaccinated with the membrane fusion HA split vaccine was significantly higher than the titer of anti-LAH antibodies in the serum of C57BL / 6 mice intraperitoneally vaccinated with the existing HA split vaccine.

[0086] 6. Cross-protection against antigenic variants

[0087] In experiments on protection against H1N1 virus infection, 200 μl of serum collected from unvaccinated mice, 200 μl of serum collected from mice vaccinated with an existing H1N1 HA split vaccine, or 200 μl of serum collected from mice vaccinated with a membrane fusion HA split vaccine was intraperitoneally administered to C57BL / 6 mice (female, 6-12 weeks old).

[0088] Three hours after the serum administration, another H1N1 influenza virus (A / Narita / 1 / 09) having different antigenicity from the vaccine strain was intranasally administered at 5 times the median lethal dose for mice (5 times the amount of virus that kills 50% of mice) under anesthesia.

[0089] The mice were observed daily for 20 days from the start of viral infection to study the survival rate. Figure 5 As shown, for C57BL / 6 mice vaccinated with membrane fusion HA split vaccine, the decline in survival rate was significantly inhibited on day 9 and thereafter after infection with other H1N1 influenza viruses with different antigenicity.

[0090] 7. Antibody binding ability to LAH epitope

[0091] The binding of anti-LAH monoclonal antibodies prepared from peripheral blood of mice or humans infected with strain X31 to existing HA split vaccines or membrane fusion HA split vaccines was measured by ELISA (enzyme-linked immunosorbent assay). The existing HA split vaccine or membrane fusion HA split vaccine of H3N2 influenza virus (strain X31) was dissolved in phosphate buffered saline (pH 7.3) and added to a 96-well plate at 50 μl each. After standing overnight at 4°C, each well was washed 3 times with phosphate buffered saline and 150 μl of phosphate buffered saline containing 1% bovine serum albumin was added. After standing for 2 hours at room temperature, each well was washed 3 times with phosphate buffered saline (containing 0.05% Tween 20) and 50 μl of LAH binding monoclonal antibodies serially diluted with phosphate buffer containing 1% bovine serum albumin were added. After standing overnight at 4°C, each well was washed three times with phosphate buffered saline (containing 0.05% Tween 20), and 100 μl of peroxidase-labeled anti-mouse IgG antibody (Southern Biotech) diluted with phosphate buffered saline containing 0.05% Tween 20 and 1% 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% Tween 20). Then, 30 mg of 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 substrates, and 50 μl of the resultant was added to each well. After color development, 25 μl of 1 mol / L sulfuric acid (Wako Pure Chemical Industries, Ltd.) was added to terminate the reaction, and the absorbance value at 490 nm was measured using Microplate Reader450 (Biorad). The change in binding capacity was calculated from the absorbance values ​​measured for the existing HA split vaccine or the membrane fusion type HA split vaccine.

[0092] like Figure 6 As shown, the binding ability of LAH-binding monoclonal antibodies to membrane-fusion HA split vaccines is 1.05-21 times that of existing HA split vaccines. The results show that acidic treatment of HA split vaccines enhances the binding ability of antibodies to LAH epitopes.

[0093] Industrial Applicability

[0094] The present invention can be used to produce influenza vaccines.

[0095] [Sequence Listing Free Text]

[0096] SEQ ID NO: 1, 2: Synthetic peptide

[0097] [Sequence Listing]

Claims

1. A method for producing an influenza membrane fusion type HA split vaccine, wherein the vaccine induces the production of antibodies that bind to LAH in the HA stem region in vivo, the method comprising: The influenza HA split vaccine was prepared by treating the whole virus vaccine with ether to remove the lipid components; and The influenza HA split vaccine is subjected to acid treatment to produce the influenza membrane fusion type HA split vaccine, wherein the acid treatment is performed at a pH of 4.0-6.0 and changes the structure of the HA protein to a structure called a membrane fusion type, The influenza HA split vaccine is of H3N2 type or H1N1 type.

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

8.

3. An influenza membrane fusion type HA split vaccine, which induces the production of antibodies in vivo that bind to LAH in the HA stem region, wherein the influenza membrane fusion type HA split vaccine is produced by the method according to any one of claims 1-2.

4. Use of the influenza membrane fusion type HA split vaccine according to claim 3 in the preparation of a vaccine for protecting against H3N2 or H1N1 influenza infection.

5. Use of the influenza membrane fusion type HA split vaccine according to claim 3 in the preparation of a vaccine for protecting against H3N2 influenza infection, wherein the influenza membrane fusion type HA split vaccine has an H3N2 type.

6. Use of the influenza membrane fusion type HA split vaccine according to claim 3 in the preparation of a vaccine for protecting against H1N1 influenza infection, wherein the influenza membrane fusion type HA split vaccine has an H1N1 type.

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