TWI857017B - Method for producing influenza HA split vaccine - Google Patents

Abstract

The present invention provides a method for manufacturing an influenza HA split vaccine, which produces antibodies that bind to the influenza HA stem region that is not prone to antigenic variation. The influenza HA split vaccine is subjected to acid treatment. The influenza HA split vaccine obtained by the acid treatment produces antibodies that bind to LAH in the HA stem region. The influenza HA split vaccine has excellent protection against other influenza virus infections with different antigenicity.

Description

Method for producing influenza HA split vaccine

The present invention relates to a method for producing an influenza HA split vaccine.

The current influenza hemagglutinin (hereinafter referred to as "HA") vaccine works by inducing HA antibodies to prevent infection. Although HA antibodies bind to the part of the virus envelope that is exposed to the outside, called the head region, this part is the region with the most structural variations between virus strains. As a result, there are situations in which HA antibodies cannot bind to antigenically variant viruses that are different from the vaccine strain, and the vaccine is ineffective.

In recent years, it has become clear that antibodies that bind to the stem region that is less susceptible to antigenic variation include antibodies that protect against infection (Patent Document 1). In order to efficiently induce stem antibodies, an HA stem protein has been developed by artificially mutating or binding a linker. This HA stem protein successfully stabilizes the unstable stem portion and has been put into human clinical trials.

However, there are problems in manufacturing for practical use, and we look forward to developing HA vaccine antigens that can more easily induce stem antibodies.

【Previous technical literature】

【Patent Literature】

Patent document 1: Japanese Patent Publication No. 2016-516090.

The present invention was developed in view of this problem, and its purpose is to provide a method for producing an influenza HA split vaccine, which produces antibodies that bind to the stem region of influenza HA that is less likely to cause antigenic variation.

The manufacturing method of the HA split vaccine of the present invention is to subject the influenza HA split vaccine to acid treatment, thereby generating antibodies that bind to LAH (long alpha helix) in the HA stem region. The influenza HA split vaccine is also effective against antigenically variant influenza viruses.

That is, the present invention is related to the following.

[Item 1]

The method for manufacturing influenza HA split vaccine is to subject the influenza HA split vaccine to acid treatment to generate antibodies that bind to LAH in the HA stem region.

[Item 1a]

A method for producing an influenza HA split vaccine comprises subjecting an influenza HA split vaccine that has not been treated with formalin to acid treatment, thereby generating antibodies that bind to LAH in the HA stem region.

[Item 1b]

A method for producing an influenza HA split vaccine comprises the steps of subjecting the influenza HA split vaccine to an acid treatment and then subjecting the vaccine to a formalin treatment to produce antibodies that bind to LAH in the HA stem region.

[Item 1c]

A method for producing an influenza HA split vaccine comprises the steps of subjecting an influenza HA split vaccine that has not been treated with formalin to acid treatment and then subjecting the vaccine to formalin treatment to generate antibodies that bind to LAH in the HA stem region.

[Item 2]

A manufacturing method as described in any one of Items 1, 1a to 1c, wherein the influenza HA split vaccine is also effective against antigenically variant influenza viruses.

[Item 3]

A method for producing an influenza HA split vaccine, wherein the influenza HA split vaccine targets antigenically mutated influenza viruses and is subjected to acid treatment to generate antibodies that bind to LAH in the HA stem region.

[Item 4]

A method for producing an influenza HA split vaccine as described in any one of items 1 to 3, 1a to 1c, wherein the aforementioned acid treatment is carried out at pH 4.4 to 5.8.

[Item 5]

A method for producing an influenza HA split vaccine as described in any one of items 1 to 4, 1a to 1c, wherein the influenza HA split vaccine is of the H3N2 type or the H1N1 type.

[Item 5a]

A method for producing an influenza HA split vaccine as described in any one of items 1 to 5, 1a to 1c, wherein the aforementioned influenza HA split vaccine is a single HA subtype influenza HA split vaccine.

[Item 5b]

The method for producing an influenza HA split vaccine as described in any one of items 1 to 5, 1a to 1c, wherein the method comprises the step of mixing two or more influenza HA split vaccine antigens, wherein the influenza HA split vaccine antigens are produced by subjecting a single subtype of influenza HA split vaccine to acid treatment.

[Item 6]

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

[Item 7]

The influenza HA split vaccine as described in item 6, wherein the influenza HA split vaccine is also effective against antigenically variant influenza viruses.

[Item 8]

An influenza HA split vaccine as described in item 6 or 7, wherein the influenza HA split vaccine is in a shape in which the HA stem region is exposed to the outside.

[Item 9]

The influenza HA split vaccine described in any one of items 6 to 8 is in a shape in which the HA stem region of the influenza HA split vaccine antigen is exposed to the outside, thereby increasing the antigenicity of the LAH in the HA stem region and generating antibodies that bind to the LAH in the HA stem region.

[Item 10]

An influenza HA split vaccine is produced by subjecting the influenza HA split vaccine to acid treatment to produce antibodies that bind to LAH in the HA stem region.

[Item 10a]

An influenza HA split vaccine is produced by subjecting an influenza HA split vaccine that has not been treated with formalin to acid treatment to produce antibodies that bind to LAH in the HA stem region.

[Item 10b]

An influenza HA split vaccine is produced by a manufacturing process comprising a step of subjecting the influenza HA split vaccine to acid treatment and a subsequent step of subjecting the vaccine to formalin treatment, thereby generating antibodies that bind to LAH in the HA stem region.

[Item 10c]

An influenza HA split vaccine is produced by a manufacturing process comprising a step of subjecting an influenza HA split vaccine that has not been treated with formalin to acid treatment and a subsequent step of subjecting the vaccine to formalin treatment to produce antibodies that bind to LAH in the HA stem region.

[Item 10d]

An influenza HA split vaccine is produced by subjecting a split influenza HA vaccine containing a single subtype to acid treatment to generate antibodies that bind to LAH in the HA stem region.

[Item 10e]

An influenza HA split vaccine is a vaccine antigen that mixes two or more influenza HA split vaccine antigens to produce antibodies that bind to LAH in the HA stem region, and the influenza HA split vaccine antigens are produced by acidifying influenza HA split vaccines containing individual subtypes.

[Item 10f]

An influenza HA split vaccine as described in any one of items 10 to 10e, wherein the influenza HA split vaccine is also effective against antigenically variant influenza viruses.

[Item 11]

An influenza HA split vaccine is produced by subjecting the influenza HA split vaccine to acid treatment to produce antibodies that bind to LAH in the HA stem region and are also effective against antigenically variant influenza viruses.

According to the present invention, an influenza HA split vaccine can be obtained by a simple method, which is to produce antibodies that bind to the HA stem region of influenza that is not easy to cause antigenic variation. Therefore, an influenza HA split vaccine that is also effective against antigenically mutated influenza viruses can be obtained.

The schematic diagram in Figure 1 illustrates the influenza virus.

Figure 2 shows the increase in LAH antibody titer in the serum of mice vaccinated with H3N2 membrane fusion HA split vaccine.

Figure 3 shows the improvement in cross-defense ability of mice vaccinated with H3N2 membrane fusion HA split vaccine against antigen variant strains.

Figure 4 shows the increase in LAH antibody titer in the serum of mice vaccinated with H1N1 membrane fusion HA split vaccine.

Figure 5 shows the improvement in cross-defense ability of mice vaccinated with H1N1 membrane fusion HA split vaccine against antigen variant strains.

Figure 6 shows that compared to the current HA split vaccine, the LAH-binding monoclonal antibody binds more strongly to the membrane fusion HA split vaccine.

Figure 7 shows that LAH-binding monoclonal antibodies strongly bind to membrane-fused HA split vaccines that were treated with formalin after acid treatment.

Figure 8 shows the increase in LAH antibody titer in the serum of mice vaccinated with H3N2 membrane fusion HA split vaccine ((pre-fix) and (post-fix)).

The following specifically describes the implementation form of the present invention with reference to the attached drawings, but the implementation form is used to facilitate the understanding of the principle of the present invention. The scope of the present invention is not limited to the following implementation form. The scope of the present invention also includes other implementation forms formed by those with common knowledge in the relevant technical field who appropriately replace the following implementation form.

The method for producing the influenza HA split vaccine of this embodiment includes a step of subjecting the influenza HA split vaccine to acid treatment.

The influenza HA split vaccine is obtained by treating the whole particle vaccine with ether and removing the lipid components that become pyrogenic substances from the whole particle vaccine treated with ether. In addition, it is produced by recovering the HA protein on the surface of the virus particles required for immunization by density gradient centrifugation, so the HA protein is the main component.

There is a glycoprotein called spike protein protruding from the surface of influenza virus (Figure 1). Influenza A virus has two types of spike proteins, HA and NA (neuraminidase), which have the function of causing viral infection. HA binds to the cells to be infected and has the function of allowing the virus to enter the cells. HA will frequently cause antigenic variation. The function of NA is as follows: cut off the binding of infected cells and HA, and release the replicating virus from the cells.

The HA of influenza A virus is divided into two regions: the globular region and the stem region (Figure 1). The globular region contains the receptor binding site, which is used to bind the virus to the target cell. The stem region includes the fusion peptide sequence, which is required for the membrane fusion of the viral envelope and the cell membrane of the target cell.

The influenza HA split vaccine is subjected to acid treatment, thereby changing the HA protein into a structure called membrane fusion type. Accompanied by the significant change in the stereostructure of the antigen stem, the shape of the membrane fusion type HA protein changes to the stem region replacing the globular region and is exposed to the outside from the viral envelope. When the inventors of this case used the membrane fusion type HA as a vaccine, they induced antibodies to LAH that bind to the stem region. In vivo, the antibodies have a protective effect against antigen-mutated virus strains. This is a new discovery, and the present invention was completed based on this new discovery.

The acid treatment is not particularly limited, for example, the pH is 2.0~6.5, preferably 3.0~6.5, more preferably 4.0~6.0, and even more preferably 4.4~5.8. Specifically, for example, pH 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 or 6.0~6.5. The acid used for the acid treatment is not particularly limited, for example, phosphoric acid, citric acid, maleic acid, hydrochloric acid, etc. can be used.

The temperature during the 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. Specifically, for example, the temperature may be 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 or 45°C to 75°C. The treatment time is, for example, 5 minutes to 120 minutes, preferably 15 minutes to 60 minutes, and more preferably 20 minutes to 45 minutes. Specifically, for example, 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, or 60 minutes to 120 minutes.

According to antigenic differences, influenza A virus has 18 HA subtypes (H1~H18) and 9 NA subtypes (N1~N9), but the influenza HA split vaccine of the present invention can target all of these subtypes. Moreover, the method for producing the influenza HA split vaccine of the present invention can not only produce type A vaccines, but also type B vaccines with HA.

The influenza HA split vaccine obtained by the manufacturing method of the present invention will produce antibodies that bind to LAH with less variation, so if within the same HA subtype, cross-defense can be achieved against influenza viruses with known antigenic variants. Moreover, cross-reactivity may also be shown between HA subtypes with similar LAH amino acid sequences (such as H3 and H7).

In this case, "single HA subtype, i.e. influenza HA split vaccine" refers to an influenza HA split vaccine containing one HA subtype selected from the 18 subtypes (H1~H18) of influenza A virus or influenza B virus. If it is a single HA subtype, the NA subtype can be the same or different. Preferred HA subtypes include H1, H3 and B.

A mixed vaccine containing two or more HA subtypes can be produced by subjecting influenza HA split vaccines composed of individual HA subtypes to acid treatment and mixing multiple (two or more) of the resulting influenza HA split vaccines. In addition, a mixed vaccine containing two or more HA subtypes can also be produced by subjecting influenza HA split vaccines that have been mixed with two or more HA subtypes to acid treatment. When vaccinating as a vaccine containing multiple subtypes, it is preferred to contain 1 to 3 subtypes selected from the group consisting of H1, H3, and B.

Preferably, the binding of the influenza HA split vaccine obtained by the manufacturing method of the present invention to the LAH-binding monoclonal antibody is stronger than that of the existing HA split vaccine. For example, the binding to the LAH-binding monoclonal antibody is 1.05 times stronger than that of the existing HA split vaccine, preferably 1.1 times stronger, more preferably 1.5 times stronger, and even more preferably 2 times stronger. Here, 1.05 times, 1.1 times, 1.5 times or 2 times stronger than the existing HA split vaccine, for example, means that when the absorbance obtained by regression shows 0.7, the reciprocal of the antibody concentration is 1.05 times, 1.1 times, 1.5 times or 2 times the reciprocal of the antibody concentration of the existing HA split vaccine. Compared with the existing HA split vaccine, the influenza HA split vaccine of the present invention has a higher binding affinity with the LAH binding monoclonal antibody, which is better, and the upper limit is not particularly limited, for example, it is in the range of 1.05-200 times, 1.1-150 times, 1.5-100 times, and 2-50 times. Alternatively, compared with the existing HA split vaccine, the influenza HA split vaccine of the present invention and the LAH binding monoclonal antibody can be 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. The method for measuring the binding of influenza HA split vaccine and LAH binding monoclonal antibody is not particularly limited and can be performed by general methods known to those with ordinary knowledge in the relevant technical field, for example, it can be measured according to the method of the embodiment of this case.

In this case, LAH-binding monoclonal antibodies refer to monoclonal antibodies that bind to LAH. The production method is not particularly limited and can be produced by general methods known to those with ordinary knowledge in the relevant technical field. When measuring the binding of influenza HA split vaccine to LAH-binding monoclonal antibodies, LAH-binding monoclonal antibodies refer to those that can bind to a peptide that is equivalent to at least a portion of LAH of the influenza virus, which is the source of the influenza HA split vaccine.

In this case, the current HA split vaccine refers to a vaccine obtained by treating a whole particle vaccine with ether and removing the lipid component that becomes a pyrogenic substance from the whole particle vaccine that has been treated with ether, and can be produced by the method of Example 1 of this case, for example. Moreover, compared to the influenza HA split vaccine of the present invention produced by a method having the following acid treatment step, the current HA split vaccine is an influenza HA split vaccine produced without acid treatment.

When manufacturing the influenza HA split vaccine of the present invention, formalin treatment can also be performed. The time point when the influenza HA split vaccine is subjected to acid treatment is preferably before the formalin treatment. When manufacturing the influenza HA split vaccine antigen of the present invention (capable of producing antibodies that bind to LAH in the HA stem region), the HA portion used in the current influenza HA split vaccine is subjected to acid treatment and then subjected to formalin treatment. This can obtain the influenza HA split vaccine antigen, which has a stronger effect of producing cross-reactive antibodies and is therefore better as a global influenza vaccine antigen. That is, in this case, it is better to treat the HA portion of the virus particles with ether or the like and remove the liposol before subjecting it to formalin treatment.

In this case, the influenza HA split vaccine before acid treatment is preferably a split vaccine that has not been treated with formalin.

The commercially available influenza HA vaccine (trade name) is described in the Biological Preparation Standard (March 30, 2004, Japan Ministry of Health, Labor and Welfare Notice No. 155, final revision: November 30, 2018, Japan Ministry of Health, Labor and Welfare Notice No. 409), which is a step of treating the virus with ether, etc., removing the fat solvent, and then treating it with formaldehyde or a substance with an equivalent effect. Although the commercially available influenza HA vaccine (trade name) is an influenza HA split vaccine, it has been treated with formaldehyde, etc., so it is better not to use the commercially available influenza HA vaccine to manufacture the influenza HA split vaccine of the present invention.

When the influenza HA split vaccine that has been subjected to acid treatment is subjected to formalin treatment, the formalin concentration in the formalin treatment solution is, for example, 0.0005v/v%~10v/v%, preferably 0.001v/v%~1v/v%, more preferably 0.003v/v%~0.5v/v, and even more preferably 0.005v/v%~0.1v/v%. The time for formalin treatment is, for example, 1 hour to 10 days. Preferably, it is 2 hours to 5 days, and more preferably, it is 12 hours to 3 days. The formalin treatment temperature is, for example, 0℃~75℃. Preferably, it is 1℃~37℃, and more preferably, it is 1℃~30℃.

The grade of formalin used should preferably be medically acceptable.

The method for producing the influenza HA split vaccine of the present invention may include a step of including an adjuvant. The adjuvant is not particularly limited, and for example, aluminum hydroxide, aluminum phosphate and other aluminum salts, chitosan, oligodeoxynucleotides, water-in-oil emulsions, etc. may be used. Aluminum hydroxide is preferred, and the use of aluminum hydroxide as an adjuvant can improve immunogenicity.

The influenza HA split vaccine obtained by the manufacturing method of the present invention can be used, for example, in the form of a booster vaccination after a specific period after the initial vaccination. The period from the initial vaccination to the booster vaccination is not particularly limited, for example, 20 days to 3 years, preferably 3 months to 2 years, and more preferably 6 months to 1 year. The amount of influenza HA split vaccine for the initial vaccination and the booster vaccination is not particularly limited, and the single dose is, for example, 1μg to 200μg, preferably 10μg to 30μg, and more preferably 15μg. The single dose is, for example, 0.5mL. When performing the initial vaccination and the booster vaccination, the method of administration is not particularly limited, for example, nasal, subcutaneous, intradermal, transdermal, intraocular, mucosal, or oral administration, preferably intramuscular administration.

The influenza HA split vaccine obtained by the production method of the present invention has a protective effect against antigenically variant virus strains. For example, when the current HA split vaccine is prepared from H3N2 influenza virus particles (A/Fujian/411/02(H3N2)) and subjected to acid treatment, it has a protective effect not only against A/Fujian/411/02(H3N2), but also against 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/Shangdong/9/93(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) also have the effect of preventing infection. Moreover, when the current HA split vaccine is prepared from H1N1 influenza virus particles (A/Puerto Rico/8/34(H1N1)) and subjected to acid treatment, it has the effect of preventing infection not only against A/Puerto Rico/8/34(H1N1), but also against 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) or A/Mexico/4108/2009(H1N1) etc. also have the effect of preventing infection.

[Implementation Example]

1. Prepare HA split vaccine

Tween80 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 suspended. Diethyl ether was added for further suspension, and the diethyl ether layer was removed after standing until the water layer and the diethyl ether layer were completely separated. After repeating the ether extraction, the diethyl ether remaining in the recovered water layer was removed by atmospheric distillation, and the HA split vaccine was made.

2. Acid treatment

After suspending the HA split vaccine in phosphate-buffered saline, 0.15M citric acid buffer (pH3.5) was added as an acidic treatment to adjust the pH to 5.0. After standing at room temperature for 30 minutes, 1M Tris buffer (pH8.0) was added to return the pH to 7.3. Centrifugation was then performed and the membrane fusion type HA split vaccine was prepared. Formalin was added to the prepared membrane fusion type HA split vaccine in a manner such that the final concentration was 0.05v/v% and the vaccine was allowed to stand for several days.

It should be noted that the current HA split vaccine is treated in the same manner as above, except that the HA split vaccine prepared in 1. is not subjected to acid treatment.

3. Measure LAH antibody titer by ELISA method

3-1. Get the H3N2 influenza vaccine

BALB/c mice (female, 6-12 weeks old) were intraperitoneally vaccinated with H3N2 type current HA split vaccine or membrane fusion HA split vaccine (10μg vaccine + 10v/v% AddaVax adjuvant (InvivoGen) dissolved in phosphate buffered saline to a volume of 200μl). 28 days after the first vaccination, the membrane fusion HA vaccine (10μg vaccine only dissolved in phosphate buffered saline to a volume of 200μl) was intraperitoneally vaccinated. 14 days after the booster vaccination, blood was collected from the vaccinated mice and serum was recovered.

3-2. Measurement by ELISA method

As described below, the LAH antibody concentration in the serum of BALB/c mice intraperitoneally vaccinated with the H3N2 type current HA split vaccine or membrane fusion HA split vaccine was measured by ELISA (Enzyme-Linked Immuno Sorbent Assay).

That is, a synthetic peptide (H3; Ac-RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENADYKDDDDKC) (sequence number 1) corresponding to a portion of the stem (long alpha helix) was dissolved in phosphate-buffered saline (pH 7.3) at 10 μg/ml, 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 at room temperature for 2 hours, each well was washed three times with phosphate-buffered saline, and mouse serum was obtained by stepwise dilution with phosphate-buffered solution containing 0.05 v/v% Tween20 and 1 v/v% bovine serum albumin. 100 μl of the mouse serum and 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 three times with phosphate-buffered saline (containing 0.05 v/v% Tween 20), and peroxidase-labeled anti-mouse IgG antibody (Southern Biotech) was diluted with phosphate-buffered saline containing 0.05 v/v% Tween 20 and 1 v/v% bovine serum albumin, and 100 μl of peroxidase-labeled anti-mouse IgG antibody 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 v/v% Tween20), and 60 ml of citric acid buffer (pH 5.0) as the base was prepared by adding 30 mg of o-phenylendiamine tablet (SIGMA) and 24 μl of 30% hydrogen peroxide (30% w/w; SIGMA), and 100 μl was added to each well. After color development, 50 μl of 1 mol/L sulfuric acid (Wako Pure Chemical Industries) was used to stop the reaction, and the absorbance at 490 nm was measured using Microplate Reader 450 (Biorad).

As shown in Figure 2, compared with the LAH antibody titer in the serum of BALB/c mice intraperitoneally vaccinated with the current HA split vaccine, the LAH antibody titer in the serum of BALB/c mice intraperitoneally vaccinated with the membrane fusion HA split vaccine was significantly increased.

4. Cross-defense against antigenic variants

In the H3N2 virus infection protection experiment, 200 μl of serum collected from unvaccinated mice or mice vaccinated with H3N2 current HA split vaccine or membrane fusion HA split vaccine was intraperitoneally injected into BALB/c mice (female, 6-12 weeks old).

Three hours after the serum administration, five mice were nasally injected with another H3N2 influenza virus (A/Guizhou/54/89) with different antigenicity from the vaccine strain at a lethal dose of 50 (5 times the amount of virus that causes lethal infection in 50% of mice) under anesthesia, thereby infecting the mice with the virus.

From the 21st day after virus infection, the mice were weighed and observed every day to analyze weight changes and survival rates. When a 25% weight loss was observed in the mice, the mice were euthanized.

As shown in Figure 3, BALB/c mice vaccinated with the membrane fusion HA split vaccine were able to significantly suppress the decrease in survival rate from day 9 after infection with other antigenically different H3N2 influenza viruses.

5. Measure LAH antibody titer by ELISA method

5-1.H1N1 influenza virus particles

C57BL/6 mice (female, 6-12 weeks old) were intraperitoneally inoculated with the H1N1 type current HA split vaccine or membrane fusion HA split vaccine (10μg vaccine + 10μg CpG-ODN1760 suspended in phosphate-buffered saline and mixed with an equal amount of Freund’s incomplete adjuvant (ROCKLAND) to a liquid volume of 200μl). 28 days after the first vaccination, the membrane fusion HA split vaccine was intraperitoneally inoculated (similar to the first vaccination, 10μg vaccine + 10μg CpG-ODN suspended in phosphate-buffered saline and mixed with an equal amount of Freund’s incomplete adjuvant (ROCKLAND) to a liquid volume of 200μl). 14 days after the booster vaccination, blood was collected from the vaccinated mice and serum was recovered.

5-2. Measurement by ELISA method

As described below, the LAH antibody concentration in the serum of C57BL/6 mice vaccinated intraperitoneally with the H1N1 type current HA split vaccine or membrane fusion HA split vaccine was measured by ELISA.

The same method as above was used except that a synthetic peptide (H1; Ac-RIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNADYKDDDDKC) (sequence number 2) corresponding to a portion of the stem (long alpha helix) and a standard monoclonal antibody (H1; clone name F2) with known concentration were used.

As shown in Figure 4, the LAH antibody titer in the serum of C57BL/6 mice intraperitoneally inoculated with the current HA split vaccine was significantly increased compared to the LAH antibody titer in the serum of C57BL/6 mice intraperitoneally inoculated with the membrane fusion HA split vaccine.

6. Cross-defense against antigenic variants

In the H1N1 virus infection protection experiment, 200 μl of serum collected from unvaccinated mice or mice vaccinated with H1N1 current HA split vaccine or membrane fusion HA split vaccine was intraperitoneally injected into C57BL/6 mice (female, 6-12 weeks old).

Three hours after the serum administration, five mice were nasally injected with another H1N1 influenza virus (A/Narita/1/09) with different antigenicity from the vaccine strain at a lethal dose of 50 (5 times the amount of virus that causes lethal infection in 50% of mice) under anesthesia to carry out virus infection.

During the 20 days from the time of virus infection, the mice were observed daily and the survival rate was investigated. As shown in Figure 5, the C57BL/6 mice inoculated with the membrane fusion HA split vaccine were able to significantly suppress the decrease in survival rate from the 9th day after infection with other antigenically different H1N1 influenza viruses.

7. Antibody binding relative to LAHepitope

The binding of LAH-binding monoclonal antibodies (#1 to #5 in Figure 6) produced from peripheral blood of mice or humans infected with the X31 strain was measured by ELISA (Enzyme-Linked Immuno Sorbent Assay) relative to the current HA split vaccine or membrane-fusion HA split vaccine. The current HA split vaccine or membrane-fusion HA split vaccine of the H3N2 influenza virus (X31 strain) was dissolved in phosphate-buffered saline (pH 7.3) and 50 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 at room temperature for 2 hours, each well was washed 3 times with phosphate-buffered saline (containing 0.05 v/v% Tween20), and 50 μl of LAH-binding monoclonal antibody diluted in phosphate buffer containing 1 v/v% bovine serum albumin was added. After standing at 4°C overnight, each well was washed 3 times with phosphate-buffered saline (containing 0.05 v/v% Tween20), and peroxidase-labeled anti-mouse IgG antibody (Southern Biotech) was diluted with phosphate-buffered saline containing 0.05 v/v% Tween20 and 1 v/v% bovine serum albumin, and 100 μl of peroxidase-labeled anti-mouse IgG antibody 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 v/v% Tween20), and 60 ml of citric acid buffer (pH 5.0) as the base was added with 30 mg of o-phenylendiamine tablet (SIGMA) and 24 μl of 30% hydrogen peroxide (30% w/w; SIGMA), and 50 μl was added to each well. After color development, the reaction was stopped with 25 μl of 1 mol/L sulfuric acid (Wako Pure Chemical Industries), and the absorbance at 490 nm was measured using Microplate Reader 450 (Biorad). The binding change was calculated based on the absorbance relative to the measured current HA split vaccine or membrane fusion HA split vaccine.

As shown in Figure 6, the binding of the membrane fusion HA split vaccine to the LAH-binding monoclonal antibody is 1.05 to 21 times stronger than that of the current HA split vaccine. These results indicate that the antibody binding to the LAH epitope can be promoted by acid treatment of the HA split vaccine.

8. Effect of formalin treatment sequence on antibody binding and antibody induction ability

8-1. Preparation of formalin pre-treated HA split vaccine

Tween80 was added to H3N2 influenza virus particles (X31 strain) suspended in phosphate-buffered saline to a final concentration of 0.1 v/v% and suspended. Diethyl ether was added for further suspension, and the diethyl ether layer was removed after the water layer and the diethyl ether layer were completely separated. After repeating the ether extraction, the diethyl ether remaining in the recovered water layer was removed by atmospheric distillation. Formalin was added to a final concentration of 0.05 v/v% and left to stand for several days, and used as formalin pre-treatment HA split vaccine.

8-2. Acid treatment of HA split vaccine before formalin treatment

After formalin pre-treated HA split vaccine was suspended in phosphate-buffered saline, 0.15M citric acid buffer (pH 3.5) was added as an acidic treatment to adjust the pH to 5.0. After standing at room temperature for 30 minutes, 1M Tris buffer (pH 8.0) was added to return the pH to 7.3. Centrifugation was then performed.

9. Antibody binding relative to LAH epitope

The antibody binding to LAH epitope was performed in the same manner as in step 7 above, and the binding changes were calculated. Here, the monoclonal antibodies used were the same antibodies #2, #4, and #5 in Figure 6, and the control group used the monoclonal antibody #6 that binds to the HA head region.

10. Measure LAH antibody titer by ELISA Tween

10-1. Get the H3N2 influenza vaccine

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

10-2. Measurement by ELISA method

The LAH antibody titer was measured by the same operation as in 3-2 above.

As shown in Figure 7, in the preparation steps of the membrane fusion HA split vaccine, compared with the vaccine before acid treatment (Pre-fix), the vaccine that is formalin-treated after acid treatment (Post-fix) is different from the vaccine before acid treatment (Pre-fix). LAH-binding monoclonal antibodies bind more strongly. These results show that when acidic treatment of HA split vaccine promotes antibody binding to LAHepitope, the time point at which formalin treatment is performed will affect its effect. It is preferable that formalin treatment is after acidic treatment. Here, in FIG. 7 and FIG. 8, the membrane fusion type split vaccine in which the HA split vaccine is subjected to acid treatment after formalin treatment according to the above-mentioned sequence of 8. is referred to as "(Pre-fix)"; The membrane fusion split vaccine in which the HA split vaccine is treated with formalin after acid treatment according to the sequence of Example 1 is called "(Post-fix)".

As shown in Figure 8, the LAH antibody titer in the serum of BALB/c mice intraperitoneally inoculated with the current HA split vaccine was higher than that of BALB/c mice intraperitoneally inoculated with the membrane fusion HA split vaccine. The LAH antibody titer of the membrane fusion HA split vaccine (post-fix) was higher than that of the membrane fusion split vaccine (Pre-fix).

11. Preparation of HA split vaccine

Use H3N2 influenza virus particles (X31 strain) to prepare HA split vaccine according to the method described in 1. above.

12. Prior review of acid treatment conditions when using a stirring tank

In the acid treatment step, 0.15M citric acid buffer (pH 3.5) or dilute hydrochloric acid was added to the phosphate-buffered saline to adjust the pH. The stirring speed after the addition of citric acid buffer and the change in color after the reaction time in the stirring tank to which the pH indicator was added were used to test the conditions for achieving uniformity after the addition of citric acid buffer. Using pH indicator methyl red in a 100mL, 20L stirring tank, the color change was observed at stirring speeds of 100rpm, 200rpm, 300rpm, 400rpm, and 500rpm from the time of addition to 10 minutes, and recorded with a video.

13. Acid treatment

After suspending the HA split vaccine in phosphate-buffered saline, 0.15M citric acid buffer (pH 3.5) or dilute hydrochloric acid was added as an acidic treatment to adjust the pH to 5 different values (2.0, 3.0, 4.0, 5.0, or 6.0). After standing for 3 times (10 minutes, 30 minutes, or 1 hour) at five temperature conditions (10°C, 25°C, 35°C, 45°C, or 55°C), 1M Tris buffer (pH 8.0) was added to return the pH to 7.3. After that, centrifugation was performed to separate it and use it as a membrane-fusion type HA split vaccine. Formalin was added to the membrane-fusion type HA split vaccine to a final concentration of 0.05v/v%, and it was left to stand for several days.

It should be noted that in the case of the current HA split vaccine, except for not subjecting the HA split vaccine prepared according to 11. above to acid treatment, the other treatments implemented are the same as above.

14. Antibody binding to LAH epitope

Using the H3N2 influenza virus X31 strain, the ELISA method according to 7. above was used to detect the binding of LAH-binding monoclonal antibodies (#1~#5 in Figure 6) prepared from mice infected with the X31 strain or human peripheral blood to the current HA split vaccine or membrane fusion HA split vaccine.

-Industrial Utilization-

Can be used to make influenza vaccines.

【Sequence Listing】

Sequence number 1, 2: synthetic peptide

<110> JAPAN as represented by DIRECTOR GENERAL of National Institute of Infectious Diseases

<120> Method for manufacturing influenza HA split vaccine

<130> H19-109TW2

<150> 108107147

<151> 2019-03-04

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 64

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptides

<220>

<221> MOD_RES

<222> (1)..(1)

<223> Acetylation

<400> 1

<210> 2

<211> 64

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptides

<220>

<221> MOD_RES

<222> (1)..(1)

<223> Acetylation

<400> 2

Claims (21)

A method for producing an influenza HA split vaccine, wherein the influenza HA split vaccine induces antibodies that bind to LAH in the HA stem region, and the method comprises the step of subjecting the influenza HA split vaccine that has not been treated with formalin to acid treatment. A method for producing an influenza HA split vaccine, wherein the influenza HA split vaccine induces antibodies that bind to LAH in the HA stem region, and the method comprises a step of subjecting the influenza HA split vaccine that has not been treated with formalin to acid treatment and a subsequent step of subjecting the vaccine to formalin treatment. The method for manufacturing the influenza HA split vaccine as described in claim 1 or 2, and the aforementioned influenza HA split vaccine is also effective against antigenically variant influenza viruses. A method for producing an influenza HA split vaccine, which is to produce an influenza HA split vaccine against antigenically variant influenza viruses by subjecting an influenza HA split vaccine that has not been treated with formalin to an acid treatment, wherein the influenza HA split vaccine induces antibodies that bind to LAH in the HA stem region. A method for producing an influenza HA split vaccine as described in any one of claim items 1, 2, and 4, wherein the aforementioned acid treatment is performed at pH 3.0-6.5. A method for producing an influenza HA split vaccine as described in any one of claim items 1, 2, and 4, wherein the aforementioned acid treatment is performed at pH 4.4-5.8. A method for manufacturing an influenza HA split vaccine as described in any one of claim items 1, 2, and 4, wherein the aforementioned acid treatment is carried out at 10°C to 60°C. A method for manufacturing an influenza HA split vaccine as described in any of claim items 1, 2, and 4, wherein the influenza HA split vaccine is of the H3N2 type or the H1N1 type. A method for manufacturing an influenza HA split vaccine as described in any of claim items 1, 2, and 4, wherein the influenza HA split vaccine is a single HA subtype influenza HA split vaccine. The method for producing an influenza HA split vaccine as described in any one of claim 1, 2, and 4, wherein the method comprises the step of mixing two or more influenza HA split vaccine antigens, wherein the influenza HA split vaccine antigens are produced by subjecting a single subtype of influenza HA split vaccine to acid treatment. An influenza HA split vaccine is produced by the method for producing an influenza HA split vaccine described in any one of claims 1 to 10, and induces antibodies that bind to LAH in the HA stem region. The influenza HA split vaccine as described in claim 11, wherein the aforementioned influenza HA split vaccine is also effective against antigenically variant influenza viruses. The influenza HA split vaccine as described in claim 11 or 12, wherein the shape of the influenza HA split vaccine is that the HA stem region is exposed to the outside. The influenza HA split vaccine described in claim 11 or 12 is in a shape in which the HA stem region of the influenza HA split vaccine antigen is exposed to the outside, thereby increasing the antigenicity of the LAH in the HA stem region and inducing antibodies that bind to the LAH in the HA stem region. An influenza HA split vaccine is produced by subjecting an influenza HA split vaccine that has not been treated with formalin to acid treatment to induce antibodies that bind to LAH in the HA stem region. An influenza HA split vaccine is produced by a manufacturing process comprising a step of subjecting an influenza HA split vaccine that has not been treated with formalin to acid treatment and a subsequent step of subjecting the vaccine to formalin treatment, thereby inducing antibodies that bind to LAH in the HA stem region. An influenza HA split vaccine is produced by subjecting a non-formalin-treated influenza HA split vaccine containing a single subtype to acid treatment to induce antibodies that bind to LAH in the HA stem region. An influenza HA split vaccine is a vaccine antigen produced by mixing two or more influenza HA split vaccine antigens to induce antibodies that bind to LAH in the HA stem region, wherein the aforementioned influenza HA split vaccine antigen is produced by acid treatment of influenza HA split vaccines containing individual subtypes that have not been treated with formalin. The influenza HA split vaccine described in any of claim items 15 to 18, wherein the influenza HA split vaccine is also effective against antigenically variant influenza viruses. An influenza HA split vaccine, the aforementioned influenza HA split vaccine is effective against antigenically mutated influenza viruses, and the aforementioned influenza HA split vaccine is produced by subjecting the influenza HA split vaccine that has not been treated with formalin to acid treatment to induce antibodies that bind to LAH in the HA stem region. A use of an influenza HA split vaccine as described in any one of claim items 11 to 20, which is used to manufacture a vaccine for preventing influenza infection.

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