CN113730566B

CN113730566B - Influenza new corona combined vaccine and preparation method thereof

Info

Publication number: CN113730566B
Application number: CN202111311110.6A
Authority: CN
Inventors: 阮承迈; 高春润; 刘淑惠; 高辉; 李新旺; 高金榜; 张靖丽; 崔艳; 李凤凯
Original assignee: Tianjin Zhongyi Anjian Biotechnology Co ltd
Current assignee: Liaoning Maokangyuan Biotechnology Co ltd; Zhongyi Anke Biotechnology Co ltd
Priority date: 2021-11-08
Filing date: 2021-11-08
Publication date: 2022-03-01
Anticipated expiration: 2041-11-08
Also published as: CN113730566A

Abstract

The invention discloses an influenza Xinguan combined vaccine, which comprises the following raw materials in mass concentration: 1-100 mug/mL of recombinant new crown vaccine containing RBD-Fc fusion protein; 1-50 mug/mL of influenza subunit vaccine containing H1N1 type influenza virus; 1-50 mug/mL of influenza subunit vaccine containing H3N2 type influenza virus; 1-50 mug/mL of influenza subunit vaccine containing B type influenza virus; the final concentration of aluminum ions in the influenza new crown combined vaccine is 0.5-2.0 mg/mL; the balance was PBS phosphate buffer. The preparation method comprises the following steps: weighing the raw materials; diluting the four vaccines with PBS phosphate buffer solution respectively, and mixing with aluminum hydroxide solution; mixing according to equal volume proportion. The combined vaccine of the novel coronavirus vaccine containing the aluminum adjuvant and the influenza subunit vaccine containing the aluminum adjuvant is combined, and after the combined vaccine is combined, two antigen component vaccines are not mutually inhibited, so that the two antigen component vaccines can be well compatible.

Description

Influenza new corona combined vaccine and preparation method thereof

Technical Field

The invention relates to the technical field of biological products, in particular to an influenza new corona combined vaccine and a preparation method thereof.

Background

The spread of new coronary pneumonia is fast, the fatality rate is high, and an effective vaccine is urgently needed to control the pandemic.

By 12 months 2020, 60 candidate vaccines based on 6 different technical routes including inactivated vaccine, nucleic acid vaccine (including DNA vaccine and mRNA vaccine), vector vaccine, protein subunit vaccine, attenuated live vaccine, virus-like particle vaccine, etc. have been approved for clinical trials globally, and 7 of them (3 inactivated vaccines, 2 mRNA nucleic acid vaccines, 2 vector vaccines) were approved for emergency use or for inclusion in the market.

At present, the new coronavirus protein subunit vaccine has 18 items to enter clinical trials, which accounts for 30 percent of vaccines entering clinical trials and is the vaccine type with the largest amount in research. Of these vaccines, an NVX-CoV2373 vaccine has entered the phase IIb trial in south africa and the phase III trial in uk, NVX-CoV2373 contains a stable full length spike protein supplemented with proprietary saponin adjuvants. Neutralizing antibodies were induced in all participants of the phase I trial, and a dose-sparing effect of the adjuvant was also observed. The adjuvant-supplemented test group induced a significant increase in neutralizing antibody titers compared to the placebo and non-adjuvanted groups. Another vaccine that entered phase III trials was the recombinant novel coronavirus vaccine of Anhui Zhifeilongkang, which did not use full-length S protein, but only contained RBD of SARS-CoV-2S protein. To date, no further data has been provided.

As global scientists have actively developed new crown vaccines, a study in the uk has found that antibodies in patients with new crown pneumonia disappear within months, as may be the case with vaccines providing antibodies. Thus, the new coronaviruses may cause repeated infections every year as do the common cold. The investigators showed that infection usually produces the strongest antibody response by the body, so if the antibody levels subside after two to three months after infection, the vaccine may do so. Therefore, people may need to inject a new crown vaccine reinforcing needle to continuously strengthen the immunity.

The influenza vaccine is used for preventing influenza (called influenza for short) caused by influenza virus, and belongs to self-fee vaccination. The current influenza vaccine is an inactivated influenza virus vaccine, and consists of 3 viruses. Influenza vaccines are one of the major measures for the prevention and control of influenza, and vaccination before or during the influenza pandemic season can reduce the chances of contracting influenza or alleviate influenza symptoms in the vaccinee.

Due to the possibly existing repeated infection characteristic of the new coronavirus, if the influenza vaccine and the new coronavirus can be jointly developed and prepared, a large amount of manpower and material resources can be saved, and the method is beneficial to the nation and people.

Adjuvants are short for immunological adjuvants, i.e., nonspecific immunostimulants, and refer to auxiliary substances that are injected into the body together with or in advance of an antigen to enhance the body's ability to respond to the antigen or to modify the type of immune response. The adjuvant is added into the vaccine, so that the immunogenicity of the antigen can be enhanced, the dosage of the vaccine is saved, the range of vaccine induced immune response can be expanded, and cross protection is induced. The aluminum adjuvant is a classic human influenza vaccine adjuvant, can improve the titer of antigen-specific antibodies and induce strong humoral immune response, but the application of the aluminum adjuvant in the influenza new crown combined vaccine is not reported yet.

Therefore, how to provide a novel aluminum-adjuvant-containing influenza corona combination vaccine is a problem to be solved urgently by the technical personnel in the field.

Disclosure of Invention

In view of the above, the present invention aims to provide a new influenza corona combined vaccine and a preparation method thereof, so as to solve the defects in the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides an influenza Xinguan combined vaccine which comprises the following raw materials in mass concentration: the recombinant new corona vaccine containing RBD-Fc fusion protein has a mass concentration of 1-100 mug/mL; the subunit vaccine containing the H1N1 influenza virus has the mass concentration of 1-50 mug/mL; the influenza subunit vaccine containing the H3N2 type influenza virus has the mass concentration of 1-50 mug/mL; the influenza subunit vaccine containing the influenza B virus has the mass concentration of 1-50 mug/mL; the final concentration of aluminum ions in the influenza new crown combined vaccine is 0.5-2.0 mg/mL; the balance was PBS phosphate buffer.

Furthermore, the mass concentration of the recombinant new corona vaccine containing the RBD-Fc fusion protein is 5-80 mu g/mL.

The RBD-Fc fusion protein has the beneficial effects that the RBD-Fc fusion protein selected by the invention

The recombinant new crown vaccine is a recombinant subunit vaccine with the receptor structural domain of the new crown virus SARS-CoV-2S protein fused with the human lgG Fc structural domain, the vaccine can protect the organism from being infected by the new crown virus, the Fc segment can promote antigen presentation, and compared with other types of vaccines, the vaccine has higher safety and higher cost benefit.

Further, the subunit vaccine containing the H1N1 influenza virus has the mass concentration of 5-20 mug/mL; the mass concentration of the influenza subunit vaccine containing the H3N2 type influenza virus is 5-20 mug/mL; the mass concentration of the influenza subunit vaccine containing the influenza B virus is 5-20 mug/mL.

The trivalent influenza virus subunit vaccine containing H1N1 type, H3N2 type and B type is prepared by selecting a new cracking process and a purification technology on the basis of influenza virus cracking vaccine, the antigen purity of the high-purity influenza vaccine reaches more than 85 percent, further nucleoprotein and M protein are removed, only the antigen with immunogenicity is reserved, the inoculation side reaction is low, the antibody rises quickly, the maintenance time is long, the good immune effect is achieved, and meanwhile, the high biological safety is achieved.

Furthermore, in the aluminum hydroxide solution, the final concentration of aluminum ions in the influenza new crown combination vaccine is 0.6-1.0 mg/mL.

The further technical scheme has the beneficial effects that the aluminum hydroxide is an aluminum adjuvant, and the antigen is retained on the surface of the aluminum hydroxide through surface adsorption, so that the titer of the antigen-specific antibody can be improved, strong humoral immune response can be induced, and the dosage of the vaccine can be saved. The FDA sets the content of aluminum adjuvant in a prophylactic vaccine to be used clinically to be not more than 0.85 mg/dose in terms of aluminum ion, and the three parts (2015 edition) of the chinese pharmacopoeia stipulates that the maximum aluminum content in a vaccine containing aluminum adjuvant is 1.04mg/mL, and the final concentration of aluminum ions in the influenza corona-new combination vaccine of the present invention is 0.6 to 1.0mg/mL, converted by the ratio of the molecular weight of aluminum ions to that of aluminum hydroxide in aluminum hydroxide being 1: 2.89.

The preparation method of the influenza new corona combined vaccine specifically comprises the following steps:

(1) weighing the raw materials according to the mass fraction of the influenza Xinguan combined vaccine;

(2) diluting a recombinant new corona vaccine containing RBD-Fc fusion protein with PBS phosphate buffer solution, and mixing with an aluminum hydroxide solution to obtain a vaccine A for later use;

(3) diluting an influenza subunit vaccine containing H1N1 type influenza virus with PBS phosphate buffer solution, and mixing with an aluminum hydroxide solution to obtain a vaccine B for later use;

(4) diluting an influenza subunit vaccine containing H3N2 type influenza virus with PBS phosphate buffer solution, and mixing with an aluminum hydroxide solution to obtain a vaccine C for later use;

(5) diluting an influenza subunit vaccine containing the influenza B virus by using PBS phosphate buffer solution, and mixing the diluted influenza subunit vaccine with an aluminum hydroxide solution to obtain a vaccine D for later use;

(6) and mixing the vaccine B, the vaccine C and the vaccine D according to an equal volume ratio, and then mixing the mixture with the vaccine A according to an equal volume ratio to obtain the influenza new crown combined vaccine.

Further, the preparation method of the aluminum hydroxide solution comprises the following steps:

62.05g of aluminum chloride and 6.8g of sodium acetate are weighed and dissolved in 1L of distilled water, filtered and sterilized by a 0.2 mu m filter membrane to obtain a solution A, and stored at 20-30 ℃ for later use;

weighing 10.25g of sodium hydroxide, dissolving in 1L of distilled water, filtering and sterilizing by a 0.2 mu m filter membrane to obtain a solution B, and storing at 20-30 ℃ for later use;

thirdly, adding the solution B into a mixing container, continuously stirring at the speed of 40-60rpm, and adding sterile distilled water to obtain a solution C;

adding the solution A into the solution C at the speed of 1-2L/min, and controlling the pH value of the solution to be 5.5-6.5 to obtain a solution D;

adding sterile distilled water into the solution D to adjust the final volume, mixing the suspension for 2 hours, and finally adding 5mol/L NaOH and/or 5mol/L acetic acid to adjust the pH value to 5.9-6.1, thus obtaining the aluminum hydroxide solution.

Furthermore, in the third step, the addition amount of the solution B is 20 percent of the volume of the aluminum hydroxide solution, and the addition amount of the sterile distilled water is 50 to 55 percent of the volume of the aluminum hydroxide solution; in the step IV, the adding amount of the solution A is 20 percent of the volume of the aluminum hydroxide solution.

According to the technical scheme, compared with the prior art, the invention has the following beneficial effects: the invention provides a combined vaccine of a novel coronavirus vaccine containing an aluminum adjuvant and an influenza subunit vaccine containing the aluminum adjuvant, after combination, two antigen component vaccines are not mutually inhibited, and can be well compatible.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the following examples, the required agents of the present invention were conventional experimental agents purchased from commercially available sources; the experimental methods not mentioned are conventional experimental methods.

The recombinant new crown vaccine stock solution containing the RBD-Fc fusion protein and the trivalent influenza virus subunit vaccine stock solution containing H1N1 type, H3N2 type and B type are provided by Zhongyi Anke biotechnology member company.

Example 1

The influenza corona combined vaccine comprises the following raw materials in mass concentration: the recombinant new corona vaccine containing the RBD-Fc fusion protein has the mass concentration of 80 mug/mL; the influenza subunit vaccine containing the H1N1 type influenza virus has the mass concentration of 18 mug/mL; the influenza subunit vaccine containing the H3N2 type influenza virus has the mass concentration of 18 mug/mL; influenza subunit vaccine containing B type influenza virus, wherein the mass concentration of the influenza subunit vaccine is 18 mug/mL; the final concentration of aluminum ions in the influenza new crown combined vaccine is 0.6 mg/mL; the balance was PBS phosphate buffer.

the preparation method of the aluminum hydroxide solution comprises the following steps:

62.05g of aluminum chloride and 6.8g of sodium acetate are weighed and dissolved in 1L of distilled water, filtered and sterilized by a 0.2 mu m filter membrane to obtain a solution A, and stored at 25 ℃ for later use;

weighing 10.25g of sodium hydroxide, dissolving in 1L of distilled water, filtering and sterilizing by a 0.2-micron filter membrane to obtain a solution B, and storing at 25 ℃ for later use;

③ adding the solution B (accounting for 20 percent of the volume of the final aluminum hydroxide solution) into a mixing container, then continuously stirring at the speed of 50rpm, and adding sterile distilled water (accounting for 50 percent of the volume of the final aluminum hydroxide solution) to obtain a solution C;

adding the solution A (accounting for 20% of the volume of the final aluminum hydroxide solution) into the solution C at the rate of 1L/min, and controlling the pH value of the solution to be 6.0 to obtain a solution D;

adding sterile distilled water into the solution D to adjust the volume to the final volume, mixing the suspension for 2 hours, and finally adding 5mol/L NaOH and/or 5mol/L acetic acid to adjust the pH value to 6.0, thus obtaining an aluminum hydroxide solution;

(2) diluting the recombinant new corona vaccine containing the RBD-Fc fusion protein by using a PBS phosphate buffer solution, and then mixing the diluted recombinant new corona vaccine with an aluminum hydroxide solution to ensure that the mass concentration of the recombinant new corona vaccine containing the RBD-Fc fusion protein is 80 mu g/mL, the final concentration of aluminum ions is 0.6mg/mL, and the balance is the PBS phosphate buffer solution to obtain a vaccine A for later use;

(3) diluting an influenza subunit vaccine containing H1N1 influenza virus with PBS phosphate buffer solution, and mixing with an aluminum hydroxide solution to ensure that the mass concentration of the influenza subunit vaccine containing H1N1 influenza virus is 18 mug/mL, the final concentration of aluminum ions is 0.6mg/mL, and the balance is PBS phosphate buffer solution to obtain a vaccine B for later use;

(4) diluting an influenza subunit vaccine containing H3N2 influenza virus with PBS phosphate buffer solution, and mixing with an aluminum hydroxide solution to ensure that the mass concentration of the influenza subunit vaccine containing H3N2 influenza virus is 18 mug/mL, the final concentration of aluminum ions is 0.6mg/mL, and the balance is PBS phosphate buffer solution to obtain a vaccine C for later use;

(5) diluting an influenza subunit vaccine containing the influenza B virus by using PBS phosphate buffer solution, and mixing the diluted influenza subunit vaccine with an aluminum hydroxide solution to ensure that the mass concentration of the influenza subunit vaccine containing the influenza B virus is 18 mug/mL, the final concentration of aluminum ions is 0.6mg/mL, and the balance is the PBS phosphate buffer solution to obtain a vaccine D for later use;

(6) and mixing the vaccine B, the vaccine C and the vaccine D according to an equal volume ratio to obtain a vaccine E, and mixing the vaccine E and the vaccine A according to an equal volume ratio to obtain the influenza Xinguan combined vaccine.

Example 2

The influenza corona combined vaccine comprises the following raw materials in mass concentration: the recombinant new corona vaccine containing the RBD-Fc fusion protein has the mass concentration of 5 mug/mL; the influenza subunit vaccine containing the H1N1 type influenza virus has the mass concentration of 20 mug/mL; the influenza subunit vaccine containing the H3N2 type influenza virus has the mass concentration of 20 mug/mL; influenza subunit vaccine containing B type influenza virus, wherein the mass concentration of the influenza subunit vaccine is 20 mug/mL; the final concentration of aluminum ions in the aluminum hydroxide solution is 0.6 mg/mL; the balance was PBS phosphate buffer.

(2) diluting the recombinant new corona vaccine containing the RBD-Fc fusion protein by using a PBS phosphate buffer solution, and then mixing the diluted recombinant new corona vaccine with an aluminum hydroxide solution to ensure that the mass concentration of the recombinant new corona vaccine containing the RBD-Fc fusion protein is 5 mu g/mL, the final concentration of aluminum ions is 0.6mg/mL, and the balance is the PBS phosphate buffer solution to obtain a vaccine A for later use;

(3) diluting an influenza subunit vaccine containing H1N1 influenza virus with PBS phosphate buffer solution, and mixing with an aluminum hydroxide solution to ensure that the mass concentration of the influenza subunit vaccine containing H1N1 influenza virus is 20 mug/mL, the final concentration of aluminum ions is 0.6mg/mL, and the balance is PBS phosphate buffer solution to obtain a vaccine B for later use;

(4) diluting an influenza subunit vaccine containing H3N2 influenza virus with PBS phosphate buffer solution, and mixing with an aluminum hydroxide solution to ensure that the mass concentration of the influenza subunit vaccine containing H3N2 influenza virus is 20 mug/mL, the final concentration of aluminum ions is 0.6mg/mL, and the balance is PBS phosphate buffer solution to obtain a vaccine C for later use;

(5) diluting an influenza subunit vaccine containing the influenza B virus by using PBS phosphate buffer solution, and mixing the diluted influenza subunit vaccine with an aluminum hydroxide solution to ensure that the mass concentration of the influenza subunit vaccine containing the influenza B virus is 20 mug/mL, the final concentration of aluminum ions is 0.6mg/mL, and the balance is the PBS phosphate buffer solution to obtain a vaccine D for later use;

Performance testing

1. Example 1 immunogenicity analysis of influenza neo-corona combination vaccine

25 female BALB/c mice of 6-8 weeks of SFP grade were selected and randomly divided into 5 groups, and each group was immunized by intraperitoneal injection for the influenza neo-corona combined vaccine prepared in example 1 (80. mu.g/mL aluminum-adjuvanted neo-corona vaccine + 18. mu.g/mL aluminum-adjuvanted influenza subunit vaccine), the vaccine A prepared in example 1 (80. mu.g/mL aluminum-adjuvanted neo-corona vaccine), the vaccine E prepared in example 1 (18. mu.g/mL aluminum-adjuvanted influenza subunit vaccine) and the 18. mu.g/mL influenza subunit vaccine (different from the vaccine E prepared in example 1 only in not containing aluminum hydroxide solution), and a PBS phosphate buffer control group was prepared, and the immunization volume was 0.5 mL.

The first immunization is carried out for 14 days with the same vaccine protein concentration and volume, the first immunization is carried out for 35 days, blood is collected and serum is separated, and anti-influenza antibody in mouse serum is determined by hemagglutination inhibition method

The titer. The results are shown in Table 1.

Table 1 example 1 anti-influenza antibody levels following immunization with a novel corona influenza combination vaccine

Group of	H1N1 antibody mean (GMT)	H3N2 antibody mean (GMT)	B antibody mean (GMT)
				Example 1 New crown combination influenza vaccine	6144	8192	3584
Example 1 vaccine A	<5	<5	<5
				Example 1 vaccine E	7424	6272	704
18 ug/mL influenza subunit vaccine	4352	10240	224
				PBS phosphate buffer control group	<5	<5	<5

Note: values in table 1 are serum dilution fold (n = 5).

As can be seen from table 1, 35 days after immunization, the anti-influenza antibody titer in mouse peripheral serum of the influenza H1N1 type antibody titer was slightly decreased, the influenza H3N2 type antibody titer was slightly increased, and the influenza B type antibody titer was significantly increased in the influenza neo-corona combined vaccine of example 1, compared to the vaccine E of example 1 after immunization. The above experiments show that the new corona vaccine component has no inhibitory effect on the influenza vaccine component.

After 35 days after immunization, compared with the vaccine E of the 18 mu g/mL influenza subunit without the aluminum adjuvant, the vaccine E of the example 1 containing the aluminum adjuvant has the advantages that the antibody titer of the H1N1 type influenza virus is obviously increased, the antibody titer of the H3N2 type influenza virus is slightly reduced, and the antibody titer of the B type influenza virus is obviously increased in mice after immunization. The above experiments show that the aluminum adjuvant has an immune promoting effect on influenza vaccines.

2. Example 2 immunogenicity analysis of influenza neo-corona combination vaccine

20 female BALB/c mice of 6-8 weeks of SFP grade were selected and randomly divided into 4 groups, and each group was immunized by intraperitoneal injection for the influenza neo-corona combined vaccine prepared in example 2 (5. mu.g/mL aluminum-adjuvant-containing neo-corona vaccine + 20. mu.g/mL aluminum-adjuvant-containing influenza subunit vaccine), the vaccine A prepared in example 2 (5. mu.g/mL aluminum-adjuvant-containing neo-corona vaccine) and the 20. mu.g/mL neo-corona vaccine (without aluminum hydroxide solution), and a PBS phosphate buffer solution control group was prepared, and the immunization volume was 0.5 mL.

The method is characterized in that the primary immunization is carried out for 14 days, the boosting immunization is carried out once by using the same vaccine protein concentration and volume, blood is collected and serum is separated when the primary immunization is carried out for 35 days, and the anti-new crown antibody titer in the mouse serum is determined by using a mouse anti-new coronavirus (2019-nCoV) S-RBD protein IgG antibody detection kit (an enzyme linked immunosorbent assay). The results are shown in Table 2.

Table 2 example 2 anti-neocorona antibody levels following immunization with influenza neocorona combination vaccine

Group of	Anti-neocorona antibody level (GMT)
		Example 2 New crown combination influenza vaccine	4842
Example 2 vaccine A	1902
		New corona vaccine of 20 mug/mL	1024
PBS phosphate buffer control group	Negative of

Note: values in table 2 are serum dilution fold (n = 5).

As can be seen from table 2, after 35 days after immunization, the anti-neocorona antibody titer in peripheral serum of mice was significantly increased after the immunization with the influenza neocorona combined vaccine in example 2, compared with that after the immunization with the vaccine a in example 2. The above experiments show that the influenza vaccine components have no inhibitory effect on the new corona vaccine component.

The anti-neocorona antibody titer in mice was increased after immunization with the aluminum-adjuvanted low dose neocorona vaccine of 5 μ g/mL compared to the mice immunized with the high dose of 20 μ g/mL neocorona vaccine. The experiments show that the aluminum adjuvant can save the dosage of the vaccine and has an immune promoting effect on the vaccine.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The influenza Xinguan combined vaccine is characterized by being prepared from the following raw materials in mass concentration:

the recombinant new corona vaccine containing RBD-Fc fusion protein has the mass concentration of 5-80 mug/mL;

the influenza subunit vaccine containing the H1N1 type influenza virus has the mass concentration of 5-20 mug/mL;

the influenza subunit vaccine containing the H3N2 type influenza virus has the mass concentration of 5-20 mug/mL;

the influenza subunit vaccine containing the influenza B virus has the mass concentration of 5-20 mug/mL;

an aluminum hydroxide solution, wherein the final concentration of aluminum ions in the aluminum hydroxide solution in the influenza neo-corona combination vaccine is 0.6-1.0 mg/mL;

the balance was PBS phosphate buffer.

2. A preparation method of a new coronary influenza combined vaccine is characterized by comprising the following steps:

(1) weighing the raw materials according to the mass fraction of the influenza Xinguan combined vaccine of claim 1;

(6) and mixing the vaccine B, the vaccine C and the vaccine D according to an equal volume ratio to obtain a vaccine E, and mixing the vaccine E and the vaccine A according to an equal volume ratio to obtain the influenza new crown combined vaccine.

3. The method for preparing the influenza corona co-vaccine according to claim 2, wherein the aluminum hydroxide solution is prepared by:

adding sterile distilled water into the solution D to adjust the final volume, mixing the suspension for 2 hours, and finally adding 5mol/L of LNaOH and/or 5mol/L of acetic acid to adjust the pH value to 5.9-6.1, thus obtaining the aluminum hydroxide solution.

4. The method for preparing the influenza new crown combined vaccine according to claim 2, wherein in the third step, the addition amount of the solution B is 20% of the volume of the aluminum hydroxide solution, and the addition amount of the sterile distilled water is 50-55% of the volume of the aluminum hydroxide solution.

5. The method for preparing the influenza corona new combination vaccine according to claim 2, wherein in the step (iv), the amount of the solution A added is 20% of the volume of the aluminum hydroxide solution.