An adjuvanted influenza vaccine, its method of preparation and use

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to an adjuvant influenza vaccine and a preparation method and application thereof. The adjuvant influenza vaccine comprises an influenza virus monovalent stock solution, an adjuvant and a PBS buffer solution; the adjuvant is prepared by using mixed oil and an auxiliary agent, the mixed oil comprises squalene, medium-chain triglyceride and alpha-tocopherol; and the auxiliary agent comprises sorbitan trioleate Span-85, Tween 80 and vitamin E succinate polyethylene glycol ester. The adjuvant influenza vaccine prepared by the application has good immunization effect, and can especially improve the overall effect of a tetravalent adjuvant influenza vaccine on four viruses.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an adjuvanted influenza vaccine, its preparation method, and its application. Background Technology

[0002] Influenza, commonly known as the flu, is an acute respiratory infectious disease caused by the influenza virus. It can cause various complications such as myocarditis, pneumonia, and bronchitis. Vaccination remains the most effective means of preventing and controlling influenza epidemics and reducing the disease burden caused by influenza. Currently, influenza vaccines used globally mainly include split vaccines, live attenuated vaccines, and recombinant subunit vaccines. The influenza vaccines sold in the Chinese market are mainly split vaccines, produced using chicken embryo media. Vaccine purification processes primarily include ultracentrifugation or gel (column) chromatography. Most commercially available influenza virus split vaccines employ a two-step purification process (a combination of ultracentrifugation and gel (column) chromatography, or a combination of two ultracentrifugation processes). Vaccines produced using this two-step purification process have relatively lower antigen purity and relatively higher levels of ovalbumin (the main allergen in influenza vaccines), which may significantly increase the risk of adverse reactions in recipients.

[0003] Due to immunostagnation, older adults often have a weakened response to traditional vaccines, resulting in less than ideal immune responses after receiving existing split vaccines. This means that traditional vaccines are insufficient to provide adequate protection for the elderly, necessitating the development of a vaccine that can effectively enhance their immune response.

[0004] Adjuvanted influenza vaccines are a combination of influenza antigens and adjuvants. Adjuvants are substances that can non-specifically enhance the body's immune response to vaccine antigens. They can enhance immunogenicity, and even in elderly people with relatively weak immune function, adjuvants can effectively stimulate them to produce a stronger and more lasting immune response, thus providing better protection against influenza viruses and reducing the risk of infection and the severity of the disease.

[0005] While existing influenza vaccine adjuvants have some effect in enhancing immune responses, they still have significant shortcomings, particularly in their insufficient ability to regulate and synergistically enhance immune responses to H1N1, H3N2, BY, and BV influenza virus strains. Specifically, traditional adjuvant formulations often struggle to simultaneously address the immune effects of multiple influenza strains, such as H3N2 and BV (B / Victoria lineage). Existing adjuvant systems exhibit varying adaptability and response mechanisms to different antigens (especially H3N2 and BV), lacking a universal regulatory mechanism capable of simultaneously, evenly, and efficiently enhancing immune responses to multiple strains. Furthermore, existing adjuvants often focus on enhancing the immunity of a single strain, neglecting the interactions between components in a multivalent vaccine. This leads to a "cost-benefit" phenomenon in practical applications—optimizing the immune effect of one component may come at the expense of the efficacy of other components, failing to achieve synergistic immune enhancement of multivalent antigens.

[0006] Therefore, there is an urgent need to develop a novel adjuvant formulation system that can simultaneously enhance the immunogenicity of key circulating strains without mutual interference, thereby achieving synergistic regulation and overall optimization of the immunogenicity of each antigen component in a multivalent influenza vaccine. Summary of the Invention

[0007] The purpose of this invention is to provide an adjuvanted influenza vaccine, its preparation method, and its application.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] An adjuvanted influenza vaccine comprises a monovalent influenza virus stock solution, an adjuvant, and a PBS buffer.

[0010] Preferably, the monovalent influenza virus stock solution includes at least one of H1N1 monovalent stock solution, H3N2 monovalent stock solution, BY monovalent stock solution, and BV monovalent stock solution.

[0011] Preferably, the adjuvant is prepared by using a mixture of oils and auxiliaries, the oils including squalene, medium-chain triglycerides and α-tocopherol; the auxiliaries including sorbitan trioleate Span-85, Tween 80 and polyethylene glycol succinate.

[0012] Preferably, the adjuvant accounts for 12.5%-50% of the volume of the adjuvant influenza vaccine.

[0013] The preparation method of the monovalent influenza virus stock solution includes the following steps:

[0014] S1. Preparation of harvest fluid: Healthy chicken embryos aged 9-11 days are inoculated with a popular virus strain, cultured at 33-35℃ for 48-72 hours, and then chilled at 2-8℃ to harvest the allantoic fluid containing the virus, which is the harvest fluid.

[0015] S2. Virus inactivation: Formaldehyde with a final concentration of 200µg / mL was added to the harvested liquid to inactivate the virus;

[0016] S3. Ultrafiltration Concentration: The inactivated harvest solution is concentrated by ultrafiltration using a 1000KD ultrafiltration membrane. The product retained by the ultrafiltration membrane is collected to obtain the virus concentrate.

[0017] S4. One-step purification: The virus concentrate is purified by centrifugation using the sucrose density gradient centrifugation method. Specifically, the concentrated virus solution is subjected to continuous flow sucrose rate zonal centrifugation in a 0-55% sucrose solution at a speed of 30,000-35,000 rpm, and the liquid fraction with a sucrose concentration of 18%-47% is collected.

[0018] S5. Two-step purification: First, the virus solution purified in the first step is ultrafiltered with a 300KD ultrafiltration membrane to remove sugar. Then, it is purified by column chromatography using Sepharose-4FF gel as the medium. The sample volume loaded each time does not exceed 8% of the column volume, and the sample loading rate is 80-350 mL / min. After the sample is injected, pH 7.2 PBS solution is pumped in for elution at a rate of 353-424 mL / min (elution linear flow rate 5-6 mm / min). The virus peak is collected using a 280 nm UV detector. The purified solution collected after chromatography is sampled for protein content detection. The protein content is controlled to be no higher than 1200 μg / mL to obtain the purified virus solution after two steps.

[0019] S6. Virus lysis: Add lysis agent to the purified virus solution after two-step purification for lysis; the lysis agent is Triton X-100, and the final concentration of Triton X-100 is 0.5wt%. Place it in a constant temperature (22℃) shaker for lysis for 16-18 hours to obtain virus lysis solution.

[0020] S7. Three-step purification: The virus lysate is subjected to continuous flow sucrose rate zonal centrifugation in a 0-55% sucrose solution at a speed of 30,000-35,000 rpm. The liquid segment with a sucrose concentration of 18%-42% is collected to obtain the purified virus lysate.

[0021] S8. After ultrafiltration to remove sugar from the purified virus lysate, it is then sterilized and filtered to obtain the monovalent stock solution.

[0022] Preferably, the method for preparing the adjuvant includes the following steps:

[0023] (1) Preparation of crude emulsion: Mix the mixed oil and the additives. The mixed oil includes squalene, medium-chain triglycerides and α-tocopherol in a mass ratio of (1.2-1.4):1:(0.5-0.7); the additives include sorbitan trioleate Span-85, Tween80 and vitamin E succinate polyethylene glycol in a mass ratio of (1.4-1.6):1:(0.3-0.5). Stir at 100rpm-300rpm until a homogeneous oil phase is formed. Add citrate buffer to the emulsifier and set the speed of the emulsifier between 10000-18000rpm. Inject the oil phase into the citrate buffer and continuously emulsify at high speed for 15-30min to obtain crude emulsion.

[0024] (2) High-pressure emulsification: The crude emulsion is transferred to a high-pressure microfluidic device and the parameters are set as follows: microfluidic pressure 10000-15000psi, flow rate 80-150mL / min; temperature control 20-30℃; homogenization cycle 4-6 times; after completion, it is filtered through a 0.22μm sterile filter to obtain the adjuvant.

[0025] The second aspect of the present invention provides a method for preparing an adjuvanted influenza vaccine, comprising the following steps: mixing monovalent stock solutions of each subtype, adding adjuvant, and supplementing with PBS buffer to obtain a semi-finished product, and passing the semi-finished product through a 0.22 μm sterile filter to obtain an adjuvanted influenza vaccine.

[0026] Preferably, the amount of adjuvant added to the semi-finished product is 12.5-50 (v / v)%.

[0027] A third aspect of the present invention provides the use of an adjuvanted influenza vaccine in the preparation of an adjuvanted influenza vaccine that enhances the immune response in the elderly population.

[0028] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0029] 1. Based on the traditional "two-step" purification method for influenza antigen preparation, this invention adds an extra purification step, namely, a "three-step purification method" to prepare influenza vaccine antigens with higher purity and fewer impurities. After that, the antigens and adjuvants are mixed to finally prepare an adjuvant influenza vaccine with good safety and high efficacy.

[0030] 2. This invention develops an adjuvanted influenza vaccine that solves the safety issues caused by low hemagglutinin purity and high impurity content in elderly people after receiving adjuvanted influenza vaccines. It reduces the amount of antigen used, significantly improves the protective effect of influenza vaccines, and has synergistic effects in inducing a higher immune response.

[0031] 3. The adjuvanted influenza vaccine prepared by this invention has good immunization effect, especially it can simultaneously improve the overall effect of the quadrivalent adjuvanted influenza vaccine against four viruses: H1N1, H3N2, BY and BV. Attached Figure Description

[0032] Figure 1 This is a schematic diagram showing the particle size of the adjuvants prepared in Examples 2-4.

[0033] Figure 2 This is a schematic diagram illustrating the effect of the vaccine prepared using the monovalent stock solution from Example 1 and the adjuvant from Example 2. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] All raw materials used in the following embodiments of the present invention are commercially available products:

[0036] The pH of the citrate buffer is 6.0.

[0037] The pH of the PBS buffer is 7.2.

[0038] Vitamin E succinate polyethylene glycol ester, CAS No. 9002-96-4, Shaanxi Changji Fu Biotechnology Co., Ltd., Vitamin E succinate polyethylene glycol 600 ester.

[0039] Example 1

[0040] This embodiment provides a method for preparing a monovalent influenza virus stock solution, including the following steps:

[0041] S1. Preparation of harvest fluid: 10-day-old healthy chicken embryos are inoculated with a popular virus strain, cultured at 35°C for 60 hours, and then chilled at 4°C to harvest the allantoic fluid of the chicken embryo containing the virus, which is the harvest fluid.

[0042] S2. Virus inactivation: Formaldehyde with a final concentration of 200µg / mL was added to the harvested liquid to inactivate the virus;

[0043] S3. Ultrafiltration Concentration: The inactivated harvest solution is concentrated by ultrafiltration using a 1000KD ultrafiltration membrane. The product retained by the ultrafiltration membrane is collected to obtain the virus concentrate.

[0044] S4. One-step purification: The virus concentrate is purified by centrifugation using a sucrose density gradient centrifugation method; specifically, the concentrated virus solution is subjected to continuous flow sucrose rate zonal centrifugation in a 0-55% sucrose solution. The centrifugation speed is 32,000 rpm, and the fraction with a sucrose concentration of 18%-47% is collected.

[0045] S5. Two-step purification: First, the virus solution purified in the first step is ultrafiltered with a 300KD ultrafiltration membrane to remove sugar. Then, it is purified by column chromatography using Sepharose-4FF gel as the medium. The sample volume loaded each time does not exceed 8% of the column volume, and the sample loading rate is 150mL / min. After the sample is injected, pH7.2 PBS solution is pumped in for elution at a rate of 400mL / min (elution linear flow rate of 6mm / min). The virus peak is collected using a 280nm UV detector. The purified solution collected after chromatography is sampled for protein content detection. The protein content is controlled to be no higher than 1200μg / mL to obtain the purified virus solution after two steps.

[0046] S6. Virus lysis: The purified virus solution after two-step purification is lysed by adding a lysing agent; the lysing agent is Triton X-100, and the final concentration of Triton X-100 is 0.5wt%. The solution is placed in a constant temperature (22℃) shaker for 17 hours to obtain the virus lysate.

[0047] S7. Three-step purification: The virus lysate is subjected to continuous flow sucrose rate zonal centrifugation in a 0-55% sucrose solution at a speed of 32,000 rpm. The liquid segment with a sucrose concentration of 18%-42% is collected to obtain the purified virus lysate.

[0048] S8. After ultrafiltration to remove sugar from the purified virus lysate, it is then sterilized and filtered to obtain the monovalent stock solution.

[0049] Following the above method, monovalent stock solutions of prevalent viral types H1N1, H3N2, BY, and BV were prepared, yielding monovalent stock solutions of H1N1, H3N2, BY, and BV, respectively. The purity of each type's monovalent stock solution was determined by SDS-PAGE. The results are shown in Tables 1-2.

[0050] Table 1. Test results of unit price of each type of stock solution

[0051]

[0052] Table 2 Purity Analysis of Each Type of Stock Solution (SDS-PAGE Method)

[0053]

[0054] As shown in Table 1, the influenza antigen prepared in Example 1 has high purity and few impurities.

[0055] Example 2

[0056] This embodiment provides an adjuvant, the preparation method of which includes the following steps:

[0057] (1) Preparation of crude emulsion: Mixed oil and additives were mixed at a mass ratio of 9:1. The mixed oil included squalene, medium-chain triglycerides and α-tocopherol in a mass ratio of 1.3:1:0.6. The additives included sorbitan trioleate Span-85, Tween80 and vitamin E succinate polyethylene glycol in a mass ratio of 1.5:1:0.4. The mixture was stirred at 200 rpm until a homogeneous oil phase was formed. Citrate buffer was added to the emulsifier and the speed of the emulsifier was set to 15000 rpm. The oil phase (citrate buffer: oil phase mass ratio of 19:1) was injected into the citrate buffer and emulsified at high speed for 20 min to obtain crude emulsion.

[0058] (2) High-pressure emulsification: The crude emulsion was transferred to a high-pressure microfluidic device and the parameters were set as follows: microfluidic pressure 12000psi, flow rate 100mL / min; temperature control 25℃; homogenization cycle 5 times; after completion, it was filtered through a 0.22μm sterile filter to obtain the adjuvant.

[0059] Example 3

[0060] The difference between this embodiment and Example 2 is that the mixed oil includes squalene, medium-chain triglycerides and α-tocopherol in a mass ratio of 1.2:1:0.7; the additives include sorbitan trioleate Span-85, Tween80 and vitamin E succinate polyethylene glycol ester in a mass ratio of 1.4:1:0.5.

[0061] Example 4

[0062] The difference between this embodiment and Example 2 is that the mixed oil includes squalene, medium-chain triglycerides and α-tocopherol in a mass ratio of 1.4:1:0.5; the additives include sorbitan trioleate Span-85, Tween80 and vitamin E succinate polyethylene glycol ester in a mass ratio of 1.6:1:0.3.

[0063] The particle size of the adjuvants in Examples 2-4 was determined using a nanoparticle size and zeta potential analyzer (results are shown in Table 3 and ). Figure 1 The particle size ranges from 169.8 to 170.9 nm, with an average of about 170.2 nm; the polydispersity index is between 0.103 and 0.172, which is less than 0.2, indicating a highly uniform particle distribution; the D50 (median particle size) is 155.6-161.8 nm, with an average of 159.4 nm, indicating a concentrated particle size.

[0064] Table 3. Adjuvant particle size detection results for Examples 2-4

[0065]

[0066] Comparative Example 1

[0067] The difference between this comparative example and Example 2 is that the mixed oil is squalene.

[0068] Comparative Example 2

[0069] The difference between this comparative example and Example 2 is that the mixed oil includes squalene and medium-chain triglycerides in a mass ratio of 1.4:1.

[0070] Comparative Example 3

[0071] The difference between this comparative example and Example 2 is that the mixed oil includes squalene, medium-chain triglycerides and α-tocopherol in a mass ratio of 1:1:1.

[0072] Comparative Example 4

[0073] The difference between this comparative example and Example 2 is that the additive is sorbitan trioleate Span-85.

[0074] Comparative Example 5

[0075] The difference between this comparative example and Example 2 is that the additives include sorbitan trioleate Span-85 and Tween80 in a mass ratio of 1.5:1.

[0076] Comparative Example 6

[0077] The difference between this comparative example and Example 2 is that the additives include sorbitan trioleate Span-85, Tween80 and vitamin E succinate polyethylene glycol ester in a mass ratio of 1:1:1.

[0078] Comparative Example 7

[0079] The difference between this comparative example and Example 2 is that the crude emulsion was transferred to a high-pressure microfluidic device, and the parameters were set as follows: microfluidic pressure 12000psi, flow rate 60mL / min.

[0080] Comparative Example 8

[0081] The difference between this comparative example and Example 2 is that the crude emulsion was transferred to a high-pressure microfluidic device, and the parameters were set as follows: microfluidic pressure 12000psi, flow rate 180mL / min.

[0082] Example 10: Quadrivalent adjuvant influenza vaccine with standard antigen dosage

[0083] This embodiment provides an adjuvanted influenza vaccine. Based on a preparation volume of 200 mL, its composition is as follows: the hemagglutinin (H1N1 and H3N2 types) from the monovalent influenza A stock solution prepared in Example 1 and the hemagglutinin (BV and BY types) from the monovalent influenza B stock solution are mixed evenly at 60 μg / mL / subtype. 100 mL is then added to an equal volume of the adjuvant from Example 2, and after being mixed evenly, the mixture is passed through a 0.22 μm sterile filter to obtain the adjuvanted influenza vaccine.

[0084] Example 11: 1 / 2 standard antigen dose quadrivalent adjuvant influenza vaccine

[0085] This embodiment provides an adjuvant influenza vaccine, which is composed of the following components based on a 200mL preparation volume: the hemagglutinin (H1N1 and H3N2 types) from the monovalent influenza A stock solution prepared in Example 1 and the hemagglutinin (BV and BY types) from the monovalent influenza B stock solution are mixed evenly at 60ug / mL / subtype. 100mL is taken and 50mL of the adjuvant from Example 2 is added, and PBS buffer (pH 7.2) is added to make up to 200mL. The mixture is then mixed evenly and passed through a 0.22μm sterile filter.

[0086] Example 12: 1 / 4 of the standard antigen dose of quadrivalent adjuvant influenza vaccine

[0087] This embodiment provides an adjuvant influenza vaccine, which is composed of the following components based on a 200mL preparation volume: the hemagglutinin (H1N1 and H3N2 types) from the monovalent influenza A stock solution prepared in Example 1 and the hemagglutinin (BV and BY types) from the monovalent influenza B stock solution are mixed evenly at 60ug / mL / subtype. 100mL is taken and 25mL of the adjuvant from Example 2 is added, and PBS buffer (pH 7.2) is added to make up to 200mL. After mixing evenly, the mixture is filtered through a 0.22μm sterile filter.

[0088] Example 13: Trivalent Adjuvant Influenza Vaccine with Standard Antigen Dosage

[0089] This embodiment provides an adjuvant influenza vaccine. Based on a preparation volume of 200 mL, its composition is as follows: the hemagglutinin (H1N1 and H3N2 types) from the monovalent influenza A stock solution prepared in Example 1 and the hemagglutinin (BV type) from the monovalent influenza B stock solution are mixed evenly at a ratio of 60 μg / mL / subtype. 100 mL is then added to an equal volume of the adjuvant from Example 2, and after being mixed evenly, the mixture is passed through a 0.22 μm sterile filter.

[0090] Example 14: 1 / 2 standard antigen dose trivalent adjuvant influenza vaccine

[0091] This embodiment provides an adjuvant influenza vaccine, which is composed of the following components based on a 200mL preparation volume: the hemagglutinin (H1N1 and H3N2 types) from the monovalent influenza A stock solution prepared in Example 1 and the hemagglutinin (BV type) from the monovalent influenza B stock solution are mixed evenly at 40ug / mL / subtype. 100mL is taken and 50mL of the adjuvant from Example 2 is added, and PBS buffer (pH 7.2) is added to make up to 200mL. After mixing evenly, the mixture is filtered through a 0.22μm sterile filter.

[0092] Example 15: 1 / 4 of the standard antigen dose trivalent adjuvant influenza vaccine

[0093] This embodiment provides an adjuvant influenza vaccine, which is composed of the following components based on a 200mL preparation volume: the hemagglutinin (H1N1 and H3N2 types) from the monovalent influenza A stock solution prepared in Example 1 and the hemagglutinin (BV type) from the monovalent influenza B stock solution are mixed evenly at 60ug / mL / subtype. 100mL is taken and 25mL of the adjuvant from Example 2 is added, and PBS buffer (pH 7.2) is added to make up to 200mL. After mixing evenly, the mixture is filtered through a 0.22μm sterile filter.

[0094] Comparative Example 10

[0095] The difference between this comparative example and Example 12 is that the adjuvant in Example 2 was replaced with the adjuvant in Comparative Example 1.

[0096] Comparative Example 11

[0097] The difference between this comparative example and Example 12 is that the adjuvant in Example 2 was replaced with the adjuvant in Comparative Example 2.

[0098] Comparative Example 12

[0099] The difference between this comparative example and Example 12 is that the adjuvant in Example 2 was replaced with the adjuvant in Comparative Example 3.

[0100] Comparative Example 13

[0101] The difference between this comparative example and Example 12 is that the adjuvant in Example 2 was replaced with the adjuvant in Comparative Example 4.

[0102] Comparative Example 14

[0103] The difference between this comparative example and Example 12 is that the adjuvant in Example 2 was replaced with the adjuvant in Comparative Example 5.

[0104] Comparative Example 15

[0105] The difference between this comparative example and Example 12 is that the adjuvant in Example 2 was replaced with the adjuvant in Comparative Example 6.

[0106] Comparative Example 16

[0107] The difference between this comparative example and Example 12 is that the adjuvant in Example 2 was replaced with the adjuvant in Comparative Example 7.

[0108] Comparative Example 17

[0109] The difference between this comparative example and Example 12 is that the adjuvant in Example 2 was replaced with the adjuvant in Comparative Example 8.

[0110] Performance testing

[0111] Animal immune evaluation experiment

[0112] (1) Select 6-8 week old female BALB / c mice, divide them into groups of 10 mice each, and immunize them according to the table below. GDK-AD01 adjuvant is the adjuvant prepared in Example 2.

[0113] Table 4 Immunization methods

[0114] (2) Blood collection from the orbital cavity of mice

[0115] Before blood collection during the experiment, the weight of each mouse was measured and their physical condition was observed and recorded. Blood was collected from the orbital rim of each group of mice 28 days after the initial immunization. Each group consisted of 10 mice, divided into 5 pairs, with two mice per pair. The blood was mixed with serum at a 1:1 ratio and stored at -80℃ for later use.

[0116] (3) Immunogenicity evaluation

[0117] Serum was separated from the collected whole blood, and its titer was determined using the hemagglutination inhibition test. Serum seroconversion rate (hemagglutination inhibition titer ≥40, considered positive serum; titer <40, considered negative serum) and other indicators were calculated. Table 2 shows the GDK-AD01 adjuvant prepared in Example 2, used to determine the efficacy of vaccines prepared using the monovalent stock solution from Example 1 and the adjuvant from Example 2. See Table 5 for details.

[0118] Table 5 shows the average titers of vaccines prepared using the adjuvant from Example 2.

[0119]

[0120] Note: The "-" in the table indicates that the trivalent influenza vaccine has not undergone By-type hemagglutination inhibition antibody titer determination.

[0121] 28 days after immunization, all animals in each group showed normal growth, and their weight was higher than before immunization. (Based on Table 5 and...) Figure 2 It can be seen that the antibody titers of different GDK-AD01 (Example 2) adjuvant groups (experimental group) and those who received the quadrivalent influenza virus split vaccine and the trivalent influenza virus vaccine (control group) were significantly increased, and the 1 / 4 GDK-AD01 adjuvant group could significantly enhance immunogenicity.

[0122] 2. The average titer of the 1 / 4 standard antigen amount quadrivalent adjuvant influenza vaccine from Comparative Examples 10-18 was tested and compared with that of the vaccine prepared in Example 12. The results are shown in Table 6.

[0123] Table 6 shows the average titers of 1 / 4 standard antigen dose quadrivalent adjuvant influenza vaccines (Comparative Examples 10-18) against different monovalent stock solutions.

[0124]

[0125] The results show that the vaccine prepared with 1 / 4 of the conventional antigen amount in Example 12 has excellent efficacy against all four viruses.

[0126] Comparative examples 1-6 show that the composition of the oil phase and the adjuvants have a significant impact on vaccines. Analysis indicates that the mixed oil combines the immunostimulatory properties of squalene, the process-friendly nature of medium-chain triglycerides, and the stability and immunomodulatory effects of α-tocopherol, resulting in a superior and more stable composite oil phase system with better synergistic effects. The compounded adjuvants not only ensure basic emulsification but also endow the adjuvants with active immunomodulation and efficient delivery capabilities. Through the synergistic effect of the composite oil phase and adjuvants, HAI titers are significantly improved, producing high levels of protective antibodies against all four subtypes: H1N1, H3N2, BV, and BY.

[0127] Furthermore, as shown in Table 6, changes in the oil phase significantly reduced the vaccine's titer against H3N2, while changes in the adjuvants significantly reduced the vaccine's titer against BV. Only under the formulation conditions of Example 12 of this invention could the synergistic regulation of the vaccine's effects against four viruses, especially H3N2 and BV, be achieved. This is because the physicochemical interactions between adjuvants and different antigens differ, and the addition of adjuvants has varying effects on enhancing the immune response to different subtypes.

[0128] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An adjuvanted influenza vaccine, characterized in that, The product includes a tetravalent influenza virus stock solution, an adjuvant, and a PBS buffer solution; the tetravalent influenza virus stock solution comprises four monovalent influenza virus stock solutions, namely H1N1 monovalent stock solution, H3N2 monovalent stock solution, BY monovalent stock solution, and BV monovalent stock solution; The preparation method of the adjuvant includes the following steps: (1) Preparation of crude emulsion: Mixed oil and additives were mixed at a mass ratio of 9:

1. The mixed oil included squalene, medium-chain triglycerides and α-tocopherol in a mass ratio of 1.3:1:0.

6. The additives included sorbitan trioleate Span-85, Tween80 and vitamin E succinate polyethylene glycol in a mass ratio of 1.5:1:0.

4. The mixture was stirred at 200 rpm until a homogeneous oil phase was formed. Citrate buffer was added to the emulsifier and the speed of the emulsifier was set to 15000 rpm. The oil phase was injected into the citrate buffer. The mass ratio of citrate buffer to oil phase was 19:

1. The mixture was sheared and emulsified for 20 min to obtain crude emulsion. (2) High-pressure emulsification: The crude emulsion was transferred to a high-pressure microfluidic device and the parameters were set as follows: microfluidic pressure 12000psi, flow rate 100mL / min; temperature control 25℃; homogenization cycle 5 times; after completion, it was filtered through a 0.22μm sterile filter to obtain the adjuvant.

2. The adjuvanted influenza vaccine according to claim 1, characterized in that, The preparation method of the monovalent influenza virus stock solution includes the following steps: S1. Prepare harvest fluid using popular virus strains; S2. Inactivate the virus in the harvested liquid; S3. Ultrafiltration Concentration: The inactivated harvest liquid is concentrated by ultrafiltration using an ultrafiltration membrane, and the product retained by the ultrafiltration membrane is collected to obtain a concentrated virus solution. S4. One-step purification: The virus concentrate was purified by centrifugation using the sucrose density gradient centrifugation method to obtain the virus solution purified in one step. S5. Two-step purification: First, the virus solution purified in the first step is ultrafiltered through an ultrafiltration membrane to remove sugar, and then purified by column chromatography to obtain the purified virus solution after two steps. S6. Virus lysis: Add lysis agent to the purified virus solution after two-step purification to lyse and obtain virus lysis solution; S7. Three-step purification: The viral lysate is purified again by centrifugation using a sucrose density gradient to obtain the purified viral lysate. S8. After ultrafiltration to remove sugar from the purified virus lysate, it is then sterilized and filtered to obtain the monovalent stock solution.

3. A method for preparing an adjuvanted influenza vaccine according to any one of claims 1-2, characterized in that, Includes the following steps: Adjuvants and PBS buffer were added to the tetravalent influenza virus stock solution to obtain a semi-finished product. The semi-finished product was then filtered to obtain the adjuvanted influenza vaccine.

4. The method for preparing an adjuvanted influenza vaccine according to claim 3, characterized in that, The amount of adjuvant added to the semi-finished product is 12.5-50 (v / v)%.

5. The use of the adjuvanted influenza vaccine of any one of claims 1-2 in the preparation of an adjuvanted influenza vaccine that enhances the immune effect on the elderly population.

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