Tetravalent influenza virus subunit vaccine and preparation method thereof

Abstract

The invention discloses a tetravalent influenza virus subunit vaccine and a preparation method thereof, and relates to the technical field of biology, each dose of the tetravalent influenza virus subunit vaccine comprises two influenza A viruses and two influenza B viruses, the tetravalent influenza virus subunit vaccine is prepared by virus inoculation, virus multiplication culture, allantoic fluid harvesting, clarification, inactivation, ultrafiltration concentration, splitting and ultracentrifugation purification, mixing, filtration sterilization, split charging and packaging. Inactivation comprises the following steps: adding a cross-linked carboxymethyl glucan solution into the monovalent virus harvesting liquid, adding formaldehyde, and inactivating at 5 DEG C for 45 hours; then genipin is mixed into the virus liquid, the stirring speed is 200 rpm, the stirring time is 8 h under the conditions that the pH is 7.2 and the temperature is 25 DEG C, residues of free methanol can be further reduced, migration of small molecules is inhibited, and the long-term storage stability is guaranteed.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a tetravalent influenza virus subunit vaccine and a preparation method thereof. Background Art

[0002] The existing vaccines on the market are inactivated whole influenza virus vaccines and split influenza virus vaccines. The traditional purification method for split vaccines is as follows: the clarified inactivated monovalent harvest fluid is ultrafiltered and concentrated, purified by sucrose density gradient centrifugation, ultrafiltered to remove sucrose, lysed, purified again by sucrose density gradient centrifugation, and ultrafiltered again to remove sucrose, followed by sterile filtration, and thimerosal is added as a preservative. The process is complex, the steps are cumbersome, it consumes a large amount of manpower and material resources, and increases the risk of microbial contamination.

[0003] For example, in a tetravalent influenza virus subunit vaccine and a preparation method thereof with the Chinese patent application number CN202010392775.3. In the inactivated virus stage of the described tetravalent influenza virus subunit vaccine, a carboxymethyl dextran solution with a substitution degree of 0.5 - 0.7 is first added, and then low-concentration formaldehyde is added for short-time inactivation. The carboxyl group (-COOH) of carboxymethyl dextran can react with the aldehyde group (-CHO) of formaldehyde to reduce the excessive cross-linking of free formaldehyde and virus antigens, thereby protecting antigenic epitopes. Compared with the traditional high-concentration formaldehyde long-time inactivation, this step reduces antigen damage through chemical synergy and reduces the residual free formaldehyde.

[0004] However, due to the random distribution of carboxymethyl substitution on the dextran molecular chain during the synthesis process, the substitution degree may be too high (>0.7) in some local areas while insufficient (<0.5) in other areas, resulting in uneven overall function. Moreover, the Schiff base formed by the condensation reaction of the carboxyl group in carboxymethyl glucose and formaldehyde is easily hydrolyzed in an acidic or high-temperature environment after inactivation, releasing formaldehyde again, resulting in a rebound in the content of free formaldehyde in the final vaccine. Summary of the Invention

[0005] By providing a tetravalent influenza virus subunit vaccine and a preparation method thereof in the embodiments of the present application, the problems of excessive formaldehyde caused by carboxymethyl chitosan and unstable vaccine properties caused by unstable substitution degree in the prior art are solved, and a safer and more stable vaccine is achieved.

[0006] The embodiments of the present application provide a preparation method of a quadrivalent influenza virus subunit vaccine, each dose of which contains two influenza A viruses and two influenza B viruses. The quadrivalent influenza virus subunit vaccine is prepared by virus inoculation, virus proliferation culture, allantoic fluid harvesting, clarification, inactivation, ultrafiltration concentration, lysis and ultracentrifugation purification, mixing, filtration sterilization, subpackaging and packaging; inactivation is carried out by adding a cross-linked carboxymethyl dextran solution to the monovalent virus harvest fluid, and then adding formaldehyde to inactivate at 5°C for 45 h; then genipin is mixed into the virus solution, the stirring speed is 200 rpm, and the stirring time is 8 h under the conditions of pH 7.2 and 25°C.

[0007] Further, the concentration of the cross-linked carboxymethyl dextran solution is 1-3% w / v.

[0008] Further, the final concentration of genipin is 0.05-0.5% w / v.

[0009] Further, the preparation method of the cross-linked carboxymethyl dextran solution is as follows: dextran and chloroacetic acid are reacted at a molar ratio of 1:2 under alkaline conditions at 50°C for 5 hours; after neutralization, dialysis purification is carried out, and the degree of substitution is controlled at 0.6 to obtain carboxymethyl dextran. Genipin is added to the carboxymethyl dextran solution, and the reaction is stirred at pH 7.4 and 37°C for 12 hours. After the reaction, unreacted genipin is removed by ultrafiltration to prepare the cross-linked carboxymethyl dextran solution.

[0010] Further, liposome-encapsulated α-tocopherol is added before ultrafiltration concentration after inactivation: the fully inactivated influenza virus allantoic harvest fluid is added with liposome-encapsulated α-tocopherol and stirred for 45 minutes.

[0011] Further, the final concentration of the liposome is 0.5-2.0% w / v.

[0012] Further, the preparation method of the liposome-encapsulated α-tocopherol is as follows: hydrogenated soy phosphatidylcholine, cholesterol, and α-tocopherol are weighed and dissolved in a chloroform-methanol mixed solvent. The solution is transferred to a round-bottom flask, and the solvent is removed by rotary evaporation to form a uniform lipid film; pH 7.0 phosphate buffer solution is added, and hydration is carried out at 55°C for 30 minutes. Probe sonication is carried out, and sterilization is carried out through a 0.22 μm filter membrane to obtain liposome-encapsulated α-tocopherol.

[0013] Further, the liposome-encapsulated α-tocopherol includes large liposomes and small liposomes; the average particle size of the large liposomes is 200 nm, which is made of DSPC, cholesterol, and α-tocopherol, and the α-tocopherol loading amount is 6% w / w. The average particle size of the small liposomes is 80 nm, which is made of DOPE, cholesterol, and mannose-PEG-DSPE, and the mannose density is 12-15 molecules / liposome.

[0014] Furthermore, the lysis step uses lysis agent micelles, including small lysis agent micelles and large lysis agent micelles. The small lysis agent micelles have a particle size of 5 - 10 nm and are made of vitamin E polyethylene glycol succinate and nonoxynol. The lysis agent concentration is 0.3 - 0.8% w / v. The large lysis agent micelles have a particle size of 15 - 25 nm and are made of nonoxynol-9 and phosphatidylglycerol. The lysis agent concentration is 0.5 - 1.0% w / v. The volume ratio of large liposomes: small liposomes: small lysis agent micelles: large lysis agent micelles is 3:2:1:1.

[0015] A tetravalent influenza virus subunit vaccine is prepared by any of the above preparation methods.

[0016] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages: First, Genipin is the product of the hydrolysis of geniposide by β-glucosidase. Through an atomic-level nucleophilic crosslinking network, it forces the CM-dextran molecular chain to stretch, improves the uniformity of carboxymethyl substitution, converts the Schiff base into a stable secondary amine structure, and locks formaldehyde through hydrogen bonding and hydrophobic interaction.

[0017] Second, α-tocopherol encapsulated in liposomes forms a multifunctional network with Genipin and CM-dextran through free radical scavenging and interfacial synergy. The dual mechanisms of physical isolation and chemical neutralization reduce the residual risk; the combination of antioxidant and conformational stability enhances immunogenicity; the interpenetrating structure inhibits the migration of small molecules and ensures long-term storage stability.

[0018] Third, the long-chain phospholipids (C16 of DSPC) of large liposomes are preferentially adsorbed on the lipid layer of the virus envelope through hydrophobic interaction, reducing contact with the tails of small lysis agent micelles (short-chain C12) and lowering the probability of membrane damage. The phytol side chain (C20) of α-tocopherol inserts into the hydrophobic core of large liposomes to stabilize the structure through van der Waals forces; the mannose modification (negative charge) on the surface of small liposomes and the dendritic cell surface receptor (positive charge) enhance targeting through electrostatic attraction, and at the same time form charge repulsion with large lysis agent micelles (neutral), reducing non-specific binding. Detailed implementation manners

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this invention belongs; the terms used in the description of the present invention in this specification are only for the purpose of describing specific implementation manners and are not intended to limit the present invention; the term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0020] Example 1: A quadrivalent influenza virus subunit vaccine, each dose of which contains two influenza A viruses (H1N1 and H3N2) and two influenza B viruses (Victoria lineage and Yamagata lineage). The quadrivalent influenza virus subunit vaccine is prepared by virus inoculation, virus propagation and culture, allantoic fluid harvest, clarification, inactivation, ultrafiltration and concentration, lysis and ultracentrifugation purification, mixing, filtration sterilization, filling and packaging; Specifically: S1. Virus inoculation: Inoculate the influenza virus working seed into the allantoic cavity of 10-day-old healthy chicken embryos; S2. Virus propagation and culture: Transfer the inoculated chicken embryos to an incubator at 34 °C and culture for 48 hours to propagate the influenza virus; S3. Allantoic fluid harvest: Screen the live chicken embryos, place them at 2 °C for 16 hours for cold embryos, and then harvest the allantoic fluid; S4. Clarification: Remove most impurities such as chicken embryo erythrocytes by centrifugation; S5. Inactivation: Add a cross-linked carboxymethyl dextran solution (final concentration 1-3% w / v) to the monovalent virus harvest fluid, and then add formaldehyde (final concentration 100 mg / ml) to inactivate at 5 °C for 45 h; then mix genipin into the virus solution, stir at a speed of 200 rpm, and stir at pH 7.2 and 25 °C for 8 h. The final concentration of genipin is 0.05-0.5% (w / v).

[0021] The preparation method of the cross-linked carboxymethyl dextran solution is as follows: React dextran (molecular weight 50-100 kDa) with chloroacetic acid at a molar ratio of 1:2 under alkaline conditions (NaOH concentration 2.5 M) at 50 °C for 5 hours; after neutralization, dialyze and purify to control the degree of substitution at 0.6 to obtain carboxymethyl dextran. Add genipin (0.1-0.5% w / w) to the carboxymethyl dextran (CM-dextran) solution, stir and react at pH 7.4 and 37 °C for 12 hours, and ultrafilter (cut-off molecular weight 10 kDa) after the reaction to remove the unreacted genipin to obtain cross-linked CM-dextran (X-CM-dextran); S6. Ultrafiltration and concentration: Ultrafilter and concentrate the fully inactivated influenza virus allantoic harvest fluid with an ultrafiltration membrane; among them, influenza A H1N1 uses an ultrafiltration membrane with a cut-off molecular weight of 1000 KD for ultrafiltration and concentration, influenza A H3N2 uses a 300 KD ultrafiltration membrane for ultrafiltration and concentration, and influenza B virus uses an ultrafiltration membrane with a cut-off molecular weight of 1000 KD for ultrafiltration and concentration; S7. Lysis and ultracentrifugation purification: Add the lysis agent nonoxynol-9 to the ultrafiltered concentrate, mix evenly and load the sample, and perform synchronous lysis during sucrose density zone centrifugation, and harvest the lysate according to the sucrose concentration; Among them, the H1N1 virus is lysed with 1% lysis solution for 4 hours and finally 10-20% of the lysis solution is harvested; the H3N2 virus is lysed with 0.5% lysis solution for 10 hours and finally 15-30% of the lysis solution is harvested; the B virus is lysed with 1.5% lysis solution for 8 hours and finally 10-30% of the lysis solution is harvested. S8. Gel filtration chromatography purification: The solution obtained from the previous step of sucrose density gradient centrifugation is further purified by a Sephadex G-25 dextran gel filtration chromatography system to obtain the monovalent stock solution of the influenza virus subunit vaccine.

[0022] Then, the monovalent stock solutions of H1N1, H3N2 and two B-type influenza viruses prepared are mixed according to the relationship of hemagglutinin concentration of 1:1:1:1. The hemagglutinin concentration of each type of virus is controlled between 40-48 μg / ml. Then, after filtration and sterilization through a 0.22 μm filter membrane, it is filled into ampoules. Each ampoule is 0.5 ml, and the single human dose is 0.5 ml. After passing the lamp inspection, the tetravalent influenza virus subunit vaccine is obtained.

[0023] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages: Genipin is the product of the hydrolysis of geniposide by β-glucosidase. Through an atomic-level nucleophilic cross-linking network, the CM-dextran molecular chain is forced to stretch, the uniformity of carboxymethyl substitution is improved, the Schiff base is converted into a stable secondary amine structure, and formaldehyde is locked through hydrogen bonds and hydrophobic interactions.

[0024] Genipin contains an epoxide group, an ester group, and an allylic hydroxyl group. The oxygen atom in the epoxide group (C1-O-C2) has a high electronegativity, making C1 and C2 strong electrophilic centers and easily attacked by nucleophilic groups (such as amino groups and carboxyl groups). When the carboxyl group (-COOH) of CM-dextran attacks C1 of the epoxide group, the C1-O bond breaks, generating an ether bond (C1-O-CM-dextran) and a hydroxyl group (C2-OH). The carbonyl oxygen (O) in the ester group (C10-O-CO-R) polarizes the C-O bond through the inductive effect, causing it to hydrolyze to generate a carboxylic acid group (-COOH) under alkaline conditions, enhancing the reaction activity. In an inactivated environment with a pH of 7.4, the ester group hydrolyzes to generate a carboxylic acid group (-COOH), and the oxygen atom of the carboxylic acid forms a hydrogen bond network with the hydroxyl group (-OH) of CM-dextran. The hydroxyl group (C9-OH) binds to the carboxyl group of CM-dextran or water molecules through hydrogen bonds to assist in the directional arrangement of molecules.

[0025] The epoxide of genipin reacts with the carboxyl groups of two CM-dextran chains to form an intermolecular crosslink (CM-dextran-O-C1-C2-OH-CM-dextran), forcing the dextran chains to change from a coiled conformation to an extended conformation. In the extended state, the C6-OH sites (carboxymethyl substitution sites) of dextran are fully exposed, improving the uniformity of the substitution reaction. The crosslinking network restricts the local folding of dextran chains, avoiding the formation of high-substitution regions (>0.7) and low-substitution regions (<0.5). In the Schiff base (R-N=CH2) formed by the condensation of CM-dextran and formaldehyde, the nitrogen atom of the imino group (-N=CH2) attacks the C2 of the genipin epoxide to form a secondary amine bond (R-NH-CH2-O-C11H13O4), blocking the hydrolysis path. This reaction converts the unstable Schiff base into an irreversible secondary amine-epoxy adduct, completely fixing formaldehyde. The network formed by genipin crosslinking wraps the Schiff base sites, preventing water molecules from contacting and inhibiting the hydrolysis reaction, preventing formaldehyde from reverting to the free state.

[0026] The carboxylic acid groups (-COOH) generated by the hydrolysis of genipin form hydrogen bonds with the hydroxyl groups (-OH) of CM-dextran, enhancing the rigidity of the crosslinked structure. The cyclopentane hydrophobic core of genipin binds to the hydrophobic region (unsubstituted glucose units) of CM-dextran through van der Waals forces, stabilizing the crosslinked structure. During the inactivation stage (pH 7.2), the crosslinking network remains stable. If the system is accidentally acidified, the genipin crosslinking bonds can still maintain the structural integrity. The crosslinking network contracts under storage conditions of 2-8°C, further compressing the formaldehyde diffusion path. It expands slightly at 37°C (the temperature of immune response), without affecting antigen release.

[0027] The crosslinking network forces the CM-dextran chains to be linearly arranged, reducing the steric hindrance of the substitution reaction and improving the uniformity of carboxymethyl distribution. Genipin consumes free metal ions (such as Fe 3+ in the reaction system, reducing the deviation of the degree of substitution caused by oxidative side reactions, and converting the Schiff base into an irreversible secondary amine structure.

[0028] Verify the ability of genipin to improve the uniformity of the degree of substitution of carboxymethyl dextran (CM-dextran) and the ability of genipin to stabilize the Schiff base formed by formaldehyde (HCHO) and CM-dextran, through 1H-NMR analysis (400 MHz) of cross-linked carboxymethyl dextran solution was performed to calculate the degree of carboxymethyl substitution and standard deviation (SD). Among them, in the control group (without genipin): dextran (50 kDa) and chloroacetic acid (molar ratio 1:2.5) were reacted under alkaline conditions (2.5 M NaOH, 50 °C) for 5 hours, and then purified by dialysis. In the experimental groups: after the above reaction was completed, 0.1%, 0.3%, 0.5% (w / w) genipin were added respectively, and the cross-linking reaction was carried out at 37 °C for 12 hours, followed by ultrafiltration purification. The results are shown in Table 1; Free formaldehyde in the allantoic fluid harvest of inactivated influenza virus was quantified by HPLC (C18 column, mobile phase acetonitrile - water = 6:4, detection wavelength 360 nm). The experimental procedure was to react CM-dextran (degree of substitution 0.6) with formaldehyde (final concentration 100 mg / mL) in a pH 7.4 buffer for 24 hours to form Schiff base. The control group was not treated with genipin, and 0.05%, 0.1%, 0.2% (w / v) genipin were added to the experimental groups respectively, and the reaction was carried out at 25 °C for 8 hours. The results are shown in Table 2; Table 1 Uniformity of the degree of substitution of carboxymethyl dextran (CM-dextran)

[0029] Table 2 Stabilization ability of Schiff base

[0030] Then, the antibody titer experiment was carried out. The virus solution prepared from the medium-dose experimental group (cross-linking dose 0.3%, inactivation dose 0.1%) was diluted with PBS to make the hemagglutinin content 30 μg / ml. Ten healthy and clean-grade ISR mice weighing 16 - 18 g, with 5 males and 5 females, were inoculated intraperitoneally with 0.2 ml each. At the same time, another 10 mice were injected with PBS buffer as a negative control. After 21 days, blood was collected from the orbital cavity, serum was separated, non-specific inhibitors were removed by treatment with potassium periodate, the antibody titer was determined by hemagglutination inhibition test, and the HA content was determined by single radial immunodiffusion (SRID) method. The results are shown in Table 3. Among them, in the control group (without genipin): the H1N1 titer log2 = 7.5, the H3N2 titer log2 = 7.4, and the average titer of type B log2 = 7.3; Protection efficiency: (titer of genipin group - titer of control group) / titer of control group × 100%.

[0031] Table 3 Virus titer

[0032] Example 2: In the above example, genipin was used to force the CM-dextran molecular chain to stretch, improving the uniformity of carboxymethyl substitution. The Schiff base was converted into a stable secondary amine structure, and formaldehyde was locked through hydrogen bonding and hydrophobic interaction, reducing substitution non-uniformity on the one hand and reducing the free methanol on the other hand. However, as a crosslinking agent, genipin residue may also cause allergies. When the genipin residue content > 0.01%, it may cause redness and swelling at the injection site. Therefore, based on Example 1, further improvements were made.

[0033] Before ultrafiltration concentration after inactivation, liposome-encapsulated α-tocopherol (vitamin E) was also added: The allantoic fluid harvest of inactivated influenza virus was added to liposome-encapsulated α-tocopherol and stirred for 45 minutes (150 rpm, 25 °C). The final concentration of liposome: 0.5 - 2.0% (w / v) (α-tocopherol content was 0.1 - 0.4% w / w), and ultrafiltration concentration was carried out using an ultrafiltration membrane; The preparation method of liposome-encapsulated α-tocopherol was: Weigh 40 mg of hydrogenated soy phosphatidylcholine (HSPC), 10 mg of cholesterol, and 5 mg of α-tocopherol, dissolve them in a chloroform-methanol (3:1 v / v) mixed solvent, transfer the solution to a round-bottom flask, and remove the solvent by rotary evaporation (40 °C, 100 rpm) to form a uniform lipid film; Add phosphate buffer (pH 7.0) containing 5% sucrose, hydrate at 55 °C for 30 minutes, and treat with probe sonication (200 W, 5 s on / 5 s off, 5 cycles), and sterilize through a 0.22 μm filter membrane to obtain liposome-encapsulated α-tocopherol with an encapsulation efficiency > 85% and a particle size of 80 - 150 nm.

[0034] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages: Liposome-encapsulated α-tocopherol forms a multifunctional network with genipin and CM-dextran through free radical scavenging and interfacial synergy, reducing the residual risk through the dual mechanisms of physical isolation and chemical neutralization; jointly improving immunogenicity through antioxidant and conformational stability; and inhibiting the migration of small molecules through an interpenetrating structure to ensure long-term storage stability.

[0035] The phenolic hydroxyl group (-OH) on the chroman ring of α-tocopherol reduces the free radical (ROO·) in the lipid peroxidation chain reaction to a stable product (ROOH) through a single electron transfer (SET) mechanism, and is oxidized to tocopherol quinone itself; the phytyl side chain of tocopherol is embedded in the hydrophobic region of the liposome bilayer (the C16 alkyl chain of phosphatidylcholine) through van der Waals forces, stabilizing the liposome structure and prolonging the release time.

[0036] α-tocopherol captures the free radical generated in the genipin crosslinking reaction (·O generated by epoxy ring opening)- ), blocking its attack on the lipid bilayer of the cell membrane; the hydrophobic region of the liposome bilayer adsorbs unreacted genipin molecules through π-π stacking, restricting its contact with cell components and isolating allergens. The cross-linked network of CM-dextran (pore size 2 - 5 nm) and liposomes (particle size 50 - 100 nm) form an interpenetrating structure, with liposomes filling the network pores and blocking the diffusion path of formaldehyde. α-Tocopherol inhibits the oxidative cross-linking side reaction (disulfide bond formation) in the CM-dextran cross-linked network, protecting the conformational integrity of the viral antigen (HA protein).

[0037] The negative charge on the liposome surface (phosphate group of the phospholipid head) and the carboxyl group (-COO - of CM-dextran bind through electrostatic attraction. At the same time, the hydroxyl groups of the phospholipids and CM-dextran form hydrogen bonds, enhancing the stability of the complex. The hydrophobic microdomains of the CM-dextran cross-linked network and the hydrophobic tails of the liposomes bind through van der Waals forces, forming a continuous hydrophobic barrier to inhibit the migration of small molecules (formaldehyde, free radicals). The CM-dextran free radicals (COO·) are further neutralized by GSH (glutathione) in the aqueous phase, forming an antioxidant cycle.

[0038] The carboxyl group of CM-dextran and the phospholipid head of the liposome jointly encapsulate the antigenic epitope of the HA protein, reducing formaldehyde cross-linking damage and enhancing the lymphatic targeting of the antigen.

[0039] Based on the medium-dose group in Example 1 (cross-linking dose 0.3%, inactivation dose 0.1%), the antioxidant capacity and toxicity reduction were verified through experiments, including the free radical scavenging rate: the ability of α-tocopherol to scavenge free radicals detected by the DPPH method, cytotoxicity: the effect of genipin residue on the survival rate of HEK293 cells detected by the MTT method, and formaldehyde residue: HPLC quantitative analysis. The experimental sample was the allantoic harvest fluid of inactivated influenza virus mixed with liposomes for 45 minutes (150 rpm, 25°C) without purification, and the residue was simulated with a final genipin concentration of 0.01%. The results are shown in Table 4; Table 4 Antioxidant and Toxicity Reduction Experiments

[0040] Then, the antigen protection and immunogenicity of the vaccine were verified. The activity of the inactivated HA protein was detected by ELISA, and the hemagglutination inhibition test (HI) was performed 21 days after immunizing mice. The experimental procedure was the same as the antibody titer experiment in Example 1. The difference was that the same amount of genipin was used in this example (cross-linking dose 0.3%, inactivation dose 0.1%), but different concentrations of liposomes were used, and the vaccine titer was detected. The results are shown in Table 5; Table 5 Antigen Protection and Immunogenicity Verification

[0041] Example 3: In Example 2, α-tocopherol encapsulated in liposomes forms a multifunctional network with genipin and CM-dextran through free radical scavenging and interfacial synergy, reducing the residual risk through dual mechanisms of physical isolation and chemical neutralization; the combination of antioxidant and conformational stability enhances immunogenicity; the interpenetrating structure inhibits the migration of small molecules and ensures long-term storage stability. However, the subsequent addition of the lysing agent nonoxynol-9 will cause the hydrophobic tails of the liposome phospholipid bilayer (such as the C16 chain of DSPC) to bind through hydrophobic interactions, destroying the integrity of the liposome membrane, resulting in liposome rupture and α-tocopherol leakage. Therefore, based on Example 2, further improvements are made.

[0042] The liposome-encapsulated α-tocopherol includes large liposomes and small liposomes; the large liposomes have an average particle size of 200 nm and are made of DSPC, cholesterol, and α-tocopherol (molar ratio 4:1:0.5), and the α-tocopherol loading amount is 6% (w / w). The small liposomes have an average particle size of 80 nm and are made of DOPE, cholesterol, and mannose-PEG-DSPE (molar ratio 3:1:0.2), and the mannose density is 12 - 15 molecules / liposome; The lysis solution is replaced with lysis agent micelles, including small lysis agent micelles and large lysis agent micelles. The small lysis agent micelles have a particle size of 5 - 10 nm and are made of vitamin E polyethylene glycol succinate (TPGS) and nonoxynol (molar ratio 1:3), and the lysis agent concentration is 0.3 - 0.8% (w / v); the large lysis agent micelles have a particle size of 15 - 25 nm and are made of nonoxynol-9 and phosphatidylglycerol (molar ratio 2:1), and the lysis agent concentration is 0.5 - 1.0% (w / v); Large liposomes: small liposomes: small lysis agent micelles: large lysis agent micelles (volume ratio) = 3:2:1:1; The small liposomes are prepared by dissolving DOPE, cholesterol, and mannose-PEG-DSPE in chloroform-methanol (3:1 v / v), evaporating to form a film by rotary evaporation, adding pH 6.5 citrate buffer, hydrating at 40°C for 30 minutes, and controlling the particle size through microfluidic technology (flow rate ratio of aqueous phase: organic phase = 3:1). The small lysis agent micelles are prepared by dissolving nonoxynol-9 and TPGS in deionized water, magnetically stirring (500 rpm, 25°C) for 2 hours, and ultrasonic treatment (50 W, 30 seconds) to promote micelle formation; The large lysis agent micelles are prepared by dissolving nonoxynol-9 and phosphatidylglycerol in pH 7.0 Tris buffer, with a flow rate ratio of aqueous phase (buffer) to organic phase (ethanol) of 5:1, and dialyzing to remove ethanol after mixing.

[0043] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages: The long-chain phospholipids (C16 of DSPC) of the large liposomes are preferentially adsorbed on the lipid layer of the viral envelope through hydrophobic interaction, reducing contact with the tail of the small lytic agent micelles (short-chain C12) and lowering the probability of membrane damage. The phytyl side chain (C20) of α-tocopherol is inserted into the hydrophobic core of the large liposomes to stabilize the structure through van der Waals forces. The mannose modification (negative charge) on the surface of the small liposomes and the surface receptors (positive charge) of the dendritic cells enhance targeting through electrostatic attraction, while forming charge repulsion with the large lytic agent micelles (neutral) to reduce nonspecific binding.

[0044] Small lytic agent micelles rely on their high specific surface area and diffusion rate to preferentially penetrate the gaps in the viral envelope and release internal antigens; large lytic agent micelles are limited in volume and slowly release the lytic agent after binding to the viral envelope, reducing interference with liposomes. The α-tocopherol encapsulated in the large liposomes forms an "antioxidant barrier" to block free radical chain reactions. Small liposomes (80 nm) carry antigen fragments and target dendritic cells through mannose receptors to improve antigen presentation efficiency.

[0045] The size difference between large liposomes and small lytic agent micelles forms steric hindrance, reducing the probability of collision between the two. Small liposomes and large lytic agent micelles achieve selective binding through size matching to avoid mutual interference. Lytic agent micelles are preferentially enriched in the viral envelope area (high curvature surface), while liposomes are enriched in the low curvature area, forming functional partitions; large liposomes stabilize viral fragments through the "anchoring effect" to prevent antigen aggregation.

[0046] Small lytic agent micelles preferentially adsorb and lyse viruses due to their size matching the pores of the viral envelope, thereby reducing the contact time with liposomes; the difference in hydrophobic interaction between large liposomes and small lytic agent micelles triggers dynamic phase separation, forming a "liposome-lytic agent" bicontinuous phase, reducing molecular collisions; the thick membrane structure of large liposomes resists lytic agent insertion through high-density phospholipid arrangement, and α-tocopherol further fills the membrane gap to improve mechanical stability.

[0047] The small lytic agent micelles quickly release antigens, and the large liposomes simultaneously scavenge free radicals to protect the integrity of the HA epitope. The mannose targeting of the small liposomes is combined with the sustained release effect of the large lytic agent micelles to extend the antigen presentation window and increase the antibody titer.

[0048] The liposome leakage rate was tested by detecting free α-tocopherol in the lysate after lysis, and the overall antigen retention rate and antibody titer were verified simultaneously. The results are shown in Table 6; Table 6 Performance verification of Example 3

[0049] Example 4: In Example 3, liposomes of various particle sizes were combined with lysing agent micelles, reducing the breakage of liposomes. It was found in the study that controllable pores could be formed by utilizing the breakage of liposomes, achieving precise release of antigens in space and time.

[0050] Slowly add small lysing agent micelles (0.5% w / v) to the small liposome suspension (1.0% w / v) in advance, stir at 200 rpm at 25°C for 10 minutes, heat up to 37°C (±1°C), and continue stirring for 20 minutes to promote the insertion of the hydrophobic tail of the lysing agent into the liposome membrane. Add α-tocopherol (0.8% w / v) and let it stand at 4°C for 30 minutes to stabilize the pore structure. The insertion depth of the lysing agent is 2 - 5 nm to obtain modified small liposomes.

[0051] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages: The lysing agent micelles are adsorbed on the surface of the liposomes (electrostatic interaction). At this time, the membrane fluidity is low and the insertion is slow. After heating up to 37°C, the liposome membrane enters the liquid crystal state (phase transition temperature 32°C), the fluidity increases, and the hydrophobic tail of the lysing agent vertically inserts into the membrane to form a pore prototype. The insertion depth is regulated by the lysing agent and the heating rate (1°C / min) to limit the expansion of the pores (2 - 5 nm).

[0052] The pores respond to the low pH (5.0) environment of the endocytic vesicles on the surface of dendritic cells (DCs). The hydrophilic head of the micelles dissociates, and the instantaneous opening frequency increases to 5 times per second to achieve antigen release. The hydrophilic head of the lysing agent micelles is electrostatically bound to the antigen and dissociates through pH response during DC endocytosis, driving the efficient export of the antigen (non-passive diffusion); the hydrophobic chain of tocopherol cross-links with the membrane lipids to form a "molecular rivet" structure, enhancing the mechanical strength of the pores. Trehalose forms a glassy hydrated layer outside the liposomes to inhibit membrane fusion; tocopherol blocks the oxidative chain reaction, and the HA activity retains 93% after storage at 4°C for 6 months. Single particles achieve the integration of "carrier - releasing agent - targeting molecule" with three functions, increasing the uptake efficiency of dendritic cells, and ultimately making the protection duration of a single dose exceed 12 months, with the vaccine titer increasing by 26% compared to the vaccine prepared in Example 3.

[0053] The above is only the preferred embodiment of the present invention and is not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A preparation method of a tetravalent influenza virus subunit vaccine, characterized in that, Each dose contains two influenza A viruses and two influenza B viruses. The quadrivalent influenza virus subunit vaccine is prepared by virus inoculation, virus propagation and culture, allantoic fluid harvest, clarification, inactivation, ultrafiltration and concentration, lysis and ultracentrifugation purification, mixing, filtration and sterilization, filling and packaging; inactivation is carried out by adding a cross-linked carboxymethyl dextran solution to the monovalent virus harvest fluid, and then adding formaldehyde for inactivation at 5 °C for 45 h; then genipin is mixed into the virus solution, and the stirring speed is 200 rpm, and the stirring time is 8 h under the conditions of pH 7.2 and 25 °C.

2. The preparation method of a tetravalent influenza virus subunit vaccine according to claim 1, characterized in that, The concentration of the cross-linked carboxymethyl dextran solution is 1-3% w / v.

3. The preparation method of a tetravalent influenza virus subunit vaccine according to claim 1, characterized in that, The final concentration of genipin is 0.05-0.5% w / v.

4. The preparation method of a tetravalent influenza virus subunit vaccine according to claim 1, wherein, The preparation method of the cross-linked carboxymethyl dextran solution is as follows: dextran and chloroacetic acid are reacted at a molar ratio of 1:2 under alkaline conditions at 50 °C for 5 hours; after neutralization, dialysis purification is carried out to control the degree of substitution to 0.6 to obtain carboxymethyl dextran. Genipin is added to the carboxymethyl dextran solution, and the reaction is stirred at pH 7.4 and 37 °C for 12 hours. After the reaction, unreacted genipin is removed by ultrafiltration to prepare the cross-linked carboxymethyl dextran solution.

5. The preparation method of a tetravalent influenza virus subunit vaccine according to claim 1, characterized in that, Liposome-encapsulated α-tocopherol is also added before ultrafiltration and concentration after inactivation: the fully inactivated influenza virus allantoic harvest fluid is added with liposome-encapsulated α-tocopherol and stirred for 45 minutes.

6. The preparation method of a tetravalent influenza virus subunit vaccine according to claim 5, wherein The final concentration of the liposome is 0.5-2.0% w / v.

7. The preparation method of a tetravalent influenza virus subunit vaccine according to claim 5, wherein The preparation method of liposome-encapsulated α-tocopherol is as follows: hydrogenated soy phosphatidylcholine, cholesterol, and α-tocopherol are weighed and dissolved in a chloroform-methanol mixed solvent. The solution is transferred to a round-bottom flask, and the solvent is removed by rotary evaporation to form a uniform lipid film; phosphate buffer solution with pH 7.0 is added, and it is hydrated at 55 °C for 30 minutes, and then sonicated with a probe. Sterilization is carried out through a 0.22 μm filter membrane to obtain liposome-encapsulated α-tocopherol.

8. The preparation method of a tetravalent influenza virus subunit vaccine according to claim 5, characterized in that, Liposome-encapsulated α-tocopherol includes large liposomes and small liposomes; the average particle size of the large liposomes is 200 nm, which is made of DSPC, cholesterol, and α-tocopherol, and the α-tocopherol loading amount is 6% w / w. The average particle size of the small liposomes is 80 nm, which is made of DOPE, cholesterol, and mannose-PEG-DSPE, and the mannose density is 12-15 molecules / liposome.

9. The preparation method of a tetravalent influenza virus subunit vaccine according to claim 8, characterized in that, The lysis step uses lysis agent micelles, including small lysis agent micelles and large lysis agent micelles. The particle size of the small lysis agent micelles is 5-10 nm, which is made of vitamin E polyethylene glycol succinate and nonoxynol. The lysis agent concentration is 0.3-0.8% w / v; the particle size of the large lysis agent micelles is 15-25 nm, which is made of nonoxynol-9 and phosphatidylglycerol, and the lysis agent concentration is 0.5-1.0% w / v; the volume ratio of large liposomes: small liposomes: small lysis agent micelles: large lysis agent micelles is 3:2:1:

1.

10. A quadrivalent influenza virus subunit vaccine, characterized in that, Prepared by the preparation method described in any one of claims 1-9.

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