A quadrivalent influenza virus subunit vaccine and its preparation method

Abstract

The present application discloses a quadrivalent influenza virus subunit vaccine and a preparation method thereof, which relate to the field of biotechnology. Each dose contains two influenza A viruses and two influenza B viruses. The quadrivalent influenza virus subunit vaccine is prepared by virus inoculation, virus proliferation and culture, allantoic fluid harvesting, clarification, inactivation, ultrafiltration concentration, lysis and ultracentrifugation purification, mixing, filtration sterilization, subpackaging and packaging; inactivation is performed by adding a cross-linked carboxymethyl dextran solution to the monovalent virus harvest liquid, and then adding formaldehyde to inactivate at 5°C for 45 hours; then, genipin is mixed into the virus liquid, stirred at a speed of 200 rpm, and stirred for 8 hours at pH 7.2 and 25°C, which can further reduce the residual free methanol, inhibit the migration of small molecules, and ensure long-term storage stability.

Description

Technical Field

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

[0002] Available vaccines on the market include whole-virus inactivated influenza vaccines and split influenza virus vaccines. Traditional split vaccine purification methods involve ultrafiltration and concentration of the clarified inactivated monovalent harvest, purification by sucrose density zonal centrifugation, ultrafiltration to remove sucrose, lysis, further purification by sucrose density zonal centrifugation, ultrafiltration to remove sucrose again, sterilization filtration, and the addition of thimerosal as a preservative. This complex process, with numerous steps, consumes significant labor and resources, and increases the risk of microbial contamination.

[0003] For example, Chinese patent application number CN202010392775.3 describes a quadrivalent influenza virus subunit vaccine and its preparation method. The quadrivalent influenza virus subunit vaccine, during the virus inactivation stage, first adds a carboxymethyl dextran solution with a degree of substitution of 0.5-0.7, followed by a short-term inactivation with low-concentration formaldehyde. The carboxyl groups (-COOH) of the carboxymethyl dextran condense with the aldehyde groups (-CHO) of formaldehyde, reducing excessive cross-linking between free formaldehyde and viral antigens, thereby protecting antigenic epitopes. Compared to traditional high-concentration formaldehyde inactivation for a long time, this step reduces antigen damage and free formaldehyde residues through chemical synergy.

[0004] However, during the synthesis process, the carboxylmethyl substitution on the glucan molecular chain may be randomly distributed, resulting in high substitution in some local areas (>0.7) and insufficient substitution in other areas (<0.5), resulting in uneven overall functionality. In addition, the Schiff base generated by the condensation reaction of the carboxyl group in carboxymethyl glucose with formaldehyde is easily hydrolyzed in an acidic or high-temperature environment after inactivation, re-releasing formaldehyde, resulting in a rebound in the free formaldehyde content in the final vaccine. Summary of the Invention

[0005] The embodiments of the present application provide a quadrivalent influenza virus subunit vaccine and a preparation method thereof, thereby solving the problems of excessive formaldehyde caused by carboxymethyl chitosan and unstable substitution degree leading to unstable vaccine properties in the prior art, thereby achieving a safer and more stable vaccine.

[0006] The embodiments of the present application provide a method for preparing a quadrivalent influenza virus subunit vaccine, wherein each dose 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 performed by adding a cross-linked carboxymethyl dextran solution to the monovalent virus harvest liquid, and then adding formaldehyde to inactivate at 5°C for 45 hours; then, genipin is mixed into the virus liquid, stirred at a speed of 200 rpm, and stirred for 8 hours at pH 7.2 and 25°C.

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

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

[0009] Furthermore, 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, dialyzed and purified to control the degree of substitution to 0.6 to obtain carboxymethyl dextran, and genipin is added to the carboxymethyl dextran solution, stirred and reacted at pH 7.4 and 37°C for 12 hours, and after the reaction, unreacted genipin is removed by ultrafiltration to obtain a cross-linked carboxymethyl dextran solution.

[0010] Furthermore, liposome-encapsulated α-tocopherol was added after inactivation and before ultrafiltration concentration: the fully inactivated influenza virus allantoic cyst harvest was added to the liposome-encapsulated α-tocopherol and stirred for 45 minutes.

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

[0012] Furthermore, the preparation method of liposome-encapsulated α-tocopherol is as follows: hydrogenated soybean phosphatidylcholine, cholesterol, and α-tocopherol are weighed, 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 is added, hydrated at 55°C for 30 minutes, probe ultrasonicated, and sterilized through a 0.22 μm filter membrane to obtain liposome-encapsulated α-tocopherol.

[0013] Furthermore, 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, with an α-tocopherol loading of 6% w / w; the small liposomes have an average particle size of 80 nm and are made of DOPE, cholesterol, and mannose-PEG-DSPE, with a mannose density of 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, are made of vitamin E polyethylene glycol succinate and nonoxynol, 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, are 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.

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

[0016] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages:

[0017] First, genipin is the product of Gardenia jasminoides hydrolysis by β-glucosidase. Through the atomic-level nucleophilic cross-linking 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 bonds and hydrophobic interactions.

[0018] Secondly, liposome-encapsulated α-tocopherol forms a multifunctional network with genipin and CM-dextran through free radical scavenging and interface synergy, and the dual mechanisms of physical isolation and chemical neutralization reduce the risk of residues; antioxidant and conformational stabilization jointly enhance immunogenicity; the interpenetrating structure inhibits the migration of small molecules and ensures long-term storage stability.

[0019] Third, 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 reducing the probability of membrane damage. The phytyl side chain (C20) of α-tocopherol is inserted into the hydrophobic core of the large liposomes, stabilizing 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. DETAILED DESCRIPTION

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains; the terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only 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 associated listed items.

[0021] Example 1: A quadrivalent influenza virus subunit vaccine, each dose comprising two influenza A viruses (H1N1 and H3N2) and two influenza B viruses (Victoria strain and Yamagata strain), the quadrivalent influenza virus subunit vaccine being prepared by virus inoculation, virus proliferation culture, allantoic fluid harvesting, clarification, inactivation, ultrafiltration concentration, lysis and ultracentrifugation purification, mixing, filtration sterilization, aliquoting, and packaging;

[0022] Specifically:

[0023] S1. Virus inoculation: Inoculate the influenza virus working seed into the allantoic cavity of 10-day-old healthy chicken embryos;

[0024] S2. Virus proliferation culture: The inoculated chicken embryos were transferred to a 34°C incubator for 48 hours to propagate the influenza virus.

[0025] S3. Allantoic fluid harvesting: screen live chicken embryos and place them in a 2°C cold place for 16 hours to harvest the allantoic fluid;

[0026] S4, clarification: remove most impurities such as chicken embryo red blood cells by centrifugation;

[0027] S5. Inactivation: Add cross-linked carboxymethyl dextran solution (final concentration 1-3% w / v) to the monovalent virus harvest solution, then add formaldehyde (final concentration 100 mg / ml) to inactivate at 5°C for 45 hours; then mix genipin into the virus solution, stirring at 200 rpm, stirring for 8 hours at pH 7.2 and 25°C, to a final genipin concentration of 0.05-0.5% (w / v).

[0028] The cross-linked carboxymethyl dextran solution was prepared by reacting dextran (molecular weight 50-100 kDa) with chloroacetic acid in a molar ratio of 1:2 at 50°C under alkaline conditions (NaOH concentration 2.5 M) for 5 hours. After neutralization, the solution was purified by dialysis to control the degree of substitution to 0.6 to obtain carboxymethyl dextran. Genipin (0.1-0.5% w / w) was added to the carboxymethyl dextran (CM-dextran) solution, and the mixture was stirred at pH 7.4 and 37°C for 12 hours. After the reaction, unreacted genipin was removed by ultrafiltration (molecular weight cut-off 10 kDa) to obtain cross-linked CM-dextran (X-CM-dextran).

[0029] S6. Ultrafiltration and concentration: The fully inactivated influenza virus allantoic fluid was ultrafiltered and concentrated using an ultrafiltration membrane; for influenza A (H1N1), an ultrafiltration membrane with a molecular weight cutoff of 1000 kD was used; for influenza A (H3N2), an ultrafiltration membrane with a molecular weight cutoff of 300 kD was used; and for influenza B, an ultrafiltration membrane with a molecular weight cutoff of 1000 kD was used;

[0030] S7, lysis and ultracentrifugation purification: The ultrafiltration concentrate was added with the lysis agent nonoxynol-9, mixed evenly, and then loaded onto the sample. Synchronous lysis was performed during sucrose density zonal centrifugation, and the lysate was harvested according to the sucrose concentration.

[0031] The influenza A (H1N1) virus was lysed using a 1% lysis buffer for 4 hours, yielding a lysate of 10-20%; the influenza A (H3N2) virus was lysed using a 0.5% lysis buffer for 10 hours, yielding a lysate of 15-30%; and the influenza B virus was lysed using a 1.5% lysis buffer for 8 hours, yielding a lysate of 10-30%.

[0032] S8. Gel filtration chromatography purification: The solution obtained by sucrose density gradient centrifugation in the previous step is further purified by Sephadex G-25 dextran gel filtration chromatography system to obtain the influenza virus subunit vaccine monovalent stock solution.

[0033] The prepared single-dose stock solutions of influenza A (H1N1), influenza A (H3N2) and two influenza B (B) viruses are then mixed in a hemagglutinin concentration ratio of 1:1:1:1. The hemagglutinin concentration of each type of virus is controlled between 40-48µg / ml. The mixture is then sterilized by filtration through a 0.22µm filter membrane and sealed in vials, each containing 0.5ml. The human dose is 0.5ml per time. After passing the lamp inspection, the quadrivalent influenza virus subunit vaccine is obtained.

[0034] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0035] Genipin is the product of Gardenia jasminoides hydrolysis by β-glucosidase. Through the atomic-level nucleophilic cross-linking 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 bonds and hydrophobic interactions.

[0036] Genipin contains epoxide, ester group, and allylic hydroxyl group. The oxygen atom in the epoxide (C1-O-C2) has high electronegativity, making C1 and C2 strong electrophilic centers, which are easily attacked by nucleophilic groups (such as amino and carboxyl groups). When the carboxyl group (-COOH) of CM-dextran attacks the C1 of the epoxide, the C1-O bond is broken, forming an ether bond (C1-O-CM-dextran) and a hydroxyl group (C2-OH).

[0037] The carbonyl oxygen (O) in the ester group (C10-O-CO-R) polarizes the CO bond through an inductive effect, causing it to hydrolyze into a carboxylic acid group (-COOH) under alkaline conditions, thereby enhancing the reaction activity. In an inactivating environment of pH 7.4, the ester group hydrolyzes into 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.

[0038] The hydroxyl group (C9-OH) combines with the carboxyl group or water molecules of CM-dextran through hydrogen bonds, assisting the directional arrangement of the molecules.

[0039] The epoxide of genipin reacts with the carboxyl groups of two CM-dextran chains to form intermolecular crosslinks (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 the dextran are fully exposed, improving the uniformity of the substitution reaction. The crosslinked network restricts the local folding of the dextran chains, preventing the formation of high-substitution regions (>0.7) and low-substitution regions (<0.5).

[0040] In the Schiff base (RN=CH2) generated by the condensation of CM-dextran and formaldehyde, the nitrogen atom of the imine 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 the formaldehyde.

[0041] The network formed by cross-linking of genipin wraps around the Schiff base sites, preventing water molecules from contacting, inhibiting the hydrolysis reaction, and preventing formaldehyde from becoming free again.

[0042] The carboxylic acid group (-COOH) generated by the hydrolysis of genipin forms hydrogen bonds with the hydroxyl group (-OH) of CM-dextran, enhancing the rigidity of the cross-linked structure. The cyclopentane hydrophobic core of genipin and the hydrophobic region (unsubstituted glucose unit) of CM-dextran are bound by van der Waals forces to stabilize the cross-linked structure. During the inactivation stage (pH 7.2), the cross-linked network remains stable. If the system is accidentally acidified, the genipin cross-links can still maintain structural integrity. The cross-linked network shrinks under storage conditions of 2-8°C, further compressing the formaldehyde diffusion path. It expands slightly at 37°C (immune response temperature) and does not affect antigen release.

[0043] The cross-linked network forces the CM-dextran chains to be arranged linearly, the steric hindrance of the substitution reaction is reduced, the uniformity of the carboxymethyl distribution is improved, and genipin consumes the free metal ions (such as Fe 3+), reducing the substitution degree deviation caused by oxidative side reactions, and converting the Schiff base into an irreversible secondary amine structure.

[0044] The uniformity of the degree of substitution of genipin on carboxymethyl dextran (CM-dextran) and the stabilization ability of genipin on the Schiff base formed by formaldehyde (HCHO) and CM-dextran were verified. 1 H-NMR analysis (400 MHz) of cross-linked carboxymethyl dextran solutions was performed to calculate the degree of carboxymethyl substitution and standard deviation (SD). For the control group (genipin-free), dextran (50 kDa) was reacted with chloroacetic acid (molar ratio 1:2.5) under alkaline conditions (2.5 M NaOH, 50°C) for 5 hours, followed by purification by dialysis. For the experimental groups, genipin (0.1%, 0.3%, or 0.5% (w / w)) was added after the reaction, followed by cross-linking at 37°C for 12 hours, and then purified by ultrafiltration. The results are shown in Table 1.

[0045] Free formaldehyde in inactivated influenza virus allantoic fluid was quantified by HPLC (C18 column, mobile phase acetonitrile-water = 6:4, detection wavelength 360 nm). CM-dextran (degree of substitution 0.6) was reacted with formaldehyde (final concentration 100 mg / mL) in pH 7.4 buffer for 24 hours to generate a Schiff base. A control group was treated with no genipin, while experimental groups were treated with 0.05%, 0.1%, and 0.2% (w / v) genipin, respectively, at 25°C for 8 hours. The results are shown in Table 2.

[0046] Table 1 Uniformity of substitution degree of carboxymethyl dextran (CM-dextran)

[0047]

[0048] Table 2 Stabilization ability of Schiff base

[0049]

[0050] Antibody titer testing was then performed. Virus suspension prepared using the medium-dose experimental group (cross-linking dose 0.3%, inactivation dose 0.1%) was diluted in PBS to a hemagglutinin content of 30 μg / ml. Ten healthy, clean-grade ISR mice weighing 16-18 g (half male and half female) were inoculated intraperitoneally with 0.2 ml of the suspension. Ten other mice were injected with PBS buffer as negative controls. After 21 days, orbital blood was collected, serum separated, and treated with potassium periodate to remove nonspecific inhibitors. Antibody titers were determined by hemagglutination inhibition assays and HA content by single-directional immunodiffusion (SRID) assays. The results are shown in Table 3. For the control group (without genipin), the H1N1 titer was log2 = 7.5, the H3N2 titer was log2 = 7.4, and the average B titer was log2 = 7.3. The protective efficacy was calculated as (genipin group titer - control group titer) / control group titer × 100%.

[0051] Table 3 Virus titer

[0052]

[0053] Example 2: The above example uses genipin to force the CM-dextran molecular chain to extend, thereby improving the uniformity of carboxymethyl substitution, converting the Schiff base into a stable secondary amine structure, and locking formaldehyde through hydrogen bonds and hydrophobic interactions. On the one hand, this reduces the unevenness of substitution, and on the other hand, it reduces the free methanol. However, at the same time, genipin is used as a cross-linking agent, and its residue may also cause allergies. When the residual amount of genipin is greater than 0.01%, it may cause redness and swelling at the injection site. In this regard, further improvements are made based on Example 1.

[0054] After inactivation and before ultrafiltration concentration, liposome-encapsulated α-tocopherol (vitamin E) was added: the fully inactivated influenza virus allantoic fluid was added to the liposome-encapsulated α-tocopherol and stirred for 45 minutes (150 rpm, 25°C) to a final liposome concentration of 0.5-2.0% (w / v) (α-tocopherol content of 0.1-0.4% w / w) and concentrated by ultrafiltration using an ultrafiltration membrane;

[0055] The preparation method of liposome-encapsulated α-tocopherol is as follows: 40 mg of hydrogenated soybean phosphatidylcholine (HSPC), 10 mg of cholesterol, and 5 mg of α-tocopherol were weighed and dissolved in a chloroform-methanol (3:1 v / v) mixed solvent. The solution was transferred to a round-bottom flask and the solvent was removed by rotary evaporation (40°C, 100 rpm) to form a uniform lipid film. pH 7.0 phosphate buffer (containing 5% sucrose) was added and hydrated at 55°C for 30 minutes. The solution was treated with probe ultrasound (200 W, 5s on / 5s off, 5 cycles) and sterilized by passing through a 0.22 μm filter membrane to obtain liposome-encapsulated α-tocopherol with an encapsulation efficiency of >85% and a particle size of 80-150 nm.

[0056] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0057] Liposome-encapsulated α-tocopherol forms a multifunctional network with genipin and CM-dextran through free radical scavenging and interface synergy. The dual mechanisms of physical isolation and chemical neutralization reduce the risk of residues; antioxidant and conformational stabilization jointly enhance immunogenicity; the interpenetrating structure inhibits the migration of small molecules and ensures long-term storage stability.

[0058] 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 the single electron transfer (SET) mechanism, and then oxidizes itself to tocopherol quinone; the phytyl side chain (phytyl chain) of tocopherol is embedded in the hydrophobic region of the liposome bilayer (C16 alkyl chain of phosphatidylcholine) through van der Waals forces, stabilizing the liposome structure and prolonging the release time.

[0059] α-Tocopherol captures the free radicals generated during the cross-linking reaction of genipin (·O - ), blocking it from attacking the lipid bilayer of the cell membrane; the hydrophobic region of the liposome bilayer adsorbs unreacted genipin molecules through π-π stacking, limiting their contact with cellular components and isolating allergens. The cross-linked network of CM-dextran (pore size 2-5 nm) and the liposomes (particle size 50-100 nm) form an interpenetrating structure. The liposomes fill the network pores and block the formaldehyde diffusion path. 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).

[0060] The negative charge on the liposome surface (phospholipid head phosphate group) and the carboxyl group (-COO - ) are bound by electrostatic attraction, while the hydroxyl groups of phospholipids form hydrogen bonds with the hydroxyl groups of CM-dextran, enhancing the stability of the complex; the hydrophobic microdomains of the CM-dextran cross-linked network are bound to the hydrophobic tails of the liposomes through van der Waals forces, forming a continuous hydrophobic barrier that inhibits 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.

[0061] 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 while enhancing the lymphatic targeting of the antigen.

[0062] Based on the medium dose group (cross-linking dose 0.3%, inactivation dose 0.1%) of Example 1, experiments were conducted to verify the antioxidant capacity and toxicity reduction, including free radical scavenging rate: DPPH method was used to detect the free radical scavenging ability of α-tocopherol, cytotoxicity: MTT method was used to detect the effect of genipin residue on the viability of HEK293 cells, and formaldehyde residue: HPLC quantitative analysis. The experimental sample was inactivated and unpurified influenza virus allantoic fluid harvested and mixed with liposomes for 45 minutes (150 rpm, 25°C) to simulate residue. The final genipin concentration was 0.01%. The results are shown in Table 4.

[0063] Table 4 Antioxidant and toxicity reduction experiments

[0064]

[0065] The vaccine was then tested for antigenic protection and immunogenicity. The activity of the inactivated HA protein was detected by ELISA, and a hemagglutination inhibition (HI) test was performed on mice 21 days after immunization. The experimental process was consistent with the antibody titer experiment in Example 1. The difference was that the experiment in this example used the same amount of genipin (cross-linking dose 0.3%, inactivation dose 0.1%) and different liposome concentrations. The vaccine titer was tested, and the results are shown in Table 5.

[0066] Table 5 Antigen protection and immunogenicity verification

[0067]

[0068] Example 3: In Example 2, α-tocopherol encapsulated in liposomes forms a multifunctional network with genipin and CM-dextran through free radical scavenging and interface synergy, and the dual mechanisms of physical isolation and chemical neutralization reduce the residual risk; antioxidant and conformational stability jointly enhance immunogenicity; the interpenetrating structure inhibits small molecule migration and ensures long-term storage stability. However, the subsequent addition of the cleavage agent nonoxynol-9 causes the hydrophobic tails of the phospholipid bilayer of the liposome (such as the C16 chain of DSPC) to bind through hydrophobic interactions, destroying the integrity of the liposome membrane, resulting in liposome rupture and leakage of α-tocopherol. In this regard, further improvements are made on the basis of Example 2.

[0069] 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 of 4:1:0.5), with an α-tocopherol loading of 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 of 3:1:0.2), with a mannose density of 12-15 molecules per liposome;

[0070] The lysis buffer was replaced with lysis agent micelles, including small lysis agent micelles and large lysis agent micelles. The small lysis agent micelles had a particle size of 5-10 nm and were made of vitamin E polyethylene glycol succinate (TPGS) and nonoxynol (molar ratio 1:3), with a lysis agent concentration of 0.3-0.8% (w / v); the large lysis agent micelles had a particle size of 15-25 nm and were made of nonoxynol-9 and phosphatidylglycerol (molar ratio 2:1), with a lysis agent concentration of 0.5-1.0% (w / v);

[0071] Large liposome: small liposome: small lytic agent micelle: large lytic agent micelle (volume ratio) = 3:2:1:1;

[0072] Small liposomes were prepared by dissolving DOPE, cholesterol, and mannose-PEG-DSPE in chloroform-methanol (3:1 v / v), rotary evaporating the film, adding pH 6.5 citrate buffer, hydrating at 40°C for 30 minutes, and controlling the particle size using microfluidics (flow rate ratio of aqueous phase: organic phase = 3:1).

[0073] Small cleavage agent micelles were prepared by dissolving nonoxynol-9 and TPGS in deionized water, stirring magnetically (500 rpm, 25 °C) for 2 h, and ultrasonicating (50 W, 30 s) to promote micelle formation;

[0074] The large lysing agent micelles were prepared by dissolving nonoxynol-9 and phosphatidylglycerol in pH 7.0 Tris buffer, with a flow rate ratio of 5:1 between the aqueous phase (buffer) and the organic phase (ethanol), and then dialyzing to remove the ethanol.

[0075] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0076] 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, stabilizing the structure through van der Waals forces. The mannose modification (negative charge) on the surface of the small liposomes enhances targeting through electrostatic attraction with the surface receptors of dendritic cells (positive charge), while forming charge repulsion with the large lytic agent micelles (neutral) to reduce nonspecific binding.

[0077] Small lytic agent micelles, with their high specific surface area and diffusion rate, preferentially penetrate the gaps in the viral envelope and release internal antigens; large lytic agent micelles, due to volume limitations, slowly release the lytic agent after binding to the viral envelope, reducing interference with liposomes. The α-tocopherol wrapped 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, thereby improving antigen presentation efficiency.

[0078] 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.

[0079] Small lytic agent micelles, because of their size matching the pores of the viral envelope, preferentially adsorb and lyse viruses, 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.

[0080] 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.

[0081] The liposome leakage rate was determined by measuring 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 ;

[0082] Table 6 Performance Verification of Example 3

[0083]

[0084] Example 4: Example 3 reduces the damage of liposomes by combining liposomes of various particle sizes with lytic agent micelles. In the study, it was found that by utilizing the damage of liposomes, controllable pores can be formed to achieve precise release of antigens in time and space.

[0085] Small lytic agent micelles (0.5% w / v) were slowly added to the small liposome suspension (1.0% w / v) in advance, stirred at 25°C (200 rpm) for 10 minutes, heated to 37°C (±1°C), and stirred for 20 minutes to promote the insertion of the hydrophobic tail of the lytic agent into the liposome membrane. α-Tocopherol (0.8% w / v) was added and the mixture was allowed to stand at 4°C for 30 minutes to stabilize the pore structure. The lytic agent insertion depth was 2-5 nm, and modified small liposomes were obtained.

[0086] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0087] The lytic agent micelles adsorb onto the liposome surface (electrostatic interaction), resulting in low membrane fluidity and slow insertion. Upon heating to 37°C, the liposome membrane enters a liquid crystal state (phase transition temperature 32°C), enhancing fluidity. The hydrophobic tail of the lytic agent vertically inserts into the membrane, forming the initial pore. The depth of insertion is controlled by the lytic agent and the heating rate (1°C / min), limiting pore expansion (2-5 nm).

[0088] The pores respond to the low pH (5.0) environment of endocytic vesicles on the surface of dendritic cells (DCs), the hydrophilic heads of the micelles dissociate, and the instantaneous opening frequency increases to 5 times / second, realizing antigen release. The hydrophilic heads of the lytic agent micelles electrostatically bind to the antigens and dissociate through pH response during DC endocytosis, driving the efficient export of antigens (non-passive diffusion); the tocopherol hydrophobic chains cross-link with membrane lipids to form a "molecular rivet" structure, the mechanical strength of the pores is improved, and trehalose forms a glassy hydration layer outside the liposomes to inhibit membrane fusion; tocopherol blocks the oxidative chain reaction, and the HA activity is retained at 93% after storage at 4°C for 6 months. The single particle realizes the integration of the three functions of "carrier-releaser-targeting molecule", and the dendritic cell uptake efficiency is increased, ultimately making the single-dose protection duration exceed 12 months, which is 26% higher than the vaccine prepared in Example 3.

[0089] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Various modifications and variations are readily apparent to those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing a quadrivalent influenza virus subunit vaccine, characterized in that: Each dose of quadrivalent influenza virus subunit vaccine contains two influenza A viruses and two influenza B viruses. The quadrivalent influenza virus subunit vaccine is prepared by virus inoculation, virus proliferation culture, influenza virus allantoic fluid harvesting, clarification, inactivation, ultrafiltration concentration, lysis and ultracentrifugation purification, mixing, filtration sterilization, packaging and packaging. The inactivation step is to add cross-linked carboxymethyl dextran solution to the influenza virus allantoic fluid, and then add formaldehyde to inactivate at 5°C for 45 hours; then, genipin is mixed into the influenza virus allantoic fluid, stirred at 200 rpm, at pH 7.2, and at 25°C for 8 hours; 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, dialyzed and purified to control the degree of substitution to 0.6 to obtain carboxymethyl dextran; genipin is added to the carboxymethyl dextran solution, and the mixture is stirred and reacted at pH 7.4 and 37°C for 12 hours; after the reaction, unreacted genipin is removed by ultrafiltration to obtain a cross-linked carboxymethyl dextran solution.

2. The method for preparing a quadrivalent influenza virus subunit vaccine according to claim 1, wherein: The concentration of the cross-linked carboxymethyl dextran solution is 1-3% w / v.

3. The method for preparing a quadrivalent influenza virus subunit vaccine according to claim 1, wherein: During the inactivation step, the final genipin concentration was 0.05-0.5% w / v.

4. The method for preparing a quadrivalent influenza virus subunit vaccine according to claim 1, wherein: After inactivation and before ultrafiltration concentration, liposome-encapsulated α-tocopherol was added: the fully inactivated influenza virus allantoic cyst harvest was added to the liposome-encapsulated α-tocopherol and stirred for 45 minutes.

5. The method for preparing a quadrivalent influenza virus subunit vaccine according to claim 4, wherein: The final liposome concentration was 0.5-2.0% w / v.

6. The method for preparing a quadrivalent influenza virus subunit vaccine according to claim 4, wherein: The preparation method of liposome-encapsulated α-tocopherol is as follows: hydrogenated soybean 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 is added, and the mixture is hydrated at 55°C for 30 minutes. The mixture is ultrasonicated with a probe and sterilized by passing through a 0.22 μm filter membrane to obtain liposome-encapsulated α-tocopherol.

7. The method for preparing a quadrivalent influenza virus subunit vaccine according to claim 4, wherein: 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, with an α-tocopherol loading of 6% w / w; the small liposomes have an average particle size of 80 nm and are made of DOPE, cholesterol and mannose-PEG-DSPE, with a mannose density of 12-15 molecules / liposome.

8. The method for preparing a quadrivalent influenza virus subunit vaccine according to claim 7, wherein: 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 and is made of vitamin E polyethylene glycol succinate and nonoxynol; the particle size of the large lysis agent micelles is 15-25 nm and is made of nonoxynol-9 and phosphatidylglycerol; the volume ratio of large liposomes: small liposomes: small lysis agent micelles: large lysis agent micelles is 3:2:1:

1.

9. A quadrivalent influenza virus subunit vaccine, characterized in that: Prepared by the preparation method according to any one of claims 1 to 8.

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