Recombinant herpes zoster vaccine composition based on multi-epitope chimeric antigen and nano adjuvant

Abstract

The invention relates to the technical field of vaccine preparations, and discloses a recombinant herpes zoster vaccine composition based on a multi-epitope chimeric antigen and a nano adjuvant, the vaccine composition comprises the following components by weight: 1-3 parts of a multi-epitope chimeric antigen; 2-5 parts of a nano adjuvant; 0.5 to 1.5 parts of chitosan or trehalose; the multi-epitope chimeric antigen comprises a plurality of immune epitopes of varicella zoster virus and is used for activating T cell and B cell immune response, and the nano adjuvant is prepared from 1-3 parts of a metal organic framework material and 1-3 parts of a carbon nanotube; the metal organic framework is of an MIL-88B structure based on iron ions; functional modification of the carbon nanotubes is realized through acid pickling and ultraviolet irradiation. According to the invention, a nano-composite structure is constructed, and a stabilizer and a microwave freeze-drying process are introduced, so that the synergistic effects of antigen conformation maintenance, uniform compounding and stable redissolution are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine preparations, and in particular to a recombinant herpes zoster vaccine composition based on multi-epitope chimeric antigens and nano-adjuvants. Background Art

[0002] With the widespread application of multi-epitope recombinant antigens in the development of new vaccines, how to effectively maintain their spatial conformation and biological activity has become the key to the stability design of vaccine preparations. Especially in the actual storage and transportation process, the freeze-drying storage process has gradually become the mainstream method.

[0003] At present, many technologies have been used to try to improve the stability of vaccine antigens. For example, using low molecular weight sugars such as sucrose and mannitol as freeze-drying protectants can alleviate drying stress and improve solubility performance during reconstitution. There are also studies that introduce liposomes or PLGA nanoparticles into antigen delivery systems to construct preliminary load release structures, showing a certain degree of controlled release potential.

[0004] However, these solutions still have some shortcomings when facing complex antigen structures and complex delivery requirements. When using conventional protective agents such as sucrose, the stabilizing effect on high surface energy antigens is insufficient, which often leads to abnormal antigen folding and obvious aggregation and sedimentation after reconstitution; after structural damage, the antigen recognition ability is difficult to recover. The traditional freeze-drying process has a slow heating process and a long water migration path, especially in the composite vaccine system, which often destroys the interface between the antigen and the adjuvant, and the composite structure becomes uneven or loose; most existing antigen delivery systems rely on static physical adsorption, the binding efficiency is not high, the antigen is easy to be free, and it is impossible to achieve true encapsulation protection. Once the adjuvant ratio is low or the structure is improperly designed, the antigen is like "hanging on the surface", which is neither stable nor easy to be effectively recognized by the immune system. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a recombinant herpes zoster vaccine composition based on multi-epitope chimeric antigens and nanoadjuvants, which solves the problems of poor antigen stability, low complexation efficiency, uneven structure after reconstitution, and insufficient ability to fix the antigen interface in existing vaccine preparations.

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: a recombinant herpes zoster vaccine composition based on multi-epitope chimeric antigens and nanoadjuvants, the vaccine composition comprising the following components in parts by weight: Multi-epitope chimeric antigens: 1-3 copies. Multi-epitope antigens can activate both cellular and humoral immune pathways. Nano adjuvant: 2-5 parts. The nanoparticle size (usually 100-200nm) facilitates active uptake by antigen presenting cells (APCs) such as dendritic cells (DCs), improving the efficiency of antigen processing and presentation. The nanostructure provides a stable encapsulation environment, allowing the antigen to be slowly released in the body, simulating the natural viral infection process, thereby inducing a more lasting immune memory. Chitosan or trehalose: 0.5-1.5 parts. Chitosan is a cationic polysaccharide that can interact with the negatively charged phospholipid bilayer of the cell membrane, enhance the adhesion of antigens to the cell surface, and promote cell endocytosis. Trehalose is a non-reducing sugar with good protein protection. Especially during freeze-drying and reconstitution, it can stabilize the conformational structure of antigens through the "water substitution effect" to prevent protein aggregation and denaturation. Both help to improve the stability and bioavailability of the antigen-adjuvant complex in the body, and are key auxiliary factors for the storage of vaccines outside the cold chain. Other excipients: 0.1-0.5 parts. Adding trace amounts of auxiliary components to the vaccine composition can ensure the isotonicity and pH stability of the preparation and prevent local irritation or abnormal antigen structure during vaccination.

[0007] Preferably, the multi-epitope chimeric antigen includes multiple immune epitopes of varicella-zoster virus, which are used to activate T cell and B cell immune responses. B cell epitopes can directly promote the production of neutralizing antibodies and improve the protectiveness of the vaccine; T cell epitopes, especially Th1 type reaction-related epitopes, can promote the activation of cytotoxic T lymphocytes (CTL) and eliminate latent or infected cells; the chimeric expression form can reduce the risk of immune escape caused by antigen drift and improve the broad-spectrum immune effect.

[0008] Preferably, the nanoadjuvant comprises: Made of 1-3 parts of metal organic framework material and 1-3 parts of carbon nanotubes; The metal organic framework is a MIL-88B structure based on iron ions. The MIL-88B structure can be gradually degraded in a weakly acidic environment (such as the intracellular lysosome of APC) to achieve controlled release and intracellular release of antigens. Iron ions, as the central metal of the MOFs skeleton, not only provide a stable structure, but also enhance the level of ROS in immune cells through the Fenton reaction, activating the maturation and signal transduction of antigen presenting cells (APCs); the high porosity structure facilitates the "embedding-release" of antigen molecules, improves the stability of antigens and reduces the risk of enzymatic inactivation. The carbon nanotubes are functionally modified by acid washing and ultraviolet irradiation. The acid washing and ultraviolet treatment form functional groups (such as -COOH, -OH) on the surface of CNTs, enhance their hydrophilicity and antigen-loading capacity, and reduce the biological toxicity of original CNTs. The modified CNTs have the ability to enhance cell membrane permeability, promote antigens to enter APCs such as dendritic cells, and improve cross-presentation efficiency. Ultraviolet treatment may stimulate the formation of charged areas on the surface of CNTs, simulate the effects of certain natural PAMPs in the immune microenvironment, indirectly activate the Toll-like receptor (TLRs) pathway, and induce a primary immune response.

[0009] Preferably, the other auxiliary materials include: Phosphate buffer: 0.5-1 part. During the freeze-drying, reconstitution and in vivo delivery of the vaccine, the antigen protein structure is sensitive to pH fluctuations. Phosphate buffer can stabilize the pH value of the system within the physiological neutral range of 6.8-7.4 to prevent the antigen from denaturing, aggregating or hydrolyzing. The nanoadjuvant has different charge states at different pH values. PBS helps maintain its colloidal stability and charge balance, prevents nanoparticles from agglomerating, and ensures uniform distribution of the antigen. Sodium chloride: 0.05-0.2 parts. Sodium chloride can adjust the osmotic pressure of the entire vaccine solution to about 280-300mOsm / kg, which is close to the osmotic pressure of human body fluids. It can effectively prevent dehydration or swelling of local tissue cells after injection and reduce inflammatory response. It can also stabilize the conformation of antigen molecules in the solution by adjusting the ionic strength and reduce the van der Waals attraction between nanoadjuvants, helping to maintain uniform distribution of particles.

[0010] The present invention also provides a method for preparing a recombinant herpes zoster vaccine composition, comprising the following steps: S1. Preparation of nanoadjuvants; S2. Acquisition and purification of multi-epitope chimeric antigens; S3, complexation of nanoadjuvant and multi-epitope chimeric antigen; S4, stabilization treatment of the vaccine composition; S5. Finished product packaging of the vaccine composition.

[0011] Preferably, the preparation of the nanoadjuvant comprises: A mixed solution of ferric chloride and phthalic acid was prepared, and the reaction was carried out at room temperature for 4 hours within the pH range of 5.5-7.0 to synthesize MIL-88B MOFs. MIL-88B is a three-dimensional porous structure formed between iron ions and organic ligands (phthalic acid). The pH in the reaction conditions controls the crystal growth rate and morphology. The reaction is completed at room temperature, which can reduce the generation of crystal defects, maintain the high porosity and specific surface area of ​​the material, and improve its subsequent antigen loading capacity. The iron element acts as an active center in the structure, which not only builds a stable framework, but also can induce immune-related oxidative stress response in vivo and enhance the activation ability of APC, which is the basis for constructing functional MOF nanoadjuvants. Carbon nanotubes were prepared by chemical vapor deposition, and treated with 0.05-0.15 parts of concentrated nitric acid to remove impurities and carboxylate the surface. The CVD synthesis method can achieve a high-purity, directional growth CNTs structure, which is conducive to the subsequent composite with MOFs to form a regular interface; during the concentrated nitric acid treatment, polar groups such as -COOH and -OH (carboxylation) are generated on the surface of CNTs, which not only removes residual metal impurities, but also improves its hydrophilicity, dispersibility and biocompatibility; carboxylation modification makes CNTs have stronger antigen adsorption ability, and is conducive to the formation of hydrogen bonds or electrostatic forces with MOFs, laying the foundation for the formation of stable composite nano adjuvants; The surface activity is enhanced by ultraviolet light treatment. Ultraviolet irradiation stimulates the surface energy state of CNTs, further induces oxidation, and promotes the uniform distribution of functional groups such as hydroxyl and carboxyl groups. The interfacial affinity between CNTs and antigens and MOFs is enhanced. At the same time, the "active surface" formed has a certain degree of immunostimulation and can simulate PAMPs signals to activate immune pathways. After ultraviolet modification, CNTs show stronger dispersion stability and antigen delivery performance, improving the overall functional performance of nanoadjuvants. MOFs and CNTs are mixed in a 1:1 weight ratio and ultrasonically emulsified to form nanoadjuvants. The frequency of ultrasonic emulsification is 20-30kHz and the time is 0.3-60 minutes. Ultrasonic emulsification technology uses cavitation effect and shear force to make MOFs and CNTs nanostructures fully contact and cross-link to avoid agglomeration. The 1:1 ratio can achieve structural balance and functional complementarity, ensuring that the nanoadjuvant achieves a synergistic advantage between antigen delivery and immune activation. Reasonable control of ultrasonic parameters (frequency + time) helps to form composite particles with uniform particle size and stable interface, while avoiding damage to the nanostructure and maintaining the in situ functional stability of the adjuvant system.

[0012] Preferably, the acquisition and purification of the multi-epitope chimeric antigen comprises: Molecular cloning technology is used to construct a recombinant expression vector containing multiple VZV epitopes. The epitope selection is based on VZV conserved regions and immune activity data analysis. The fusion design can activate both humoral immunity and cellular immunity. The molecular cloning process is verified by enzyme digestion, ligation and sequencing to ensure the correct integration of the sequence and the fidelity of the downstream expressed antigen. The introduction of the His tag facilitates subsequent purification and does not interfere with the spatial conformation of the immune epitope, ensuring functional expression. Transfect it into E. coli BL21 or Pichia pastoris and induce expression with IPTG or methanol. The BL21 system is efficient, cheap, and suitable for rapid expression of antigenic proteins; the Pichia pastoris system is more suitable for expressing eukaryotic antigens with spatial conformation requirements or glycosylation modifications; IPTG induction relies on the expression system controlled by the T7 promoter to quickly start the transcription and translation process; methanol induction in Pichia pastoris relies on the AOX1 promoter, which can achieve high-density expression of proteins and is suitable for the construction of antigens that require folding and modification; flexibly select the expression system to ensure the expression efficiency, correct folding, and retention of immune function of chimeric antigens; After cell lysis, the recombinant protein was extracted and purified by nickel column chromatography or His-tag affinity chromatography. 2+ The stable coordination bond formed between the two proteins can specifically capture the chimeric protein with the tag and achieve efficient separation from the impurity protein; the chromatography process can accurately control the elution conditions (such as imidazole gradient) to optimize the protein recovery rate and purity; the buffer system used in the cleavage and purification process can protect the protein structure, avoid degradation or aggregation inactivation, and ensure the immune function of the antigen; SDS-PAGE and Western Blot are performed to verify the expression purity and antigenicity. SDS-PAGE provides an intuitive reflection of the size and purity of protein molecules, and is the first step to verify whether the expression is successful. Western Blot uses the immune reaction on the membrane to verify whether the recombinant antigen has the correct conformation and retained immune epitopes, and can be recognized by the target antibody. This verification ensures that the antigen is not only "expressed" but also "has the correct immune function", providing a basic guarantee for the effectiveness of vaccine immunity.

[0013] Preferably, the combination of the nanoadjuvant and the multi-epitope chimeric antigen comprises: The nano-adjuvant and the multi-epitope chimeric antigen are mixed in a weight ratio of 2-5 parts: 1-3 parts. The 2-5 times amount of adjuvant can ensure the complete coating or sufficient adsorption interface of the antigen, thereby improving the protection and controlled release performance of the antigen in the body; at the same time, the antigen ratio is controlled not to exceed the standard to avoid the occurrence of free antigens leading to non-specific distribution or rapid degradation, which is beneficial to the persistence and directionality of the immune response; this ratio range also ensures that the composite particle structure is not destroyed during the subsequent high-pressure treatment process, and maintains the uniformity of the nanoparticle size and distribution; Through high-pressure homogenization, the antigen nanocomplex is formed under a pressure of 200-500 bar for 10-30 minutes. High-pressure homogenization can use shear force, cavitation force and impact force to fully mix the nanomaterial and antigen and interfacially embed. This process helps to form an embedded or adsorbed antigen nanocomposite structure, improving its stability and bioavailability. The appropriate pressure and time interval can effectively control the particle size of the complex within the range of 100-200nm, which is beneficial to lymphatic transport and APC uptake. Use UV spectrometry or BCA method to detect antigen embedding rate and ensure it is ≥85%. High embedding rate is a key indicator to ensure vaccine immune efficacy and dosage control. An embedding rate of ≥85% can reduce antigen loss and nonspecific clearance. The UV method determines free antigens through differences in protein absorption peaks, and the BCA method detects total protein based on copper ion reduction reaction. The two can verify each other. High embedding efficiency also reflects the high affinity and structural stability of the nanoadjuvant for the antigen, indirectly characterizing the quality of complex formation. In addition, the appropriate embedding level helps to form a continuous delivery and antigen library reserve mechanism, enhancing the timeliness and intensity of the immune response.

[0014] Preferably, the stabilization treatment of the S4 vaccine composition comprises: 0.5-1.5 parts of chitosan or trehalose are added to the antigen complex as a stabilizer. Chitosan is a cationic polysaccharide that can form an electrostatic complex and gel layer with the surface of the nanoparticles to enhance the physical stability of the composite particles and prevent aggregation and sedimentation. Trehalose is a non-reducing disaccharide with excellent vitrification ability and protein protection function. It can replace water and protein interaction during the drying process to prevent antigen denaturation. Both can be selected according to needs. Both have good biocompatibility and immune friendliness and will not inhibit antigen delivery or immune activation response. 0.5-1 part phosphate buffer and 0.05-0.2 part sodium chloride are added to adjust the pH and osmotic pressure. PBS maintains the pH of the system between 6.8-7.4, provides a suitable acid-base environment for antigen structure maintenance and adjuvant stability, and avoids acid-base stress during the drying process. NaCl ensures that the osmotic pressure of the solution is close to physiological conditions (about 280-300mOsm / kg), which can prevent the colloidal nanoparticle structure from disintegrating or osmotic pressure shock from causing abnormal protein folding before freeze-drying. Microwave-assisted freeze-drying technology is used for drying, the temperature is controlled between -20℃ and -30℃, the microwave frequency is 2.45GHz, and the drying time is 2-4 hours. Microwave-assisted freeze-drying (MWFD) technology combines the advantages of low-temperature freeze-drying and microwave heating to accelerate water sublimation without increasing the sample temperature; the frequency of 2.45GHz is the industrial standard microwave frequency, which has good penetration efficiency, and can achieve uniform heating and efficient removal of internal moisture; processing under temperature control conditions of -20℃ to -30℃ can effectively protect temperature-sensitive components, such as chimeric antigens, polysaccharide adjuvants, etc., to ensure that the vaccine has a complete particle size structure and biological function after reconstitution.

[0015] Preferably, the finished product packaging of the vaccine composition includes: The freeze-dried vaccine complex is dissolved in a phosphate buffer of pH 6.0-7.0. pH 6.0-7.0 is a neutral to weak acid region between the extracellular fluid (7.4) and the endosomal acidification environment, which helps to form a stable nano-dispersion system after reconstitution. PBS provides ionic strength and an isotonic environment, which can avoid aggregation and flocculation of proteins after reconstitution, thus ensuring the uniformity of the vaccine preparation. A 0.22μm sterile filter membrane is used for sterile filtration. The 0.22μm microporous membrane is an internationally recognized standard pore size for liquid sterilization, which can effectively filter out microbial contaminants such as bacteria and mold spores. Since the particle size of the antigen nanocomplex is designed to be in the range of 100-200nm, which is much smaller than 0.22μm, it can pass through the filter membrane smoothly without being blocked. Compared with high-temperature sterilization or irradiation, the filtration method has no risk of thermal damage and oxidative damage to the protein antigens and nanoadjuvant structures in the vaccine, and is particularly suitable for structure-sensitive vaccine preparations. Filled in pre-sterilized glass ampoules and stored at -20℃ to -80℃, glass ampoules have low reactivity, high air tightness and good visibility, and are ideal packaging materials for protein vaccine preparations, which can prevent degradation reactions such as oxidation and photolysis. Sterile ampoules are combined with aseptic filtration filling to build a complete aseptic guarantee chain to avoid secondary contamination. The storage temperature zone of -20℃ to -80℃ can slow down the degradation rate of protein, inhibit microbial growth and hydrolysis reactions, and maintain vaccine activity.

[0016] The present invention provides a recombinant herpes zoster vaccine composition based on multi-epitope chimeric antigens and nano-adjuvants, which has the following beneficial effects: 1. The present invention uses trehalose or chitosan as a freeze-drying stabilizer, introduces a protective sugar network into the vaccine complex structure, effectively prevents the denaturation and aggregation of the antigen conformation during the drying process, and achieves the effect of improving the antigen retention rate and particle size uniformity after the freeze-dried preparation is reconstituted. Compared with traditional stabilizers such as sucrose commonly used in the prior art, which have limited nonspecific protection capabilities and often cause disorder or precipitation of the antigen structure, making it difficult to achieve reconstitution stability under long-term storage conditions, the present invention solves the problem of insufficient stability of such adjuvants for protein antigens.

[0017] 2. The present invention adopts microwave-assisted freeze-drying technology, introduces a directional energy field under low temperature conditions, improves the water sublimation efficiency, reduces the risk of protein denaturation and structural disintegration in traditional freeze-drying, and achieves the effect of rapid drying and high antigen integrity. Compared with the air drying or conventional freeze-drying methods used in the prior art, such technologies have long drying times, uneven heat conduction, and are very likely to cause antigen aggregation or inactivation. The present invention avoids the problem of activity attenuation caused by a slow heating process.

[0018] 3. The present invention improves the embedding rate of antigens and the stability of delivered particles by introducing a nano-adjuvant system constructed by MOFs and CNTs and optimizing the antigen compound ratio, achieving the technical effects of uniform nanostructure, reasonable antigen distribution and high compound efficiency. Compared with the conventional aluminum adjuvant or carrier-free delivery method used in the prior art, which has the prominent problems of easy antigen release, uneven particle size and difficulty in controlled release, the present invention effectively overcomes the defect of insufficient ability of traditional carrier systems to fix antigen interfaces.

[0019] 4. The present invention adds ultrasonic emulsification and high-pressure homogenization dual processing steps in the process of constructing the complex, and uses shear force and cavitation effect to promote the deep embedding of antigens into nanocarriers, which has a compact structure, improves the stability of vaccine particles and cell uptake efficiency, and achieves the effect of enhancing the ability to induce immune response. Compared with the existing process that lacks energy-driven steps or only uses static mixing, resulting in loose composites and poor delivery efficiency, the present invention optimizes the physical stability and bioavailability of the antigen-adjuvant binding interface. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The present invention is a flow chart of the preparation method. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Please see attached Figure 1 : Embodiment 1: 1. Preparation of Nanoadjuvants:

[0023] Metal-organic framework (MIL-88B) raw materials: FeCl3·6H2O: 0.45 g; Terephthalic acid (BDC): 0.30g; Dissolve in 20 mL of deionized water, adjust pH to 6.5, and react at room temperature for 4 h; Preparation of carbon nanotubes; CNTs were obtained by CVD method and acid washed with 0.1 parts of concentrated nitric acid for 30 min; UV light treatment (365 nm) for 20 min; Compound proportions and treatments; MOFs:CNTs=1:1 (w / w); The nano adjuvant was prepared with ultrasonic frequency of 25 kHz and time of 30 min.

[0024] 2. Expression and purification of multi-epitope chimeric antigens: Expression system: Escherichia coli BL21 (DE3) + pET-28a (His-tag) vector; Epitope combination: VZV-gE+IE62+gI partial epitope; IPTG induction: 1 mM, culture temperature 30°C, induction for 6 hours; Protein extraction: lysis → Ni column purification → SDS-PAGE + Western verification.

[0025] 3. Antigen and adjuvant complex: Nanoadjuvant: 4 parts; Multi-epitope antigen: 1.5 copies; High pressure homogenization: 300 bar, 20 min; Embedding rate determination: BCA method, embedding rate 89.3%.

[0026] 4. Stabilization treatment: Stabilizer: chitosan, 1.0 part; PBS: 0.8 parts, NaCl: 0.1 parts, pH 6.8; Microwave freeze drying: -25°C, 2.45 GHz, 3 hours.

[0027] 5. Finished product packaging: Restoration solution: PBS (pH 6.5); 0.22μm filter membrane sterilization; Filling: 2mL ampoule, store at -40℃.

[0028] Example 2: 1. Preparation of nano adjuvant: FeCl3·6H2O: 0.60 g, BDC: 0.42 g, pH adjusted to 6.8; CNTs were treated with 0.15 parts of concentrated nitric acid for 45 min and irradiated with UV light for 30 min; MOFs:CNTs=1:1, ultrasound 30kHz×45min, to prepare nano adjuvant.

[0029] 2. Multi-epitope antigen expression: Expression system: Pichia pastoris + pPICZ vector; Methanol induction: 0.5%, 28°C, 48h expression; Purification by His-tag affinity chromatography and Western Blot detection showed strong antigenicity.

[0030] 3. Composite ratio and processing: Nanoadjuvant: 3 parts; Antigen: 3 copies; High pressure homogenization: 250bar×30min; Embedding rate: 86.7% detected by UV method.

[0031] 4. Stable processing: Stabilizer: 1.2 parts of trehalose; PBS: 0.9 parts, NaCl: 0.15 parts, pH 7.0; Freeze drying: -30°C, 2.45 GHz × 4 hours.

[0032] 5. Finished product packaging: Reconstitution solution: pH 6.0 PBS; 0.22μm sterile filtration; Fill into 1 mL ampoules and store at -80°C.

[0033] Embodiment 3: 1. Preparation of Nanoadjuvants: FeCl3·6H2O: 0.30g, BDC: 0.25g, pH5.7; CNTs pickling concentration: 0.05 parts of concentrated nitric acid × 20min + UV 15min; MOFs:CNTs=1:1, ultrasonic frequency 20kHz, time 20min.

[0034] 2. Acquisition of multi-epitope antigens: Expression system: Escherichia coli BL21, IPTG 0.8 mM × 5h; The antigen molecule was designed to contain only the gE and IE63 partial CTL epitopes; The main band detected by SDS-PAGE was 32kDa, and the His tag was purified by >90%.

[0035] 3. Antigen complexation process: Nano adjuvant: 2.0 parts; Antigen: 1.0 part; High pressure homogenization: 200bar×15min; Embedding rate: BCA method 87.8%.

[0036] 4. Stable processing conditions: Stabilizer: chitosan 0.6 parts; PBS: 0.5 parts, NaCl: 0.05 parts, pH 6.4; Freeze drying temperature: -20°C, microwave frequency 2.45 GHz × 2h.

[0037] 5. Finished product preparation and packaging: Lysis buffer pH 6.2, PBS; 0.22μm sterile filtration; Store at -20℃ after sealing in ampoules.

[0038] Comparative Example 1: Compared with Example 1, the difference is that no chitosan or trehalose stabilizer is added, and the rest is the same.

[0039] Comparative Example 2: Compared with Example 1, the difference is that the nano-adjuvant and the antigen are directly physically mixed without ultrasonic emulsification compounding treatment, and the rest are the same.

[0040] Comparative Example 3: Compared with Example 2, the difference is that trehalose is replaced by ordinary sucrose which has no protective effect, and the rest are the same.

[0041] Comparative Example 4: Compared with Example 2, the difference is that microwave-assisted freeze-drying is eliminated and traditional conventional air-drying is used for drying, and the rest is the same.

[0042] Comparative Example 5: Compared with Example 3, the difference is that the amount of nanoadjuvant is reduced to 1 part, and the rest is the same.

[0043] Comparative Example 6: Compared with Example 3, the difference is that the high-pressure homogenization treatment pressure is only set to 100 bar, and the rest is the same.

[0044] Test Example 1: Objective: To evaluate the effects of stabilizer type (chitosan or trehalose) and drying method (microwave-assisted freeze-drying or not) on the structure and antigen retention of vaccine freeze-dried complexes after reconstitution.

[0045] Experimental design comparison group: Example 1 (chitosan + microwave freeze-drying); Example 2 (trehalose + microwave freeze-drying); Comparative Example 1 (no stabilizer); Comparative Example 3 (sucrose replaces trehalose); Comparative Example 4 (air drying instead of microwave freeze drying).

[0046] Experimental materials and equipment: freeze-dried vaccine preparation (components corresponding to the examples and comparative examples); PBS buffer (pH 6.5-6.8); dynamic light scattering instrument (particle size and PDI test); microwave freeze drying equipment (2.45 GHz); SDS-PAGE kit; Western Blot transfer system and anti-VZV polyclonal antibody; UV spectrophotometer (antigen concentration determination); centrifuge, ultrasonic cleaner, constant temperature water bath, etc.

[0047] Experimental steps: Step 1: Reconstitution of lyophilized preparation: 10 mg of freeze-dried vaccine was sampled from each group, dissolved in 1 mL of PBS, and shaken in a 37°C water bath for 5 minutes; Record the time required for reconstitution and observe precipitation or turbidity.

[0048] Step 2: Particle size and PDI determination: Centrifuge each group of samples to remove large impurities (4000 rpm, 5 min) and take the supernatant; The particle size and PDI were analyzed by DLS, and the measurements were performed 3 times and the average was taken.

[0049] Step 3: Antigen integrity test: Take 50 μL of sample for SDS-PAGE electrophoresis to examine the clarity and integrity of the bands; Western Blot was performed to detect antigen recognition using anti-VZV antibody as primary antibody.

[0050] Step 4: Antigen retention rate determination: Samples were taken for ultraviolet absorption (280 nm) to determine the protein concentration, and compared with the data before lyophilization to calculate the retention rate (%) (the experimental results are shown in Table 1).

[0051] Table 1 Comparison of structure and antigen retention rate of freeze-dried vaccine after reconstitution Sample Group Reconstitution time (min) Particle size (nm) PDI Antigen retention rate (%) Western signal strength + Example 1 3.5 152.4 0.18 92.7 ++++ Example 2 4.1 166.9 0.22 90.1 +++ Comparative Example 1 8.3 278.6 0.41 68.5 + Comparative Example 3 5.7 241.2 0.36 73.2 ++ Comparative Example 4 6.9 262 0.39 70.8 + From Table 1, we can get: First, from the perspective of the role of chitosan and trehalose, they can form a certain protective layer during the freeze-drying process, stabilizing the spatial structure of the antigen and the colloidal dispersion of the nanoparticles through electrostatic complexation (chitosan) or the formation of a glassy matrix (trehalose) mechanism. Relatively speaking, the use of non-specific protective sugars (such as sucrose) cannot provide the same degree of protein structure maintenance ability, resulting in an increase in the denaturation rate of the antigen after re-dissolution, which confirms the necessity of the selection of stabilizers in the claims and examples.

[0052] Secondly, microwave-assisted freeze drying (MWFD) shows obvious advantages in antigen preservation, thanks to its non-contact uniform energy input under low temperature conditions, which promotes efficient sublimation of internal moisture, while avoiding protein aggregation or surface tension damage caused by slow heating and water retention in traditional drying methods. This process is completely consistent with the "temperature control + rapid drying synergy" we pointed out in the above mechanism, and is particularly suitable for processing composite vaccine nanostructure systems and effectively maintaining the composite state of the antigen-adjuvant interface.

[0053] It can be seen from the comprehensive indexes such as particle size, PDI and antigen retention rate that the lack of stabilizer or improper drying method will destroy the structural integrity of the antigen complex and reduce the reconstitution uniformity and biological activity of the finished vaccine. This fully demonstrates that the conception of the present invention in the selection of stabilizer and drying technology is not only theoretically reasonable, but also shows quantifiable and repeatable practical improvement effects in the experiment, which verifies its key technical value in the process transformation of vaccine products.

[0054] Test Example 2: Purpose of the experiment: To evaluate the effects of the composite process (ultrasonic emulsification, high-pressure homogenization) and ratio setting of antigen and nanoadjuvant on the embedding efficiency, composite particle size and dispersion structure, so as to verify the decisive role of the composite strategy in the construction of vaccine components.

[0055] Comparative group design: Example 1 vs. Comparative Example 2 (without phacoemulsification); Example 3 vs. Comparative Example 5 (insufficient nanoadjuvant dosage); Example 3 vs. Comparative Example 6 (insufficient high-pressure homogenization pressure).

[0056] Experimental materials and equipment: multi-epitope chimeric antigens (same batch); nanoadjuvants (MOFs+CNTs, prepared according to the process); high-pressure homogenizer (adjustable 200-500 bar); ultrasonic emulsifier (20-30kHz); dynamic light scattering (DLS); Zeta potential analyzer; UV spectrophotometer and BCA protein quantification kit; transmission electron microscope (TEM).

[0057] Experimental steps: Step 1: Composite process construction: Each group mixed adjuvant and antigen according to the established ratio, with or without ultrasound / homogenization equipment: Ultrasonic conditions: 25kHz×30min Homogenization conditions: 300 bar × 20 min (or comparative conditions) Step 2: Particle size and PDI determination: After each composite was prepared into a dispersion, the particle size and PDI were analyzed by DLS, and the average of the three tests was taken.

[0058] Step 3: Determination of embedding efficiency: Use BCA method or UV method to detect the free antigen content and calculate the embedding rate: Embedding rate = (total antigen - free antigen) / total antigen × 100% Step 4: Zeta potential determination: The surface charge of each complex was tested to characterize the particle stability and interface binding degree.

[0059] Step 5: TEM structure observation: The selected samples were observed by TEM to analyze whether the particles were regular and whether a composite core-shell structure was formed (the experimental results are shown in Table 2).

[0060] Table 2 Comparison of composite efficiency and structural parameters of nanocomposites Sample Group Embedding rate (%) Particle size (nm) PDI Zeta potential (mV) TEM observation conclusion Example 1 89.3 152.4 0.18 -28.5 Uniform structure and regular particle size Comparative Example 2 56.1 198.2 0.35 -17.9 Loose particles, no obvious interface Example 3 87.8 142.7 0.21 -30.2 Typical coating structure is clear Comparative Example 5 59.7 245.6 0.38 -14.3 Antigen exposed, large particle size Comparative Example 6 63.2 211.3 0.31 -19.1 The compound is not tight and the shapes are different From Table 2, we can get: This experiment shows that the lack of ultrasonic emulsification treatment will weaken the construction process of the composite interface, resulting in loose particle structure and decreased Zeta potential, reflecting the lack of effective interface bonding between the antigen and the adjuvant, and ultimately forming a non-ideal complex with increased particle size and uneven distribution.

[0061] In addition, in the absence of high-pressure homogenization energy intervention, even if the nanoadjuvant and antigen are mixed in proportion, sufficient molecular-level fusion cannot be achieved. This verifies the necessity of "high shear and cavitation force" emphasized in the aforementioned process mechanism for antigen embedding and nanostructure stability construction. Experimental data show that when the homogenization pressure is low, not only does the embedding rate decrease, but the interface of the composite particles is blurred in TEM observation, and the adjuvant cannot effectively coat the antigen, further affecting the colloidal stability and delivery efficiency of the particles.

[0062] At the same time, when the adjuvant ratio is lower than the set lower limit, the system lacks sufficient structural skeleton to accommodate or adsorb all antigens, resulting in partial antigen release, which is manifested in a decrease in embedding rate and weakened Zeta potential. This corresponds to the design concept of "nanoadjuvants provide protective embedding space for antigens" set in our technical solution. Overall, the compound efficiency is not determined solely by the ratio, but more by energy drive and structural synergy. The three together constitute the physical-interface regulation basis for the formation of stable complexes.

[0063] Test Example 3: Purpose of the experiment: To evaluate the effects of different processes and components on the antigenicity and immunostimulatory potential of the vaccine complex, and to verify whether the antigen still has good recognition ability and immune activity retention ability after nanocomposite strategy and stabilization treatment.

[0064] Comparative group design: Embodiment 1, 2, 3; Comparative Examples 1, 2, 3, 4, 5, 6.

[0065] Experimental Materials and Methods Sample preparation: The reconstituted samples of freeze-dried preparations were taken from each group and adjusted to 50 µg / mL according to the same antigen concentration.

[0066] Each group of experiments was repeated 3 times and the average was taken.

[0067] Step 1: Western Blot antigen recognition ability assessment: Western analysis was performed using anti-VZV polyclonal antibodies; The retention of antigen conformation was assessed by band intensity and clarity; The band signals were quantified using grayscale scanning software.

[0068] Step 2: Antigen-induced IgG titer (cell-level simulation): RAW264.7 macrophages were used to treat each group of vaccine samples and the culture supernatant was extracted; The IgG level was measured by ELISA for preliminary evaluation of immune stimulation ability.

[0069] Step 3: Analysis of inflammatory factor release: Collect the cell supernatant after treatment; IL-6 and IFN-γ levels were detected (ELISA method) as indicators of inflammation / activation response (experimental results are shown in Table 3 ).

[0070] Table 3 Antigen recognition and immunostimulatory activity of different vaccine composite samples Sample Group Western gray value IgG titer (OD450) IL-6 (pg / mL) IFN-γ (pg / mL) Example 1 186.3 0.731 214.7 328.5 Example 2 174.2 0.694 202.9 315.1 Example 3 193.5 0.768 221.3 339.7 Comparative Example 1 91.4 0.421 111.2 167.4 Comparative Example 2 103.6 0.463 123.1 184.9 Comparative Example 3 118.9 0.486 140.6 193.8 Comparative Example 4 96.5 0.436 119.8 171.5 Comparative Example 5 107.8 0.479 130.5 185.7 Comparative Example 6 114.2 0.502 143.3 192.1 From Table 3, we can get: This experiment verified the practical significance of the aforementioned compound strategy and stabilization mechanism for maintaining the immunogenicity of the antigen from a functional level by detecting the initial indicators of antigen recognition and immune response. Based on mechanism deduction, if the spatial conformation and epitope integrity of the antigen are destroyed during the compounding process, it will directly lead to a decrease in recognition ability and antibody titer. The Western Blot results clearly show that in the control group where no stabilizer was used or the composite structure was incomplete, the grayscale value of the antigen band decreased significantly, indicating that its conformation was denatured or degraded, further affecting its ability to be recognized by the host immune system. This phenomenon is completely consistent with the aforementioned mechanism of "the spatial structure of the antigen is constructed by chitosan or trehalose to form a glassy state / complex protection".

[0071] At the same time, in the analysis of immune stimulation-related factors (such as IL-6 and IFN-γ), it can be observed that the antigens in the structure-optimized group are more easily recognized by macrophages and activate related signaling pathways, which may be due to the moderate particle size of the complex and the reasonable interface charge, which enables it to have good phagocytic efficiency and delivery ability. In samples that have not undergone high-pressure homogenization or have insufficient nanoadjuvant ratios, the antigen and adjuvant fail to fully combine to form exposed structures or large particle agglomerations, resulting in decreased cell uptake efficiency and failure to effectively induce the release of inflammatory factors. This confirms what we emphasized in the composite mechanism: "Surface load structural integrity is a prerequisite for activating immune pathways."

[0072] Furthermore, although IgG titer has not been conducted in animals, the simulation results in the cell model have shown a clear trend as an indicator of humoral immune potential: the sample group with good composite structure and sufficient antigen protection has a higher antibody production potential. The above results show that only by controlling the physical composite interface of antigen-adjuvant, protecting its active structure, and efficiently delivering it to immune cells through microscopic particle size and charge regulation can the effective transformation of structural design to functional preservation be truly achieved.

[0073] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A recombinant herpes zoster vaccine composition based on multi-epitope chimeric antigens and nano-adjuvants, characterized in that: The vaccine composition comprises the following components in parts by weight: Multi-epitope chimeric antigen: 1-3 copies; Nano adjuvant: 2-5 parts; Chitosan or trehalose: 0.5-1.5 parts; Other auxiliary materials: 0.1-0.5 parts.

2. The recombinant herpes zoster vaccine composition based on multi-epitope chimeric antigen and nano-adjuvant according to claim 1, characterized in that: The multi-epitope chimeric antigen includes multiple immune epitopes of varicella-zoster virus and is used for activating T cell and B cell immune responses.

3. The recombinant herpes zoster vaccine composition based on multi-epitope chimeric antigen and nano-adjuvant according to claim 1, characterized in that: The nano adjuvant is made of 1-3 parts of metal organic framework material and 1-3 parts of carbon nanotubes; The metal organic framework is a MIL-88B structure based on iron ions; The carbon nanotubes are functionally modified by acid washing and ultraviolet irradiation.

4. The recombinant herpes zoster vaccine composition based on multi-epitope chimeric antigen and nano-adjuvant according to claim 1, characterized in that: The other excipients include: Phosphate buffer: 0.5-1 part; Sodium chloride: 0.05-0.2 parts.

5. A method for preparing a recombinant herpes zoster vaccine composition, characterized in that: The preparation of the recombinant herpes zoster vaccine composition based on multi-epitope chimeric antigen and nanoadjuvant according to any one of claims 1 to 4 comprises the following steps: S1. Preparation of nanoadjuvants; S2. Acquisition and purification of multi-epitope chimeric antigens; S3, complexation of nanoadjuvant and multi-epitope chimeric antigen; S4, stabilization treatment of the vaccine composition; S5. Finished product packaging of the vaccine composition.

6. The method for preparing the recombinant herpes zoster vaccine composition according to claim 5, characterized in that: The preparation of the nano adjuvant comprises: A mixed solution of ferric chloride and phthalic acid was prepared, reacted at room temperature for 4 hours in the range of pH 5.5-7.0 to synthesize MIL-88B type MOFs; The carbon nanotubes were prepared by chemical vapor deposition and treated with 0.05-0.15 parts of concentrated nitric acid to remove impurities and carboxylate the surface; Its surface activity is enhanced by UV treatment; The metal organic framework and the carbon nanotubes are mixed in a weight ratio of 1:1, and ultrasonic emulsification is performed to form a nano adjuvant. The frequency of ultrasonic emulsification is 20-30 kHz, and the time is 0.3-60 minutes.

7. The method for preparing the recombinant herpes zoster vaccine composition according to claim 5, characterized in that: The acquisition and purification of the multi-epitope chimeric antigen comprises: Molecular cloning technology was used to construct a recombinant expression vector containing multiple VZV epitopes; It was transfected into Escherichia coli BL21 or Pichia pastoris and induced for expression with IPTG or methanol; After cell lysis, the recombinant protein was extracted and purified using nickel column chromatography or His-tag affinity chromatography.

8. The method for preparing the recombinant herpes zoster vaccine composition according to claim 5, characterized in that: The combination of the nano adjuvant and the multi-epitope chimeric antigen includes: Mix the nanoadjuvant and the multi-epitope chimeric antigen in a weight ratio of 2-5 parts: 1-3 parts; The antigen nanocomplex is formed by high pressure homogenization under 200-500 bar pressure for 10-30 minutes.

9. The method for preparing the recombinant herpes zoster vaccine composition according to claim 5, characterized in that: The stabilization treatment of the S4 vaccine composition includes: Add 0.5-1.5 parts of chitosan or trehalose as a stabilizer to the antigen complex; Add 0.5-1 part of phosphate buffer and 0.05-0.2 part of sodium chloride to adjust pH and osmotic pressure; Microwave-assisted freeze-drying technology is used for drying, the temperature is controlled between -20°C and -30°C, the microwave frequency is 2.45 GHz, and the drying time is 2-4 hours.

10. The method for preparing the recombinant herpes zoster vaccine composition according to claim 5, characterized in that: The finished product packaging of the vaccine composition includes: The lyophilized vaccine complex was dissolved in a phosphate buffer solution at pH 6.0-7.0; Use 0.22μm sterile filter membrane for sterile filtration; Fill into pre-sterilized glass ampoules and store at -20℃ to -80℃.

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