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

Abstract

The present application relates to the technical field of vaccine preparation, and discloses a recombinant herpes zoster vaccine composition based on a multi-epitope chimeric antigen and a nano adjuvant, which comprises the following components in parts by weight: 1-3 parts of a multi-epitope chimeric antigen; 2-5 parts of a nano adjuvant; 0.5-1.5 parts of chitosan or trehalose; and 0.1-0.5 parts of other auxiliary materials, wherein the multi-epitope chimeric antigen comprises multiple immune epitopes of varicella zoster virus and is used for activating T cell and B cell immune responses, and the nano adjuvant comprises: 1-3 parts of a metal organic framework material and 1-3 parts of a carbon nanotube; the metal organic framework is an MIL-88B structure based on iron ions; and the carbon nanotube is functionally modified through acid pickling and ultraviolet light irradiation. The present application realizes the synergistic effect of antigen conformation maintenance, uniform compounding and stable reconstitution by constructing a nano composite structure and introducing a stabilizer and a microwave freeze-drying process.

Description

Technical Field

[0001] This invention relates to the field of vaccine formulation technology, specifically to a recombinant herpes zoster vaccine composition based on multi-epitope chimeric antigens and nano-adjuvants. Background Technology

[0002] With the widespread application of multi-epitope recombinant antigens in the development of novel vaccines, effectively maintaining their spatial conformation and biological activity has become crucial for vaccine formulation stability design. Especially in actual storage and transportation, freeze-drying has gradually become the mainstream method.

[0003] Currently, numerous technologies are being explored 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 during reconstitution. Other studies have introduced liposomes or PLGA nanoparticles into antigen delivery systems, constructing preliminary load-release structures and demonstrating a 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, often leading to abnormal antigen folding and significant aggregation and sedimentation after reconstitution. After structural damage, antigen recognition ability is difficult to recover. Traditional freeze-drying processes involve slow heating and long water migration paths, especially in complex vaccine systems, which often damages the antigen-adjuvant interface, resulting in an uneven or loose complex structure. Most existing antigen delivery systems rely on static physical adsorption, which has low binding efficiency, making antigens easy to detach and unable to achieve true encapsulation protection. If the adjuvant ratio is too low or the structural design is inappropriate, the antigen is like "hanging on the surface," which is neither stable nor easily recognized by the immune system. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a recombinant herpes zoster vaccine composition based on multi-epitope chimeric antigens and nano-adjuvants, which solves the problems of poor antigen stability, low compounding efficiency, uneven structure after reconstitution, and insufficient ability to fix antigen interfaces in existing vaccine formulations.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a recombinant herpes zoster vaccine composition based on multi-epitope chimeric antigen and nano-adjuvant, wherein the vaccine composition comprises the following components in parts by weight:

[0007] Multi-epitope chimeric antigen: 1-3 copies. Multi-epitope antigens can simultaneously activate cellular and humoral immune pathways.

[0008] Nanoadjuvant: 2-5 parts, nanoparticle size (usually 100-200nm) is easy for antigen-presenting cells (APCs) such as dendritic cells (DCs) to be actively taken up, improving the efficiency of antigen processing and presentation; the nanostructure provides a stable encapsulation environment, allowing the antigen to be slowly released in vivo, mimicking the natural viral infection process, thereby inducing a more lasting immune memory.

[0009] 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, enhancing the adhesion of antigens to the cell surface and promoting endocytosis. Trehalose is a non-reducing sugar with good protein protection, especially in freeze-drying and reconstitution, where it can stabilize the conformational structure of antigens through the "water substitution effect," preventing protein aggregation and denaturation. Both contribute to improving the stability and bioavailability of antigen-adjuvant complexes in vivo and are key cofactors for vaccine storage outside the cold chain.

[0010] Other excipients: 0.1-0.5 parts. Adding trace amounts of excipients to the vaccine composition can ensure the isotonicity and pH stability of the formulation and prevent local irritation or abnormal antigen structure during vaccination.

[0011] Preferably, the multi-epitope chimeric antigen includes multiple immunoepitaxes of varicella-zoster virus, used to activate T cell and B cell immune responses. B cell epitopes can directly promote the production of neutralizing antibodies and improve the protective efficacy of the vaccine; T cell epitopes, especially Th1 response-associated epitopes, can promote the activation of cytotoxic T lymphocytes (CTLs) to clear 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.

[0012] Preferably, the nano-adjuvant comprises:

[0013] It is made of 1-3 parts metal-organic framework material and 1-3 parts carbon nanotubes;

[0014] The metal-organic framework is an iron-based MIL-88B structure. The MIL-88B structure can be gradually degraded in a weakly acidic environment (such as intracellular lysosomes of APCs), enabling controlled release and intracellular release of antigens. As the central metal of the MOF backbone, iron ions not only provide structural stability but also enhance the ROS level in immune cells through the Fenton reaction, activating the maturation and signal transduction of antigen-presenting cells (APCs). The high porosity structure facilitates the "intercalation-release" of antigen molecules, improving antigen stability and reducing the risk of enzymatic inactivation.

[0015] The carbon nanotubes are functionalized through acid washing and ultraviolet irradiation. Acid washing and ultraviolet treatment form functional groups (such as -COOH, -OH) on the surface of CNTs, enhancing their hydrophilicity and antigen loading capacity, and reducing the biotoxicity of the original CNTs. The modified CNTs have the ability to enhance cell membrane permeability, which can promote the entry of antigens into dendritic cells and other APCs, and improve cross-presentation efficiency. Ultraviolet treatment may stimulate the formation of charged regions on the surface of CNTs, which can mimic the function of certain natural PAMPs in the immune microenvironment, indirectly activating the Toll-like receptor (TLR) pathway and triggering a primary immune response.

[0016] Preferably, the other excipients include:

[0017] Phosphate buffer: 0.5-1 part. During the lyophilization, reconstitution and in vivo delivery of vaccines, the structure of antigen proteins is sensitive to pH fluctuations. Phosphate buffer can stabilize the pH of the system within the physiological neutral range of 6.8-7.4, avoiding antigen denaturation, aggregation or hydrolysis. Nanoadjuvants have different charge states at different pH levels. PBS helps maintain their colloidal stability and charge balance, prevents nanoparticle aggregation, and ensures uniform antigen distribution.

[0018] Sodium chloride: 0.05-0.2 parts. Sodium chloride can adjust the osmotic pressure of the entire vaccine solution to about 280-300 mOsm / kg, which is close to the osmotic pressure of human body fluids. It can effectively prevent the dehydration or swelling of local tissue cells at the injection site, reduce inflammatory response, stabilize the conformation of antigen molecules in solution by adjusting ionic strength, and reduce van der Waals attraction between nano-adjuvants, which helps to maintain uniform particle distribution.

[0019] The present invention also provides a method for preparing a recombinant herpes zoster vaccine composition, comprising the following steps:

[0020] S1, preparation of nano-adjuvants;

[0021] S2. Acquisition and purification of multi-epitope chimeric antigens;

[0022] S3, a combination of nano-adjuvants and multi-epitope chimeric antigens;

[0023] S4. Stabilization treatment of vaccine composition;

[0024] S5. Finished packaging of vaccine compositions.

[0025] Preferably, the preparation of the nano-adjuvant includes:

[0026] A mixed solution of ferric chloride and phthalic acid was prepared and reacted at room temperature for 4 hours within the pH range of 5.5-7.0 to synthesize MIL-88B-type MOFs. MIL-88B is a three-dimensional porous structure formed between iron ions and organic ligands (phthalic acid). The pH in the reaction conditions controlled the crystal growth rate and morphology. The reaction was completed at room temperature, which can reduce the formation of crystal defects, maintain the high porosity and specific surface area of ​​the material, and improve its subsequent antigen loading capacity. Iron element acts as an active center in this structure, not only building a stable framework, but also inducing immune-related oxidative stress response in vivo and enhancing APC activation ability, which is the basis for constructing functional MOF nanoadjuvants.

[0027] Carbon nanotubes (CNTs) were prepared by chemical vapor deposition (CVD), and impurities and carboxylated surfaces were removed by treatment with 0.05-0.15 parts of concentrated nitric acid. CVD synthesis can achieve high-purity, directionally grown CNT structures, which is beneficial for subsequent composite formation with MOFs to form regular interfaces. During the concentrated nitric acid treatment, polar groups such as -COOH and -OH were generated on the CNT surface (carboxylation), which not only removed residual metal impurities but also improved their hydrophilicity, dispersibility, and biocompatibility. Carboxylation modification gives CNTs a stronger antigen adsorption capacity and facilitates the formation of hydrogen bonds or electrostatic interactions with MOFs, laying the foundation for the formation of stable composite nanoadjuvants.

[0028] UV irradiation enhances the surface activity of CNTs. UV irradiation excites the surface energy states of CNTs, further inducing oxidation and promoting the uniform distribution of functional groups such as hydroxyl and carboxyl groups. It also enhances the interfacial affinity between CNTs and antigens and MOFs. At the same time, the formed "active surface" has a certain degree of immunostimulatory properties and can simulate PAMP signaling to activate immune pathways. After UV modification, CNTs exhibit stronger dispersion stability and antigen delivery performance, improving the overall functional performance of nanoadjuvants.

[0029] MOFs and CNTs were mixed in a 1:1 weight ratio and ultrasonically emulsified to form nano-adjuvants. The ultrasonic emulsification frequency was 20-30 kHz and the time was 0.3-60 minutes. The ultrasonic emulsification technology enabled the MOFs and CNTs nanostructures to fully contact and cross-link through cavitation effect and shear force, avoiding aggregation. The 1:1 ratio can achieve structural balance and functional complementarity, ensuring that the nano-adjuvants achieve synergistic advantages in 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.

[0030] Preferably, the acquisition and purification of the multi-epitope chimeric antigen includes:

[0031] Recombinant expression vectors containing multiple VZV epitopes were constructed using molecular cloning technology. Epitope selection was based on the analysis of conserved VZV regions and immune activity data. The fusion design can simultaneously activate humoral and cellular immunity. The molecular cloning process was verified by enzyme digestion, ligation, and sequencing to ensure correct sequence integration and guarantee the antigen fidelity of downstream expression. The introduction of the His tag facilitated subsequent purification without interfering with the spatial conformation of the immune epitopes, ensuring functional expression.

[0032] Transfecting the chimeric antigen into *E. coli* BL21 or *Pichia pastoris* and inducing expression with IPTG or methanol offers several advantages. The BL21 system is efficient and inexpensive, making it suitable for rapid expression of antigen proteins. The *Pichia pastoris* system is more suitable for expressing eukaryotic antigens with spatial conformational requirements or glycosylation modifications. IPTG-induced expression systems, controlled by the T7 promoter, rapidly initiate transcription and translation. Methanol-induced expression in *Pichia pastoris* depends on the AOX1 promoter, enabling high-density protein expression and making it suitable for constructing antigens requiring folding modifications. Flexible selection of the expression system ensures the expression efficiency, correct folding, and preservation of immune function of the chimeric antigen.

[0033] After cell lysis, recombinant proteins were extracted and purified using nickel column chromatography or His-tag affinity chromatography. His-tag and Ni 2+ Stable coordination bonds are formed between them, which can specifically capture tagged chimeric proteins and achieve efficient separation from impurities; the elution conditions (such as imidazole gradient) can be precisely controlled during the chromatography process to optimize protein recovery and purity; the buffer system used in the lysis and purification process can protect the protein structure, avoid degradation or aggregation and inactivation, and ensure the immune function of the antigen.

[0034] SDS-PAGE and Western Blot are used to verify the purity and antigenicity of the expression. SDS-PAGE provides a direct reflection of the protein molecule size and purity, and is the first step in verifying whether the expression is successful. Western Blot uses membrane immune reactions to verify whether the recombinant antigen has the correct conformation and retains the 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 immunization.

[0035] Preferably, the combination of the nano-adjuvant and the multi-epitope chimeric antigen comprises:

[0036] The nano-adjuvant is mixed with the multi-epitope chimeric antigen at a weight ratio of 2-5 parts: 1-3 parts. The 2-5 times amount of adjuvant ensures complete coating or sufficient adsorption interface for the antigen, improving the protective and controlled release performance of the antigen in vivo. At the same time, controlling the antigen ratio does not exceed the limit to avoid non-specific distribution or rapid degradation caused by free antigen, 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, maintaining the uniformity of nanoparticle size and distribution.

[0037] Antigen nanocomposites are formed by high-pressure homogenization at 200-500 bar for 10-30 minutes. High-pressure homogenization utilizes shear force, cavitation force, and impact force to fully mix and interfacially integrate the nanomaterials with the antigen. This process helps to form embedded or adsorbed antigen nanocomposites, improving their stability and bioavailability. The appropriate pressure and time range can effectively control the particle size of the composite within the range of 100-200 nm, which is beneficial for lymphatic transport and APC uptake.

[0038] Antigen encapsulation efficiency should be detected using ultraviolet spectrophotometry or the BCA method, ensuring it is ≥85%. High encapsulation efficiency is a key indicator for ensuring vaccine efficacy and dosage control. An encapsulation efficiency of ≥85% can reduce antigen loss and non-specific clearance. The ultraviolet method measures free antigen by measuring the difference in protein absorption peaks, while the BCA method detects total protein based on the copper ion reduction reaction. The two methods can be used to verify each other. High encapsulation efficiency also reflects the high affinity and structural stability of the nano-adjuvant for the antigen, indirectly characterizing the quality of complex formation. In addition, an appropriate encapsulation level helps to form a sustained delivery and antigen library reserve mechanism, enhancing the timeliness and intensity of the immune response.

[0039] Preferably, the stabilization treatment of the S4 vaccine composition includes:

[0040] Add 0.5-1.5 parts of chitosan or trehalose to the antigen complex as a stabilizer. Chitosan is a cationic polysaccharide that can form electrostatic complexes and gel layers on the surface of nanoparticles, improving the physical stability of the composite particles and preventing aggregation and sedimentation. Trehalose is a non-reducing disaccharide with excellent vitrification ability and protein protection function. It can replace water in the interaction with proteins during the drying process to prevent antigen denaturation. Both can be selected according to needs and have good biocompatibility and immunofriendliness, and will not inhibit antigen delivery or immune activation response.

[0041] Add 0.5-1 parts phosphate buffer and 0.05-0.2 parts sodium chloride to adjust pH and osmotic pressure. PBS maintains the pH of the system between 6.8 and 7.4, providing a suitable acid-base environment for antigen structure preservation and adjuvant stability, and avoiding acid-base stress during the drying process. NaCl ensures that the osmotic pressure of the solution is close to physiological conditions (about 280-300 mOsm / kg), which can prevent the disintegration of colloidal nanoparticle structure or osmotic pressure shocks from causing abnormal protein folding before lyophilization.

[0042] Microwave-assisted freeze-drying (MWFD) technology was used for drying, with the temperature controlled between -20°C and -30°C, the microwave frequency at 2.45 GHz, and the drying time at 2-4 hours. MWFD technology combines the advantages of low-temperature freeze-drying and microwave heating, accelerating water sublimation without raising the sample temperature. The frequency of 2.45 GHz is an industrial standard microwave frequency with good penetration efficiency, enabling uniform heating and efficient removal of internal moisture. Processing under temperature control conditions of -20°C to -30°C effectively protects temperature-sensitive components, such as chimeric antigens and polysaccharide adjuvants, ensuring that the vaccine retains its complete particle size structure and biological function after reconstitution.

[0043] Preferably, the finished product packaging of the vaccine composition includes:

[0044] The lyophilized vaccine complex was dissolved in phosphate buffer at pH 6.0-7.0. pH 6.0-7.0 is a neutral to slightly acidic region between extracellular fluid (7.4) and endosomal acidification environment, which helps to form a stable nano-dispersion system after reconstitution. PBS provides ionic strength and isotonic environment, which can avoid protein aggregation and flocculation after reconstitution and ensure the homogeneity of vaccine formulation.

[0045] Aseptic filtration is performed using a 0.22μm sterile filter membrane. The 0.22μm microporous membrane is the internationally recognized standard pore size for liquid sterilization, which can effectively filter out microbial contaminants such as bacteria and fungal spores. Since the particle size of the antigen nanocomposite is designed 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 does not pose a risk of thermal or oxidative damage to the protein antigens and nano-adjuvant structures in the vaccine, making it particularly suitable for structurally sensitive vaccine formulations.

[0046] Filled into pre-sterilized glass ampoules and stored at -20°C to -80°C, glass ampoules, with their low reactivity, high airtightness, and good visibility, are ideal packaging materials for protein vaccine preparations, preventing degradation reactions such as oxidation and photolysis. The sterilized ampoules, combined with aseptic filtration filling, form a complete aseptic protection chain, avoiding secondary contamination. The storage temperature range of -20°C to -80°C can slow down the protein degradation rate, inhibit microbial growth and hydrolysis reactions, and maintain vaccine activity.

[0047] This invention provides a recombinant herpes zoster vaccine composition based on multi-epitope chimeric antigen and nano-adjuvant. It possesses the following beneficial effects:

[0048] 1. This invention uses trehalose or chitosan as a freeze-drying stabilizer, introducing a protective sugar network into the vaccine complex structure. This effectively prevents antigen conformational denaturation and aggregation during the drying process, thereby improving the antigen retention rate and particle size uniformity after reconstitution of the freeze-dried formulation. Compared to traditional stabilizers such as sucrose commonly used in the prior art, their non-specific protective ability is limited, often leading to antigen structural disorder or precipitation, making it difficult to achieve reconstitution stability under long-term storage conditions. This invention solves the problem of insufficient stability of such adjuvants for protein antigens.

[0049] 2. This invention employs a microwave-assisted freeze-drying process, introducing a directional energy field at low temperatures to enhance water sublimation efficiency and reduce the risks of protein denaturation and structural disintegration in traditional freeze-drying. This achieves rapid drying with high antigen integrity. Compared to existing technologies using air drying or conventional freeze-drying methods, which have long drying times and uneven heat conduction, easily causing antigen aggregation or inactivation, this invention avoids the activity attenuation problem caused by slow heating.

[0050] 3. This invention improves the antigen encapsulation rate and delivery particle stability by introducing a nano-adjuvant system constructed from MOFs and CNTs and optimizing the antigen-to-particle ratio. This achieves the technical effects of uniform nanostructure, reasonable antigen distribution, and high compounding efficiency. Compared to conventional aluminum adjuvants or carrier-free delivery methods, which suffer from problems such as easy antigen release, uneven particle size, and difficulty in controlled release, this invention effectively overcomes the shortcomings of traditional carrier systems in terms of insufficient antigen interfacial fixation.

[0051] 4. This invention incorporates a dual-processing step of ultrasonic emulsification and high-pressure homogenization during the complex construction process. Utilizing shear force and cavitation effects, it promotes deep embedding of the antigen into the nanocarrier, resulting in a compact structure. This enhances the stability of vaccine particles and cellular uptake efficiency, thereby improving the ability to induce an immune response. Compared to existing processes that lack an energy-driven step or rely solely on static mixing, leading to loose composites and poor delivery efficiency, this invention optimizes the physical stability and bioavailability of the antigen-adjuvant binding interface. Attached Figure Description

[0052] Figure 1 This is a flowchart of the preparation method of the present invention. Detailed Implementation

[0053] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] Please see the appendix Figure 1 : Example 1:

[0055] 1. Preparation of nano-adjuvants:

[0056] Metal-Organic Framework (MIL-88B) Raw Materials:

[0057] FeCl3·6H2O: 0.45g;

[0058] Terephthalic acid (BDC): 0.30g;

[0059] Dissolve in 20 mL of deionized water, adjust pH to 6.5, and react at room temperature for 4 h;

[0060] Carbon nanotube preparation;

[0061] CNTs were obtained by CVD and then acid-washed with 0.1 part concentrated nitric acid for 30 min.

[0062] UV irradiation (365nm) for 20 min;

[0063] Composite ratio and processing;

[0064] MOFs:CNTs=1:1 (w / w);

[0065] Nano-adjuvants were prepared by ultrasonication at a frequency of 25 kHz for 30 minutes.

[0066] 2. Expression and purification of multi-epitope chimeric antigens:

[0067] Expression system: Escherichia coli BL21(DE3) + pET-28a (His-tag) vector;

[0068] Epitope combination: VZV-gE+IE62+gI partial epitopes;

[0069] IPTG induction: 1 mM, culture temperature 30℃, induction for 6 hours;

[0070] Protein extraction: lysis → Ni column purification → SDS-PAGE + Western spectroscopy verification.

[0071] 3. Antigen and adjuvant combination:

[0072] Nano adjuvant: 4 parts;

[0073] Multiepitope antigen: 1.5 samples;

[0074] High-pressure homogenization: 300 bar, 20 min;

[0075] Encapsulation efficiency determination: BCA method, encapsulation efficiency 89.3%.

[0076] 4. Stabilization treatment:

[0077] Stabilizer: Chitosan, 1.0 part;

[0078] PBS: 0.8 parts, NaCl: 0.1 parts, pH 6.8;

[0079] Microwave freeze drying: -25℃, 2.45GHz, 3 hours.

[0080] 5. Finished product packaging:

[0081] Reconstitution solution: PBS (pH 6.5);

[0082] Sterilization via 0.22μm filter membrane;

[0083] Filling: 2mL ampoules, store at -40℃.

[0084] Example 3:

[0085] 1. Preparation of nano-adjuvants:

[0086] FeCl3·6H2O: 0.30g, BDC: 0.25g, pH5.7;

[0087] CNT pickling concentration: 0.05 parts concentrated nitric acid × 20 min + UV 15 min;

[0088] MOFs:CNTs=1:1, ultrasound frequency 20kHz, time 20min.

[0089] 2. Acquisition of multi-epitope antigens:

[0090] Expression system: Escherichia coli BL21, IPTG 0.8mM × 5h;

[0091] The antigen molecule was designed to contain only gE and IE63 CTL epitopes;

[0092] SDS-PAGE analysis showed the main band to be 32 kDa, and His tag purification was >90%.

[0093] 3. Antigen complexation process:

[0094] Nano-adjuvant: 2.0 parts;

[0095] Antigen: 1.0 sample;

[0096] High-pressure homogenization: 200 bar × 15 min;

[0097] Encapsulation rate: 87.8% using the BCA method.

[0098] 4. Stabilization conditions:

[0099] Stabilizer: 0.6 parts chitosan;

[0100] PBS: 0.5 parts, NaCl: 0.05 parts, pH 6.4;

[0101] Freeze-drying temperature: -20℃, microwave frequency 2.45GHz×2h.

[0102] 5. Finished product preparation and packaging:

[0103] Dissolve in PBS (pH 6.2).

[0104] 0.22μm sterilization filtration;

[0105] Store the ampoules at -20°C after sealing.

[0106] Comparative Example 1: The difference from Example 1 is that chitosan or trehalose stabilizers were not added, but everything else is the same.

[0107] Comparative Example 2: Compared with Example 1, the difference is that ultrasonic emulsification and compounding were not performed. Instead, the nano-adjuvant and antigen were directly mixed physically. All other aspects were the same.

[0108] Comparative Example 3: Compared with Example 2, the difference is that trehalose was replaced with ordinary sucrose which does not have a protective effect, otherwise they are the same.

[0109] Comparative Example 4: Compared with Example 2, the difference is that microwave-assisted freeze drying was cancelled, and conventional air drying was used instead; otherwise, they are the same.

[0110] Comparative Example 5: Compared with Example 3, the difference is that the amount of nano-adjuvant was reduced to 1 part, and all other aspects were the same.

[0111] Comparative Example 6: Compared with Example 3, the difference is that the high-pressure homogenization pressure is only set to 100 bar, while the rest are the same.

[0112] Test Example 1:

[0113] Experimental objective: To evaluate the effects of stabilizer type (chitosan or trehalose) and drying method (whether microwave-assisted freeze-drying is used) on the structure and antigen retention of the vaccine freeze-dried complex after reconstitution.

[0114] Experimental design comparison group:

[0115] Example 1 (Chitosan + Microwave Freeze-drying);

[0116] Example 2 (trehalose + microwave freeze-drying);

[0117] Comparative Example 1 (without stabilizer);

[0118] Comparative Example 3 (Sucrose instead of trehalose);

[0119] Comparative Example 4 (Air drying instead of microwave freeze drying).

[0120] Experimental materials and equipment: freeze-dried vaccine formulations (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 blotting transfer system and anti-VZV polyclonal antibody; ultraviolet spectrophotometer (antigen concentration determination); centrifuge, ultrasonic cleaner, constant temperature water bath, etc.

[0121] Experimental steps:

[0122] Step 1: Reconstitute the lyophilized formulation:

[0123] 10 mg of the freeze-dried vaccine from each group was taken, dissolved in 1 mL of PBS, and shaken in a 37°C water bath for 5 minutes.

[0124] Record the time required for reconstitution and observe any precipitation or turbidity.

[0125] Step 2: Particle size and PDI determination:

[0126] Centrifuge each group of samples to remove large particulate impurities (4000 rpm, 5 min), and collect the supernatant;

[0127] Particle size and PDI were analyzed using DLS, with measurements taken three times and the average value calculated.

[0128] Step 3: Antigen integrity test:

[0129] Take 50µL of sample and perform SDS-PAGE electrophoresis to examine the clarity and integrity of the bands;

[0130] Western blotting was performed to detect antigen recognition using anti-VZV antibody primary antibody.

[0131] Step 4: Antigen retention rate determination:

[0132] Samples were taken for UV absorption (280 nm) to determine protein concentration, and the retention rate (%) was calculated by comparing the data with that before lyophilization (see Table 1 for experimental results).

[0133] Table 1 Comparison of structure and antigen retention rate of freeze-dried vaccines after reconstitution

[0134] 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 +

[0135] From Table 1, we can obtain:

[0136] First, considering the functions of chitosan and trehalose, both can form a protective layer during freeze-drying, stabilizing the spatial structure of the antigen and the colloidal dispersion of nanoparticles through electrostatic complexation (chitosan) or the formation of a glassy matrix (trehalose), respectively. In contrast, using non-specific protective sugars (such as sucrose) cannot provide the same level of protein structure maintenance, leading to an increased denaturation rate after antigen reconstitution, thus confirming the necessity of the stabilizer selection specified in the claims and examples.

[0137] Secondly, microwave-assisted freeze-drying (MWFD) exhibits significant advantages in antigen preservation due to its ability to provide non-contact, uniform energy input at low temperatures, promoting efficient sublimation of internal moisture while avoiding protein aggregation or surface tension damage caused by slow heating and moisture retention in traditional drying methods. This process is entirely consistent with the "synergistic effect of temperature control and rapid drying" described in the aforementioned mechanism, making it particularly suitable for processing complex vaccine nanostructure systems and effectively maintaining the antigen-adjuvant interface complex state.

[0138] The combined analysis of particle size, PDI, and antigen retention rate reveals that the absence of stabilizers or improper drying methods can disrupt the structural integrity of the antigen complex, reducing the reconstitution homogeneity and bioactivity of the finished vaccine. This demonstrates that the present invention's concept regarding stabilizer selection and drying technology is not only theoretically sound but also exhibits quantifiable and repeatable practical improvement effects in experiments, validating its key technological value in vaccine product process transformation.

[0139] Test Example 2:

[0140] Experimental objective: To evaluate the effects of antigen-nanoadjuvant composite process (ultrasonic emulsification, high-pressure homogenization) and ratio settings on encapsulation efficiency, composite particle size, and dispersion structure, thereby verifying the decisive role of composite strategy in vaccine component construction.

[0141] Control group design:

[0142] Example 1 vs. Comparative Example 2 (without ultrasonic emulsification);

[0143] Example 3 vs Comparative Example 5 (insufficient nanozole dosage);

[0144] Example 3 vs Comparative Example 6 (Insufficient pressure for high-pressure homogenization).

[0145] Experimental materials and equipment: multi-epitope chimeric antigen (uniform batch); nano-adjuvants (MOFs+CNTs, prepared according to process); high-pressure homogenizer (adjustable 200-500 bar); ultrasonic emulsifier (20-30 kHz); dynamic light scattering instrument (DLS); Zeta potential analyzer; ultraviolet spectrophotometer and BCA protein quantification kit; transmission electron microscope (TEM).

[0146] Experimental steps:

[0147] Step 1: Construction of the composite process:

[0148] Each group mixes adjuvant and antigen in a predetermined ratio, with or without using ultrasound / homogenization equipment:

[0149] Ultrasonic conditions: 25kHz × 30min

[0150] Homogenization conditions: 300 bar × 20 min (or comparative conditions)

[0151] Step 2: Particle size and PDI determination:

[0152] After each complex was prepared into a dispersion, its particle size and PDI were analyzed by DLS, and the average value was taken from three tests.

[0153] Step 3: Encapsulation rate determination:

[0154] The content of free antigen was detected using the BCA method or ultraviolet method, and the encapsulation efficiency was calculated.

[0155] Encapsulation efficiency = (Total antigen - Free antigen) / Total antigen × 100%

[0156] Step 4: Zeta potential measurement:

[0157] The surface charge of each composite was tested to characterize particle stability and interfacial bonding.

[0158] Step 5: TEM structural observation:

[0159] Selected samples were observed using TEM to analyze whether the particles were regular and whether they formed a composite core-shell structure (experimental results are shown in Table 2).

[0160] Table 2 Comparison of nanocomposite composite efficiency and structural parameters

[0161] Sample group Encapsulation rate (%) Particle size (nm) PDI Zeta potential (mV) TEM observation conclusions 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 The particles are loose and have no obvious interface. Example 3 87.8 142.7 0.21 -30.2 Typical encapsulation structure is clear Comparative Example 5 59.7 245.6 0.38 -14.3 Exposed antigens, large particle size Comparative Example 6 63.2 211.3 0.31 -19.1 The composite is not tightly packed and the shapes are inconsistent.

[0162] From Table 2, we can obtain:

[0163] This experiment shows that the absence of ultrasonic emulsification weakens the construction process of the complex interface, resulting in loose particle structure and decreased zeta potential, reflecting the lack of effective interfacial binding between antigen and adjuvant, and ultimately forming a non-ideal complex with increased particle size and uneven distribution.

[0164] Furthermore, in the absence of high-pressure homogenization energy, even when the nano-adjuvant and antigen are mixed in the correct proportions, sufficient molecular-level fusion cannot be achieved. This verifies the necessity of "high shear and cavitation forces" emphasized in the aforementioned process mechanism for antigen embedding and the construction of nanostructure stability. Experimental data show that when the homogenization pressure is low, not only does the encapsulation rate decrease, but the interface of the composite particles becomes blurred in TEM observation, and the adjuvant cannot effectively encapsulate the antigen, further affecting the colloidal stability and delivery efficiency of the particles.

[0165] Meanwhile, when the adjuvant ratio falls below a set lower limit, the system lacks a sufficient structural framework to accommodate or adsorb all the antigen, leading to partial antigen release, manifested in decreased encapsulation efficiency and weakened zeta potential. This corresponds to our design concept in the technical solution: "nanoadjuvants provide protective encapsulation space for antigens." Overall, the composite efficiency is not solely determined by the ratio, but relies more on energy-driven processes and structural synergy; these three factors together constitute the physical-interface regulation basis for stable composite formation.

[0166] Test Example 3:

[0167] Experimental objective: To evaluate the effects of different processes and components on the antigenicity and immunostimulatory potential of vaccine complexes, and to verify whether antigens still possess good recognition ability and immune activity retention after nanocomposite strategies and stabilization treatment.

[0168] Control group design:

[0169] Examples 1, 2, and 3;

[0170] Comparative examples 1, 2, 3, 4, 5, and 6.

[0171] Experimental Materials and Methods:

[0172] Sample preparation:

[0173] For each group, samples were reconstituted from the lyophilized formulation and adjusted to 50 µg / mL according to the same antigen concentration.

[0174] Each experiment was repeated 3 times, and the average was taken.

[0175] Step 1: Western Blot antigen recognition capability assessment:

[0176] Western spectroscopy was performed using anti-VZV polyclonal antibody;

[0177] Antigen conformation retention was assessed by measuring band intensity and clarity.

[0178] The strip signal is quantized using grayscale scanning software.

[0179] Step 2: Antigen-induced IgG titer (cellular-level simulation):

[0180] The vaccine samples from each group were treated with RAW264.7 macrophages, and the culture supernatant was extracted.

[0181] IgG levels were detected by ELISA and used as a preliminary assessment of immune stimulation capacity.

[0182] Step 3: Analysis of inflammatory factor release:

[0183] Collect the cell supernatant after processing;

[0184] The levels of IL-6 and IFN-γ were measured using an ELISA method as indicators of inflammation / activation response (the experimental results are shown in Table 3).

[0185] Table 3. Antigen recognition and immunostimulatory activity of different vaccine composite samples

[0186] Sample group Western grayscale 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

[0187] From Table 3, we can obtain:

[0188] This experiment, by detecting preliminary indicators of antigen recognition and immune response, functionally verified the practical significance of the aforementioned complexation strategy and stabilization mechanism for maintaining antigen immunogenicity. Based on mechanistic deduction, if the spatial conformation and epitope integrity of the antigen are disrupted during the complexation process, it will directly lead to a decrease in recognition ability and a reduction in antibody titer. Western blotting results clearly show that in the comparison samples without stabilizers or with incomplete complex structures, the gray value of the antigen band decreased significantly, indicating that its conformation had degenerated or degraded, further affecting its ability to be recognized by the host immune system. This phenomenon is completely consistent with the aforementioned mechanism of "antigen spatial structure being protected by a glassy / complex structure constructed from chitosan or trehalose".

[0189] Meanwhile, in the analysis of immune-stimulating factors (such as IL-6 and IFN-γ), it was observed that antigens in the optimized structure group were more easily recognized by macrophages and activated related signaling pathways. This may be due to the moderate particle size and reasonable interfacial charge of the complex, giving it good phagocytic efficiency and delivery capability. In contrast, in samples that were not homogenized under high pressure or had insufficient nanoadjuvant ratios, the antigen and adjuvant failed to bind sufficiently, forming exposed structures or large particle aggregates, resulting in decreased cellular uptake efficiency and an inability to effectively induce the release of inflammatory factors. This confirms our emphasis in the complex mechanism that "the integrity of the surface-loaded structure is a prerequisite for activating immune pathways."

[0190] Furthermore, although IgG titer, as a characterization indicator of humoral immune potential, was not measured in animals, simulations in cell models showed a clear trend: sample groups with well-designed complex structures and adequate antigen protection exhibited higher antibody production potential. These results suggest that only by controlling the physical interface between the antigen and adjuvant, protecting their active structure, and regulating their efficient delivery to immune cells through microscopic particle size and charge modulation can the effective transformation from structural design to functional preservation be truly achieved.

[0191] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a recombinant herpes zoster vaccine composition, characterized in that, Includes the following steps: S1. Preparation of nano-adjuvants; S2. Acquisition and purification of multi-epitope chimeric antigens; S3. Combination of nano-adjuvants and multi-epitope chimeric antigens; S4. Stabilization treatment of vaccine composition; S5. Finished packaging of vaccine compositions; The preparation of the nano-adjuvant includes: dissolving 0.45g FeCl3·6H2O and 0.30g terephthalic acid in 20mL deionized water, adjusting the pH to 6.5, and reacting at room temperature for 4 hours to synthesize the MIL-88B type metal-organic framework; Carbon nanotubes were prepared by chemical vapor deposition and treated with 0.1 part of concentrated nitric acid for 30 min; then treated with 365 nm ultraviolet light for 20 min; MIL-88B metal-organic framework and carbon nanotubes were mixed at a weight ratio of 1:1 and ultrasonically emulsified to form a nano-adjuvant. The ultrasonic emulsification frequency was 25 kHz and the time was 30 min. The preparation of the multi-epitope chimeric antigen includes: constructing a recombinant expression vector containing a partial epitope of VZV-gE+IE62+gI using molecular cloning technology; transfecting it into Escherichia coli BL21(DE3) for pET-28a vector-induced expression; extracting and purifying the recombinant protein by nickel column chromatography after cell lysis, and verifying the recombinant protein by SDS-PAGE+Western. The process of combining the nano-adjuvant with the multi-epitope chimeric antigen involves: mixing 4 parts of nano-adjuvant with 1.5 parts of multi-epitope chimeric antigen; processing the mixture under high pressure homogenization at 300 bar for 20 minutes; and determining the encapsulation rate to be 89.3% using the BCA method to form an antigen nanocomposite. The stabilization treatment of the S4 vaccine composition includes: adding 1.0 part of chitosan as a stabilizer to the antigen complex; adding 0.8 parts of phosphate buffer and 0.1 parts of sodium chloride to adjust the pH to 6.8; and drying using microwave-assisted freeze-drying technology, with the temperature controlled at -25°C, the microwave frequency at 2.45 GHz, and the drying time at 3 hours. The finished product packaging of the vaccine composition includes: dissolving the lyophilized vaccine complex in a phosphate buffer solution at pH 6.5; performing aseptic filtration using a 0.22 μm sterile filter membrane; filling it into pre-sterilized 2 mL ampoules; and storing it at -40°C.

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