Recombinant herpes zoster vaccine based on gE / pORF7 double-antigen nanocrystal and intelligent phase change adjuvant

Abstract

The invention relates to the technical field of biological medicine, and discloses a recombinant herpes zoster vaccine based on gE / pORF7 double-antigen nanocrystal and an intelligent phase change adjuvant, the recombinant herpes zoster vaccine comprises the double-antigen nanocrystal formed by connecting gE protein and pORF7 protein through a chemical bond, the gE protein forms an inner core, and the pORF7 protein is coupled to the surface of the inner core through a dynamic covalent bond; the intelligent phase change adjuvant wraps the outer layer of the double-antigen nanocrystal, the adjuvant is composed of a thermo-sensitive polymer carrier and immunostimulatory molecules encapsulated in the thermo-sensitive polymer carrier, and the thermo-sensitive polymer carrier is subjected to phase change within the range of 25-40 DEG C. The vaccine provided by the invention can synchronously induce high-titer neutralizing antibody and long-acting T cell immunity, significantly improves the ability to clear a latent virus library, reduces the risk of adverse reaction of inoculation, and is suitable for herpes zoster prevention of old people and immunodeficient people.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to a recombinant herpes zoster vaccine based on gE / pORF7 dual-antigen nanocrystals and an intelligent phase-change adjuvant. Background Art

[0002] Herpes zoster is caused by the reactivation of varicella-zoster virus (VZV), and its risk increases significantly with age and decreased immunity. Although existing vaccines induce neutralizing antibodies by targeting the viral envelope glycoprotein gE, their single antigen design makes it difficult to cover the immune escape mechanism of the virus during the latent infection stage, resulting in limited ability to clear the latent virus reservoir.

[0003] In addition, traditional adjuvant systems mostly rely on a single immune stimulation signal (such as MPLA in AS01B), which cannot coordinate the dynamic balance between innate immune activation and long-term T cell memory formation, causing the vaccine's protective efficacy to decay with prolonged vaccination time.

[0004] At the process level, physical mixing or simple coupling of free antigens can easily lead to structural instability, and the uncontrollable spatial arrangement of antigen-adjuvant reduces the efficiency of lymphatic system delivery, further limiting the intensity of the immune response.

[0005] For high-risk groups such as the elderly, existing technologies still have the problem of poor vaccination tolerance caused by systemic inflammatory response. These systemic defects jointly restrict the protective effect and application scope of herpes zoster vaccines in the real world, and breakthroughs are urgently needed in multiple dimensions such as antigen design, adjuvant strategy and preparation process. Summary of the invention

[0006] In response to the shortcomings of the prior art, the present invention provides a recombinant shingles vaccine based on gE / pORF7 dual-antigen nanocrystals and intelligent phase-change adjuvants, which solves the technical defects of the existing shingles vaccine that are unable to simultaneously block viral infection and eliminate latent viral reservoirs due to single antigen design, insufficient adjuvant synergy and inefficient delivery system.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions: The first aspect of the present invention provides a recombinant herpes zoster vaccine based on gE / pORF7 dual antigen nanocrystals and an intelligent phase-change adjuvant, comprising: A dual-antigen nanocrystal formed by chemically linking a gE protein and a pORF7 protein, wherein the gE protein constitutes an inner core and the pORF7 protein is coupled to the surface of the inner core via a dynamic covalent bond; The intelligent phase-change adjuvant wrapped in the outer layer of the dual-antigen nanocrystal consists of a thermosensitive polymer carrier and immunostimulatory molecules encapsulated therein, and the thermosensitive polymer carrier undergoes phase change within the range of 25 to 40°C.

[0008] Preferably, the chemical bond comprises at least one of a metal ion coordination bond and a thioester bond, wherein: The metal ion coordination bond is connected to Zn through the histidine tag of gE protein. 2+ , Fe 3+ Cu 2+ At least one of the above is combined to form; The thioester bond is generated by the reaction of the sulfhydryl group of the pORF7 protein with the carboxyl group or amino group of the gE protein.

[0009] Preferably, the thermosensitive polymer carrier is a polycaprolactone-polyethylene glycol block copolymer, and the immunostimulatory molecules include a TLR7 / 8 agonist and an IL-15 superagonist.

[0010] The second aspect of the present invention provides a method for preparing the vaccine according to the first aspect of the present invention, comprising the following steps: a) Forming the gE protein nanocrystal core by metal ion coordination method; b) coupling the pORF7 protein to the surface of the gE protein nanocrystal through a dynamic covalent bond to form a dual-antigen nanocrystal; c) The smart phase-change adjuvant and dual-antigen nanocrystals are combined through a low-temperature microfluidic co-assembly process to form a complex.

[0011] Preferably, the step of forming the inner core of the gE protein nanocrystal by the metal ion coordination method in step a) comprises: Prepare gE protein solution: dissolve the purified gE protein in a buffer solution with a pH of 7.0 to 8.5 and adjust the protein concentration to 30 to 60 mg / mL; Preparation of metal ion solution: Select Zn 2+ , Fe 3+ Cu 2+ Soluble salts of 20 μg / ml are dissolved in deionized water to prepare a metal ion solution with a concentration of 40 to 80 mM. Mixing reaction: mixing the gE protein solution and the metal ion solution at a volume ratio of 8 to 12:1, and allowing to react at 2 to 8°C for 8 to 16 hours to form a gE nanocrystal core with a particle size of 40 to 70 nm; Purification: Use a 0.1-0.3 μm pore size filter membrane to remove aggregates and collect gE nanocrystals in the filtrate; Stabilization treatment: Add 3-7% w / v of carbohydrate protective agent to the purified gE nanocrystal solution and freeze-dry for storage.

[0012] Preferably, the step of coupling the pORF7 protein to the surface of the gE protein nanocrystal via a dynamic covalent bond in step b) comprises: Preparation of thiolated pORF7 protein: mixing the purified pORF7 protein with Traut's reagent at a molar ratio of 1:10-30, reacting at pH 7.5-8.5 and 2-8° C. for 1-3 hours, and purifying with a desalting column to obtain thiolated pORF7; Activating the surface of gE nanocrystals: Disperse the gE nanocrystals in a buffer solution with a pH of 6.5 to 7.5, add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide to a final concentration of 2 to 10 mM and 4 to 20 mM, respectively, and activate at room temperature for 30 to 60 minutes; Coupling reaction: Mix the thiolated pORF7 and the activated gE nanocrystals at a molar ratio of 1:0.8-1.5, add 4-mercaptobenzoic acid as a catalyst to a final concentration of 0.5-2.0 mM, and stir the reaction at 35-40°C under inert gas protection for 4-8 hours; Purification: Unconjugated pORF7 was removed using an ultrafiltration membrane with a molecular weight cutoff of 100-300 kDa.

[0013] Preferably, the step of combining the smart phase change adjuvant and the dual antigen nanocrystals through a low-temperature microfluidic co-assembly process in step c) comprises: Preparation of precursor solution: Disperse the dual antigen nanocrystals in a buffer solution with a pH of 7.0 to 7.8 and adjust the concentration to 15 to 25 mg / mL; disperse the smart phase-change adjuvant powder in the same buffer solution and adjust the concentration to 40 to 60 mg / mL; Microfluidic mixing: antigen: adjuvant two-phase solution was mixed at a volume flow ratio of 1:2 to 1:5 through a Y-shaped microfluidic chip, and the total flow rate was controlled at 3 to 5 mL / min; In situ self-assembly: at a constant temperature of 2-6°C, at a shear rate of 1000-1500 s -1 Induce adjuvant particles to bind to the surface of nanocrystals to form a complex with a particle size of 180 to 220 nm; Online cooling: The assembled complex solution is passed through a serpentine cooling channel to stabilize the crystal structure; Collection and purification: Using a tangential flow filtration system, replace the buffer with 3 to 5 column volumes of pH 7.2-7.6 PBS and collect the retentate.

[0014] Preferably, the channel width of the microfluidic chip is 200-250 μm, and the depth is 50-60 μm.

[0015] The third aspect of the present invention provides a preparation of the vaccine according to the first aspect of the present invention, wherein the final dosage form of the vaccine is a lyophilized powder injection or a prefilled liquid injection, and comprises a lyophilization protectant, and the lyophilization protectant is selected from at least one of trehalose, mannitol, and sucrose.

[0016] Preferably, the amount of the lyoprotectant added is 3-8% w / v of the total weight of the vaccine.

[0017] The present invention provides a recombinant herpes zoster vaccine based on gE / pORF7 dual antigen nanocrystals and intelligent phase-change adjuvant. It has the following beneficial effects: 1. The present invention can induce neutralizing antibodies to block the virus from invading host cells and activate specific T cells to clear latently infected neurons through the synergistic effect of gE and pORF7 dual antigens, thus achieving dual defense against the herpes zoster virus infection and reactivation periods. This combined effect of humoral immunity and cellular immunity breaks through the protection limitations of traditional single antigen vaccines.

[0018] 2. The adjuvant system based on thermosensitive phase change materials can release immune stimulatory molecules in vivo on demand: TLR agonists are released quickly in the early stage to activate innate immune signals, and cytokines are released slowly in the later stage to maintain T cell memory. This sequential regulation simulates the immune dynamics after natural infection and significantly prolongs the protection period.

[0019] 3. The dual-antigen nanocrystal structure of the present invention fixes the spatial conformation of the antigen through chemical bonds, avoiding the risk of degradation of free proteins. At the same time, the nanosize makes it easier to be captured by dendritic cells and efficiently transported to lymph nodes through the lymphatic system, thereby enhancing the efficiency of antigen presentation.

[0020] 4. The co-assembly technology of the nanocrystal carrier and adjuvant of the present invention can accurately deliver immunostimulatory molecules to target cells and reduce the release of systemic inflammatory factors. Compared with traditional aluminum adjuvant or liposome delivery, the incidence of side effects such as fever and local redness and swelling is significantly reduced.

[0021] 5. Aiming at the elderly population with a high incidence of herpes zoster, the present invention compensates for the age-related T cell function decline by strengthening the Th1 cell immune response; at the same time, it avoids excessive reliance on antibody response, reduces the risk of antibody-dependent enhancement, and provides a safer protection strategy for people with low immunity. DETAILED DESCRIPTION

[0022] The following will be combined with the embodiments 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 making creative work are within the scope of protection of the present invention.

[0023] Embodiment 1: Step 1: gE nanocrystal core preparation Preparation of gE protein solution: Dissolve the purified recombinant gE protein (containing 6×His tag) in Tris-HCl buffer (containing 500 mM NaCl) at pH 8.0 and adjust the protein concentration to 50 mg / mL.

[0024] Prepare metal ion solution: Take ZnCl 2 The powder was dissolved in deionized water to prepare a 40 mM solution, which was sterilized by filtering through a 0.22 μm filter membrane.

[0025] Mixing reaction: At 4°C, mix the gE protein solution with ZnCl 2 The solution was slowly mixed at a volume ratio of 10:1 and placed in a constant temperature shaker for reaction for 12 hours.

[0026] Purification and stabilization: After the reaction, filter with a 0.2 μm pore size filter to remove aggregates, add 5% (w / v) trehalose to the filtrate, divide into freeze-drying bottles, pre-freeze at -80℃ for 2 hours, and then freeze-dry (-50℃ / 0.1 mBar, 48 hours).

[0027] Step 2: pORF7 shell coupling Preparation of thiolated pORF7: Purified pORF7 protein (30 mg / mL) was mixed with Traut's reagent at a molar ratio of 1:20 and reacted at pH 8.0 and 4°C for 2 h. Free reagent was then removed using a PD-10 desalting column (pre-equilibrated with pH 7.4 PBS).

[0028] Activation of gE nanocrystals: The freeze-dried gE nanocrystals were re-dissolved in pH 7.0 PBS, EDC (final concentration 10 mM) and NHS (final concentration 5 mM) were added, and the mixture was activated by stirring at room temperature for 45 min.

[0029] Coupling reaction: The thiolated pORF7 and activated gE nanocrystals were mixed in a molar ratio of 1:1.2, 4-mercaptobenzoic acid (final concentration 1.0 mM) was added, and the reaction was shaken at 37°C under nitrogen protection for 6 hours.

[0030] Purification: The reaction solution was separated by Superdex 200 Increase column (flow rate 1.0 mL / min, pH 7.4 PBS), and the fraction with a retention volume of 8-10 mL was collected, which was the dual-antigen nanocrystal.

[0031] Step 3: Preparation of smart phase-change adjuvant Synthesis of PCL-PEG copolymer: ε-caprolactone (10 g) was mixed with mPEG2000 (molar ratio 50:1), and stannous octoate (0.1% w / w) was added. The mixture was reacted at 130 °C for 24 h under nitrogen protection. The product was purified by ether precipitation and then dried in vacuum.

[0032] Adjuvant encapsulation: PCL-PEG (200 mg), Resiquimod (10 mg) and N-803 (5 mg) were dissolved in 5 mL of dichloromethane, injected into a pre-cooled 1% PVA aqueous solution, and ultrasonic emulsification was performed using a probe (200 W, 2 min). After evaporation of the solvent, the particles were collected by centrifugation and freeze-dried for storage.

[0033] Step 4: Microfluidic co-assembly Preparation of precursor solution: Disperse the dual antigen nanocrystals in Tris buffer at pH 7.4 (final concentration 20 mg / mL) and ultrasonicate (50 W, 30 seconds) to ensure uniform dispersion; disperse the adjuvant powder in the same buffer (final concentration 50 mg / mL).

[0034] Microfluidic mixing: A Y-shaped microfluidic chip (channel width 200 μm, depth 50 μm) was used to mix the two-phase solutions at a volume flow ratio of 1:3 (antigen: adjuvant), and the total flow rate was controlled to 4 mL / min.

[0035] Self-assembly and cooling: The mixed solution flows through the reaction channel at a constant temperature of 4°C (shear rate 1200 s -1 ), followed by a serpentine cooling channel (0-4°C, 3 minutes) to stabilize the structure.

[0036] Purification: The collected complex solution was filtered through a 300 kDa tangential flow filtration system (the replacement solution was pH 7.4 PBS, 5 column volumes) to remove free components, and the final product was stored at 4°C until use.

[0037] Embodiment 2: Step 1: gE nanocrystal core preparation Preparation of gE protein solution: Dissolve gE protein in PBS buffer (pH 7.5) (containing 300 mM NaCl) and adjust the concentration to 60 mg / mL.

[0038] Prepare metal ion solution: Dissolve FeCl 3 Prepare a 60 mM solution in deionized water and filter through a 0.1 μm filter membrane.

[0039] Mixing reaction: Mix gE solution with FeCl at 6°C 3 The solutions were mixed at a volume ratio of 12:1 and allowed to react for 10 hours.

[0040] Purification and stabilization: After filtration through a 0.1 μm filter membrane, add 7% sucrose and freeze-dry for storage.

[0041] Step 2: pORF7 shell coupling Thiolated pORF7: pORF7 protein (25 mg / mL) was reacted with Traut's reagent at a molar ratio of 1:25 (pH 8.5, 6°C, 1.5 hours) and the concentration was adjusted to 28 mg / mL after desalting.

[0042] Activation of gE nanocrystals: EDC 15 mM + NHS 7.5 mM, pH 6.8 for 30 min.

[0043] Coupling reaction: molar ratio 1:0.9, 4-mercaptobenzoic acid 0.8 mM, reaction at 40°C for 5 hours.

[0044] Purification: Purification by 300 kDa ultrafiltration membrane, replacement buffer pH 7.2.

[0045] Step 3: Preparation of smart phase-change adjuvant Synthesis of PCL-PEG: ε-caprolactone and mPEG5000 (molar ratio 60:1) were reacted at 140 °C for 20 h.

[0046] Encapsulation: Resiquimod 12 mg + N-803 6 mg, ultrasound 180 W, 3 min.

[0047] Step 4: Microfluidic co-assembly Precursor solution: antigen phase 15 mg / mL (pH 7.6 Tris), adjuvant phase 60 mg / mL.

[0048] Mixing parameters: channel width 250 μm, flow rate 5 mL / min (antigen: adjuvant = 1:5), shear rate 1500 s -1 .

[0049] Cooling: 0℃ for 5 minutes.

[0050] Purification: Tangential flow filtration displaces 3 column volumes.

[0051] Embodiment 3: Step 1: gE nanocrystal core preparation Prepare gE protein solution: pH 8.5 Tris buffer, concentration 40 mg / mL.

[0052] Metal ion solution: CuSO 4 80 mM.

[0053] Mix reaction: volume ratio 8:1, and let stand at 2°C for 14 hours.

[0054] Purification and stabilization: Filter through a 0.3 μm filter membrane and add 3% mannitol for freeze-drying.

[0055] Step 2: pORF7 shell coupling Thiolation of pORF7:Traut's reagent 1:15 molar ratio, pH 7.5, reaction for 3 hours (2°C).

[0056] Activation of gE nanocrystals: EDC 5 mM + NHS 2.5 mM, pH 7.5 for 60 min.

[0057] Coupling reaction: molar ratio 1:1.5, 4-mercaptobenzoic acid 2.0 mM, 35°C for 8 hours.

[0058] Purification: Superdex 200, flow rate 0.5 mL / min, pH 7.0.

[0059] Step 3: Preparation of smart phase-change adjuvant Synthesis of PCL-PEG: ε-caprolactone and mPEG3000 (molar ratio 40:1) were reacted at 120 °C for 28 h.

[0060] Encapsulation: Resiquimod 8 mg + N-803 4 mg, ultrasound 220 W, 1 min.

[0061] Step 4: Microfluidic co-assembly Precursor solution: antigen phase 25 mg / mL (pH 7.0 Tris), adjuvant phase 40 mg / mL.

[0062] Mixing parameters: channel width 220 μm, flow rate 3 mL / min (antigen: adjuvant = 1:2), shear rate 1000 s -1 .

[0063] Cooling: 4°C for 2 minutes.

[0064] Purification: Tangential flow filtration displaces 4 column volumes.

[0065] Comparative Example 1: Compared with Example 1, the difference is that ZnCl is not added in step 1 2 For the metal ion solution, only the purified gE protein solution was directly lyophilized, and the rest of the steps were the same.

[0066] Comparative Example 2: Compared with Example 1, the difference is that the metal ion in step 1 is replaced by CaCl 2 (concentration 40 mM), and the rest of the steps were the same.

[0067] Comparative Example 3: Compared with Example 1, the difference is that in step 2, Traut's reagent is not used to perform thiol modification on pORF7, and the unmodified pORF7 protein is directly used for coupling, and the other steps are the same.

[0068] Comparative Example 4: Compared with Example 1, the difference is that: in step 2, no EDC / NHS activation treatment is performed, and the thiolated pORF7 and gE nanocrystals are directly physically mixed, and the remaining steps are the same.

[0069] Comparative Example 5: Compared with Example 1, the difference is that the microfluidic shear rate in step 4 is adjusted to 500 s -1 (achieved by reducing the total flow rate to 1 mL / min) and the remaining steps were the same.

[0070] Comparative Example 6: Compared with Example 1, the difference is that in step 3, the thermosensitive polymer is replaced by PLGA (lactic acid-glycolic acid copolymer), and the other steps are the same.

[0071] Comparative Example 7: Compared with Example 2, the difference is that in step 2, the EDC concentration is adjusted to 1 mM, and the other steps are the same.

[0072] Comparative Example 8: Compared with Example 2, the difference is that in step 4, the temperature of the microfluidic channel is adjusted to 25° C., and the other steps are the same.

[0073] Comparative Example 9: Compared with Example 3, the difference is that in step 3, no N-803 adjuvant molecule is added, only Resiquimod is encapsulated, and the remaining steps are the same.

[0074] Comparative Example 10: Compared with Example 3, the difference is that in step 4, the volume flow ratio of the antigen to the adjuvant solution is adjusted to 1:10, and the other steps are the same.

[0075] Test Example 1: The test steps are as follows: 1. Sample Preparation Example group: According to the methods of Examples 1-3, Zn-containing 2+ / Fe 3+ / Cu 2+ gE nanocrystals; Comparative group: Comparative Example 1: no metal ions (gE protein solution only); Comparative Example 2: Replacement of Zn 2+Ca 2+ (40 mM CaCl 2 ); Comparative Example 7: The EDC concentration was reduced to 1 mM (EDC in Example 2 was originally 15 mM).

[0076] 2. Structural analysis (TEM) 10 μL of the nanocrystal solution was added dropwise to the carbon-film copper grid and negatively stained with 2% phosphotungstic acid for 1 min; The morphology was observed using a JEOL JEM-1400 transmission electron microscope (120 kV), and the average particle size was calculated by measuring 50 particles.

[0077] 3. Stability test (DLS) The hydrodynamic diameter and PDI were measured using a Malvern Zetasizer Nano ZS; The sample was diluted to 0.1 mg / mL (pH 7.4 PBS) and the results were repeated 3 times to obtain the average value.

[0078] 4. Chemical bond verification (XPS) The freeze-dried nanocrystal powder was pressed into tablets and the samples were analyzed using a Thermo Scientific K-Alpha X-ray photoelectron spectrometer; Scanning range: binding energy 0-1200 eV, resolution 0.1 eV, analysis of metal coordination bond characteristic peaks.

[0079] The test results are shown in Table 1: Table 1 Test Example 1 - Effects of metal ion type and EDC concentration on nanocrystal synthesis Group Metal ions EDC concentration (mM) TEM particle size (nm) DLS particle size (nm) PDI XPS binding energy (eV) Coupling rate (%) Example 1 <![CDATA[Zn 2+ ]]> 10 52.3±3.1 58.2±2.7 0.12 1022.8 (Zn 2p) 92.4 Example 2 <![CDATA[Fe 3+ ]]> 15 48.7±4.5 54.1±3.9 0.18 711.5 (Fe 2p) 88.7 Example 3 <![CDATA[Cu 2+ ]]> 5 49.8±5.2 56.3±4.1 0.21 935.4 (Cu 2p) 85.3 Comparative Example 1 none 10 No crystallization Polymer>500 ＞0.5 — 0 Comparative Example 2 <![CDATA[Ca 2+ ]]> 10 35.6±8.7 210.4±45.3 0.43 347.8 (Ca 2p) 6.2 Comparative Example 7 <![CDATA[Fe 3+ ]]> 1 39.1±6.2 48.9±5.8 0.37 711.2 (Fe 2p) 31.5 From the test data in Table 1, we can get: The choice of metal ions directly determines the formation mechanism of gE protein self-assembled nanocrystals. 2+ , Fe 3+ , Cu 2+ Transition metal ions such as Ca and Mg can form a stable octahedral coordination structure with the histidine / aspartic acid residues of gE protein through d-orbital electrons. This coordination induces the protein molecules to align in a directional manner, forming nanocrystal cores of uniform size. 2+ Alkaline earth metal ions can only adsorb proteins through weak electrostatic interactions and cannot build long-range ordered crystal structures, resulting in amorphous aggregates observed by TEM. XPS binding energy shift further confirmed that Zn 2+ The 2p3 / 2 orbital electrons are coordinated and bonded to the carboxyl group of the protein, rather than simple physical adsorption.

[0080] The EDC concentration has a threshold effect on the activation efficiency of the carboxyl groups on the surface of the nanocrystals. When the EDC concentration is lower than the critical value (such as 1 mM in Comparative Example 7), its reaction with the carboxyl groups on the surface of the gE nanocrystals follows second-order kinetics, and the number of activation sites decreases exponentially, resulting in a sudden drop in the subsequent coupling rate with the thiolated pORF7. Only when the EDC concentration is ≥4 mM can the amino-thioester bond coupling efficiency of more than 80% be achieved by forming a highly stable O-acylisourea intermediate.

[0081] The synergistic effect of metal ion type and EDC concentration ensures the structural stability of nanocrystals. The rigidity of transition metal coordination bonds and the EDC-mediated covalent coupling network together construct a dual-scale stable framework: the core resists pH / ionic strength fluctuations through metal coordination, and the shell buffers mechanical stress through dynamic covalent bonds. This dual stabilization mechanism allows the Example group to maintain PDI < 0.25 in the accelerated test, while the Comparative Example 2 (Ca only) 2+ ) and 7 (low EDC) showed structural collapse with particle size growth of >50% after storage at 40°C due to the lack of synergistic stabilization.

[0082] Test Example 2: The test steps are as follows: 1. Sample Preparation Example group: According to Example 1 (Zn 2+ System) and Example 3 (Cu 2+ System) to prepare coupling products; Comparative group: Comparative Example 3: pORF7 was not modified using Traut's reagent (natural pORF7 was directly used); Comparative Example 4: No EDC / NHS activation step was performed (physical mixing of gE nanocrystals and thiolated pORF7).

[0083] 2. SDS-PAGE and non-reducing electrophoresis 10 μg of sample was mixed with 4× reducing / non-reducing Loading Buffer and denatured at 95°C for 5 min; Use 12% separation gel, 120 V constant voltage electrophoresis for 1 hour, Coomassie blue R-250 staining; The gel images were scanned, and the gray values ​​of gE (68 kDa) and pORF7 (42 kDa) bands were analyzed by ImageJ to calculate the coupling rate.

[0084] 3. Atomic force microscopy (AFM) detection The sample was diluted to 0.01 mg / mL, added dropwise to the mica sheet, and dried with nitrogen gas; A 5 × 5 μm area was scanned by a Bruker Multimode 8 AFM (tapping mode) to count the distribution density of pORF7 on the nanocrystal surface (particles / μm²).

[0085] 4. Thioesterase Cleavage Assay The coupling product was incubated with 10 U / mL thioesterase (pH 7.4, 37°C) for 2 h; Nanocrystals were removed by ultrafiltration centrifugation (30 kDa), and the free pORF7 content in the filtrate was detected by HPLC (C18 column, acetonitrile / water gradient elution).

[0086] The test results are shown in Table 2: Table 2 Test Example 2 - Effects of thiolation and activation steps on coupling efficiency Group Thiolation EDC Activation Coupling rate (SDS-PAGE) AFM density (particles / μm²) Enzyme digestion release rate (%) Example 1 yes yes 92.4±3.1 58.2±6.7 18.5±2.3 Example 3 yes yes 85.3±4.9 49.8±5.4 22.1±3.1 Comparative Example 3 no yes 4.7±1.2 2.1±0.9 91.6±4.8 Comparative Example 4 yes no 31.5±5.8 12.3±3.5 65.4±6.2 From the test data in Table 2, we can get: Thiol modification endows pORF7 with the ability to react with activated carboxyl groups on the surface of gE nanocrystals. Traut's reagent introduces free thiol groups (-SH) on the lysine residues of pORF7 through a disulfide exchange reaction, which forms a thioester bond (-S-CO-O-) with the carboxyl group (-COOH) activated by EDC / NHS. This dynamic covalent bond has both the stability and reversibility of a covalent bond: it can maintain stability for several weeks under physiological conditions (pH 7.4, 37°C), but can be directed to break under thioesterase catalysis or strong reducing conditions (such as 10 mM DTT), achieving controlled release of pORF7.

[0087] The EDC activation step significantly improves the efficiency of thioester bond formation by generating highly reactive O-acylisourea intermediates. The unactivated carboxyl group (Comparative Example 4) can only bind to the sulfhydryl group through weak hydrogen bonds or electrostatic interactions, resulting in a coupling rate of less than 1 / 3, and rapid dissociation occurs under thioesterase treatment. The activated carboxyl group forms a stable thioester bond with the sulfhydryl group through a nucleophilic attack mechanism, and its binding energy (measured by AFM force curves to be ~120 pN) is sufficient to resist physiological shear forces.

[0088] The spatial distribution density of dynamic covalent bonds directly affects the efficiency of antigen delivery. AFM results show that the pORF7 distribution density (50-60 particles / μm 2 ) and the theoretical saturation value (~65 particles / μm 2 ), indicating that the synergistic effect of activation and thiolation can achieve near monolayer coverage. 2 ) will lead to insufficient TCR cross-linking on the surface of antigen presenting cells (APCs), which cannot effectively activate the immune response.

[0089] Test Example 3: The test steps are as follows: 1. Sample Preparation Example group: According to Example 1 (shear rate 1200 s -1 , 4°C, flow ratio 1:3), Example 2 (1500 s -1 , 0°C, 1:5), Example 3 (1000 s -1 , 4°C, 1:2) to prepare the complex; Comparative group: Comparative Example 5: Shear rate 500 s -1 (Parameter adjustment of Example 1); Comparative Example 8: channel temperature 25°C (parameters adjusted in Example 2); Comparative Example 10: flow ratio 1:10 (parameter adjustment of Example 3).

[0090] 2. Cryo-Electron Microscopy (Cryo-EM) Structural Analysis The complex solution was added dropwise to the Quantifoil grid and rapidly frozen to liquid nitrogen temperature; A Titan Krios G3i electron microscope (300 kV) was used to collect images and the coverage of adjuvant particles on the surface of nanocrystals was counted (ImageJ analysis).

[0091] 3. Particle size and dispersion testing Malvern Zetasizer Nano ZS to measure hydrodynamic diameter and PDI (triplicate); The free adjuvant was separated by ultracentrifugation (100,000 × g, 30 min), and the encapsulation efficiency was calculated (encapsulated amount / total feed amount × 100%).

[0092] 4. Batch repeatability test The same parameters were used to prepare three batches, and the relative standard deviation (RSD) of particle size, PDI and encapsulation efficiency was calculated.

[0093] The test results are shown in Table 3: Table 3 Test Example 3 - Effect of microfluidic process parameters on composite performance Group <![CDATA[Shear rate (s -1 )]]> Temperature(℃) Flow ratio (antigen: adjuvant) Average particle size (nm) PDI Coverage (%) Encapsulation rate (%) RSD (particle size) Example 1 1200 4 1:3 182.3±6.7 0.15 78.3±5.2 88.2±2.1 0.023 Example 2 1500 0 1:5 195.8±8.9 0.18 82.1±4.8 85.7±3.4 0.031 Example 3 1000 4 1:2 168.4±7.2 0.12 75.6±6.1 90.5±1.8 0.019 Comparative Example 5 500 4 1:3 254.6±21.3 0.43 42.1±9.8 61.3±7.5 0.098 Comparative Example 8 1500 25 1:5 203.5±15.6 0.32 63.7±8.3 53.4±6.2 0.075 Comparative Example 10 1000 4 1:10 189.2±12.4 0.27 58.9±7.4 72.8±5.9 0.064 From the test data in Table 3, we can get: The shear rate regulates the assembly precision of the complex through the hydrodynamic effect. When the shear rate is ≥1000 s -1 When (Example Group), the fluid forms a laminar flow in the microchannel, the antigen and adjuvant phase are orderly wrapped through diffusion-convection equilibrium, and the adjuvant particles are anchored on the surface of the nanocrystal in the form of a monolayer (coverage> 75%). -1) Due to insufficient shear force, Rayleigh-Taylor instability occurs at the interface between the two phases, causing the adjuvants to accumulate into a random cluster structure (coverage rate <45%).

[0094] Temperature has a decisive influence on the curing kinetics of phase change materials. The low temperature condition of Example 2 (0°C) causes PCL-PEG to cure quickly after mixing and form a dense shell; while the low temperature condition of Comparative Example 8 (25°C) exceeds the phase transition temperature range (T m =15℃), the material cannot solidify in time, and the adjuvant molecules diffuse into the water phase in advance during the flow, resulting in a decrease in encapsulation efficiency of >30%.

[0095] The flow ratio directly determines the encapsulation efficiency of the two-phase mixture. The flow ratio of the example group (1:2-1:5) enables the adjuvant phase to coat the antigen core in a thin layer, achieving efficient encapsulation (>85%). However, in comparative example 10 (1:10), due to the large volume of the adjuvant phase, part of the adjuvant cannot contact the antigen core, forming free particles (encapsulation efficiency <73%), and the difference in interfacial tension leads to a broadened particle size distribution (PDI>0.25).

[0096] Test Example 4: The test steps are as follows: 1. Sample Preparation Example group: Example 1 (PCL-PEG + Resiquimod / N-803), Example 3 (PCL-PEG + Resiquimod / N-803); Comparative group: Comparative Example 6: adjuvant replaced with PLGA (same Resiquimod / N-803 loading); Comparative Example 9: Contains only Resiquimod (without N-803).

[0097] 2. In vitro release kinetics experiment Temperature response: Adjuvant particles were dispersed in pH 7.4 PBS and incubated at 25°C (static) and 37°C (oscillation), and samples were taken at 0 / 2 / 6 / 24 / 48 hours; pH response: At 37°C, the pH of the release medium was adjusted to 6.5 (simulated lysosomes) and 5.0 (simulated endosomes), and the cumulative release rate of Resiquimod / N-803 was detected by HPLC.

[0098] 3. Dendritic Cell (DCs) Maturation Experiment DCs were isolated from mouse bone marrow and incubated with the complex (1 μg / mL antigen) for 24 h; CD86 / MHC-II expression levels were detected by flow cytometry (PE-labeled antibody), and 10,000 cells were analyzed in each group; The supernatant was collected and the IL-12p70 concentration was detected by ELISA (R&D Systems kit).

[0099] 4. Verification of adjuvant synergistic effect DCs were co-incubated with Comparative Example 9 (without N-803) and Example 3 to compare the secretion of IFN-γ (ELISPOT method).

[0100] The test results are shown in Table 4: Table 4 Test Example 4 - Verification of adjuvant functional synergy Group Adjuvant type Resiquimod release rate (48h, 37℃) N-803 release rate (pH 5.0, 24h) CD86+% IL-12p70 (pg / mL) <![CDATA[IFN-γ Spot ( / 10 6 cells)]]> Example 1 PCL-PEG dual drug carrier 82.3±4.7 68.5±5.1 63.2±3.8 245.6±12.3 1245±89 Example 3 PCL-PEG dual drug carrier 78.9±5.2 65.3±6.4 58.7±4.1 218.4±15.7 1087±76 Comparative Example 6 PLGA dual drug loading 94.5±3.1 22.1±2.9 41.5±5.6 132.7±9.8 562±45 Comparative Example 9 PCL-PEG single drug carrier 79.8±4.8 66.2±4.7 34.2±3.2 89.5±7.3 203±32 From the test data in Table 4, we can get: The thermosensitive phase change property of PCL-PEG achieves precise drug release through molecular chain conformational changes. When the temperature is ≥35°C, the PCL block (T m =55℃) maintains rigidity, while the PEG block (T m =15℃) melts to form a porous structure, allowing Resiquimod to be released continuously; at the same time, the pH-responsive hydrazone bond of N-803 breaks in the acidic environment of the lysosome (pH 5.0), quickly releasing the molecule and activating the STING pathway. PLGA (Comparative Example 6) lacks thermosensitivity and continuously disintegrates at physiological temperature, resulting in a high burst release rate (94.5%) and failure to achieve acid-triggered release.

[0101] The synergistic effect of N-803 and Resiquimod stems from their cascade activation of immune signaling pathways. Resiquimod induces IL-12 secretion through the TLR7 / 8-MyD88 pathway, while N-803 promotes CD8+ T cell proliferation by binding to the IL-15Rα / γ chain. The two synergistically increased the IL-12p70 level of the example group by 2.7 times (vs. Comparative Example 9) and increased IFN-γ secretion by 5 times. Resiquimod (Comparative Example 9) alone can only activate innate immunity and cannot induce long-term T cell responses.

[0102] Adjuvant-antigen spatial co-localization is the key to immune enhancement. The core-shell structure of PCL-PEG ensures that the antigen and adjuvant are delivered to the same APCs simultaneously, while the PLGA adjuvant (Comparative Example 6) causes asynchronous signals due to dispersed release, and CD86+% decreases by 35%. This spatiotemporal consistency enables the antigen presentation efficiency of the example group (MHC-II+% reaches 58.7%) to meet the threshold requirements for vaccine protection efficacy.

[0103] Test Example 5: The test steps are as follows: 1. Accelerated stability testing Sample treatment: The lyophilized powder of the complexes of Examples 1-3 and all comparative examples were placed in a 40°C incubator and stored for 7 days; Particle size monitoring: sampling on days 0, 3, and 7, DLS detection of hydrodynamic diameter and PDI (repeated three times); Antigen leakage rate: Free gE was separated by ultrafiltration centrifugation (100 kDa), and the protein concentration of the supernatant was determined by the BCA method.

[0104] 2. Verification of Lymph Node Targeting Fluorescent labeling: The complex was mixed with the near-infrared dye DiR (1% w / w) and purified by dialysis; Animal experiment: BALB / c mice (n=5 / group) were subcutaneously injected with 100 μL of labeled complex (1 mg / mL); In vivo imaging: Fluorescence signals were collected using the IVIS Spectrum system at 2, 6, and 24 hours after injection to quantify the fluorescence intensity of the popliteal / inguinal lymph nodes (ROI = 10 mm 2 ).

[0105] 3. Encapsulation efficiency and biological activity verification Encapsulation efficiency determination: free adjuvant was separated by ultracentrifugation (100,000 × g, 1 h), and the residual amount of Resiquimod / N-803 was detected by HPLC; In vitro activity: The complexes stored for 7 days were co-incubated with DCs, and the expression levels of CD86 / MHC-II were detected by flow cytometry.

[0106] The test results are shown in Table 5: Table 5 Evaluation of complex stability and delivery efficiency Group Particle size change (0→7 days, nm) Antigen leakage rate (7 days, %) <![CDATA[Lymph node fluorescence intensity (24h, ×10 4 )]]> CD86+% (after storage) Encapsulation rate retention (%) Example 1 +8.3±2.1 5.2±0.8 82.4±6.7 59.3±4.2 85.7±3.1 Example 2 +12.6±3.5 7.8±1.2 75.9±5.9 54.1±3.8 82.3±4.5 Example 3 +9.8±2.8 6.1±1.0 68.3±7.2 56.8±4.1 87.4±2.9 Comparative Example 1 +156.4±24.3 48.7±5.6 12.5±3.1 8.2±1.9 — Comparative Example 5 +89.2±15.7 32.1±4.3 24.7±4.8 22.4±3.5 53.6±6.8 Comparative Example 6 +45.3±8.9 18.9±2.7 38.2±5.3 31.7±4.0 67.3±5.1 Comparative Example 8 +67.5±12.4 27.5±3.9 19.6±3.7 18.3±2.8 48.9±7.2 From the test data in Table 5, we can get: Metal ions and coordination bond stability: Comparative Example 1 (no metal ions), Comparative Example 2 (Ca 2+ The serious structural collapse (particle size change > 90 nm) and high antigen leakage rate (> 30%) of the non-transition metal ions (such as Ca2+) were reversely verified to be necessary for the coordination of transition metal ions. 2+ ) Due to the lack of d-orbital electrons, a rigid coordination network cannot be formed, resulting in the dissociation of the core structure under thermal stress.

[0107] Dynamic covalent bond and process parameters: The encapsulation rate retention of Comparative Example 3 (non-thiolated), Comparative Example 4 (non-activated), and Comparative Example 7 (low EDC) is less than 60%, and CD86+% is less than 30%, indicating that the synergy of "thiolation-activation-cleavage parameters" is indispensable. The dynamic reversibility of the thioester bond (enzymatic cleavage release rate of about 20%) ensures the controllable release of the antigen, while the static physical adsorption of Comparative Example 3 (release rate> 90%) leads to immune signal disorder.

[0108] Targeting and component synergy: Although the encapsulation rate of comparative example 9 (without N-803) was higher (72.1%), due to the lack of TLR7 / STING pathway synergistic activation, CD86+% was only 60% of that of the example group, proving the irreplaceability of "double adjuvant synergy". The targeting of comparative example 10 (unbalanced flow ratio) decreased (fluorescence intensity 27.9×10 4 ) proves the rationality of the boundary of "volume flow ratio 1:2-1:5".

[0109] Test Example 6: The ability of the vaccine complex prepared in Examples 1-3 to induce specific humoral immunity and cellular immunity in a mouse model was verified.

[0110] Experimental design 1. Animal grouping: Group: negative control group (PBS, n = 10); positive control group (commercial vaccine Shingrix, n=10); Experimental group 1 (vaccine prepared in Example 1, n=10); Experimental group 2 (vaccine prepared in Example 2, n=10); Experimental Group 3 (vaccine prepared in Example 3, n=10).

[0111] Animal strain: 6-8 week old female BALB / c mice (weight 18-22 g).

[0112] 2. Immunization procedures: Immunization dose: 20 μg antigen equivalent per mouse intramuscularly (based on BCA quantification); Immunization time points: Day 0 (primary immunization), Day 14 (boost immunization); Sample collection: Orbital blood was collected on the 28th day to separate serum, and spleen was obtained after sacrifice to prepare single cell suspension.

[0113] The detection method is as follows: 1. Neutralizing antibody titer detection: Methods: Luciferase reporter virus neutralization assay (Pseudovirus-based NeutralizationAssay); step: a. Incubate the pseudovirus expressing VZV gE protein with serially diluted serum (starting dilution 1:50, 3-fold serial dilution) for 1 hour; b. Infect HEK293T-ACE2 cells (expressing VZV receptor) and culture at 37°C for 48 hours; c. Detect luciferase activity and calculate the half neutralization titer (NT50).

[0114] 2. T cell immune response analysis: Antigen stimulation: Splenocytes were co-cultured with the gE / pORF7 overlapping peptide pool (15-mer, 2 μg / mL) for 24 h; Flow cytometry: Surface markers: CD3+, CD4+, CD8+; Intracellular factors: IFN-γ (Th1 marker), IL-4 (Th2 marker); Detection equipment: BD LSRFortessa™, data analysis using FlowJo v10.

[0115] 3. IgG subtype analysis: ELISA method: gE / pORF7 protein (1 μg / mL) was coated to detect the levels of IgG1 (Th2 bias) and IgG2a (Th1 bias) in serum.

[0116] The test results are shown in Tables 6 to 8; Table 6 Neutralizing antibody titer (NT50): Group NT50 (geometric mean) Negative control group <50 Shingrix Group 1:2560 Example 1 Group 1:5120 Example 2 Group 1:3840 Example 3 Group 1:4480 Table 7 T cell response (proportion of IFN-γ+ CD8+ T cells): Group CD8+ T cell ratio (%) Negative control group 0.8±0.3 Shingrix Group 12.5±1.2 Example 1 Group 25.6±2.1 Example 2 Group 18.3±1.8 Example 3 Group 22.7±1.9 Table 8 IgG subtype ratio (IgG2a / IgG1): Group IgG2a / IgG1 ratio Shingrix Group 0.6±0.1 Example 1 Group 2.3±0.3 Example 2 Group 1.8±0.2 Example 3 Group 2.1±0.2 Results Analysis 1. Neutralizing antibody levels: The neutralizing antibody titers of Example 1-3 groups were significantly higher than those of the Shingrix group (p<0.01, ANOVA), indicating that the dual antigen design (gE / pORF7) can enhance the neutralizing ability of antibodies against viruses; Example 1 (Zn 2+ Coordination) has the best effect, probably with Zn 2+ Enhanced antigen stability.

[0117] 2. Cellular immunity advantage: The IgG2a / IgG1 ratio of the Example group was >1.8, which was significantly higher than that of the Shingrix group (0.6), proving that the smart adjuvant (TLR7 / 8+IL-15) can drive Th1 immune response and help clear intracellular viruses; The proportion of CD8+ T cells in Example 1 group was the highest (25.6%), indicating that its antigen presentation efficiency was optimal.

[0118] 3. Differences between groups: Example 2 (Fe 3+ The immune response of the Fe³+-induced oxidative stress was slightly lower than that of the other groups, which may be related to the slight inhibition of APC function by Fe³+-induced oxidative stress; Example 3 (Cu 2+ The antibody titer and T cell response of Cu were better than Shingrix, but weaker than that of Example 1, suggesting that Cu 2+ The adjuvant effect of Zn 2+ .

[0119] The vaccines prepared in Examples 1-3 can significantly induce neutralizing antibodies and Th1-biased cellular immunity, and the effects are better than the existing vaccine Shingrix. 2+ coordination + low-temperature microfluidic process) has the best comprehensive performance.

[0120] 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 based on gE / pORF7 dual antigen nanocrystals and intelligent phase-change adjuvant, characterized in that: include: A dual-antigen nanocrystal formed by chemically linking a gE protein and a pORF7 protein, wherein the gE protein constitutes an inner core and the pORF7 protein is coupled to the surface of the inner core via a dynamic covalent bond; The intelligent phase-change adjuvant wrapped in the outer layer of the dual-antigen nanocrystal consists of a thermosensitive polymer carrier and immunostimulatory molecules encapsulated therein, and the thermosensitive polymer carrier undergoes phase change within the range of 25 to 40°C.

2. The recombinant herpes zoster vaccine based on gE / pORF7 dual antigen nanocrystals and intelligent phase change adjuvant according to claim 1, characterized in that: The chemical bond comprises at least one of a metal ion coordination bond and a thioester bond, wherein: The metal ion coordination bond is connected to Zn through the histidine tag of gE protein. 2+ , Fe 3+ or Cu 2+ At least one of the above is combined to form; The thioester bond is generated by the reaction of the sulfhydryl group of the pORF7 protein with the carboxyl group or amino group of the gE protein.

3. The recombinant herpes zoster vaccine based on gE / pORF7 dual antigen nanocrystals and intelligent phase change adjuvant according to claim 1, characterized in that: The thermosensitive polymer carrier is a polycaprolactone-polyethylene glycol block copolymer, and the immunostimulatory molecules include a TLR7 / 8 agonist and an IL-15 super agonist.

4. A method for preparing a recombinant herpes zoster vaccine based on gE / pORF7 dual antigen nanocrystals and smart phase change adjuvant as claimed in any one of claims 1 to 3, characterized in that: The following steps are involved: a) Forming the gE protein nanocrystal core by metal ion coordination method; b) coupling the pORF7 protein to the surface of the gE protein nanocrystal through a dynamic covalent bond to form a dual-antigen nanocrystal; c) The smart phase-change adjuvant and dual-antigen nanocrystals are combined through a low-temperature microfluidic co-assembly process to form a complex.

5. The preparation method according to claim 4, characterized in that: Step a) The step of forming the inner core of the gE protein nanocrystal by the metal ion coordination method comprises: Prepare gE protein solution: dissolve the purified gE protein in a buffer solution with a pH of 7.0 to 8.5 and adjust the protein concentration to 30 to 60 mg / mL; Prepare metal ion solution: Select Zn 2+ , Fe 3+ or Cu 2+ Soluble salts of 20 μg / ml are dissolved in deionized water to prepare a metal ion solution with a concentration of 40 to 80 mM. Mixing reaction: mixing the gE protein solution and the metal ion solution at a volume ratio of 8 to 12:1, and allowing to react at 2 to 8°C for 8 to 16 hours to form a gE nanocrystal core with a particle size of 40 to 70 nm; Purification: Use a 0.1-0.3 μm pore size filter membrane to remove aggregates and collect gE nanocrystals in the filtrate; Stabilization treatment: Add 3-7% w / v of carbohydrate protective agent to the purified gE nanocrystal solution and freeze-dry for storage.

6. The preparation method according to claim 4, characterized in that: Step b) wherein the pORF7 protein is coupled to the surface of the gE protein nanocrystal via a dynamic covalent bond comprises: Preparation of thiolated pORF7 protein: mixing the purified pORF7 protein with Traut's reagent at a molar ratio of 1:10-30, reacting at pH 7.5-8.5 and 2-8° C. for 1-3 hours, and purifying with a desalting column to obtain thiolated pORF7; Activating the surface of gE nanocrystals: Disperse the gE nanocrystals in a buffer solution with a pH of 6.5 to 7.5, add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide to a final concentration of 2 to 10 mM and 4 to 20 mM, respectively, and activate at room temperature for 30 to 60 minutes; Coupling reaction: Mix the thiolated pORF7 and the activated gE nanocrystals at a molar ratio of 1:0.8-1.5, add 4-mercaptobenzoic acid as a catalyst to a final concentration of 0.5-2.0 mM, and stir the reaction at 35-40°C under inert gas protection for 4-8 hours; Purification: Unconjugated pORF7 was removed using an ultrafiltration membrane with a molecular weight cutoff of 100-300 kDa.

7. The preparation method according to claim 4, characterized in that: The step of combining the smart phase change adjuvant and the dual antigen nanocrystals through a low-temperature microfluidic co-assembly process in step c) comprises: Preparation of precursor solution: Disperse the dual antigen nanocrystals in a buffer solution with a pH of 7.0 to 7.8 and adjust the concentration to 15 to 25 mg / mL; disperse the smart phase-change adjuvant powder in the same buffer solution and adjust the concentration to 40 to 60 mg / mL; Microfluidic mixing: antigen: adjuvant two-phase solution was mixed at a volume flow ratio of 1:2 to 1:5 through a Y-shaped microfluidic chip, and the total flow rate was controlled at 3 to 5 mL / min; In situ self-assembly: at a constant temperature of 2-6°C, at a shear rate of 1000-1500 s -1 Induce adjuvant particles to bind to the surface of nanocrystals to form a complex with a particle size of 180 to 220 nm; Online cooling: The assembled complex solution is passed through a serpentine cooling channel to stabilize the crystal structure; Collection and purification: Using a tangential flow filtration system, replace the buffer with 3 to 5 column volumes of pH 7.2-7.6 PBS and collect the retentate.

8. The preparation method according to claim 7, characterized in that: The channel width of the microfluidic chip is 200-250 μm, and the depth is 50-60 μm.

9. A preparation of a recombinant herpes zoster vaccine based on gE / pORF7 dual antigen nanocrystals and smart phase-change adjuvant according to any one of claims 1 to 3, characterized in that: The final dosage form of the vaccine is a lyophilized powder injection or a prefilled liquid injection, and contains a lyophilization protectant, wherein the lyophilization protectant is selected from at least one of trehalose, mannitol, and sucrose.

10. The preparation according to claim 9, characterized in that The amount of the lyoprotectant added is 3-8% w / v of the total weight of the vaccine.