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

Abstract

The present invention relates to the field of biomedical technologies, and discloses a recombinant herpes zoster vaccine based on gE / pORF7 dual-antigen nanocrystals and an intelligent phase-transition adjuvant, which includes dual-antigen nanocrystals formed by chemically bonding gE protein and pORF7 protein, wherein the gE protein forms the core, and the pORF7 protein is coupled to the surface of the core through dynamic covalent bonds; and an intelligent phase-transition adjuvant wrapped around the outer layer of the dual-antigen nanocrystals, and the adjuvant is composed of a thermosensitive polymer carrier and immunostimulatory molecules encapsulated therein, and the thermosensitive polymer carrier undergoes a phase transition within the range of 25-40°C. The vaccine of the present invention can simultaneously induce high-titer neutralizing antibodies and long-term T cell immunity, significantly improve the ability to clear the latent virus reservoir, and at the same time reduce the risk of vaccination adverse reactions, and is suitable for the prevention of herpes zoster in the elderly and immunodeficient populations.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical 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 incidence risk increases significantly with age and declining immunity. Although existing vaccines induce neutralizing antibodies by targeting the viral envelope glycoprotein gE, their single-antigen design is difficult to cover the immune escape mechanism during the latent infection stage of the virus, resulting in limited ability to clear the latent virus reservoir.

[0003] In addition, traditional adjuvant systems mostly rely on a single immune-stimulating signal (such as MPLA in AS01B), and are unable to coordinate the dynamic balance between innate immune activation and the formation of long-term T cell memory, resulting in the attenuation of the vaccine's protective efficacy over time.

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

[0005] For high-risk groups such as the elderly, existing technologies also have problems with poor vaccination tolerance caused by systemic inflammatory reactions. These systemic deficiencies jointly restrict the protective effect and application scope of herpes zoster vaccines in the real world, and there is an urgent need to achieve breakthroughs in multiple dimensions such as antigen design, adjuvant strategies, and preparation processes. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides a recombinant herpes zoster vaccine based on gE / pORF7 dual-antigen nanocrystals and an intelligent phase-change adjuvant, which solves the technical defects of existing herpes zoster vaccines that are unable to simultaneously block virus infection and clear the latent virus reservoir due to single-antigen design, insufficient adjuvant synergy, and low efficiency of the delivery system.

[0007] To achieve the above objectives, the present invention is realized through the following technical solutions:

[0008] 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:

[0009] A dual-antigen nanocrystal formed by chemically bonding gE protein and pORF7 protein, wherein the gE protein forms the core, and the pORF7 protein is covalently coupled to the surface of the core through a dynamic covalent bond;

[0010] An intelligent phase change adjuvant encapsulated in the outer layer of the double-antigen nanocrystal. The adjuvant consists of a thermosensitive polymer carrier and an immunostimulatory molecule encapsulated therein. The thermosensitive polymer carrier undergoes a phase change within the range of 25 to 40 °C.

[0011] Preferably, the chemical bond includes at least one of a metal ion coordination bond and a thioester bond, wherein:

[0012] The metal ion coordination bond is formed by binding with at least one of Zn 2+ , Fe 3+ or Cu 2+ through the histidine tag of the gE protein;

[0013] 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.

[0014] Preferably, the thermosensitive polymer carrier is a polycaprolactone-polyethylene glycol block copolymer, and the immunostimulatory molecule includes a TLR7 / 8 agonist and an IL-15 superagonist.

[0015] The second aspect of the present invention provides a preparation method of the vaccine described in the first aspect of the present invention, including the following steps:

[0016] a) Forming a gE protein nanocrystal core by the metal ion coordination method;

[0017] b) Coupling the pORF7 protein to the surface of the gE protein nanocrystal through a dynamic covalent bond to form a double-antigen nanocrystal;

[0018] c) Combining the intelligent phase change adjuvant with the double-antigen nanocrystal through a low-temperature microfluidic co-assembly process to form a complex.

[0019] Preferably, the step of forming the gE protein nanocrystal core by the metal ion coordination method in step a) includes:

[0020] Preparing a gE protein solution: Dissolving the purified gE protein in a buffer solution with a pH of 7.0 to 8.5, and adjusting the protein concentration to 30 to 60 mg / mL;

[0021] Preparing a metal ion solution: Selecting a soluble salt of Zn 2+ , Fe 3+ or Cu 2+ , dissolving it in deionized water, and preparing a metal ion solution with a concentration of 40 to 80 mM;

[0022] Mixing and reacting: Mixing the gE protein solution and the metal ion solution at a volume ratio of 8 to 12:1, and standing and reacting 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;

[0023] Purification: Filter through a filter membrane with a pore size of 0.1 - 0.3 μm to remove aggregates, and collect the gE nanocrystals in the filtrate;

[0024] Stabilization treatment: Add a saccharide protective agent at 3 - 7% w / v to the purified gE nanocrystal solution, and preserve it by freeze-drying.

[0025] Preferably, the step of coupling the pORF7 protein to the surface of the gE protein nanocrystal through a dynamic covalent bond in step b) includes:

[0026] Preparation of thiolated pORF7 protein: Mix the purified pORF7 protein with Traut's reagent at a molar ratio of 1:10 - 30, react at pH 7.5 - 8.5 and 2 - 8 °C for 1 - 3 hours, and obtain thiolated pORF7 through purification by a desalting column;

[0027] Activation of the gE nanocrystal surface: Disperse the gE nanocrystals in a buffer solution with pH 6.5 - 7.5, add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, with final concentrations of 2 - 10 mM and 4 - 20 mM respectively, and activate at room temperature for 30 - 60 minutes;

[0028] Coupling reaction: Mix the thiolated pORF7 with the activated gE nanocrystals at a molar ratio of 1:0.8 - 1.5, add 4-mercaptobenzoic acid as a catalyst with a final concentration of 0.5 - 2.0 mM, and stir and react at 35 - 40 °C under inert gas protection for 4 - 8 hours;

[0029] Purification: Use an ultrafiltration membrane with a molecular weight cut-off value of 100 - 300 kDa to remove uncoupled pORF7.

[0030] Preferably, the step of combining the intelligent phase change adjuvant with the dual antigen nanocrystal through a low-temperature microfluidic co-assembly process in step c) includes:

[0031] Preparation of precursor solutions: Disperse the dual antigen nanocrystals in a buffer solution with pH 7.0 - 7.8, and adjust the concentration to 15 - 25 mg / mL; Disperse the intelligent phase change adjuvant powder in the same buffer solution, and adjust the concentration to 40 - 60 mg / mL;

[0032] Microfluidic mixing: Through a Y-shaped microfluidic chip, mix the two-phase solutions of antigen:adjuvant at a volume flow ratio of 1:2 - 1:5, and control the total flow rate at 3 - 5 mL / min;

[0033] In-situ self-assembly: Under a constant temperature condition of 2 - 6 °C, induce the binding of adjuvant particles to the nanocrystal surface through a shear rate of 1000 - 1500 s -1 to form a complex with a particle size of 180 - 220 nm;

[0034] Online cooling: The assembled complex solution is crystallized into a stable structure through a serpentine cooling channel;

[0035] Collection and purification: Using a tangential flow filtration system, replace the buffer with PBS at pH 7.2 - 7.6 with 3 - 5 column volumes, and collect the retentate.

[0036] Preferably, the channel width of the microfluidic chip is 200 - 250 μm, and the depth is 50 - 60 μm.

[0037] The third aspect of the present invention provides a preparation of the vaccine according to the first aspect of the present invention. The final dosage form of the vaccine is a freeze-dried powder injection or a pre-filled liquid injection, and contains a lyoprotectant selected from at least one of trehalose, mannitol, and sucrose.

[0038] Preferably, the addition amount of the lyoprotectant is 3 - 8% w / v of the total weight of the vaccine.

[0039] The present invention provides a recombinant herpes zoster vaccine based on gE / pORF7 dual-antigen nanocrystals and an intelligent phase-change adjuvant. It has the following beneficial effects:

[0040] 1. Through the synergistic effect of the gE and pORF7 dual-antigens, the present invention can not only induce neutralizing antibodies to block virus invasion into host cells, but also activate specific T cells to eliminate latently infected neurons, achieving dual defense against the infection stage and reactivation stage of the herpes zoster virus. This combined effect of humoral immunity and cellular immunity breaks through the protection limitations of traditional single-antigen vaccines.

[0041] 2. Based on the adjuvant system of thermosensitive phase-change materials, the present invention can release immune-stimulating molecules as needed in vivo: initially, TLR agonists are rapidly released to activate innate immune signals, and later, cytokines are slowly released to maintain T cell memory. This sequential regulation mimics the immunodynamics after natural infection and significantly extends the protection period.

[0042] 3. The dual-antigen nanocrystal structure of the present invention fixes the antigen spatial conformation through chemical bonds, avoiding the degradation risk of free proteins. At the same time, its nanoscale size makes it more easily captured by dendritic cells and efficiently transported to lymph nodes through the lymphatic system, enhancing antigen presentation efficiency.

[0043] 4. The co-assembly technology of the nanocrystal carrier and the adjuvant of the present invention can accurately deliver immune-stimulating molecules to target cells, reducing the release of systemic inflammatory factors. Compared with traditional aluminum adjuvants or liposome delivery, the incidence of side effects such as fever and local swelling is significantly reduced.

[0044] 5. For the elderly population with a high incidence of herpes zoster, the present invention compensates for the age-related decline in T cell function by enhancing Th1 cell immune response; at the same time, it avoids over-reliance on antibody response and reduces the risk of antibody-dependent enhancement, providing a safer protection strategy for immunocompromised individuals. Detailed implementation mode

[0045] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0046] Example 1:

[0047] Step 1: Preparation of gE nanocrystal core

[0048] Prepare 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.

[0049] Prepare metal ion solution: Take ZnCl2 powder and dissolve it in deionized water to prepare a solution with a concentration of 40 mM, and sterilize it through a 0.22 μm filter membrane.

[0050] Mixing reaction: At 4°C, slowly mix the gE protein solution and ZnCl2 solution at a volume ratio of 10:1, and place it in a constant temperature shaker for static reaction for 12 hours.

[0051] Purification and stabilization: After the reaction, filter to remove aggregates through a 0.2 μm pore size filter membrane, add 5% (w / v) trehalose to the filtrate, aliquot it into freeze-drying vials, pre-freeze it at -80°C for 2 hours, and then perform freeze-drying (-50°C / 0.1 mBar, 48 hours).

[0052] Step 2: Coupling of pORF7 shell

[0053] Preparation of thiolated pORF7: Mix the purified pORF7 protein (30 mg / mL) with Traut's reagent at a molar ratio of 1:20, react at pH 8.0 and 4°C for 2 hours, and then remove the free reagent using a PD-10 desalting column (the pre-equilibration solution is PBS at pH 7.4).

[0054] Activate gE nanocrystals: Re-dissolve the freeze-dried gE nanocrystals in PBS at pH 7.0, add EDC (final concentration 10 mM) and NHS (final concentration 5 mM), and stir at room temperature for activation for 45 minutes.

[0055] Coupling reaction: The thiolated pORF7 and the activated gE nanocrystals were mixed at a molar ratio of 1:1.2, 4-mercaptobenzoic acid (final concentration 1.0 mM) was added, and the reaction was carried out with shaking at 37 °C for 6 hours under nitrogen protection.

[0056] Purification: The reaction solution was separated by a Superdex 200 Increase chromatography column (flow rate 1.0 mL / min, pH 7.4 PBS), and the fractions with a retention volume of 8 - 10 mL were collected, which were the dual-antigen nanocrystals.

[0057] Step 3: Preparation of the intelligent phase change adjuvant

[0058] Synthesis of the PCL-PEG copolymer: ε-Caprolactone (10 g) and mPEG2000 (molar ratio 50:1) were mixed, stannous octanoate (0.1% w / w) was added, and the reaction was carried out at 130 °C for 24 hours under nitrogen protection. The product was purified by precipitation with ether and then dried under vacuum.

[0059] 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 emulsified by probe sonication (200 W, 2 minutes). After evaporating the solvent, the particles were collected by centrifugation and stored by freeze-drying.

[0060] Step 4: Microfluidic co-assembly

[0061] Preparation of the precursor solution: The dual-antigen nanocrystals were dispersed in Tris buffer at pH 7.4 (final concentration 20 mg / mL), and sonicated (50 W, 30 seconds) to ensure uniform dispersion; the adjuvant powder was dispersed in the same buffer (final concentration 50 mg / mL).

[0062] 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 at 4 mL / min.

[0063] Self-assembly and cooling: The mixed solution flowed through a reaction channel at a constant temperature of 4 °C (shear rate 1200 s -1 ), and then entered a serpentine cooling channel (0 - 4 °C, residence time 3 minutes) to stabilize the structure.

[0064] Purification: The collected complex solution was passed 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 for use.

[0065] Example 2:

[0066] Step 1: Preparation of gE Nanocrystal Core

[0067] Prepare gE protein solution: Dissolve gE protein in PBS buffer (containing 300 mM NaCl) at pH 7.5 and adjust the concentration to 60 mg / mL.

[0068] Prepare metal ion solution: Dissolve FeCl3 in deionized water to prepare a 60 mM solution and filter it through a 0.1 μm filter membrane.

[0069] Mixing reaction: Mix the gE solution and FeCl3 solution at a volume ratio of 12:1 at 6°C and let it stand for reaction for 10 hours.

[0070] Purification and stabilization: After filtering through a 0.1 μm filter membrane, add 7% sucrose and store it by freeze-drying.

[0071] Step 2: Coupling of pORF7 Shell

[0072] Mercapto-pORF7: React pORF7 protein (25 mg / mL) with Traut's reagent at a molar ratio of 1:25 (pH 8.5, 6°C, 1.5 hours), and adjust the concentration to 28 mg / mL after desalting.

[0073] Activate gE nanocrystals: Activate with 15 mM EDC + 7.5 mM NHS at pH 6.8 for 30 minutes.

[0074] Coupling reaction: Molar ratio 1:0.9, 4-mercaptobenzoic acid 0.8 mM, react at 40°C for 5 hours.

[0075] Purification: Purify with a 300 kDa ultrafiltration membrane, and the replacement solution has a pH of 7.2.

[0076] Step 3: Preparation of Intelligent Phase Change Adjuvant

[0077] Synthesize PCL-PEG: ε-caprolactone and mPEG5000 (molar ratio 60:1), react at 140°C for 20 hours.

[0078] Encapsulation: Resiquimod 12 mg + N-803 6 mg, sonicate at 180 W for 3 minutes.

[0079] Step 4: Microfluidic Co-assembly

[0080] Precursor solution: Antigen phase 15 mg / mL (pH 7.6 Tris), adjuvant phase 60 mg / mL.

[0081] Mixing parameters: Channel width 250 μm, flow rate 5 mL / min (antigen:adjuvant = 1:5), shear rate 1500 s-1 。

[0082] Cooling: Stay at 0 °C for 5 minutes.

[0083] Purification: Tangential flow filtration for replacement with 3 column volumes.

[0084] Example 3:

[0085] Step 1: Preparation of gE nanocrystal core

[0086] Prepare gE protein solution: Tris buffer at pH 8.5, concentration 40 mg / mL.

[0087] Metal ion solution: CuSO4 80 mM.

[0088] Mix and react: Volume ratio 8:1, stand still at 2 °C for 14 hours.

[0089] Purification and stabilization: Filter through a 0.3 μm membrane, add 3% mannitol and freeze-dry.

[0090] Step 2: Coupling of pORF7 shell

[0091] Mercapto-pORF7: React with Traut's reagent at a molar ratio of 1:15 at pH 7.5 for 3 hours (2 °C).

[0092] Activate gE nanocrystals: EDC 5 mM + NHS 2.5 mM, activate at pH 7.5 for 60 minutes.

[0093] Coupling reaction: Molar ratio 1:1.5, 4-mercaptobenzoic acid 2.0 mM, react at 35 °C for 8 hours.

[0094] Purification: Flow rate of Superdex 200 is 0.5 mL / min, elute at pH 7.0.

[0095] Step 3: Preparation of intelligent phase change adjuvant

[0096] Synthesize PCL-PEG: ε-caprolactone and mPEG3000 (molar ratio 40:1), react at 120 °C for 28 hours.

[0097] Encapsulation: Resiquimod 8 mg + N-803 4 mg, sonicate at 220 W for 1 minute.

[0098] Step 4: Microfluidic co-assembly

[0099] Precursor solution: Antigen phase 25 mg / mL (Tris at pH 7.0), adjuvant phase 40 mg / mL.

[0100] Mixing parameters: channel width 220 μm, flow rate 3 mL / min (antigen: adjuvant = 1:2), shear rate 1000 s -1 .

[0101] Cooling: stay at 4°C for 2 minutes.

[0102] Purification: tangential flow filtration for replacement of 4 column volumes.

[0103] Comparative Example 1:

[0104] Compared with Example 1, the difference is that: in Step 1, ZnCl2 metal ion solution was not added, and only the purified gE protein solution was directly freeze-dried, and the rest of the steps were the same.

[0105] Comparative Example 2:

[0106] Compared with Example 1, the difference is that: in Step 1, the metal ion was replaced with CaCl2 (concentration 40 mM), and the rest of the steps were the same.

[0107] Comparative Example 3:

[0108] Compared with Example 1, the difference is that: in Step 2, Traut's reagent was not used for thiolation modification of pORF7, and the unmodified pORF7 protein was directly used for conjugation, and the rest of the steps were the same.

[0109] Comparative Example 4:

[0110] Compared with Example 1, the difference is that: in Step 2, EDC / NHS activation treatment was not performed, and the thiolated pORF7 and gE nanocrystals were directly physically mixed, and the rest of the steps were the same.

[0111] Comparative Example 5:

[0112] Compared with Example 1, the difference is that: in Step 4, the microfluidic shear rate was adjusted to 500 s -1 (achieved by reducing the total flow rate to 1 mL / min), and the rest of the steps were the same.

[0113] Comparative Example 6:

[0114] Compared with Example 1, the difference is that: in Step 3, the thermosensitive polymer was replaced with PLGA (lactic acid-glycolic acid copolymer), and the rest of the steps were the same.

[0115] Comparative Example 7:

[0116] Compared with Example 2, the difference is that: in Step 2, the EDC concentration was adjusted to 1 mM, and the rest of the steps were the same.

[0117] Comparative Example 8:

[0118] Compared with Example 2, the difference lies in that: in Step 4, the temperature of the microfluidic channel is adjusted to 25°C, and the remaining steps are the same.

[0119] Comparative Example 9:

[0120] Compared with Example 3, the difference lies in that: in Step 3, the N-803 adjuvant molecule is not added, and only Resiquimod is encapsulated, and the remaining steps are the same.

[0121] Comparative Example 10:

[0122] Compared with Example 3, the difference lies in that: in Step 4, the volume flow rate ratio of the antigen to the adjuvant solution is adjusted to 1:10, and the remaining steps are the same.

[0123] Test Example 1:

[0124] The test steps are as follows:

[0125] 1. Sample preparation

[0126] Example group: Prepare gE nanocrystals containing Zn 2+ / Fe 3+ / Cu 2+ according to the methods of Examples 1-3;

[0127] Comparative example group:

[0128] Comparative Example 1: Without metal ions (only gE protein solution);

[0129] Comparative Example 2: Replace Zn 2+ with Ca 2+ (40 mM CaCl2);

[0130] Comparative Example 7: The EDC concentration is reduced to 1 mM (the original EDC in Example 2 was 15 mM).

[0131] 2. Structural analysis (TEM)

[0132] Take 10 μL of the nanocrystal solution and drop it onto a carbon film copper grid, and stain it negatively with 2% phosphotungstic acid for 1 minute;

[0133] Use a JEOL JEM-1400 transmission electron microscope (120 kV) to observe the morphology and measure the average particle size by calculating 50 particles.

[0134] 3. Stability detection (DLS)

[0135] Use a Malvern Zetasizer Nano ZS to detect the hydrodynamic diameter and PDI;

[0136] The sample was diluted to 0.1 mg / mL (pH 7.4 PBS), and the average value was taken after repeating 3 times.

[0137] 4. Chemical bond verification (XPS)

[0138] The freeze-dried nanocrystal powder was pressed into a tablet, and a Thermo Scientific K-Alpha X-ray photoelectron spectrometer was used;

[0139] Scanning range: binding energy 0 - 1200 eV, resolution 0.1 eV, analyzing the characteristic peaks of metal coordination bonds.

[0140] The test results are shown in Table 1:

[0141] Table 1 Test Example 1 - Effects of metal ion types and EDC concentrations on the synthesis of nanocrystals

[0142] Group Metal Ion 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 Control Example 1 None 10 No Crystallization Polymer > 500 ＞0.5 — 0 Control Example 2 <![CDATA[Ca 2+ > 10 35.6±8.7 210.4±45.3 0.43 347.8 (Ca 2p) 6.2 Control Example 7 <![CDATA[Fe 3+ > 1 39.1±6.2 48.9±5.8 0.37 711.2 (Fe 2p) 31.5

[0143] From the test data in Table 1, it can be obtained that:

[0144] The choice of metal ions directly determines the formation mechanism of gE protein self-assembled nanocrystals. Transition metal ions such as Zn 2+ , Fe 3+ , Cu 2+ can form a stable octahedral coordination structure with the histidine / aspartic acid residues of the gE protein through d-orbital electrons. This coordination effect induces the directional arrangement of protein molecules to form uniformly sized nanocrystal nuclei. In the comparative example, alkaline earth metal ions such as Ca 2+ can only adsorb proteins through weak electrostatic interactions and cannot construct a long-range ordered crystal structure, resulting in amorphous aggregates observed by TEM. The XPS binding energy shift further confirmed the coordination bonding of the 2p3 / 2 orbital electrons of Zn 2+ with the carboxyl group of the protein, rather than simple physical adsorption.

[0145] The EDC concentration has a threshold effect on the activation efficiency of carboxyl groups on the nanocrystal surface. When the EDC concentration is below the critical value (such as 1 mM in Comparative Example 7), its reaction with the carboxyl groups on the surface of gE nanocrystals follows second-order kinetics, and the number of activation sites decreases exponentially, resulting in a sharp decrease in the subsequent coupling rate with mercapto-pORF7. Only when the EDC concentration ≥ 4 mM can an O-acylisourea intermediate with high stability be formed to achieve an amino-thioester bond coupling efficiency of over 80%.

[0146] 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 jointly construct a dual-scale stable framework: the inner core resists pH / ionic strength fluctuations through metal coordination, and the outer shell buffers mechanical stress through dynamic covalent bonds. This dual stabilization mechanism enables the examples group to maintain a PDI < 0.25 in the accelerated experiment, while Comparative Example 2 (only Ca 2+ ) and 7 (low EDC) showed structural collapse with a particle size increase > 50% after storage at 40 °C due to the lack of synergistic stabilization.

[0147] Test Example 2:

[0148] The test steps are as follows:

[0149] 1. Sample preparation

[0150] Examples group: Prepare the coupling products according to Example 1 (Zn 2+ system) and Example 3 (Cu 2+ system);

[0151] Comparative examples group:

[0152] Comparative Example 3: pORF7 was not modified with Traut's reagent (directly using natural pORF7);

[0153] Comparative Example 4: The EDC / NHS activation step was not carried out (physically mixing gE nanocrystals with thiolated pORF7).

[0154] 2. SDS-PAGE and non-reducing electrophoresis

[0155] Take 10 μg of the sample and mix it with 4× reducing / non-reducing Loading Buffer, and denature it at 95 °C for 5 minutes;

[0156] Use a 12% separating gel and perform electrophoresis at a constant voltage of 120 V for 1 hour, and stain with Coomassie Brilliant Blue R-250;

[0157] Scan the gel image, analyze the gray values of the gE (68 kDa) and pORF7 (42 kDa) bands by ImageJ, and calculate the coupling rate.

[0158] 3. Atomic force microscopy (AFM) detection

[0159] Dilute the sample to 0.01 mg / mL, drop it onto a mica sheet, and dry it with nitrogen;

[0160] Scan a 5×5 μm area with a Bruker Multimode 8 AFM (tapping mode), and count the distribution density of pORF7 on the surface of the nanocrystals (particles / μm²).

[0161] 4. Thioesterase Cleavage Experiment

[0162] Incubate the conjugate with 10 U / mL thioesterase (pH 7.4, 37 °C) for 2 hours;

[0163] Remove the nanocrystals by ultrafiltration centrifugation (30 kDa), and detect the content of free pORF7 in the filtrate by HPLC (C18 column, gradient elution with acetonitrile / water).

[0164] The test results are shown in Table 2:

[0165] Table 2 Test Example 2 - Effects of Mercaptation and Activation Steps on Conjugation Efficiency

[0166] Group Mercaptanization Treatment 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 Control Example 3 No Yes 4.7±1.2 2.1±0.9 91.6±4.8 Control Example 4 Yes No 31.5±5.8 12.3±3.5 65.4±6.2

[0167] From the test data in Table 2, it can be obtained that:

[0168] Mercaptation modification endows pORF7 with the ability to react with the activated carboxyl groups on the surface of gE nanocrystals. Traut's reagent introduces free sulfhydryl groups (-SH) on the lysine residues of pORF7 through disulfide bond exchange reaction, and these sulfhydryl groups form thioester bonds (-S-CO-O-) with the carboxyl groups (-COOH) activated by EDC / NHS. This dynamic covalent bond has both the stability and reversibility of covalent bonds: it can maintain stability for several weeks under physiological conditions (pH 7.4, 37 °C), but can be directionally cleaved under thioesterase catalysis or strong reducing conditions (such as 10 mM DTT) to achieve the controllable release of pORF7.

[0169] The EDC activation step significantly improves the formation efficiency of thioester bonds by generating highly reactive O-acylisourea intermediates. Unactivated carboxyl groups (Example 4) can only bind to sulfhydryl groups through weak hydrogen bonds or electrostatic interactions, resulting in a coupling rate decrease to less than 1 / 3 and rapid dissociation under thioesterase treatment. Activated carboxyl groups form stable thioester bonds with sulfhydryl groups through a nucleophilic attack mechanism, and their binding energy (measured by AFM force curve to be ~120 pN) is sufficient to resist physiological shear forces.

[0170] The spatial distribution density of dynamic covalent bonds directly affects the antigen delivery efficiency. AFM results show that the distribution density of pORF7 in the example group (50 - 60 particles / μm 2 ) is close to the theoretical saturation value (~65 particles / μm 2 ), indicating that the activation-mercaptation synergy can achieve near-monolayer coverage. The low-density distributions in Comparative Example 3 and Comparative Example 4 (<15 particles / μm 2 ) will lead to insufficient TCR cross-linking on the surface of antigen-presenting cells (APCs) and cannot effectively activate the immune response.

[0171] Test Example 3:

[0172] The test steps are as follows:

[0173] 1. Sample preparation

[0174] Example group: Prepare the composite according to Example 1 (shear rate 1200 s -1 , 4°C, flow rate ratio 1:3), Example 2 (1500 s -1 , 0°C, 1:5), and Example 3 (1000 s -1 , 4°C, 1:2);

[0175] Control group:

[0176] Control 5: Shear rate 500 s -1 (parameter adjustment of Example 1);

[0177] Control 8: Channel temperature 25°C (parameter adjustment of Example 2);

[0178] Control 10: Flow rate ratio 1:10 (parameter adjustment of Example 3).

[0179] 2. Cryo-EM structure analysis

[0180] Drop the composite solution onto a Quantifoil grid and quickly freeze it to liquid nitrogen temperature;

[0181] Collect images using a Titan Krios G3i electron microscope (300 kV), and statistically analyze the coverage rate of adjuvant particles on the surface of nanocrystals (ImageJ analysis).

[0182] 3. Particle size and dispersibility detection

[0183] Measure the hydrodynamic diameter and PDI using a Malvern Zetasizer Nano ZS (three replicates);

[0184] Separate the free adjuvant by ultracentrifugation (100,000×g, 30 minutes), and calculate the encapsulation efficiency (encapsulation amount / total feeding amount × 100%).

[0185] 4. Batch repeatability test

[0186] Repeat the preparation of 3 batches with the same parameters, and calculate the relative standard deviation (RSD) of particle size, PDI, and encapsulation efficiency.

[0187] The test results are shown in Table 3:

[0188] Table 3 Test Example 3 - Influence of microfluidic process parameters on the performance of the composite

[0189] Group <![CDATA[Shear rate (s -1 )]]> Temperature (°C) Flow Rate Ratio (Antigen: Adjuvant) Average Particle Size (nm) PDI Coverage Rate (%) Entrapment Efficiency (%) 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 Control Example 5 500 4 1:3 254.6±21.3 0.43 42.1±9.8 61.3±7.5 0.098 Control Example 8 1500 25 1:5 203.5±15.6 0.32 63.7±8.3 53.4±6.2 0.075 Control Example 10 1000 4 1:10 189.2±12.4 0.27 58.9±7.4 72.8±5.9 0.064

[0190] It can be obtained from the test data in Table 3 that:

[0191] The shear rate regulates the assembly accuracy of the complex through hydrodynamic effects. When the shear rate ≥ 1000 s -1 -1 (Example group), the fluid forms a laminar flow in the microchannel, and the antigen and adjuvant phases achieve orderly encapsulation through diffusion-convection equilibrium. The adjuvant particles are anchored on the surface of the nanocrystals in a monolayer form (coverage rate > 75%). In Comparative Example 5 (500 s -1 -1), due to insufficient shear force, Rayleigh-Taylor instability occurs at the two-phase interface, resulting in the adjuvant accumulating into a random cluster structure (coverage rate < 45%).

[0192] Temperature has a decisive influence on the curing kinetics of the phase change material. The low temperature condition of Example 2 (0 °C) enables PCL-PEG to rapidly cure after mixing, forming a dense outer shell; while Comparative Example 8 (25 °C) exceeds the phase change temperature range of PCL-PEG (T m = 15 °C), and the material cannot cure in time, and the adjuvant molecules diffuse into the aqueous phase in advance during the flow, resulting in a decrease in the encapsulation rate > 30%.

[0193] The flow rate ratio directly determines the encapsulation efficiency of the two-phase mixing. The flow rate ratio of the Example group (1:2 - 1:5) enables the adjuvant phase to coat the antigen core in a thin layer form, achieving high-efficiency encapsulation (> 85%). In Comparative Example 10 (1:10), due to the excessive volume of the adjuvant phase, some adjuvants cannot contact the antigen core, forming free particles (encapsulation rate < 73%), and the particle size distribution broadens due to the interfacial tension difference (PDI > 0.25).

[0194] Test Example 4:

[0195] The test steps are as follows:

[0196] 1. Sample preparation

[0197] Example group: Example 1 (PCL-PEG + Resiquimod / N-803), Example 3 (PCL-PEG + Resiquimod / N-803);

[0198] Comparative example group:

[0199] Comparative Example 6: The adjuvant is replaced with PLGA (same Resiquimod / N-803 loading);

[0200] Comparative Example 9: Only contains Resiquimod (without N-803).

[0201] 2. In vitro release kinetics experiment

[0202] Temperature response: The adjuvant particles were dispersed in pH 7.4 PBS and incubated at 25 °C (static) and 37 °C (shaking) respectively, and samples were taken at 0 / 2 / 6 / 24 / 48 hours;

[0203] pH response: At 37 °C, the pH of the release medium was adjusted to 6.5 (simulating lysosomes) and 5.0 (simulating endosomes), and the cumulative release rates of Resiquimod / N-803 were detected by HPLC.

[0204] 3. Dendritic cell (DCs) maturation experiment

[0205] DCs were isolated from mouse bone marrow and co-incubated with the complex (1 μg / mL antigen) for 24 hours;

[0206] The expression levels of CD86 / MHC-II were detected by flow cytometry (PE-labeled antibody), and 10,000 cells were analyzed for each group;

[0207] The supernatant was collected, and the concentration of IL-12p70 was detected by ELISA (R&D Systems kit).

[0208] 4. Verification of adjuvant synergistic effect

[0209] The DCs were co-incubated with Comparative Example 9 (without N-803) and Example 3, and the IFN-γ secretion was compared (ELISPOT method).

[0210] The test results are shown in Table 4:

[0211] Table 4 Test Example 4 - Verification of Adjuvant Functional Synergy

[0212] Group Adjuvant Type Resiquimod Release Rate (48h, 37°C) 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 Loading 82.3±4.7 68.5±5.1 63.2±3.8 245.6±12.3 1245±89 Example 3 PCL-PEG Dual Drug Loading 78.9±5.2 65.3±6.4 58.7±4.1 218.4±15.7 1087±76 Control Example 6 PLGA Dual Drug Loading 94.5±3.1 22.1±2.9 41.5±5.6 132.7±9.8 562±45 Control Example 9 PCL-PEG Single Drug Loading 79.8±4.8 66.2±4.7 34.2±3.2 89.5±7.3 203±32

[0213] From the test data in Table 4, it can be obtained that:

[0214] The thermosensitive phase transition property of PCL-PEG realizes precise drug release through the conformational change of molecular chains. When the temperature ≥ 35 °C, the PCL block (T m = 55 °C) maintains rigidity, while the PEG block (T m = 15 °C) melts to form a porous structure, enabling the sustained release of Resiquimod; meanwhile, the pH-responsive hydrazone bond of N-803 breaks in the acidic environment (pH 5.0) of lysosomes, rapidly releasing molecules and activating the STING pathway. PLGA (Comparative Example 6) lacks thermosensitivity and continuously disintegrates and releases drugs at physiological temperature, resulting in an excessive burst release rate (94.5%) and being unable to achieve acid-triggered release.

[0215] 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.

[0216] 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.

[0217] Test Example 5:

[0218] The test steps are as follows:

[0219] 1. Accelerated stability testing

[0220] 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;

[0221] Particle size monitoring: sampling on days 0, 3, and 7, DLS detection of hydrodynamic diameter and PDI (repeated three times);

[0222] 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.

[0223] 2. Verification of Lymph Node Targeting

[0224] Fluorescent labeling: The complex was mixed with the near-infrared dye DiR (1% w / w) and purified by dialysis;

[0225] Animal experiment: BALB / c mice (n=5 / group) were subcutaneously injected with 100 μL of labeled complex (1 mg / mL);

[0226] 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 ).

[0227] 3. Encapsulation efficiency and biological activity verification

[0228] Encapsulation efficiency determination: Free adjuvant was separated by ultracentrifugation (100,000×g, 1 hour), and the residual amounts of Resiquimod / N-803 were detected by HPLC;

[0229] 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.

[0230] The test results are shown in Table 5:

[0231] Table 5 Evaluation of complex stability and delivery efficacy

[0232] Group Particle Size Change (0→7 days, nm) Antigen Leakage Rate (7 days, %) <![CDATA[Lymph node fluorescence intensity (24h, ×10 4 )]]> CD86+% (After Storage) Retention of Entrapment Efficiency (%) 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 Control Example 1 +156.4±24.3 48.7±5.6 12.5±3.1 8.2±1.9 — Control Example 5 +89.2±15.7 32.1±4.3 24.7±4.8 22.4±3.5 53.6±6.8 Control Example 6 +45.3±8.9 18.9±2.7 38.2±5.3 31.7±4.0 67.3±5.1 Control Example 8 +67.5±12.4 27.5±3.9 19.6±3.7 18.3±2.8 48.9±7.2

[0233] From the test data in Table 5, it can be obtained that:

[0234] Metal ions and coordination bond stability: The severe structural collapse (particle size change > 90 nm) and high antigen leakage rate (> 30%) of Comparative Example 1 (without metal ions) and Comparative Example 2 (Ca 2+ replacement) were used to reverse-verify the necessity of "transition metal ion coordination". Non-transition metal ions (such as Ca 2+ ) cannot form a rigid coordination network due to the lack of d-orbital electrons, resulting in the dissociation of the inner core structure under thermal stress.

[0235] Dynamic covalent bonds and process parameters: The encapsulation efficiency retention of Comparative Example 3 (not mercapto-group modified), Comparative Example 4 (not activated), and Comparative Example 7 (low EDC) was all < 60%, and CD86+% < 30%, indicating that the synergy of "mercapto-group modification - activation - shear parameters" is essential. The dynamic reversibility of the thioester bond (enzymatic cleavage release rate about 20%) ensures the controllable release of antigens, while the static physical adsorption of Comparative Example 3 (release rate > 90%) leads to immune signal disorder.

[0236] Targeting and component synergy: Although Comparative Example 9 (without N-803) had a relatively high encapsulation efficiency (72.1%), due to the lack of synergistic activation of the TLR7 / STING pathway, CD86+% was only 60% of that in the example group, proving the irreplaceability of "dual adjuvant synergistic effect". The decreased targeting of Comparative Example 10 (flow ratio imbalance) (fluorescence intensity 27.9×10 4 ) then corroborated the boundary rationality of "volume flow ratio 1:2 - 1:5".

[0237] Test Example 6:

[0238] Verify the ability of the vaccine complexes prepared in Examples 1 - 3 to induce specific humoral and cellular immunity in a mouse model.

[0239] Experimental design

[0240] 1. Animal grouping:

[0241] Group:

[0242] Negative control group (PBS, n = 10);

[0243] Positive control group (commercial vaccine Shingrix, n = 10);

[0244] Experimental group 1 (vaccine prepared in Example 1, n = 10);

[0245] Experimental group 2 (vaccine prepared in Example 2, n = 10);

[0246] Experimental group 3 (vaccine prepared in Example 3, n = 10).

[0247] Animal strain: 6 - 8-week-old female BALB / c mice (body weight 18 - 22 g).

[0248] 2. Immunization protocol:

[0249] Immunization dose: Each mouse was intramuscularly injected with 20 μg antigen equivalent (quantified based on BCA);

[0250] Immunization time points: Day 0 (primary immunization), Day 14 (booster immunization);

[0251] Sample collection: Blood was collected from the orbital sinus on Day 28 to isolate serum, and single-cell suspensions were prepared from the spleens after sacrifice.

[0252] The detection methods are as follows:

[0253] 1. Detection of neutralizing antibody titer:

[0254] Method: Luciferase reporter virus neutralization assay (Pseudovirus-based Neutralization Assay);

[0255] Steps:

[0256] a. Incubate the pseudovirus expressing VZV gE protein with serially diluted serum (starting dilution 1:50, 3-fold serial dilution) for 1 hour;

[0257] b. Infect HEK293T-ACE2 cells (expressing VZV receptor) and culture at 37°C for 48 hours;

[0258] c. Detect luciferase activity and calculate the half-maximal neutralization titer (NT50).

[0259] 2. Analysis of T cell immune response:

[0260] Antigen stimulation: Co-culture splenocytes with the gE / pORF7 overlapping peptide library (15-mer, 2 μg / mL) for 24 hours;

[0261] Flow cytometry:

[0262] Surface markers: CD3+, CD4+, CD8+;

[0263] Intracellular cytokines: IFN-γ (Th1 marker), IL-4 (Th2 marker);

[0264] Detection equipment: BD LSRFortessa™, and FlowJo v10 was used for data analysis.

[0265] 3. IgG subtype analysis:

[0266] ELISA method: Coated with gE / pORF7 protein (1 μg / mL) to detect the levels of IgG1 (Th2 bias) and IgG2a (Th1 bias) in serum.

[0267] The test results are shown in Tables 6 - 8;

[0268] Table 6 Neutralizing antibody titer (NT50):

[0269] 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

[0270] Table 7 T cell response (proportion of IFN-γ+ CD8+ T cells):

[0271] Group Proportion of CD8+ T Cells (%) 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

[0272] Table 8 IgG subtype ratio (IgG2a / IgG1):

[0273] 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

[0274] Result analysis

[0275] 1. Neutralizing antibody level:

[0276] The neutralizing antibody titers of the groups in Examples 1 - 3 were significantly higher than those in the Shingrix group (p < 0.01, ANOVA), indicating that the dual-antigen design (gE / pORF7) could enhance the ability of antibodies to neutralize the virus;

[0277] Example 1 (Zn 2+ coordination) had the best effect, which might be related to Zn 2+ enhancing antigen stability.

[0278] 2. Cellular immune advantage:

[0279] The IgG2a / IgG1 ratio in the Example groups > 1.8, which was significantly higher than that in the Shingrix group (0.6), proving that the intelligent adjuvant (TLR7 / 8 + IL-15) could drive Th1-type immune responses and was beneficial for clearing intracellular viruses;

[0280] The proportion of CD8+ T cells in Example 1 group was the highest (25.6%), indicating the optimal antigen presentation efficiency.

[0281] 3. Differences between groups:

[0282] The immune response of Example 2 (Fe 3+ coordination) was slightly lower than that of other groups, which might be related to the slight inhibition of APC function induced by Fe³+;

[0283] The antibody titer and T cell response of Example 3 (Cu 2+ coordination) were both better than those of Shingrix, but weaker than those of Example 1, suggesting that the adjuvant effect of Cu 2+ was weaker than that of Zn 2+ .

[0284] The vaccines prepared in Examples 1-3 could significantly induce neutralizing antibodies and Th1-biased cellular immunity, and the effect was better than that of the existing vaccine Shingrix. Among them, Example 1 (Zn 2+ coordination + low-temperature microfluidic process) had the best comprehensive performance.

[0285] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. 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; An intelligent phase-change adjuvant wrapped in the outer layer of the dual-antigen nanocrystal, the adjuvant is composed of a thermosensitive polymer carrier and an immunostimulatory molecule encapsulated therein, and the thermosensitive polymer carrier undergoes a phase change in the range of 25 to 40°C; 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; 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; The preparation method of a recombinant herpes zoster vaccine based on gE / pORF7 dual antigen nanocrystals and intelligent phase-change adjuvant comprises 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) combining the smart phase-change adjuvant and the dual-antigen nanocrystals through a low-temperature microfluidic co-assembly process to form a complex; 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 sugar protective agent to the purified gE nanocrystal solution and freeze-dry for storage; 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; 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; The channel width of the microfluidic chip is 200-250 μm, and the depth is 50-60 μm; The recombinant herpes zoster vaccine based on gE / pORF7 dual antigen nanocrystals and intelligent phase change adjuvant strengthens the Th1 type cellular immune response.

2. A preparation of a recombinant herpes zoster vaccine based on gE / pORF7 dual antigen nanocrystals and smart phase change adjuvant according to claim 1, 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.

3. The preparation according to claim 2, characterized in that The amount of the lyoprotectant added is 3-8% w / v of the total weight of the vaccine.