A varicella-zoster virus virus-like particle vaccine gE-eVLP, a recombinant adenovirus vector vaccine, an mRNA vaccine, their preparation methods and applications

Abstract

This invention provides a virus-like particle vaccine gE-eVLP for varicella-zoster virus, a recombinant adenovirus vector vaccine, an mRNA vaccine, and their preparation methods and applications, belonging to the field of vaccine technology. The nucleotide sequence of the virus-like particle vaccine gE-eVLP provided by this invention includes a signal peptide sequence, a truncated and / or mutated sequence of the gE domain without the original signal peptide, and an EGE motif; the recombinant adenovirus vector vaccine and mRNA vaccine contain gE-eVLP. This invention innovatively integrates EABR self-assembled particle technology into the VZV adenovirus vector vaccine and mRNA vaccine technology platform, and systematically evaluates the immunogenicity of three novel vaccines: Ad26-tPA-gE-eVLP, AdC68-tPA-gE-eVLP, and LNP-tPA-gE-eVLP, providing important experimental evidence for the development of a new generation of highly effective and safe VZV vaccines.

Description

Technical Field

[0001] This invention relates to the field of vaccine technology, and in particular to a varicella-zoster virus virus-like particle vaccine gE-eVLP, a recombinant adenovirus vector vaccine, an mRNA vaccine, and their preparation methods and applications. Background Technology

[0002] Varicella-zoster virus (VZV) is a pathogen of significant clinical importance. Primary infection typically causes chickenpox in children, but the virus can remain latent in nerve ganglia long after recovery. When the body's immunity declines, VZV reactivates, causing herpes zoster (HZ). Epidemiological statistics show that 30% of chickenpox patients experience VZV reactivation in old age, leading to HZ. The overall average incidence rate is approximately 3.40–4.82 per 1000 people, increasing to over 11 per 1000 people in those over 80 years of age. The VZV genome encodes 67 different proteins, with glycoprotein gE being the most abundant on the viral surface. It is one of the main antigens for inducing humoral and cellular immunity in the host and is also an important target antigen for VZV vaccine development. The currently marketed Shingrix vaccine uses a truncated gE (1–546 aa) as its main antigen. Given the large domestic market demand, accelerating the development of VZV vaccines based on gE protein is an effective way to solve the problems of strong immune side effects, low protective efficiency of live attenuated vaccines, and insufficient supply of Shingrix vaccines.

[0003] In recent years, virus-like particle (VLP) vaccine technology has attracted widespread attention due to its potential to stimulate both innate and adaptive immune responses in the host. Studies have shown that VZV gE-based VLP vaccines (gE-VLPs) can induce high levels of anti-VZV antibodies. Furthermore, the combination of VLP vaccine technology with other advanced vaccine platforms has opened up new directions for vaccine development. In particular, enveloped virus-like particle (eVLP) technology, by mimicking the natural structure of the virus, can significantly enhance antigen presentation efficiency and induce a more comprehensive immune response. Professor Bjorkman's team combined mRNA vaccines with EABR self-assembly particle technology to develop an innovative platform capable of encoding self-assembling eVLPs. This research team inserted the ESCRT- and ALIX-binding domains (EABR) into the cytoplasmic tail region of the novel coronavirus S protein, successfully enabling the S protein to form S-eVLPs budding from host cells using the ESCRT pathway. This technology provides a new approach for VZV vaccine development.

[0004] Adenovirus vector vaccines have become another important research direction due to their excellent T-cell immune induction capabilities. However, the current high level of host anti-vector immune response has adversely affected the effectiveness of Ad vector-based gene delivery, while rare serotype adenovirus vectors (such as Ad26 and AdC68) can effectively circumvent pre-existing immunity and have shown good protective effects in the elderly population. This provides an important reference for the development of adenovirus vector-based VZV vaccines. Meanwhile, mRNA vaccine technology has gradually attracted attention due to its advantages such as rapid development and efficient expression. This technology can not only induce strong humoral and cellular immunity but also has a self-adjuvant effect. However, when designing mRNA vaccines, it is necessary to focus on the optimization of the mRNA sequence and the regulation of the 5'UTR and 3'UTR, which are crucial to the antigen expression level and immune protection effect mediated by mRNA vaccines. Recent studies show that the protective effect of HZ mRNA vaccines is comparable to Shingrix, with better safety. These research results indicate that mRNA vaccine platforms have broad application prospects in VZV vaccine development.

[0005] The rapid development of vaccine technology platforms has provided multiple innovative pathways for the design of novel VZV vaccines. eVLP vaccines can present viral antigens in their natural conformation, inducing a strong immune response; adenovirus vector vaccines are renowned for their superior T-cell immune induction capabilities; and mRNA vaccine technology, with its advantages of rapid development and efficient antigen expression, is emerging in the field of infectious disease control. However, how to integrate these technological advantages to develop more effective VZV vaccines remains a key focus and challenge in current research.

[0006] Based on this, the present invention is proposed. Summary of the Invention

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a varicella-zoster virus-like particle vaccine gE-eVLP, wherein the nucleotide sequence of the gE-eVLP includes a signal peptide sequence, a truncated and / or mutated sequence of the gE domain without the original signal peptide, and an EGE motif. The truncation of the gE domain includes: truncation of the TGN target sequence or retention of the TGN target sequence but truncation of the subsequent sequence, and truncation of the internalized signal motif ET; The mutations in the gE domain include: the Y569A mutation that disrupts the TGN targeting sequence localization, the Y582G mutation that eliminates the endocytic signal motif ET, or the S593A, S595A, T596A, and T598A mutations that regulate phosphorylation modification clusters. The nucleotide sequence of the EGE motif is shown in SEQ ID NO: 25.

[0008] Preferably, the TGN targeting sequence deletion is based on gE-△SP as shown in SEQ ID NO: 2, by removing amino acids 568 to 623, resulting in gE-△TGN, as shown in SEQ ID NO: 9; The method of retaining the TGN target sequence but truncating the subsequent sequence is to truncate amino acids 572 to 623 from gE-△SP as shown in SEQ ID NO: 2, and the truncated result is gE-TGN, as shown in SEQ ID NO: 10; The Y569A mutation that disrupts the TGN target sequence localization is a Y569A mutation performed on the basis of gE-TGN as shown in SEQ ID NO: 10, resulting in gE-mTGN as shown in SEQ ID NO: 11; The endocytic signal motif ET truncation is based on gE-△SP as shown in SEQ ID NO: 2, by removing amino acids 582 to 623, resulting in gE-△ET, as shown in SEQ ID NO: 12; The elimination of the endocytosis signal motif Y582G mutation is achieved by removing amino acids 589 to 623 from the gE-△SP amino acid sequence shown in SEQ ID NO: 2 and performing a Y582G mutation, resulting in gE-mET, as shown in SEQ ID NO: 13. The S593A, S595A, T596A, and T598A mutations of the phosphorylation modification clusters are performed on the basis of gE-ΔSP as shown in SEQ ID NO: 2, with simultaneous Y569A mutations, resulting in gE-dM, as shown in SEQ ID NO: 14.

[0009] Preferably, the signal peptide is tPA, and its nucleotide sequence is shown in SEQ ID NO: 3.

[0010] The present invention also provides an expression plasmid containing the virus-like particle vaccine gE-eVLP, wherein the expression vector is pcDNA3.1-empty; the constructed expression plasmid includes pcDNA3.1-tPA-gE-dM-EGE, pcDNA3.1-tPA-gE-△ET-EGE, pcDNA3.1-tPA-gE-mET-EGE, pcDNA3.1-tPA-gE-TGN-EGE, pcDNA3.1-tPA-gE-mTGN-EGE, and pcDNA3.1-tPA-gE-△TGN-EGE.

[0011] The present invention also provides a method for preparing the virus-like particle vaccine gE-eVLP, wherein the expression plasmid is transfected into cells, and the virus-like particle vaccine gE-eVLP is produced by secretory expression of the cells.

[0012] The present invention also provides a recombinant adenovirus vector vaccine based on the varicella-zoster virus of the gE-eVLP, wherein the nucleotide sequence of the gE-eVLP is introduced into an adenovirus vector to obtain a recombinant adenovirus.

[0013] Preferably, the nucleotide sequence of the gE-eVLP is a codon-optimized nucleotide sequence; the gE domain of the gE-eVLP is a codon-optimized thick nucleotide sequence of gE-ΔET as shown in SEQ ID NO: 31; The adenovirus vector is pkAd26-empty or pkAdC68-empty.

[0014] The present invention also provides a method for preparing the recombinant adenovirus vector vaccine, wherein the recombinant adenovirus is linearized and then transfected into cells to prepare a seed virus, the seed virus is expanded and cultured, purified, and the recombinant adenovirus vector vaccine is obtained.

[0015] This invention also provides an mRNA vaccine based on the gE-eVLP varicella-zoster virus. The method involves amplifying 5U1-gE-eVLP using mRNA plasmid primers, inserting 5U1-gE-eVLP into the vector pcDNA3.1 T7-Nluc-3U1 to obtain a recombinant plasmid for the mRNA vaccine. After linearization, in vitro transcription, capping, and purification, the recombinant plasmid yields the mRNA vaccine. The mRNA vaccine is then encapsulated in liposomes to obtain an LNP-mRNA vaccine. The mRNA plasmid primers are as shown in SEQ ID NO: 34~37; The nucleotide sequence of the amplified 5U1-gE-eVLP with the addition of 3U1 and polyA tails is shown in SEQ ID NO: 33.

[0016] The present invention also provides the application of the virus-like particle vaccine gE-eVLP, the recombinant adenovirus vector vaccine, or the mRNA vaccine described herein in the preparation of varicella-zoster virus vaccine drugs.

[0017] This invention replaces the original signal peptide of the gE protein with the tPA signal peptide. Results show that replacing the tPA signal peptide significantly increases the expression level of the gE protein. This invention also systematically optimizes the mutation and truncation strategies for the VZV gE protein, further improving its expression level. Furthermore, in constructing the gE-eVLP target antigen sequence with potential vaccine application value, different modification strategies for the gE gene, upon binding to the EGE motif, significantly affected the expression pattern of the gE protein. In summary, this invention, through systematic optimization of the gE protein expression strategy and combined with eVLP self-assembly particle technology, successfully constructed a gE-eVLP target antigen sequence with potential vaccine application value.

[0018] This invention also innovatively integrates EABR self-assembled particle technology into VZV adenovirus vector vaccine and mRNA vaccine technology platforms, systematically evaluates the immunogenicity of three novel vaccines—Ad26-tPA-gE-eVLP, AdC68-tPA-gE-eVLP, and LNP-tPA-gE-eVLP—and compares them with Shingrix, providing important experimental evidence for the development of a new generation of highly effective and safe VZV vaccines. Attached Figure Description

[0019] Figure 1 The diagram shows the sequence structure and modification of the gE protein, including the A-gE protein sequence structure and the B-gE protein sequence modification strategy.

[0020] Figure 2 The electrophoresis results for the recombinant plasmid are as follows: A—PCR amplification result of the target gene; B—agarose gel electrophoresis identification result of pcDNA 3.1-empty digestion; M—DNA relative molecular mass standard; 1—gE-ΔSP (size 1782bp); 2—tPA (size 103bp); 3—pcDNA 3.1-empty; 4—pcDNA 3.1-empty KpnI .

[0021] Figure 3 To verify the effect of tPA signal peptide on gE protein expression using Western blot, A—Western blot was used to identify the expression of gE and tPA-gE in HEK293A cells; B—ImageJ was used to calculate grayscale values; negative—pcDNA3.1-empty negative control; M—pre-stained protein marker; : P <0.05, : P <0.01.

[0022] Figure 4Western blot analysis of gE protein truncation and mutant expression: A—gE truncation and mutant in vitro expression; B—in vitro expression after adding the EGE sequence; M—prestained protein marker; negative—pcDNA3.1-empty; 1—pcDNA3.1-tPA-gE; 2—pcDNA3.1-tPA-gE-dM; 3—pcDNA3.1-tPA-gE-ΔET; 4—pcDNA3.1-tPA-gE-mET; 5—pcDNA3.1-tPA-gE-ΔTGN; 6—pcDNA3.1-tPA-gE-TGN; 7—pcDNA3.1-tPA-gE-mTG N; 8—pcDNA3.1-tPA-gE-△CT; 9—pcDNA3.1-tPA-gE-EGE; 10—pcDNA3.1-tPA-gE-dM-EGE; 11—pcDNA3.1-tPA-gE-△ET-EGE; 12—pcDNA3.1-tPA-gE- mET-EGE; 13—pcDNA3.1-tPA-gE-ΔTGN-EGE; 14—pcDNA3.1-tPA-gE-TGN-EGE; 15—pcDNA3.1-tPA-gE-mTGN-EGE; 16—pcDNA3.1-tPA-gE-ΔCT-EGE.

[0023] Figure 5 Expression verification of virus-like particle plasmid formation: M—pre-stained protein marker; 1—pcDNA3.1-empty; 2 / 7—pcDNA3.1-tPA-gE; 3 / 8—pcDNA3.1-tPA-gE-△TM; 4 / 9—pcDNA3.1-tPA-gE-△ET; 5 / 10—pcDNA3.1-tPA-gE-△ET-GE; 6 / 11—pcDNA3.1-tPA-gE-△ET-EGE; 1~6 are protein samples collected 24h after transfection; 7~11 are protein samples collected 48h after transfection.

[0024] Figure 6 Immunoelectron microscopy structural characterization of gE-eVLP: A—control group (pcDNA3.1-tPA-gE-△ET transfection group, 6000×); BC—immunoelectron microscopy analysis of gE-eVLP (pcDNA3.1-tPA-gE-△ET-EGE transfection group, 200000×).

[0025] Figure 7 A schematic diagram of constructing a plasmid expression cassette for an mRNA vaccine.

[0026] Figure 8 This is a schematic diagram of the expression cassette of a non-replicating recombinant adenovirus.

[0027] Figure 9 The images show the results of multiple enzyme digestion identification of recombinant adenovirus plasmids. The left image of each image is a simulation of multiple enzyme digestion of the recombinant adenovirus plasmid, and the right image is a comparison of the agarose gel electrophoresis image.

[0028] Figure 10 Observation of pathological features of HEK293A cells infected with recombinant adenovirus (10×).

[0029] Figure 11 Purification and morphological identification of recombinant adenovirus.

[0030] Figure 12 Recombinant adenovirus genome enzyme digestion identification analysis, with the left image of each figure being a simulation of multiple enzyme digestion of the recombinant adenovirus genome and the right image being a comparison of the agarose gel electrophoresis image.

[0031] Figure 13 M-prestained protein marker was used to detect the infectious expression of recombinant adenovirus in Western blot.

[0032] Figure 14 For PCR amplification and linearization verification of the target gene fragment, A—Agarose gel electrophoresis identification of the target fragment; B—Verification of plasmid linearization treatment; M—DNA gradient marker; 1~2—5U1-tPA-gE; 3~4—ET; 5—pcDNA 3.1-5U1-tPA-gE-3U1-polyA; 6— BsaI -pcDNA3.1-5U1-tPA-gE-3U1-polyA; 7—pcDNA3.1-tPA-gE-eVLP-3U1-polyA; 8— BsaI -pcDNA3.1-tPA-gE-eVLP-3U1-polyA.

[0033] Figure 15 M—prestained protein marker—was used to validate Cap-mRNA expression using Western blot.

[0034] Figure 16 The following data represent the immune responses in mice 2–6 weeks after primiparity with recombinant adenovirus vector vaccines: serum gE-specific IgG antibodies in mice immunized with A-Ad26 vector vaccine 2–6 weeks after primiparity; serum gE-specific IgG antibodies in mice immunized with B-AdC68 vector vaccine 2–6 weeks after primiparity; serum antibody subtype ratios in mice in the high-dose groups of C-Ad26 and AdC68 recombinant adenoviruses; and the number of spleen lymphocytes secreting specific IFN-γ in the high-dose groups of D-Ad26 and AdC68 recombinant adenoviruses. P < 0.05 P < 0.01 P < 0.001 P < 0.0001.

[0035] Figures 17-20 The levels of specific binding antibodies in the serum of mice in the mRNA vaccine group at weeks 2-6. Figure 17 —IgG; Figure 18 —IgG1; Figure 19 —IgG2a; Figure 20 —Ratio of antibody subtypes IgG2a to IgG1; P < 0.05 P < 0.01 P < 0.001 P < 0.0001.

[0036] Figure 21 The ability of spleen lymphocytes in mice 6 weeks after primary immunization with mRNA vaccine to secrete cytokines: A—number of specific spleen lymphocytes secreting IFN-γ; B—number of specific spleen lymphocytes secreting IL-4. P < 0.05 P < 0.01 P < 0.001 P < 0.0001.

[0037] Figures 22-26 The immunization schedule for mice and the level of specific binding antibodies in mouse serum. Figure 22 —Schematic diagram of mouse immunization program; Figure 23 —IgG; Figure 24 —IgG2a; Figure 25 —IgG1; Figure 26 —Ratio of IgG2a to IgG; P < 0.05 P < 0.01 P < 0.001 P < 0.0001.

[0038] Figures 27-29 To detect the ability of mouse splenic lymphocytes to secrete cytokines using ELISpot, Figure 27 —The number of specific splenic lymphocytes that secrete IFN-γ; Figure 28 —The number of specific splenic lymphocytes that secrete IL-2; Figure 29 —Number of IL-4-specific splenic lymphocytes; ns: P>0.05; P < 0.05 P < 0.01 P < 0.001 P < 0.0001. Detailed Implementation

[0039] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1

[0041] 1. Modification of the gE intracellular region and efficient formation of self-assembled enveloped virus-like particles

[0042] 1.1 Experimental Materials

[0043] 1.1.1 Cells and Plasmids

[0044] The amino acid sequence of gE from the VZV oka strain was derived from Uiprot (Q9J3M8), with humanized codons and the removal of the PmeI restriction site. The pcDNA3.1-gE gene sequence carrying the target gene was synthesized by GenScript Biotech Ltd. pcDNA3.1-empty, tPA signal peptide sequences, pkAd5-S-EGE, and HEK293A cell lines were provided by the Diarrhea Laboratory of the Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention. Stbl2 competent cells were purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd., and DH5α chemocompetent cells were purchased from Shanghai Yisheng Biotechnology Co., Ltd.

[0045] 1.1.2 Main Reagents and Consumables

[0046] Table 1.1 Main Reagents and Consumables

[0047] 1.1.3 Main Instruments

[0048] Table 1.2 Main Instruments

[0049] 1.1.4 Preparation of Commonly Used Reagents

[0050] 1) LB liquid culture medium: Weigh 3g Tryptone, 3g NaCl, and 1.5g Yeast Extract, add distilled water to a final volume of 300mL, autoclave, and store at 4℃ for later use. 2) LB solid medium: Weigh 4.5g agar powder, 3g Tryptone, 3g NaCl, 1.5g Yeast Extract, add distilled water to a final volume of 300mL, and autoclave. When the temperature drops to about 50℃, add 100mg / mL ampicillin or 50mg / mL kanamycin stock solution at a ratio of 1:1000, pour into a petri dish and spread in a single layer. Allow it to solidify and store at 4℃ for later use. 3) 50% sterile glycerol: Weigh 50g of glycerol, add 50mL of ddH2O, autoclave, and store at 4℃; 4) Complete culture medium for HEK293A cells: Add 50 mL of FBS to 450 mL of DMEM medium; 5) Cell cryopreservation solution: Dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), and DMEM are prepared in a ratio of 1:1:8 and should be used immediately after preparation. 6) Complete cell lysis buffer: Add 1 μL of 100×PMSF protein inhibitor to every 99 μL of RIPA lysis buffer; 7) Transfer buffer: Mix 100 mL of 10× rapid transfer buffer, 200 mL of anhydrous ethanol and 700 mL of ddH2O thoroughly; 8) Electrophoresis buffer: Dissolve one packet of Precast Running Buffer in 2L of ddH2O; 9) 20% sucrose: Weigh 20g of sucrose, add 100mL of PBS, autoclave, and store at 4℃ for later use.

[0051] 1.2 Experimental Methods

[0052] 1.2.1 Construction of pcDNA3.1-tPA-gE recombinant plasmid

[0053] Based on the pcDNA3.1-tPA-gE gene map, upstream and downstream primers with homologous arms were designed using SnapGene software. The primer sequences are shown in Table 1.3. Using pcDNA3.1-gE and tPA as templates, the gE-ΔSP (without the original gE signal peptide sequence SP (1-30aa: MGTVNKPVVGVLMGFGIITGTLRITNPVRA)) and tPA fragment with homologous arms were amplified by polymerase chain reaction. The reaction system and conditions are shown in Tables 1.4 and 1.5.

[0054] The nucleotide sequence of gE-△SP (SEQ ID NO: 1, 1782bp):

[0055] The amino acid sequence of gE-△SP (SEQ ID NO: 2): .

[0056] tPA nucleotide sequence (SEQ ID NO: 3, 66bp): ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCC; tPA amino acid sequence (SEQ ID NO: 4): MDAMKRGLCCVLLLCGAVFVSP.

[0057] Table 1.3 PCR Amplification Primer Information Table

[0058] Table 1.4 PCR amplification reaction system for target fragments

[0059] Table 1.5 PCR amplification reaction procedure

[0060] Note: Steps 2 to 4 need to be repeated 30 times. The value of Tm is the melting temperature.

[0061] PCR products were identified by 1% agarose gel electrophoresis. The target amplified fragment with the correct band size and specificity was excised and recovered into EP tubes. The target fragment was then purified using a purification gel. The specific steps are as follows: 1) Add 400 μL of XP2 buffer to the gel block and melt the gel block completely in a metal bath at 55°C; 2) Transfer the melted gel solution to the purification column and centrifuge at 10,000 rpm for 1 min; 3) Discard the filtrate, recover the purification column, and add 500 μL of XP2 to the purification column; 4) Centrifugation: 13000 rpm, 1 min; 5) Discard the filtrate, recover the purification column, and add 700 μL of SPW to the purification column; 6) Centrifugation: 13000 rpm, 1 min; 7) Discard the filtrate, recover the purification column, and centrifuge the empty column at 13000 rpm for 2 min; 8) Transfer the filter column to a new EP tube, add ddH2O preheated at 65℃, and let it stand at room temperature for 2 minutes; 9) Centrifuge to elute DNA: 13000 rpm, 1 min. After mixing the elution buffer, measure the concentration using Nanodrop.

[0062] The pcDNA3.1-empty vector was double-digested with ApaI and KpnI. The digestion system and procedure are shown in Table 1.6.

[0063] Table 1.6 pcDNA 3.1-empty double digestion system

[0064] Reaction program: 37℃, 2h; 65℃, 20min; 4℃, ∞.

[0065] Homologous recombination of the target fragment and the digested vector was performed according to the ClonExpress® Ultra One Step Cloning Kit manual. The homologous recombination system and reaction procedure are shown in Table 1.7. After the homologous recombination procedure, the homologous recombination product was added to DH5α competent cells. After incubating on ice for 5 min, 20 μL was spread onto a preheated 37°C solid LB plate containing ampicillin and incubated upside down at 37°C for 14 h. The next day, a single colony was picked and placed in liquid LB medium containing ampicillin and shaken overnight at 37°C and 220 rpm for 12 h. The bacterial culture was sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were confirmed to be correct and stored at -80°C with sterile glycerol to a final concentration of 25%.

[0066] Table 1.7 Homologous recombination reaction system of pcDNA3.1-tPA-gE

[0067] Reaction program: 37℃, 15 min; 4℃, ∞.

[0068] The bacterial culture was added to 300 mL of ampicillin-resistant liquid LB medium at a 1:1000 inoculation ratio and incubated overnight at 37°C and 220 rpm for 14 hours. Large-scale extraction of the plasmid (pcDNA3.1-tPA-gE recombinant plasmid) was then performed using the EndoFree Plasmid Maxi Kit (QIAGEN, 12362). The specific operating steps are as follows: 1) Centrifugation of bacterial culture: 8000 rpm, 10 min; 2) Discard the supernatant, add 10 mL of P1 solution to resuspend the bacterial precipitate, and transfer it to a 50 mL centrifuge tube; 3) Add 10 mL of P2 solution, gently invert and mix 5 times, then let stand at room temperature for 5 minutes; 4) Add 10 mL of P3 solution, immediately invert and mix 5 times, the solution will turn from blue to white; 5) Centrifugation: 4000 rpm for 10 min; 6) Place the QIA filter cartridges onto 50mL centrifuge tubes; 7) Pour the centrifuged liquid directly into the QIAfilter Cartridges filter column (do not pour in the flocculent material), and incubate at room temperature for 10 minutes; 8) Insert the QIAfilter Plungers and use them to filter the liquid into 50mL centrifuge tubes; 9) Add 2.5 mL of ER reagent to the centrifuge tube, mix well, and incubate in an ice blender for 30 min; 10) Take out QIAGEN-tip 500, add 10mL QBT, and wait for it to filter out completely to activate the filter column; 11) Add the sample solution from step 9) to a QIAGEN-tip 500 filter column and filter until complete; 12) Add 30mL of QC, filter until completely filtered, then add another 30mL of QC and wait for it to filter completely. 13) Place the filter column into a high-speed centrifuge tube, add 15 mL of QN to the filter column to filter out the DNA; 14) Add 10.5 mL of isopropanol to the high-speed centrifuge tube and mix well; 15) Centrifugation: 13000 rpm, 30 min, 4℃; 16) After centrifugation, discard the supernatant as soon as possible and add 5 mL of 70% ethanol solution; 17) Centrifugation: 13000 rpm, 10 min, 4℃; 18) Discard 4 mL of supernatant, resuspend the precipitate in the remaining liquid and transfer it to a sterile 1.5 mL EP tube; 19) Centrifugation: 13000 rpm, 5 min, 4℃; 20) Discard the supernatant, place the plasmid precipitate in a fume hood to dry until the precipitate changes from white to transparent; 21) Add 300 μL of TEB buffer and incubate at 4°C overnight to dissolve the plasmid precipitate. Measure the concentration with Nanodrop the next day.

[0069] 1.2.2 Modification of the intracellular region of gE

[0070] Modification diagram as follows Figure 1 As shown. By integrating mutation modification and domain truncation strategies, the focus is on engineering key domains that affect protein localization and endocytosis function: 1) Functional site mutation strategy: Based on the intracellular transport regulation mechanism of gE protein reported in the literature, site-directed mutations are implemented in three key functional regions: disrupting the TGN (trans-Golginetwork, TGN) targeting sequence localization (Y569A), eliminating the endocytosis signal motif (ET) (Y582G), and regulating phosphorylation modification clusters (S593A, S595A, T596A, and T598A). 2) Domain truncation strategy: intracellular region truncation (△CT), TGN targeting sequence truncation (△TGN), endocytosis signal motif (ET) truncation (△ET), dual truncation of transmembrane and intracellular regions (△TM), and retention of TGN but truncation of subsequent sequences (TGN).

[0071] Based on the above strategy, the following recombinant plasmids with mutant / truncated variants were constructed: 1) Functional mutant: pcDNA3.1-tPA-gE-dM (Y569A+SSTT mutation), 2) Internalization motif modified: pcDNA3.1-tPA-gE-△ET (truncated 582~623aa) / mET (truncated 589~623aa + Y582G mutation), 3) Positional regulation: pcDNA3.1-tPA-gE-△TGN (truncated 568~623aa) / TGN (truncated 572~623aa) / mTGN (truncated 572~623aa + Y569A mutation), 4) Transmembrane structure modified: pcDNA3.1-tPA-gE-△CT (truncated 559~623aa) / △TM (truncated 547~623aa). Furthermore, the eVLPs technology developed by the Bjorkman team was introduced, and the self-assembly element EGE (EPM-GSlinker-EABR) was fused to the C-terminus of the gE protein. Through the synergistic action of the ESCRT pathway, the self-assembly of nanoparticles was promoted, and a novel gE-eVLP vaccine candidate was constructed.

[0072] According to pcDNA3.1-tPA-gE-EGE, pcDNA3.1-tPA-gE-dM, pcDNA3.1-tPA-gE-dM-EGE, pcDNA3.1-tPA-gE-△ET, pcDNA3.1-tPA-gE-△ ET-GE, pcDNA3.1-tPA-gE-ΔET-EGE, pcDNA3.1-tPA-gE-mET, pcDNA3.1-tPA-gE-mET-EGE, pcDNA3.1-tPA-gE-TGN, pcDNA3. Gene maps of pcDNA3.1-tPA-gE-TGN-EGE, pcDNA3.1-tPA-gE-mTGN, pcDNA3.1-tPA-gE-mTGN-EGE, pcDNA3.1-tPA-gE-△TGN, pcDNA3.1-tPA-gE-△TGN-EGE, pcDNA3.1-tPA-gE-△CT, pcDNA3.1-tPA-gE-△CT-EGE, and pcDNA3.1-tPA-gE-△TM were constructed, and upstream and downstream primers with homologous arms were designed using Snapgene software. Using pcDNA3.1-tPA-gE and pkAd5-S-EGE as templates, the target fragment with homologous arms was amplified by polymerase chain reaction. The experimental steps for constructing recombinant plasmids were the same as in "1.2.1 Construction of pcDNA3.1-tPA-gE recombinant plasmid".

[0073] The amino acid sequence of the base sequence gE-△SP described above is shown in SEQ ID NO: 2.

[0074] gE-ΔTGN: Amino acid sequence after truncation of the TGN targeting sequence (SEQ ID NO: 9): SVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVK。

[0075] gE-TGN: Amino acid sequence that retains TGN but truncates the subsequent sequence (SEQ ID NO: 10): .

[0076] gE-mTGN: The Y569A mutation that disrupts the TGN targeting sequence localization is a Y569A mutation based on the sequence shown in SEQ ID NO: 10 (SEQ ID NO: 11, the underlined site is the mutation site Y569A): SVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKA A RV.

[0077] gE-ΔET: Amino acid sequence with the endocytosis signal motif ET truncated (SEQ ID NO: 12): .

[0078] gE-mET: The amino acid sequence after the Y582G mutation with the endocytosis signal motif removed (SEQ ID NO: 13, underlined site is the mutation site Y582G): SVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQ GQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRG SDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCL GISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAYRVDKSPYNQSMY G AGLPVD.

[0079] gE-dM: The amino acid sequence regulating the SSTT+Y569A mutation of the phosphorylation modification cluster (SEQ ID NO: 14, underlined sites are mutation sites Y569A, S593A, S595A, T596A and T598A): SVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKA A RVDKSPYNQSMYYAGLPVDDFED A E AA D A EEEFGNAIGGSHGGSSYTVYIDKTR。

[0080] gE-△CT: Amino acid sequence after intracellular region deletion (SEQ ID NO: 15): SVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLI。

[0081] gE-ΔTM: Amino acid sequence with transmembrane region and intracellular region doubly truncated (SEQ ID NO: 1:6): SVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLA。

[0082] EPM nucleotide sequence (SEQ ID NO: 17): gctctgcctggcaatcctgatcaccgggaaatgggcgagacactgcccgaggaagtgggcgagtacagacagcctagcggcggcagcgtgcccgtgtcccctggccctccatctggactcgagcctaccagcagcagcccctac; EPM amino acid sequence (SEQ ID NO: 18): ALPGNPDHREMGETLPEEVGEYRQPSGGSVPVSPGPPSGLEPTSSSPY; EABR nucleotide sequence (SEQ ID NO: 19): ttcaacagctctatcaacaacatccacgagatggaaatccagctgaaggacgccctggaaaagaaccagcagtggctggtctacgaccagcaaagagaggtgtatgtgaagggcctgctggccaagatcttcgagctggaaaaaaagaccgagaccgccgcc; EABR amino acid sequence (SEQ ID NO: 20): FNSSINNIHEMEIQLKDALEKNQQWLVYDQQREVYVKGLLAKIFELEKKTETAA; GS linker nucleotide sequence (SEQ ID NO: 21): ggcggaggcggaagc; GS linker amino acid sequence (SEQ ID NO: 22): GGGGS.

[0083] Therefore, the nucleotide sequence of the GE motif is (SEQ ID NO: 23): ggcggaggcggaagcttcaacagctctatcaacaacatccacgagatggaaatccagctgaaggacgccctggaaaagaaccagcagtggctggctacgaccagcaaagagaggtgtatgtgaagggcctgctggccaagatcttcgagctggaaaaaaagaccgagaccgccgcc; The amino acid sequence of the GE motif is (SEQ ID NO: 24): GGGGSFNSSINNIHEMEIQLKDALEKNQQWLVYDQQREVYVKGLLAKIFELEKKTETAA; The nucleotide sequence of the EGE motif is (SEQ ID NO: 25): gctctgcctggcaatcctgatcaccgggaaatgggcgagacactgcccgaggaagtgggcgagtacagacagcctagcggcggcagcgtgcccgtgtcccctggccctccatctggactcgagcctaccagcagcagcccctacggcggaggcggaagctt caacagctctatcaacaacatccacgagatggaaatccagctgaaggacgccctggaaaagaaccagcagtggctggtctacgaccagcaaagagaggtgtatgtgaagggcctgctggccaagatcttcgagctggaaaaaaagaccgagaccgccgcc; The amino acid sequence is (SEQ ID NO: 26): ALPGNPDHREMGETLPEEVGEYRQPSGGSVPVSPGPPSGLEPTSSSPYGGGGSFNSSINNIHEMEIQLKDALEKNQQWLVYDQQREVYVKGLLAKIFELEKKTETAA.

[0084] 1.2.3 pcDNA 3.1 Transfection and Protein Extraction of Recombinant Plasmids

[0085] Collect the supernatant secretory proteins. Before transfection, replace the culture medium in each well with 1 mL of serum-free DMEM. Transfect pcDNA3.1 and the recombinant plasmid mentioned above into 6-well HEK293A cells at a cell density of 80-90% using Lipo3000 transfection reagent. Incubate at 37℃ in a 5% CO2 cell culture incubator for 24 h and 48 h, then extract protein samples. Specific operating steps are as follows: 1) The premixed solution of the fully lysed solution is now prepared on ice; 2) Collect cell culture medium, centrifuge at 800 rpm for 4 min, discard cell debris and other precipitates, and collect the supernatant. This is the cell culture supernatant sample. 3) Wash HEK293A cells with pre-cooled PBS, discard the PBS, and place the cells on ice; 4) Add 200 μL of complete lysis buffer to each well and lyse on ice for 30 min; 5) Transfer the lysed sample to an EP tube; 6) Sample centrifugation: 10000 rpm, 10 min; 7) Add 5× protein loading buffer to the supernatant, mix well, and place in a metal bath at 100°C for 10 min for denaturation. 8) Store the samples at -80℃ and avoid repeated freeze-thaw cycles.

[0086] 1.2.4 Western blot identification

[0087] Western blot validation was performed using Yeasen 10% SDS-PAGE gel. The specific steps are as follows: 1) Remove the pre-cast gel, fix it to the electrophoresis tank, add the electrophoresis solution, and then add the protein sample into the sample well; 2) Set constant voltage electrophoresis: 150V, 38min; 3) After electrophoresis, place the electrophoresis gel in the following order: negative electrode transfer plate - absorbent paper - electrophoresis gel - PVDF membrane - absorbent paper - positive electrode transfer plate; 4) Set constant current transfer: 0.45mA, 28min; 5) After the transfer is complete, cover the PVDF membrane with 5% skim milk and seal it on a shaker at low speed at room temperature for 2 hours. 6) Washing the membrane: Wash the membrane 6 times with PBST, shaking on a shaker at high speed for 3 minutes each time; 7) Primary antibody incubation: Dilute anti-GAPDH antibody (1:5000) and anti-VZV gE antibody (1:8000) with 2% BSA+PBS solution and incubate on a shaker at room temperature for 2 hours; 8) Repeat the membrane washing step; 9) Secondary antibody incubation: Dilute HRP-labeled goat anti-mouse IgG with 2% skim milk (1:5000) and incubate at room temperature for 1 hour; 10) Repeat the membrane washing step; 11) Development: Add ECL developer to cover the film and place it in a Tanon 4800 imager for exposure and development.

[0088] 1.2.5 Purification and immunoelectron microscopy of eVLP samples

[0089] The enrichment and purification of eVLP samples followed the protocol of Professor Bjorkman's team, and the specific steps are as follows: 1) pcDNA3.1-tPA-gE-△ET-EGE was transfected into HEK293A, and the cell culture supernatant was collected after 48 hours; 2) Centrifugation: 800 rpm, 10 min, 4℃; 3) Collect the supernatant, discard the cell debris precipitate, and filter the supernatant through a 0.45μm filter membrane; 4) Add 2 mL of 20% sterile sucrose solution to the lower layer of the ultracentrifuge tube, and add the supernatant from step 3) to the upper layer to maintain the liquid surface separation; 5) Vacuum ultracentrifuge centrifugation: 50,000 rpm, 2 hours, 4°C, with speed increase set to 3 and speed decrease set to 4; 6) After centrifugation, discard the liquid, resuspend the precipitate in 100 μL of sterile PBS, and incubate overnight at 4°C to obtain the purified eVLP sample. 7) Add an equal volume of 4% paraformaldehyde to the eVLP sample for fixation for at least 4 hours; 8) Drip 30 μL of liquid onto the wax film and perform negative staining with a nickel mesh with carbon film for 30 min; 9) Absorb excess liquid with filter paper, wash three times with 0.05M PBS, 1 min each time; 10) Block with 0.05M glycine for 10 min, block with 1% BSA for 1 h, add anti-VZV gE antibody (1:100), and incubate overnight at 4°C; 11) The nickel mesh was washed 6 times with PBS for 1 min each time; blocked with 1% BSA for 10 min; and incubated with secondary antibody (1:100, 10nm colloidal gold) for 3 h. 12) Wash with PBS 6 times, 1 min each time; fix with 2.5% glutaraldehyde for 15 min, and wash with ddH2O 4 times; 13) After staining with 2% uranium acetate for 5 minutes, wash with ddH2O 4 times, air dry naturally, and observe and photograph with JEOL-1200.

[0090] 1.2.6 Statistical Methods

[0091] Data processing, analysis, and plotting were performed using GraphpadPrism 9.5. One-way ANOVA was used to analyze the significance of differences between experimental groups. P < 0.05 indicates statistical significance, and ns indicates P > 0.05. This indicates that P < 0.05; This indicates that P < 0.01; This indicates that P < 0.001; This means P < 0.0001.

[0092] 1.3 Experimental Results

[0093] 1.3.1 Obtaining recombinant plasmids that form gE-eVLP

[0094] The agarose gel electrophoresis results of the target bands gE-ΔSP (1782bp, as shown in SEQ ID NO: 1), tPA (103bp, carrying a homologous arm compared to the tPA sequence described in SEQ ID NO: 3), and pcDNA3.1-empty after KpnI digestion are as follows: Figure 2As shown, the electrophoretic bands are consistent with the target gene. After inserting the target fragment into pcDNA3.1-empty, the recombinant plasmid pcDNA3.1-tPA-gE was obtained. Figure 2 M—DNA relative molecular mass standard; 1—gE-ΔSP (size 1782bp); 2—tPA (size 103bp); 3—pcDNA3.1-empty; 4—pcDNA3.1-empty KpnI .

[0095] 1.3.2 Replacement of the tPA signal peptide increased gE expression.

[0096] To evaluate the effect of the signal peptide on gE protein expression efficiency, recombinant plasmids pcDNA3.1-gE (containing the natural signal peptide SP) and pcDNA3.1-tPA-gE (with the signal peptide tPA replacing the natural signal peptide SP) were transfected into HEK293A cells, with three replicate wells set up. Simultaneously, pcDNA3.1-empty was transfected as a negative control. Western blot validation was performed. Figure 3 A) The results showed that the negative control group had no corresponding band, while both experimental groups showed specific bands in the range of 72-100 kDa, indicating that the recombinant gE protein was successfully expressed and the molecular weight was consistent with the expected molecular weight of the gE protein after glycosylation modification. Gray values ​​were compared using ImagJ. Figure 3 (B) The results showed that the tPA signal peptide increased the expression level of gE protein by about 1 time compared with the wild-type signal peptide.

[0097] 1.3.3 Secretory expression of gE protein using recombinant plasmids carrying the EGE motif

[0098] To verify the expression of gE and its truncated and mutant forms, we analyzed the expression of different constructs using Western blot. Figure 4The results showed that: 1) the expression levels of pcDNA3.1-tPA-gE (1), pcDNA3.1-tPA-gE-EGE (9), pcDNA3.1-tPA-gE-dM (2) and pcDNA3.1-tPA-gE-dM-EGE (10) were similar. Among them, pcDNA3.1-tPA-gE (1) and pcDNA3.1-tPA-gE-dM (2) were not detected in the cell culture supernatant, while the expression levels of pcDNA3.1-tPA-gE-EGE (9) and pcDNA3.1-tPA-gE-dM-EGE (10) in the cell culture supernatant were low. 2) The expression of pcDNA3.1-tPA-gE-△ET(3), pcDNA3.1-tPA-gE-mET(4), pcDNA3.1-tPA-gE-△ET-EGE(11), and pcDNA3.1-tPA-gE-mET-EGE(12) is similar. pcDNA3.1-tPA-gE-△ET(3) and pcDNA3.1-tPA-gE-mET(4) are expressed in small amounts in the cell culture supernatant, while pcDNA3.1-tPA-gE-△ET-EGE(11) and pcDNA3.1-tPA-gE-mET-EGE(12) have the highest expression levels in the cell culture medium. 3) A comparison between pcDNA3.1-tPA-gE-TGN (6) and pcDNA3.1-tPA-gE-TGN-EGE (14), pcDNA3.1-tPA-gE-△TGN (5) and pcDNA3.1-tPA-gE-△TGN-EGE (13), pcDNA3.1-tPA-gE-mTGN (7) and pcDNA3.1-tPA-gE-mTGN-EGE (15) shows that pcDNA3.1-tPA-gE-△TGN (5) will form a small amount of secretion in the cell culture supernatant. After adding the EGE sequence, the secretion of pcDNA3.1-tPA-gE-TGN-EGE (14), pcDNA3.1-tPA-gE-△TGN-EGE (13), and pcDNA3.1-tPA-gE-mTGN-EGE (15) in the supernatant was not as high as that of pcDNA3.1-tPA-gE-△ET-EGE (11), and the expression level of the TGN mutant mTGN-EGE (15) in the cell culture supernatant was even lower. 4) pcDNA3.1-tPA-gE-△CT (8) will form secretory expression, which is almost completely expressed in the cell culture supernatant, but after adding the EGE sequence, pcDNA3.1-tPA-gE-△CT-EGE (16) shows non-secretory expression, which is mostly expressed in the cell lysate.The above results indicate that different modification strategies significantly affect the expression level of gE protein. In addition, the binding of gE genes with different modification strategies to the EGE motif also significantly affects the expression pattern of gE protein.

[0099] To further clarify the functions of the EPM and EABR motifs proposed by Professor Bjorkman's team, samples from the gE and gE-ΔET groups were collected at 24h and 48h time points and simultaneously transfected with the same antigen sequence pcDNA3.1-tPA-gE-ΔTM as the Shingrix marketed vaccine. Western blot was used to verify protein expression. The results showed that... Figure 5 As shown, pcDNA3.1-tPA-gE (2 / 7) and pcDNA3.1-tPA-gE-△ET (4 / 9) were not detected in the cell culture supernatant. pcDNA3.1-tPA-gE-△ET-GE (5 / 10) and pcDNA3.1-tPA-gE-△ET-EGE (6 / 11) with added EABE sequence were both detected in the supernatant. The band of pcDNA3.1-tPA-gE-△ET-EGE (6 / 11) with added EPM was higher than that of pcDNA3.1-tPA-gE-△ET-GE (5 / 10), and the protein expression level at 48 h was higher than that at 24 h. Furthermore, pcDNA3.1-tPA-gE-△ET (9) was expressed in small amounts in the supernatant at 48 h, which may be related to protein release caused by cell death and rupture. No protein was detected in the pcDNA3.1-tPA-gE-△TM (3 / 8) cell lysate; it was almost completely secreted into the cell culture supernatant. These results indicate that the EGE motif enables the secretion of gE protein as virus-like particles, signifying the successful preparation of the virus-like particle vaccine gE-eVLP.

[0100] 1.3.4 Electron microscopy confirmed the formation of gE-eVLP

[0101] pcDNA3.1-tPA-gE-△ET negative control ( Figure 6 A) Microscopic examination revealed a few vesicle-like structures with little or no colloidal gold labeling. The pcDNA3.1-tPA-gE-△ET-EGE experimental group ( Figure 6 B and Figure 6 C) Under a microscope, a substance with an exosome-like structure in the size of 50-100 nm was detected, and multiple gE-specifically labeled colloidal gold particles were distributed on the surface, indicating that the gE protein was highly exposed on the surface of the eVLP.

[0102] In summary, this invention replaces the original signal peptide of the gE protein with the tPA signal peptide and clones it into the eukaryotic expression vector pcDNA3.1-empty. Transfection of HEK293A cells and in vitro Western blot analysis showed that replacing the tPA signal peptide significantly increased the expression level of the gE protein, consistent with previous observations. This finding provides new experimental evidence for further research on the role of signal peptides in protein expression and immune responses.

[0103] This invention, through systematically optimizing the mutation and truncation strategy of the VZV gE protein, further confirms the effectiveness of target protein structural modification in improving eVLP yield. First, in the gE protein mutation and truncation strategy, we found that truncating the endocytosis signal sequence (ET) and linking it to the EGE sequence significantly enhanced the secretory expression of the gE protein. This may be because the removal of the ET region effectively prevents the endocytosis of the gE protein, prolonging its residence time on the cell membrane, thus providing more favorable conditions for ESCRT pathway-driven eVLP self-assembly. However, truncating the TGN target sequence did not significantly promote the formation of gE-eVLPs, and the Y569A mutation of TGN actually inhibited the self-assembly effect of EGE. This indicates that the TGN region may play a complex role in the intracellular transport and localization of the gE protein, and its specific mechanism still needs further investigation. Second, this study observed that removing the transcellular and intracellular regions (ΔTM) of the gE protein significantly enhanced its secretory expression, which is consistent with the modification in the HZ-marketed protein vaccine Shingrix, mainly used to improve protein purification. This phenomenon suggests that the intracellular region of the gE protein may play a dual role in its secretion and self-assembly: on the one hand, removal of the intracellular region may reduce the interaction between the gE protein and intracellular transport mechanisms, thereby promoting its secretion; on the other hand, the addition of the EGE sequence may alter the conformation of the gE protein or its interaction with other cellular components, thus affecting its secretory behavior. In summary, the results of this study indicate that truncation and mutation of the gE protein have a significant impact on its expression, secretion, and self-assembly behavior, providing important experimental evidence and theoretical reference for the development of gE-eVLP-based vaccines. This discovery also provides a new perspective for the study of the functional domains of the gE protein, and future structural biology techniques can be used to further elucidate the structure-activity relationship of the gE protein. Furthermore, immunoelectron microscopy results further confirmed the formation of the gE-eVLP.

[0104] In summary, this embodiment successfully constructed a gE-eVLP target antigen sequence with potential vaccine application value by systematically optimizing the expression strategy of the gE protein and combining it with eVLP self-assembly particle technology. Next, the immunogenicity and protective efficacy of gE-eVLP can be explored, and its safety and efficacy in animal models can be evaluated, providing scientific evidence and laying the experimental foundation for further clinical development and application. Simultaneously, the mechanism of action of the gE protein functional domain revealed in this study also provides new insights for the research of related viral proteins. The research results not only provide a novel technical pathway for the development of next-generation shingles vaccines but also offer important references and guidance for the design and optimization of other virus-like particle vaccines.

[0105] Example 2

[0106] 2. Preparation of VZV recombinant adenovirus vector vaccine and mRNA vaccine

[0107] 2.1 Experimental Materials

[0108] 2.1.1 Cells and Plasmids

[0109] The gene sequences of 5U1-tPA-gE-3U1 and 5U1-tPA-gE-△ET-EGE-3U1 carrying the target gene were optimized using the LinearDesign platform developed by Beijing Baidu Netcom Technology Co., Ltd. The nucleotide sequences of tPA-gE and tPA-gE-△ET-EGE after codon optimization are shown in SEQ ID NO: 28 and SEQ ID NO: 31, respectively. The plasmids pcDNA3.1-5U1-ET (gE 582~623aa) and pcDNA3.1-5U1-tPA-gE-△ET-EGE were synthesized by GenScript Biotech Co., Ltd. The pcDNA3.1-T7-Nluc-3U1, the non-replicating adenovirus vector backbones pkAd26-empty, pkAdC68-empty, and the plasmids pkAd26-eGFP and pkAdC68-eGFP were provided by the Diarrhea Laboratory of the Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention. Other plasmids and cell lines are the same as in "1.1.1 Cells and Plasmids".

[0110] The amino acid sequence of tPA-gE (SEQ ID NO: 27): MDAMKRGLCCVLLLCGAVFVSPSVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAYRVDKSPYNQSMYYAGLPVDDFEDSESTDTEEEFGNAIGGSHGGSSYTVYIDKTR。

[0111] The codon-optimized tPA-gE nucleotide sequence (SEQ ID NO: 28, the underlined sequence is the codon-optimized tPA sequence): ATGGACGCAATGAAACGTGGTTTATGCTGTGTACTGTTACTGTGCGGAGCGGTATTTGTGAGCCCC

[0112] Amino acid sequence of tPA-gE-△ET-EGE (SEQ ID NO: 29): MDAMKRGLCCVLLLCGAVFVSPSVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAYRVDKSPYNQSMYALPGNPDHREMGETLPEEVGEYRQPSGGSVPVSPGPPSGLEPTSSSPYGGGGSFNSSINNIHEMEIQLKDALEKNQQWLVYDQQREVYVKGLLAKIFELEKKTETAA。

[0113] Nucleotide sequence of tAP-gE-△ET-EGE (SEQ ID NO: 30, capital letters are tPA sequence, underlined are EGE sequence): gctctgcctggcaatcctgat caccgggaaatgggcgagacactgcccgaggaagtgggcgagtacagacagcctagcggcggcagcgtgcccgtgt cccctggccctccatctggactcgagcctaccagcagcagcccctacggcggaggcggaagcttcaacagctctat caacaacatccacgagatggaaatccagctgaaggacgccctggaaaagaaccagcagtggctggtctacgaccag caaagagaggtgtatgtgaagggcctgctggccaagatcttcgagctggaaaaaaagaccgagaccgccgcc .

[0114] The nucleotide sequence of codon-optimized tAP-gE-△ET-EGE (SEQ ID NO: 31, underlined sequences are the codon-optimized tPA and EGE sequences): ATGGACGCAATGAAACGTGGTTTATGCTGTGTACTGTTACTGTGCGGAGCGGTATTTGTGAGCCCC GCTCTGCCCGGGAACCCCGAC CACAGGGAGATGGGGGAGACTCTGCCTGAGGAGGTGGGGGAGTACAGGCAGCCCAGCGGCGGGGTCCGTGCCCGTGA GCCCCGGCCCTCCCAGCGGGCTGGAGCCCACCAGCTCCAGCCCCTATGGGTGGGGGCGGGAGCTTCAACAGCTCCAT CAACAACATCCACGAGATGGAGATCCAGCTGAAGGACGCCCTGGAGAAGAACCAGCAGTGGCTGGTGTACGACCAG CAGCGGGAGGTGTACGTGAAGGGTCTGCTGGCCAAGATCTTCGAGCTGGAGAAGAAGACCGAAACCGCAGCC .

[0115] 2.1.2 Main Reagents

[0116] Table 2.1 Main Reagents

[0117] The other reagents are the same as those in "1.1.2 Main Reagents".

[0118] 2.1.3 Main Instruments

[0119] Same as "1.1.3 Main Instruments".

[0120] 2.1.4 Preparation of main reagents

[0121] 1) 1.2 mg / mL CsCl: Weigh 53 g CsCl, add 87 mL Tris-HCl (10 mM, pH 7.8), autoclave, and store at 4 °C; 2) 1.4 mg / mL CsCl: Weigh 26.8 g CsCl, add 92 mL Tris-HCl (10 mM, pH 7.8), autoclave, and store at 4 °C; 3) Desalting gel: Under aseptic conditions, add 7.5 mL of Bio-Gel P-6DG Media to a 50 mL centrifuge tube, then add PBS solution to 50 mL, invert and mix well, and store at 4 °C for later use.

[0122] 2.2 Experimental Methods

[0123] 2.2.1 Preparation of Ad26 and AdC68 recombinant adenovirus vector vaccine

[0124] 2.2.1.1 Construction and Identification of Recombinant Adenovirus Plasmids

[0125] Based on the gene sequences pkAd26-tPA-gE, pkAd26-tPA-gE-eVLP, pkAd26-eGFP, pkAdC68-tPA-gE, pkAdC68-tPA-gE-eVLP, and pkAdC68-eGFP (where gE-eVLP is an abbreviation of gE-△ET-EGE), upstream and downstream primers with homologous arms were designed using Snapgene software. Using pcDNA3.1-tPA-gE and pcDNA3.1-tPA-gE-eVLP as templates, the target fragments with homologous arms (H-tPA-gE-h and H-tPA-gE-△ET-EGE-H) were amplified by polymerase chain reaction. The PCR amplification system and program were the same as those in Tables 1.4 and 1.5. The identification and purification of the target fragment by agarose gel electrophoresis are the same as in "1.2.1 Construction of pcDNA3.1-tPA-gE recombinant plasmid".

[0126] The pkAd26-empty and pkAdC68-empty plasmid vectors were linearized. The linearization system and procedure are shown in Table 2.2.

[0127] Table 2.2 Adenovirus double enzyme digestion reaction system and procedure

[0128] Reaction program: 37℃, 2h; 65℃, 20min; 4℃, ∞.

[0129] The homologous recombination system was the same as in "1.2.1 Construction of pcDNA3.1-tPA-gE recombinant plasmid". The homologous recombination products (pkAd26-tPA-gE, pkAd26-tPA-gE-eVLP, pkAdC68-tPA-gE, pkAdC68-tPA-gE-eVLP) were added to Stbl2 competent cells.

[0130] 2.2.1.2 Packaging and Amplification of Recombinant Adenovirus

[0131] 1) Cell preparation

[0132] HEK293A cells were passaged into six-well plates 24 hours before transfection with the recombinant adenovirus plasmid, and then cultured overnight at 37°C in a 5% CO2 cell culture incubator.

[0133] 2) Plasmid linearization

[0134] The recombinant adenovirus plasmid extracted using the endotoxin-free kit was linearized with PacI enzyme to remove the resistance gene sequence before use. The linearization system and procedure are shown in Table 2.3.

[0135] Table 2.3 PacI linearized recombinant adenovirus plasmid reaction system and procedure

[0136] Reaction program: 37℃, 2h; 65℃, 20min; 4℃, ∞.

[0137] 3) Transfection with recombinant adenovirus plasmids

[0138] The PacI linearized recombinant adenovirus system was added to 180 μL of serum-free medium, followed by 5 μL of X-tremeGENE HP DNA Transfection Reagent. The mixture was gently stirred and allowed to stand at room temperature for 20 min before being added to HEK293A cells at a cell density of 70%–80%. After 24 h, the medium was replaced with 2% FBS-DMEM maintenance medium, and the cells were continued to be cultured in a cell culture incubator.

[0139] 4) Harvesting of P0 generation virus

[0140] Add approximately 200 μL of 2% FBS-DMEM medium every 3 days. Once the cells have fully developed cytopathic effects, collect the cells and culture them in sterile centrifuge tubes. This is the P0 generation virus, which should be stored at -80°C.

[0141] 5) Virus amplification and culture

[0142] P0 to P1 generation amplification: The P0 generation virus seed was subjected to three freeze-thaw cycles at -80℃ / room temperature, vortexed for 1 min, and then inoculated into HEK293A cells (T75 culture flasks) at a density of 90%. The cells were cultured at 37℃ and 5% CO2 for 48 h until complete cytopathic effect was achieved. The cell suspension was then collected to obtain the P1 generation virus seed.

[0143] P1 to P2 generation expansion: After freeze-thaw shaking, the P1 generation virus seed was used to infect HEK293A cells (4 150mm culture dishes) with a 90% inoculation density. The cells were cultured for 48 hours until complete cytopathic effect was achieved, and the suspension was collected as the P2 generation virus seed.

[0144] Large-scale preparation of P2 to P3 generations: After freeze-thaw shaking, P2 generation virus was used to infect HEK293A cells (32 150mm culture dishes) with a 90% inoculation density. After complete cytopathic effect, the cells were collected. The supernatant was discarded by centrifugation at 1000×g for 10 min at 4℃. The pellet was resuspended in 4mL DMEM and subjected to freeze-thaw shaking three times to obtain P3 generation virus solution, which was stored at -80℃ for later use.

[0145] 2.2.1.3 Purification and electron microscopy detection of recombinant adenovirus

[0146] The virus solution was purified using CsCl density gradient ultracentrifugation, requiring aseptic technique throughout the process. The specific steps are as follows: 1) Centrifuge the P3 generation virus solution at 4°C for 10,000 rpm for 10 min, and store the virus supernatant on ice. 2) Pipette 2 mL of 1.2 mg / mL CsCl into the lower layer of an ultracentrifuge tube, then pipette 2 mL of 1.4 mg / mL cesium chloride into the upper layer, and then place the virus supernatant into the upper layer of the 1.4 mg / mL cesium chloride. Each liquid layer must be separated into layers. 3) Slowly place the ultracentrifuge tubes into the vacuum ultracentrifuge, centrifuge at 4°C for 25,000 rpm for 2.5 hours, and set the speed increase to 3 and the speed decrease to 4. 4) After centrifugation, use a 1mL syringe to extract the lower layer of “cloudy” virus particles and place them in an EP container on ice for later use; 5) Add 6 mL of desalting gel (4°C) to the empty column tube of the affinity chromatography column and wash 3 times with sterile PBS; 6) Add the virus particle solution to the chromatography column and collect the filtrate using an EP tube at the bottom, collecting 6 drops in one tube; 7) Continue adding 1 mL of sterile PBS to the chromatography column and wait for it to filter out completely; 8) Repeat step 7) 3-4 times, collecting the filtrate during the addition of PBS, and collecting 6 drops into one tube; 9) Measure the OD260 absorbance of each tube, collect those with OD260>3 into one tube, add sterile glycerol to a final concentration of 10%, and mix thoroughly. 10) Using PBS containing 10% glycerol as a control, measure OD260 again and calculate the viral concentration using the formula: OD260 × 1.1 × 10⁻⁶. 12 VP / mL; 11) Aliquot the virus solution into 100μL tubes, label them, and store them at -80℃ to avoid repeated freeze-thaw cycles; 12) The purified virus solution was negatively stained with phosphotungstic acid and its morphology was observed under a transmission electron microscope (FEI TECNAI 12).

[0147] 2.2.1.4 Identification of the titer of recombinant adenovirus

[0148] The hexagonal protein is an important component of the adenovirus capsid and is required for adenovirus replication. However, the expression of this protein depends on the E1 gene product. HEK293A cells are a trans-complementary cell type of the E1 gene, and infection of these cells with recombinant adenovirus can express the hexagonal protein. Therefore, the detection of the hexagonal protein can reflect the viral infectivity titer. The specific experimental steps are as follows: 1) Virus dilution: Dilute the virus to be tested 10 times with DMEM + 10% FBS to a final concentration of 10. -3 ~10 -8 Place on ice for later use; 2) Cell plating: Dilute HEK293A cells to 5 × 10⁻⁶. 5 Cells / mL: Seed HEK293A cells into 12-well plates, 1 mL per well, incubate at 37℃ with 5% CO2 for 20 min, then add 100 μL of virus dilution buffer. Perform one replicate for each dilution. 3) Continue culturing in a 37℃ 5% CO2 cell culture incubator for 48 hours; 4) Carefully discard the culture medium and allow the cells to dry at room temperature until they are visibly dry; 5) Add 1 mL of pre-cooled methanol solution to the wall and fix at -20℃ for 10 min; 6) Discard the methanol, wash three times with PBS + 1% BSA, 1 mL per well; 7) Primary antibody incubation: Dilute Mouse Anti-Hexon Antibody (1:1000) with PBS + 1% BSA, 0.5 mL per well, and incubate at 37℃ for 1 h; 8) Washing: Discard the primary antibody and wash three times with PBS + 1% BSA, 1 mL per well; 9) Secondary antibody incubation: Dilute HPR-labeled Anti-Mouse Antibody (1:1000) with PBS + 1% BSA, 0.5 mL per well, and incubate at 37℃ for 1 h; 10) DAB preparation: Dilute 10× DAB to 1× using 1× Stable Peroxidase Buffer and equilibrate to room temperature; 11) Washing: Discard the primary antibody and wash three times with PBS + 1% BSA, 1 mL per well; 12) Color development: Add 500 μL of DAB color development solution to each well and incubate at room temperature for 10 min; 13) Terminate color development: Discard DAB and add 1 mL PBS; 14) Counting: Select at least four fields of view, with 5 to 50 spots in each field of view; 15) Titer calculation: IFU / mL = .

[0149] 2.2.1.5 Identification of recombinant adenovirus genome

[0150] The genome of the purified adenovirus was extracted using a Qiagen kit and then sequenced and identified by multiple enzyme digestion. The specific steps are as follows: 1) Genome extraction Take 100 μL of purified adenovirus (5 × 10¹² VP / mL), add 140 μL of Buffer ATL, 30 μL of proteinase K, and 30 μL of proteinase E (1 mg / mL) sequentially, mix well, and digest at 55 °C for 3 hours. After digestion, add 300 μL of Buffer AL and incubate at 70 °C for 10 minutes. Then add 300 μL of anhydrous ethanol, mix well, transfer to an adsorption column, and centrifuge at 8000 rpm for 1 minute. Wash sequentially with 500 μL of Buffer AW1 and 500 μL of Buffer AW2, and finally elute with 60 μL of preheated ddH₂O (55 °C). Collect the genome by centrifugation at 12000 rpm for 2 minutes.

[0151] 2) PCR amplification and sequencing

[0152] Primers designed using Snapgene software (targeting upstream and downstream sequences of the target gene) were used for PCR amplification according to the procedure described in Section 1.2.1. The PCR products were directly sent to Qingke Biotechnology Co., Ltd. for sequencing analysis.

[0153] 3) Identification by multiple enzyme digestion

[0154] use Sma I, Nco I, Eco NI or Ale I, Xho I, Xcm The extracted genome was digested with restriction endonucleases of type I, and the digestion pattern was analyzed by 8% agarose gel electrophoresis.

[0155] 2.2.1.6 Identification of infectious expression of recombinant adenovirus

[0156] 1) Cell preparation

[0157] 24 hours before the experiment, HEK293A cells were seeded at an appropriate density in six-well plates and cultured overnight in a 37°C, 5% CO2 incubator to allow the cells to reach approximately 70-80% confluence at the time of infection.

[0158] 2) Viral infection

[0159] Take the purified recombinant adenovirus and administer it at 1×10⁻⁶ ppm. 8 VP, 1×10 9 VP and 1×10¹ 0 VP was added to six-well plates at three concentration gradients and cultured for 24 hours. Ad26-empty and AdC69-empty virus-infected cells were used as negative controls. All experiments were performed in triplicate to ensure the reliability of the results.

[0160] 3) Membrane protein extraction

[0161] The methods are described in “1.2.3 pcDNA 3.1 Transfection and Protein Extraction of Recombinant Plasmids” and “1.2.4 Western Blot Identification”.

[0162] 2.2.2 Preparation of mRNA vaccines

[0163] 2.2.2.1 Construction strategy of mRNA recombinant plasmid

[0164] The plasmid maps of pcDNA3.1-5U1-tPA-gE-3U1 and pcDNA3.1-5U1-tPA-gE-eVLP-3U1 were designed using SnapGene software, and upstream and downstream primers were designed (Table 2.4). After double digestion with KpnI and EcoRV, the target sequences (5U1-tPA-gE, 5U1-tPA-gE-△ET-EGE) were inserted into pcDNA3.1 T7-Nluc-3U1. Figure 7 As shown. The agarose gel electrophoresis identification and gel purification steps for the target fragment are the same as in "1.2.1 Construction of pcDNA3.1-tPA-gE recombinant plasmid". The inserted mRNA vaccine sequence is shown in SEQ ID NO: 32 and SEQ ID NO: 33.

[0165] gE-mRNA vaccine sequence (5U1-tPA-gE-3U1-poly(A), SEQ ID NO: 32, the first underlined segment is the 5U1 sequence, the bolded segment is the KOZAK sequence, the second underlined segment is the 3U1 sequence, the middle segment is the tPA-gE sequence, and the last segment is the poly(A) tail): GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGACCCCGGCGCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGT GATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCAC ACCCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGCTACCCCGAGTCTCCCCCGACCTCGGG TCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCC AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA.

[0166] gE-eVLP-mRNA vaccine sequence (5U1-tPA-gE-△ET-EGE-3U1-poly(A), SEQ ID NO: 33, the first underlined segment is the 5U1 sequence, the bolded segment is the KOZAK sequence, the second underlined segment is the 3U1 sequence, the middle segment is the tPA-gE-△ET-EGE sequence, and the last segment is the poly(A) tail): GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGACCCCGGCGCCCAAGCACGCAGCAATGCAGC TCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACT AAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGTACTGCATGCACGCAATGCTAGCTGC CCCTTTCCCGTCCTGGCTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCAC TCACCACCTCTGCTAGTTCCAGACACCTCC AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA.

[0167] Table 2.4 mRNA plasmid primer sequences

[0168] 2.2.2.2 Linearization of mRNA plasmids

[0169] The correctly identified plasmids (pcDNA3.1-5U1-tPA-gE-3U1 and pcDNA3.1-5U1-tPA-gE-eVLP-3U1) were linearized using BsaI enzyme, and the enzyme digestion system and procedure are shown in Table 2.5.

[0170] Table 2.5 Linearization system and reaction procedure of mRNA plasmid with BsaI enzyme

[0171] Reaction procedure: 37℃, 12~16h.

[0172] After the enzyme digestion was completed, the results were verified by agarose gel electrophoresis. The enzyme digestion system that was correctly identified was then purified using the EZNA Gel Extraction Kit. The specific experimental steps are as follows: 1) Add an equal volume of XP2, mix well, and then shake off the liquid from the cap; 2) Column chromatography: Add the sample to the filter column and centrifuge at 10000×g for 10 min; 3) Change the collection tube, add 700 μL SPW, centrifuge at 13000×g for 1 min, and discard the filtrate; 4) Add 700 μL of SPW and wash again, centrifuge at 13000×g for 1 min, and discard the filtrate; 5) Centrifugation of empty column tubes: 13000g, 2min; 6) Insert the filter column into the EP tube; 7) Elution: Add 20 μL of preheated DEPC water and let stand for 2 min. 8) Centrifuge: 13000×g, 1min, collect the purified sample; 9) Use NanoDrop to measure concentration and absorbance. An absorbance of 1.8 to 1.9 is considered the correct range.

[0173] 2.2.2.3 In vitro transcription, capping, and purification

[0174] Thaw 5× Transcription Buffer at room temperature, thoroughly mix NTPs and centrifuge. Store 5× Transcription Buffer at room temperature and keep NTPs on ice for later use. The order of preparation for the transcription system is as follows: add water, buffer and NTPs first, and add template and enzyme last (the order must be strictly followed). See Table 2.6 for the specific system.

[0175] Table 2.6 In vitro transcription system

[0176] Reaction procedure: Transcription at 37℃ for 4 hours.

[0177] After transcription, 2 μL of DNase I was added to the system, mixed well, and incubated at 37°C for 15 min to remove the template. Then, an appropriate amount of RNA was diluted to 67 μL with RNase-free water. The diluted RNA was heated at 65°C for 5 min, and immediately placed on ice for 5 min after the process. The mRNA capping system is shown in Table 2.7.

[0178] Table 2.7 mRNA Capping System

[0179] Reaction procedure: 42℃ for 60 min.

[0180] After capping, the mRNA was purified using 7.5M LiCl. The specific experimental steps are as follows: 1) Preparation of purification system: 100 μL reaction mixture + 500 μL RNase-free H2O + 300 μL 7.5M LiCl; 2) After mixing thoroughly, place at -20℃ for 30 minutes; 3) Centrifugation: Centrifuge at 13000 rpm, 4℃ for 15 min, and collect the precipitate; 4) Washing: Add 500 μL of pre-cooled 70% ethanol to wash the RNA precipitate; 5) Centrifugation: Centrifuge at maximum speed, 4℃ for 5 minutes, then discard the supernatant; 6) Repeat the washing step three times; 7) Allow the RNA to air dry until it changes from white to slightly transparent; 8) Dissolve the RNA sample in 40 μL of RNase-free H2O and measure the concentration: the absorbance should be between 1.8 and 1.9. 9) Samples were identified by 1% agarose gel electrophoresis: 120mV, 20min; 10) The prepared mRNA is aliquoted into EP tubes and stored at -80℃ to avoid repeated freeze-thaw cycles.

[0181] 2.3 Experimental Results

[0182] 2.3.1 Obtaining non-replicating recombinant adenovirus expressing gE and gE-eVLP

[0183] 2.3.1.1 Successful construction of a non-replicating recurrent adenovirus plasmid

[0184] Deleting the E3 region or part of the E1 region of Ad26 and AdC68, and replacing the E4 region with the Ad5 E4 region sequence, finally obtained non-replicating Ad26-empty and AdC68-empty vectors. Figure 8 After PCR, gel electrophoresis revealed that the target fragments H-tPA-gE-H, H-tPA-gE-eVLP-H, CMV-tPA-gE-polyA, and CMV-tPA-gE-eVLP-polyA were inserted into the E1 region of the non-replicating Ad26-empty and AdC68-empty regions via homologous recombination.

[0185] The target fragment and enzyme digestion vector were homologously recombinated into pkAd26-empty and pkAdC68-empty to obtain recombinant adenovirus plasmids pkAd26-tPA-gE, pkAd26-tPA-gE-eVLP, and pkAdC68-tPA-gE, pkAdC68-tPA-gE-eVLP. The recombinant adenovirus plasmids were then subjected to... Sma I, Nco I, Eco NI or Ale I, Xho I, Xcm After multiple digestion with enzyme I, identification was performed by 0.8% agarose gel electrophoresis. The agarose gel electrophoresis pattern of the recombinant adenovirus plasmid standard simulated using SnapGene was consistent with the actual agarose gel electrophoresis results. The expected band positions were correct, no non-specific bands appeared, and no fragment deletions were observed. Figure 9 This result confirms that all recombinant adenovirus plasmids were constructed correctly, and the vector backbone and insert fragments conformed to the expected design.

[0186] 2.3.1.2 Successfully rescued recombinant adenovirus

[0187] Recombinant adenovirus plasmids with correct enzyme digestion results were linearized with restriction endonuclease PacI and transfected into HEK293 cells for recombinant adenovirus packaging. Simultaneously, pkAd26-eGFP and pkAdC68-eGFP were transfected as controls to observe vector expression. The next day, green fluorescence was observed under a fluorescence inverted microscope, indicating correct transfection. Obvious plaques appeared 5-13 days after transfection with the recombinant adenovirus plasmids. Empty vectors Ad26-empty and AdC68-empty showed plaques earliest, with 3-4 plaques observable under the microscope around day 4-5. By day 7 post-transfection, 90% of the cells showed lesions under the microscope. Figure 10 The recombinant adenovirus was successfully rescued when 90% of the cells showed cytopathic effects after 9-12 days, according to Ad26-tPA-gE, Ad26-tPA-gE-eVLP, AdC68-tPA-gE, and AdC68-tPA-gE-eVLP.

[0188] 2.3.1.3 The recombinant adenovirus has an intact structure.

[0189] After CsCl gradient ultracentrifugation, P3 generation adenovirus showed two "cloudy" bands: the upper layer was incompletely packaged adenovirus, and the lower layer was complete packaged virus particles. Figure 11 (See the red arrow in the image). The lower layer of virus particles was extracted and filtered through a desalting gel to obtain a high-purity, low-toxicity recombinant adenovirus preparation. A transmission electron microscope image (negative staining) of the recombinant adenovirus shows typical icosahedral virus particles with a diameter of approximately 90-100 nm. Figure 11 The VP / IFU ratio ranged from 67 to 205 (Table 2.8).

[0190] Table 2.8 Infectivity titers of recombinant adenovirus

[0191] 2.3.1.4 The recombinant adenovirus genome is intact.

[0192] The genome of the recombinant adenovirus was extracted for identification by multiple enzyme digestion and sequencing of the target fragment. Comparison with a simulated standard multiple enzyme digestion pattern showed that the relative positions and sizes of the bands were consistent. Figure 12 The target fragment was correctly sequenced after PCR amplification. Despite five mutations and deletions in the recombinant adenovirus genome, the target fragment was successfully and accurately inserted into the adenovirus vector expression frame.

[0193] 2.3.1.5 Recombinant adenovirus successfully infected HEK293A cells to express gE.

[0194] Western blot was used to detect the expression of recombinant adenovirus vaccines Ad26-tPA-gE, Ad26-tPA-gE-eVLP, and AdC68-tPA-gE, AdC68-tPA-gE-eVLP in HEK293A cells 24 h after infection. (Set to 10) 10 VP, 10 9 VP and 10 8 VP infection rate, set to 10 10 VP's Ad26-empty and AdC68-empty were used as controls. Results showed that 10 8 VP-infected HEK293 cells showed relatively weak protein expression, 10 9 VP, 10 10 A strong target protein band was detected after VP recombinant adenovirus infected cells, proving that recombinant adenovirus can be expressed in mammalian cells. Figure 13 ).

[0195] 2.3.2 Successful preparation of mRNA vaccines based on gE and gE-eVLP

[0196] 2.3.2.1 Obtaining and linearizing the recombinant plasmid for the mRNA vaccine

[0197] The target gene obtained by PCR amplification was confirmed by agarose gel electrophoresis to be of the expected size, and the band was single. Figure 14 A). The target fragment was recovered using the EZNA Gel Extraction Kit, yielding concentrations of 114.4 ng / μL and 109.6 ng / μL, respectively. Homologous recombination of the target fragment with the pcDNA3.1-T7-KpnI-5U1-Nluc-EcoRV-3U1 restriction vector yielded recombinant plasmids pcDNA3.1-5U1-tPA-gE-3U1-polyA and pcDNA3.1-tPA-gE-eVLP-3U1-polyA, which were confirmed to be correct by sequencing. Figure 14 B). The linearized product of the recombinant plasmid was obtained by BsaI single enzyme digestion and compared with the undigested product. The size of the product was consistent with the expected fragment size. Figure 14 For PCR amplification and linearization verification of the target gene fragment, A—Agarose gel electrophoresis identification of the target fragment; B—Verification of plasmid linearization treatment; M—DNA gradient marker; 1~2—5U1-tPA-gE; 3~4—ET; 5—pcDNA 3.1-5U1-tPA-gE-3U1-polyA; 6— BsaI -pcDNA3.1-5U1-tPA-gE-3U1-polyA; 7—pcDNA3.1-tPA-gE-eVLP-3U1-polyA; 8—BsaI -pcDNA3.1-tPA-gE-eVLP-3U1-polyA.

[0198] 2.3.2.2 Normal in vitro expression of Cap-mRNA gE

[0199] Cap-mRNA obtained from in vitro transcription, capping with capping enzyme, and purification of 5U1-tPA-gE-3U1-polyA and 5U1-tPA-gE-eVLP-3U1-polyA was transfected into HEK-293A cells for expression verification. Figure 15 Cap-tPA-gE-eVLP was significantly secreted in the supernatant, but its expression was less pronounced compared to intracellular or membrane expression. Figure 15 (Expression in cell lysate), Cap-tPA-gE expression was higher.

[0200] 2.3.3.3 Physicochemical Properties of LNP-mRNA

[0201] Suzhou Nearshore Protein Technology Co., Ltd. was commissioned to use the Moderna encapsulation protocol, and SM102 liposomes were used for encapsulation to obtain LNP-5U1-tPA-gE-3U1 and LNP-5U1-tPA-gE-eVLP-3U1. The results were measured using a Brookhaven 90 Plus PALS high-sensitivity Zeta potential and particle size analyzer. The final results are shown in Table 2.9.

[0202] Table 2.9 Physicochemical properties of LNP-mRNA

[0203] 2.4 Discussion

[0204] In this embodiment, the E4 region of the selected Ad26 and AdC68 was replaced with the E4 region of Ad5. Furthermore, the E1 and E3 regions of Ad26 and AdC68 were deleted to obtain a non-replicating recombinant adenovirus as a vector. The antigen sequence (tPA-gE-△ET-EGE) designed in Example 1 was loaded into the E1 region of the non-replicating recombinant adenovirus for expression. Finally, recombinant adenoviruses Ad26-tPA-gE-eVLP and AdC68-tPA-gE-eVLP were successfully rescued in HEK293A cells, realizing the combination of eVLP self-assembly particle technology and adenovirus vector.

[0205] The VZV mRNA vaccine designed in this embodiment uses the LinearDesign algorithm and selects the mtRNR1+AES dual UTR (146+132nt) screened by our laboratory as the 3’UTR of this vaccine (i.e., 3U1), and Moderna designs the 5’UTR as the 5’UTR region of this vaccine (i.e., 5U1). Through biological expression and verification of various physical and chemical properties, it is proved that the vaccine can effectively express the target antigen gE in eukaryotic cells in vitro. Finally, an mRNA vaccine (LNP-tPA-gE-eVLP) based on gE-eVLP modified by EABR / EPM was successfully prepared.

[0206] Example 3

[0207] Study on the Immunogenicity of VZV Recombinant Adenovirus Vector Vaccine and mRNA Vaccine

[0208] 3.1 Experimental Materials

[0209] 3.1.1 Experimental Animals and Grouping

[0210] 6-8-week-old SPF-grade female C57BL / 6N mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and the animal license number was SCXK (Beijing) 2019-0010. All experimental mice were raised under a 12h light / 12h dark rhythm condition. The experimental protocol has passed the review of the Ethics Committee of the Chinese Center for Disease Control and Prevention (Ethics number: bdbs20240422034), and all experimental operations complied with the relevant regulations of laboratory animal ethics. The experimental mice were grouped as follows: Table 3.1 Grouping and Immunization Dose of Mice for Exploring the Immunogenicity of VZV Recombinant Adenovirus Vector Vaccine

[0211] Table 3.2 Grouping and Immunization Dose of Experimental Mice for Exploring the Immunogenicity of VZV mRNA Vaccine

[0212] Table 3.3 Grouping and Immunization Dose of Mice in the Experiment of Sequential Immunization with Recombinant Adenovirus and Vaccine Combination Comparison

[0213] 3.1.2 Main Reagents and Consumables

[0214] Goat anti-mouse IgG, IgG2a, and IgG1 were purchased from Abcam; Carbonate-Bicarbonate buffer was purchased from Sigma-Aldrich; 96-well EIA / RIA Plates were purchased from Corning; eBioscience Cell Stimulation Kit (500×) was purchased from Thermo Fisher Scientific; ELISpot Plus: Mouse IFN-γ, IL-2, and IL-4 (HRP) were purchased from Dakota; other reagents were the same as in "1.1.2 Main Reagents and Consumables".

[0215] 3.1.3 Main Instruments

[0216] Multifunctional microplate reader (EnSpire): PE Corporation; Fluorescence immunoassay system (CTL, catalog number: S6ULTRA-03-06164); other items are the same as in "1.1.3 Main Instruments".

[0217] 3.1.4 Preparation of Commonly Used Reagents

[0218] 1) 2M H2SO4 stop solution: Add 55.6mL of 98% concentrated sulfuric acid dropwise to 250mL of ddH2O in a beaker, along the glass rod and the beaker wall, stirring constantly to release heat, and then add ddH2O to make up to 500mL. 2) The VZV gE protein (31~545aa) was expressed and purified in E. coli by Nanjing GenScript Technology Co., Ltd. The rest is the same as "1.1.4 Preparation of Commonly Used Reagents".

[0219] 3.2 Experimental Methods

[0220] 3.2.1 Mouse Immunization Strategy

[0221] The vaccine was prepared using sterile PBS, and 50 μL of the vaccine formulation was injected intramuscularly into each of the left and right legs of each mouse. A control group was set up, consisting of either an LNP group or an Ad26-empty or AdC68-empty group. Every two weeks after the initial immunization, 200 μL of blood was collected from the mandibular vein. After being kept at room temperature for 2 hours, the serum was separated by centrifugation at 10,000 rpm and 4°C for 20 minutes and stored at -80°C for subsequent specific antibody detection. Mice were sacrificed by cervical dislocation at week 6 or 8 after the initial immunization, and splenic lymphocytes were isolated from the spleen.

[0222] 3.2.2 Indirect ELISA detection of specific antibody titers in mouse serum

[0223] Serum from immunized mice stored at -80℃ was used to detect the titers of anti-VZV gE specific IgG, IgG1, and IgG2a antibodies in the serum using an indirect ELISA method. The specific steps are as follows: 1) Coating antigen: Prepare gE protein (2.5 ng / μL) with coating buffer, 50 μL per well, and incubate the microplate at 4°C overnight; 2) Blocking: Unseal the plate, wash the plate 3 times with PBST, and block with 5% skim milk (200 μL per well) and incubate at 37°C for 2 hours. 3) Primary antibody incubation: Unseal the plate, wash the plate 3 times with PBST, dilute the serum to be tested with 2% skim milk, serially dilute 3 or 4 times, set up pre-immunization mixed serum and Ad26-empty group / AdC68-empty group / LNP group mixed serum as negative wells, add to the reaction plate, and incubate at 37℃ for 1 h; 4) Secondary antibody incubation: Unseal the plate, wash the plate 3 times with PBST, dilute GoatAnti-Mouse IgG / IgG1 / IgG2a (HRP) with 2% skim milk at a ratio of 1:1000, and incubate at 37°C for 40 min; 5) Color development: Remove the sealing film, wash 5-7 times with PBST, add 50 μL of TMB color development solution to each well, and incubate at room temperature in the dark for 5 min. 6) Termination of color development: After color development is complete, add 2M H2SO4 to terminate the process, 50μL per well. Use a microplate reader to measure the OD value of each well at wavelengths of 450nm and 630nm, and analyze the results. 7) Result determination: The final antibody titer is defined as the difference between the OD450 and OD630 absorbance of the final control blank that is greater than 2.1 times that of the negative control blank.

[0224] 3.2.3 Isolation of mouse spleen lymphocytes

[0225] 1) Under sterile conditions, mice were euthanized by cervical dislocation, immersed in disinfectant alcohol for 3-4 seconds, and then lifted to remove excess alcohol; 2) Dissect and preserve the spleen by immersing it in pre-cooled 1640 culture medium; 3) Remove the spleen and place it in a 40μm filter, add 4mL of mouse lymphocyte separation solution, and grind it thoroughly with a syringe plunger; 4) Transfer the grinding solution to a 15mL centrifuge tube; 5) Slowly add 1 mL of 1640 culture medium to cover the liquid, maintaining a clear liquid surface separation; 6) Centrifugation at room temperature: 800×g, 40min, ramp rate 3, deceleration rate 3; 7) After centrifugation, aspirate the lymphocyte layer into a new 15mL centrifuge tube; 8) Add 10 mL of 10% FBS + 1640 medium and gently invert to mix. 9) Centrifuge at room temperature: 250×g, 10 min; or 500×g, 5 min; 10) Discard the supernatant and resuspend the cell pellet in 1 mL of 10% FBS + 1640 medium. 11) Countess 3, dilute spleen cells to 3×106 Cells / mL for later use.

[0226] 3.2.4 ELISApot detection of INF-γ, IL-2, and IL-4 secretion by mouse splenic lymphocytes

[0227] After equilibrating the ELISpot assay kit to room temperature, the ELISpot plate was disassembled in a clean bench, and the number of cytokines secreted by mouse spleen lymphocytes was detected. The specific steps are as follows: 1) Activation of pre-coated plates: Wash 4 times with 200 μL / well of sterile PBS, and pat dry with sterile paper on the last wash; 2) Add 100 μL / well of 10% FBS + 1640 and incubate at room temperature for 30 min; 3) Discard the culture medium and add cell suspension: 100 μL / well.

[0228] 4) Set up a positive control: 5 × 10⁵ Cells / well; a negative control / experimental group: 3 × 10⁵ Cells / well. 5 Cells / wells; Background negative control: Add 10% FBS + 1640 medium; 5) eBioscience cell stimulant and gE protein were prepared to a final concentration of 10× using 1640 medium, and 10 μL of stimulant was added to each well. 6) Positive control wells: Add working concentration of eBioscience positive stimulant; Negative control wells / background negative control wells: Add 10% FBS + 1640; Experimental wells: Final concentration of 10 μg / μL gE protein; 7) Incubation: Incubate at 37℃ in a 5% CO2 incubator for 48 hours; 8) Discard the culture medium and cells, wash 5 times with PBS, 200 μL / well, 1 min each time, and pat dry on absorbent paper on the last wash. 9) Add detection antibody (biotin-labeled): Dilute with 0.5% FBS-PBS to 1 μg / mL, 100 μL / well, and incubate at room temperature for 2 h; 10) Washing: Discard the liquid in the wells, wash 5 times with PBS (200 μL / well), and blot dry on absorbent paper after the last wash. 11) Add secondary antibody (enzyme-labeled avidin-incubated with Streptavidin-HRP): Dilute the secondary antibody (1:1000 dilution with 0.5% FBS-PBS), 100 μL / well, and incubate at room temperature for 1 h; 12) Washing: Pour out the liquid in the wells, wash 5 times with PBS, 200 μL / well, and blot dry on absorbent paper for the last wash; 13) Color development: Add TMB for display, 100 μL / well, incubate at room temperature in the dark for 15 min; 14) Termination of color development: After the spots have grown to a suitable size, pour out the liquid, remove the base plate, and wash the front and back sides and the base with deionized water 3 to 5 times to terminate the color development process. 15) Place the board upside down on absorbent paper, pat dry any small water droplets, and let it sit at room temperature in a well-ventilated place for 10-30 minutes to allow the film to air dry naturally. 16) Bioreader reads the ELISpot plate, records the number of spots and various data parameters for analysis.

[0229] 3.2.5 Statistical Analysis

[0230] Same as "1.2.6 Statistical Methods".

[0231] 3.3 Experimental Results

[0232] 3.3.1 Immunogenicity Study of Recombinant Adenovirus Vaccine

[0233] The immunogenicity of the recombinant adenovirus vaccine was assessed using a dose gradient method. C57BL / 6N mice were divided into low (1×10⁻⁶) groups. 6 IFU), medium (1×10 7 IFU), high (1×10 8 IFU was administered via single intramuscular injection in three dosage groups; specific experimental groupings are shown in Table 3.1. Combined with the results of 2-6 weeks of dynamic monitoring of antibody IgG, (…) Figure 16 (A~B) Serum gE-specific IgG antibody levels in mice of all vaccine groups peaked 4 weeks post-immunization, showing a clear dose-dependent effect: low-dose group (1×10⁻⁶) 6 No significant specific IgG response was detected in the IFU group; the medium-dose group (1×10) 7 IFU induced IgG antibody production at levels of 10² to 10³. Notably, the non-eVLP modified vaccine group also failed to induce detectable antibody production under medium-dose conditions. The high-dose group (10⁸ IFU) produced specific IgG antibodies against the VZV gE protein in mice, with antibody levels reaching 10⁸. 2.48 ~10 4.05 Furthermore, in both non-replicating recombinant adenovirus vaccine groups, the IgG antibody titer in the eVLP (tPA-gE-eVLP) vaccine group was significantly higher than that in the non-eVLP (tPA-gE) vaccine group, and the difference was statistically significant (P<0.0001).

[0234] IgG subtype analysis in the high-dose group showed (Figure 16 C): The IgG2a / IgG1 ratio of the two vaccines was 1.18-1.51, indicating a Th1-biased immune response; the ratio in the eVLP group was closer to 1, showing a more balanced Th1 / Th2 response; compared with the non-eVLP group, eVLP modification significantly improved the balance of the immune response.

[0235] ELISpot detection of high-dose group (1×10) 8 IFU cellular immune response results showed ( Figure 16 D): Splenic lymphocytes in the Ad28-empty and AdC68-empty control mice showed almost no specific IFN-γ secretion (11.6±5.6, 12.4±8.9). In the Ad26 and AdC68 recombinant adenovirus vector vaccines, the number of IFN-γ secreting cells in the gE-eVLP modified vaccine group was significantly higher than that in the unmodified gE group (133±37.8 vs 51±28.4; 124.7±48.1 vs 55.3±21.7; P<0.01).

[0236] 3.3.2 Immunogenicity Study of mRNA Vaccine

[0237] The mRNA vaccine was administered intramuscularly at three doses: 0.5 μg, 2.5 μg, and 5 μg. A second immunization was given 3 weeks after the initial immunization. The specific grouping of the experimental mice is shown in Table 3.2. The dynamic changes in total IgG antibody levels in mouse serum were detected using ELISA. The experimental results showed (…). Figure 17 In all three dosage groups, the mRNA vaccine effectively induced specific IgG antibody responses in mice. Antibody levels showed a significant dose-dependent increasing trend after immunization (P<0.05), reaching a plateau at week 4 and remaining at around 10. 4 ~10 6 It is noteworthy that the antibody levels among mice within each mRNA vaccine group showed relatively small differences (CV < 20%).

[0238] Further comparison of the immunization effects of LNP-tPA-gE and LNP-tPA-gE-eVLP vaccines revealed no statistically significant difference in the serum antibody levels induced by the two (P>0.05). To further evaluate the characteristics of the immune response induced by the mRNA vaccine, this study measured the levels of specific IgG antibody subtypes (IgG1 and IgG2a). The results showed ( Figures 18 - 20 See legend Figure 17 The titer ranges of IgG1 and IgG2a in each dose group were 10. 4.41 ~10 5.60The IgG2a / IgG1 ratio was between 0.86 and 0.99. The immune response induced by the RNA vaccine showed a balanced mixed response of Th1 and Th2, without obvious bias in the immune response.

[0239] ELISpot technology was used to detect splenic lymphocytes from mice in each vaccine group that secreted IFN-γ and IL-4, in order to systematically evaluate the characteristics of vaccine-induced cellular immune responses. Experimental results showed ( Figure 21 Both groups (A and B) exhibited a significant dose-dependent cellular immune response: the secretion levels of both IFN-γ and IL-4 showed a significant upward trend with increasing vaccine dose (P<0.05). Notably, the LNP-tPA-gE-eVLP vaccine group was significantly superior to the LNP-tPA-gE group in the secretion levels of both cytokines (P<0.05), with the increase in IL-4 secreting cells being particularly significant (P<0.01). This result confirms that eVLP modification can not only enhance the Th1-type immune response induced by mRNA vaccines but also significantly improve the Th2-type immune response level, indicating that the eVLP structure plays an important role in promoting a comprehensive immune response.

[0240] 3.3.3 Recombinant adenovirus sequential immunization and comparison with mRNA and Shingrix vaccines

[0241] Sixty SPF female 6-week-old mice were randomly divided into 10 groups of 6 mice each (Table 3.3), and the mice were immunized twice at three-week intervals. Figure 22 The changes in antibody endpoint titers at different time points (0-8 weeks) were detected by ELISA. The results showed that the antibody endpoint titers in the LNP-tPA-gE-eVLP and Shingrix vaccine groups exhibited similar trends. Between 2 and 4 weeks, the antibody endpoint titers in mice showed a significant upward trend, reaching a peak at 4 weeks (between 105.19 and 105.78). From 4 to 8 weeks, the titers decreased slightly, but remained above 10. 4.82 ~10 5.60 Between the heterologous booster group and the homologous booster group, the antibody endpoint titer was slightly higher, but the difference was not statistically significant (P>0.05). After secondary immunization with the recombinant adenovirus vaccine, the antibody endpoint titer in mice showed an upward trend at week 4, and the antibody endpoint titer was between 10 and 10 at weeks 4-8. 4.19 ~10 4.82 Between these levels, the changes were relatively stable. Overall, considering antibody levels: mRNA vaccine group > Shingrix group > non-replicating recombinant adenovirus sequential immunization group ( Figure 23 ).

[0242] Mouse serum gE specific antibody subtype detection ( Figures 24 - 26The results showed that IgG2a and IgG1 in the LNP-tPA-gE-eVLP group were significantly higher than those in the recombinant adenovirus heterologous booster group and the Shingrix group. The antibody subtype IgG2a / IgG1 ratio showed that the Th1 / Th2 immune responses were balanced in the mRNA vaccine group and the Shingrix group, with a ratio close to 1. In the recombinant adenovirus heterologous booster group, the IgG2a / IgG1 ratio was between 1.1 and 1.3, indicating an immune response biased towards the Th1 type.

[0243] Mice were sacrificed at week 8 after primary immunization, and spleen lymphocytes were isolated. Splenic lymphocytes were stimulated with gE protein, and the number of splenic lymphocytes secreting IFN-γ, IL-2, and IL-4 was detected using ELISApot. Two replicates were performed for each mouse. Results showed ( Figures 27 - 29 The control group showed no significant response. The LNP-tPA-gE-eVLP combination showed the best performance in inducing the number of IFN-γ (479.6±23.82), IL-2 (487.7±23.37), and IL-4 (290±70.75) secreting cells, demonstrating the strongest immune response. Shingrix showed a high level of IFN-γ, IL-2, and IL-4 secreting cells (401.6±116.8, 180.1±159.1, and 225.5±124), second only to the LNP-tPA-gE-eVLP vaccine group, with statistically significant differences (P<0.05). Among the sequential immunization combinations of recombinant adenovirus vaccines, the Ad26-eVLP+Ad26-eVLP primary immunization and the AdC68-eVLP+AdC68-eVLP booster immunization combinations had the highest IL-2 secretion level (186.7±75.1). However, the sequential immunization combination performed better overall in inducing the number of IFN-γ (199.2±135.8) and IL-2 (94.1±81.5) secreting cells, but performed only moderately in inducing the number of IL-4 (5.7±10.5) secreting cells, which was inferior to the mRNA vaccine group and the Shingrix group.

[0244] 3.4 Discussion

[0245] This embodiment systematically evaluated the immunogenicity characteristics of VZV vaccines based on different technology platforms (recombinant adenovirus vector and mRNA-LNP) in mouse models. First, referring to the dosages used in previous studies, we established specific dosages for the prepared non-replicating recombinant adenovirus vaccine and mRNA vaccine to conduct preliminary immunogenicity studies. In this embodiment, the recombinant adenovirus vector vaccine (Ad26 / AdC68) was administered at 10... 8The IFU dose showed good immunization efficacy. Specifically, the tPA-gE-eVLP-induced binding antibody levels and IFN-γ secretion levels detected by ELISpot were higher in the tPA-gE-eVLP group than in the gE group. Furthermore, the addition of eVLP brought the IgG2a / IgG1 ratio closer to 1, indicating that combining eVLP with recombinant adenovirus vector vaccine technology not only enhances humoral immune responses but also has the potential to balance Th1 and Th2 immune responses. The VZV mRNA vaccine designed in this invention induced high humoral and cellular immune responses at doses ranging from 0.5 μg to 5 μg, with minimal intra-group individual variability, indicating that mRNA vaccine-induced immunity is less affected by individual differences. The mRNA vaccine group performed well in inducing binding antibodies, with antibody titers maintaining a high plateau between 2 and 8 weeks. Although the binding antibody levels showed a dose-dependent increase in humoral immunity, this increase was not statistically significant. P >0.05). Compared to LNP-tPA-gE, the addition of EABR self-assembled particle technology enabled LNP-tPA-gE-eVLP to induce more splenocytes to secrete IFN-γ and IL-4 ( P <0.05). Notably, different vaccine platforms exhibited significant biases in immune response. The balance between Th1 and Th2 immune responses is crucial for immune homeostasis. Compared to recombinant adenovirus vector vaccines, mRNA vaccines induced an IgG2a / IgG1 ratio close to 1, indicating a balanced immune response. Furthermore, in the low-dose mRNA vaccine group (0.5 μg), the IgG2a / IgG1 ratio was 0.86 / 0.88, indicating a more Th2 humoral immune response. In the medium (2.5 μg) and high (5 μg) dose groups, the IgG2a / IgG1 ratio ranged from 0.93 to 0.99, suggesting that medium and high doses induced a more balanced Th1 / Th2 immune response in mice, indicating that higher doses of VZV mRNA vaccines can effectively balance Th1 and Th2 immune responses while inducing humoral immunity. The difference in immune response between recombinant adenovirus vaccines and mRNA vaccines may stem from the different mechanisms of action of the delivery systems: adenovirus vectors activate innate immunity through the TLR9-MyD88 pathway, while LNP vectors promote Th1 polarization through endosomal TLR7 / 8 signaling.

[0246] Preliminary evaluation of immunization efficacy indicates that the eVLP self-assembled particle technology significantly enhances the immune response of recombinant adenovirus vector vaccines compared to its combined use with mRNA vaccines. This difference primarily stems from the different immune activation characteristics of the two vaccine platforms and their synergistic mechanisms with eVLP. Specifically, both mRNA vaccines and eVLP tend to induce Th2 immune responses and promote humoral immune responses, potentially leading to overlap between the mRNA vaccine and eVLP in activating humoral immunity pathways. Furthermore, the mRNA vaccine itself can already maximize the activation of the body's immune response, which may explain why eVLP's enhancement effect is not significant. In contrast, recombinant adenovirus vector vaccines mainly induce Th1 immune responses, favoring cellular immune responses, and their combination with eVLP significantly enhances humoral immune response levels. This synergistic effect was clearly verified in our analysis of changes in binding antibody levels. These findings provide important references for the optimal combination of different vaccine platforms and adjuvants.

[0247] Preliminary results on immune responses indicate that the VZV recombinant adenovirus vector vaccine provides less protective immunity than the mRNA vaccine. However, studies suggest that heterologous immunization using different serotypes of recombinant adenovirus has proven to be an effective strategy for enhancing antigen-specific immune responses. Therefore, this study used Ad26 and AdC68 as vectors to conduct a head-to-head comparison of humoral and cellular immune responses induced by different primary-booster immunization regimens of VZV, employing recombinant adenovirus vectors of different serotypes, aiming to better understand the impact of vaccination regimens on immune responses. In exploring heterologous booster strategies, while sequential immunization with Ad26 and AdC68 reduced the antibody decay rate (a 28.6% reduction in the rate of decay over 4-8 weeks), the overall immunogenicity remained significantly lower than that of the mRNA vaccine group. This result differs from the heterologous sequential effect of adenovirus reported by Denis et al. (LIU J, XU K, XING M, et al. Heterologous prime-boost immunizations with chimpanzeeadenoviral vectors elicit potent and protective immunity against SARS-CoV-2 infection[J]. Cell Discovery, 2021, 7(1): 123. LOGUNOV DY, DOLZHIKOVA IV, SHCHEBLYAKOV DV, et al. Safety and efficacy of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine: an interim analysis of arandomised controlled phase 3 trial in Russia[J]. The Lancet, 2021, 397(10275): 671-681.), which may be related to the antigen design or the choice of experimental animal model used in this invention. Based on the combined data from single-dose and heterologous or homologous boosters, the secondary booster immunization showed some effectiveness, slowing the rate of decline in antibody titers between 4 and 8 weeks. In the recombinant adenovirus vaccine group, both ELISA results for antibody subtypes and ELISA-detected cytokine-secreting cell counts indicated a significantly Th1-type cellular immune response. Further analysis revealed that while the Ad26 and AdC68 heterologous booster strategy could enhance the immune response to some extent, its effect was limited, and the level of immune protection induced was still lower than that of the mRNA vaccine group.Notably, the mRNA vaccine designed in this invention (especially the high-dose LNP-tPA-gE-eVLP group) exhibits excellent immunogenicity, with induced binding antibody levels comparable to the marketed Shingrix vaccine, and using the same antigen dose (5 μg). More importantly, the mRNA vaccine group also demonstrated a stronger cellular immune response than Shingrix, an advantage that may be significant for preventing latent infection and achieving long-term immune protection. These findings provide strong evidence for the application of mRNA vaccines in herpesvirus control and also suggest that the mRNA platform has unique advantages in balancing humoral and cellular immune responses.

[0248] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A varicella-zoster virus virus-like particle vaccine gE-eVLP, characterized in that, The nucleotide sequence of the gE-eVLP includes a signal peptide sequence, a truncated and / or mutated sequence of the gE domain without the original signal peptide, and an EGE motif. The truncation of the gE domain includes: truncation of the TGN target sequence or retention of the TGN target sequence but truncation of the subsequent sequence, and truncation of the internalized signal motif ET; The mutations in the gE domain include: the Y569A mutation that disrupts the TGN targeting sequence localization, the Y582G mutation that eliminates the endocytic signal motif ET, or the S593A, S595A, T596A, and T598A mutations that regulate phosphorylation modification clusters. The nucleotide sequence of the EGE motif is shown in SEQ ID NO:

25.

2. The varicella-zoster virus-like particle vaccine gE-eVLP as described in claim 1, characterized in that, The TGN target sequence deletion is based on gE-△SP as shown in SEQ ID NO: 2, by removing amino acids 568 to 623, resulting in gE-△TGN, as shown in SEQ ID NO: 9; The method of retaining the TGN target sequence but truncating the subsequent sequence is to truncate amino acids 572 to 623 from gE-△SP as shown in SEQ ID NO: 2, and the truncated result is gE-TGN, as shown in SEQ ID NO: 10; The Y569A mutation that disrupts the TGN target sequence localization is a Y569A mutation performed on the basis of gE-TGN as shown in SEQ ID NO: 10, resulting in gE-mTGN as shown in SEQ ID NO: 11; The endocytic signal motif ET truncation is based on gE-△SP as shown in SEQ ID NO: 2, by removing amino acids 582 to 623, resulting in gE-△ET, as shown in SEQ ID NO: 12; The elimination of the endocytosis signal motif Y582G mutation is achieved by removing amino acids 589 to 623 from the gE-△SP amino acid sequence shown in SEQ ID NO: 2 and performing a Y582G mutation, resulting in gE-mET, as shown in SEQ ID NO:

13. The S593A, S595A, T596A, and T598A mutations of the phosphorylation modification clusters are performed on the basis of gE-ΔSP as shown in SEQ ID NO: 2, with simultaneous Y569A mutations, resulting in gE-dM, as shown in SEQ ID NO:

14.

3. The varicella-zoster virus-like particle vaccine gE-eVLP as described in claim 1, characterized in that, The signal peptide is tPA, and its nucleotide sequence is shown in SEQ ID NO:

3.

4. An expression plasmid containing the virus-like particle vaccine gE-eVLP according to any one of claims 1 to 3, characterized in that, The expression vector was pcDNA3.1-empty; the constructed expression plasmids included pcDNA3.1-tPA-gE-dM-EGE, pcDNA3.1-tPA-gE-△ET-EGE, pcDNA3.1-tPA-gE-mET-EGE, pcDNA3.1-tPA-gE-TGN-EGE, pcDNA3.1-tPA-gE-mTGN-EGE, and pcDNA3.1-tPA-gE-△TGN-EGE.

5. A method for preparing the virus-like particle vaccine gE-eVLP according to any one of claims 1 to 3, characterized in that, The expression plasmid described in claim 4 is transfected into cells to produce the virus-like particle vaccine gE-eVLP through secretory expression in the cells.

6. A recombinant adenovirus vector vaccine based on the gE-eVLP varicella-zoster virus according to any one of claims 1 to 3, characterized in that, The nucleotide sequence of gE-eVLP according to any one of claims 1 to 3 is introduced into an adenovirus vector to obtain a recombinant adenovirus.

7. The recombinant adenovirus vector vaccine as described in claim 6, characterized in that, The nucleotide sequence of the gE-eVLP is a codon-optimized nucleotide sequence; the gE domain of the gE-eVLP is a codon-optimized nucleotide sequence of gE-△ET as shown in SEQ ID NO: 31; The adenovirus vector is pkAd26-empty or pkAdC68-empty.

8. A method for preparing the recombinant adenovirus vector vaccine according to claim 7, characterized in that, The recombinant adenovirus was linearized and then transfected into cells to prepare a seed virus. The seed virus was then expanded and purified to obtain a recombinant adenovirus vector vaccine.

9. An mRNA vaccine based on the gE-eVLP varicella-zoster virus according to any one of claims 1 to 3, characterized in that, 5U1-gE-eVLP was amplified using mRNA plasmid primers and then inserted into the vector pcDNA3.1 T7-Nluc-3U1 to obtain the mRNA vaccine recombinant plasmid. The recombinant plasmid was linearized, transcribed in vitro, capped, and purified to obtain the mRNA vaccine. The mRNA vaccine was then encapsulated in liposomes to obtain the LNP-mRNA vaccine. The mRNA plasmid primers are as shown in SEQ ID NO: 34~37; The nucleotide sequence of the amplified 5U1-gE-eVLP with the addition of 3U1 and polyA tails is shown in SEQ ID NO:

33.

10. The use of the virus-like particle vaccine gE-eVLP according to any one of claims 1 to 3, the recombinant adenovirus vector vaccine according to claim 7, or the mRNA vaccine according to claim 9 in the preparation of a varicella-zoster virus vaccine drug.

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