Recombinant herpes zoster vaccine composition and preparation method thereof

Abstract

The invention relates to the field of biological medicine, in particular to a recombinant herpes zoster vaccine composition and a preparation method thereof. The recombinant herpes zoster vaccine composition comprises gE protein and an adjuvant, the adjuvant is a squalene oil-in-water adjuvant, and the squalene oil-in-water adjuvant comprises the following components by concentration: 4-5wt% of squalene, 1-5wt% of gE protein, 1-5wt% of gE protein, 1-5wt% of gE protein, 1-5wt% of gE protein, and the balance of water. 0.2 to 0.8 wt% of Tween 80; and 0.2 to 0.8 wt% of Span 85. The vaccine composition disclosed by the invention can specifically enhance cellular immune response aiming at the varicella-zoster virus, can remarkably improve expression of cytokines such as INF-gamma and IL-2 of CD4 + T cells of mice, improves the immune response level of body fluid and cells at the same time, shows high immunocompetence for preventing clinical varicella-zoster virus infection, and has good clinical application prospects. And the components in the vaccine composition are cheap and easy to obtain, so that the vaccine yield is effectively improved, and the vaccine cost is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a recombinant herpes zoster vaccine composition and a preparation method thereof. Background Art

[0002] Varicella-zoster virus (VZV) is a pathogen that is widely present in the global population, infecting more than 90% of the world's population. Primary VZV infection often occurs in childhood and can cause chickenpox characterized by systemic vesicular eruptions, and after infection, it can remain latent in the host's neuronal cells. With age, decreased immune function or other factors, the latent virus can be reactivated, spread along the peripheral nerves, and cause herpes zoster (HZ). With the popularization of vaccines, the incidence of varicella is currently basically under control, but HZ is more common in many elderly people. For people who are infected with VZV for the first time and have low immunity or VZV reactivation, they are often accompanied by complications such as meningitis, stroke, and post-HZ neuralgia, which reduce the patient's quality of life and even endanger their lives.

[0003] Globally, vaccines related to herpes zoster disease can be divided into two types: attenuated herpes zoster vaccine and recombinant subunit herpes zoster vaccine. The former plays a role in the prevention of herpes zoster, but the effect is greatly weakened with age, while the latter has a high protective effect in all age groups and can be maintained for a considerable period of time. At present, the internationally approved HZ vaccine is Merck's Zostavax, which has an effectiveness of 69.8% (50-59 years old) and 51% (over 60 years old) in preventing HZ. In addition, there is GlaxoSmithKline (GSK)'s recombinant subunit herpes zoster vaccine Shringrix, which consists of herpes zoster virus gE protein and adjuvant system AS01B. The AS01B adjuvant system is a liposome adjuvant containing monophosphoryl lipid A and saponin QS-21, which can enhance cellular immune response. Clinical studies have shown that Shringrix vaccine can significantly reduce the risk of HZ in people over 50 years old (91% to 97%), and can effectively reduce the subsequent risk of PHN (≥80%). Studies have found that the reason why Shringrix forms long-term protection in the elderly may be related to its ability to induce higher levels of IL-2 and IFN-γ. Subunit herpes zoster vaccines have great prospects in the domestic market.

[0004] With the continuous development and progress of biotechnology in the vaccine industry, 9 VZV surface glycoproteins have been identified, among which gE glycoprotein is the most expressed glycoprotein of VZV, which plays a major role in the replication and assembly of the virus and also mediates the spread of the virus between cells. Given that VZV-gE has strong immunogenicity and can induce the body to produce an immune response against VZV, it has become one of the main candidate antigens for VZV subunit vaccines and DNA vaccines.

[0005] At present, there are hundreds of adjuvants that have been confirmed and used: 1. Inorganic salt adjuvants It is mainly a salt compound of aluminum and magnesium, and is the earliest discovered and most widely used vaccine adjuvant. A large number of clinical trials believe that aluminum adjuvants can stimulate the body to produce strong humoral immunity, but will cause injection site reactions and have a weak ability to induce cellular immunity. Aluminum salts mainly include aluminum phosphate, aluminum hydroxide and aluminum sulfate. Currently, aluminum adjuvants commonly used are aluminum hydroxide and aluminum phosphate. The mechanism of action of aluminum adjuvants mainly includes storage effect and immunostimulatory effect. Aluminum adjuvants can also stimulate innate immunity and induce monocytes to secrete IL-1β and IL-18 by activating Nlrp3 inflammasomes. In addition, aluminum adjuvants can stimulate necrotic cells at the injection site to release damage-associated pattern molecules (DAMPs) such as DNA, ATP and uric acid, and the released DAMPs contribute to the interaction between dendritic cells and CD4+T cells. So far, aluminum adjuvants are still the only widely used adjuvants in vaccines. However, aluminum adjuvants have great defects: they are generally effective in increasing serum antibodies, but they can cause injection site reactions and have a weak ability to induce cellular immunity, which limits their application to a certain extent.

[0006] 2. Oil-water emulsion adjuvant Oil adjuvant is a traditional adjuvant type, among which the most famous is Freund's adjuvant, which is currently widely used in inactivated vaccines for animals, but it is highly toxic. In recent years, an emulsion adjuvant similar to Freund's adjuvant has been developed, such as squalene oil-in-water adjuvant, AS03 and AF03. The immunogenicity is relatively poor when used alone, and the dosage needs to be increased to increase the immunogenicity. However, increasing the dosage of adjuvant will face greater side effects. The side effects of oil emulsions mainly include intense pain, inflammatory reactions, granulomas and ulcers formed at the vaccination site.

[0007] 3. Adjuvants targeting pattern recognition receptors Some components in bacteria, fungi, viruses and plants can activate innate immunity by targeting pattern recognition receptors, thus becoming potential vaccine adjuvants, such as monophosphoryl lipid A (MPLA), CpG motif, etc. The immunogenicity is relatively poor when used alone, and increasing the dosage will increase the risk of side effects, including local reactions such as pain, swelling, nodules, and rashes at the injection site, as well as systemic symptoms such as flu-like symptoms.

[0008] 4. Cytokine adjuvants Cytokine adjuvants mainly regulate the response type in the Th1 / Th2 cell immune process, and affect the signal transduction and stimulation signals between cells. The earliest cytokine used as an adjuvant, IL-1, can enhance the primary and secondary responses to antigens, induce antigen-specific T cell activity enhancement and B cell proliferation. Cytokines can be divided into interleukins (IL), interferons (IFN), tumor necrosis factor superfamily, colony stimulating factors, chemokines, growth factors, etc. Summary of the invention

[0009] The present invention focuses on providing a new generation of recombinant herpes zoster preventive vaccine, which contains varicella zoster virus (VZV) antigen. Existing studies on VZV-gE protein show that the protein molecule of wild-type VZV-gE includes four regions, namely, signal peptide, extracellular region, transmembrane region and intracellular region, and the three antigenic determinants of VZV-gE are distributed in the extracellular region. Considering factors such as the effect of the hydrophobic transmembrane region on the expression of exogenous proteins, the expression level of the protein can be improved by removing the transmembrane region (hydrophobic region) and the intracellular region (such as patent CN102517302A).

[0010] In addition, the technical problem to be solved by the present invention is to further enhance the preventive effect, improve the immune memory ability, reduce complications and sequelae, selectively enhance the cell-mediated immune response, and avoid the risks of live attenuated vaccines on the basis of the existing technology, and provide a herpes zoster recombinant protein vaccine adjuvant formula.

[0011] The specific technical scheme of the present invention is: A recombinant herpes zoster vaccine composition comprises a gE protein and an adjuvant, wherein the adjuvant is a squalene oil-in-water adjuvant, and the squalene oil-in-water adjuvant comprises the following concentration components: squalene: 4-5wt%; Tween 80: 0.2-0.8wt%; Span 85: 0.2-0.8wt%.

[0012] The sequence number of the gE protein is as follows: SVLRYDDFHTDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDV FKGDLNPKPQGQRMIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPG VLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNS GCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRY.

[0013] In an embodiment, the squalene oil-in-water adjuvant comprises the following concentration components: squalene: 4.3wt%; Tween 80: 0.5wt%; Span 85: 0.5wt%, and citric acid buffer is added to adjust the pH value to 6.5.

[0014] Preferably, the content of the gE protein is 20-100 μg / mL.

[0015] Preferably, the preparation method of the gE protein is as follows: Construction of PLK002-22P20-A2: Using PLK002 as an empty vector, adding a Hind III restriction site and a KOZAK sequence at the front end of the signal peptide of the antigen DNA sequence, and adding a stop codon and a Not I restriction site at the back end; recovering the vector and the target fragment separately and connecting them, sequencing and restriction digestion identification were performed through the plasmid DNA of the positive transformants, and the PLK002-22P20-A expression vector was successfully constructed; adding a KOZAK sequence at the front end of the signal peptide of the antigen DNA sequence, and adding a stop codon at the back end, replacing the HindIII to Not I DNA sequence on the PLK002 vector with 22P20-A2 by the homologous recombination method, sequencing and restriction digestion identification were performed through the positive transformants, and finally PLK002-22P20-A2 was successfully constructed; Using CHO K1BN suspension cells as host cells, the expression vector of PLK002-22P20-A2 constructed above was electroporated into the host cells. After transfection, the cells were screened for high-expression monoclonal strains by pressure screening, step-by-step amplification, ELISA and SEC-HPLC, and cultured continuously. The culture supernatant was harvested by centrifugal filtration, and the collected supernatant was filtered through clarification, the first anion chromatography, low pH incubation + deep filtration, hydrophobic chromatography, the first ultrafiltration liquid exchange, the second deep filtration, the second anion chromatography, the second ultrafiltration liquid exchange, nanofiltration, dilution and stock solution sterilization filtration to obtain the final sample, i.e., gE protein.

[0016] Preferably, the diameter of the squalene oil-in-water adjuvant droplets is 150-180 nm; preferably, the diameter of the squalene oil-in-water adjuvant droplets is 160 nm.

[0017] Preferably, the volume of the squalene oil-in-water adjuvant is 25-50%.

[0018] Preferably, it further comprises a buffer, wherein the buffer is at least one of PBS buffer, Tris-HCl buffer and citrate buffer.

[0019] Preferably, the method further comprises a chemotactic factor; the amount of the chemotactic factor added is 5-15 μg / mL.

[0020] Furthermore, the chemokine is at least one of CCL3 / MIP-1α and CXCL10 / IP-10. Furthermore, the chemokine is a combination of 30-70wt% of CCL3 / MIP-1α and 30-70wt% of CXCL10 / IP-10.

[0021] The addition of chemokines CCL3 / MIP-1α and / or CXCL10 / IP-10 helps attract more immune cells (such as dendritic cells, T cells) to migrate to the vaccination site and local lymph nodes, enhancing antigen presentation and specific immune responses. By regulating the chemotaxis and activation state of immune cells, Th1-type immune responses are promoted, which is beneficial to cell-mediated immune defense, while maintaining a certain degree of Th2 response to promote antibody production. Enhanced immune cell activation and optimized Th1 / Th2 balance help form strong immune memory and provide a basis for long-term protection.

[0022] The preparation method of the recombinant herpes zoster vaccine composition comprises mixing squalene oil-in-water adjuvant with gE protein, adding buffer, and performing blowing and adsorption under light-proof conditions to obtain the recombinant herpes zoster vaccine composition.

[0023] Beneficial technical effects: Compared with the prior art, the present invention has the following advantages: 1. The recombinant herpes zoster vaccine composition of the present invention has an increased expression level of gE antigen-specific IgG after secondary immunization. At the humoral immune response level, the composition of gE protein and squalene water-in-oil adjuvant can effectively promote B lymphocyte response to enhance the production of specific IgG antibodies, effectively prevent the recurrence of herpes zoster, and play an important role in preventing herpes zoster.

[0024] 2. The recombinant herpes zoster vaccine composition of the present invention, at the level of cellular immune response, gE and squalene oil-in-water adjuvant can effectively promote Th1 cell response to enhance the secretion of cytokines IFN-γ and IL-2, IL-2 can effectively activate CD4+ and CD8+ T cells and form memory phenotypes, promote the differentiation of CD4+ T cells into Th1 and Th2 cells, IFN-γ promotes the killing of herpes zoster virus, and the coordinated action of the two prevents the reactivation of the virus, controls the intracellular infection of the virus, and maintains the latent state of the virus.

[0025] 3. The recombinant herpes zoster vaccine composition of the present invention uses VZV-gE protein in combination with squalene oil-in-water adjuvant, showing significant improvements in immunogenicity and cytokine expression, and has better immune effects than using aluminum hydroxide adjuvant or CpG adjuvant alone.

[0026] 4. The recombinant herpes zoster vaccine composition of the present invention also includes chemotactic factors CCL3 / MIP-1α and / or CXCL10 / IP-10. CXCL10 / IP-10, through its specific receptor CXCR3, particularly promotes the migration and activation of Th1 cells, which is essential for the formation of cell-mediated immune responses against pathogens. When these two chemokines work together, they not only recruit a wider and more diverse population of immune cells, but also particularly enhance the immune defense against specific pathogens. Therefore, the combination of CCL3 / MIP-1α and CXCL10 / IP-10 achieves a comprehensive activation of the immune system, including strengthening humoral immunity and cell-mediated immune responses, especially by promoting the production of IFN-γ, TNF-α and IL-2, which are essential for resisting infection and disease.

[0027] 5. The present invention makes full use of existing detection technology and can efficiently, accurately and stably react to immune response levels, providing good support for adjuvant formulation screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 This is the constructed plasmid map of PLK002-22P20-A2; Figure 2 is the plasmid restriction map; Figure 3 This is a flow chart of the protein purification process; Figure 4 This is a diagram of the purification results of gE protein; Figure 5 This is a comparison chart of gE-specific cell-mediated cellular immunity levels; Figure 6 It is the gE-specific cell-mediated immune response level (ELISPOT) graph; Figure 7 The results of flow cytometry detection of CD4+T cell response for single adjuvant prescription; Figure 8 This is a graph showing the gE-specific cell-mediated immune response levels (ICS) of IFN-γ, TNF-α, and IL-2. DETAILED DESCRIPTION

[0030] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are the terms and routine procedures widely used in the corresponding fields.

[0031] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0032] Unless otherwise specified, the experimental methods described in the following examples are conventional methods unless otherwise specified; the raw materials and reagents are commercially available or can be prepared by known methods.

[0033] Example 1: Production of gE protein, molecule A2 is a recombinant protein antigen for varicella-zoster virus, and DNA sequence optimization and signal peptide optimization are performed based on the amino acid sequence of CHO cells. PLK002 is selected as an empty vector, and a HindIII restriction site (AAGCTT) and a KOZAK sequence (GCCGCCACC) are added to the front end of the signal peptide of the designed and optimized antigen DNA sequence, and a stop codon (TGATAA) and a NotI restriction site (GCGGCCGC) are added to the back end. HindIII and NotI double-digest PLK002 and 22P20-A gene synthesis sequences, and the vector and the target fragment are recovered and connected respectively. Positive transformants are screened by kanamycin resistance, and the plasmid DNA of the positive transformants is sequenced and identified by restriction digestion, and finally the PLK002-22P20-A expression vector is successfully constructed. The KOZAK sequence (GCCGCCACC) is added to the front end of the signal peptide of the designed and optimized antigen DNA sequence, and a stop codon (TGATAA) is added to the back end. The DNA sequence from HindIII to NotI on the PLK002 vector was replaced by 22P20-A2 by homologous recombination, and positive transformants were screened by kanamycin resistance. The plasmid DNA of positive transformants was sequenced and identified by restriction enzyme digestion (see Figure 2 ), and finally successfully built PLK002-22P20-A2 (see Figure 1 ).

[0034] CHOK1BN suspension cells were selected as host cells, and the expression vector of PLK002-22P20-A2 successfully constructed above was transferred into the host cells by electroporation. After transfection, the cells were screened by pressure screening, step-by-step amplification, ELISA and SEC-HPLC to screen out a high-expression monoclonal strain with an expression level of 2.96 g / L. The cells were continuously subcultured, and the cell density and cell viability were monitored every day. The culture was stopped for 10-12 days when the cell viability was lower than 80%, and the culture supernatant was harvested by centrifugal filtration. The collected supernatant was clarified and filtered, the first anion chromatography, low pH incubation + deep filtration, hydrophobic chromatography, the first ultrafiltration exchange, the second deep filtration, the second anion chromatography, the second ultrafiltration exchange, nanofiltration, dilution and sterilization filtration of the original solution to obtain the final purified sample, i.e., gE protein. The process flow is as follows Figure 3 As shown, the purified gE protein Figure 4 As shown, the protein purity detected by SEC-HPLC was >97%.

[0035] The sequence number of the prepared gE protein is as follows: SVLRYDDFHTDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDV FKGDLNPKPQGQRMIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPG VLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNS GCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRY.

[0036] Table 1: PLK002-22P20-A2 carrier key component information Key Components name Location Function CMVenhancer Human cytomegalovirus early enhancer 124-503 Enhance target gene transcription CMVpromoter Human cytomegalovirus early promoter 504-707 Initiate transcription of target gene 5'UTR-intron-5'UTRelements 5'UTR-Intron-5'UTR original 722-1686 Improve translation efficiency 22P20-A2 22P20-A2 recombinant antigen 1696-3282 Encoding target gene SV40poly(A)signal Simian vacuolating virus PolyA 3412-3546 Termination of transcription Ori pBR322 replication origin 3846-4434 Plasmid replication origin AmpRpromoter Ampicillin resistance gene promoter 5421-5525 Initiation of knaR gene transcription KqA Aph (3')-Ia (kanamycin resistance gene) coding region 4605-5420 For plasmid screening SV40promoter Simian vacuolating virus promoter 5804-6133 Initiate transcription of target gene GS Glutamine synthetase gene 6172-7296 GS system selection markers SV40intron and polyA Simian vacuolating virus intron and PolyA 7348-8199 Termination of transcription Example 2: Screening of vaccine formulations and immunogenicity studies. In this example, the gE protein prepared in Example 1 was used as an immunogen, diluted with 30 mM phosphate buffer, 0.02% Tween 80 pH 7.5, and evenly mixed with aluminum hydroxide, CpG1018 adjuvant, and squalene oil-in-water adjuvant at room temperature to prepare aluminum adjuvant vaccine solutions, CpG adjuvant vaccine solutions, and squalene oil-in-water adjuvant vaccine solutions, respectively. Immunogenicity studies were carried out using C57BL / 6 mice (purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.) as an animal model.

[0037] Immunization group 1: negative control, sterile PBS buffer.

[0038] Immunization group 2: gE protein solution.

[0039] Immunization group 3: 50μg protein gE + 500μg Al(OH) 3。

[0040] A method for preparing a recombinant herpes zoster vaccine composition comprises the following steps: 500 μg of aluminum hydroxide adjuvant was mixed with 50 μg of VZV-gE protein; Sterile PBS buffer with a pH of 7.5 was added to make the solution 500 μl, and the solution was adsorbed by blowing under light-proof conditions to obtain a recombinant herpes zoster vaccine composition.

[0041] Immunization group 4: 50 μg protein gE + 50% (v / v) squalene water-in-oil adjuvant.

[0042] A method for preparing a recombinant herpes zoster vaccine composition comprises the following steps: 250 μl of squalene oil-in-water adjuvant was mixed with 50 μg of VZV-gE protein; Sterile PBS buffer with a pH of 7.5 was added to make the solution 500 μl, and the solution was adsorbed by blowing under light-proof conditions to obtain a recombinant herpes zoster vaccine composition.

[0043] Immunization group 5: 50 μg protein gE + 100% (v / v) squalene water-in-oil adjuvant.

[0044] A method for preparing a recombinant herpes zoster vaccine composition comprises the following steps: 450 μl of squalene oil-in-water adjuvant was mixed with 50 μg of VZV-gE protein; Sterile PBS buffer with a pH of 7.5 was added to make the solution 500 μl, and the solution was adsorbed by blowing under light-proof conditions to obtain a recombinant herpes zoster vaccine composition.

[0045] Immunization group 6: 50μg protein gE + 25μg CpG1018.

[0046] A method for preparing a recombinant herpes zoster vaccine composition comprises the following steps: 25 μg of CpG1018 adjuvant was mixed with 50 μg of VZV-gE protein; Sterile PBS buffer with a pH of 7.5 was added to make the solution 500 μl, and the solution was adsorbed by blowing under light-proof conditions to obtain a recombinant herpes zoster vaccine composition.

[0047] Immunization group 7: 50μg protein gE + 100μg CpG1018.

[0048] A method for preparing a recombinant herpes zoster vaccine composition comprises the following steps: 100 μg of CpG1018 adjuvant was mixed with 50 μg of VZV-gE protein; Sterile PBS buffer with a pH of 7.5 was added to make the solution 500 μl, and the solution was adsorbed by blowing under light-proof conditions to obtain a recombinant herpes zoster vaccine composition.

[0049] Immunization group 8: 50μg protein gE + 400μg CpG1018.

[0050] A method for preparing a recombinant herpes zoster vaccine composition comprises the following steps: 400 μg of CpG1018 adjuvant was mixed with 50 μg of VZV-gE protein; Sterile PBS buffer with a pH of 7.5 was added to make the solution 500 μl, and the solution was adsorbed by blowing under light-proof conditions to obtain a recombinant herpes zoster vaccine composition.

[0051] The specific operation of the immunogenicity evaluation method is as follows: The number of mice used in each group was 6, and each mouse was immunized twice, with the immunization time points being 0 days and 28 days, respectively. The single dosing volume was 50 μL / mouse, and the corresponding amount of the dosing preparation was accurately drawn for intramuscular injection. Before dosing each group, the dosing preparation was gently shaken manually to reduce the precipitation, and a small amount of the corresponding dosing preparation was drawn to rinse the syringe. The day of the first immunization was defined as the 0th day of the experiment. The mouse spleen organ was removed 56 days after dosing for the preparation of mouse spleen lymphocytes.

[0052] Mouse spleen lymphocyte preparation: On the 56th day after immunization, mouse spleen lymphocytes were prepared for enzyme-linked immunosorbent assay and flow cytometry detection.

[0053] Complete culture medium: Mix 90% RPMI1640 culture medium, 10% FBS and 1% double antibody and prepare it before use.

[0054] Spleen removal: mice were killed by cervical dislocation and soaked in 75% ethanol for 5 min. The spleen was removed from the mice under sterile environment.

[0055] Grinding: Grind the spleen in a cell sieve to collect spleen cells, and centrifuge at 300×g for 5 minutes at room temperature.

[0056] Lyse red blood cells: discard the supernatant, add RBC to resuspend the pellet, treat at 4°C for 5 minutes, centrifuge at 300×g for 5 minutes. Wash with serum-free medium, and finally resuspend the cells in 3 mL of complete medium.

[0057] Counting: Dilute 10 times for counting and determine the cell viability.

[0058] Culture: Fresh mouse spleen lymphocytes were cultured at 37°C and 5% CO 2 The cells were cultured overnight in a cell culture incubator and used for enzyme-linked immunosorbent spot assay (ELISPOT assay) and flow cytometry assay (ICS assay) to detect the immune response effect.

[0059] Test Example 1: VZV antigen-specific IgG titer test.

[0060] Enzyme-linked immunosorbent assay (ELISA assay) method for measuring VZV antigen-specific IgG titers: On the 56th day after immunization, serum samples were collected from the orbital venous plexus or submandibular vein of mice and subjected to enzyme-linked immunosorbent assay to confirm the VZV antigen-specific IgG titer.

[0061] Coating: Dilute the antigen protein with alkaline coating buffer and add it to the ELISA plate at 4°C overnight.

[0062] Blocking: On the second day, discard the coating solution, wash the ELISA plate with PBST, pat dry, add blocking solution, and incubate at 37℃ for 1h.

[0063] Serum binding: discard the blocking solution, wash the ELISA plate with PBST, pat dry, and mark the plate with group numbers. Add the diluted serum to the corresponding well plate and incubate at 37℃ for 1 hour.

[0064] Secondary antibody incubation: discard the serum diluent, wash the ELISA plate with PBST and pat dry, add the diluted secondary antibody, and incubate at 37℃ for 1h.

[0065] Color development: discard the secondary antibody, wash the ELISA plate with PBST and pat dry, add the color developing solution, and let it stand at room temperature away from light for 5 minutes. When the color of the well plate shows an obvious gradient, add the stop solution to terminate the reaction.

[0066] Reading: Detect OD450 reading using a microplate reader within 15 minutes after adding the stop solution.

[0067] Data processing: 2.1 times of the negative OD value was taken as the cutoff, and the maximum dilution of serum with an OD value greater than the cutoff was the antibody titer.

[0068] The VZV-gE antigen-specific IgG titer was measured according to the above method. As shown in Table 2, compared with the positive control group and other experimental groups, the expression level of gE antigen-specific IgG was increased after the second immunization. The results show that at the humoral immune response level, the combination of gE protein and squalene water-in-oil adjuvant can effectively promote B lymphocyte response to enhance the production of specific IgG antibodies, effectively prevent the recurrence of herpes zoster, and play an important role in preventing herpes zoster.

[0069] Table 2: VZV-gE antigen-specific IgG titer (ELISA) Group IgG(GMT) 1 ＜1:50 2 ＜1:500 3 1：64800 4 1：113400 5 1：129600 6 1：59400 7 1：75600 8 1：108000 Test Example 2: VZV antigen-specific cell-mediated immune response assay.

[0070] Enzyme-linked immunospot assay (ELISPOT assay) method for measuring VZV antigen-specific cell-mediated immune responses: On day 56 after immunization, ELISpot assay was performed to confirm VZV antigen-specific cell-mediated immune responses.

[0071] Incubation: Add peptide stimulation mixture, non-stimulation control and positive stimulation according to the plate layout. Dilute the isolated mouse spleen lymphocytes according to the counting results, and add the diluted samples to the ELISPOT plate. Set up 2 peptide stimulation wells, 2 non-stimulation wells and 2 positive stimulation wells for each sample as parallel controls. Incubate the cells at 37°C, 5% CO 2 Culture in the cell culture incubator for about 36 hours. Do not move the ELISPOT plate during this period.

[0072] Secondary antibody binding: After dilution, add the specific detection antibody (secondary antibody) to the ELISPOT plate and place it at room temperature for 2 hours. Discard the liquid in the plate and wash it with PBS 5 times.

[0073] Enzyme conjugate: Add the diluted enzyme conjugate to the ELISPOT plate and leave at room temperature for 1 hour.

[0074] Color development: Filter the substrate with a 0.45 μm filter membrane, add it to each well, react for 5 minutes and obvious spots will appear. Rinse with a large amount of distilled water to terminate the reaction.

[0075] Spot counting: After the ELISPOT plate is dried, spot counting analysis is performed using an ELISPOT image analysis system.

[0076] VZV-gE antigen-specific cell-mediated immune responses were measured according to the above method, and the experimental results are shown in Figure 5 and Figure 6 . Figure 6 Figure a is the negative control, Figure b is the IFN-γ immune response level of gE (50 μg) + squalene oil-in-water adjuvant (250 μl) in Group 4; Figure c is the IL-2 immune response level of gE (50 μg) + squalene oil-in-water adjuvant (250 μl) in Group 4. Figure 5 The results showed that compared with the negative control group and other experimental groups, the gE antigen-specific cell-mediated immune response in the example after the second immunization was significantly improved. The results showed that at the level of cellular immune response, gE and squalene water-in-oil adjuvants can effectively promote Th1 cell response to enhance the secretion of cytokines IFN-γ and IL-2. IL-2 can effectively activate CD4+ and CD8+ T cells and form memory phenotypes, promote the differentiation of CD4+ T cells into Th1 and Th2 cells, and IFN-γ promotes the killing of herpes zoster virus. The coordinated action of the two prevents the reactivation of the virus, controls the intracellular infection of the virus, and maintains the latent state of the virus, playing an important role in preventing the occurrence of herpes zoster.

[0077] Test Example 3: VZV-gE antigen-specific cell-mediated cytokine secretion assay.

[0078] Method for determining lymphocyte subsets and their cytokine profiles by flow cytometry assay (ICS assay): Culture: Fresh mouse spleen lymphocytes were cultured at 37°C and 5% CO 2 Culture in a cell culture incubator overnight.

[0079] Add stimuli: Use culture medium to prepare VZV-gE peptide stimulation mixture, non-stimulation control, and positive stimuli, where the concentration of VZV-gE peptide library is 1 μg / mL. Add the stimuli to the wells with cells, mix well, and incubate at 37°C and 5% CO 2 Culture in a cell culture incubator.

[0080] Blocking: After stimulation, the blocking agent is diluted in culture medium and added to the cells, which are then cultured in a cell culture incubator.

[0081] Live / Dead staining: transfer the cells to an EP tube, add PBS, centrifuge at 600×g for 5 min, discard the supernatant, dilute the Live / Dead staining antibody with PBS, resuspend the cells, mix well, and place at room temperature away from light for 20 min.

[0082] Surface staining: Add PBS, centrifuge at 600×g for 5 min at room temperature, and discard the supernatant. Dilute each surface fluorescent labeling antibody (CD3 antibody, CD4 antibody, CD8 antibody) with PBS, resuspend the cells, mix well, and place at room temperature away from light for 60 min.

[0083] Fixation: Dilute IC fixation buffer with PBS, resuspend cells, place at room temperature in the dark for 60 min, centrifuge at 600 × g for 5 min at room temperature, and discard supernatant.

[0084] Intracellular staining: dilute the permeabilization solution into 1× working solution with PBS, prepare each intracellular fluorescent-labeled antibody (IFN-γ antibody, IL-2 antibody, TNF-α antibody), mix well and gently resuspend the cells, and place at room temperature away from light for 60 minutes.

[0085] Washing: Wash and detect the distribution of T cells and corresponding cytokine secretion caused by antigen stimulation.

[0086] The VZV-gE antigen-specific cell-mediated lymphocyte subsets and their cytokine secretion were measured according to the above method. The experimental results are shown in Figure 7 , Figure 8 .

[0087] like Figure 7As shown, compared with the negative control group, when the adjuvant is only squalene oil-in-water adjuvant or CpG, the prepared recombinant herpes zoster vaccine composition can produce a certain level of induction of IFN-γ, TNF-α, and IL-2, among which the immune activity of the recombinant herpes zoster vaccine composition prepared when the squalene oil-in-water adjuvant is 250μl is better.

[0088] like Figure 8 Shown are the percentages of CD4+ cells in the lymphocyte population relative to the total cell population that secrete IFN-γ (IFN-γ+), IL-2 (IL-2+), and both cytokines (IFN-γ+IL-2+) after mouse splenocyte stimulation.

[0089] After the second immunization, the number of cytokines secreted by CD4+T lymphocytes mediated by gE antigen-specific cells in the embodiment was increased. The results show that at the level of cellular immune response, the combination of gE and squalene water-in-oil adjuvant can effectively promote Th1 cell response to enhance the secretion of cytokines IL-2, IFN-γ and TNF-α, activate CD4+ and CD8+T cells and form memory phenotypes, promote the differentiation of CD4+T cells into Th1 and Th2 cells, prevent virus reactivation, and play an important role in preventing herpes zoster.

[0090] VZV-specific cellular immunity is the main determinant of the risk and severity of herpes zoster. Antigen-specific T cells can inhibit VZV reactivation in infected cells, so cellular immunity can control the incidence of herpes zoster. Therefore, when analyzing the test data, we focus on the expression of cytokines such as INF-γ and IL-2 obtained by ELISPOT and flow cytometry (ICS) tests, which reflect the level of cell-mediated immune response.

[0091] The embodiments are all recombinant herpes zoster vaccine compositions in different combinations proposed by the present invention. In the embodiments, VZV-gE protein was used in combination with squalene oil-in-water adjuvant, showing a significant improvement in immunogenicity and cytokine expression. It can be seen from the comparative examples that the immune effects of aluminum hydroxide adjuvant and CpG adjuvant alone are lower than those of squalene oil-in-water adjuvant. This may be because the combination of squalene oil-in-water adjuvant can more comprehensively activate different immune cells and enhance antigen presentation.

[0092] Furthermore, in the recombinant herpes zoster vaccine composition of the present invention, a further technical solution is provided for introducing immunomodulators such as chemokines to further activate the immune system and enhance immune memory.

[0093] Immunization group 9, a method for preparing a recombinant herpes zoster vaccine composition, comprising the following steps: Mix 250 μl of squalene oil-in-water adjuvant and 50 μg of VZV-gE protein; Then mixed with 10 μg of chemokine CCL3 / MIP-1α; Sterile PBS buffer with a pH of 7.5 was added to make the solution 500 μl, and the solution was adsorbed by blowing under light-proof conditions to obtain a recombinant herpes zoster vaccine composition.

[0094] Immunization group 10, a method for preparing a recombinant herpes zoster vaccine composition, comprising the following steps: Mix 250 μl of squalene oil-in-water adjuvant and 50 μg of VZV-gE protein; Then mixed with 10 μg of chemokine CXCL10 / IP-10; Sterile PBS buffer with a pH of 7.5 was added to make the solution 500 μl, and the solution was adsorbed by blowing under light-proof conditions to obtain a recombinant herpes zoster vaccine composition.

[0095] Immunization group 11, a method for preparing a recombinant herpes zoster vaccine composition, comprising the following steps: Mix 250 μl of squalene oil-in-water adjuvant and 50 μg of VZV-gE protein; Then mixed with 5 μg of chemokine CCL3 / MIP-1α and 5 μg of chemokine CXCL10 / IP-10; Sterile PBS buffer with a pH of 7.5 was added to make the solution 500 μl, and the solution was adsorbed by blowing under light-proof conditions to obtain a recombinant herpes zoster vaccine composition.

[0096] Table 3: VZV-gE antigen-specific IgG titer (ELISA) Group IgG titer after first dose Second dose IgG titer Immunization group 4 1：4800 1：113400 Immunization group 9 1：6400 1：174960 Immunization group 10 1：18000 1：189540 Immunization group 11 1：48600 1：204120 Table 4: VZV-gE antigen-specific cell-mediated immune response (ELISPOT) Group IFN-γ (per 200,000 spleen cells) IL-2 (per 200,000 spleen cells) Immunization group 4 16 113 Immunization group 9 60 146 Immunization group 10 63 153 Immunization group 11 102 195 Table 5: VZV-gE antigen-specific cell-mediated immune response (ICS) Group IFN-γ / %(Parent proportion) TNF-α(Parent proportion) IL-2 (Parent proportion) Immunization group 4 0.004 0.215 0.208 Immunization group 9 0.101 0.318 0.304 Immunization group 10 0.135 0.312 0.298 Immunization group 11 0.153 0.336 0.322 As can be seen from the data in Table 3, the introduction of chemokines (CCL3 / MIP-1α, CXCL10 / IP-10, and a combination of the two) into immune groups 9-11 all showed an improvement effect relative to immune group 4, which indicates that the introduction of chemokines can effectively increase the IgG titer. In particular, immune group 11, which contains both CCL3 / MIP-1α and CXCL10 / IP-10, showed a higher IgG titer than other examples, indicating that the combined use of the two chemokines synergistically activates the immune system through different mechanisms, thereby producing a synergistic effect and leading to a stronger humoral immune response.

[0097] As can be seen from the data in Table 4, with the introduction of chemokines, the production of IFN-γ and IL-2 increased, with immune group 11 again showing the highest level. This suggests that the vaccine formulation of immune group 11 is able to effectively activate cell-mediated immune responses, especially in promoting Th1-type immune responses. This is consistent with the role of CCL3 / MIP-1α and CXCL10 / IP-10 in promoting the migration of Th1 cells and other immune cells to the site of infection.

[0098] From the data in Table 5, it can be seen that the introduction of chemokines (CCL3 / MIP-1α, CXCL10 / IP-10, and the combination of the two) in immune groups 9-11 all showed an improvement effect relative to immune group 4, which shows that the introduction of chemokines can effectively enhance the immune response. Immune group 11 has an advantage in activating cell-mediated immune responses, showing the highest expression of IFN-γ, TNF-α, and IL-2. This shows that immune group 11 can not only promote Th1-type immune responses, but also activate a wide range of cell-mediated immune functions, including enhancing the expression of TNF-α and IL-2, which are key factors in the immune system's resistance to infection and disease.

[0099] The combination of CCL3 / MIP-1α and CXCL10 / IP-10 showed a significant synergistic effect in immune group 11, because these two chemokines activate the immune system through different mechanisms, resulting in complementary and enhanced immune responses. CCL3 / MIP-1α mainly attracts a wide range of immune cells, such as macrophages and T cells, to the site of infection or vaccination through its receptors CCR1 and CCR5, thereby promoting a strong initial immune response and enhanced immune memory formation. CXCL10 / IP-10, through its specific receptor CXCR3, specifically promotes the migration and activation of Th1 cells, which are essential for the formation of cell-mediated immune responses against pathogens. When these two chemokines work together, they not only recruit a broader and more diverse population of immune cells, but also specifically enhance the immune defense against specific pathogens. Therefore, the combination of CCL3 / MIP-1α and CXCL10 / IP-10 achieves a comprehensive activation of the immune system, including the enhancement of humoral and cell-mediated immune responses, particularly by promoting the production of IFN-γ, TNF-α, and IL-2, which are critical for protection against infection and disease.

[0100] The present invention provides a recombinant herpes zoster vaccine composition and its preparation method and application ideas and methods. There are many methods and approaches to specifically implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant herpes zoster vaccine composition, characterized in that: The invention comprises gE protein and adjuvant, wherein the adjuvant is squalene oil-in-water adjuvant, and the squalene oil-in-water adjuvant comprises the following concentration components: squalene: 4-5wt%; Tween 80: 0.2-0.8wt%; Span 85: 0.2-0.8wt%.

2. The recombinant herpes zoster vaccine composition according to claim 1, characterized in that The content of the gE protein is 20-100 μg / mL.

3. The recombinant herpes zoster vaccine composition according to claim 2, characterized in that: The preparation method of the gE protein is as follows: Construction of PLK002-22P20-A2: Using PLK002 as an empty vector, the antigen DNA sequence signal peptide and gE protein component coding were connected into the expression empty vector to construct the PLK002-22P20-A2 expression vector; Using CHO K1BN suspension cells as host cells, the expression vector of PLK002-22P20-A2 constructed above was transferred into the host cells by electroporation. After transfection, the cells were screened by pressure screening, step-by-step amplification, ELISA and SEC-HPLC to screen out high-expressing monoclonal strains, and then cultured continuously. The culture supernatant was harvested by centrifugation and purification, and the collected supernatant was obtained by final sample, i.e., gE protein.

4. The recombinant herpes zoster vaccine composition according to claim 3, characterized in that: The construction method of PLK002-22P20-A2 is as follows: Construction of PLK002-22P20-A2: Using PLK002 as an empty vector, adding a HindIII restriction site and a KOZAK sequence at the front end of the signal peptide of the antigen DNA sequence, and adding a stop codon and a Not I restriction site at the back end; recovering the vector and the target fragment separately and connecting them, sequencing and enzyme digestion identification were performed through the plasmid DNA of the positive transformants, and the PLK002-22P20-A expression vector was successfully constructed; adding a KOZAK sequence at the front end of the signal peptide of the antigen DNA sequence, and adding a stop codon at the back end, and replacing the DNA sequence from HindIII to Not I on the PLK002 vector with 22P20-A2 by the homologous recombination method, sequencing and enzyme digestion identification were performed through the positive transformants, and finally PLK002-22P20-A2 was successfully constructed.

5. The recombinant herpes zoster vaccine composition according to claim 1, characterized in that: The diameter of the squalene oil-in-water adjuvant droplets is 150-180 nm.

6. The recombinant herpes zoster vaccine composition according to claim 1, characterized in that The volume of the squalene oil-in-water adjuvant is 25-50%.

7. The recombinant herpes zoster vaccine composition according to claim 1, characterized in that The method further comprises a buffer, which is at least one of a PBS buffer, a Tris-HCl buffer and a citrate buffer.

8. The method for preparing the recombinant herpes zoster vaccine composition according to any one of claims 1 to 7, characterized in that: The squalene oil-in-water adjuvant is mixed with the gE protein, and then a buffer solution is added, and the mixture is blown and adsorbed under light-proof conditions to obtain a recombinant herpes zoster vaccine composition.

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