A recombinant herpes zoster vaccine composition and a method for preparing the same

Abstract

The present application relates to the field of biological medicine, and particularly relates 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 squalene oil-in-water, and the squalene oil-in-water adjuvant comprises the following concentration components: squalene: 4-5 wt%; Tween 80: 0.2-0.8 wt%; Span 85: 0.2-0.8 wt%. The vaccine composition can specifically enhance the cellular immune response to varicella-zoster virus, can significantly improve the expression of cytokines such as INF-gamma and IL-2 of CD4+ T cells in mice, and can improve the level of humoral and cellular immune response, shows high immunocompetence for preventing varicella-zoster virus infection in clinic, and the components in the vaccine composition are cheap and easy to obtain, effectively improve the yield of the vaccine, and reduce the cost of the vaccine.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to a recombinant varicella-zoster vaccine composition and a preparation method thereof. BACKGROUND

[0002] Varicella-zoster virus (VZV) is a pathogen widely present in the global population, infecting more than 90% of the world's population. Primary infection of VZV often occurs in childhood, which can be characterized by varicella with systemic vesicular rash, and latent in the host's neuronal cells after infection. With age, immune function decline 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 has been basically controlled, but HZ is a common phenomenon in many elderly people. For people with primary infection of VZV and low immunity or reactivation of VZV, complications such as meningitis, stroke, and post-HZ neuralgia often occur, which reduces the quality of life of patients and even endangers life.

[0003] Globally, herpes zoster disease-related vaccines 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 reduced with age, while the latter plays a high protective role in all age groups and can maintain for a long time. At present, the international approved HZ vaccine is Zostavax of Merck Company, which has a preventive effect of 69.8% (50-59 years old) and 51% (over 60 years old) on HZ. In addition, there is a recombinant subunit herpes zoster vaccine Shringrix of GSK, which is composed of herpes zoster virus gE protein and adjuvant system AS01B. AS01B adjuvant system is a liposome adjuvant containing monophosphoryl lipid A and saponin QS-21, which can enhance the cellular immune response. Clinical studies have shown that the Shringrix vaccine can significantly reduce the risk of HZ in people over 50 years old (91% to 97%), and can effectively reduce the risk of subsequent PHN (≥80%), and studies have found that the reason why Shringrix can form long-term protection in the elderly population may be related to its ability to induce higher levels of IL-2 and IFN-γ. Subunit herpes zoster vaccine has a huge market prospect in the domestic market.

[0004] With the development and progress of biotechnology in the field of vaccine industry, there are currently nine kinds of VZV viral surface glycoproteins identified, among which the gE glycoprotein is the highest expression glycoprotein of VZV, which plays a major role in the replication and assembly of the virus, and also mediates the transmission of the virus between cells. Since 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 vaccine and DNA vaccine.

[0005] At present, there are more than one hundred kinds of adjuvants that have been confirmed and used:

[0006] 1. Inorganic salt adjuvant

[0007] Mainly aluminum and magnesium salt compounds, which are the earliest discovered and most widely used vaccine adjuvants. A large number of clinical trials have shown that aluminum adjuvants can stimulate the body to produce strong humoral immunity, but can cause injection site reactions and have weak ability to induce cellular immunity. Aluminum salts mainly include aluminum phosphate, aluminum hydroxide and aluminum sulfate. The commonly used aluminum adjuvants are aluminum hydroxide and aluminum phosphate. The mechanism of action of aluminum adjuvants mainly includes depot effect and immune stimulation effect. Aluminum adjuvants can also stimulate the release of DNA, ATP and uric acid and other damage-associated molecular patterns (DAMPs) from necrotic cells at the injection site, and the released DAMPs help the interaction of dendritic cells and CD4+ T cells. So far, aluminum adjuvants are still the only widely used adjuvant in vaccines. However, aluminum adjuvants have great defects: they are usually effective in increasing serum antibodies, but can cause injection site reactions and have weak ability to induce cellular immunity, which limits their application to some extent.

[0008] 2. Oil-water emulsion adjuvant

[0009] Oil adjuvants are traditional adjuvant types, among which the most famous is Freund's adjuvant, which is currently widely used in animal inactivated vaccines, but has high toxicity. In recent years, an emulsion adjuvant similar to Freund's adjuvant has been developed, such as squalene oil-in-water adjuvant, AS03 and AF03, etc. The immunogenicity of the immunogen alone is relatively poor, and the dosage needs to be increased to increase the immunogenicity. However, increasing the dosage of the adjuvant will face the problem of greater side effects. The side effects of oil emulsion mainly include strong pain, inflammatory reactions, granulomas and ulcers formed at the vaccination site, etc.

[0010] 3. Adjuvants targeting pattern recognition receptors

[0011] Bacteria, fungi, viruses and some components in plants can activate innate immunity by targeting pattern recognition receptors, thus becoming potential vaccine adjuvants. For example, 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, induration, and red rash at the injection site, and systemic symptoms such as flu-like symptoms.

[0012] 4. Cytokine adjuvants

[0013] Cytokine adjuvants mainly regulate the type of response in Th1 / Th2 cell immune process, and also affect intercellular signal transmission and stimulation signals. The earliest cytokine used as an adjuvant, IL-1, can enhance the primary and secondary responses to antigens, induce enhanced antigen-specific T cell activity 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

[0014] The present application focuses on providing a new generation of recombinant herpes zoster preventive vaccine, which contains varicella zoster virus (VZV) antigens. According to the existing research on VZV-gE protein, the wild-type VZV-gE protein molecule includes four regions, i.e. signal peptide, extracellular region, transmembrane region and intracellular region, and the three antigenic determinants of VZV-gE are all distributed in the extracellular region. Considering the influence of the hydrophobic transmembrane region on the expression of exogenous proteins and other factors, the expression level of the protein can be improved by removing the transmembrane region (hydrophobic region) and the intracellular region (for example, patent CN102517302A).

[0015] In addition, the technical problem to be solved by the present application is to further enhance the preventive effect, improve the immune memory ability, reduce complications and sequelae, selectively enhance the cell-mediated immune response, and at the same time avoid the risks existing in attenuated live vaccines, and to provide a herpes zoster recombinant protein vaccine adjuvant formula.

[0016] The specific technical scheme of the present application is:

[0017] A recombinant herpes zoster vaccine composition, comprising gE protein and an adjuvant, wherein the adjuvant is squalene oil-in-water adjuvant, and the squalene oil-in-water adjuvant comprises the following components at the following concentrations: squalene: 4-5 wt%; Tween 80: 0.2-0.8 wt%; Span 85: 0.2-0.8 wt%.

[0018] The sequence number of the gE protein is as follows:

[0019] SVLRYDDFHTDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYD GFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVD QRQYGDVFKGDLNPKPQGQRMIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTG DAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLF DELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAV TPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRL YSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSF GLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTA GQPPATTKPKEITPVNPGTSPLLRY.

[0020] In an embodiment, the squalene oil-in-water adjuvant comprises the following concentration of components: squalene: 4.3 wt%; Tween 80: 0.5 wt%; Span 85: 0.5 wt%, with the addition of citric acid buffer for adjusting the pH value to 6.5.

[0021] Preferably, the gE protein is present in an amount of 20-100 μg / mL.

[0022] Preferably, the gE protein is prepared by the following method:

[0023] Construction of PLK002-22P20-A2: PLK002 is used as a blank vector, a Hind III enzyme cutting site and a KOZAK sequence are added at the front of the signal peptide of the antigen DNA sequence, and a stop codon and a Not I enzyme cutting site are added at the rear; after recovering the vector and the target fragment, respectively, the connection is carried out, and the positive transformant plasmid DNA is sequenced and enzyme cut identified, and the PLK002-22P20-A expression vector is successfully constructed; a KOZAK sequence is added at the front of the signal peptide of the antigen DNA sequence, and a stop codon is added at the rear, and 22P20-A2 is replaced by the DNA sequence from Hind III to Not I on the PLK002 vector by homologous recombination, and the positive transformant is sequenced and enzyme cut identified, and finally the PLK002-22P20-A2 is successfully constructed.

[0024] The expression vector of PLK002-22P20-A2 constructed above is transferred into the host cell by electroporation, and the high expression monoclonal strain is screened out by pressure screening, step-by-step amplification, ELISA and SEC-HPLC after transfection, and the culture supernatant is harvested by centrifugal filtration, and the collected supernatant is clarified by filtration, first anion chromatography, low pH incubation + depth filtration, hydrophobic chromatography, first ultrafiltration liquid exchange, second depth filtration, second anion chromatography, second ultrafiltration liquid exchange, nanofiltration, dilution and primary solution sterilization filtration, to obtain the final sample, i.e. gE protein.

[0025] Preferably, the squalene water-in-oil adjuvant droplet diameter is 150-180nm; preferably, the squalene water-in-oil adjuvant droplet diameter is 160nm.

[0026] Preferably, the squalene water-in-oil adjuvant volume is 25-50%.

[0027] Preferably, it further comprises a buffer, and the buffer is at least one of PBS buffer, Tris-HCl buffer and citrate buffer.

[0028] Preferably, it further comprises a chemical chemotactic factor; the chemical chemotactic factor is added in an amount of 5-15μg / mL.

[0029] Further, the chemical chemotactic factor is at least one of CCL3 / MIP-1α and CXCL10 / IP-10, and more further, the chemical chemotactic factor is a combination of 30-70wt% CCL3 / MIP-1α and 30-70wt% CXCL10 / IP-10.

[0030] The addition of the chemotactic factors CCL3 / MIP-1a and / or CXCL10 / IP-10 helps to 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 modulating the chemotaxis and activation status of immune cells, promoting Th1-type immune responses, cell-mediated immune defense is facilitated, while maintaining a certain degree of Th2 response to promote antibody production. Enhanced immune cell activation and optimized Th1 / Th2 balance help to form a strong immune memory, providing a basis for long-term protection.

[0031] The preparation method of the recombinant herpes zoster vaccine composition mixes squalene oil-in-water adjuvant with gE protein, then adds a buffer solution, and blows and adsorbs in the dark to obtain the recombinant herpes zoster vaccine composition.

[0032] Beneficial technical effects: Compared with the prior art, the present application has the following advantages:

[0033] 1、The recombinant herpes zoster vaccine composition of the present application can improve the expression level of gE antigen-specific IgG after secondary immunization, and the combination of gE protein and squalene oil-in-water 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.

[0034] 2、The recombinant herpes zoster vaccine composition of the present application can effectively promote Th1 cell response to enhance the secretion of cytokines IFN-γ and IL-2 at the level of cellular immune response, IL-2 can effectively activate CD4+ and CD8+ T cells and form memory phenotype, promote the differentiation of CD4+ T cells to Th1 and Th2 cells, IFN-γ can promote the killing of herpes zoster virus, and the coordinated action of the two can prevent virus reactivation, control intracellular infection of virus and maintain latent state of virus.

[0035] 3、The recombinant herpes zoster vaccine composition of the present application uses VZV-gE protein in combination with squalene oil-in-water adjuvant, which shows a significant improvement in immunogenicity and cytokine expression, and has better immune effect than aluminum hydroxide adjuvant and CpG adjuvant alone.

[0036] 4、The recombinant zoster vaccine composition of the present application also comprises the chemotactic factors CCL3 / MIP-1a and / or CXCL10 / IP-10, which, through their specific receptor CXCR3, in particular facilitate the migration and activation of Th1 cells, which are essential for the development of a cell-mediated immune response against pathogens. When these two chemokines act together, they not only recruit a more extensive and diverse population of immune cells, but also particularly enhance the immune defense capacity against specific pathogens. Thus, the combination of CCL3 / MIP-1a and CXCL10 / IP-10 achieves a comprehensive activation of the immune system, including the strengthening of both humoral and cell-mediated immune responses, in particular by promoting the production of IFN-γ, TNF-α and IL-2, which are essential for the resistance to infection and disease.

[0037] 5、The present application makes full use of existing detection techniques, and can efficiently, accurately and stably respond to the immune response level, thereby providing good support for adjuvant formula screening. BRIEF DESCRIPTION OF DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0039] Figure 1 The PLK002-22P20-A2 plasmid map is constructed;

[0040] Figure 2 The plasmid enzyme digestion map is constructed;

[0041] Figure 3 The protein purification process flow chart is constructed;

[0042] Figure 4 The gE protein purification result chart is constructed;

[0043] Figure 5 The gE specific cell-mediated cellular immunity level comparison chart is constructed;

[0044] Figure 6 The gE specific cell-mediated immune response level (ELISPOT) chart is constructed;

[0045] Figure 7 The single adjuvant prescription flow cytometry detection CD4+T cell reaction result chart is constructed;

[0046] Figure 8Figure 1 shows the levels of IFN-γ, TNF-α, IL-2 immune response mediated by gE specific cells (ICS). DETAILED DESCRIPTION

[0047] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person of ordinary skill in the art. And the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory operation steps used herein are the terms and conventional steps widely used in the corresponding field.

[0048] The technical solutions of the present application will be further described in detail below in combination with specific examples. It should be understood that the following examples are only illustratively described and explained the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope intended to be protected by the present application.

[0049] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the raw materials and reagents are commercially available, or can be prepared by known methods.

[0050] Example 1: Production of gE protein, molecular A2 is a recombinant protein antigen against varicella-zoster virus, and the DNA sequence is optimized according to the CHO cell amino acid sequence and the signal peptide is optimized. PLK002 is selected as a blank vector, a HindIII enzyme cutting site (AAGCTT) and a KOZAK sequence (GCCGCCACC) are added at the front end of the optimized antigen DNA sequence signal peptide, and a termination codon (TGATAA) and a NotI enzyme cutting site (GCGGCCGC) are added at the rear end. The PLK002 and 22P20-A gene synthesis sequences are double enzyme cut by HindIII and NotI, and after recovering the vector and the target fragment respectively, they are connected, the positive transformants are screened by kanamycin resistance, the positive transformant plasmid DNA is sequenced and enzyme cut identified, and finally the PLK002-22P20-A expression vector is successfully constructed. A KOZAK sequence (GCCGCCACC) is added at the front end of the optimized antigen DNA sequence signal peptide, and a termination codon (TGATAA) is added at the rear end. The 22P20-A2 is replaced by the DNA sequence from HindIII to NotI on the PLK002 vector by homologous recombination method, the positive transformants are screened by kanamycin resistance, the positive transformant plasmid DNA is sequenced and enzyme cut identified (see Figure 2 ), and finally the PLK002-22P20-A2 (see Figure 1 ) is successfully constructed.

[0051] The CHOK1BN suspension cells were selected as the host cells, and the expression vector of PLK002-22P20-A2 successfully constructed above was transformed into the host cells by electroporation. After transfection, the cells were screened by pressure screening, gradually expanded, and screened by ELISA and SEC-HPLC to obtain a high-expression monoclonal strain with an expression amount of 2.96 g / L. The cell density and cell viability were monitored every day during continuous subculture, and the culture was stopped when the cell viability was lower than 80% after 10-12 days of culture. The culture supernatant was harvested by centrifugal filtration. The collected supernatant was subjected to clarification filtration, first anion chromatography, low-pH incubation + depth filtration, hydrophobic chromatography, first ultrafiltration liquid exchange, second depth filtration, second anion chromatography, second ultrafiltration liquid exchange, nanofiltration, dilution, and primary sterile filtration to obtain the final purified sample, i.e., gE protein. The process flow is shown in Figure 3 The purified gE protein is shown in Figure 4 The SEC-HPLC detection showed that the protein purity was >97%.

[0052] The sequence number of the prepared gE protein is as follows:

[0053] SVLRYDDFHTDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRMIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRY.

[0054] Table 1: PLK002-22P20-A2 vector key element information

[0055] Key Elements Name Location Function CMV enhancer Human cytomegalovirus early enhancer 124-503 Enhance transcription of gene of interest CMV promoter Human cytomegalovirus early promoter 504-707 Initiate transcription of gene of interest 5'UTR-intron-5'UTR elements 5'UTR-intron-5'UTR elements 722-1686 Improve translation efficiency 22P20-A2 22P20-A2 recombinant antigen 1696-3282 Encode gene of interest SV40 poly(A) signal SV40 poly(A) signal 3412-3546 Terminate transcription Ori Ori 3846-4434 Plasmid replication initiation AmpR promoter AmpR promoter 5421-5525 Initiate transcription of knaR gene KnaR Aph (3')-Ia (kanamycin resistance gene) coding region 4605-5420 Used for plasmid selection SV40 promoter SV40 promoter 5804-6133 Initiate transcription of gene of interest GS GS 6172-7296 Glutamine synthetase gene GS system selection marker SV40 intron and polyA 7348-8199 SV40 intron and polyA

[0056] Example 2: Screening of vaccine formulation and immunogenicity study, in this example, the gE protein prepared in Example 1 is used as immunogen, diluted with 30 mM phosphate buffer, 0.02% Tween 80 pH 7.5, mixed evenly with aluminum hydroxide, CpG1018 adjuvant, squalene oil-in-water adjuvant at room temperature, respectively prepared into aluminum adjuvant vaccine solution, CpG adjuvant vaccine solution and squalene oil-in-water adjuvant vaccine solution, and the immunogenicity study is carried out by using C57BL / 6 mice (purchased from Jiangsu Huacheng Xinnuo Pharmaceutical Technology Co., Ltd.) as animal model.

[0057] Immunization group 1: negative control, sterile PBS buffer.

[0058] Immunization group 2: gE protein solution.

[0059] Immunization group 3: 50 μg protein gE + 500 μg Al(OH) 3。

[0060] A preparation method of a recombinant herpes zoster vaccine composition, comprising the following steps:

[0061] Mix 500 μg of aluminum hydroxide adjuvant with 50 μg of VZV-gE protein;

[0062] Add sterile PBS buffer with pH 7.5 to the solution to 500 μl, and prepare the recombinant herpes zoster vaccine composition by blowing and adsorbing under light-proof conditions.

[0063] Immunization group 4: 50 μg protein gE + 50% (v / v) squalene oil-in-water adjuvant.

[0064] A preparation method of a recombinant herpes zoster vaccine composition, comprising the following steps:

[0065] Mix 250 μl of squalene oil-in-water adjuvant with 50 μg of VZV-gE protein;

[0066] Add sterile PBS buffer with pH 7.5 to the solution to 500 μl, and prepare the recombinant herpes zoster vaccine composition by blowing and adsorbing under light-proof conditions.

[0067] Immunization group 5: 50 μg protein gE + 100% (v / v) squalene oil-in-water adjuvant.

[0068] A preparation method of a recombinant herpes zoster vaccine composition, comprising the following steps:

[0069] Mix 450 μl of squalene oil-in-water adjuvant with 50 μg of VZV-gE protein;

[0070] The solution is supplemented with sterile PBS buffer at pH 7.5 to 500 μl, and the recombinant varicella vaccine composition is prepared by blowing and adsorbing under light-avoiding conditions.

[0071] Immune group 6: 50 μg protein gE + 25 μg CpG1018.

[0072] A preparation method of a recombinant varicella vaccine composition comprises the following steps:

[0073] 25 μg of CpG1018 adjuvant is mixed with 50 μg of VZV-gE protein;

[0074] The solution is supplemented with sterile PBS buffer at pH 7.5 to 500 μl, and the recombinant varicella vaccine composition is prepared by blowing and adsorbing under light-avoiding conditions.

[0075] Immune group 7: 50 μg protein gE + 100 μg CpG1018.

[0076] A preparation method of a recombinant varicella vaccine composition comprises the following steps:

[0077] 100 μg of CpG1018 adjuvant is mixed with 50 μg of VZV-gE protein;

[0078] The solution is supplemented with sterile PBS buffer at pH 7.5 to 500 μl, and the recombinant varicella vaccine composition is prepared by blowing and adsorbing under light-avoiding conditions.

[0079] Immune group 8: 50 μg protein gE + 400 μg CpG1018.

[0080] A preparation method of a recombinant varicella vaccine composition comprises the following steps:

[0081] 400 μg of CpG1018 adjuvant is mixed with 50 μg of VZV-gE protein;

[0082] The solution is supplemented with sterile PBS buffer at pH 7.5 to 500 μl, and the recombinant varicella vaccine composition is prepared by blowing and adsorbing under light-avoiding conditions.

[0083] The method for evaluating immunogenicity is specifically operated as follows:

[0084] The number of mice used in each group was 6, each mouse was immunized twice, the immunization time points were 0 days and 28 days, the single dose volume was 50 μL per mouse, and the corresponding amount of drug preparation was accurately extracted for intramuscular injection. Before administration of each group, the drug preparation was gently shaken by hand to reduce the generated precipitate, and a small amount of the corresponding drug preparation was extracted to rinse the syringe. The first immunization administration day was defined as the 0th day of the test. The mouse spleen was removed 56 days after administration for mouse spleen lymphocyte preparation.

[0085] Preparation of mouse spleen lymphocytes:

[0086] After 56 days of immunization, mouse spleen lymphocytes were prepared for enzyme-linked immunosorbent assay and flow cytometry detection.

[0087] Complete medium: 90% RPMI1640 medium, 10% FBS and 1% double antibody were mixed and prepared immediately before use.

[0088] Spleen removal: the mouse was sacrificed by cervical dislocation, soaked in 75% ethanol for 5 min, and the mouse spleen was removed in a sterile environment.

[0089] Grinding: After grinding and filtering the spleen in the cell screen, the spleen cells were collected, and centrifuged at 300 x g for 5 min at room temperature.

[0090] Red blood cell lysis: discard the supernatant, add RBC resuspension sediment, treat at 4°C for 5 min, centrifuge at 300 x g for 5 min. Wash with serum-free medium, and finally resuspend the cells with 3 mL of complete medium.

[0091] Counting: dilute 10 times for counting and determine the cell viability.

[0092] Culture: Fresh mouse spleen lymphocytes were cultured overnight at 37°C in a 5% CO2 cell incubator for enzyme-linked immunospot assay (ELISPOT assay) and flow cytometry assay (ICS assay) to detect immune response effect.

[0093] Test Example 1: VZV antigen-specific IgG titer test.

[0094] Method for measuring VZV antigen-specific IgG titer by enzyme-linked immunosorbent assay (ELISA assay):

[0095] After 56 days of immunization, the mouse orbital plexus or submandibular vein serum sample was collected for enzyme-linked immunosorbent assay to confirm the VZV antigen-specific IgG titer.

[0096] Coating: dilute the antigen protein with alkaline coating buffer and add it to the enzyme-labeled plate, and incubate at 4°C overnight.

[0097] Blocking: The next day, the plate was washed with PBST and dried, and blocking solution was added. The plate was incubated at 37°C for 1 h.

[0098] Serum binding: The blocking solution was discarded, and the plate was washed with PBST and dried. The plate was labeled with group and serial number. The diluted serum was added to the corresponding well, and the plate was incubated at 37°C for 1 h.

[0099] Secondary antibody incubation: The serum dilution was discarded, and the plate was washed with PBST and dried. The diluted secondary antibody was added, and the plate was incubated at 37°C for 1 h.

[0100] Color development: The secondary antibody was discarded, and the plate was washed with PBST and dried. The color developing solution was added, and the plate was incubated at room temperature for 5 min. When the color gradient of the well was obvious, the stop solution was added to stop the reaction.

[0101] Reading: The OD450 reading was detected by an enzyme-labeled instrument within 15 min after the addition of the stop solution.

[0102] Data processing: The OD value of 2.1 times the negative value was used as the cutoff, and the maximum dilution of the serum with an OD value greater than the cutoff was the antibody titer.

[0103] The VZV-gE antigen-specific IgG titer was measured according to the above method. As shown in Table 2, the expression level of gE antigen-specific IgG after secondary immunization was increased compared with the positive control group and other experimental groups. The results showed that, at the level of humoral immune response, the combination of gE protein and squalene oil-in-water adjuvant can effectively promote the 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.

[0104] Table 2: VZV-gE antigen-specific IgG titer (ELISA)

[0105] Terminate transcription Group 1 ＜1:50 2 ＜1:500 3 1：64800 4 1：113400 5 1：129600 6 1：59400 7 1：75600 8 1：108000

[0106] Test Example 2: VZV antigen-specific cell-mediated immune response assay.

[0107] Method for measuring VZV antigen-specific cell-mediated immune response by enzyme-linked immunospot assay (ELISPOT assay):

[0108] On the 56th day after immunization, enzyme-linked immunospot assay was performed to confirm the VZV antigen-specific cell-mediated immune response.

[0109] Incubation: According to the plate layout, add the peptide stimulation mixture, non-stimulation control and positive stimulant respectively. Dilute the separated mouse spleen lymphocytes according to the counting results, and add the diluted sample into the ELISPOT plate, with 2 peptide stimulation holes, 2 non-stimulation holes and 2 positive stimulation holes for each sample, and set up parallel controls. Incubate the cells in a 5% CO2 cell incubator at 37°C for about 36 hours, and do not move the ELISPOT plate during this period.

[0110] 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 for 5 times.

[0111] Enzyme coupling: Add the diluted enzyme conjugate to the ELISPOT plate, and place it at room temperature for 1 hour.

[0112] Color development: Filter the substrate with a 0.45 μm filter membrane, and add it to each hole, and obvious spots will appear after 5 minutes of reaction. Wash with a large amount of distilled water to terminate the reaction.

[0113] Spot counting: After the ELISPOT plate is dried, use an enzyme-linked immunospot image analysis system to count and analyze the spots.

[0114] According to the above method, the VZV-gE antigen-specific cell-mediated immune response was measured, and the experimental results are shown in IgG (GMT) and Figure 5 . 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) of group 4, and figure c is the IL-2 immune response level of gE (50 μg) + squalene oil-in-water adjuvant (250 μl) of group 4. Figure 6 The results show that the gE antigen-specific cell-mediated immune response in the example after the second immunization is significantly improved compared with the negative control group and other experimental groups. The results show that at the level of cellular immune response, gE and squalene oil-in-water adjuvant can effectively promote the 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 a memory phenotype, promote the differentiation of CD4+ T cells to Th1 and Th2 cells, IFN-γ promotes the killing of herpes zoster virus, and the coordination of the two prevents viral reactivation, controls the intracellular infection of the virus, and maintains the latent state of the virus, which plays an important role in preventing the occurrence of herpes zoster.

[0115] Test Example 3: VZV-gE antigen-specific cell-mediated cytokine secretion assay.

[0116] Flow cytometry assay (ICS assay) determines the method of lymphocyte subgroups and cytokine distribution:

[0117] Culture: Fresh mouse spleen lymphocytes were taken and cultured overnight at 37°C in a 5% CO2 cell incubator.

[0118] Stimulation: VZV-gE peptide pool was prepared at a concentration of 1 μg / mL in culture medium. The stimulator, no stimulator control, and positive stimulator were added to the wells containing the cells and mixed. The cells were incubated at 37°C in a 5% CO2 cell incubator.

[0119] Blocking: After stimulation, the blocking agent was diluted in culture medium and added to the cells, which were then incubated in the cell incubator.

[0120] Live / Dead staining: The cells were transferred to an EP tube, and PBS was added. The cells were centrifuged at 600 x g for 5 min, and the supernatant was discarded. The Live / Dead staining antibody was diluted in PBS, and the cells were resuspended and mixed. The cells were incubated at room temperature in the dark for 20 min.

[0121] Surface staining: PBS was added, and the cells were centrifuged at 600 x g for 5 min at room temperature. The supernatant was discarded. Each surface fluorescent marker antibody (CD3 antibody, CD4 antibody, and CD8 antibody) was diluted in PBS, and the cells were resuspended and mixed. The cells were incubated at room temperature in the dark for 60 min.

[0122] Fixation: IC fixation buffer was diluted in PBS, and the cells were resuspended. The cells were incubated at room temperature in the dark for 60 min, centrifuged at 600 x g for 5 min at room temperature, and the supernatant was discarded.

[0123] Intracellular staining: The membrane disrupter was diluted to 1x working solution in PBS, and each intracellular fluorescent marker antibody (IFN-γ antibody, IL-2 antibody, and TNF-α antibody) was prepared. The cells were resuspended and mixed gently, and the cells were incubated at room temperature in the dark for 60 min.

[0124] Washing: The cells were washed and examined for the distribution of T cells and the corresponding cytokine secretion caused by antigen stimulation.

[0125] The VZV-gE antigen-specific cell-mediated lymphocyte subpopulation and cytokine secretion were measured according to the above method, and the experimental results are shown in Figure 5 、 Figure 7 .

[0126] As shown in Figure 8 , compared with the negative control group, the recombinant herpes zoster vaccine composition prepared when the adjuvant was only squalene oil-in-water adjuvant or CpG could induce a certain level of IFN-γ, TNF-α, and IL-2, and the immunocompetence of the recombinant herpes zoster vaccine composition prepared when the squalene oil-in-water adjuvant was 250 μl was better.

[0127] AsFigure 7 The percentage of CD4+ cells secreting IFN-γ (IFN-γ+), IL-2 (IL-2+) and both cytokines (IFN-γ+IL-2+) after stimulation of mouse splenocytes.

[0128] The number of cytokines secreted by CD4+ T lymphocytes mediated by gE antigen-specific cells after the second immunization was increased in the examples. The results show that, at the level of cellular immune response, the combination of gE and squalene oil-in-water 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 phenotype, promote the differentiation of CD4+ T cells to Th1 and Th2 cells, prevent viral reactivation, and play an important role in preventing herpes zoster.

[0129] VZV-specific cellular immunity is a major 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.

[0130] The examples are different combinations of the recombinant herpes zoster vaccine composition proposed in the present application. In the examples, VZV-gE protein is used in combination with squalene oil-in-water adjuvant, which shows a significant increase in immunogenicity and cytokine expression. As can be seen from the comparative examples, the immunization effect of aluminum hydroxide adjuvant and CpG adjuvant alone is lower than that 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.

[0131] Further, in the recombinant herpes zoster vaccine composition of the present application, further technical solutions are provided, and immune modulators such as chemical chemotactic factors are introduced to further activate the immune system and enhance immune memory.

[0132] Example 9, a method for preparing a recombinant herpes zoster vaccine composition, comprising the following steps:

[0133] Mix 250 μl of squalene oil-in-water adjuvant and 50 μg of VZV-gE protein;

[0134] Mix with 10 μg of chemical chemotactic factor CCL3 / MIP-1α;

[0135] The solution was made up to 500 μl with sterile PBS buffer at pH 7.5, under light protection, by pipetting and adsorption, to obtain a recombinant varicella zoster vaccine composition.

[0136] Immunization group 10, a method for preparing a recombinant varicella zoster vaccine composition, comprising the following steps:

[0137] 250 μl of squalene oil-in-water adjuvant was mixed with 50 μg of VZV-gE protein;

[0138] and 10 μg of the chemokine CXCL10 / IP-10;

[0139] The solution was made up to 500 μl with sterile PBS buffer at pH 7.5, under light protection, by pipetting and adsorption, to obtain a recombinant varicella zoster vaccine composition.

[0140] Immunization group 11, a method for preparing a recombinant varicella zoster vaccine composition, comprising the following steps:

[0141] 250 μl of squalene oil-in-water adjuvant was mixed with 50 μg of VZV-gE protein;

[0142] and 5 μg of the chemokine CCL3 / MIP-1 alpha and 5 μg of the chemokine CXCL10 / IP-10;

[0143] The solution was made up to 500 μl with sterile PBS buffer at pH 7.5, under light protection, by pipetting and adsorption, to obtain a recombinant varicella zoster vaccine composition.

[0144] Table 3: VZV-gE antigen-specific IgG titers (ELISA)

[0145] Figure 8 Group First dose IgG titer Second dose IgG titer 1：4800 1：113400 Immunization group 4 1：6400 1：174960 Immunization group 9 1：18000 1：189540 Immunization group 10 1：48600 1：204120

[0146] Table 4: VZV-gE antigen-specific cell-mediated immune response (ELISPOT)

[0147] Immunization group 11 Group IFN-γ (per 200,000 spleen cells) IL-2 (per 200,000 spleen cells) 16 113 Immunization group 4 60 146 Immunization group 9 63 153 Immunization group 10 102 195

[0148] Table 5: VZV-gE antigen-specific cell-mediated immune response (ICS)

[0149] Immunization group 11 Group IFN-γ / % (Parent ratio) TNF-α (Parent ratio) IL-2 (Parent ratio) 0.004 0.215 0.208 Immunization group 4 0.101 0.318 0.304 Immunization group 9 0.135 0.312 0.298 Immunization group 10 Immunization group 11 0.153 0.336 0.322

[0150] As seen from the data in Table 3, the introduction of chemotactic factors (CCL3 / MIP-1a, CXCL10 / IP-10, and a combination of both) in immunization groups 9-11 showed an increase in IgG titers relative to immunization group 4, indicating that the introduction of chemotactic factors effectively increased IgG titers. In particular, immunization group 11, which contained both CCL3 / MIP-1a and CXCL10 / IP-10, showed higher IgG titers relative to other examples, suggesting that the combined use of both chemotactic factors synergistically activated the immune system through different mechanisms, resulting in a synergistic effect that led to a stronger humoral immune response.

[0151] As seen from the data in Table 4, the introduction of chemotactic factors increased the production of IFN-γ and IL-2, with immunization group 11 again showing the highest levels. This indicates that the vaccine formulation of immunization group 11 effectively activated cell-mediated immune responses, particularly in promoting Th1-type immune reactions. This is consistent with the role of CCL3 / MIP-1a and CXCL10 / IP-10 in promoting the migration of Th1 cells and other immune cells to the site of infection.

[0152] As seen from the data in Table 5, the introduction of chemotactic factors (CCL3 / MIP-1a, CXCL10 / IP-10, and a combination of both) in immunization groups 9-11 showed an increase in immune responses relative to immunization group 4, indicating that the introduction of chemotactic factors effectively increased immune responses. The advantage of immunization group 11 in activating cell-mediated immune responses showed the highest expression of IFN-γ, TNF-a, and IL-2. This indicates that immunization group 11 not only promotes Th1-type immune reactions but also activates a wide range of cell-mediated immune functions, including the enhancement of TNF-a and IL-2 expression, which are key factors for the immune system to resist infection and disease.

[0153] The combination of CCL3 / MIP-1a and CXCL10 / IP-10 showed a significant synergistic effect in the immune group 11, because these two chemokines activate the immune system through different mechanisms, resulting in a complementary and enhanced immune response. CCL3 / MIP-1a mainly attracts a broad 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 promotes the migration and activation of Th1 cells through its specific receptor CXCR3, which is essential for the formation of a cell-mediated immune response against pathogens. When these two chemokines act together, they not only recruit a broader and more diverse population of immune cells, but also specifically enhance the immune defense capacity against specific pathogens. Therefore, the combination of CCL3 / MIP-1a and CXCL10 / IP-10 achieves a comprehensive activation of the immune system, including strengthening the humoral and cell-mediated immune response, particularly by promoting the production of IFN-γ, TNF-α and IL-2, which are essential for fighting infections and diseases.

[0154] The present application provides a recombinant herpes zoster vaccine composition and its preparation method and application ideas and methods. There are many methods and ways to realize the technical scheme, and the above description is only the preferred embodiment of the present application. It should be pointed out that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should be considered as the protection scope of the present application.

Claims

1. A recombinant varicella vaccine composition, characterized in that, The gE protein and an adjuvant, wherein the adjuvant is a squalene oil-in-water adjuvant comprising the following components: squalene: 4-5 wt%; Tween 80: 0.2-0.8 wt%; Span 85: 0.2-0.8 wt%; further comprising a chemical chemoattractant; the total amount of the chemical chemoattractant added is 20 μg / mL; The chemical chemoattractant is a combination of 30-70 wt% CCL3 / MIP-1a and 30-70 wt% CXCL10 / IP-10; The amino acid sequence of the gE protein is as follows: SVLRYDDFHTDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRMIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRY; The content of the gE protein is 20-100 μg / mL; and the volume of the squalene oil-in-water adjuvant is 25-50%.

2. The recombinant varicella vaccine composition of claim 1, wherein The preparation method of the gE protein is as follows: Constructing PLK002-22P20-A2: taking PLK002 as an empty vector, connecting the signal peptide and the gE protein component coding of the antigen DNA sequence into the expression empty vector respectively to construct the PLK002-22P20-A2 expression vector; Taking CHO K1BN suspension cells as host cells, the expression vector of the above-constructed PLK002-22P20-A2 is transformed into the host cells by electroporation, and the cells after transfection are screened by pressure, amplified step by step, screened by ELISA and SEC-HPLC to obtain high expression monoclonal strains, which are continuously subcultured, centrifuged and filtered to harvest the culture supernatant, and the collected supernatant is purified to obtain the final sample, i.e. the gE protein.

3. The recombinant varicella vaccine composition of claim 2, wherein the recombinant varicella vaccine composition is a live attenuated vaccine. The construction method of the PLK002-22P20-A2 is as follows: The PLK002-22P20-A2 is constructed: taking PLK002 as a blank vector, adding a HindIII enzyme cutting site and a KOZAK sequence in front of an antigen DNA sequence signal peptide, and adding a termination codon and a Not I enzyme cutting site in the rear; after recovering the vector and the target fragment, the two are connected, and the positive transformant plasmid DNA is sequenced and enzyme cut to identify, and the PLK002-22P20-A expression vector is successfully constructed; the KOZAK sequence is added in front of the antigen DNA sequence signal peptide, and the termination codon is added in the rear, the 22P20-A2 replaces the DNA sequence from HindIII to Not I on the PLK002 vector through homologous recombination, and the positive transformant is sequenced and enzyme cut to identify, and finally the PLK002-22P20-A2 is successfully constructed.

4. The recombinant varicella vaccine composition of claim 1, wherein The squalene oil-in-water adjuvant droplet diameter is 150-180 nm.

5. The recombinant varicella vaccine composition as set forth in claim 1, wherein The buffer is at least one of a PBS buffer, a Tris-HCl buffer and a citrate buffer.

6. The method of preparing a recombinant varicella vaccine composition according to any one of claims 1 to 5, wherein, 250 μl of squalene oil-in-water adjuvant and 50 μg of VZV-gE protein are mixed; 5 μg of chemical chemotactic factor CCL3 / MIP-1α and 5 μg of chemical chemotactic factor CXCL10 / IP-10 are further mixed; Sterile PBS buffer with pH 7.5 is added to the solution to 500 μl, and the solution is blown and adsorbed under light shielding conditions to prepare a recombinant herpes zoster vaccine composition.

Images