阅读说明：本技术 一种重组水痘带状疱疹病毒疫苗 (Recombinant varicella zoster virus vaccine ) 是由 孔健 姜培红 彭玲 杨帅 许磊涛 张琨 于 2019-11-29 设计创作，主要内容包括：本发明公开了一种重组水痘带状疱疹病毒疫苗,其包括减毒活VZV毒株(OKA株)基因的重组糖蛋白gE胞外区的氨基酸序列和人免疫球蛋白Fc段形成的融合蛋白,本发明进一步包括所述融合蛋白的制备和应用,以及相应的重组基因,真核表达载体等,本发明的融合蛋白具有较好的免疫原性,可诱导产生高水平的血清中和抗体。(The invention discloses a recombinant varicella zoster virus vaccine, which comprises a fusion protein formed by an amino acid sequence of a recombinant glycoprotein gE extracellular region of an attenuated live VZV strain (OKA strain) gene and a human immunoglobulin Fc segment.)

1. A recombinant varicella zoster virus vaccine preparation comprises a fusion protein formed by an amino acid sequence of a recombinant glycoprotein gE extracellular region of an attenuated live VZV strain (OKA strain) gene and a human immunoglobulin Fc section, wherein the amino acid sequence of the fusion protein is SEQ ID No. 1.

2. The vaccine formulation of claim 1, further comprising a vaccine adjuvant.

3. The vaccine formulation of claim 2, the vaccine adjuvant being selected from the group consisting of: an aluminum hydroxide adjuvant, an aluminum phosphate adjuvant, or a mixture of aluminum hydroxide and aluminum phosphate adjuvants.

4. The vaccine formulation of claim 1, wherein each dosage unit contains 5-200 μ g of the fusion protein.

5. The vaccine formulation of claim 4, wherein each dosage unit contains 10-100 μ g of the fusion protein.

6. The vaccine formulation of claim 5, wherein each dosage unit contains 20-60 μ g of the fusion protein.

7. The vaccine formulation of claim 1, further comprising other substances that enhance immunogenicity, including but not limited to: phosphatidylcholine, lecithin, 3D-MPL, long chain fatty acids (esters), mineral oil, vegetable oil, sodium methylcellulose, sodium carboxymethylcellulose, and cholesterol-containing liposomes.

8. The vaccine formulation of claim 1, which is a lyophilized formulation.

9. The vaccine formulation of claim 8, wherein the lyophilized formulation is dissolved with an adjuvant suspension containing aluminum hydroxide before use, mixed well and injected intramuscularly or subcutaneously.

10. A recombinant gene capable of expressing the fusion protein of claim 1, having the DNA sequence of SEQ ID No. 2.

Technical Field

The invention relates to a vaccine preparation, in particular to a vaccine preparation which can induce an organism immune system to generate a neutralizing antibody aiming at recombinant varicella-zoster virus glycoprotein E fusion protein.

Background

Varicella-zoster virus (VZV), a member of the herpesvirus (Herpesviridae) subfamily alphaherpesvirus, is a double-stranded DNA virus with a diameter of 150-200 nm and morphologically is a concentric circular structure consisting of a nucleic acid core, a protein capsid and an envelope. The VZV genome is a linear double-stranded DNA molecule of about 125kb, comprising a unique long fragment (UL) of about 100kb and a unique short fragment (US) of about 5.4kb separated by terminal and internal repeats of 6.8kb, sharing 72 Open Reading Frames (ORFs), encoding 8 glycoproteins, gB, gC, gE, gH, gI, gK, gL and gM, in addition to protein molecules associated with biological activities such as viral replication, transcription, packaging, release, and proteins interacting with the host cell, which play an extremely important role in the maturation and packaging of viruses. Wherein gE (glycoprotein E, gE) is encoded by ORF68 gene, which consists of 1872 bases and is located in a short fragment region of VZV genome, and the encoded gE contains 623 amino acids (amino acids, AA). The N end of the polypeptide is a 544 AA hydrophilic extracellular region (containing a signal peptide), and the C end is a 17AA transmembrane hydrophobic region and a 62AA intracellular region. gE belongs to type I membrane proteins, is an essential glycoprotein for the production of infectious VZV particles, and is also the most antigenic and abundant glycoprotein on the viral envelope and infected cell membranes, and it is also widely present on the surface of VZV particles and in the cell membranes and cytoplasm of host cells, and can induce cellular and humoral immunity.

The glycoprotein E of the VZV virus comprises two antigenic determinant coding regions E1 and c1, and the antigenic determinants are stable among VZV strains from different regions, have higher conservation and are more suitable vaccine antigen candidate subunits. gE is present on the surface of VZV virions as well as on the surface and in the cytoplasm of infected cells, and it exists in different glycopolypeptide forms at different stages of virus maturation. The gE molecule contains a trafficking signal and can be involved in the assembly of viral proteins and the formation of an envelope in the golgi apparatus. In the serum of varicella and herpes zoster patients in convalescence, VZV antibodies are mainly directed against the three glycoproteins gB, gH and gE, and in particular are mainly targeted against gE. The gE induces the specific humoral immunity and cellular immunity generated by the organism, and can protect the organism from the attack of viruses. gE contains phosphorylated high mannose O-chains and N-chains of complex glycans, which coordinate gI binding to the Fc domain of human IgG, covalently link gE and gI, and function as Fc receptors on the surface of infected cells.

When VZV is primarily infected, the virus enters local lymph nodes through respiratory tract mucous epithelium and is replicated, lymphocytes infected with the virus enter blood circulation through lymph circulation to infect peripheral blood mononuclear leukocytes, and the virus spreads to the skin along with the blood flow, and the varicella is clinically shown. After the varicella heals, the virus is latent in craniocerebral ganglia, spinal cord posterior root ganglia, autonomic neurons or enteric neurons. When the body resistance is reduced, the latent virus is reactivated, replicated and migrates along the peripheral nerves to the skin, with the clinical manifestations of herpes zoster.

VZV is highly contagious, and patients with chickenpox or shingles are the only source of infection, and VZV is transmitted primarily via airborne droplets and direct contact. Varicella patients shed the virus by saliva or ocular fluid 2 days before the appearance of a rash. The blister fluid of patients with varicella or herpes zoster contains infectious virus particles, and the droplets generated during breathing can be infected after being inhaled by the susceptible people, or can be infected by directly contacting the blister fluid of skin lesions, and the more the skin lesions are, the stronger the infection is. Varicella can occur in susceptible people after infection, but does not directly cause herpes zoster.

Serum epidemiological investigation shows that 95 to 97 percent of women of childbearing age have positive serum anti-VZV antibodies, so the probability of varicella virus infection in 6 months after the birth of the infant is very low. Adults negative for serum antibodies, babies born by pregnant women negative for serum antibodies, people with immunodeficiency, babies born by pregnant women who suffer from chicken pox in the 4 th to 5 th months of pregnancy, and babies born by mothers who suffer from chicken pox before and after childbirth are all prone to serious primary infection of VZV. The risk of herpes zoster in older people, immunodeficient people, children with chickenpox in pregnancy and children with chickenpox in the last year is increased.

More than 95% of young human sera in north america and europe are positive for VZV antibodies and thus at risk for herpes zoster. The incidence of herpes zoster is 3-5/thousand-year, wherein 3-10/thousand-year in Asia-Pacific region, 10.4/thousand-year in Korea, 10.9/thousand-year in Japan, 3.4-5.8/thousand-year and 4.89-5.67/thousand-year in mainland and Taiwan, respectively. There is currently no data on the incidence of herpes zoster in africa.

Approximately 13% to 47% of patients with herpes zoster have complications or sequelae, mainly involving the nervous system and eyes. Neurological complications include postherpetic neuralgia (PHN), Ramsay-Hunt syndrome, Bell's facial paralysis, meningitis, myelitis, and transient cerebral ischemia or stroke. The ocular complications are mainly eye herpes zoster.

PHN is the most common sequela of herpes zoster, a more internationally accepted definition of pain lasting for more than 90 days after the appearance of a rash. About 30% to 50% of PHN lasts for more than 1 year, and a few can be as long as 10 years. Patients with herpes zoster develop PHN by about 5% -30%, most of the literature reports 10% -20%, and the Chinese continental region reports 8.6% -13.8%. The incidence of PHN increases with age of the patient, with approximately 8% in patients aged 50 or older and up to 33% in patients aged 80 or older. The PHN susceptibility factors are clear, such as advanced age, severe prodrome, severe rash, severe pain and low immunity. Patients with trigeminal involvement, with SLE, diabetes or neuropsychiatric abnormalities are also predisposed to PHN.

Herpes zoster eye disease (HZO) is the result of latent VZV being reactivated and replicating to involve the trigeminal nerve eye branch. Based on the population base statistics, the HZO incidence rate is 30.9/10 ten thousand, and 104.6/10 ten thousand is achieved for the population more than or equal to 65 years old. The HZO incidence rate is 10-20% based on the basal number of herpes zoster patients, and also increases with age. The clinical manifestations of HZO are, in turn, blepharitis, keratitis, conjunctivitis, scleritis, uveitis or acute progressive retinal necrosis. Approximately 2.5% of patients with HZO in the united states develop ocular lesions, of which 6% are blinding. About half of HZO patients have skin lesions, and about 21% eventually develop PHN.

Elderly or immunocompromised patients can also have recurrent herpes zoster outbreak, skin lesions spreading, bacterial infection or verrucous hyperplasia, and also can have disease and drug resistance. Even severe cases involve many organs such as lung, gastrointestinal tract and brain, and are prone to misdiagnosis due to the occurrence of hepatitis, pancreatitis, pneumonia, myocarditis, esophagitis or peptic ulcer before herpes zoster appears.

Protective immunity can be obtained after VZV infection of humans vaccinated with an attenuated strain of VZV (OKA strain) or naturally. The OKA attenuated live vaccine has been approved by the U.S. FDA, the Chinese drug administration, the European Union and other organizations for children vaccination to prevent the wild varicella virus from infecting children, and the high-dose OKA attenuated live vaccine has been approved by the U.S. FDA and the European Union and used for preventing the old people over 50 years old to prevent or reduce the diseases caused by the herpes zoster virus such as intercostal neuralgia caused by the herpes zoster virus. Currently, 60 countries and regions such as European Union, United states, etc. recommend the vaccination of people with normal immune function of 50 years or more with Zostavax to prevent herpes zoster and PHN. The vaccination was performed by injecting a single dose of vaccine (0.65mL, containing 19, 400PFU virus) subcutaneously in the deltoid region of the upper arm. Occasionally causing headache, local reaction after injection and other adverse reactions. The large-scale multi-center clinical test proves that the incidence rate of herpes zoster after the inoculation of a population with normal immune function of 50-59 years old is reduced by 69.8%, and the incidence rate of herpes zoster, the incidence rate of PHN and the disease burden of a population with the immune function of more than or equal to 60 years old are respectively reduced by 51.3%, 66.5% and 61.1%. The prevention efficiency of Zostavax decreases gradually with age of the vaccinee, severe immunosuppression and contraindication for vaccination in pregnant women. Thus, the need to prepare a safer and more effective vaccine is particularly acute. The herpes zoster subunit vaccine prepared from VZV gE recombinant protein developed by GSK company and AS01B adjuvant has the advantages that the incidence rates of herpes zoster and PHN are respectively reduced by 97.2% and 91.2% when people with normal immune function of more than or equal to 50 years old are inoculated, the incidence rates of herpes zoster and PHN are respectively reduced by 89.8% and 88.8% when people with normal immune function of more than or equal to 70 years old are inoculated, the effect is better than that of attenuated live vaccine Zostavax, and the application prospect is possibly better. Although the dilution AS01B used in the gE vaccine of the recombinant VZV virus contains QS21, 3D-MPL, phosphatidylcholine and other oily adjuvants, the effect of the dilution is obviously better than that of the vaccine Zostavax of the VZV attenuated live vaccine, but the dilution has the greatest defect of causing temporary nodules at the injection site or nodules which can be regressed after a long time.

CN102517302A discloses a method for recombinant expression of varicella-zoster virus truncated glycoprotein E and application thereof. The method comprises the steps of introducing genes of varicella-zoster virus (VZV) truncated glycoprotein E (gpE) with a transmembrane region and an intracellular region removed and a His label added into host cells, and expressing the genes to obtain the recombinant varicella-zoster virus truncated glycoprotein E. The expression method is helpful for improving the expression quantity of the target protein, simplifies downstream purification work, can easily realize large-scale production of the protein, can be used for indirect ELISA detection of the anti-varicella-zoster virus specific immunoglobulin in a plasma sample by taking the recombinant protein as a capture antigen, can improve the accuracy of clinical diagnosis of VZV infection, and is used in other fields requiring high-throughput detection of the VZV specific immunoglobulin. Since this method is a prokaryotic expressed non-glycosylated protein, it is mainly used for the detection of past infection of VZV, and is not suitable for the preparation of immune compositions or human vaccines that require complex glycosylation to produce serum-neutralizing VZV antibodies. Li Fumin et al disclose a method of amplifying VZV glycoprotein E gene by PCR, cloning it into eukaryotic expression vector pcDNA3.1 and identifying it by double digestion and sequencing. As a result, the amplified target gene includes the full-length glycoprotein E gene, and the length of the gene is about 1.9 kb; and a glycoprotein E gene recombinant expression vector is successfully constructed (clinical journal of practical hospital, 2006, stage 02). Yixing Xue et al disclose a method for constructing eukaryotic expression plasmid pCDNA3.1-gE containing VZV gE ectodomain gene, carrying out lipofection on COS-7 cell after sequencing, and screening cell strain stably expressing VZV gE by G418. RT-PCR method is adopted to detect mRNA of VZV gE, immunoblotting and indirect immunofluorescence method are adopted to detect immunoreactivity of gE, and expression products are processed by Ni⁺⁺After purification by NTA column, coating an ELISA plate, and detecting the VZV-IgG antibody level in 127 parts of serum of a normal child aged 0-10 years. The result successfully screens COS-7 cell strains capable of stably expressing VZVgE ectodomain genes, detects gE mRNA by RT-PCR, and expresses g through immune printing and dyeing and indirect immunofluorescence identificationE has immunoreactivity, and both COS-7 cell strain and culture supernatant have gE protein expression, the expression amount is about 0.632 mu g/mL, and the purity is about 90%. 127 parts of VZV-IgG antibody in serum of 0-10 year-old children are detected by an ELISA experiment, the total positive rate is 81.89%, and the specificity and the sensitivity are 93.75% and 88.24% respectively (the university of Anhui medical science, 2015, 03). The research on VZV glycoprotein E mainly focuses on realizing that eukaryotic cells express the protein, the purity of an expression product is very low, the purity of the expression product can only meet the actual detection requirement, the quality requirement of human vaccines can not be met, the subsequent research on purification of the expression product is not found, and the report on the human vaccines is not found.

Lichumin et al disclose a recombinant eukaryotic expression plasmid pCI-neo-gE537-His constructed by infecting a human diploid cell (2BS strain) with an Oka strain VZV (VZV-Oka), extracting genomic DNA, PCR-amplifying a gE537 target fragment using it as a template, cloning to a vector pCI-neo. And extracting plasmids after a large amount of amplification, transfecting 293FT cells, performing transient expression, and purifying by a nickel column to obtain the target protein gE 537-His. The purified product can be specifically combined with a band of the mouse anti-gE glycoprotein monoclonal antibody at a relative molecular mass of about 90KDa, and can be reacted with mAb-10 and mAb-12 anti-gE monoclonal antibodies (J.Biopreparation, 2016, 11). Yixing Xu discloses a method for cloning and expressing the ectodomain of VZVgE gene, which comprises the following steps: inoculating skin vesicle liquid of a patient with herpes zoster clinically collected to a monolayer of human embryonic fibroblasts for virus separation; characteristic Cytopathic effect (CPE), indirect immunofluorescence and DNA sequencing characterization was performed on the isolated virus strains. The confirmed VZV clinical isolate is cultured in vitro, the ectodomain fragment of VZV gE gene is amplified by PCR, prokaryotic expression plasmid gE-pET-32a (+) and eukaryotic expression plasmid gE-pCDNA3.1/myc-His (-) are respectively constructed, after sequencing, the prokaryotic plasmid is transformed into E.coli BL21(DE3) competent cells, and the fusion protein expressed by VZV gE prokaryotic is obtained by induction of Isopropyl thio beta-D-galactoside (IPTG). Identifying the specificity of the recombinant protein by SDS-PAGE electrophoresis and immunoblotting, and using Ni⁺⁺-NTA column purification and column renaturation of the expressed protein. The eukaryotic plasmid is mediated by liposomeIntroducing the cells to COS-7 cells, screening out a cell strain stably expressing VZV gE protein by G418, and passing an expression product through Ni⁺⁺-NTA column purification; the mRNA of the VZV gE gene was detected by RT-PCR, and the immunoreactivity of the gE fusion protein was detected by immunoblotting and indirect immunofluorescence. The purified prokaryotic expression gE protein and eukaryotic expression gE protein are respectively used for immunizing New Zealand rabbits to obtain rabbit anti-VZV gE polyclonal antibodies (university of Anhui medical science, Shuobo paper). The above results show that they achieved expression of VZV gE in animal cells, but the purity of the resulting expression product was low.

Fc receptors are expressed on the surface of many innate immune cells and, therefore, Fc fragments have been widely used in DC targeting studies, and are classified into Fc α r (iga), Fc α/γ r (iga and igm), Fc ∈ r (ige), and Fc γ r (igg) depending on the subtype of antibody to which the receptor binds. In addition, Fc receptors can be classified into high affinity receptors and low affinity receptors according to their affinity, with high affinity receptors binding monoclonal antibodies and low affinity antibodies binding polyclonal antibodies. In the research of targeting DC, the application of targeting Fc γ R was the earliest, and many published studies have shown that Fc γ R targeting can significantly improve antigen presentation efficiency in vitro, promote antigen binding to MHC II, and Fc γ R-targeted antigen is ultimately presented to CD4⁺T cells, activate the TH1 signaling pathway (Dai X, Jayapal M, Tay HK, Reghunathan R, et al. differential signal transmission, membrane diversion, and animal functions mediated by Fc γ RI veruss Fc γ RIIa. blood 2009; 114: 318-27.).

VZV gE (or antigens derived from other pathogenic microorganisms) and a monoclonal antibody Fc fragment of a DC receptor are subjected to fusion expression, the constructed fusion protein is internalized by DCs, Fc recombinant protein or Fc coupled protein is degraded by intracellular protease in an endocytosis pathway, and the formed antigen peptide can be loaded on MHC-1 and/or MHC-II molecules. The outstanding advantage of Fc mediation is that antigen can be directly delivered to antigen presenting cells, the antigen presenting efficiency is improved, and the method can selectively activate specific signal paths by targeting specific receptors on the DC surface (Caminschi I, short K.stimulating antibody responses by targeting antibodies to dendritic cells. trends in immunology.2012; 33: 71-7.).

The invention links the outer membrane region of the gE protein peptide chain of the VZV virus with the gene of the CH2-CH3 region of the coded human immunoglobulin, inserts the target gene into the eukaryotic cell expression vector, transfects the CHO cell, and successfully expresses the fusion protein containing the VZV gE-Fc. The recombinant protein is purified by affinity chromatography, ion exchange chromatographic column and molecular sieve chromatography, and possible virus pollution is removed by virus inactivation, so that highly purified fusion protein is obtained. The Fc fragment of the fusion protein can be combined with Fc receptors on the surfaces of dendritic cells of the immune system in a human body, so that the antigen presentation efficiency of the dendritic cells is enhanced, and high-titer serum neutralizing antibodies can be generated after immunization.

Disclosure of Invention

The present invention relates to a method for preventing and/or reducing the severity of herpes zoster and/or Post Herpetic Neuralgia (PHN), comprising administering to an individual an immunogenic composition comprising a VZV antigen or a recombinant immunogenic derivative gE protein thereof in combination with a live attenuated VZV (OKA strain) or a whole inactivated VZV.

The invention also relates to a method of preventing or ameliorating herpes zoster reactivation and/or post herpetic neuralgia, the method comprising delivering to an individual in need thereof an immunogenic composition or vaccine comprising a gE fusion protein or an immunogenic derivative or immunogenic fragment thereof in combination with an adjuvant.

In view of the above problems in the prior art, it is an object of the present invention to provide a varicella-zoster virus glycoprotein E-Fc gene fusion protein to achieve high purity of the expression product of the glycoprotein E of varicella-zoster virus in mammals.

In order to achieve the purpose, the invention adopts the following technical scheme:

a recombinant varicella-zoster virus vaccine preparation comprises a fusion protein formed by an amino acid sequence of a recombinant glycoprotein gE extracellular region of an attenuated live VZV strain (OKA strain) gene and a human immunoglobulin Fc segment, wherein the amino acid sequence of the fusion protein is SEQ ID No. 1.

The vaccine preparation of the invention also contains a vaccine adjuvant. The vaccine adjuvant is selected from: an aluminum hydroxide adjuvant, an aluminum phosphate adjuvant, or a mixture of aluminum hydroxide and aluminum phosphate adjuvants.

The vaccine preparation provided by the invention contains 5-200 mug of fusion protein in each dosage unit.

Preferably, each dosage unit of the vaccine preparation of the invention contains 10-100 mug of fusion protein.

The vaccine preparation provided by the invention is more preferable, wherein each dosage unit contains 20-60 mu g of fusion protein.

The vaccine formulation of the present invention may also contain other substances that enhance immunogenicity, including but not limited to: phosphatidylcholine, lecithin, 3D-MPL, long chain fatty acids (esters), mineral oil, vegetable oil, sodium methylcellulose, sodium carboxymethylcellulose, and cholesterol-containing liposomes.

The vaccine preparation is a freeze-dried preparation. The freeze-dried preparation is dissolved by adding aluminum hydroxide adjuvant suspension before use, and is injected intramuscularly or subcutaneously after being uniformly mixed.

The invention further provides a recombinant gene which can express the fusion protein, and the DNA sequence of the recombinant gene is SEQ ID No. 2.

The fusion protein (hereinafter abbreviated as gE-Fc or VZV gE) expression vector of the invention is formed by inserting a gE-Fc fusion gene into an eukaryotic mammalian cell expression vector.

The fusion protein is an exogenous antigen or a derivative thereof which can efficiently initiate the immune system of a human body to generate immune response, wherein the immune response refers to the ability of inducing the immune system of an organism to respond and generate high-titer serum neutralizing antibodies. Such a response may reduce the incidence of herpes zoster disease in the population, or may reduce the relief of pain or symptoms such as intercostal neuralgia caused by herpes zoster. The improvement in immune response is assessed using techniques common in the art, such as serum neutralizing antibody levels or VZV gE (glycosylated protein) ELISA antibody level assays, or using known clinical decision criteria to assess improvement at the level of clinical symptoms or signs.

As a preferable embodiment, the expression product can be mixed with aluminum adjuvant or other medicines for enhancing immunogenicity, and can be used for preparing medicines for preventing infectious diseases such as intercostal neuralgia caused by herpes zoster of the elderly over 50 years old, and also can be used for preventing infection caused by varicella-zoster virus of infants and young children.

Like protective antigens of various viruses, protective antigens of VZV also belong to the major glycosylated proteins of VZV, glycoproteins, particularly glycoprotein gE, whose diverse glycosylated forms form the major neutralizing antigen of VZV, and serum antibodies against gE protein have been shown to neutralize VZV.

Other suitable antigens also include various glycoproteins such as gB, gH, gC, gI, IE63 (see, e.g., Huang et al J. Virol.1992, 66: 2664, Sharp et al J. Inf. Dis.1992, 165: 852, Debrus, J Virol.1995 May; 69 (5): 3240-5 and references therein), IE62 (see, e.g., Arvin et al J. Immunol. 1991146: 257, Sabella J Virol.1993 Dec; 67 (12): 7673-6 and references therein), ORF4 or ORF 10(Arvin et al Viral Immunol. 200215: 507), but these glycoproteins are less abundant on VZV membranes than gE and do not constitute the main source of VZV-producing neutralizing antibodies for the body.

The invention realizes the high-efficiency expression of VZV glycoprotein E gene in a mammal expression system, under the condition of not changing the amino acid sequence, the VZV glycoprotein E extracellular region gene is synthesized according to codon optimization codon preferred by CHO cells, a gE-Fc fusion gene is constructed, then an eukaryotic expression vector is constructed, the eukaryotic expression vector is transfected into CHO K1 cells, the Protein expression condition is detected by Protein A affinity chromatography HPLC, the combination activity of VZV gE and human anti-varicella virus specific immunoglobulin is detected by ELISA, and the secretion expression of the VZV glycoprotein E gene in the CHO cells is successfully realized. The highly purified VZV gE is used for immunizing rabbits and BALB/C mice, and the serum neutralization test of the immune serum neutralization OKA strain virus proves that the expressed VZV gE-Fc protein has good immunogenicity, and can generate high-titer serum neutralization antibodies after 2 times of immunization.

The amount of VZV gE antigen used should be an amount that induces an immunoprotective response in the body without significant adverse side effects. The amount of antigen used will vary depending on the adjuvant used and the manner in which it is present. Generally, it is expected that each dose may contain about 2-1000 μ g of VZV gE; when used in humans, in order to reduce the number of injections, adsorption with an aluminum adjuvant may be used, and when adsorption with an aluminum adjuvant is used, it is expected that high titers of neutralizing antibodies may be produced using 5 to 200 μ g of VZV gE, with a preferred dose of 10 to 100 μ g, a suitable immunization dose of about 10 μ g, 25 μ g, 50 μ g, 100 μ g of VZV gE, or about 200 μ g of VZV gE, and an optimal dose in adults may be 50 μ g or 100 μ g of VZV gE; the optimal dose in infants may be 10 μ g, 20 μ g VZV gE.

Modes of administration of the vaccine formulations of the present invention include, for example, topical, intranasal, mucosal, intradermal, intraperitoneal, subcutaneous, and intramuscular injection administration.

The vaccine formulations of the present invention, optionally in combination with an adjuvant and/or (other) suitable carrier.

In the case of using a boosting protocol, or in the case of using a multiple vaccination protocol, 2, 3, 4 or more immunizations may be used. Protocols suitable for priming a boost include 1, 2, 3 or 6 months between each immunization.

The sequence of the outer envelope region of VZV gE is a part of the sequence as listed in appendix 1 of the present invention, and the complete sequence of VZV gE was first published in Virus Research (Virus Research, Haumont et al Vol.40, 1996p 199-. Unless the context clearly indicates otherwise, reference hereinafter to VZV gE or gE includes truncated VZV gE or other fragments or derivatives of VZV gE-Fc.

gE or its derivatives or fragments are liquid or lyophilized, in another aspect gE or its derivatives gE-Fc or its fragments, polymers may be present in suspension with aluminum adjuvant, or in solution or suspension with other immunopotentiator ingredients (e.g. containing QS21, cholesterol, mineral oil, vegetable oil, fish oil, long chain fatty acids, long chain fatty acid esters, 3D-MPL adjuvant, etc.).

gE or its derivative may be coated in polylactic acid micro carrier or the micro carrier formed with glycolide/lactide copolymer, and the coated recombinant protein medicine may be released slowly after intramuscular or subcutaneous injection to stimulate the immune system of body to produce antibody.

Compared with the prior art, the invention has the following advantages:

1. according to the invention, a mammal animal Cell (CHO) is selected to efficiently express a secretory gE-Fc fusion protein, the obtained target product is a glycosylated protein, and the expression product is mainly dimer and monomer; each dimer form molecule contains 4 molecules of gE, and the fusion protein produced by the invention has large molecular weight and strong immunogenicity.

2. The gE-Fc is a fusion Protein of the outer membrane region of VZV gE and a human immunoglobulin Fc fragment (CH2-CH3 region), can adopt commercial Protein A affinity chromatographic packing to carry out high-efficiency preliminary separation and purification, reduces the discharge of environmental pollutants to the maximum extent, is an environment-friendly separation process, and is beneficial to further purifying target Protein by adopting ion exchange chromatography, molecular size exclusion chromatography and the like in subsequent steps;

3. the recombinant gE-Fc fusion protein expressed by the CHO cells is glycosylated protein, maintains the spatial structure of natural gE protein, has good immunogenicity, and has prospect and advantage of large-scale popularization.

4. The Fc of the gE-Fc fusion protein provided by the present invention can bind to Fc receptors present on the surface of antigen-presenting cells present in the immune system in humans, and actively present gE antigen. Animal immunogenicity experiment results show that the gE-Fc protein produced by the recombinant CHO cell is used as an antigen, and can still generate high-titer serum neutralizing antibodies after rabbits and mice are immunized without using an oily adjuvant and an immunostimulant;

drawings

FIG. 1 agarose gel electrophoresis detection of VZV gE-Fc plasmid restriction products

Lane 1: DL10000 DNA Marker (10000bp, 7000bp, 4000bp, 2000bp, 1000bp, 500bp and 250bp)

Lanes 2-3: plasmid pUC57-gE-Fc cleavage product

Lanes 4-5: plasmid pXC-K383L enzyme digestion product

FIG. 2 PCR screening of Positive clones from VZV gE-Fc recombinant plasmid colonies

Lane 1: DL10000 DNA Marker (10000bp, 7000bp, 4000bp, 2000bp, 1000bp, 500bp and 250bp)

Lanes 2-7: colony PCR screening randomly picked 6 clones gE-Fc-1-6

FIG. 3 agarose gel electrophoresis detection of linearized digestion products of plasmid expression vectors

Lane 1: DL10000 DNA Marker (10000bp, 7000bp, 4000bp, 2000bp, 1000bp, 500bp and 250bp)

Lane 2: plasmid pXC4-VZV gE-Fc

Lane 3: linearized plasmid VZV gE-Fc-conc

FIG. 4 HPLC-SEC chromatogram of recombinant VZV gE protein after affinity chromatography purification

FIG. 5 HPLC-SEC chromatogram of recombinant VZV gE protein

FIG. 6 serum antibody titers (geometric mean titer GMT) following immunization of mice with each dose of VZV gE aluminum adjuvant vaccine

FIG. 7 determination of serum neutralizing antibody titer after immunization of BALB/C mice with recombinant VZV gE aluminum adjuvant adsorbed vaccine

FIG. 8 geometric mean titers of neutralizing antibodies following immunization of BALB/C mice with varying doses of VZV gE aluminum adjuvant vaccine

The specific implementation mode is as follows:

the invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto.

Example 1 construction of plasmid expression vector:

1. source of Gene sequences

The varicella zoster virus E protein (VZV gE) is an extracellular region (ECD, 31-546aa) of the gE protein, and has 516 amino acids in total; the Fc segment is human IgG1 Fc, 232 amino acids in total (appendix: amino acid sequence 1). The varicella zoster virus E protein gene and the Fc gene of human IgG1 are connected in series (appendix: DNA sequence 2), Nanjing Kingsler Biotechnology GmbH is entrusted to synthesize a VZV gE-Fc fusion protein gene sequence and is connected to a pUC57-1.8K vector, the synthesized gene comprises an enzyme cutting site, a Kozak sequence, a signal peptide, a target gene (2244bp) and a stop codon, and the total length is 2355 bp. The recombinant gene was synthesized with codon optimization to facilitate expression in the Chinese hamster ovary cell Cricetulus griseus (CHO cell).

2. Construction of VZV gE-Fc gene-containing expression plasmid

Inoculating a glycerol strain containing a recombinant gene provided by Kimura biotechnology limited into an LB (Amp +) culture medium, culturing at 37 ℃ and 180rpm for 15h, and extracting a Plasmid pUC57-gE-Fc by using TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0; utilizing HindIII and EcoR to carry out double enzyme digestion on the plasmid pUC57-gE-Fc to obtain a target gene fragment gE-Fc-H/E (the size is about 2300bp), and carrying out enzyme digestion on the mammalian cell expression plasmid pXC-K383L to obtain a carrier fragment pXC-H/E (the size is about 7000 bp); the agarose gel electrophoresis detection of the cleaved products is shown in FIG. 1.

The target fragment was recovered using the TaKaRa MiniBest Agarose Gel Extraction Kit (FIG. 1, arrow). Adopting a sticky end connection technology, connecting and reacting the recovered enzyme digestion products gE-Fc-H/E and pXC-H/E for 6H at 16 ℃ by TaKaRa DNA Ligation Kit LONG (TaKaRa), then transforming into competent DH5 alpha, and carrying out inverted culture for 15H at 37 ℃; two clones, gE-Fc-1 and gE-Fc-2, were screened by colony PCR (FIG. 2); taking pXC-F and pXC-R as primers and gE-Fc-1 as a template to amplify a target gene by PCR, wherein the size is about 2400 bp; sequencing the amplified product by Beijing Huada gene, and sequencing the amplified product by two reactions; sequencing was analyzed using the software BioEdit7.0.9.0 and the sequence of clone gE-Fc-1 was identical to the designed sequence. The sequencing primer is as follows:

pXC-F：5’-TAACAGACTGTTCCTTTCCATG-3’

pXC-R：5’GTAAAACCTCTACAAATGTGGT-3’

1-F：5’-AGCACATCTGCCTGAAGC-3’

1-F1：5’-GCTTATTGTCTGGGCATCT-3’

clone gE-Fc-1 was inoculated into 300mlLB(Amp⁺) Culturing at 37 deg.C and 180rpm in culture medium for 16h, and extracting plasmid pXC-VZV gE-Fc with large-scale/large-scale plasmid extraction kit (Beijing Bomaide Gene technology Co., Ltd.); after the plasmid was linearized with endonuclease PvuI (TaKaRa), it was extracted and purified with phenol/chloroform/isoamyl alcohol, ethanol precipitated and redissolved in 1ml of sterile TE Buffer (TaKaRa). The gel electrophoresis detection of plasmid pXC-VZV gE-Fc and linearized plasmid VZV gE-Fc-was shown in FIG. 3.

Example 2 establishment and screening of Stable clones

In the sterile laminar flow workbench, the perforation voltage of a gene pulse generator Xcell (Bio-Rad) is set to 300V, 900 muF monopulse and infinite resistance, a disposable electric cup (Bio-Rad) with a gap of 4mm is taken out, 40 mug of linearized plasmid DNA (100 muL) and 0.7ml of CHO K1 cell suspension (1.5 x 10)⁷cells/ml), the linearized plasmid VZV gE-Fc-is directly transfected into CHO K1 cells by an electrotransfection method, the cells in an electric shocking cup are transferred into a triangular culture flask, 30ml of CD CHO culture solution (GIBCO) is added, and the temperature is 36-37 ℃ and the concentration of 5% CO is controlled₂Culturing on a shaker at 135 rpm for 24 hours, collecting the cells by low speed centrifugation, changing to CD CHO culture medium containing 50. mu.M MSX (without glutamine), transferring the cells to a 96-well flat-bottom plate by limiting dilution, and placing the plate at 37 ℃ with 10% CO₂Culturing in an incubator, observing under an inverted microscope, marking monoclonal cell pores, screening out monoclonal strains with higher expression quantity by an ELISA method (sheep anti-human IgG + expression product VZV gE-Fc + sheep anti-human IgG-HRP) and a protein A HPLC method, continuously subculturing and detecting to finally obtain 3 cell clone strains with high expression target genes, wherein the clone numbers are 5B3, 8D8 and 12C3 respectively; the expression level of the recombinant protein in the culture supernatant was measured by the feed test and HPLC, and the 5B3 clone was selected for the amplification test.

Example 3 expression and purification of the target product

Inoculating the obtained 5B3 clone strain into 2L triangular flask containing 500ml CD CHO culture solution, screwing air-permeable bottle cap, and culturing at 36-37 deg.C and 5% CO₂Culturing on 135r/min rotary shaking table for 4 daysThen, 5B3 cells are transferred into a 5L full-automatic bioreactor containing 2.0L of CD Opti-CHO culture solution, the culture parameters are set as the rotating speed of 60r/min, the temperature of 36.5 ℃, the pH value of 7.0-7.4 and the dissolved oxygen DO of 40% -60%, the cell activity, the cell density and the glucose content are sampled and detected day by day, the viable cell density is increased to 5 multiplied by 10 after 4 days of culture⁶～7×10⁶Supplementing 200-300 ml of CD Efficient Feed C to cells/ml, supplementing 1 time every other day, measuring the glucose content in the culture solution every day, and if the glucose content is lower than 11.1mmole/L, supplementing 40% of glucose solution to the full-automatic bioreactor through a peristaltic pump until the glucose content reaches 22 mmole/L; culturing for 12-14 days, stopping culturing when the ratio of living cells is reduced to 60% -70%, centrifuging at 12000r/min at a high speed (or using a 3M deep filter) to remove cells and cell debris, and collecting cell culture supernatant. The culture supernatant was filtered through a 0.45 μm filter and the filtrate was passed through a 40mM PBS (pH7.4, 150mM NaCl) pre-equilibrated Protein A gel chromatography column (Mabselect)^TM Sure,MabSelect^TM Sure LX,MabCapture^TMA, AT Besterose A, etc.), and then washing 2-4 column volumes with 40mM PBS flow to A₂₈₀Return to baseline level; the eluate is exchanged with 100mM glycine-hydrochloric acid buffer (or citric acid-sodium citrate buffer, acetic acid-sodium acetate buffer, pH 3.0-4.0) to elute the conjugate, the collected eluate is left at room temperature (18-25 ℃) for 30 minutes (inactivation of virus at low pH), and then 0.2M Na is added₂HP0₄Adjusting the pH value to 7.4-8.0, and filtering through a filter membrane of 0.45 mu m to further remove insoluble particles. Protein solution obtained by Protein A affinity chromatography purification is detected by Shimadzu LC-20AT HPLC (BioCore SEC-500, 7.8mm x 30cm, Suzhou Naviz science and technology Co., Ltd)), the ratio of main peak (dimer) is about 65-78%, the ratio of monomer is about 20-30%, a part of polymer product is still existed before the main peak, the ratio is generally within 10%, and the chromatogram is shown in figure 4.

Then loading the harvested protein solution containing the target product into DEAE Sepharose 4Fast Flow (or Q Sepharose 4Fast Flow, NanoGel 50Q, Besterose DEAE, Besterose Q, POROS Q, POROS XQ, etc.) which is well balanced by 20mM PB (pH 7.4-8.0), and after loading is finished, loadingWashing the column with 20mM PB buffer to A₂₈₀Returning to the baseline level, sequentially carrying out gradient elution by NaCl solutions with different concentrations, collecting the target protein VZV gE, regenerating 1 column volume of an anion exchange chromatographic column by using 1.0M NaCl solution, and finally balancing by 20mM PB (pH 7.4-8.0) buffer solution for later use.

And purifying the harvested VZV gE by a Sephacryl S400 HR or other suitable molecular sieve chromatographic column, collecting a target product, sterilizing, filtering and storing at 2-8 ℃.

The purified VZV gE was checked for purity by Shimadzu LC-20AT HPLC with a mobile phase of 40mM PBS (containing 0.5M Na)₂SO₄pH7.5), flow rate 0.750ml/min, and a BioCore SEC500 (7.8X 300mm, Navtech, Inc., or TSK 5000SWxl, Toyo Cauda, Inc.), A, analytical column₂₈₀The detection result shows that the purity of VZV gE reaches more than 98 percent, the result is shown in figure 5, and the relative molecular weight is about 400 KDa.

Example 4 Formaldehyde inactivation of target product

The recombinant VZV gE obtained in example 3 was diluted to 100. mu.g/ml-1000. mu.g/ml with 20mM PBS (pH 7.2-8.0, 135mM NaCl), 38% formaldehyde solution was added to 0.1% (v/v) of the total volume of the solution, and the solution was left at 37 ℃ for 72 hours while shaking twice a day, mixed well, and then left at 2-8 ℃.

Example 5 beta-propiolactone inactivation of the target product

The recombinant VZV gE solution obtained in the example 3 is cooled to 2-8 ℃, after weighing, beta-propiolactone is added until the final concentration is 0.1-0.01% of the weight of the solution, the solution is continuously placed for 72 hours at the temperature of 2-8 ℃, and the solution is shaken twice a day during the period and is uniformly mixed. After 72 hours, the VZV gE solution is heated to 37 ℃ and maintained for 4 hours to completely convert beta-propiolactone into lactic acid, and then the solution is placed at 2-8 ℃.

Example 6 removal of Formaldehyde or beta-propiolactone from the target product

The recombinant VZV gE-containing protein solution obtained in example 5 or example 6 is diluted with 20mM PB (pH 7.2-8.0, or 20mM Tris-HCl solution pH 7.2-8.0) appropriately until the concentration of NaCl in the solution is reduced to below 50mM, and then the VZV gE-containing solution is passed through a filter 2DEAE Sepharose 4FF chromatographic column equilibrated with 0mM PB (or 20mM Tris-HCl, pH 7.2-8.0), followed by subsequent washing with 20mM PB (or 20mM Tris-HCl, pH 7.2-8.0) to A₂₈₀And completely returning to the baseline level, continuously washing 4 column volumes of liquid, eluting VZV gE bound on the gel by using an eluent (containing 20mM PB or 20mM Tris-HCl solution, pH7.5) containing 0.4M NaCl, collecting the solution containing the target product, filtering by using a sterilizing filter membrane with the pore diameter of 0.2 mu M, and collecting the filtrate, namely the vaccine stock solution.

Example 7 preparation of aluminum-adjuvanted vaccines

Diluting the vaccine stock solution obtained in the example 6 to 10 mu g/ml-800 mu g/ml by using 20mM Tris-HCl (pH7.2-7.5, containing 135-150 mM NaCl), uniformly mixing the diluted vaccine stock solution and an equal volume of aluminum hydroxide adjuvant suspension (the content of aluminum is 0.2-1.5 mg/ml) at room temperature, and then placing the mixture at 2-8 ℃.

Taking out the vaccine solution containing the aluminum adjuvant from 2-8 ℃, sub-packaging into 2ml penicillin bottles (or pre-filling into glass syringes) under an aseptic condition, sealing, and storing in the dark at 2-8 ℃.

The following table shows an example of preparing vaccines with different VZV gE contents in a volume of 1000ml (the 1 st column from the left is the antigen content in 1ml of vaccine prepared, and the 1 st column from the right is the antigen content in 0.5ml of vaccine used in conventional intramuscular injection), and using vaccine stock solution containing VZV gE with a concentration of 800 μ g/ml to prepare aluminum-adjuvant-containing vaccines, the preparation method is as follows:

TABLE 1 formulation of VZV gE aluminum adjuvant vaccine solutions of different antigen content

Example 8 lyophilized recombinant VZV gE fusion protein

Diluting the vaccine stock solution obtained in the example 6 to 40-800 mu g/ml by using 20mM Tris-HCl (pH 7.2-7.5, containing 135mM NaCl), adding 10% sucrose (or 10% trehalose, 10% mannitol and 10 lactose) solution to a final concentration of 3%, adjusting the concentration of VZV gE in the solution to be subpackaged to 20 mu g/ml (or 50, 80, 100, 200 and 400 mu g/ml), mixing uniformly, subpackaging into 2ml tubular bottles with 1.0ml of each bottle, adding a butyl rubber plug into a freeze-drying bin after half adding, setting a pre-freezing temperature to-40-45 ℃, freezing for 4 hours, then starting vacuum freeze-drying, controlling the temperature by adopting an automatic temperature raising program, raising the temperature from-40 ℃ to-25 ℃ for 6 hours, raising the temperature from-25 ℃ to-5 ℃ for 4 hours, maintaining the temperature from 0-5 ℃ for 1 hour, maintaining the temperature at 25 ℃ for 1 hour, and maintaining the temperature at 35 ℃ for 6-8 hours, and then pressing the butyl rubber plug (or filling a high-purity nitrogen or argon pressure plug) in a vacuum state.

Taking out the small bottle which is pressed and plugged from the freeze dryer, sending the small bottle into an automatic capping machine, and locking an aluminum cap. Then transferring to a cold storage at 2-8 ℃ for storage. Before use, 1.0ml of water for injection or aluminum hydroxide adjuvant suspension is sucked by a disposable sterile syringe and injected into a vial containing the freeze-dried VZV gE, and the mixture is gently mixed for about 5 minutes to obtain the vaccine without macroscopic particles. The vaccine should be used immediately after dissolution, or within 30 minutes at the latest. The vaccine is used for subcutaneous or intramuscular injection, and is strictly forbidden to be used for intravenous injection.

Example 9 animal immunization experiment an aluminum adjuvant-adsorbed vaccine containing VZV gE 100. mu.g/ml was taken out from a freezer at 2-8 ℃, diluted with 20mM Tris-HCl to a solution containing VZV gE 8. mu.g/ml and 2. mu.g/ml, and then an equal volume of aluminum adjuvant was added to the solution to prepare an animal experimental aluminum adjuvant vaccine containing VZV gE 4. mu.g/ml and 1. mu.g/ml (or VZV gE 2. mu.g/ml and 0.5. mu.g/0.5 ml).

Randomly dividing female BALB/C of 4-6 weeks into 5 groups, wherein each group comprises 8 mice, and injecting 0.5ml of aluminum adjuvant into the abdominal cavity of each mouse in a control group; 4 experimental groups are injected with 0.5ml of VZV gE antigen adsorbed by aluminum-containing adjuvant 50, 10, 2 and 0.5 mu g in the abdominal cavity respectively, and each dosage group is injected with 8 mice; immunizing for 1 time at an interval of two weeks after the primary immunization, immunizing for 4 times totally, collecting blood from tail vein 7 days after 2 and 3 times of immunization, separating serum, freezing at-70 deg.C, collecting blood from heart 7 days after 4 times of immunization, separating serum, freezing at-70 deg.C.

And (3) ELISA antibody titer detection: diluting the recombinant VZV gE-His protein to 1 mu g/ml by using a carbonate buffer solution, coating a 96-hole enzyme label plate (Costar), placing 100 mu l of the recombinant VZV gE-His protein in each hole at 37 ℃ for 1 hour, and placing the recombinant VZV gE-His protein at 2-8 ℃ overnight; the liquid in the 96-well plate was discarded, washed 3 times with 20mM PBS, and then 200. mu.l of blocking solution (2%)Bovine serum albumin, component V), blocked for 60 minutes at room temperature; sucking the blocking liquid in the holes, washing the holes for 3 times by using 20mM PBS-T solution, adding the mouse serum pre-diluted by 1:50 (or 1:500 or 1:1000) into the first row of holes of a 96-hole enzyme label plate, then carrying out 2-fold serial dilution by multiple, reacting the negative control by using the mouse serum (1:100) only immunized by using an aluminum adjuvant in an abdominal cavity for 60 minutes at 37 ℃, sucking the blocking liquid in the holes, and washing the holes for 3 times by using the 20mM PBS-T solution; taking out goat anti-mouse IgG-HRP conjugate, diluting with an enzyme conjugate diluent 1:100 (the diluent contains 1mg/ml of human IgG), pre-reacting for 30 minutes at room temperature, then adding into a 96-hole enzyme label plate, reacting for 30 minutes at 37 ℃ with 100 mu l of each hole; the blocking solution in the well was aspirated, washed 3 times with 20mM PBS-T solution, 100. mu.l of TMB developing solution was added to each well, 50. mu.l of stop solution was added after 10 minutes to terminate the reaction, and then A was measured with a TECAN Infinit 200 microplate reader₄₅₀Absorbance, mixing with negative control group serum A₄₅₀3 times of the amount of the total of₄₅₀Values below 0.100, calculated as 0.100), the titer of the sera after immunization is determined. The geometric mean and standard deviation of the titers of anti-VZV gE antibodies in the sera of the mice of each experimental group are shown in Table 2 below; after 2 immunizations, positive transfer occurred except in the lowest dose group (0.5 μ g)1 mouse serum antibody which did not positive transfer; the statistical analysis of the antibody titer determined by each group of experimental animals shows that after 3 times of immunization of the experimental mice of each dose group, the serum antibody titer is obviously higher than that after 2 times of immunization, the serum antibody titer is still partially increased but not obviously increased after 4 th immunization, and after 2, 3 and 4 times of immunization, the serum titers of the mice of the three high dose groups have no significant difference, and the antibody titers in the serum have no significant difference. Serum antibody titers after immunization of BALB/C mice with each dose of VZV gE aluminum adjuvant vaccine are shown in FIG. 6.

Serum neutralizing antibody titer assay: VZV is a virus which can generate cell fusion lesion on human embryonic lung diploid cells, so that the virus plaque reduction neutralization experiment can be used for detecting the virus neutralization capacity of antibodies with different serum dilutions, and calculating the serum titer which can reduce the virus number by 50%. The serum antibody neutralization virus experiment is the most direct experiment for detecting whether the neutralizing VZV virus antibody exists in the serum after immunizationThe experiment has the defects of complex operation, low sensitivity, more manpower consumption, longer time consumption, incapability of using instruments and equipment for automatic interpretation and the like, and ED₅₀The above inconveniences lead to the use of this experiment for the determination of serum neutralizing antibodies by very few people, requiring specialized data processing software for the calculations. Since the experiment used too large amounts of serum, we only performed neutralizing antibody assays on mouse sera taken after the last immunization.

The assay was a VZV serum neutralization assay performed on flat-bottom 96-well microplates with the cell matrix being MRC-5 cells (purchased from ATCC) sensitive to VZV. Taking out frozen serum from a refrigerator at the temperature of-70 ℃, thawing the frozen serum at room temperature, diluting the serum to be detected with 199 culture solution (GIBCO) containing 10% FBS in a sterile purification workbench at a ratio of 1:10, then carrying out 4-fold serial dilution in a 96-well plate, measuring 7 dilutions in total, wherein each dilution is used for carrying out two-well measurement, positive control serum is rabbit anti-VZV gE serum, negative control serum is mouse mixed serum (diluted at a ratio of 1: 10) immunized by using an aluminum adjuvant, and each 96-well plate is provided with 6-well virus solution control holes and 6-well cell control; mixing diluted blood serum to be detected with a certain amount of OKA strain virus (purchased from ATCC in America), covering with 96-well microplate, shaking on a microplate oscillator for 30 seconds, and placing in 10% CO₂The reaction was carried out at 37 ℃ in an incubator (10%) for 30 minutes.

Taking out the MRC-5 cells (ATCC, generation 25-38) growing in a full monolayer from a carbon dioxide incubator, sucking out a culture solution in a T75 culture bottle (Corning) in a hundred-grade purification workbench, adding 5ml of a 0.25% pancreatin (GIBCO) solution to digest the MRC-5 cells growing in a monolayer, acting for 3 minutes at room temperature, sucking out the pancreatin solution, adding 10ml of a cell culture solution, slightly blowing the inner surface of the cell culture bottle, dispersing the MRC-5 cells, and adding a certain volume of the cell culture solution to prepare a cell suspension. Taking out a 96-hole microporous plate containing serum to be detected from the incubator, adding cell suspension by using a multichannel pipettor, covering a plate cover, oscillating the plate cover on a microporous plate oscillator for 30 seconds, putting the plate cover into a carbon dioxide incubator (10%) with the temperature of 37 ℃ for culturing for 3-4 days, observing cytopathic effect of each hole by using an inverted microscope every day after 48 hours, and accurately counting and recording the number of plaques in each hole. After 96 hours, 12-channel pipettor is usedSucking out the liquid in the hole to a waste liquid tank containing 0.1% sodium hypochlorite, adding 0.1% crystal violet for dyeing for 1 hour, decoloring, reversely buckling the microporous plate on absorbent paper, and drying at room temperature. Recording the number of the plaques of each dilution of each serum into an EXCEL 2016 table, calculating the reduction number of the plaques of each dilution of the serum by taking the average number of the plaques in the virus solution holes (6-hole average value, 25-30/hole) as 100%, converting the reduction number into percentage, and calculating ED (immune deficiency) of the data by using Prism 5.0 software₅₀Value, this ED₅₀The values are the serum titer at which the number of plaques is reduced by 50%, and the neutralizing antibody titer ED of the serum of each mouse in the four dose groups₅₀The measurement curve of (A) is shown in FIG. 7, and the geometric mean titer of the serum neutralizing antibody titer and the distribution of the neutralizing antibody titer of each dose group are shown in Table 3; a comparison of the geometric mean titers of the VZV serum neutralizing antibody titers of the dose groups is shown in FIG. 8, from which it can be seen that the serum neutralizing antibody titers of the other 3 groups reached higher levels, except that the neutralizing antibody titer of the lowest dose group was significantly lower than that of the other 3 groups.

TABLE 2 serum antibody titers (ELISA) following immunization of BALB/C mice with different doses of VZV gE aluminum adjuvant vaccine

TABLE 3 serum neutralizing antibody titers after different doses of VZV gE aluminum adjuvant-adsorbed vaccine immunization of BALB/C mice

Sequence listing

<110> Beijing Green bamboo Biotechnology Ltd

<120> a recombinant varicella zoster virus vaccine

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<213> Artificial sequence

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Ser Val Leu Arg Tyr Asp Asp Phe His Ile Asp Glu Asp Lys Leu

5 10 15

Asp Thr Asn Ser Val Tyr Glu Pro Tyr Tyr His Ser Asp His Ala

20 25 30

Glu Ser Ser Trp Val Asn Arg Gly Glu Ser Ser Arg Lys Ala Tyr

35 40 45

Asp His Asn Ser Pro Tyr Ile Trp Pro Arg Asn Asp Tyr Asp Gly

50 55 60

Phe Leu Glu Asn Ala His Glu His His Gly Val Tyr Asn Gln Gly

65 70 75

Arg Gly Ile Asp Ser Gly Glu Arg Leu Met Gln Pro Thr Gln Met

80 85 90

Ser Ala Gln Glu Asp Leu Gly Asp Asp Thr Gly Ile His Val Ile

95 100 105

Pro Thr Leu Asn Gly Asp Asp Arg His Lys Ile Val Asn Val Asp

110 115 120

Gln Arg Gln Tyr Gly Asp Val Phe Lys Gly Asp Leu Asn Pro Lys

125 130 135

Pro Gln Gly Gln Arg Leu Ile Glu Val Ser Val Glu Glu Asn His

140 145 150

Pro Phe Thr Leu Arg Ala Pro Ile Gln Arg Ile Tyr Gly Val Arg

155 160 165

Tyr Thr Glu Thr Trp Ser Phe Leu Pro Ser Leu Thr Cys Thr Gly

170 175 180

Asp Ala Ala Pro Ala Ile Gln His Ile Cys Leu Lys His Thr Thr

185 190 195

Cys Phe Gln Asp Val Val Val Asp Val Asp Cys Ala Glu Asn Thr

200 205 210

Lys Glu Asp Gln Leu Ala Glu Ile Ser Tyr Arg Phe Gln Gly Lys

215 220 225

Lys Glu Ala Asp Gln Pro Trp Ile Val Val Asn Thr Ser Thr Leu

230 235 240

Phe Asp Glu Leu Glu Leu Asp Pro Pro Glu Ile Glu Pro Gly Val

245 250 255

Leu Lys Val Leu Arg Thr Glu Lys Gln Tyr Leu Gly Val Tyr Ile

260 265 270

Trp Asn Met Arg Gly Ser Asp Gly Thr Ser Thr Tyr Ala Thr Phe

275 280 285

Leu Val Thr Trp Lys Gly Asp Glu Lys Thr Arg Asn Pro Thr Pro

290 295 300

Ala Val Thr Pro Gln Pro Arg Gly Ala Glu Phe His Met Trp Asn

305 310 315

Tyr His Ser His Val Phe Ser Val Gly Asp Thr Phe Ser Leu Ala

320 325 330

Met His Leu Gln Tyr Lys Ile His Glu Ala Pro Phe Asp Leu Leu

335 340 345

Leu Glu Trp Leu Tyr Val Pro Ile Asp Pro Thr Cys Gln Pro Met

350 355 360

Arg Leu Tyr Ser Thr Cys Leu Tyr His Pro Asn Ala Pro Gln Cys

365 370 375

Leu Ser His Met Asn Ser Gly Cys Thr Phe Thr Ser Pro His Leu

380 385 390

Ala Gln Arg Val Ala Ser Thr Val Tyr Gln Asn Cys Glu His Ala

395 400 405

Asp Asn Tyr Thr Ala Tyr Cys Leu Gly Ile Ser His Met Glu Pro

410 415 420

Ser Phe Gly Leu Ile Leu His Asp Gly Gly Thr Thr Leu Lys Phe

425 430 435

Val Asp Thr Pro Glu Ser Leu Ser Gly Leu Tyr Val Phe Val Val

440 445 450

Tyr Phe Asn Gly His Val Glu Ala Val Ala Tyr Thr Val Val Ser

455 460 465

Thr Val Asp His Phe Val Asn Ala Ile Glu Glu Arg Gly Phe Pro

470 475 480

Pro Thr Ala Gly Gln Pro Pro Ala Thr Thr Lys Pro Lys Glu Ile

485 490 495

Thr Pro Val Asn Pro Gly Thr Ser Pro Leu Leu Arg Tyr Ala Ala

500 505 510

Trp Thr Gly Gly Leu Ala Glu Pro Lys Ser Cys Asp Lys Thr His

515 520 525

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

530 535 540

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

545 550 555

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

560 565 570

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

575 580 585

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

590 595 600

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

605 610 615

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

620 625 630

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

635 640 645

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

650 655 660

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

665 670 675

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

680 685 690

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

695 700 705

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

710 715 720

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

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His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

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agcgtgctgc ggtacgacga tttccacatc gacgaggata agctggacac caatagcgtg 60

tatgagcctt actatcactc cgaccacgcc gagtccagct gggtgaacag gggcgagtct 120

tcccggaagg cttacgacca caactctccc tatatctggc ctagaaatga ctacgatggc 180

tttctggaga acgcccatga gcaccatggc gtgtataatc agggccgggg catcgactct 240

ggcgagagac tgatgcagcc tacacagatg tccgctcagg aggatctggg cgacgataca 300

ggcatccacg tgatcccaac cctgaatggc gacgacaggc ataagatcgt gaacgtggat 360

cagaggcagt acggcgacgt gttcaagggc gatctgaatc ccaagcctca gggccagcgc 420

ctgatcgagg tgtccgtgga ggagaaccat ccattcaccc tgcgcgcccc tatccagcgc 480

atctacggcg tgaggtatac cgagacatgg tcctttctgc ctagcctgac ctgcacaggc 540

gacgctgctc cagctatcca gcacatctgc ctgaagcata ccacatgttt tcaggacgtg 600

gtggtggacg tggattgtgc cgagaataca aaggaggatc agctggctga gatcagctac 660

agattccagg gcaagaagga ggccgatcag ccatggatcg tggtgaacac ctctacactg 720

tttgacgagc tggagctgga cccccctgag atcgagcctg gcgtgctgaa ggtgctgcgc 780

accgagaagc agtacctggg cgtgtatatc tggaacatga ggggcagcga cggcacctcc 840

acatacgcta ccttcctggt gacatggaag ggcgatgaga agacccggaa tccaacacca 900

gctgtgaccc ctcagccaag aggcgctgag tttcacatgt ggaactatca cagccacgtg 960

ttctccgtgg gcgacacctt ttccctggcc atgcacctgc agtacaagat ccatgaggct 1020

ccattcgacc tgctgctgga gtggctgtat gtgcccatcg atcctacatg ccagcccatg 1080

cgcctgtaca gcacctgtct gtatcaccca aatgcccccc agtgcctgtc ccatatgaac 1140

agcggctgta cctttacatc cccacacctg gcccagaggg tggctagcac agtgtaccag 1200

aactgcgagc atgccgacaa ttacaccgct tattgtctgg gcatctctca catggagcct 1260

tccttcggcc tgatcctgca tgacggcggc accacactga agtttgtgga tacccctgag 1320

agcctgtctg gcctgtacgt gttcgtggtg tacttcaacg gccacgtgga ggccgtggct 1380

tacacagtgg tgtctaccgt ggatcatttc gtgaacgcca tcgaggagag gggatttcca 1440

ccaacagctg gacagcctcc agctaccaca aagcccaagg agatcacacc tgtgaaccca 1500

ggcacctccc ctctgctgag atatgccgct tggaccggcg gcctggctga gcccaaatct 1560

tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 1620

gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 1680

acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 1740

gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 1800

taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 1860

aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1920

aaagggcagc cccgagaacc acaggtgtac accctgcctc catctcggga tgagctgacc 1980

aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 2040

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgccccc cgtgctggac 2100

tccgacggct ccttcttcct ctatagcaag ctcaccgtgg acaagagcag gtggcagcag 2160

gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 2220

agcctctccc tgtctccggg taaa 2244