Varicella-zoster virus nanoparticle protein, and preparation method therefor and use thereof

Abstract

A varicella-zoster virus (VZV) nanoparticle protein, and a preparation method therefor and a use thereof. The VZV protein comprises a partial or full sequence of an amino acid sequence of an extracellular region of a VZV gE glycoprotein, with W at position 200 mutation to C and L at position 245 mutation to C in the amino acid sequence of the extracellular region of the VZV gE glycoprotein. By means of the rational optimization design of the amino acid sequence of the VZV gE protein by means of protein genetic engineering, the VZV gE recombinant protein modified with amino acid mutations has increased stability and immunogenicity compared with the VZV gE protein. Moreover, by further designing the protein structure, the VZV gE protein is repeatedly displayed on the surface of ferritin nanoparticles with the desired epitopes exposed, thereby further enhancing immunogenicity.

Description

A varicella-zoster virus nanoparticle protein and a preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a varicella-zoster virus nanoparticle protein and a preparation method and application thereof. BACKGROUND

[0002] Varicella-zoster virus (VZV) is also known as human herpesvirus 3 (HHV-3), belongs to the human herpesvirus alpha subfamily, and is one of the eight human herpesviruses. It is the smallest among all known herpesviruses, about half the size of human cytomegalovirus (HCMV). VZV virus is composed of a linear, double-stranded DNA wrapped by a nucleocapsid, and a protein layer separates the nucleocapsid from the lipid envelope. This protein layer forms the main viral glycoprotein. VZV produces about six glycoproteins, now designated gB, gC, gE, gH, gI, and gL. These proteins are also expressed on the surface of infected cell membranes during viral replication. In infected cells, the gE protein is the most abundant protein, which is adsorbed to gI in a non-covalent manner, and can be adsorbed to the Fc fragment of antibody G (IgG). The gB protein is the target of neutralizing antibodies and plays an important role in viral entry into cells. The gH protein has a fusion function, facilitating the spread of viruses between cells; gH protein requires gL protein for glycosylation and transfer to the cell surface. The gC protein has no functional role in VZV replication.

[0003] VZV-gE glycoprotein (glycoprotein E, gE) is the key antigen protein currently studied. gE is encoded by the open reading frame (ORF) 68 gene of the virus, which consists of 1872 bases located in the short fragment region of the VZV genome. This protein contains 623 amino acid sequences including a signal peptide, a protein main body, a hydrophobic anchor region, and a C-terminal tail region. gE exists on the surface of viral particles and infected cells and in the cytoplasm, is an essential glycoprotein for the production of infectious viral particles, and is the most abundant envelope glycoprotein on the viral envelope and host cell membrane. In the serum of patients with varicella and herpes zoster in the recovery period, VZV antibodies are mainly directed against gE, gB, and gH, especially the cellular immunity and humoral immunity induced by gE as the main target, which can protect animals from viral attack. Therefore, gE protein is an important target for effectively inducing cellular immune and humoral immune responses.

[0004] VZV virus is a virus that is prevalent worldwide, with strong infectivity, and only one serotype has been found so far. In nature, VZV only infects humans. The primary infection caused by VZV is varicella, which is common in childhood. After the primary infection, the virus can establish a lifelong latent period in the host sensory ganglion. In addition, studies have found that the FDA-approved Oka strain attenuated live vaccine for varicella prevention and wild-type virus can also establish latent infection. As age increases, immune function is impaired, and other factors can lead to weakened cellular immune response, which can induce VZV reactivation and trigger herpes zoster (HZ). Herpes zoster is more common in adults and the elderly. Its symptoms are unilateral vesicular skin rash, usually accompanied by symptoms such as fever and fatigue, and local skin begins to turn red with a burning and nerve pain. About 9% to 34% of patients with herpes zoster will develop post-herpetic neuralgia (PHN), with pain ratings of 7 or higher, which is severe pain, and the pain can last for 3 months to several years, seriously affecting the patient's quality of life. Some elderly people also develop herpes zoster ophthalmia, which is characterized by unilateral eyelid swelling, conjunctival hyperemia, and accompanied by headache symptoms, and in severe cases, even permanent loss of independent living ability.

[0005] The incidence of herpes zoster is on the rise.

[0006] Herpes zoster and post-herpetic neuralgia are currently mainly treated with antiviral and symptomatic treatment, and there is no specific drug. Most clinical use broad-spectrum antiviral drugs such as acyclovir for treatment, and some anesthetic or non-anesthetic analgesics and anticonvulsant, antidepressant drugs are also used to relieve the severe neuralgia caused by herpes zoster. Herpes zoster may relapse even after recovery, so vaccination with VZV vaccine is the most effective, simplest, and most economical means of controlling infectious diseases.

[0007] Currently, there are four types of shingles vaccines approved for marketing worldwide, one is attenuated live vaccine: Merck's Zostavax, SK Chemical's NBP608 (2017, only in the Korean market) and China Changchun Baige (domestic market in June 2023); the other is recombinant protein vaccine: GSK's Shingrix. Among them, Merck's Zostavax was approved by the FDA in May 2006, followed by the European Union, Canada, Australia and most countries in Asia and the Middle East (> 50 countries), but has not been approved in China. Zostavax is an attenuated live vaccine based on the Oka strain. In people aged 60-69, Zostavax is used as a single subcutaneous injection, and the vaccine's preventive efficacy is 64%. With age, Zostavax efficacy decreases, and in adults over 60 years of age, the vaccine's effectiveness against PHN is only 39%.

[0008] Another vaccine approved by the FDA in the United States, Shingrix (GSK), is a shingles recombinant subunit vaccine containing gE glycoprotein and AS01B adjuvant. It was approved by the FDA in October 2017 for the prevention of shingles in people aged ≥50 years, and is the second shingles vaccine approved for marketing by the FDA. It was approved for marketing in China in May 2019 and became the first shingles vaccine in the country. Shingrix uses AS01B adjuvant, which is composed of monophosphoryl lipid A (MPL) and QS-21. MPL is an immune-enhancing substance isolated from the surface of bacteria, and QS-21 is a natural compound extracted from Quillaja saponaria Molina in Chile. Shingrix is administered by intramuscular injection, and vaccination in people aged ≥50 years with normal immune function reduces the incidence of shingles and PHN by 97.2% and 91.2%, respectively, and in people aged ≥70 years, it reduces the incidence by 89.8% and 88.8%, respectively, which is better than Merck's attenuated live vaccine Zostavax. Although Shingrix is significantly better than Zostavax, the vaccine formulation is complex, the antigen needs to be freeze-dried and separated from the adjuvant, and the two are mixed when used, and its ASO1B adjuvant system, especially QS-21, cannot be chemically synthesized, and the raw materials are relatively scarce, the production cost is relatively high, the price is expensive, and the domestic supply is severely insufficient. Therefore, it is particularly important to further explore and develop new VZV vaccines with small side effects, high effectiveness, and simple formulation and low cost.

[0009] With the continuous development of science and technology, nanoparticle vaccines as a new vaccine technology concept are an important supplement and development of existing vaccine technologies, aiming to improve the safety and effectiveness of vaccines through innovative vaccine design, and are expected to provide new solutions for the prevention and control of viral infections worldwide, especially in resource-limited environments. However, for VZV viruses, how to tap the potential of nanoparticle vaccines, how to optimize antigen presentation and improve the immunogenicity of vaccines to solve the problems faced by existing vaccines, and how to design nanoparticle proteins to enhance the recognition and response of the immune system while ensuring stable antigen delivery and reducing storage and transportation challenges, all of which still need further research and exploration. SUMMARY

[0010] In order to overcome the deficiencies in the prior art, the present application provides a new type of VZV nanoparticle recombinant protein vaccine, which is developed to solve the problems faced by existing vaccines by optimizing antigen presentation and improving the immunogenicity of vaccines, and to further enhance the recognition and response of the immune system by reasonably optimizing the design of VZV key antigen protein (gE protein) combined with ferritin nanoparticle carrier.

[0011] On the one hand, the vaccine can maintain the structural stability and antigen cluster function of the protein under different temperature environments through amino acid mutation modification of VZV gE protein, which helps to stabilize antigen delivery and reduce the challenge of temperature changes to protein structure during storage and transportation. On the other hand, the present application can present VZV gE protein repeatedly on the surface of the nanoparticle by connecting the amino acid mutation modified VZV gE protein to the ferritin nanoparticle carrier, greatly increasing the density of antigens and neutralizing epitopes, and being able to more effectively stimulate the body to produce an immune response.

[0012] In addition, the VZV nanoparticle recombinant protein vaccine provided by the present application can also quickly activate antigen-presenting cells and promote the uptake and cross-presentation of antigens to strengthen the immune response level under the condition of reducing the addition of adjuvants, and has better antigen cluster function and obvious advantages in neutralizing antibody induction effect compared with the VZV recombinant protein vaccine. Moreover, the preparation process of the VZV nanoparticle recombinant protein vaccine provided by the present application is relatively simple, without the need for a complex adjuvant system (such as ASO1B), which reduces production costs and facilitates future large-scale production and supply.

[0013] The new type of VZV nanoparticle recombinant protein vaccine provided by the present application plays a very important role in making up for the deficiencies of existing interventions for VZV infection in high-risk populations, and in increasing the research and development of domestic vaccine products and accelerating the development of vaccine clinical trials in China.

[0014] The technical solutions of the present application for solving the technical problems are as follows:

[0015] In the first aspect of the present application, a varicella-zoster virus (VZV) protein is provided, which comprises part or all of the amino acid sequence of the extracellular region of VZV gE protein, and the W at position 200 and the L at position 245 in the amino acid sequence of the extracellular region of VZV gE protein are mutated to C, and the amino acid sequence of the extracellular region of VZV gE protein is 1-546 of the sequence shown in SEQ ID NO. 1.

[0016] Further, the protein comprises all of the amino acid sequence of the extracellular region of VZV gE protein, and the W at position 200 and the L at position 245 in the sequence shown in SEQ ID NO. 2 are mutated to C.

[0017] Further, the protein is any one of gE-monomer, gE-dimer and gE-ferritin nanoparticle protein, which are modified by amino acid mutation and designed by structure optimization to enhance stability and immunogenicity, and the amino acid sequences of the gE-monomer, gE-dimer and gE-ferritin nanoparticle protein are shown in SEQ ID NO: 4-6, respectively.

[0018] Further, when the protein is gE-ferritin nanoparticle protein, the amino acid mutation and structure optimization are designed as follows: the transmembrane region and intracellular region in the full-length sequence of VZV gE protein shown in SEQ ID NO. 1 are deleted to obtain the extracellular region sequence of gE protein shown in SEQ ID NO. 2, and then point mutation of amino acid is performed, the W at position 200 and the L at position 245 in the VZV gE protein are mutated to C, and the ferritin sequence shown in SEQ ID NO. 7 is connected to the C-terminus, and then the full-length mutant sequence of VZV gE-ferritin nanoparticle is obtained, and the amino acid sequence is shown in SEQ ID NO. 6.

[0019] Further, when the protein is a gE-monomer nanoparticle protein, the amino acid mutation modification and structure optimization design is as follows: the transmembrane region and intracellular region in the full-length sequence of VZV gE protein with the amino acid sequence shown as SEQ ID NO. 1 are deleted to obtain the extracellular region sequence of gE protein shown as SEQ ID NO. 2, and on this basis, point mutation of amino acids is performed, W at position 200 of VZV gE protein is mutated to C, L at position 245 is mutated to C, and Throm and 6his sequences are connected at the C-terminal end, and then the full-length mutant sequence of VZV gE-monomer is obtained, and the amino acid sequence is shown as SEQ ID NO. 4.

[0020] Further, when the protein is a gE-dimer nanoparticle protein, the amino acid mutation modification and structure optimization design is as follows: the transmembrane region and intracellular region in the full-length sequence of VZV gE protein with the amino acid sequence shown as SEQ ID NO. 1 are deleted to obtain the extracellular region sequence of gE protein shown as SEQ ID NO. 2, and on this basis, point mutation of amino acids is performed, W at position 200 of VZV gE protein is mutated to C, L at position 245 is mutated to C, and the dimer sequence shown as SEQ ID NO. 8 is designed to be connected at the C-terminal end, and then the full-length mutant sequence of VZV gE-dimer is obtained, and the amino acid sequence is shown as SEQ ID NO. 5.

[0021] In the second aspect of the present application, a biological material is provided, which is at least one of the following (1)-(4):

[0022] (1) a nucleic acid molecule encoding the varicella-zoster virus protein according to the first aspect;

[0023] (2) a recombinant expression vector containing the nucleic acid molecule according to (1);

[0024] (3) a recombinant microorganism containing the nucleic acid molecule according to (1) or a recombinant microorganism containing the recombinant expression vector according to (2);

[0025] (4) a recombinant cell line containing the nucleic acid molecule according to (1) or a recombinant cell line containing the recombinant expression vector according to (2).

[0026] In the third aspect of the present application, a preparation method of the varicella-zoster virus protein according to the first aspect is provided, which comprises the following steps: expressing the nucleic acid molecule encoding the varicella-zoster virus protein according to the first aspect in a biological organism or a biological cell to obtain the varicella-zoster virus protein.

[0027] Further, the method for preparing the varicella-zoster virus protein comprises the following steps: introducing the nucleic acid molecule encoding the varicella-zoster virus protein as described in the first aspect into CHO cells to obtain recombinant cells; culturing the recombinant cells to obtain the varicella-zoster virus protein.

[0028] In a fourth aspect of the present application, there is provided the use of the method for preparing the varicella-zoster virus protein as described in the first aspect or the biological material as described in the second aspect or the method for preparing the varicella-zoster virus protein prepared according to the method as described in the third aspect in any one of the following (1)-(3):

[0029] (1) preparing a product against VZV;

[0030] (2) preparing a product for preventing and / or treating VZV infection;

[0031] (3) preparing a product for preventing and / or treating a disease caused by VZV.

[0032] In a fifth aspect of the present application, there is provided an immunogenic composition comprising the varicella-zoster virus protein as described in the first aspect or the biological material as described in the second aspect or the method for preparing the varicella-zoster virus protein prepared according to the method as described in the third aspect.

[0033] In a sixth aspect of the present application, there is provided a varicella-zoster vaccine comprising the varicella-zoster virus protein as described in the first aspect or the biological material as described in the second aspect or the method for preparing the varicella-zoster virus protein prepared according to the method as described in the third aspect or the immunogenic composition as described in the fifth aspect, and optionally a pharmaceutically acceptable adjuvant.

[0034] Further, in the varicella-zoster vaccine, the adjuvant comprises any one or a combination of any multiple of aluminum salt adjuvant, CpG adjuvant, immune stimulating complex, and liposome adjuvant.

[0035] Further, in the varicella-zoster vaccine, the formulation form of the vaccine is aqueous or lyophilized.

[0036] In a seventh aspect of the present application, there is provided the use of the varicella-zoster vaccine as described in the sixth aspect in any one of the following (1)-(3):

[0037] (1) preparing a product against VZV;

[0038] (2) preparing a product for preventing and / or treating VZV infection;

[0039] (3) preparing a product for preventing and / or treating a disease caused by VZV.

[0040] The present application has the following technical effects:

[0041] (1) The present application uses protein genetic engineering means to reasonably optimize the amino acid sequence of VZV gE protein. The VZV gE recombinant protein modified by amino acid mutation has increased stability and immunogenicity compared with VZV gE protein. And through further design of protein structure, the VZV gE protein is repeatedly displayed on the surface of ferritin nanoparticles, and the required antigen epitopes are exposed, further enhancing the immunogenicity.

[0042] (2) The present application can make the protein maintain its structural stability and antigen cluster function under different temperature environments by mutating the amino acid of VZV gE protein. Even after chemical reaction, it can still maintain good antigenicity. Experiments have proved that the stability of the modified gE protein prepared by the present application under different temperatures is significantly better than that of the unmodified gE protein, and the modified gE protein after high temperature treatment still shows high antigen binding activity.

[0043] (3) The present application designs amino acid mutation of VZV gE protein, and fuses and expresses gE mutant protein and ferritin particles in eukaryotic cells to obtain VZV-gE ferritin recombinant protein nanoparticles repeatedly displayed on the surface of ferritin nanoparticles. The gE ferritin nanoparticles stabilize and expose the required antigen epitopes, destroy or hide the unnecessary antigen epitopes, and effectively improve the immunogenicity of the antigen. Experiments have shown that: on the one hand, compared with VZV-gE monomer recombinant protein vaccine, VZV-gE ferritin nanoparticle recombinant protein vaccine immunizes mice with the highest antibody titer value, and can obtain serum with higher protective titer, indicating that the VZV-gE ferritin nanoparticle recombinant protein vaccine prepared by the present application has the best immunization effect; on the other hand, compared with VZV-gE protein (GSK) and VZV-gE dimer recombinant protein vaccine, VZV-gE ferritin nanoparticle recombinant protein vaccine immunizes mice with the highest antibody titer value, and can obtain serum with higher protective titer, indicating that the VZV-gE ferritin nanoparticle recombinant protein vaccine prepared by the present application has the best immunization effect, and the VZV-gE dimer recombinant protein vaccine is the second.

[0044] (4) The VZV-gE ferritin nanoparticle recombinant protein vaccine prepared by the application can achieve good immune effect with less adjuvant, thereby reducing potential side effects caused by the use of adjuvant and being safer; the preparation process of the VZV nanoparticle recombinant protein vaccine provided by the application is relatively simple, and a complex adjuvant system (such as ASO1B) is not needed, thereby reducing the production cost of the vaccine, being conducive to large-scale production and supply in the future, and providing an effective and low-cost option for the research and development of VZV vaccines. It is shown by experiments that: on the one hand, for the VZV-gE monomer recombinant protein, when the content of the adjuvant in the immunization preparation is added in a certain proportion, the serum protection titer obtained will increase, and the immune effect is better, which shows that the addition of the adjuvant has a certain enhancing and assisting effect on the immune effect of the vaccine; and the results in Figure 5 show that although the content of the CpG adjuvant added in the preparation M1 is 10 times that of the CpG adjuvant in the preparation F, the antibody titer of the VZV-gE ferritin nanoparticle recombinant protein vaccine (preparation F) immunized mice is still higher than that of the VZV-gE monomer recombinant protein vaccine (preparation M1). On the other hand, in the case that only one kind of CpG adjuvant is added in the gE ferritin nanoparticle recombinant protein vaccine, the protein antibody titer IC50 (Log10) value is still higher than that of the VZV-gE protein (GSK) and gE dimer recombinant protein vaccine to which two kinds of adjuvants (Alum+CpG) are added. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 is a transmission electron microscope image of the VZV gE nanoparticle protein.

[0046] Figure 2 is a temperature stability detection of the gE protein before and after modification (0.5 ug / mL).

[0047] Figure 3 is a temperature stability detection of the gE protein before and after modification (0.125 ug / mL).

[0048] Figure 4 is a temperature stability detection of the gE protein before and after modification (0.02 ug / mL).

[0049] Figure 5 is a protein antibody titer detection in the mouse immune serum of the preparation A (before modification) and the preparation B (after modification).

[0050] Figure 6 is a protein antibody titer detection in the mouse immune serum of the preparation M1, the preparation M2 and the preparation F.

[0051] Figure 7 is a protein antibody titer detection in the mouse immune serum of the preparation G1, the preparation D1 and the preparation F1.

[0052] Figure 8 is a protein antibody titer detection in the mouse immune serum of the preparation G2, the preparation D2 and the preparation F2.

[0053] Figure 9 is a detection of protein antibody titers in mouse immune sera of preparation G0, preparation D0 and preparation F0.

[0054] Figure 10 is an IC50 (Log10) analysis value of protein antibody titers in mouse immune sera of preparations V1-V9, wherein: V1: preparation G0; V2: preparation G1; V3: preparation G2; V4: preparation D0; V5: preparation D1; V6: preparation D2; V7: preparation F0; V8: preparation F1; V9: preparation F2. DETAILED DESCRIPTION

[0055] In order to more clearly show the technical solutions, objectives and advantages of the present application, the technical solutions of the present application will be described in detail below in conjunction with specific examples and drawings. The test methods used in the following examples are conventional methods unless otherwise specified; the instruments, reagents, materials, etc. used are conventional commercial means unless otherwise specified. Example 1: Preparation of VZV protein

[0056] Varicella zoster virus (VZV) is a member of the alpha herpesvirus subfamily of the Herpesviridae family, i.e., Human herpesvirus 3.

[0057] gE (glycoprotein E, gE) is encoded by the ORF68 gene and located in the short segment region of the VZV genome. The encoded gE contains 623 amino acids, and the gE protein molecule is mainly composed of a hydrophilic extracellular region (containing a signal peptide) composed of the first 546 amino acids, a hydrophobic transmembrane region composed of the 547th-623rd amino acids and an intracellular tail.

[0058] (I) Construction of the protein

[0059] The full-length sequence of the VZV gE protein mentioned in the present application refers to NCBI Reference Sequence: NP_040190.1, and the specific sequence is shown in SEQ ID NO. 1 (envelope glycoprotein E [Human alphaherpesvirus 3] / strain="Dumas" / 623aa).

[0060] The VZV gE protein used in the present application is different from the original full-length protein, but is a truncated protein. The present application selects a conserved truncated gE protein amino acid sequence as a design optimization template. In order to improve the expression efficiency and the stability of the protein structure, and preserve the antigen cluster function, only the sequence of the signal peptide region and the mature antigen region is selected, and the specific sequence is shown in SEQ ID NO. 2.

[0061] The VZV gE-monomer protein mutant provided by the application is designed based on a large number of experiments and layer-by-layer screening by means of protein genetic engineering, mainly the mutation and modification of the amino acid sequence of the mature antigen region of the selected truncated protein, the optimization of protein stability and immunogenicity, and the mutant is different from the amino acid sequence of the VZV gE protein included in the NCBI database, and the specific sequence is shown as SEQ ID NO. 4. In addition, the specific sequence of the VZV gE-monomer protein before modification provided by the application is shown as SEQ ID NO. 3.

[0062] In addition, the protein structure is further optimized and designed based on the VZV gE-monomer protein modified by amino acid mutation, which is designed as gE-dimer (the specific sequence is shown as SEQ ID NO. 5) and gE-ferritin (the specific sequence is shown as SEQ ID NO. 6), so as to realize higher antigen expression amount and stability, and greatly enhance the immunogenicity of the VZV gE protein.

[0063] (1) Amino acid sequence design

[0064] The application provides a VZV gE-ferritin nanoparticle recombinant protein with enhanced stability and immunogenicity obtained by amino acid mutation modification, and the amino acid sequence design method is as follows:

[0065] The transmembrane region and intracellular region in the full-length sequence of the VZV gE protein are deleted to obtain a truncated gE protein sequence without a carboxy-terminal hydrophobic anchor region as shown in SEQ ID NO. 2, and then amino acid point mutation is performed on the basis, and the specific mutation method is that the W at the 200th position of the VZV gE protein is mutated to C, the L at the 245th position is mutated to C, and the ferritin sequence as shown in SEQ ID NO. 7 is connected to the C terminal, and then the VZV gE-ferritin full-length mutant sequence is obtained, and the amino acid sequence is shown as SEQ ID NO. 6.

[0066] The application also relates to a VZV gE-monomer recombinant protein with enhanced stability and immunogenicity obtained by amino acid mutation modification, and the amino acid sequence design method is as follows:

[0067] The transmembrane region and intracellular region in the full-length sequence of VZV gE protein are deleted to obtain a truncated gE protein sequence without a carboxy-terminal hydrophobic anchor region as shown in SEQ ID NO. 2, and on this basis, amino acid point mutations are further performed, and the specific mutation mode is that W at position 200 of VZV gE protein is mutated to C, L at position 245 is mutated to C, and Throm and 6his sequences are connected at the C-terminal end, and then a VZV gE-monomer full-length mutant sequence is obtained, and the amino acid sequence is shown in SEQ ID NO. 4.

[0068] The present application also relates to another VZV gE-dimer recombinant protein with enhanced stability and immunogenicity obtained by amino acid mutation modification, and the design mode of the amino acid sequence comprises the following:

[0069] The transmembrane region and intracellular region in the full-length sequence of VZV gE protein are deleted to obtain a truncated gE protein sequence without a carboxy-terminal hydrophobic anchor region as shown in SEQ ID NO. 2, and on this basis, amino acid point mutations are further performed, and the specific mutation mode is that W at position 200 of VZV gE protein is mutated to C, L at position 245 is mutated to C, and Throm and 6his sequences are connected at the C-terminal end, and then a VZV gE-monomer full-length mutant sequence is obtained, and the amino acid sequence is shown in SEQ ID NO. 4.

[0070] SEQ ID NO. 1-SEQ ID NO. 8 sequences are shown as follows:

[0071] SEQ ID NO. 1:

[0072] > VZV gE 1-623 (NP_040190.1 / 623aa / envelope glycoprotein E [Human alphaherpesvirus 3] / strain="Dumas")

[0073] SEQ ID NO. 2:

[0074] > VZV gE 1-546

[0075] SEQ ID NO. 3:

[0076] > VZV modified gE-monomer

[0077] SEQ ID NO. 4:

[0078] > VZV modified gE-monomer W200C L245C

[0079] SEQ ID NO. 5:

[0080] > VZV modified gE-dimer W200C L245C

[0081] SEQ ID NO. 6:

[0082] > VZV modified gE-ferritin W200C L245C

[0083] SEQ ID NO. 7:

[0084] > ferritin

[0085] SEQ ID NO. 8:

[0086] > dimer

[0087] (2) Synthesis of the gene of the VZV gE protein of interest

[0088] According to the VZV gE protein amino acid sequences SEQ ID NO. 3-SEQ ID NO. 6 designed above and the codon bias of the host cell, the corresponding gene coding sequence is determined, and the restriction endonuclease EcoRI sequence is added at the C-terminal of the segment gene, and the restriction endonuclease XbaI sequence is added at the N-terminal, and the designed nucleotide sequence is chemically synthesized.

[0089] (3) Plasmid amplification and extraction of the gene of interest

[0090] The pUC19 plasmid vector is double digested by EcoRI and XbaI restriction enzymes, and then connected with the above synthesized gene, introduced into the amplification host DH5a, and screened for single clones using LB(Amp+) agar solid medium; the single clone containing the gene of interest is inoculated in LB(Amp+) liquid medium, cultured and amplified at 37°C, 200 rpm, and the Sigma-Aldrich GenElute™ HP plasmid medium preparation kit is used to extract the plasmid pUC19-gE; the extracted plasmid is double digested by EcoRI and XbaI restriction enzymes, and the gE gene fragment of interest is recovered using the TaKaRa MiniBest Agarose Gel Extraction Kit.

[0091] (4) Construction of a eukaryotic expression vector

[0092] The mammalian cell expression plasmid pGN-M containing CMV promoter and dihydrofolate reductase (DHFR) gene was double digested by EcoRI and XbaI restriction enzymes, and the vector DNA fragment was recovered by TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0; the vector DNA fragment and the target gene fragment were connected by sticky end method and introduced into the DH5α amplification host, and a single clone containing the eukaryotic expression plasmid pGN-M_gE was obtained by screening; inoculation was performed in LB (Amp+) for amplification culture, the amplified plasmid was extracted by using the endotoxin-free plasmid extraction kit TaKaRa MidiBEST Endo-free Plasmid Purification Kit, and the plasmid was named as VZVgE.

[0093] (II) Expression of VZV gE protein in CHO K1 cells and clone screening

[0094] CHO K1 (ATCC) cells were used as host cells, and after cell recovery, the cells were cultured in 10% newborn calf serum DMEM medium (Sigma-Aldrich), and subcultured once every 3 days. After subculture for 2 generations, it was observed that the cells grew well, and then the CHO K1 cells were inoculated at 0.75×10 6 cells / well of three 9.6 cm 2The cells were incubated in a humidified incubator at 5% C02 and 37°C with 4 μg of pcDNAVZVE vector in each well of the well, with IMDM + FBS (Gibco) added to two of the wells after mixing the DNA with Lipofectamine 2000 (Sigma-Aldrich), and Lipofectamine 2000 alone added to the third well as a negative control. After 48 hours, the medium was removed and centrifuged at 200 x g for 5 minutes, and the supernatant was stored at -20°C. IMDM + FBS medium and 10 μg / mL Blasticidin-HCl (Invitrogen) were added to one of the wells of transfected cells, and the other well of transfected cells was washed with PBS and then lysed with 50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% (v / v) Triton X-100 containing complete, EDA-free protease inhibitor cocktail (Roche Diagnostics). The lysate was centrifuged at 16,000 x g for 10 minutes at 4°C and stored at -20°C. Western blot was used to detect the presence of recombinant protein in the supernatant and lysate. After 5 days in selective medium, the cells were detached with trypsin (Invitrogen) and then seeded onto 9 cm Petri dishes for isolation of single clones by serial dilution. Forty-two single clones were selected and transferred to wells of a 96-well plate over the next 7-11 days. The culture supernatants were tested by Western blot to screen for high expression of VZV gE protein. The clone that secreted the highest amount of VZV gE protein was selected for the next round of screening, and the cells were expanded and 30 new clones were selected for preservation.

[0095] The selected clones were expanded into three T175 flasks (NEXUS). Trypsin was added, the cells were washed with PBS and resuspended in 100 mL of ProCHO4 (Lonza) with lx ProHT, 4 mM L-glutamine and 2% FBS (Lonza) in 250 mL spinner flasks. The cells were incubated in a humidified incubator at 37°C with 5% C02 at 90 rpm agitation with the lid slightly ajar to ensure air diffusion. Samples were taken daily, stained with trypan blue (Sigma-Aldrich) and counted, and the cells were passaged every 3-5 days. When the viable cell concentration was greater than 0.3 x 106 cells / mL and the viable cell number was greater than 90% at plateau phase, the cells were considered to be well adapted to serum-free suspension growth.

[0096] (Three) Production of VZV gE protein in bioreactor

[0097] A 1.5 liter perfusion culture was set up in a bioreactor with a spin filter (10 μιη) separator. The culture parameters were set as follows: temperature was controlled at 37°C by a heating blanket, pH was adjusted at 6.9 by CO2or 0.3 M sodium hydroxide, agitation speed was set at 200-300 RPM, and dissolved oxygen (dO2) was adjusted to 40% of saturated air with a mixture of N2and O2gas at a maximum flow rate of 200 mL / min. The perfusion rate was set at 0.3 to 0.8 V dilution per day, and cell counts were performed daily by sampling the culture broth. Trypan blue staining was used, and the glucose and lactate concentrations in the supernatant were measured off-line.

[0098] A total of 12.5 liters of cell-free broth was collected, centrifuged at 8000 x g for 30 minutes at 4°C, filtered with a 0.45 μιη membrane, and concentrated by ultrafiltration with a 10 kDa membrane package. The sample solution was concentrated to 0.5 liter, and 0.5 liter of PBS was added. The solution was concentrated to 0.5 liter again. The above steps were repeated 5 times.

[0099] (Four) Purification of VZV gE protein

[0100] The sample solution was loaded onto a Q-Sepharose fast flow (GE Bioscience) column, and the column was washed with 20 mM Tris-HCl pH 7.5. The column was then washed with 20 mM Tris-HCl pH 7.5 with 200 mM sodium chloride added, to further remove adsorbed protein impurities. The gE protein was eluted with a solution in which the concentration of sodium chloride was increased to 300 mM. Ammonium sulfate was added to the combined eluate to a concentration of 800 mM, and the solution was loaded onto a Butyl-Sepharose (GE Bioscience) column. The column was washed with phosphate buffered saline (PBS, 6 mM Na2HPO4, 1.5 mM KH2PO4, 0.15 M sodium chloride pH 6.8) with 800 mM ammonium sulfate added, and then with PBS containing 400 mM ammonium sulfate. The gE protein was finally eluted with purified water. The final sample was loaded onto a Sephacryl S-400 HR (GE Bioscience) column, which was washed with PBS. The protein peak was collected, and a cosolvent was added. The sample was lyophilized in a vacuum lyophilizer and stored at -70°C until use.

[0101] The VZV pre-modification gE-monomer, VZV post-modification gE-monomer, VZV post-modification gE-dimer, and VZV post-modification gE-ferritin nanoparticle proteins were each prepared using the above-described method.

[0102] The electron microscope image of Figure 1 shows that the VZV-gE ferritin nanoparticle protein can form uniform nanoparticles of nanometer size after being rationally designed and constructed, which makes the protein structure more stable and exposes more required antigen epitopes for display, thereby effectively improving the immunogenicity and stability of the VZV gE protein antigen.

[0103] Example 2: Temperature stability detection of the gE protein before and after modification obtained in Example 1

[0104] 1) The protein to be tested (the gE-monomer protein before modification prepared in Reference Example 1 and the gE-monomer protein after modification prepared in Example 1, respectively) was diluted to 20 ug / mL with 1*PBS pH7.4 buffer and placed in a 1.5 mL centrifuge tube with a total volume of 1 mL.

[0105] 2) Different temperature incubations were performed according to the following table.

[0106] 3) After incubation, each sample was placed at 4°C for temporary storage.

[0107] 4) Preparation of detection antibody (E5-G6)

[0108] BALB / c mice were immunized with gE protein vaccine (GSK) at a protein dose of 100 ug per mouse, and B lymphocytes were extracted from the spleens of the BALB / c mice after they were immunized twice. Then, the monoclonal antibody mAb (number: E5-G6) against VZV-gE was prepared and cloned by hybridoma cell technology.

[0109] 5) ELISA detection was performed according to the following steps:

[0110] The samples stored at 4°C were diluted to 1 ug / mL with 1*PBS pH7.4, and 100 uL / well was added to the enzyme-labeled plate (NUNC 442404) for coating at 4°C overnight. The enzyme-labeled plate was spun dry, 1% BSA-PBS was added at 150 uL / well, and incubation was performed at 37°C for 1 hour. The plate washer was programmed to wash the plate 3 times, and different concentrations of detection antibody (i.e., primary antibody) were added according to the design: 0.5 ug / mL E5-G6, 0.125 ug / mL E5-G6, and 0.02 ug / mL E5-G6, 100 uL / well, and incubation was performed at 37°C for 2 hours. The plate washer was programmed to wash the plate 3 times, and anti-mouse secondary antibody was added at a dilution of 1:2000, 100 uL / well, and incubation was performed at 37°C for 1 hour. The plate washer was programmed to wash the plate 3 times, pNPP substrate solution was added, 100 uL / well, and the enzyme-labeled instrument was set at a wavelength of 405 nm for reading.

[0111] The results of the temperature stability detection of the protein before and after modification are shown in Figures 2-4.

[0112] As can be seen from FIGS. 2-4, the gE protein before and after modification has significant differences in binding activity with the monoclonal antibody at different temperatures. The OD value of the gE protein after modification detected at 4°C, 37°C, 60°C and 80°C using different concentrations of detection antibodies (0.5 ug / mL E5-G6, 0.125 ug / mL E5-G6 and 0.02 ug / mL E5-G6) is obviously higher than that of the gE protein before modification, indicating that the gE protein after modification prepared in the application can still maintain high antigen binding activity after being treated at different temperatures, that is, compared with the gE protein before modification, the temperature stability of the gE protein after modification prepared in the application is significantly enhanced.

[0113] Example 3: Preparation of immunological preparation

[0114] (1) Preparation A / Preparation B:

[0115] The VZV gE monomer before modification / VZV gE monomer after modification protein obtained by the method of Example 1 was 0.1 mg / mL, phosphate buffer pH 5.8 buffer was added, sterilized by 0.22 μm membrane filtration, sterile aluminum phosphate gel (BeneTag) was added, stirred at 4°C for 1 hour, sterilely divided into 0.8 mL / bottle, stored at 4°C, and used for immunization, that is, Preparation A (VZV gE monomer before modification) and Preparation B (VZV gE monomer after modification).

[0116] (2) Preparation M1:

[0117] The VZV gE monomer after modification protein obtained by the method of Example 1 was 5 μg / dose, 10 μg / dose of CpG (Genscript) was added, phosphate buffer pH 5.8 buffer was added, sterilized by 0.22 μm membrane filtration, stirred at 4°C for 1 hour, sterilely divided into 0.8 mL / bottle, stored at 4°C, and used for immunization.

[0118] (3) Preparation M2 / Preparation F:

[0119] The VZV gE monomer after modification / VZV gE ferritin protein obtained by the method of Example 1 was 5 μg / dose, 1 μg / dose of CpG (Genscript) was added, respectively, phosphate buffer pH 5.8 buffer was added, sterilized by 0.22 μm membrane filtration, stirred at 4°C for 1 hour, sterilely divided into 0.8 mL / bottle, stored at 4°C, and used for immunization, that is, Preparation M2 (VZV gE monomer after modification+CpG1) and Preparation F (VZV gE ferritin+CpG1).

[0120] (4) Formulation G1, formulation D1 and formulation F1:

[0121] Take VZV-gE protein (GSK) and VZV modified gE dimers / VZV modified gE ferritin protein obtained by the method of Example 1, respectively 5 μg / dose, respectively add phosphate buffer pH 5.8 buffer, filter sterilization with 0.22 μm membrane, add sterile aluminum phosphate gel (Benetag) 10 μg / dose, stir at 4°C for 1 hour, sterilely divide 0.8 mL / bottle, store at 4°C, wait for immunization, namely formulation G1 (VZV gE+Alum), formulation D1 (VZV modified gE dimers+Alum), formulation F1 (VZV modified gE ferritin+Alum).

[0122] (5) Formulation G2, formulation D2 and formulation F2:

[0123] Take VZV-gE protein (GSK) and VZV modified gE dimers / VZV modified gE ferritin protein obtained by the method of Example 1, respectively 5 μg / dose, respectively add 1 μg / dose of CpG (Genscript), then add phosphate buffer pH 5.8 buffer, filter sterilization with 0.22 μm membrane, then add 10 μg / dose of sterile aluminum phosphate gel (Benetag), stir at 4°C for 1 hour, sterilely divide 0.8 mL / bottle, store at 4°C, wait for immunization, namely formulation G2 (VZV gE+Alum+CpG), formulation D2 (VZV modified gE dimers+Alum+CpG), formulation F2 (VZV modified gE ferritin+Alum+CpG).

[0124] (6) Formulation G0, formulation D0 and formulation F0:

[0125] Take VZV-gE protein (GSK) and VZV modified gE dimers / VZV modified gE ferritin protein obtained by the method of Example 1, respectively 5 μg / dose, respectively add phosphate buffer pH 5.8 buffer, filter sterilization with 0.22 μm membrane, add AS01B (GSK) 50 μl / dose, stir at 4°C for 1 hour, sterilely divide 0.8 mL / bottle, store at 4°C, wait for immunization, namely formulation G0 (VZV gE+AS01B), formulation D0 (VZV modified gE dimers+AS01B), formulation F0 (VZV modified gE ferritin+AS01B).

[0126] Example 4: Immunization of small white mice with formulation A / formulation B and blood collection

[0127] Take 4-6 weeks female BALB / c mice, randomly divided into 2 groups, each group of immunization preparation is prepared by using example 3: preparation A and preparation B, every two weeks subcutaneous immunization once, 0.1 mL each time, a total of two times, immunization 35 days after blood, and then blood at room temperature for 4 hours, at 10000 RPM, room temperature centrifugation, take the supernatant serum, -70℃ preservation for detection.

[0128] Example 5: ELISA method for detecting antibody titer of preparation A / preparation B mouse immune serum

[0129] Prepare the purified gE protein stock solution 1 μg / mL (1xPBS solution), stored in 4℃ refrigerator. Dilute the protein stock solution to 4 μg / mL in coating buffer, add 100 μL coating solution to each well to coat the ELISA plate, incubate at room temperature overnight. Wash 3 times with plate washing buffer, add 150 μL blocking buffer, incubate at 37℃ for 1 hour, wash 3 times with plate washing buffer, 300 μl per well, and store at 4℃ for one week.

[0130] Dilute the corresponding serum to be tested obtained by injecting the vaccine into the mice in example 4 into working sample serum, dilute appropriately, add to the first row of holes in the ELISA plate, 100 μl per well, 2-fold serial dilution from the first row, incubate at 37℃ for 2 hours. Wash 3 times with plate washing buffer, 300 μl per well, add AP labeled goat anti-mouse secondary antibody at a dilution of 1:1000, 100 uL per well, incubate at 37℃ for 1 hour. Wash the plate 3 times according to the program, add pNPP substrate solution, 100 uL per well, set the wavelength to 405 nm on the enzyme label instrument, and read the results.

[0131] The detection results of figure 5 show that there is a significant difference in antibody titer between the gE protein vaccine before and after modification, and the antibody titer of the gE-monomer protein vaccine after amino acid modification (preparation B) is significantly higher than that of the gE-monomer protein vaccine before modification (preparation A); and after dilution of the serum of the immunized mice by different multiples, at each concentration, the antibody titer of the gE-monomer protein vaccine after amino acid modification (preparation B) is still significantly higher than that of the gE-monomer protein vaccine before modification (preparation A), and with the gradual increase of the serum dilution multiple, the antibody titers of the gE-monomer protein before and after modification show a decreasing trend.

[0132] Therefore, compared with the gE-monomer vaccine (preparation A) without amino acid modification, the antibody titer detection value of the gE-monomer (preparation B) recombinant protein vaccine prepared by the application after amino acid modification is higher, and serum with higher protection titer can be obtained, indicating that the immune effect of the gE-monomer recombinant protein vaccine prepared by the application after amino acid modification is significantly enhanced.

[0133] Example 6: Immunization of mice with preparation M1 / preparation M2 / preparation F and blood sampling

[0134] 4-6 week old female BALB / c mice were randomly divided into 3 groups, 10 mice in each group, and the immunization preparations were preparation M1, preparation M2 and preparation F prepared by Example 3, respectively. The mice were subcutaneously immunized once every two weeks, 0.1 mL each time, a total of two times, and blood was sampled after the second immunization. The blood was then placed at room temperature for 4 hours, centrifuged at 10,000 RPM at room temperature, and the supernatant serum was collected and stored at -70°C for detection.

[0135] Example 7: ELISA method for detecting protein antibody titer in mouse serum immunized with preparation M1 / preparation M2 / preparation F recombinant protein vaccine

[0136] Prepare a purified gE protein stock solution of 1 μg / mL (1×PBS solution) and store it in a 4°C refrigerator. Dilute the protein stock solution to 4 μg / mL in coating buffer, add 100 μL of coating solution to each well of the ELISA plate, and incubate at room temperature overnight. Wash 3 times with plate washing buffer, add 150 μL of blocking buffer, and incubate at 37°C for 1 hour. Wash 3 times with plate washing buffer at 300 μl per well, and store at 4°C for up to one week.

[0137] Dilute the corresponding serum to be tested obtained by injecting the vaccine into the mice in Example 6 to obtain working sample serum, dilute it to an appropriate multiple, and add it to the first row of holes in the ELISA plate, 100 μl per well. Start from the first row and proceed to 2-fold serial dilution, and incubate at 37°C for 2 hours. Wash 3 times with plate washing buffer at 300 μl per well, add 100 μL of HRP-labeled goat anti-mouse antibody (1:2000 dilution), and incubate at 37°C for 1 hour. Wash 3 times with plate washing buffer at 300 μl per well, add TMB substrate solution, 100 μl per well, and read the plate at 450 nm.

[0138] The results of Fig. 6 show that the antibody titers of the mice immunized with the gE monomer and gE ferritin nanoparticle recombinant protein vaccines modified by amino acid mutation and added with different contents of CpG adjuvant are obviously different, wherein the antibody titer detection value of the nanoparticle recombinant protein of preparation F (gE ferritin+CpG1) is obviously higher than that of preparation M1 (gE monomer+CpG10) and preparation M2 (gE monomer+CpG1), and the antibody titer of preparation M1 (gE monomer+CpG10) is the second; and the serum of the immunized mice is diluted by different times, and at each concentration, the antibody titer of the mice immunized with the nanoparticle recombinant protein of preparation F (gE ferritin+CpG1) is still obviously higher than that of preparation M1 (gE monomer+CpG10) and preparation M2 (gE monomer+CpG1), the antibody titer of preparation M1 (gE monomer+CpG10) is higher than that of preparation M2 (gE monomer+CpG1), and with the gradual increase of the serum dilution times, the antibody titers of the preparations all show a gradually decreasing trend.

[0139] It can be seen that, under the condition of adding different contents of adjuvant in each immunization preparation, compared with the VZV-gE monomer recombinant protein vaccine, the antibody titer value of the mice immunized with the VZV-gE ferritin nanoparticle recombinant protein vaccine is the highest, and the serum with a higher protection titer can be obtained, which indicates that the VZV-gE ferritin nanoparticle recombinant protein vaccine prepared in the application has a better immunization effect; and for the VZV-gE monomer recombinant protein, when the content of the adjuvant in the immunization preparation is added according to a certain proportion, the protection titer of the obtained serum will increase, and the immunization effect is better, which indicates that the addition of the adjuvant has a certain enhancing and assisting effect on the immunization effect of the vaccine; and the results of Fig. 6 show that although the content of the CpG adjuvant added in preparation M1 is 10 times of that in preparation F, the antibody titer of the mice immunized with the VZV-gE ferritin nanoparticle recombinant protein vaccine (preparation F) is still higher than that of the VZV-gE monomer recombinant protein vaccine (preparation M1), which indicates that the VZV-gE ferritin nanoparticle recombinant protein vaccine prepared in the application can still achieve a good immunization effect under the condition of adding a small amount of adjuvant, which also indicates from another aspect that the nanoparticle vaccine developed in the application can reduce the use of adjuvant in the vaccine, thereby reducing the potential side effects after immunization of the vaccine, and the safety is higher, and the production cost of the vaccine can be further reduced.

[0140] Example 8: Immunization of white mice with preparation G0 / preparation D0 / preparation F0 / preparation G1 / preparation D1 / preparation F1 / preparation G2 / preparation D2 / preparation F2 and blood sampling

[0141] Take 4-6 weeks female BALB / c mice randomly divided into 9 groups, 10 in each group, and each group of immunization preparation is prepared by using Example 3: preparation G0, preparation D0, preparation F0, preparation G1, preparation D1, preparation F1, preparation G2, preparation D2 and preparation F2, subcutaneous immunization every two weeks, 0.1 mL each time, a total of two times, and blood is collected after the second immunization, then the blood is placed at room temperature for 4 hours, centrifuged at 10000 RPM at room temperature, and the supernatant serum is aspirated and stored at -70℃ for detection.

[0142] Example 9: ELISA method for detecting protein antibody titer in mouse immune serum of preparation G0 / preparation D0 / preparation F0 / preparation G1 / preparation D1 / preparation F1 / preparation G2 / preparation D2 / preparation F2

[0143] Prepare a 1 μg / mL stock solution of purified gE protein (1×PBS solution) and store it in a 4℃ refrigerator. Dilute the protein stock solution to 4 μg / mL in coating buffer, add 100 μL of coating solution to each well of the ELISA plate, and incubate at room temperature overnight. Wash 3 times with plate washing buffer, add 150 μL of blocking buffer, and incubate at 37℃ for 1 hour. Wash 3 times with 300 μl / well of plate washing buffer, and store at 4℃ for up to one week.

[0144] Dilute the corresponding serum to be tested obtained from the mouse injection vaccine in Example 8 to working sample serum, dilute it to the appropriate dilution, and add it to the first row of wells of the ELISA plate, 100 μl / well. Perform 4-fold serial dilution starting from the first row, and incubate at 37℃ for 2 hours. Wash 3 times with 300 μl / well of plate washing buffer, add 100 μL of HRP-labeled goat anti-mouse antibody (1:2000 dilution), and incubate at 37℃ for 1 hour. Wash 3 times with 300 μl / well of plate washing buffer, add TMB substrate solution, 100 μl / well, and then measure the OD value using an enzyme marker (450 nm wavelength), and analyze and calculate the IC50 (Log10) value using GraphPad Prism software.

[0145] The detection results are shown in Figures 7-10.

[0146] The results of FIG. 7 show that the antibody titers of the mice immunized with the VZV-gE protein (GSK), the modified gE dimers and the modified gE ferritin nanoparticle recombinant protein vaccine respectively are obviously different when the same alum adjuvant is added in the immunization formulations, the antibody titer detection value of the formulation F1 (gE ferritin + alum) nanoparticle recombinant protein is obviously higher than that of the formulation G1 (gE + alum) and the formulation D1 (gE dimers + alum), the antibody titer of the formulation D1 (gE dimers + alum) is the second, and the serum of the immunized mice is diluted by different times, the antibody titer of the mice immunized with the formulation F1 (gE ferritin + alum) nanoparticle recombinant protein is still significantly higher than that of the mice immunized with the formulation G1 (gE + alum) and the formulation D1 (gE dimers + alum) at each concentration, the antibody titer of the mice immunized with the formulation D1 (gE dimers + alum) is significantly higher than that of the mice immunized with the formulation G1 (gE + alum), and the antibody titers of the formulations gradually decrease with the gradual increase of the serum dilution times.

[0147] The results of FIG. 8 show that the antibody titers of the mice immunized with the VZV-gE protein (GSK), the modified gE dimers and the modified gE ferritin nanoparticle recombinant protein vaccine respectively are obviously different when the same two adjuvants (alum + CpG) are added in the immunization formulations, the antibody titer detection value of the formulation F2 (gE ferritin + alum + CpG) nanoparticle recombinant protein is obviously higher than that of the formulation G2 (gE + alum + CpG) and the formulation D2 (gE dimers + alum + CpG), the antibody titer of the formulation D2 (gE dimers + alum + CpG) is the second, and the serum of the immunized mice is diluted by different times, the antibody titer of the mice immunized with the formulation F2 (gE ferritin + alum + CpG) nanoparticle recombinant protein is still significantly higher than that of the mice immunized with the formulation G2 (gE + alum + CpG) and the formulation D2 (gE dimers + alum + CpG) at each concentration, the antibody titer of the mice immunized with the formulation D2 (gE dimers + alum + CpG) is higher than that of the mice immunized with the formulation G2 (gE + alum + CpG), and the antibody titers of the formulations gradually decrease with the gradual increase of the serum dilution times.

[0148] The results of FIG. 8 show that the antibody titers of the mice immunized with the VZV-gE protein (GSK), the modified gE dimers and the modified gE ferritin nanoparticle recombinant protein vaccine respectively are obviously different when the same two adjuvants (alum + CpG) are added in the immunization formulations, the antibody titer detection value of the formulation F2 (gE ferritin + alum + CpG) nanoparticle recombinant protein is obviously higher than that of the formulation G2 (gE + alum + CpG) and the formulation D2 (gE dimers + alum + CpG), the antibody titer of the formulation D2 (gE dimers + alum + CpG) is the second, and the serum of the immunized mice is diluted by different times, the antibody titer of the mice immunized with the formulation F2 (gE ferritin + alum + CpG) nanoparticle recombinant protein is still significantly higher than that of the mice immunized with the formulation G2 (gE + alum + CpG) and the formulation D2 (gE dimers + alum + CpG) at each concentration, the antibody titer of the mice immunized with the formulation D2 (gE dimers + alum + CpG) is higher than that of the mice immunized with the formulation G2 (gE + alum + CpG), and the antibody titers of the formulations gradually decrease with the gradual increase of the serum dilution times.

[0149] The results of Figure 9 show that there are differences in antibody titers of mice immunized with VZV-gE protein (GSK), modified gE dimers and modified gE ferritin nanoparticle recombinant protein vaccines, respectively, with the same AS01b adjuvant (GSK) added to the immunization formulations, and that the antibody titers of mice immunized with the formulations F0 (gE ferritin + AS01b) nanoparticle recombinant protein are significantly higher than those of G0 (gE + AS01b) and D0 (gE dimers + AS01b) at different dilution multiples of the serum, especially at dilution multiples of 1:6400, 1:25600, 1:102400 and 1:409600, and the antibody titers of D0 (gE dimers + AS01b) are significantly higher than those of G0 (gE + AS01b), and the antibody titers of each formulation gradually decrease with increasing dilution multiples of the serum.

[0150] The results of Figure 10 show that there are obvious differences in IC50 (Log10) values of protein antibody titers in the mouse immune sera of formulations V1-V9, wherein V1: formulation G0; V2: formulation G1; V3: formulation G2; V4: formulation D0; V5: formulation D1; V6: formulation D2; V7: formulation F0; V8: formulation F1; and V9: formulation F2. The IC50 (Log10) values of protein antibody titers in the mouse immune sera of V7 are higher than those of V1 and V4; the IC50 (Log10) values of protein antibody titers in the mouse immune sera of V8 are significantly higher than those of V2 and V5; the IC50 (Log10) values of protein antibody titers in the mouse immune sera of V9 are significantly higher than those of V3 and V6; which indicates that the IC50 (Log10) values of protein antibody titers in the mouse immune sera of VZV-gE ferritin nanoparticle recombinant protein formulations (V7 / V8 / V9) are higher than those of VZV-gE protein (V1 / V2 / V3) and VZV-gE dimers recombinant protein formulations (V4 / V5 / V6), respectively, regardless of the adjuvant added. In addition, the results in the figure also show that the IC50 (Log10) values of protein antibody titers in the mouse immune sera of V8 are significantly higher than those of V3 and V6, i.e. the IC50 (Log10) values of protein antibody titers of V8: formulation F1 (gE ferritin + Alum) nanoparticle recombinant protein are still higher than those of V3: formulation G2 (gE + Alum + CpG) and V6: formulation D2 (gE dimers + Alum + CpG), although only one adjuvant is added to the V8: formulation F1 (gE ferritin + Alum) nanoparticle recombinant protein.

[0151] Therefore, when different types of adjuvants are added to various immunizing agents, the modified gEferritin nanoparticle recombinant protein vaccine produces the highest antibody titer in mice immunized with VZV-gE protein (GSK) and the modified gEdimer recombinant protein vaccine, resulting in serum with higher protective titers. This indicates that the VZV-gEferritin nanoparticle recombinant protein vaccine prepared in this invention has a superior immunizing effect, followed by the modified gEdimer recombinant protein vaccine. Meanwhile, even with only one adjuvant added to the gEferritin nanoparticle recombinant protein vaccine, its protein antibody titer IC50 (Log10) value is still higher than that of VZV-gE protein (GSK) and the gEdimer recombinant protein vaccine, both of which have two adjuvants added. This demonstrates that the VZV-gEferritin nanoparticle recombinant protein vaccine prepared in this invention can still achieve a good immunizing effect with reduced adjuvant addition. Furthermore, this also shows that the nanoparticle vaccine developed in this invention can reduce the use of adjuvants in vaccines, thereby reducing potential side effects after immunization, increasing safety, and lowering vaccine production costs.

[0152] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention pending approval.

Claims

1. A VZV protein, characterized in that, The protein contains the complete amino acid sequence of the extracellular region of the VZV gE protein, as shown in SEQ ID NO.2, with the W at position 200 and the L at position 245 of the sequence shown in SEQ ID NO.2 mutated to C.

2. The VZV protein according to claim 1, characterized in that, The protein is any one of gE-monomer, gE-dimer, and gE-ferritin nanoparticle proteins obtained through amino acid mutation modification and structural optimization design, and the amino acid sequences of the gE-monomer, gE-dimer, and gE-ferritin nanoparticle proteins are shown in SEQ ID NO: 4-6.

3. The VZV protein according to claim 2, characterized in that, The protein is gE-ferritin nanoparticle protein, and the amino acid mutation modification and structural optimization design methods are as follows: The transmembrane and intracellular regions of the full-length VZV gE protein sequence shown in SEQ ID NO.1 were deleted to obtain the extracellular region sequence of the gE protein shown in SEQ ID NO.

2. Then, amino acid point mutations were performed on this sequence, with W at position 200 of the VZV gE protein being mutated to C and L at position 245 being mutated to C. At the same time, the ferritin sequence shown in SEQ ID NO.7 was attached to the C-terminus to obtain the full-length VZV gE-ferritin mutant sequence, the amino acid sequence of which is shown in SEQ ID NO.

6.

4. The VZV protein according to claim 2, characterized in that, The protein is a gE-monomer nanoparticle protein, and the amino acid mutation modification and structural optimization design methods are as follows: The transmembrane and intracellular regions of the full-length VZV gE protein sequence, as shown in SEQ ID NO.1, were deleted to obtain the extracellular region sequence of the gE protein, as shown in SEQ ID NO.

2. Further amino acid point mutations were then performed, changing W at position 200 of the VZV gE protein to C and L at position 245 to C. Simultaneously, Throm and 6his sequences were linked to the C-terminus to obtain the full-length VZV gE-monomer mutant sequence, whose amino acid sequence is shown in SEQ ID NO.

4.

5. The VZV protein according to claim 2, characterized in that, The protein is a gE-dimer nanoparticle protein, and the amino acid mutation modification and structural optimization design methods are as follows: The transmembrane and intracellular regions of the full-length VZV gE protein sequence, as shown in SEQ ID NO.1, were deleted to obtain the extracellular region sequence of the gE protein, as shown in SEQ ID NO.

2. Further amino acid point mutations were then performed, changing W at position 200 of the VZV gE protein to C and L at position 245 to C. Simultaneously, a dimer sequence, as shown in SEQ ID NO.8, was designed and linked to the C-terminus to obtain the full-length VZV gE-dimer mutant sequence, whose amino acid sequence is shown in SEQ ID NO.

5.

6. A biomaterial, characterized in that, It is at least one of the following (1)-(4): (1) A nucleic acid molecule encoding the VZV protein as described in any one of claims 1-5; (2) A recombinant expression vector containing the nucleic acid molecule described in (1); (3) Recombinant microorganisms containing the nucleic acid molecules described in (1) or recombinant microorganisms containing the recombinant expression vector described in (2); (4) A recombinant cell line containing the nucleic acid molecule described in (1) or a recombinant cell line containing the recombinant expression vector described in (2).

7. A method for preparing the VZV protein according to any one of claims 1-5, comprising the following steps: expressing a nucleic acid molecule encoding the VZV protein according to any one of claims 1-5 in a biological cell to obtain the VZV protein.

8. The method for preparing VZV protein according to claim 7, characterized in that: The method includes the following steps: introducing a nucleic acid molecule encoding the VZV protein as described in any one of claims 1-5 into CHO cells to obtain recombinant cells; culturing the recombinant cells to obtain the VZV protein.

9. The use of the VZV protein as described in any one of claims 1-5, or the biomaterial as described in claim 6, or the VZV protein prepared according to the method described in claim 7 or 8, in any one of the following (1)-(3): (1) Preparation of products resistant to VZV; (2) Prepare products for the prevention and / or treatment of VZV infection; (3) Prepare products for the prevention and / or treatment of diseases caused by VZV.

10. An immunogenic composition, characterized in that, The immunogenic composition comprises the VZV protein as described in any one of claims 1-5, the biomaterial as described in claim 6, or the VZV protein prepared according to the method described in claim 7 or 8.

11. A varicella-zoster vaccine, characterized in that, The vaccine comprises the VZV protein as described in any one of claims 1-5, or the biomaterial as described in claim 6, or the VZV protein prepared according to the method described in claim 7 or 8, or the immunogenic composition as described in claim 10, and optionally a pharmaceutically acceptable adjuvant.

12. The varicella-zoster vaccine according to claim 11, characterized in that, The adjuvant comprises any one or any combination of aluminum salt adjuvants, CpG adjuvants, immunostimulatory complexes, and liposome adjuvants.

13. The varicella-zoster vaccine according to claim 11, characterized in that, The vaccine is prepared in the form of an aqueous solution or a lyophilized solution.

14. The use of the varicella-zoster vaccine as described in claim 11 in any one of the following (1)-(3): (1) Preparation of products resistant to VZV; (2) Prepare products for the prevention and / or treatment of VZV infection; (3) Prepare products for the prevention and / or treatment of diseases caused by VZV.

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