Immune composition product for preventing or treating varicella zoster virus-related diseases and preparation method therefor

Abstract

Provided is a varicella zoster vaccine. Specifically, the vaccine comprises an immune composition, and the immune composition comprises an antigen component and a particle protein component. The particle protein component comprises a nanoparticle protein, and the antigen component and the particle protein component covalently bind to each other by means of a binding peptide 1 and a binding peptide 2 to form an immunogenic complex. Provided is a preparation method for the varicella zoster vaccine.

Description

An immune composition product for preventing or treating varicella-zoster virus-related diseases and its preparation method Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an immune composition product for preventing or treating varicella-zoster virus (VZV)-related diseases and a production method thereof. Background Art

[0002] Shingles is a segmentally distributed, erythematous, vesicular skin disease caused by the varicella zoster virus (VZV), which remains dormant in neurons in the dorsal horn of the spinal cord after initial infection with chickenpox. It is more common in patients with advanced age, immunodeficiency, or those taking immunosuppressants. Its symptoms and residual neuralgia are highly distressing. To date, there is no effective treatment for this disease, and vaccination remains the only preventable treatment.

[0003] In most people with VZV, the virus remains dormant within neurons. After varying periods of latency, it can be released and infect other cells. The primary protective mechanism of the shingles vaccine is to induce a cellular immune response, inhibiting viral activation within neurons and its spread through nerves to other cells. Antibodies produced by humoral immunity also play a role in protection.

[0004] There are currently two shingles vaccines available worldwide: recombinant (GSK) and attenuated vaccines (MERCK), both vaccines cover people aged 50 and above. gE protein is the most abundant glycoprotein expressed by VZV and has high immunogenicity. Recombinant vaccine The product consists of active ingredients, AS01B adjuvant system, and other excipients. The active ingredient is VZV glycoprotein E (gE), which is produced by transfecting protein coding sequences and expressing specific antigens in Chinese hamster ovary (CHO) cells through DNA recombination technology, and then purified and freeze-dried. The gE protein for injection is a sterile white powder. The AS01B adjuvant system suspension is a liposome preparation containing two immune-enhancing ingredients (3-O-deacylated-4'-monophosphoryl lipid A (MPL) and Quillaja saponin QS-21). The injection suspension (AS01B adjuvant system) is a colorless to light brown liquid with a milky luster. CD4 + The reduction of T cell numbers and the decline of immune response are key factors in activating VZV virus. The enhancement of specific T cell immunity is the core competitiveness of herpes zoster vaccine. The adjuvant AS01B contained in it can effectively and continuously promote the specific CD4 +T cell development and differentiation. It has stronger protective effects against shingles and postherpetic neuralgia caused by shingles, and both have been approved for use in multiple countries. It became the first shingles vaccine to be marketed in China. It has not yet been launched in the country.

[0005] Although using AS01B Shingles vaccine can The vaccine's 51% protection increased to over 90%, but However, it also has significant drawbacks. Adjuvant production capacity is limited, and the side effects of the adjuvant are significant. The AS01B adjuvant is known to induce a strong inflammatory response, and recipients often experience symptoms such as generalized muscle and joint pain, fatigue, and fever after vaccination. Consequently, many eligible individuals are reluctant to receive the vaccine, significantly reducing vaccination rates and compliance. Therefore, developing new vaccines with fewer side effects to increase vaccination rates and compliance among eligible individuals is of profound clinical significance.

[0006] Summary of the Invention

[0007] The present invention provides a varicella-zoster vaccine and a preparation method thereof. The vaccine is a nanoparticle vaccine that can avoid the use of AS01B adjuvant, thereby eliminating or reducing the side effects caused by such adjuvants; at the same time, the vaccine In comparison, the T cell immunogenicity and antibody immunogenicity of the nanoparticle vaccine were higher than This provides a The higher protective efficacy has far-reaching clinical value. At the same time, it also solves the current problems of insufficient varicella-zoster vaccine technology and supply types, filling the current gap in the supply of nanoparticle-based varicella-zoster vaccines.

[0008] Nanoparticle vaccines: vaccines based on the formation of nanoparticle proteins, which are mainly used to display antigens.

[0009] The present invention provides an immunogenic complex comprising a protein formed by a covalent binding reaction between an antigen component and a particle protein component.

[0010] The present invention provides an immune composition comprising the immunogenic complex of the present invention and a pharmaceutically acceptable carrier, which can be in the form of a lyophilized preparation or an injection preparation.

[0011] The present invention provides a vaccine comprising the immune composition of the present invention and an adjuvant.

[0012] The present invention provides an immunogenic complex comprising:

[0013] (1) an antigenic component comprising varicella-zoster virus (VZV) gE protein or an immunogenic fragment thereof;

[0014] (2) A granular protein component comprising nanoparticle protein.

[0015] The present invention provides an immunogenic complex comprising:

[0016] (1) an antigenic component comprising varicella-zoster virus (VZV) gE protein or an immunogenic fragment thereof, a connecting peptide 1 and a binding peptide 1;

[0017] (2) a particle protein component comprising nanoparticle protein, connecting peptide 2 and binding peptide 2;

[0018] The antigen component and the granule protein component are covalently bound to each other via binding peptide 1 and binding peptide 2.

[0019] The present invention provides an immunogenic complex comprising:

[0020] (1) an antigenic component consisting of varicella-zoster virus (VZV) gE protein or an immunogenic fragment thereof, a connecting peptide 1, and a binding peptide 1;

[0021] (2) granule protein component, consisting of nanoparticle protein, connecting peptide 2 and binding peptide 2;

[0022] The antigen component and the granule protein component are covalently bound to each other via binding peptide 1 and binding peptide 2.

[0023] In some embodiments, in any immunogenic complex provided by the present invention, the antigenic component is formed by fusing VZV gE protein to binding peptide 1 via connecting peptide 1 at the C-terminus.

[0024] In some embodiments, an "immunogenic fragment" refers to a portion of an oligopeptide, polypeptide, or protein that is immunogenic and elicits a protective immune response when administered to a subject.

[0025] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the particle protein component is formed by fusing the nanoparticle protein to the binding peptide 2 via the connecting peptide 2 at the N-terminus.

[0026] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the antigen component, from N-terminus to C-terminus, is sequentially: varicella-zoster virus (VZV) gE protein or an immunogenic fragment thereof, connecting peptide 1, and binding peptide 1; the granule protein component, from N-terminus to C-terminus, is sequentially: binding peptide 2, connecting peptide 2, and nanoparticle protein; the antigen component and the granule protein component are covalently bound to each other through binding peptide 1 and binding peptide 2 to form an immunogenic complex.

[0027] In some embodiments, in any one of the immunogenic complexes provided herein, the antigen component and / or the particle protein component comprises a histidine tag.

[0028] The present invention provides an immunogenic complex comprising:

[0029] (1) an antigenic component comprising varicella-zoster virus (VZV) gE protein or an immunogenic fragment thereof, and a connecting peptide 1;

[0030] (2) The granule protein component comprises nanoparticle protein subunits.

[0031] In some embodiments, the VZV gE protein is linked to one subunit of the nanoparticle protein to form a fusion protein, which is then bound to another subunit of the nanoparticle protein.

[0032] In some embodiments, in any of the immunogenic complexes provided herein, the particle protein component comprises a nanoparticle protein. Preferably, the nanoparticle protein can be a virus-like particle protein, which is formed by a viral structural protein, preferably, by a bacteriophage capsid protein AP205. The particle protein component and the antigen component can be covalently bound to form a particle structure.

[0033] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the nanoparticle protein used can also be selected from: NPM particles, ferritin particles (Ferritin), I53-50 particles, etc.

[0034] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the nanoparticle protein I53-50 particles used are composed of two subunits, I53-50A and I53-50B.

[0035] In some embodiments, in any immunogenic complex provided by the present invention, the binding peptide 1 comprises the amino acid sequence shown in SEQ ID NO: 1.

[0036] In some embodiments, in any immunogenic complex provided by the present invention, the binding peptide 2 comprises the amino acid sequence shown in SEQ ID NO:2.

[0037] In some embodiments, in any one of the immunogenic complexes provided herein, the connecting peptide 1 comprises (GGGGS) n or (EAAAK) n wherein n is an integer greater than 0 and less than or equal to 5. In some embodiments, in any one of the immunogenic complexes provided by the present invention, the connecting peptide 1 is preferably (GGGGS)3 (SEQ ID NO: 3), (EAAAK)3 (SEQ ID NO: 4) or GGSGGSGSEKAAKAEEAAR (SEQ ID NO: 5).

[0038] In some embodiments, in any one of the immunogenic complexes provided herein, the connecting peptide 2 comprises (GGS) n 、(SGGSGG) n or (GSGGSGGSG) n wherein n is an integer greater than 0 and less than or equal to 10. In some embodiments, in any one of the immunogenic complexes provided by the present invention, the connecting peptide 2 is preferably GGSGGSGGS (SEQ ID NO: 6), GGSGGSGGSGGS (SEQ ID NO: 7), SGGSGG (SEQ ID NO: 8), or GSGGSGGSG (SEQ ID NO: 9).

[0039] Preferably, the varicella-zoster virus (VZV) gE protein of the present invention is expressed using a signal peptide having an amino acid sequence as shown in SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12; more preferably, the signal peptide sequence is SEQ ID NO: 12.

[0040] Specifically, since the first 544 amino acids of the VZV gE protein do not belong to the transmembrane region and are antigenic, the design of the VZV gE protein of the present invention is based on the VZV gE amino acid sequence 1-544 (VZV gE amino acids 1-544 are shown in SEQ ID NO: 13), wherein amino acids 1-30 are the natural secretion signal peptide inherent to VZV gE, and VZV gE amino acid sequence 31-544 is an amino acid sequence without a signal peptide (such as the amino acid sequence shown in SEQ ID NO: 14). The present invention uses the VZV gE sequence 31-544, or uses non-natural signal peptides of other sequence structures to replace the above-mentioned natural structure signal peptide in order to increase protein expression. The above-mentioned signal peptide is not connected to the signal peptide or is connected to amino acids 31-544 of VZV gE using a signal peptide with other non-natural structures, so that VZV gE is connected to the binding peptide 1 (the binding peptide 1 is named "4T") at the C-terminus through a specific linker peptide 1 (linker 1). At the same time, a histidine (e.g., 6His) purification tag can be added to the C-terminus of the fusion protein; the coding gene for the above-mentioned fusion protein is inserted into a eukaryotic cell expression vector (e.g., pcDNA3.4) and expressed in CHO cells to obtain a fusion protein composed of VZV gE and binding peptide 1. The antigen component is subjected to nickel column affinity chromatography and molecular sieve chromatography to obtain a high-purity protein, which is VZV gE-4T.

[0041] In some embodiments, in any one of the immunogenic complexes provided herein, the varicella-zoster virus (VZV) gE protein comprises the amino acid sequence shown in SEQ ID NO:14.

[0042] In some embodiments, in any of the immunogenic complexes described above, the particle protein component is a fusion protein formed by linking peptide 2 at the N-terminus of a nanoparticle protein with binding peptide 2. Preferably, the nanoparticle protein is NPM, AP205 capsid protein 3 (AP205), or Ferritin. Specifically, in some alternative approaches, binding peptide 2 (designated "4C") is linked to the gene encoding the nanoparticle protein via linker peptide 2, inserted into a prokaryotic expression vector (e.g., pET-28a(+) or pET-30a(+)), and expressed in E. coli cells to obtain a fusion protein of binding peptide 2 and the nanoparticle protein. The fusion protein can be purified by chromatography, such as anion exchange chromatography or hydrophobic chromatography, to obtain a product. The nanoparticle protein is preferably NPM, AP205, or Ferritin; the resulting particle protein components are designated NPM-4C, AP205-4C, or Ferritin-4C.

[0043] Specifically, in some alternative embodiments, under suitable reaction conditions, any of the aforementioned antigen components is conjugated to a granule protein component, whereby the immunogenic complex is formed by covalently binding the binding peptide 1 of the antigen component to the binding peptide 2 of the granule protein component. Different immunogenic complexes can be formed using different nanoparticle proteins, and these immunogenic complexes are designated VZV gE-NPM, VZV gE-AP205, or VZV gE-Ferritin, respectively.

[0044] Preferably, in any immunogenic complex provided by the present invention, the antigen component is expressed using the signal peptide MEFGLSWVFLVAIIKGVQC (SEQ ID NO: 12), and comprises VZV gE protein (SEQ ID NO: 14), connecting peptide 1 (EAAAK) 3 (SEQ ID NO: 4), binding peptide 1 (SEQ ID NO: 1), and a histidine tag; more preferably, the sequence of the antigen component is as shown in SEQ ID NO: 15.

[0045] Preferably, in any one of the immunogenic complexes provided by the present invention, the antigenic component is expressed using the signal peptide MEFGLSWVFLVAIIKGVQC (SEQ ID NO: 12), and comprises VZV gE protein (SEQ ID NO: 14), connecting peptide 1 (GGGGS) 3 (SEQ ID NO: 3), binding peptide 1 (SEQ ID NO: 1), and a histidine tag; more preferably, the sequence of the antigenic component VZV gE-4T is as shown in SEQ ID NO: 16.

[0046] In some embodiments, the present invention provides an immunogenic complex comprising:

[0047] (1) an antigenic component comprising varicella-zoster virus (VZV) gE protein, connecting peptide 1 and binding peptide 1;

[0048] (2) A particle protein component comprising nanoparticle protein, connecting peptide 2 and binding peptide 2.

[0049] The connecting peptide 1 is any connecting peptide commonly used in the art, including but not limited to (GGGGS) n or (EAAAK) n The amino acid sequence of n can be an integer greater than 0 and less than or equal to 5, preferably SEQ ID NO: 3 or SEQ ID NO: 4; the connecting peptide 2 is any connecting peptide commonly used in the art, including but not limited to (GGS) n 、(SGG) n or (GSGGSGGSG) nThe amino acid sequence of n can be an integer greater than 0 and less than or equal to 10, preferably SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9; the nanoparticle protein is NPM, AP205 or Ferritin.

[0050] In some embodiments, in any one of the immunogenic complexes provided herein, the nanoparticle protein NPM comprises the amino acid sequence shown in SEQ ID NO:17.

[0051] Preferably, in any immunogenic complex provided by the present invention, the particle protein component comprises NPM-4C, as shown in SEQ ID NO: 18, which is a fusion protein obtained by connecting the binding peptide 2 shown in SEQ ID NO: 2 to the nanoparticle protein NPM shown in SEQ ID NO: 17 via the connecting peptide 2 shown in SEQ ID NO: 7.

[0052] In other embodiments, the present invention provides an immunogenic complex comprising:

[0053] (1) an antigenic component comprising varicella-zoster virus (VZV) gE protein and a connecting peptide 1, wherein the connecting peptide 1 comprises the amino acid sequence shown in SEQ ID NO: 5;

[0054] (2) a granule protein component comprising nanoparticle protein subunits; preferably, the nanoparticle protein subunits are I53-50A and / or I53-50B subunits.

[0055] In some embodiments, in any one of the immunogenic complexes provided herein, the nanoparticle protein I53-50 comprises I53-50A and / or I53-50B subunits; preferably, I53-50A comprises the amino acid sequence shown in SEQ ID NO: 19, and I53-50B comprises the amino acid sequence shown in SEQ ID NO: 20.

[0056] Specifically, in any of the immunogenic complexes provided herein, the varicella-zoster virus (VZV) gE protein is linked to a subunit of a nanoparticle protein to form a fusion protein, which is then bound to another subunit of the nanoparticle protein. Preferably, the subunit of the nanoparticle protein is I53-50A or I53-50B. Furthermore, in some alternative embodiments, the VZV gE protein in the antigenic component is linked to the nanoparticle protein I53-50A subunit at the C-terminus via linker peptide 1 to form a VZVgE-I53-50A fusion protein; this fusion protein is then bound to the nanoparticle protein I53-50B subunit.

[0057] As described above, when I53-50 is selected as the nanoparticle protein, I53-50 comprises two subunits, I53-50A and I53-50B. The extramembrane region of the VZV gE protein containing a specific signal peptide or not containing a signal peptide is connected to I53-50A through a connecting peptide 1, and a histidine (e.g., 6H) purification tag can be added to the C-terminus. The gene encoding the fusion protein is inserted into a eukaryotic cell expression vector (e.g., pcDNA3.4), expressed and purified in CHO cells, and the resulting fusion protein is named VZV gE-I53-50A; at the same time, a histidine (e.g., 6H) purification tag can be added to the C-terminus of I53-50B, and the gene encoding the protein is inserted into a prokaryotic cell expression vector (e.g., pET-30a(+)), expressed and purified in E. coli cells, and the resulting protein is named I53-50B. Then, under appropriate reaction conditions, VZV gE-I53-50A and I53-50B were covalently bound to form varicella-zoster nanoparticles, named VZV gE-I53-50.

[0058] Preferably, any immunogenic complex provided by the present invention comprises VZV gE-I53-50A (a fusion protein obtained by linking the VZV gE protein as shown in SEQ ID NO: 14 to I53-50A as shown in SEQ ID NO: 19 via a linker peptide 1), as shown in SEQ ID NO: 21.

[0059] In some embodiments, in any one of the immunogenic complexes provided herein, the nanoparticle protein Ferritin comprises the amino acid sequence shown in SEQ ID NO:22.

[0060] Preferably, in any immunogenic complex provided by the present invention, the particle protein component comprises Ferritin-4C (a fusion protein formed by binding peptide 2 as shown in SEQ ID NO: 2 through connecting peptide 2 as shown in SEQ ID NO: 8 and nanoparticle protein Ferritin as shown in SEQ ID NO: 22), as shown in SEQ ID NO: 23.

[0061] In some embodiments, in any one of the immunogenic complexes provided herein, the nanoparticle protein AP205 comprises the amino acid sequence shown in SEQ ID NO:24.

[0062] Preferably, in any immunogenic complex provided by the present invention, the particle protein component comprises AP205-4C (a fusion protein formed by binding peptide 2 as shown in SEQ ID NO: 2 and connecting peptide 2 as shown in SEQ ID NO: 9 with nanoparticle protein AP205 as shown in SEQ ID NO: 24), as shown in SEQ ID NO: 25.

[0063] In some embodiments, the present invention provides an immunogenic complex comprising any one or more of the following (1)-(7):

[0064] (1) The amino acid sequence of the varicella-zoster virus (VZV) gE protein is shown in SEQ ID NO: 14;

[0065] (2) the amino acid sequence of the connecting peptide 1 is shown in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5;

[0066] (3) The amino acid sequence of the binding peptide 1 is shown in SEQ ID NO: 1;

[0067] (4) the nanoparticle protein is selected from NPM, AP205 or Ferritin;

[0068] (5) the nanoparticle protein subunit is selected from I53-50A and / or I53-50B;

[0069] (6) The connecting peptide 2 includes an amino acid sequence of (GGS)n, (SGGSGG)n or (GSGGSGGSG)n, where n can be an integer greater than 0 and less than or equal to 10;

[0070] (7) The amino acid sequence of the binding peptide 2 is shown in SEQ ID NO: 2.

[0071] In some embodiments, the present invention provides an immunogenic complex comprising any one or more of the following (1)-(3):

[0072] (1) The amino acid sequence of NPM is shown in SEQ ID NO: 17; the amino acid sequence of Ferritin is shown in SEQ ID NO: 22; and the amino acid sequence of AP205 is shown in SEQ ID NO: 24;

[0073] (2) The amino acid sequence of I53-50A is shown in SEQ ID NO: 19; the amino acid sequence of I53-50B is shown in SEQ ID NO: 20;

[0074] (3) The amino acid sequence of the connecting peptide 2 is shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0075] In some embodiments, the present invention provides an immunogenic complex comprising any one or more of the following (1)-(6):

[0076] (1) The amino acid sequence of varicella-zoster virus (VZV) gE protein is shown in SEQ ID NO: 14;

[0077] (2) the amino acid sequence of connecting peptide 1 is shown in SEQ ID NO: 3 or SEQ ID NO: 4;

[0078] (3) the amino acid sequence of binding peptide 1 is shown in SEQ ID NO: 1;

[0079] (4) The nanoparticle protein is selected from NPM, AP205, or Ferritin; wherein the amino acid sequence of NPM is shown in SEQ ID NO: 17, the amino acid sequence of Ferritin is shown in SEQ ID NO: 22, and the amino acid sequence of AP205 is shown in SEQ ID NO: 24;

[0080] (5) the amino acid sequence of connecting peptide 2 is shown in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9;

[0081] (6) the amino acid sequence of binding peptide 2 is shown in SEQ ID NO: 2;

[0082] In some embodiments, the present invention provides an immunogenic complex comprising any one or more of the following (1)-(6):

[0083] (1) The amino acid sequence of varicella-zoster virus (VZV) gE protein is shown in SEQ ID NO: 14;

[0084] (2) the amino acid sequence of connecting peptide 1 is shown in SEQ ID NO: 4;

[0085] (3) the amino acid sequence of binding peptide 1 is shown in SEQ ID NO: 1;

[0086] (4) the nanoparticle protein is NPM, whose amino acid sequence is shown in SEQ ID NO: 17;

[0087] (5) the amino acid sequence of connecting peptide 2 is shown in SEQ ID NO: 7;

[0088] (6) The amino acid sequence of binding peptide 2 is shown in SEQ ID NO: 2.

[0089] In some embodiments, the present invention provides an immunogenic complex, wherein the varicella-zoster virus (VZV) gE protein is expressed using a signal peptide having an amino acid sequence as shown in any one of SEQ ID NOs: 10-12; and the antigen component and / or the granule protein component comprises a histidine tag.

[0090] In some embodiments, the present invention provides an immunogenic complex consisting of an antigen component and a granule protein component, wherein the amino acid sequence of the antigen component is shown in SEQ ID NO: 15, and the amino acid sequence of the granule protein component is shown in SEQ ID NO: 18.

[0091] Furthermore, the present invention also provides a method for preparing any of the above-mentioned immunogenic complexes, comprising the following steps:

[0092] (1) The antigen component and granule protein component encoding genes are respectively connected into expression vectors to construct expression recombinant plasmids and expression host strains, express the target protein, and purify it;

[0093] (2) The antigen component obtained in step (1) is co-incubated with the granule protein component to obtain an immunogenic complex.

[0094] The present invention provides a method for preparing an immunogenic complex for preventing or treating varicella-zoster virus-related diseases:

[0095] (1) ligating the genes encoding the varicella-zoster virus antigen component and the granule protein component into expression vectors to construct expression recombinant plasmids;

[0096] (2) constructing a recombinant strain capable of expressing the varicella-zoster virus antigen component and the granule protein component in a host cell;

[0097] (3) using the recombinant strain to express the fusion protein and purify the fusion protein;

[0098] (4) The antigen component and the granule protein component are co-incubated to produce a conjugation reaction and obtain an immunogenic complex.

[0099] Preferably, the immunogenic complex obtained in the above step (4) is purified to obtain a vaccine stock solution.

[0100] Preferably, in the method for preparing an immunogenic complex for preventing or treating varicella-zoster virus-related diseases, in step (1), the plasmid expressing the varicella-zoster virus antigen component can be pcDNA3.4, and the plasmid expressing the granule protein component can be pET-28a(+) or pET-30a(+).

[0101] In the method for preparing an immunogenic complex for preventing or treating varicella-zoster virus-related diseases described in the present invention, in step (2), the host cell expressing the varicella-zoster virus antigen is CHO, and the host cell expressing the granule protein component vector is E. coli.

[0102] The immunogenic complex for preventing or treating varicella-zoster virus-related diseases of the present invention comprises an antigenic component comprising a fusion protein formed by the VZV gE-binding peptide 1.

[0103] In the immunogenic complex for preventing or treating varicella-zoster virus-related diseases described in the present invention, the binding ratio of VZV gE-4T to NPM-4C is 6:1, and the binding conditions are pH 7.4 0.1M Tris-HCl, 25% (w / v) Sucrose, and reaction at 22°C for 48 hours; the binding ratio of VZV gE-I53-50A to I53-50B is 1:3, and the binding conditions are pH 7.4, 20mM Tris-HCl, 150mM NaCl, 25°C, and reaction for 2 hours; the binding ratio of VZV gE-4T to AP205-4C is 2:1, and the binding conditions are pH 6.2 40mM The reaction was performed in the presence of Na₂HPO₄, 25% (w / v) sucrose, and 200 mM sodium citrate (Na₃C₆H₅O) at 22°C for 24 hours. VZVgE-4T and Ferritin-4C were bound in a 6:1 ratio using 0.1 M Tris-HCl (pH 7.4) and 25% (w / v) sucrose at 22°C for 48 hours. Endotoxin levels in both assays were less than 100 EU / ml, meeting the requirements for large-scale production.

[0104] The present invention also provides an immune composition, which comprises any one of the above-mentioned immunogenic complexes and a pharmaceutically acceptable carrier; preferably, the pharmaceutically acceptable carrier comprises a stabilizer, an excipient, a surfactant, a buffer, and a pH regulator, wherein the stabilizer is sucrose and arginine, the excipient is mannitol, the surfactant is Tween 80, the buffer is disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate, and the pH regulator is hydrochloric acid.

[0105] In some embodiments, the immunogenic complex of the immune composition of the present invention is contained in an amount of 0.25-100 μg / dose, preferably 0.5-50 μg / dose, and more preferably 0.5 μg / dose, 1 μg / dose, 2 μg / dose, 3 μg / dose, 4 μg / dose, 5 μg / dose, 10 μg / dose, 15 μg / dose, 20 μg / dose, 25 μg / dose, 30 μg / dose, 35 μg / dose, 40 μg / dose, 45 μg / dose, or 50 μg / dose. The dose used in mouse experiments is 1 / 10 of the human dose.

[0106] In some embodiments, the immune composition provided by the present invention is an injection or a lyophilized preparation, preferably a lyophilized preparation.

[0107] In some embodiments, the immune composition provided by the present invention is a lyophilized preparation, which comprises a VZV gE-NPM immunogenic complex, a stabilizer, an excipient, a surfactant, a buffer, and a pH adjuster; preferably, the stabilizer is sucrose or arginine, the excipient is mannitol, the surfactant is Tween 80, the buffer is disodium hydrogen phosphate dihydrate or sodium dihydrogen phosphate dihydrate, and the pH adjuster is hydrochloric acid.

[0108] In some embodiments, the immune composition provided by the present invention is a lyophilized formulation comprising a VZV gE-NPM immunogenic complex, sucrose, arginine, mannitol, Tween 80, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, and hydrochloric acid. Each unit dose of the lyophilized formulation comprises: VZV gE-NPM 0.5-50 μg, preferably 25 μg-50 μg; sucrose 10-20 mg, preferably 12-15 mg; mannitol 10-30 mg, preferably 20-25 mg; Tween 80 0.1-0.5 mg, preferably 0.2-0.3 mg; arginine 2-8 mg, preferably 3-5 mg; disodium hydrogen phosphate dihydrate 0.5-1.5 mg, preferably 1-1.2 mg; sodium dihydrogen phosphate dihydrate 0.5-1 mg, preferably 0.6-0.8 mg; and hydrochloric acid 8.0-9.5 mg, preferably 8.2-9.0 mg.

[0109] In some embodiments, the immune composition provided by the present invention is a lyophilized preparation, which contains 25 μg or 50 μg of VZV gE-NPM immunogenic complex, 12.5 mg of sucrose, 25 mg of mannitol, 0.25 mg of Tween 80, 4.35 mg of arginine, 1.085 mg of disodium hydrogen phosphate dihydrate, 0.62 mg of sodium dihydrogen phosphate dihydrate, and 8.66 mg of hydrochloric acid.

[0110] In some embodiments, the immune composition provided by the present invention is an injection, which comprises a VZV gE-NPM immunogenic complex, a stabilizer, a surfactant, a buffer, and a pH adjuster; preferably, the stabilizer is sucrose, the surfactant is Tween 80, the buffer is disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, and the pH adjuster is hydrochloric acid.

[0111] In some embodiments, the immunogenic composition provided by the present invention is an injectable solution comprising the VZV gE-NPM immunogenic complex, sucrose, Tween 80, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, and hydrochloric acid. Each unit dose of the injectable solution comprises: 0.5-50 μg, preferably 25 μg-50 μg, of the VZV gE-NPM immunogenic complex; 10-30 mg, preferably 15-25 mg, of sucrose; 0.05-0.5 mg, preferably 0.1-0.3 mg, of Tween 80; 0.2-1 mg, preferably 0.3-0.8 mg, of disodium hydrogen phosphate dihydrate; 0.1-0.5 mg, preferably 0.2-0.4 mg, of sodium dihydrogen phosphate dihydrate; and 0.2-0.5 mg, preferably 0.25-0.35 mg, of hydrochloric acid.

[0112] In some embodiments, the immune composition provided by the present invention is an injection solution, which contains 25 μg or 50 μg of VZV gE-NPM immunogenic complex, 20 mg of sucrose, 0.125 mg of Tween 80, 0.5425 mg of disodium hydrogen phosphate dihydrate, 0.31 mg of sodium dihydrogen phosphate dihydrate, and 0.3 mg of hydrochloric acid.

[0113] The present invention further provides a varicella-zoster vaccine, which comprises any one of the above-mentioned immune compositions and an adjuvant, wherein the adjuvant is selected from at least one of: aluminum salt adjuvants, Freund's complete adjuvant, propolis adjuvant, water-oil adjuvant, cytokine, CpG DNA, genetically engineered attenuated toxin, immunostimulatory complex, and liposome.

[0114] In the varicella-zoster vaccine described herein, the water-oil adjuvant is a squalene-based adjuvant containing squalene. In mouse experiments, a 0.5 μg / dose dose of the immunogenic complex (e.g., VZVgE-NPM) combined with 25 μl / dose of the squalene-based adjuvant achieved a good immune response.

[0115] The varicella-zoster vaccine of the present invention contains 5-50 μg / dose of the immunogenic complex per unit dose of the vaccine for human use, preferably 5 μg, 25 μg or 50 μg, and 0.105 mg to 10.5 mg of squalene.

[0116] The squalene adjuvant of the present invention contains: (w / w) squalene 0.5%-5%, Span 85 0.05%-1%, Tween 80 0.05%-1%, and 10mM citrate buffer.

[0117] The squalene adjuvant of the present invention contains (w / w) 1.5%-5% of squalene, 0.05%-1% of Span 85, 0.05%-1% of Tween 80, and 10mM citrate buffer.

[0118] The squalene adjuvant of the present invention preferably contains: (w / w) squalene 2%-4.5%, Span 85 0.2%-0.5%, Tween 80 0.2%-0.5%, and 10 mM citrate buffer. Among them, the more preferred amount of squalene is 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4% (w / w), the more preferred amount of Span 85 is 0.3%-0.4% (w / w), and the more preferred amount of Tween 80 is 0.3%-0.4% (w / w).

[0119] The squalene water-oil adjuvant used in the specific embodiment of the present invention may contain the following components: (w / w) squalene 3.9%, Span 85 0.47%, Tween 80 0.47%, and 10 mM citrate buffer.

[0120] The squalene water-oil adjuvant used in the specific embodiment of the present invention may include the following components: (w / w) squalene 4.3%, Span 85 0.5%, Tween 80 0.5%, and 10 mM citrate buffer.

[0121] The squalene water-oil adjuvant used in the specific embodiment of the present invention may contain the following components: (w / w) squalene 4.03%, Span 85 0.5%, Tween 80 0.5%, citric acid 0.016%, and sodium citrate 0.264%.

[0122] The squalene water-oil adjuvant used in the specific embodiment of the present invention may contain the following components: (w / w) squalene 3.0225%, Span 85 0.375%, Tween 80 0.375%, citric acid 0.012%, and sodium citrate 0.198%.

[0123] The squalene water-oil adjuvant used in the specific embodiment of the present invention may include the following components: (w / w) squalene 2.015%, Span 85 0.25%, Tween 80 0.25%, citric acid 0.08%, and sodium citrate 0.132%.

[0124] The squalene water-oil adjuvant used in the specific embodiment of the present invention may contain the following components: (w / w) squalene 0.403%, Span 85 0.05%, Tween 80 0.05%, citric acid 0.0016%, and sodium citrate 0.0264%.

[0125] Furthermore, the squalene adjuvant (Adjuvant 1) used in the specific embodiment of the present invention preferably comprises: squalene 10.50 mg (4.2%), Span 85 1.25 mg (0.5%), Tween 80 1.25 mg (0.5%), citric acid 0.04 mg (0.264%), sodium citrate 0.66 mg (0.016%) (w / w). This adjuvant can be used in conjunction with the injection formulation of VZV gE-NPM. Optionally, the amount of Adjuvant 1 in the unit dose vaccine for mouse experiments can be 25 μl / dose, and the amount of Adjuvant 1 in the unit dose vaccine for human use can be 250 μl / dose (0.25 ml / dose).

[0126] As mentioned above, the dosage of the immunogenic complex VZV gE-NPM and adjuvant is different for humans and mice. The corresponding relationship is: when used as a human dose, the dosage of VZV gE-NPM and adjuvant is 10 times the dosage used in mice. For example, if VZV gE-NPM is 5 μg / dose for mice, the human dose must be 50 μg / dose; if the adjuvant is 50 μl / dose for mice, the human dose must be 500 μl / dose (0.5 ml / dose); if the adjuvant is 25 μl / dose for mice, the human dose must be 250 μg / dose (0.25 ml / dose), and so on.

[0127] The control vaccine in the present invention involves the adjuvant AS01B adjuvant, and the AS01B composition is: each 0.5mL of AS01B adjuvant contains 50μg of Quillaja saponin QS-21, 50μg of 3-O-deacyl-4'-monophosphoryl lipid A (MPL), 1mg of dioleoylphosphatidylcholine (DOPC), 0.25mg of cholesterol, 4.385mg of sodium chloride, 0.15mg of anhydrous disodium hydrogen phosphate, and 0.54mg of potassium dihydrogen phosphate. The AS01B adjuvant used in the present invention is a commercially available vaccine from GSK. An adjuvant product sold in combination with VZV gE protein.

[0128] The present invention further provides a complete kit, characterized in that it comprises the varicella-zoster vaccine described in the present invention, and the instruments and containers required for vaccinating the vaccine.

[0129] The present invention provides a varicella-zoster vaccine, comprising a VZV gE-NPM immune composition (i.e., an immune combination containing VZV gE-NPM, which can be prepared into a lyophilized preparation or an injection preparation) and an adjuvant (in liquid form). The VZV gE-NPM immune composition and the adjuvant are packaged in separate bottles. The adjuvant contains 10.50 mg of squalene, 1.25 mg of Span 85, 1.25 mg of Tween 80, 0.04 mg of sodium citrate, and 0.66 mg of citric acid. When the adjuvant is used in conjunction with the VZV gE-NPM lyophilized preparation and the injection, the concentration of each adjuvant component in the adjuvant bottle is 1 / 2 of that in the latter case (diluted by half). The VZV gE-NPM immune complex content is 50 μg / dose or 25 μg / dose (two specifications).

[0130] For the VZV gE-NPM immune composition in the form of a lyophilized preparation: before clinical vaccination, all the liquid must be extracted from the adjuvant bottle into the bottle containing the VZV gE-NPM lyophilized preparation, mixed well and used. After reconstitution, the human dose is 0.5 ml each time, containing 50 μg or 25 μg of the VZV gE-NPM immunogenic complex, respectively.

[0131] The VZV gE-NPM immune composition is formulated as an injectable solution: the human dose of VZV gE-NPM injection is 0.25 ml, and the human dose of adjuvant is 0.25 ml. Prior to clinical vaccination, all liquid should be withdrawn from the adjuvant bottle and transferred to the bottle containing VZV gE-NPM injection. Mix thoroughly before use. After reconstitution, the human dose is 0.5 ml, containing 50 μg or 25 μg of the VZV gE-NPM immunogenic complex, respectively.

[0132] The present invention provides use of a varicella-zoster nanoparticle immunogenic complex, an immune composition or a vaccine in the preparation of a medicament for preventing or treating herpes zoster.

[0133] All reagents used in the present invention can be purchased commercially.

[0134] Compared with the prior art, the present invention has the following beneficial effects:

[0135] (1) The immunogenic complex of the present invention fills the current global gap in the supply of nanoparticle-type varicella-zoster vaccines. After trying different recombinant particle proteins, it was found that the varicella-zoster nanoparticle immunogenic complexes VZV gE-NPM, VZV gE-I53-50, VZV gE-AP205, and VZV gE-Ferritin obtained by using the different nanoparticle proteins used in the present invention can achieve relatively ideal technical effects: uniform particle size, even distribution without aggregation, stable product performance, qualified endotoxin, suitable for non-clinical development and antibody immunogenicity testing, and thus suitable for use as varicella-zoster vaccines.

[0136] (2) Compared with the currently marketed recombinant protein varicella-zoster vaccine The varicella-zoster vaccine of the present invention further improves the level of antibodies produced in the body and the immune effect of T cells. After the varicella-zoster nanoparticle immunogenic complex provided by the present invention is successfully prepared, Different non-clinical cell and antibody immunogenicity experiments were conducted for comparison. The T cell immunogenicity and antibody immunogenicity induced by the varicella-zoster nanoparticle immunogenic complex provided by the present invention are higher than This shows that the varicella-zoster nanoparticle immunogenic complex provided by the present invention can induce a vaccine that is superior to the commercial vaccine. The varicella-zoster nanoparticle immunogenic complex provided by the present invention is more effective than the varicella-zoster recombinant protein vaccine already marketed by GSK when using a specific squalene adjuvant. Earlier intervention in the immune system induction process can provide better immune protection.

[0137] More importantly, the varicella-zoster nanoparticle immunogenic complex provided by the present invention can achieve the same efficacy as the varicella-zoster nanoparticle immunogenic complex provided by the present invention while significantly reducing the dosage. Similar effects. For example, in mice primed with attenuated varicella vaccine (simulated infection) followed by two subsequent injections, VZV gE-NPM induced an excellent humoral immune response and significantly higher levels of IFN-γ and IL-2 than the control group with the same antigen dose. A 1 / 10 dose of VZV gE-NPM, or 0.5 μg, achieved the same effect as a full dose of Shingrix, or 5 μg.

[0138] (3) The present invention uses a specific linker peptide (EAAAK)3, which has a positive effect on both the expression of the fusion protein formed by VZV gE and binding peptide 1 and the immunogenicity of the immune composition. Western blot, SDS-PAGE, and immunogenicity tests revealed that the optimal effect was achieved by linking VZV gE and binding peptide 1 with (EAAAK)3.

[0139] (4) The present invention selects the specific signal peptide MEFGLSWVFLVAIIKGVQC for transfection expression to obtain the relatively highest expression level.

[0140] (5) The present invention combines squalene adjuvants with different squalene contents with VZV gE-NPM. Observation of the effects shows that the vaccine provided by the present invention can exert an ideal immunogenic effect even at very low squalene content, thereby greatly saving the amount of expensive squalene and thus reducing costs.

[0141] (6) The present invention selects stabilizers, excipients, surfactants, buffers, and pH regulators of specific types and proportions, thereby achieving the most ideal stability in the freeze-dried formulation of the immune complex.

[0142] (7) The method for preparing the nanoparticle-type varicella-zoster vaccine provided by the present invention is low-cost and suitable for large-scale production.

[0143] The granular protein component of the present invention is produced using Escherichia coli fermentation and chromatographic purification, while the VZV gE antigen is produced using CHO cell reactor culture and chromatographic purification. Both are suitable for industrial large-scale production and offer advantages such as high expression, stable process and yield, and simple operation. The yield of a single batch of recombinant granular protein component can be combined with multiple batches of VZV gE antigen, improving production efficiency. Compared to conventional recombinant protein vaccines, the nanoparticle vaccine of the present invention offers higher levels of immune protection at equivalent or lower doses, potentially reducing the cost of large-scale production.

[0144] The method for preparing the recombinant granular protein component provided by the present invention does not require special equipment, is easy to scale up, is suitable for industrial production, has a short production time, and a simple and stable process, which can reduce the cost of large-scale industrial production. The protein product prepared using the recombinant granular protein component method provided by the present invention effectively reduces the side effects caused by the residues of impurities, host proteins, exogenous DNA, antibiotics, bacterial endotoxins and other substances in the granules, thereby improving safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0145] FIG1 shows the effects of different linker peptides 1 (linker 1) on the expression and immunogenicity of the VZV gE-binding peptide 1 protein sequence, wherein:

[0146] a is the result of SDS-PAGE of the supernatant of the protein sequence of VZV gE-binding peptide 1 with different structures, i.e., (G4S)3, (EAAAK)3, and no linker peptide, expressed in CHO cells on day 8. The arrow points to the position of the target protein.

[0147] b and c are the target protein contents in the supernatant of the harvested fluid when CHO cells were transiently transfected and stably transfected, respectively (detected by ELISA);

[0148] d, e, and f show the immune test effects of VZV gE-NPM formed by VZV gE-binding peptide 1 containing different connecting peptides 1 after preparation into VZV gE-NPM in animals.

[0149] FIG2 shows the relevant detection results of VZV gE binding to nanoparticle protein NPM to form VZV gE-NPM, wherein:

[0150] a is the result of SDS-PAGE of VZV gE-NPM;

[0151] b is the result of Western Blot of VZV gE-NPM (primary antibody is anti-VZV gE antigen);

[0152] c is the result of SEC identification of samples collected during the VZV gE-NPM purification process.

[0153] FIG3 shows another related detection result of VZV gE combining with nanoparticle protein NPM to form VZV gE-NPM, wherein:

[0154] a is the result of SDS-PAGE of samples collected during the purification process of VZV gE-NPM;

[0155] b is the DLS detection result of NPM empty particles;

[0156] c is the DLS detection result of VZV gE-NPM.

[0157] FIG4 shows the relevant detection results of VZV gE binding to nanoparticle protein I53-50 to form VZV gE-I53-50, wherein:

[0158] a is the result of SEC identification of VZV gE-I53-50A purified by molecular sieve;

[0159] b is the result of SDS-PAGE of purified VZV gE-I53-50A;

[0160] c is the DLS detection result of I53-50 empty particles.

[0161] FIG5 shows another related detection result of VZV gE binding to nanoparticle protein I53-50 to form VZV gE-I53-50, wherein:

[0162] a is the SEC identification result of the combined formation of VZV gE-I53-50;

[0163] b is the SDS-PAGE results of purified I53-50A, I53-50B, and VZV gE-I53-50A;

[0164] c is the DLS detection result of VZV gE-I53-50.

[0165] FIG6 shows the relevant detection results of VZV gE binding to the nanoparticle protein Ferritin to form VZV gE-Ferritin, wherein:

[0166] a is the SDS-PAGE identification result after purification of Ferritin-4C;

[0167] b is the SDS-PAGE identification result after purification of VZV gE-Ferritin;

[0168] c is the DLS detection result of VZV gE-Ferritin.

[0169] FIG7 shows the results of the purification and particle size detection of the VZV gE-AP205 binding product, wherein:

[0170] a is the SDS-PAGE identification result of the combined AP205-4C after purification;

[0171] b shows the DLS detection results of VZV gE-AP205.

[0172] FIG8 shows the electron microscopy results of VZV gE-NPM.

[0173] FIG9 shows the electron microscopy results of VZV gE-I53-50.

[0174] FIG10 shows the results of electron microscopy detection of VZV gE-Ferritin.

[0175] FIG11 shows the electron microscopy results of VZV gE-AP205.

[0176] FIG12 shows the cellular and humoral immune responses of four particle vaccines, VZV gE-NPM, VZV gE-I53-50, VZV gE-Ferritin, and VZV gE-AP205, in a mouse model, wherein:

[0177] a is the IgG antibody titer produced by the four nanoparticle vaccines in the mouse model on day 13 after immunization;

[0178] b is the IgG antibody titer produced by the four nanoparticle vaccines in the mouse model on day 28 after immunization;

[0179] c is the detection results of spleen cytokine IL-2 produced by four nanoparticle vaccines in the mouse model on day 28 after immunization;

[0180] d is the detection result of spleen cytokine IFN-γ produced by four nanoparticle vaccines in the mouse model on day 28 after immunization.

[0181] FIG13 shows the cellular and humoral immune responses induced by VZV gE-NPM particle vaccine in a varicella attenuated vaccine primed mouse model, wherein:

[0182] a is the IgG antibody titer produced by VZV gE-NPM in the mouse model on day 58 after immunization;

[0183] b is the detection result of spleen cytokine IFN-γ produced by VZV gE-NPM in the mouse model on day 58 after immunization;

[0184] c is the detection result of spleen cytokine IL-2 produced by VZV gE-NPM in the mouse model on day 58 after immunization.

[0185] FIG14 shows the immune response results induced by VZV gE-NPM particle vaccine combined with adjuvants containing different squalene contents. DETAILED DESCRIPTION

[0186] The principles and features of the present invention are described below with reference to examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments and are not intended to limit the scope of protection of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers. When numerical ranges are given in the examples, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints may be used. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the examples, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials described in the examples of the present invention may also be used to implement the present invention, based on the prior art knowledge of those skilled in the art and the description of the present invention. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional reagent companies.

[0187] Example 1 Determination of Connector Peptide 1 and Signal Peptide

[0188] 1. Materials - Connector Peptide 1 and Signal Peptide:

[0189] (1) Connector peptide 1:

[0190] Design 1 - no linker 1 (no linker 1); Design 2 - GGGGSGGGGSGGGGS (SEQ ID NO: 3); Design 3 - EAAAKEAAAKEAAAK (SEQ ID NO: 4)

[0191] (2) Signal peptide:

[0192] MGWSLILLFLVAVATRVLS(SEQ ID NO:10), MEWSWVFLFFLSVTTGVHS(SEQ ID NO:11), MEFGLSWVFLVAIIKGVQC(SEQ ID NO:12)

[0193] 2. Experimental methods:

[0194] (1) Confirmation experiments without using linker peptide 1 or using linker peptide 1 with a different structure:

[0195] The first 544 amino acids of the VZV gE protein do not belong to the transmembrane region and are antigenic. Therefore, the VZV gE vaccine is designed based on the amino acid sequence of positions 1-544, and the original signal peptide consisting of amino acids 1-30 is replaced with the specific signal peptide selected and determined by the present invention as shown in SEQ ID NO: 12, and connected to the extramembrane region of the varicella-zoster virus (VZV) gE protein VZV gE31-544 as shown in SEQ ID NO: 14, and then connected to the binding peptide 1 (i.e., "4T") as shown in SEQ ID NO: 1 through a linker peptide 1 (linker 1) as shown in SEQ ID NO: 3 or SEQ ID NO: 4 to form a fusion protein, and a histidine 6 His purification tag is added to the C-terminus of the fusion protein. The coding gene encoding the above fusion protein is inserted into the eukaryotic cell expression vector pcDNA3.4 and expressed in CHO cells to obtain the fusion protein VZV gE-binding peptide 1, i.e., VZV gE-4T, as an antigen component.

[0196] (2) Confirmation experiments using different signal peptides:

[0197] The connecting peptide 1 is shown in SEQ ID NO: 3, and the signal peptide is shown in SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. The signal peptide is connected to the N-terminus of VZV gE31-544 to express VZV gE-4T and VZV gE-I53-50A. The details are as follows:

[0198] Preparation of VZV gE-4T: VZV gE31-544 is shown in SEQ ID NO: 14, the signal peptide connected to the N-terminus of VZV gE31-544 is shown in SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, and the connecting peptide 1 shown in SEQ ID NO: 3 connected to the C-terminus of VZV gE31-544 so that VZV gE is connected to the binding peptide 1 (the binding peptide 1 is named "4T") at the C-terminus, and a 6His purification tag is added to the C-terminus of the fusion protein; the gene encoding the above fusion protein is inserted into the eukaryotic cell expression vector pcDNA3.4, and expressed in CHO cells to obtain the fusion protein VZV gE-4T as an antigen component.

[0199] Preparation of VZV gE-I53-50A: VZV gE31-544 is shown in SEQ ID NO:14, and the signal peptide connected to the N-terminus of VZV gE31-544 is shown in SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12. Then, the VZV gE-I53-50A is fused with the I53-50A shown in SEQ ID NO:19 via the connecting peptide 1 shown in SEQ ID NO:3. The gene encoding the above fusion protein is inserted into the eukaryotic cell expression vector pcDNA3.4, and expressed in CHO-S cells to obtain the fusion protein VZV gE-I53-50A as an antigen component.

[0200] 3. Experimental results:

[0201] (1) Experimental results using different connecting peptides 1:

[0202] When transiently transfected into CHO cells using the three VZV gE-4T designs (no linker, (G4S)3, and (EAAAK)3), all expressed the target antigen, as shown in Figure 1a and Figure 1b. Figure 1a shows the SDS-GAGE analysis of the CHO transient transfection supernatant, while Figure 1b shows the ELISA analysis of the supernatant. The results demonstrate that all three designs can be transiently expressed in CHO cells, with the (EAAAK)3 design achieving the highest expression, followed by the no linker design, and the (G4S)3 linker design achieving the lowest expression.

[0203] Furthermore, as shown in Figure 1b and Figure 1c, the target protein content was assessed by ELISA in the expression supernatant under transient and stable transfection conditions in CHO cells, respectively. The results were consistent with the SDS-PAGE trend shown in Figure 1a. The expression level of the target product was the highest when (EAAAK)3 was used as a linker, and similar when no linker was used. When (G4S)3 was used as a linker, the expression level of the target product was not as high as when no linker was used or when (EAAAK)3 was used as a linker.

[0204] When linker1 containing different structures was made into VZV gE-4T and combined with NPM-4C to prepare VZV gE-NPM, immune tests were conducted in animals and corresponding effects were obtained, as shown in Figure 1d, Figure 1e, and Figure 1f (see Example 10 for details). It can be seen that the use of (EAAAK)3 achieved the best immunological effect, (EAAAK)3 was better than (G4S)3, and both (EAAAK)3 and (G4S)3 were better than the design without linker.

[0205] In summary, the present invention uses (EAAAK)3 and (G4S)3 to connect VZV gE and binding peptide 1 (ie, 4T), and most preferably uses (EAAAK)3 (SEQ ID NO: 4) to prepare the fusion protein VZV gE-binding peptide 1 (ie, VZV gE-4T).

[0206] (2) Experimental results using different signal peptides:

[0207] The transient transfection results of the experiments using the above different signal peptides showed that the signal peptide with the highest expression level was that represented by SEQ ID NO: 12. Therefore, the present invention uses the signal peptide represented by SEQ ID NO: 12 for stable transfection.

[0208] Table 1 Detection of expression levels of target proteins with different signal peptides

[0209] Table 2 Amino acid sequences of binding peptides, linker peptides, and signal peptides in the examples of this application

[0210] Example 2 Expression of the gene encoding the VZV gE-binding peptide 1 fusion protein

[0211] The original signal peptide consisting of amino acids 1-30 was replaced with a specific signal peptide selected and determined by the present invention as shown in SEQ ID NO: 12, and connected to the extracellular region of varicella-zoster virus (VZV) gE protein VZV gE31-544 as shown in SEQ ID NO: 14 (the signal peptide was connected to the N-terminus of VZV gE31-544), and then connected to the binding peptide 1 (i.e., "4T") as shown in SEQ ID NO: 1 via linker 1 (G4S) 3 (as shown in SEQ ID NO: 3) or (EAAAK) 3 (as shown in SEQ ID NO: 4), and a histidine 6 His purification tag was added to the C-terminus. The gene encoding the above fusion protein was inserted into the eukaryotic cell expression vector pcDNA3.4 and expressed in CHO cells to obtain the fusion protein VZV gE-binding peptide 1, i.e., "VZV gE-4T", as an antigen component. The steps for selecting and determining the signal peptide and linker peptide 1 are as shown in Example 1. The signal peptide linked to the N-terminus of VZV gE31-544 produced during intracellular expression is cleaved in the host cell, and the VZV gE-4T secreted from the host cell does not contain the signal peptide.

[0212] The VZV gE-4T (without signal peptide) formed using the connecting peptide 1 shown in SEQ ID NO: 4 is shown in SEQ ID NO: 15, and the VZV gE-4T (without signal peptide) formed using the connecting peptide 1 shown in SEQ ID NO: 3 is shown in SEQ ID NO: 16, as shown in Table 3.

[0213] Table 3 VZV gE-binding peptide 1 fusion protein sequence in the examples of this application

[0214] Example 3: Purification of VZV gE-binding peptide 1 fusion protein

[0215] The antigen component of the fusion protein VZV gE-4T (as shown in SEQ ID NO: 15 or SEQ ID NO: 16) expressed in Example 2 was purified by nickel column affinity chromatography and molecular sieve chromatography to obtain a high-purity protein. The specific steps are as follows:

[0216] 1. Experimental materials: Capsule filter (Bricap C01: 180cm 2 ) was purchased from Cobetter, Pellicon2 ultrafiltration membrane package was purchased from Millipore, nickel ion affinity filler Ni Bestarose FF was purchased from Bogelon, molecular sieve (HiLoad 16 / 600 Superdex 200pg) was purchased from Cytiva, ultrafiltration fixture and peristaltic pump Masterflex L / S

[0217] Ultrafiltration consumables: Millipore 10kDa Pellicon2 regenerated cellulose micromembrane (0.1m 2 surface area)

[0218] 2. Experimental methods:

[0219] (1) Sample processing: The CHO cell culture supernatant containing the above-mentioned VZV gE-binding peptide 1 fusion protein was centrifuged at 12000g for 60 minutes to harvest about 2.35L of supernatant. The supernatant was then filtered through a Cobetter 0.45+0.2μm filter (Model: Bricap C01: 180cm 2 ) for clarification and filtration.

[0220] (2) Tangential flow filtration

[0221] The purpose of this step is to concentrate the clarified liquid and replace it with affinity chromatography buffer to reduce the effect of EDTA in the culture medium on the nickel affinity filler. It is carried out at room temperature.

[0222] Ultrafiltration equipment and related methods used include:

[0223] Ultrafiltration process: Wash with water, rinse with 0.1M sodium hydroxide and circulate for 30 minutes for disinfection, use a peristaltic pump flow rate of 400mL / min, wash with 6L of water, test the pH to approximately 10, equilibrate the ultrafiltration membrane, rinse the membrane with 1L of TBS buffer (20mM Tris-HCl, 150mM NaCl, pH7.4), and equilibrate the membrane with 1L of TBS buffer until the permeate pH reaches 7.38.

[0224] Concentration of VZV gE-4T culture supernatant: Concentrate approximately 2.3 L of the clarified liquid using a peristaltic pump at a flow rate of 500 mL / min, a sample agitation rate of approximately 150 r / min, and monitored inlet pressures of 13 psi and reflux pressures of 5 psi (permeate flow rate of approximately 52 ml / min). Following these process parameters, concentrate the liquid to 0.46 L. Fluid exchange: Perform 6x diafiltration using 2.8 L of TBS buffer, monitoring inlet pressures of 12 psi and reflux pressures of 5 psi.

[0225] Concentrated sample recovery: After closing the filtration end, control the peristaltic pump flow rate to 200 mL / min to recover the VZV gE-4T ultrafiltration fraction, totaling approximately 0.5 L. The ultrafiltration membrane package was disinfected with water and 0.1 M sodium hydroxide, respectively, and circulated for 60 minutes before finally storing in 0.1 M sodium hydroxide.

[0226] (3) Nickel ion affinity chromatography

[0227] Affinity purification was performed using a nickel ion affinity chromatography column with a 10 mL column volume and a flow rate of 5 mL / min, loading 70 mL of the ultrafiltration sample.

[0228] Chromatographic procedure: CIP and washing of the chromatography column, equilibration of the column with TBS buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4), loading of the sample, washing with TBS solution, washing with 20 mM imidazole + TBS buffer to remove impurities, and elution of the VZV gE-binding peptide 1 (i.e., VZV gE-4T) component with 500 mM imidazole + TBS buffer.

[0229] (4) Molecular sieve purification

[0230] HiLoad 16 / 600 Superdex 200 pg was used for purification, the molecular sieve column volume was 120 mL, and the loading amount of the VZV gE-4T affinity purified sample was controlled at approximately 4% of the column volume.

[0231] Chromatographic procedure: CIP and washing of the chromatography column; equilibration of the column with TBS buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4), loading of the sample, elution with TBS solution, and collection of the VZV gE-4T fraction.

[0232] Example 4: Expression and purification of binding peptide 2-NPM fusion protein

[0233] The nanoparticle protein NPM (as shown in SEQ ID NO: 17) is connected to the binding peptide 2 (as shown in SEQ ID NO: 2, sequence shown in Table 1) at the N-terminus through the connecting peptide 2 (as shown in SEQ ID NO: 7, sequence shown in Table 1), thereby forming the binding peptide 2-NPM fusion protein, namely NPM-4C (as shown in SEQ ID NO: 18). The relevant sequences of NPM and NPM-4C are shown in Table 4.

[0234] Table 4 NPM, NPM-4C fusion protein sequences in the examples of this application

[0235] The coding gene of the fusion protein is expressed in Escherichia coli. After the bacteria are harvested, they need to be crushed by high-pressure homogenization to release the target protein and clarify the liquid. The main purpose is to remove bacterial fragments and impurity proteins. The liquid clarification is mainly completed by heat treatment. A two-step heating method is used for heat treatment. The supernatant after E. coli crushing is subjected to the first step heating and the second step heating (ie, "two-step heating"), and the impurity removal effect of the two-step heating step and the purity of the recombinant granular protein component are measured.

[0236] 60 g of wet E. coli cells collected by centrifugation were resuspended in 240 ml of buffer (20 mM Tris-HCl, 2 mM PMSF, pH 9.0) and disrupted using a high-pressure homogenizer at 1000 bar. After centrifugation, 280 ml of supernatant was collected, and 40 ml of this was subjected to a two-step heating procedure. The supernatant after disruption, the supernatant from the first heating centrifugation step, and the resuspension of the pellet from the second heating centrifugation step were analyzed by SDS-PAGE.

[0237] As shown in Table 5, in the first heating step, adjust the pH to 9.0, heat in an 80°C water bath for 1 hour, return to room temperature, and centrifuge to collect approximately 35 ml of supernatant. In the second heating step, add 35 ml of 100 mM Tris-HCl, 5 mM EDTA, 4% Triton, pH 7.4 buffer, followed by 7 ml of 1 M Tris-HCl, pH 7.4, and mix thoroughly. Heat in a 60°C water bath for 10 minutes, then immediately centrifuge to collect the precipitate, and reconstitute the precipitate with 20 mM Tris-HCl, 5 mM EDTA, pH 9.0 buffer.

[0238] Table 5 Two-step heating extraction method for recombinant granular protein component products

[0239] After the two-step heating process, adding different concentrations of urea and sodium chloride before chromatographic purification can significantly reduce impurities other than the target recombinant granule protein. The preferred process conditions for pretreatment of recombinant granule protein component samples before Fractogel DEAE M chromatography are soaking in 8M urea and 50-200mM sodium chloride.

[0240] The recombinant granule protein fraction sample was purified using ion exchange and hydrophobic chromatography. The first chromatographic purification step utilized a Fractogel DEAE M chromatography process. Specific steps and parameters are shown in Table 6. The Fractogel DEAE M elution pool was first diluted with buffer and then stabilized with 50% (w / v) sucrose. Specific parameters are shown in Table 7. The protein fraction was then purified using a hydrophobic Octyl Bestarose 4FF chromatography process (second chromatographic purification step). Specific steps and parameters are shown in Table 8.

[0241] First step chromatography method: chromatography filler - Fractogel DEAE M, retention time - 12.5min

[0242] Table 6 First step chromatography method

[0243] Table 7 Sample dilution method before the second step chromatography

[0244] Second step chromatography method: Chromatographic filler - Octyl Bestarose 4FF, retention time - 12.5min

[0245] Table 8 Second step chromatography method

[0246] Results and Analysis:

[0247] Purity testing revealed that after further purification using the above chromatographic medium combination, the purity of the obtained product could reach over 99.0%.

[0248] Example 5: Binding of VZV gE and NPM, and Particle Characterization

[0249] 1. Combination of VZV gE and NPM:

[0250] 1. Production of VZV gE-NPM binding products:

[0251] VZV gE-binding peptide 1 (VZV gE-4T) set forth in SEQ ID NO:15 or SEQ ID NO:16 and binding peptide 2 (NPM-4C) set forth in SEQ ID NO:18 were mixed at a BCA protein ratio of 6:1. A 50% (w / v) sucrose stock solution was added to a final concentration of approximately 25% (w / v) sucrose. A 1M Tris-HCl pH 7.4 stock solution was added to stabilize the pH at approximately 10% of the total reaction volume. The binding reaction was performed at 22°C for 48 hours. For example, a VZV gE-NPM binding system can be: 6 mL of VZV gE-4T (1 mg / mL), 1 mL of NPM-4C (1 mg / mL), 8.75 mL of 50% sucrose, and 1.75 mL of 1M Tris-HCl 7.4, for a total volume of 17.5 mL.

[0252] 2. Purification of VZV gE-NPM binding product:

[0253] Use Cytiva HiLoad 16 / 600 Superdex 200 pg (120 mL column volume) or Cytiva Superdex 200 Increase 10 / 300 GL (23 mL column volume) to purify the VZV gE-NPM binding product and remove VZV gE antigen not bound to NPM-4C. If using the HiLoad 16 / 600 Superdex 200 pg molecular sieve, the sample loading volume for the VZV gE-NPM binding sample should be controlled at approximately 3% to 6%. If using the Superdex 200 Increase 10 / 300 GL molecular sieve, the sample loading volume for the VZV gE-NPM binding sample should be controlled at 0.5 mL to 1 mL.

[0254] Chromatographic procedure: equilibrate the column with CIP and washing buffer 12.5% ​​(w / v) sucrose TBS solution (20 mM Tris-HCl, 150 mM NaCl, 12.5% ​​(w / v) sucrose pH 7.4), load the sample, and elute with 12.5% ​​(w / v) sucrose TBS solution to collect the VZV gE-NPM fraction.

[0255] 2. Particle Characterization

[0256] 1. Experimental materials: VZV gE-NPM particles prepared in the above examples

[0257] 2. Detection method:

[0258] (1) TEM detection:

[0259] Negatively stained samples were prepared using the flotation method. A 400-mesh support film was used, pre-treated for hydrophilicity, and deionized water and 2% uranyl formate negative staining solution were prepared. 3 μL of the prepared protein sample (0.12 mg / mL) was dripped directly onto one side of the support film. After a one-minute timer, excess liquid was removed from the edge of the support film using clean filter paper. After drying slightly, the support film was quickly rinsed twice with a drop of deionized water. Then, 5 μL of negative staining solution was added dropwise, followed by a one-minute timer. After the sample was removed, the support film was lifted with tweezers and the stain removed using filter paper. A thin layer was left to air dry for examination. Examination was performed under a 120 kV transmission electron microscope (FERRITINI Tecnai Spirit). The overall staining of the support film was observed at low magnification, with wells of appropriate thickness selected for observation. At high magnification, a suitable area was photographed and stored.

[0260] (2) SDS-PAGE and Western blot methods

[0261] The SDS-PAGE test sample was prepared with LDS sample loading buffer (4×) plus reducing agent DTT, heated at 70°C for 5 minutes, cooled to room temperature, centrifuged at 10,000 rpm for 20 seconds, vortexed to mix, and the final loading amount was 5 μg. The test sample and non-prestained protein molecular weight standard were loaded onto a 4-12% Bis-Tris gel, matched with MES electrophoresis buffer, set the voltage to 150V, and the electrophoresis lasted for about 60 minutes. After the electrophoresis, the gel was removed and placed in a clean container. An appropriate amount of Coomassie Brilliant Blue staining solution was added until the gel was covered, and the gel was stained on a shaker for 2 hours. After the staining was completed, the staining solution was poured out, and the gel was destained by soaking in purified water. The decolorization was continued on a shaker until the gel background color was completely removed, and the gel was photographed using a GelDoc Go gel imager. The loading amount of the Western blot test sample was 0.5 μg, and the gel running method was the same as that of SDS-PAGE. The membrane was transferred using a Turbo instrument and corresponding reagents, incubated using an iBind instrument with Anti-gE mouse monoclonal antibody and goat anti-mouse secondary antibody conjugated with AP enzyme, and then developed with a colorimetric solution. The GelDoc Go was used for photography.

[0262] (3)DLS method

[0263] Dilute the purified test sample to a concentration of 0.25 mg / mL. Using a Zetasizer Lab instrument, inject ≥1 mL of the sample into the sample cell. Run the instrument for detection. Analyze the data based on the Z-Average (nm) and Polydispersity Index (PI) values, as well as the Size Distribution by Intensity / Volume curve, and report the results.

[0264] 3. Results

[0265] According to the above method, VZV gE-4T represented by SEQ ID NO:15 and NPM-4C represented by SEQ ID NO:18 were conjugated. The binding efficiency was determined by SDS-PAGE grayscale analysis to be 79.2%. As shown in Figure 2, Figure 2a shows the SDS-PAGE analysis of the VZV gE-NPM prepared according to the present invention, Figure 2b shows the Western Blot analysis of the VZV gE-NPM according to the present invention, and Figure 2c shows the binding and stability of the VZV gE-NPM according to the present invention. In the SEC analysis results in Figure 2c, Peak 1 represents the nanoparticles formed after a 6:1 mixture of VZV gE and NPM, and Peak 2 represents the VZV gE antigen protein remaining after conjugation. Figure 3a shows the purification and separation of VZV gE and NPM after conjugation, and Figure 3b shows the particle size analysis of empty NPM particles. DLS analysis shows a particle diameter of 27.6 nm, indicating that the product is stable and meets the endotoxin standard. Samples B2-B6, shown in Figure 3a, are included and collected from Peak 1 in Figure 2c, and samples B8-C1 are included and collected from Peak 2 in Figure 2c. Figure 3c shows the particle size analysis results after gE and NPM are combined. DLS results show a particle diameter of 34.4 nm, indicating that the product is stable and endotoxin-free.

[0266] Figure 8 is a photo of VZV gE-NPM particles under electron microscopy (VZV gE-NPM 0.13 mg / mL, 18500×). The photo shows that the particles are evenly distributed without aggregation.

[0267] The VZV gE-4T shown in SEQ ID NO: 16 was combined with NPM-4C to obtain the same technical effect as the above result.

[0268] The above results indicate that the VZV gE and NPM provided by the present invention assemble normally and have a reasonable molecular weight range.

[0269] Example 6: Expression and purification of VZV gE-I53-50A and I53-50B, their combination, and particle characterization

[0270] Unlike other particles, VZV gE and I53-50 are combined by expressing and purifying the VZV gE-I53-50A fusion protein and I53-50B separately and combining them to obtain VZV gE-I53-50. The sequence structures of I53-50A, I53-50B, and VZV gE-I53-50A in the examples of this application are shown in Table 9.

[0271] 1. Expression and purification of I53-50B

[0272] 1. Experimental Materials:

[0273] Capsule filter (Bricap C01: 180cm 2 ) was purchased from Cobetter, membrane package was purchased from Millipore, Ni Bestarose FF was purchased from Bogelon, and molecular sieve (HiLoad 16 / 600 Superdex 200 pg) was purchased from Cytiva.

[0274] 2. Experimental methods:

[0275] 1) Induced expression: The I53-50B coding gene was expressed in Escherichia coli. The I53-50B BL21 Condon-plus / BL21(DE3) monoclonal strain was inoculated into 50 mL of LB(Kan+) medium and cultured at 37°C, 250 rpm for 4-5 h. When the OD600 of the culture was approximately 0.6-0.8, the culture was transferred to 18°C ​​and IPTG was added to a final concentration of 0.5 mM. Protein expression was induced at 200 rpm for 16 h.

[0276] 2) Harvesting the Cultured Cells by Centrifugation: Transfer the cultured cells to a clean, labeled 50 mL centrifuge tube and centrifuge at 6000 rpm at room temperature to collect the cells. Discard the culture medium and resuspend the cells in 10 mL of 300 mM NaCl, 50 mM Tris 7.4, 1 mM DTT, and 0.75% CHAPS solution. Vortex thoroughly on a shaker to mix.

[0277] 3) Ultrasonication: Place the 50 mL tube containing the resuspended bacterial suspension on ice and ultrasonicate. Use a #2 horn, 100% power, and sonicate for 5 seconds, then 5 seconds on. The total sonication time for each sample is 10 minutes.

[0278] 4) Collect the target protein by centrifugation: 15000 rpm, 4°C, 30 min, and collect the supernatant of cell disruption. The sequence of the target protein I53-50B is shown in SEQ ID NO: 20.

[0279] 5) Histrap purification of target protein: Wash buffer: 300 mM NaCl, 50 mM Tris pH 7.4, 1 mM DTT, 0.75% CHAPS, 30 mM Imidazole; elution buffer: 300 mM NaCl, 50 mM Tris pH 7.4, 1 mM DTT, 0.75% CHAPS, 300 mM Imidazole. Purify using a Histrap Excel-5 mL or Histrap Bogelong-10 mL column. Equilibrate the column with 5 CV of wash buffer. Filter the target protein through a 0.22 μm filter and dilute to 45 mL with wash buffer. Inject the protein using S1. Wash away contaminants with 10 CV of wash buffer. Elute the target protein with 5 CV of elution buffer.

[0280] 6) Buffer exchange: Use a concentrator tube to replace the target protein solution eluted by Histrap with 300mM NaCl, 50mM Tris pH7.4, 0.75% CHAPS solution, then determine the protein concentration and store at an appropriate temperature for subsequent binding reactions.

[0281] 2. Expression, purification and binding reaction of VZV gE-I53-50A target protein

[0282] 1. Experimental materials: Capsule filter (Bricap C01: 180cm 2 ) was purchased from Cobetter, membrane package was purchased from Millipore, Ni Bestarose FF was purchased from Bogelon, molecular sieve (HiLoad 16 / 600 Superdex 200 pg) was purchased from Cytiva

[0283] 2. Experimental methods:

[0284] 1) The gene encoding VZV gE-I53-50A was inserted into the eukaryotic expression vector pcDNA3.4 and expressed in CHO-S cells. The CHO-S cells were revived and cultured in suspension in ExpiCHO medium. The culture conditions were as follows: temperature: 37°C, humidity: 80%, CO2 concentration: 8%, and rotation speed: 120 rpm.

[0285] 2) Expand the culture of CHO-S cells, with a doubling time of approximately 16 h / generation. When the density reaches 6×10 6 When the concentration reaches 100 μg / mL, the cells are ready for transfection.

[0286] 3) Use cold OptiPRO TM Medium (4°C) to dilute the plasmid carrying the VZV gE target gene.

[0287] 4)ExpiFectamine TM Before use, mix CHO Reagent by inverting 4-5 times to ensure thorough mixing. TM Dilute ExpiFectamine in Medium (4°C) TM After dilution, let CHO Reagent stand for 2-3 minutes and immediately mix with the diluted DNA.

[0288] 5) Let the mixed solution from steps 3 and 4 stand for 2-3 minutes (no longer than 5 minutes), then add it to the prepared CHO-S cells. While adding the transfection mixture, shake the cells continuously to ensure that the transfection complex is fully mixed with the cells.

[0289] 6) After transfection, the cells were placed in a shaker at 37°C, 80% humidity, 8% CO2, and 120 rpm for expression culture.

[0290] 7) Add ExpiCHO on expression day 1 TM Enhancer, select Max Titer expression mode and add ExpiCHO TM Feed solution was added slowly and the culture bottle was shaken continuously. Then the culture temperature was lowered to 32℃.

[0291] 8) Expression day 5: Add ExpiCHO according to the Max Titer expression mode TM Feed solution was added slowly and the culture bottle was shaken continuously.

[0292] 9) Record cell viability and cell number on expression day 5, 7, 9, or 11, depending on the actual situation, and take samples for SDS-PAGE analysis on day 7.

[0293] 10) Collect the cell expression supernatant: centrifuge at 8000 rpm for 30 min, collect the supernatant, filter with a 0.22 μm filter membrane, and label and retain.

[0294] 11) The cell supernatant was diluted 3-fold with equilibration buffer (20 mM PBS, pH 6.0) to reduce conductivity, the pH was adjusted to 6.0 with phosphoric acid, and VZV gE-I53-50A was purified using a DEAE chromatography column. The equilibration buffer was 20 mM PBS, pH 6.0, and the elution buffer was 20 mM PBS, pH 6.0, 1 M NaCl.

[0295] 12) VZV gE-I53-50A was further purified using molecular sieves as shown in Figure 4(a). The elution buffer was 20 mM Tris-HCl, 150 mM NaCl, pH 7.4. The purified gE-I53-50A was characterized by SDS-PAGE as shown in Figure 4(b). The protein concentration of VZV gE-I53-50A was determined and the protein was frozen at -80°C. The target protein sequence, VZV gE-I53-50A, is shown in SEQ ID NO:21.

[0296] 13) VZV gE-I53-50A and I53-50B were mixed at a mass ratio of 1:3 for binding experiment. The binding conditions were pH 7.4, 20 mM Tris-HCl, 150 mM NaCl, 25°C, and room temperature for 2 hours to obtain the bound product VZV gE-I53-50.

[0297] 14) The product after molecular sieve separation and purification is shown in Figure 5a. The purified VZV gE-I53-50 was identified by SDS-PAGE and compared with I53-50A and I53-50B as shown in Figure 5b. The particle size of VZV gE-I53-50 was detected and compared with I53-50 as shown in Figure 4c and Figure 5c.

[0298] 3. Particle Characterization

[0299] 1. Experimental materials: VZV gE-I53-50 particles prepared in the above examples

[0300] 2. Detection method: Same as Example 5.

[0301] 4. Results

[0302] Figures 4a through 5c show the binding and stability results of the VZV herpes simplex gE-I53-50 particles claimed in the present invention. Figures 4a and 4b show the results of the preparation and purification of the VZV gE-I53-50A antigen. Peak 1 in Figure 4a represents the purified VZV gE-I53-50A, and lanes 2 through 8 in Figure 4b represent the sample collected from peak 1 in Figure 4a. DLS results in Figure 4c show that the I53-50 particles have a diameter of 30.7 nm and are stable, with acceptable endotoxin levels. Figures 5a and 5b show the binding and post-binding purification results of the VZV gE-I53-50 claimed in the present invention. Peak 2 in Figure 5a represents the nanoparticles formed by the binding of VZV gE-I53-50A and I53-50B in a 1:3 ratio, and Peak 3 represents the remaining I53-50B protein after binding. Figure 5b shows the analysis and identification results of VZV gE-I53-50A after purification in SDS-PAGE and compared with I53-50A and I53-50B; the DLS results in Figure 5c show that the diameter of VZV gE-I53-50 particles is 60.15 nm, and the product is stable and endotoxin-qualified.

[0303] Electron microscopy results of gE-I53-50 particles are shown in Figure 9. A photograph of VZV gE-I53-50 particles (VZV gE-I53-50 0.13 mg / mL, 18500×) shows that VZV gE-I53-50A reacts with I53-50B to form nanoparticles with the expected molecular weight. The image shows uniform particle distribution with no aggregation.

[0304] Table 9 I53-50A, I53-50B, VZV gE-I53-50A in the examples of this application

[0305] Example 7: Expression and purification of VZV gE and Ferritin, their combination, and particle characterization

[0306] The recombinant nanoparticle protein Ferritin is fused at its N-terminus with the connecting peptide 2 of SEQ ID NO:8 and the binding peptide 2 of SEQ ID NO:2, thereby forming a binding peptide 2-Ferritin fusion protein, i.e., "Ferritin-4C." The sequences of Ferritin and Ferritin-4C in the examples of this application are shown in Table 10.

[0307] Table 10 Ferritin and Ferritin-4C in Examples of the present application

[0308] 1. Expression and Purification of Ferritin-4C

[0309] 1. Experimental Materials:

[0310] Capsule filter (Bricap C01: 180cm 2 ) was purchased from Cobetter, membrane package was purchased from Millipore, HisTrap excel was purchased from Cytiva, and molecular sieve (HiLoad 16 / 600 Superdex 200 pg) was purchased from Cytiva.

[0311] 2. Experimental methods:

[0312] 1) Induction expression conditions: The coding gene of Ferritin-4C was expressed in E. coli, and Ferritin-4C BL21 (DE3) monoclonal bacteria were inoculated into 400 mL LB (Amp + ) medium, cultured at 37°C, 220 rpm for 4-5 hours. When the culture OD600 reached approximately 0.6-0.8, the culture was transferred to 18°C, IPTG was added to a final concentration of 0.5 mM, and protein expression was induced at 200 rpm for 16 hours. The sequence of the target protein, Ferritin-4C, is shown in SEQ ID NO: 23.

[0313] 2) Harvest the bacteria by centrifugation: The culture medium was centrifuged at 7000 g at room temperature to collect the bacteria. The culture medium was discarded and the bacteria were resuspended in 40 mL of 150 mM NaCl, 20 mM Tris pH 7.4 solution.

[0314] 3) Ultrasonic disruption: Place the resuspended bacterial solution in an ice-water bath for ultrasonic disruption. Use a #2 horn, 50% power, and sonicate for 3 seconds, 7 seconds on, for a total of 12 minutes.

[0315] 4) Collect the target protein by centrifugation: 13,000 g, 4°C, 30 min. Discard the supernatant and solubilize the inclusion bodies with 20 mM Tris-HCl, 150 mM NaCl, 8 M Urea, 2% Triton X-100, pH 7.4 for 1 h.

[0316] 5) Histrap purification of target protein: Wash buffer 1 is 20 mM Tris-HCl, 150 mM NaCl, 8 M Urea, pH 7.4; Wash buffer 2 is 20 mM Tris-HCl, 150 mM NaCl, 8 M Urea, 2% Triton X-100, pH 7.4; Elution buffer is 20 mM Tris-HCl, 150 mM NaCl, 1 M Imidazole, pH 7.4. After the inclusion bodies are dissolved, centrifuge at 13000g for 30 minutes, collect the supernatant, and load it onto a Histrap excel-5ml NI column; use Wash buffer 2 to equilibrate the Histrap excel-5ml for 10CV, then load the sample; after loading, rinse the column with Wash buffer 2 for 40CV to remove endotoxins; use Wash buffer 1 to rinse the column for 10CV to remove Triton X-100; use 2% Elution buffer to rinse for 10CV to wash away impurities; use 2%-100% Elution buffer to linearly elute the target protein for 15CV; after elution, detect the protein purity by SDS-PAGE.

[0317] 6) Dilution and Renaturation: The dilution and renaturation buffer is 20mM Tris-HCl, 150mM NaCl, 25% (w / v) Sucrose, pH 7.4. After nickel column purification, the target protein is collected and concentrated using a concentrator. Meanwhile, the sample is graded and diluted with SEC buffer to 0.25M. The sample is concentrated to 1ml and then purified by molecular sieve separation. The elution buffer is SEC buffer. After elution, protein purity is determined by SDS-PAGE. The purified Ferritin-4C is shown in Figure 6(a). The protein concentration is determined by BCA assay. The sample is stored at an appropriate temperature for subsequent binding reactions.

[0318] II. Binding of VZV gE-binding peptide 1 and binding peptide 2-ferritin and purification of the binding product

[0319] VZV gE-binding peptide 1 (SEQ ID NO:15 or SEQ ID NO:16) and binding peptide 2-ferritin (Ferritin-4C) were mixed at a BCA protein ratio of 6:1. A 50% (w / v) sucrose stock solution was added to a final concentration of approximately 25% (w / v). A 1M Tris-HCl pH 7.4 stock solution (10% of the total reaction volume) was added to stabilize the pH. The binding reaction was performed at 22°C for 48 hours.

[0320] The binding rate between the VZV gE-binding peptide 1 and the binding peptide 2-Ferritin was calculated to be 80% using the SDS-PAGE grayscale method.

[0321] The bound product was separated and purified using molecular sieve separation (purification buffer: 20 mM Tris-HCl, 150 mM NaCl, 25% (w / v) Sucrose, pH 7.4) to collect the VZV gE-ferritin fraction. The purified VZV gE-ferritin nanoparticles were characterized by SDS-PAGE, as shown in Figure 6b.

[0322] 3. Particle Characterization

[0323] 1. Experimental materials: VZV gE-Ferritin particles prepared in the above examples.

[0324] 2. Detection method: Same as Example 5.

[0325] 4. Results

[0326] Figure 6a shows the SDS-PAGE results of the purified Ferritin-4C nanoparticles claimed in the present invention; Figure 6b shows the SDS-PAGE results of the purified VZV gE-Ferritin-bound vesicle nanoparticles claimed in the present invention; Figure 6c shows the particle size measurement results of the VZV gE-Ferritin nanoparticles, with DLS results showing a particle diameter of 34.17 nm, indicating product stability; Figure 10 is a photograph of the negative staining electron microscopy results of the VZV gE-Ferritin nanoparticles, showing uniform particle distribution and no aggregation. These results demonstrate that the VZV gE-Ferritin particles provided by the present invention are properly assembled and have a reasonable molecular weight range.

[0327] Figures 6 and 10 show the effects obtained by the binding reaction between VZV gE-4T shown in SEQ ID NO: 15 and Ferritin-4C; the binding reaction between VZV gE-4T shown in SEQ ID NO: 16 and Ferritin-4C achieved the same technical effects as the above results.

[0328] Example 8: Expression and purification of AP205 fusion protein, binding to VZV gE, and particle characterization

[0329] The recombinant nanoparticle protein AP205 is fused at its N-terminus with the linker peptide 2 of SEQ ID NO:9 and the binding peptide 2 of SEQ ID NO:2, thereby forming the binding peptide 2-AP205 fusion protein, i.e., "AP205-4C." The sequences of AP205 and AP205-4C in the examples of this application are shown in Table 11.

[0330] Table 11 AP205 and AP205-4C sequences in the examples of this application

[0331] 1. Expression and purification of AP205-4C:

[0332] 1. Experimental Materials:

[0333] Ni-NTA was purchased from QIAGEN, nuclease (Benzonase) was purchased from Sigma, and dialysis bags (300 KD) were purchased from Spectrum Labs.

[0334] 1. Experimental methods:

[0335] The gene encoding AP205-4C was expressed in Escherichia coli. A 2.4 L culture was resuspended in 50 ml of lysis buffer and incubated at room temperature for 15 minutes. The mixture was then placed on ice for 10 minutes and sonicated 12 times for 30 seconds each, with 60 seconds between each (ultrasound power ratio of 30%). The sonicated lysate was centrifuged for 20 minutes (15,000 g, 4°C), the supernatant discarded, and the precipitate collected and resuspended in urea buffer to break up inclusion bodies. The precipitate was stirred at 300 rpm at room temperature overnight. The next day, the urea suspension was centrifuged for 80 minutes (15,000 g, 25°C), the supernatant collected, filtered with a syringe (0.45 μm), and incubated at room temperature for 5 minutes with 250 U of nuclease (Benzonase, Sigma). The sequence of AP205-4C is shown in SEQ ID NO: 25.

[0336] Wash the Ni-NTA (QIAGEN) column material (3 ml) twice with ultrapure water (5CV, 15 ml), wash it twice with equilibration buffer 2 (5CV, 15 ml), add the sample resuspension solution, shake at room temperature for 10 minutes, centrifuge at 4700g for 4 minutes and remove the supernatant. Dilute the column material with 45 ml equilibration buffer 1 for renaturation, let the column material stand and remove the supernatant, repeat the operation 3 times. Wash with 45 ml elution buffer 1, let the column material stand and remove the supernatant, repeat the operation 3 times. Wash the column material with 45 ml elution buffer 2, let the column material stand and remove the supernatant, a total of 6 times. Wash the column material with 15 ml elution buffer 3, let the column material stand and remove the supernatant, repeat the operation 3 times. Use 1 ml elution buffer for the column material, shake at room temperature (900 rpm) for 10 minutes, centrifuge for 10 minutes (13000g, 4°C), remove the upper wash buffer and set aside. Place 1 ml of eluate in a dialysis bag (300 kD, Spectrumlabs), seal it, and dialyze it against 1 L of dialysis buffer for 3 hours. Then, dialyze it into 1 L of fresh dialysis buffer overnight. Remove the eluate the next day, centrifuge, and store at 4°C until needed. See Table 12 below for specific parameters.

[0337] Table 12 Chromatography method

[0338] Results and Analysis:

[0339] Purity testing revealed that the purity of the product obtained after refinement by the above combination of purification and dialysis can reach over 95.0%.

[0340] II. Binding of VZV gE-binding peptide 1 and binding peptide 2-AP205 and purification of the binding product

[0341] 1. Production of VZV gE-AP205 binding products:

[0342] VZV gE-4T (SEQ ID NO:15 or SEQ ID NO:16) and AP205-4C were mixed at a BCA protein ratio of 2:1. A 50% (w / v) sucrose stock solution was added to a final concentration of approximately 25% (w / v) sucrose. A 200 mM sodium citrate (Na3C6H5O7·2H2O) stock solution, 40 mM Na2HPO4, pH 6.2, was added to stabilize the pH. The binding reaction was performed at 22°C for 24 hours.

[0343] 2. Purification of VZV gE-AP205 binding product:

[0344] The VZV gE-AP205 binding product was purified using a dialysis bag (300 kD, Spectrumlabs). Unbound gE antigen was removed using a magnetic stirrer (350 rpm). Dialysis was performed in four cycles, each lasting at least 4 hours. The dialysis buffer consisted of 50 mM glycine, 25 mM sodium citrate, and 0.1% (v / v) Tween 20, pH 6.2. After dialysis, the binding product was removed and centrifuged (13,000 g, 4°C) for 10 minutes. The supernatant was stored at 4°C.

[0345] 3. Particle Characterization

[0346] 1. Experimental materials: VZV gE-AP205 particles prepared in the above examples.

[0347] 2. Detection method: Same as Example 5.

[0348] IV. Results

[0349] As shown in Figure 7 , Figure 7a shows the results of SDS-PAGE analysis of the VZV gE-AP205 prepared according to the present invention. Figure 7b shows the DLS analysis of AP205-4C, which shows a particle diameter of 17.31 nm, indicating an endotoxin-free standard. Figure 11 shows a negative-staining electron microscopy image of VZV gE-AP205 nanoparticles, demonstrating uniform particle distribution and absence of aggregation.

[0350] Figures 7 and 11 show the effects obtained by the binding reaction between VZV gE-4T shown in SEQ ID NO: 15 and AP205-4C; the binding reaction between VZV gE-4T shown in SEQ ID NO: 16 and AP205-4C achieved the same technical effects as the above results.

[0351] The above results indicate that the VZV-gE provided by the present invention assembles normally with AP205 and has a reasonable molecular weight range.

[0352] Summarize:

[0353] It can be seen from Examples 5 to 8 that the four prepared nanoparticle products can all be used as the varicella-zoster nanoparticle immunogenic complexes claimed in the present invention, and can achieve relatively ideal technical effects: the particles have uniform particle size, uniform distribution without aggregation, and qualified endotoxin content, and are suitable for non-clinical development and antibody immunogenicity test testing, and are thus suitable as candidate vaccines for varicella-zoster nanoparticles.

[0354] Example 9: Particle Thermal Stability

[0355] 1. Experimental methods:

[0356] 1. Experimental instrument: UNcle universal protein stability analyzer

[0357] 2. Operation method: Take protein samples of the same concentration (0.1 mg / ml) and add 9 μl to each well of the UNi tube. Repeat 3 wells for each sample. Set the temperature range from 25 to 95°C at a heating rate of 1°C / min. Measure the Tm and Tagg266 values ​​of each protein 3 times. Analyze the BCM trend to obtain the results.

[0358] 2. Experimental Results

[0359] The thermal stability of the four particles of VZV gE-NPM (formed by VZV gE-4T shown in SEQ ID NO: 15 and NPM-4C shown in SEQ ID NO: 18), VZV gE-I53-50, VZV gE-Ferritin, and VZV gE-AP205 obtained in the above example was measured: the Tagg266 (°C) of VZV gE-NPM, VZV gE-I53-50, and VZV gE-AP205 were measured by SLS at a wavelength of 266 nm using UNcle, and were 61.5±4.88, 53.1±6.02, and 82.3±2.50, respectively (the Tagg266 (°C) of gE-Ferritin could not be measured using the Uncle instrument). The Tm (°C) values ​​of VZV gE-I53-50, VZV gE-Ferritin, and VZV gE-AP205 were measured to be 59.0±0, 60.3±0.45, and 69.7±0.95, respectively. (The Tm (°C) value of gE-NPM could not be measured using the Uncle instrument.) The VZV gE-NPM formed by VZV gE-4T represented by SEQ ID NO:16 and NPM-4C represented by SEQ ID NO:18 achieved the same technical results as described above.

[0360] The above data show that the thermal stability of the four particles is good.

[0361] Example 10: Immunogenicity Test of VZVgE-NPM, VZVgE-I53-50, VZVgE-Ferritin, and VZVgE-AP205 in Balb / c Mice

[0362] After the varicella-zoster nanoparticle immunogenic complex provided by the present invention is successfully prepared, the above composition is used as a vaccine against the varicella-zoster recombinant protein vaccine already marketed by GSK. Various non-clinical cellular and antibody immunogenicity assays were performed.

[0363] 1. Experimental Materials

[0364] (1) Experimental animals

[0365] Recombinant varicella-zoster vaccine Female 5-6-week-old SPF Balb / c mice were purchased from GSK and were purchased from Vital River. VZV gE peptide was synthesized by Nanjing GenScript. After passing quarantine, mice were ear-tagged and randomly grouped by weight, with four mice per cage. Animals were housed in a standard SPF animal facility with sterile feed and sterile deionized water. The room was maintained under a 12-hour light cycle, a temperature of 21 ± 2°C, and a humidity of 30-70%.

[0366] (2) Test article and control article

[0367] ① Test vaccine stock solution

[0368] The tested vaccine protein stock solutions were prepared by Guangzhou Painuo Biotechnology Co., Ltd.: VZV gE-NPM (Examples 4-5), VZV gE-I53-50 (Example 6), VZV gE-Ferritin (Example 7), VZV gE-AP205 (Example 8), and VZV gE-4T (Examples 2-3).

[0369] ②Test vaccine adjuvant

[0370] Adjuvant 1: Squalene 10.50 mg (4.2%), Span 85 1.25 mg (0.5%), Tween 80 1.25 mg (0.5%), citric acid 0.04 mg (0.264%), sodium citrate 0.66 mg (0.016%) (w / w)

[0371] Adjuvant 1 was prepared by Guangzhou Painuo Biotechnology Co., Ltd. according to the following steps:

[0372] Weigh squalene and Span 85, stir evenly with a magnetic stirrer to form the oil phase. Weigh Tween 80 and water for injection, stir with a magnetic stirrer until the Tween 80 is completely dissolved. Take sodium citrate buffer and add it to the water for injection and stir evenly. Then add the prepared Tween 80 solution and stir evenly to form the aqueous phase. Immerse the disperser in the aqueous phase, turn on the disperser, and slowly drip the oil phase into the aqueous phase to form colostrum. Pour the colostrum into the microjet and homogenize at 12000psi until the average particle size is less than 180nm, and the average particle size is about 160nm. After sterilization through a 0.2μm filter membrane, it is squalene adjuvant 1.

[0373] ③Vaccine reference substance

[0374] (Varicella attenuated vaccine), (Recombinant Varicella-Zoster Vaccine)

[0375] 2. Experimental methods

[0376] (1) Vaccine preparation

[0377] ① Preparation of the vaccine of the present invention

[0378] The immunogenic complex stock solution (VZV gE-NPM) was diluted to 25 μL with TBS (pH 7.4) according to the dose, and then mixed with 25 μL of adjuvant 1. Care was taken to protect from light and avoid oxidation.

[0379] ②Control vaccine preparation

[0380] Varilrix (varicella vaccine):

[0381] This product contains borosilicate glass tube injection bottles and butyl rubber stoppers (pre-filled syringes, containing 0.5ml of diluent / vial), and penicillin vials (containing vaccine lyophilized powder). Connect the pre-filled syringe and penicillin vial, inject all the diluent into the penicillin vial to dissolve the lyophilized powder, shake well, and the powder should be clear and free of foreign matter after complete dissolution.

[0382] (Recombinant Varicella-Zoster Vaccine):

[0383] After reconstitution, one dose (0.5 mL) contains 50 μg of gE protein. Use a disposable sterile syringe to extract all the AS01B adjuvant in the vial to dissolve the lyophilized powder, shake well, and the powder should be clear and free of foreign matter after complete dissolution.

[0384] Shingrix 0.5μg: Dissolve Shingrix antigen protein in 2500μL TBS, mix thoroughly, and then pipette 250μL of this solution. Add 250μL of AS01B adjuvant and mix thoroughly. In the mouse model test, each dose contains 50μL volume and 0.5ug antigen protein.

[0385] (2) Animal experimental immunization procedures

[0386] a. Two-shot immunization—comparison of different structural linker peptides 1 (VZV gE-NPM no linker, VZV gE-NPM (G4S) 3linker, VZV gE-NPM (EAAAK) 3linker, where both (G4S) 3linker and (EAAAK) 3linker are linker peptides 1)

[0387] Eighteen female BALB / c mice that passed the adaptive observation test were randomly divided into three groups of six animals each. Each mouse received a 50 μL intramuscular injection of the tibial fluid (25 μL per leg) into the caudal or cranial tibialis of both legs. Table 13 shows the experimental groupings and dosing. The immunization schedule was as follows: primary immunization on D0, secondary immunization on D14, and blood collection and serum isolation on D28 after the primary immunization for specific IgG antibody determination. The spleen was then harvested on D28 for white blood cell isolation and determination of cellular immune markers. The results are shown in Figures 1, d, e, and f.

[0388] Table 13 Experimental groups and dosages

[0389] b. Two-shot immunization—four immunogenic complexes and (VZV gE-NPM, VZV gE-I53-50, VZV gE-Ferritin, VZV gE-AP205, )

[0390] Forty female BALB / c mice that passed the adaptive observation test were randomly divided into five groups of eight animals each. Each mouse received a 50 μL intramuscular injection of the drug into the caudal or cranial tibialis of both legs (25 μL per leg). Table 14 shows the experimental groupings and dosing. The immunization schedule was as follows: primary immunization on D0, secondary immunization on D14. Blood was collected on D13 and D28 after the primary immunization for serum analysis and specific IgG antibody determination. The spleen was harvested on D28 for leukocyte isolation and determination of cellular immune markers.

[0391] This part of the experiment ("b. Two-shot immunization - four immunogenic complexes and ”), VZV gE-NPM is an immunogenic complex formed by VZV gE-4T (i.e., SEQ ID NO: 15) in Example 5, wherein linker 1 is (EAAAK)3, and NPM-4C shown in SEQ ID NO: 18.

[0392] The results are shown in Figure 12.

[0393] Table 14 Experimental groups and dosages

[0394] *: The dose of the group refers to VZV gE protein

[0395] c. Attenuated primary immunization + two additional immunizations

[0396] Twenty-four female BALB / c mice that had passed the adaptive observation observation were randomly divided into three groups of eight animals each, with a 50 μL / animal dose. The immunization schedule was as follows: Varilrix (attenuated varicella vaccine) was administered subcutaneously on D-35, with 50 μL injected subcutaneously at the back of the neck. The test articles were VZV gE-NPM (0.5 μg VZV gE-NPM, 25 μL Adjuvant 1), Shingrix (5 μg, 50 μL AS01B adjuvant), and Shingrix 0.5 (0.5 μg, 25 μL AS01B adjuvant). Immunizations were given once on D0 and D28, with 50 μL injected intramuscularly into the caudal or cranial tibialis of each leg (25 μL per leg). The first immunization with the test article was performed on day 0. Blood was collected on days 14 and 58 for serum analysis and specific IgG antibody determination. On day 58, the spleen was harvested for leukocyte isolation and cellular immune marker determination. The results are shown in Figure 13. VZV gE-NPM is an immunogenic complex formed by VZV gE-4T represented by SEQ ID NO: 15 and NPM-4C represented by SEQ ID NO: 18. The preparation method is as described in Example 5. The linker used for VZV gE-4T shown in Figure 13 is (EAAAK)3.

[0397] (3) Specific IgG antibody detection

[0398] The whole blood collected in the centrifuge tube was placed at room temperature for 2 hours or in a 4°C refrigerator overnight. After the blood coagulated and the clot shrank, it was centrifuged at 4000 rpm for 10 minutes. The supernatant was collected and placed in a clean centrifuge tube and stored at -20°C.

[0399] A 96-well microtiter plate (Thermo Fisher Scientific) was coated with VZV gE protein (2 μg / mL) at 100 ng / 50 μL / well overnight at 4°C. The plates were then washed twice with PBST (0.05% Tween 20) and blocked at room temperature (25°C ± 3°C) for 1–4 hours. The plates were then washed twice. Diluted immune serum was then added and incubated at room temperature for 1 hour, followed by four washes. A 1:5000 dilution of secondary antibody, Goat Anti-Mouse IgG H&L (HRP) working solution, was then added at 50 μL / well. The plates were incubated at room temperature for 1 hour, followed by six washes. 100 μL of color development solution was then added to each well. Color development was performed at room temperature in the dark for 10 minutes, followed by the addition of 100 μL of 1 M HCl to each well for termination. The plate reader was set at a dominant wavelength of 450 nm and a reference wavelength of 620 nm. Sample absorbance was calculated as OD450 minus OD620. The measurement was completed within 5 minutes after termination.

[0400] Data processing:

[0401] The data is reliable if the following conditions are met:

[0402] The OD value of the control serum is ±0.2, the OD value corresponding to the sample initial concentration is <3.0, the OD value corresponding to the blank well is less than 0.1, and the coefficient of variation of the replicate wells (response value) should be less than 20%.

[0403] Transfer the raw sample data into the Excel Endpoint ELISA template to calculate the antibody titer. Use a cutoff value of 0.15. Display the data as a GMT (geometric mean titer) bar chart.

[0404] (4) Mouse cytokine ELISpot detection

[0405] After euthanasia, mice were bled and sterile procedures were performed in a cleanroom. The mice were immobilized, the abdominal cavity exposed, and the spleen isolated. Forceps were used to place the spleen in a sample tube containing an appropriate amount of pre-chilled sterile 1× PBS, ensuring complete immersion. The spleen was dissociated into single cells using a tissue dissociator (Miltenyi Biotec) and then added to a 96-well plate from the kit that had been washed four times with PBS and conditioned with α-MEM complete medium for 1-4 hours.

[0406] The cells were set at three densities, 5×10 6 / 50μL / well, 2.5×10 6 / 50μL / well, 1.25×10 6 Then, 100 ng / 50 μL / well of peptide stimulator was added, and the negative control well (set up for each animal) and the positive control well contained 5×10 cells. 6 The negative control wells were stimulated with 50 μL of complete medium. For the positive control wells, 50 μL of positive stimulator (PMA final concentration of 6 μg / mL, ION final concentration of 2 μg / mL) was added. Incubate in a 37°C, 5% CO2 incubator for approximately 20 hours. Subsequent steps were performed according to the kit instructions (MABTECH). Biotinylated monoclonal antibody, Streptavidin-ALP, and the chromogenic substrate BCIP / NBT-plus were added in sequence. After 10 minutes, color development was stopped by slowly rinsing with tap water. The reaction strips were air-dried at room temperature in the dark, and the spots were counted using an ELISpot reader.

[0407] Data processing:

[0408] Number of cytokine spots = number of spots in peptide stimulation wells (cell density 5×10 6 / 50μL / well)-the number of spots in the negative control well (cell density 5×10 6 cells / 50 μL / well)

[0409] The data are presented as mean values ​​using bar graphs.

[0410] (5) Statistical analysis

[0411] The results were analyzed using Graphpad Prism 9.1.2 software. Unpaired t test or One-Way ANOVA were used to analyze the differences, and the data between the two groups were defined as having significant differences when P < 0.05.

[0412] 2. Test results:

[0413] (1) Combined with the results shown in d, e, and f in Figure 1, the results obtained by using "a. Two-shot immunization" are as follows:

[0414] Figure 1d shows IFN-γ levels on day 28, Figure 1e shows IL-2 levels on day 28, and Figure 1f shows IgG levels on day 28. For linker 1 (linker 1), molecules designed with a linker, whether (G4S)3 or (EAAAK)3, showed stronger immunogenicity than designs without linker 1. This was demonstrated by molecules with linker 1 inducing significantly higher cellular immune responses than those without linker 1, and by an increased antibody response. Furthermore, it was found that using (EAAAK)3 as linker 1 induced a significantly stronger immune response than (G4S)3.

[0415] The results obtained using "b. Two-shot immunization" are shown in Figure 12.

[0416] Antibody Response: As shown in Figure 12 (a), the comprehensive evaluation results of the four nanoparticle groups on D13 (day 13) are all superior to those of the control group. The VZV gE-NPM, VZV gE-I53-50, and VZV gE-Ferritin nanoparticle systems all produced significantly higher antibody titers than the control group, while the antibody titer of the VZV gE-AP205 group also showed a trend of being higher than the control group. (b) shows the antibody titers on D28 (day 28), with all nanoparticle groups showing significant differences compared to the control group.

[0417] Cellular Responses: (c) and (d) show spleen cytokine assay results on day 28 (D28). These results demonstrate that VZV gE-NPM induced significantly higher IFN-γ and IL-2 responses than the control group. VZV gE-I53-50 and VZV gE-Ferritin groups induced significantly higher IL-2 responses than the control group. Overall, all nanoparticle groups exhibited higher cytokine responses than the control vaccine.

[0418] It can be seen that when using non-potent adjuvants, the nanoparticle platform of the present invention (four immunogenic complexes, VZV gE-NPM, VZV gE-I53-50, VZV gE-Fe, VZV gE-AP205) can induce cellular and humoral immune responses that are superior to Shringrix. At the same time, in terms of side effects or safety, the varicella-zoster vaccine prepared by the nanoparticle platform combined with a non-potent adjuvant has obvious advantages.

[0419] (2) The results obtained by using "c. attenuated primary immunization + two-dose supplementary immunization" are shown in Figure 13.

[0420] In mice immunized with a primary attenuated varicella vaccine (simulated infection) followed by two subsequent doses, VZV gE-NPM induced a superior humoral immune response, with significantly higher IFN-γ and IL-2 cytokine responses than the control group, which received the same antigen dose. As shown in Figure 13, a 1 / 10 dose of VZV gE-NPM achieved the same efficacy as a full dose of Shingrix (5 μg). This demonstrates that the particle vaccine is superior to the control vaccine across different immunization schedules.

[0421] Example 11: Immunogenicity test of VZV gE-NPM combined with adjuvants containing different squalene contents in Balb / c mice

[0422] 1. Experimental materials and experimental methods refer to Example 10.

[0423] 1. Adjust the squalene content in the adjuvant used in different experimental groups (the corresponding contents of the other components in the adjuvant were also adaptively adjusted, as shown below, but the changes in the other components had no effect on the immune effect). The contents of the various components of the squalene adjuvant used in different groups are as follows:

[0424] Adjuvant 25μL group (Group 1), in which the squalene content was 4.03% (w / w), equivalent to 1.01mg, i.e. 40.3mg / ml; Span 85 content was 0.5% (w / w), equivalent to 0.125mg, i.e. 5mg / ml; Tween 80 content was 0.5% (w / w), equivalent to 0.125mg, i.e. 5mg / ml; citric acid content was 0.016% (w / w), equivalent to 0.004mg, i.e. 0.16mg / ml; sodium citrate content was 0.264% (w / w), equivalent to 0.066mg, i.e. 2.64mg / ml.

[0425] Adjuvant 2.5 μL group (Group 2), in which squalene content was 0.403% (w / w), equivalent to 0.01% of the mass of squalene in Group 1, equivalent to 0.101 mg, or 4.03 mg / ml; Span 85 content was 0.05% (w / w), equivalent to 0.01% of the mass of Span in Group 1, equivalent to 0.0125 mg, or 0.5 mg / ml; Tween 80 content was 0.05% (w / w), equivalent to Tween 1 in Group 1. 80 mass, equivalent to 0.0125 mg, i.e. 0.5 mg / ml; citric acid content 0.0016% (w / w) is equivalent to 0.01 of the mass of citric acid in group 1, equivalent to 0.0004 mg, i.e. 0.016 mg / ml; sodium citrate content 0.0264% (w / w) is equivalent to 0.01 of the mass of sodium citrate in group 1, equivalent to 0.0066 mg, i.e. 0.264 mg / ml.

[0426] Adjuvant 18.75 μL group (Group 3), wherein the content of hornene is 3.0225% (w / w), equivalent to 0.75% of the mass of hornene in Group 1, equivalent to 0.7575 mg, or 30.225 mg / ml; the content of Span 85 is 0.375% (w / w), equivalent to 0.75% of the mass of Span in Group 1, equivalent to 0.0938 mg, or 3.75 mg / ml; the content of Tween 80 is 0.375% (w / w), equivalent to The mass of Tween 80 in group 1 is 0.75, which is equivalent to 0.0938 mg, or 3.75 mg / ml; the citric acid content of 0.012% (w / w) is equivalent to the mass of citric acid in group 1, which is equivalent to 0.003 mg, or 0.12 mg / ml; the sodium citrate content of 0.198% (w / w) is equivalent to the mass of sodium citrate in group 1, which is equivalent to 0.75, which is equivalent to 0.0795 mg, or 1.98 mg / ml.

[0427] Adjuvant 12.5 μL group (Group 4), in which the squalene content was 2.015% (w / w), equivalent to 0.5 times the mass of squalene in Group 1, equivalent to 0.505 mg, or 20.15 mg / ml; Span 85 content was 0.25% (w / w), equivalent to 0.5 of the mass of Span 85 in Group 1, equivalent to 0.0625 mg, or 2.5 mg / ml; Tween 80 content was 0.25% (w / w), equivalent to 0.5 of the mass of Tween 80 in Group 1, equivalent to 0.0625 mg, or 2.5 mg / ml; citric acid content was 0.08% (w / w), equivalent to 0.5 of the mass of citric acid in Group 1. Equivalent to 0.002 mg, i.e. 0.08 mg / ml; the sodium citrate content of 0.132% (w / w) is equivalent to 0.5 of the mass of sodium citrate in group 1, equivalent to 0.033 mg, i.e. 1.32 mg / ml.

[0428] Control group (NA): contained only VZV gE-NPM without adjuvant.

[0429] 2. Each group received a 5 μg dose of VZV gE-NPM antigen protein in the animal model. A primary immunization was performed on day 0, an intermediate blood sample was collected on day 14, a booster immunization was performed on day 14, and whole blood was collected for serum separation and spleen removal on day 28. VZV gE-NPM is an immunogenic complex formed by VZV gE-4T represented by SEQ ID NO:15 and NPM-4C represented by SEQ ID NO:18, and was prepared as described in Example 5.

[0430] 2. Experimental results:

[0431] As shown in Figure 14, the VZV gE-NPM samples used in this experiment were used for primary immunization on days 0 and 14, with the animal model receiving a dose of 5 μg. Sampling, testing, and analysis were performed on day 28. Graphs were analyzed using standard one-way analysis of variance with Dunnett's multiple comparison test.

[0432] At the same antigen dose (5 μg gE-NPM protein), the use of different squalene adjuvant contents revealed that the 18.75 μL and 12.5 μL groups achieved comparable efficacy to the 25 μL group, with the 18.75 μL group even achieving superior efficacy compared to the 25 μL group. Since each group received a 5 μg dose of VZV gE-NPM antigen protein, the mass ratios of VZV gE-NPM to squalene were 202:1 (Group 1), 20.2:1 (Group 2), 151.5:1 (Group 3), and 101:1 (Group 4).

[0433] It can be seen that the classification results after sampling on day 28 show that the vaccine represented by VZV gE-NPM in the present invention can also exert an ideal immune effect under the conditions of adjuvants with different degrees of low squalene content.

[0434] The squalene adjuvant component in the unit dose of the recombinant herpes zoster vaccine for human use of the present invention is preferably: squalene 10.50 mg (4.2%), Span 85 1.25 mg (0.5%), Tween 80 1.25 mg (0.5%), citric acid 0.04 mg (0.264%), and sodium citrate 0.66 mg (0.016%) (w / w).

[0435] Example 12: Stability Study of VZV gE-NPM Vaccine Freeze-dried Formulations with Different Compositions and Screening of the Optimal Freeze-dried Formulation

[0436] 1. Experimental Methods: Different formulations were designed, with varying amounts of alcohol, amino acids, and surfactants. SEC-HPLC and SDS-PAGE were used to test the purity of the vaccine compositions and to compare the stability of the different formulations. The VZV gE-NPM protein concentration in the formulation was 0.1 mg / ml. The lyophilized product was placed in a 40°C incubator, and physical and chemical analysis by SDS-PAGE and SEC-HPLC was performed after 3, 7, 14, and 28 days.

[0437] 2. Experimental results: as shown in Table 15 below.

[0438] Based on the above experimental results, it can be seen that various factors have different effects on the stability of the particle protein vaccine of the present invention. Maintaining a constant immunogenic complex content (VZV gE-NPM 0.1 mg / ml), the optimal types and concentrations of sugars, alcohols, amino acids, and surfactants were determined through screening to be: sucrose 25 mg / ml, mannitol 50 mg / ml, arginine 8.7 mg / ml, and Tween 80 0.5 mg / ml. VZV gE-NPM is an immunogenic complex formed by VZV gE-4T represented by SEQ ID NO:15 and NPM-4C represented by SEQ ID NO:18, and its preparation method is described in Example 5.

[0439] Thus, the formula of the VZV gE-NPM lyophilized preparation can be determined: containing VZV gE-NPM 25 μg or 50 μg, sucrose 12.5 mg, mannitol 25 mg, Tween 80 0.25 mg, arginine 4.35 mg, disodium hydrogen phosphate dihydrate 1.085 mg, sodium dihydrogen phosphate dihydrate 0.62 mg, and hydrochloric acid 8.66 mg.

[0440] The experimental results show that the key temperatures of the optimal freeze-dried preparation formula are as follows: collapse temperature Tc is -39 degrees Celsius, glass transition temperature Tg is 51.6 degrees Celsius, and glass transition temperature Tg' is -55 (Tc, Tg, and Tg' are the key temperatures of the freeze-dried formula, which are used to guide the pre-freezing and primary drying temperature settings of the freeze-drying process and the maximum storage temperature. Generally, the pre-freezing temperature should be lower than Tg', the primary drying temperature should be lower than Tc, and the finished product storage temperature should be lower than Tg).

[0441] Taking into account the extreme conditions that the finished freeze-dried vaccine preparation may be subjected to during transportation and storage, the freeze-dried formula preparation was placed under high temperature (40°C for 7d and 14d), shaking (the sample was fixed on a decolorization shaker, the speed was set to 240rpm, and the time was 24h), and illumination (the sample was placed upright in a 4°C light box for 3d). The in vivo potency test 24h after reconstitution showed that the activity of the preparation samples after the above treatment could remain stable (5-6 week old BALB / c female mice were used, primary immunization on D0, secondary immunization on D14, blood collection on D14, and blood and spleen collection on D28), and the immunological activity remained good.

[0442] Thus, the formulation composition of the unit dose recombinant herpes zoster vaccine (lyophilized preparation) for human use was finally determined. The vaccine is prepared by reconstituting the injectable VZV gE-NPM lyophilized preparation formula in an adjuvant, 0.5 ml / dose. The formulation of the lyophilized preparation is shown in Table 16 below.

[0443] Among them, the dosage of VZV gE-NPM and adjuvant is different for humans and mice during vaccination: when used as a human dose, the dosage of VZV gE-NPM and adjuvant is 10 times that of mice. For example, 5 μg / dose of VZV gE-NPM for mice corresponds to 50 μg / dose for humans, and 50 μl / dose of adjuvant for mice corresponds to 500 μl / dose (0.5 ml / dose) for humans.

[0444] Table 15 Prescription screening of recombinant herpes zoster vaccine freeze-dried VZV gE-NPM preparations

[0445] Table 16: Composition of the freeze-dried formulation of VZV gE-NPM vaccine

[0446] In summary, the above embodiments and drawings are only preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An immunogenic complex, characterized in that Include: The antigen component consists of varicella-zoster virus (VZV) gE protein or an immunogenic fragment thereof, connecting peptide 1 and binding peptide 1; The granule protein component consists of nanoparticle protein, connecting peptide 2 and binding peptide 2; The antigen component and the granule protein component are covalently bound to each other via binding peptide 1 and binding peptide 2.

2. The immunogenic complex according to claim 1, characterized in that Any one or more of the following (1)-(6): (1) The amino acid sequence of varicella-zoster virus (VZV) gE protein is shown in SEQ ID NO: 14; (2) the amino acid sequence of connecting peptide 1 is shown in SEQ ID NO: 3 or SEQ ID NO: 4; (3) the amino acid sequence of binding peptide 1 is shown in SEQ ID NO: 1; (4) The nanoparticle protein is selected from NPM, AP205, or Ferritin; wherein the amino acid sequence of NPM is shown in SEQ ID NO: 17, the amino acid sequence of Ferritin is shown in SEQ ID NO: 22, and the amino acid sequence of AP205 is shown in SEQ ID NO: 24; (5) the amino acid sequence of connecting peptide 2 is shown in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9; (6) The amino acid sequence of binding peptide 2 is shown in SEQ ID NO:

2.

3. The immunogenic complex according to claim 1 or 2, characterized in that: The varicella-zoster virus (VZV) gE protein is expressed using a signal peptide having an amino acid sequence as shown in any one of SEQ ID NOs: 10-12; the antigen component and / or the granule protein component comprises a histidine tag.

4. The immunogenic complex according to claim 3, characterized in that The amino acid sequence of the antigen component is shown in SEQ ID NO: 15, and the amino acid sequence of the granule protein component is shown in SEQ ID NO:

18.

5. The method for preparing the immunogenic complex according to any one of claims 1 to 4, characterized in that: include: (1) The antigen component and granule protein component encoding genes are respectively connected into expression vectors to construct expression recombinant plasmids and expression host strains, express the target protein, and purify it; (2) The antigen component obtained in step (1) is co-incubated with the granule protein component to obtain an immunogenic complex.

6. An immune composition, characterized in that The immunogenic complex comprises the immunogenic complex of any one of claims 1 to 4, further comprising a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a stabilizer, an excipient, a surfactant, a buffer, and a pH adjuster, wherein the stabilizer is sucrose and arginine, the excipient is mannitol, the surfactant is Tween 80, the buffer is disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate, and the pH adjuster is hydrochloric acid.

7. The immune composition according to claim 6, characterized in that The immune composition is a lyophilized preparation, and each unit dose of the lyophilized preparation contains: VZV gE-NPM 25μg-50μg, sucrose 12-15mg, mannitol 20-25mg, arginine 3-5mg, polysorbate 80 0.2-0.3mg, disodium hydrogen phosphate dihydrate 1-1.2mg, sodium dihydrogen phosphate dihydrate 0.6-0.8mg, and hydrochloric acid 8.0-9.5mg.

8. A varicella-zoster vaccine, characterized in that: The invention comprises the immune composition according to claim 7 and an adjuvant, wherein the adjuvant comprises (w / w) squalene 1.5%-5%, Span 85 0.05%-1%, Tween 80 0.05%-1%, and 10 mM citrate buffer.

9. The vaccine according to claim 8, characterized in that Each unit dose of the vaccine for human use comprises 5 μg, 25 μg or 50 μg of the immunogenic complex according to any one of claims 1 to 4 and 0.105 mg to 10.5 mg of squalene.

10. Use of the immunogenic complex according to any one of claims 1 to 4, the immune composition according to any one of claims 6 to 7, or the vaccine according to any one of claims 8 to 9 in the preparation of a medicament for preventing or treating herpes zoster.

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