Immunogenic composition as well as preparation method and application thereof

Abstract

The invention discloses an immunogenic composition as well as a preparation method and application thereof. The immunogenic composition comprises self-assembled gE nanoparticles, an immunopotentiator, namely, saponin QS-21, and a neutral liposome, the self-assembled gE nanoparticles are formed by polymerizing and assembling a plurality of monomers, and the monomers comprise VZV gE, a linker peptide (SGS) and VZV gI polypeptide containing Th epitopes. The immunogenic composition can be applied to VZV vaccines, and solves the technical problems of weak gE immunogenicity and serious vaccine side reaction in vaccines prepared in the prior art. The immunogenicity of the vaccine is equivalent to that of Xinanliei, and the use (variety and dosage) of an immunopotentiator can be reduced, so that the clinical side reaction of the vaccine is lower; in addition, the vaccine can induce a high-level gI specific CMI reaction, which is beneficial to further improvement of the effectiveness of the vaccine. In short, the immunogenic composition has good clinical application potential.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, and particularly relates to an immunogenic composition and a preparation method and application thereof. BACKGROUND

[0002] Varicella zoster virus (VZV) is an alpha herpesvirus that infects humans and causes varicella (chickenpox), zoster (shingles) and postherpetic neuralgia (PHN). The genome of varicella zoster virus is about 125 kb, which encodes 67 kinds of proteins, of which the envelope glycoprotein includes gB, gC, gE, gH, gI, gL, etc. Glycoprotein E (gE) is the most abundant and immunogenic glycoprotein on the envelope and host cell membrane of varicella zoster virus, which contains a large number of B cell and T cell epitopes. Glycoprotein I (gI) is a relatively abundant glycoprotein on the envelope of varicella zoster virus, which has strong immunogenicity and contains a confirmed Th (T helper) cell epitope.

[0003] Shingrix, a recombinant protein zoster vaccine of GlaxoSmithKline ® (Shingrix ®) was approved by FDA in 2017 for the prevention of herpes zoster in adults 50 years of age and older and in immunocompromised adults 18 years of age and older; the vaccine can induce higher gE-specific CMI (Cell-Mediated Immunity) response (the vaccine can induce a higher number of gE-specific IL2+ and / or IFN-g+ CD4+ T cells) and gE-specific antibody levels than the live attenuated vaccine; the vaccine is composed of the extracellular domain of glycoprotein E (gE) and the AS01B adjuvant system; the AS01B adjuvant system (complex adjuvant) contains 50 pg of the immune enhancer / adjuvant 3-O-deacylated-4'-monophosphoryl lipid A (3D-MPLA), 50 pg of the immune enhancer / adjuvant Quillaja saponaria Molina saponin QS-21, dioleoylphosphatidylcholine (DOPC) and cholesterol; Quillaja saponaria Molina saponin QS-21 can activate NLRP3 (NLR family Pyrin domain protein 3) inflammasome, thereby releasing caspase-1 (caspase-1) dependent cytokines IL-1b and IL-18, thereby promoting Th17 cell maturation or driving INF-g mediated Th1 response; 3D-MPLA activates MyD88 (Myeloid Differentiation Factor 88) and TRIF (TIR domain-containing adaptor inducing interferon-beta) dependent signaling pathways by interacting with TLR-4, inducing the production of inflammatory cytokines and interferons, thereby enhancing immune response. Compared with the live attenuated vaccines of Merck and GSK, the GSK Shingrix ® has higher protection rate, but has higher rate of serious adverse reactions in the clinic.

[0004] The current clinical review of Shingrix ® reveals that Shingrix ® can induce high levels of inflammatory marker C-reactive protein (CRP) after being injected into rabbits, up to 9 times in male animals and up to 5 times in female animals; high levels of CRP indicate the induction of acute phase reactions and the increase of systemic inflammatory response, which may be related to clinical adverse reactions such as prostration, fatigue, nausea, etc. A study published in Vaccine in 2025 shows that the adverse reactions (tolerance) of the vaccine have become a core obstacle for Americans to vaccinate Shingrix ® ; the vaccination rate of the vaccine after completing 1 dose vaccination in people over 50 years old (17%) is significantly lower than that of influenza vaccine (47.4% in people aged 50-64 years old; 71.3% in people aged 65 years old and above) and pneumonia vaccine (69% in people aged 65 years old and above). In China, the adverse reaction problem of Shingrix ® has a significant negative impact on its vaccination rate.

[0005] Shingrix ®The main reason for the high incidence of clinical adverse reactions and serious adverse reactions (grade 3 AE) of the vaccine is that two immune enhancers (QS-21 and 3D-MPLA) are used; the use of two immune enhancers / adjuvants may overactivate the immune system, leading to the release of a large amount of inflammatory factors, enhancing local or systemic inflammatory reactions, and causing symptoms such as fever, fatigue, and muscle soreness.

[0006] There is a clinical need for a new generation of vaccines that can induce gE-specific CMI and gE-specific antibodies that are not inferior to those of Zostavax, while having lower clinical adverse reactions. Therefore, using only one immune enhancer (50 μg of QS-21) while forming nanoparticles of gE and increasing Th epitopes in the vaccine antigen to improve the levels of gE-specific CMI and gE-specific antibodies is an ideal technical route for developing new generation vaccines. Nanoparticle antigens retain the natural conformation of viral antigens but do not contain genetic material and are not infectious or pathogenic, with a particle size of 10-200 nm; nanoparticle antigens can activate the innate immune system and induce strong humoral and cellular immune responses, and their antigen presentation efficiency is significantly better than that of traditional vaccines.

[0007] Patent CN117100850A improves the immunogenicity of gE and the level of VZV-specific CMI by fusing and expressing gE with multiple predicted Th epitopes of gB and / or gC and / or gH and / or gI and / or gK and / or gL and / or gM and / or gN, but most of the predicted Th epitopes have not been confirmed by research, and the immune enhancement ability is weak; the process for preparing gE-containing nanoparticles in patent CN116983403B is particularly complex: gE fusion protein (consisting of gE, linker 1, and binding peptide 1) is expressed and purified in CHO cells, and granule protein (consisting of nanoparticle protein, linker 2, and binding peptide 2) is expressed and purified in E. coli, then the gE fusion protein and the granule protein are mixed to form a gE-containing nanoparticle protein mixture, and then the mixture is purified to prepare gE-containing nanoparticle protein; although the gE-containing nanoparticle protein prepared by this process improves the immunogenicity of gE, the granule protein and the binding complex protein (consisting of binding peptide 1 and binding peptide 2) that granulate gE are non-VZV and have strong antigenicity, and can stimulate strong non-VZV-specific (granule protein and binding complex protein-specific) immune responses, which are not required for varicella-zoster vaccine and may exacerbate the clinical adverse reactions of the vaccine. SUMMARY

[0008] The present application aims to provide an immunogenic composition, a preparation method and application thereof, to solve the problem of large clinical side effects of vaccines in the prior art. To achieve the above-mentioned purpose, the present application provides the following technical solutions. In the first aspect, the present application provides an immunogenic composition, which comprises: self-assembled gE nanoparticles, an immune enhancer and neutral liposomes. The self-assembled gE nanoparticles are assembled by polymerization of monomers; the monomers comprise a VZV glycoprotein E extracellular region, a connecting peptide and a VZV glycoprotein I extracellular region polypeptide containing Th epitopes.

[0009] Further, the amino acid sequence of the gE is shown in SEQ ID NO. 1; further, the amino acid sequence of the gI is shown in SEQ ID NO. 2; further, the gI polypeptide is gI AA82-AA154, and the amino acid sequence is shown in SEQ ID NO. 3; further, the amino acid sequence of the monomer is shown in SEQ ID NO. 4.

[0010] Further, the VZV glycoprotein I extracellular region (gI) polypeptide containing Th epitopes comprises at least Th epitope 1 (epitope ID: 839316), Th epitope 2 (epitope ID: 2224791) and Th epitope 3 (epitope ID: 1597827); further, the amino acid sequence of the Th epitope 1 is FCFRSVQVIRYDGCPRIRTS; further, the amino acid sequence of the Th epitope 2 is RYDGCPRIRTSAFISCRYKH; further, the amino acid sequence of the Th epitope 3 is TSAFISCRYKHSWHYGNSTD.

[0011] Further, the Th epitope 1 is located at positions of AA82-AA101 in the extracellular domain polypeptide sequence of VZV glycoprotein I, and has an amino acid sequence of FCFRSVQVIRYDGCPRIRTS; further, the Th epitope 1 has been confirmed by Ying Ying Kong in 2016 to be an HLA-DRB1*03:01, HLA-DRB1*13:01, HLA-DRB3*03:01 and HLA-DRB3*02:02 restricted epitope, and the IDs (IEDB IDs) in the Immune Epitope Database (IEDB) are 2884824, 2884827, 2884826 and 2884825, respectively; further, the Th epitope 1 has been confirmed by Ying Ying Kong in 2017 to be an HLA-DRB3*01:01 and HLA-DR restricted epitope, and the IDs (IEDB IDs) in the Immune Epitope Database (IEDB) are 3497311 and 3497310, respectively; further, the Th epitope 1 has been confirmed by Kerry J. Laing in 2019 to be an HLA-DRB3*03:01 restricted epitope, and the ID (IEDB ID) in the Immune Epitope Database (IEDB) is 5003302.

[0012] Further, the Th epitope 2 is located at positions of AA91-AA110 in the extracellular domain polypeptide sequence of VZV glycoprotein I, and has an amino acid sequence of RYDGCPRIRTSAFISCRYKH; further, the Th epitope 2 has been confirmed by Kerry J. Laing in 2020 to be a Th epitope, and the ID (IEDB ID) in the Immune Epitope Database (IEDB) is 8326833.

[0013] Further, the Th epitope 3 is located at positions of AA100-AA119 in the extracellular domain polypeptide sequence of VZV glycoprotein I, and has an amino acid sequence of TSAFISCRYKHSWHYGNSTD; further, the Th epitope 3 has been confirmed by Ying Ying Kong in 2017 to be an HLA-DPB1*02:01 restricted Th epitope, and the ID (IEDB ID) in the Immune Epitope Database (IEDB) is 3497348.

[0014] Further, according to the self-assembled gE nanoparticle, the monomer thereof can contain various purification tags for facilitating protein purification; further, the purification tag includes but is not limited to any one or a combination of more of the following: MBP tag, GST tag, GFP tag, Fc tag, Halo tag, HA tag, Myc tag, Flag tag, His tag, Strep-tag II tag; further, the purification tag is characterized by being located at the N-terminus and / or C-terminus of the monomer of the gE nanoparticle, preferably the C-terminus; further, when the purification tag is a 6X His tag and located at the C-terminus of the monomer, the amino acid sequence of the monomer containing the purification tag is shown as SEQ ID NO. 5, and the DNA sequence of the monomer includes but is not limited to SEQ ID NO. 6.

[0015] Further, the neutral liposome includes dioleoylphosphatidylcholine (DOPC) and cholesterol; further, the ratio of the dioleoylphosphatidylcholine (DOPC) and cholesterol is 2:1 to 8:1.

[0016] Further, according to the immunogenic composition, the concentration of the self-assembled gE nanoparticle in the composition is 5 to 400 μg / ml, the concentration of the immune enhancer Quillaja saponin QS-21 is 25 to 200 μg / ml, the concentration of the dioleoylphosphatidylcholine (DOPC) is 500 to 8000 μg / ml, and the concentration of the cholesterol is 62.5 to 4000 μg / ml.

[0017] In a second aspect, the present application provides a method for preparing an immunogenic composition, including the following steps: S1, preparing a self-assembled gE nanoparticle solution; S2, preparing an immune enhancer Quillaja saponin QS-21 solution; S3, preparing a neutral liposome; S4, preparing a composite adjuvant with the neutral liposome and the immune enhancer Quillaja saponin QS-21 solution; S5, preparing an immunogenic composition with the self-assembled gE nanoparticle solution and the composite adjuvant.

[0018] Further, the self-assembled gE nanoparticle solution in S1 has a concentration of 10 to 10000 μg / ml; further, the self-assembled gE nanoparticle solution contains a buffer; further, the buffer includes but is not limited to any one or a combination of more of the following: phosphate buffer, histidine buffer, glycine buffer, citric acid buffer, acetic acid buffer, Tris buffer, HEPES buffer.

[0019] Further, the preparation of the self-assembled gE nanoparticle solution in S1 includes but is not limited to the following steps: Step 1, construction of an expression vector or viral vector containing a nucleic acid sequence encoding the monomer; Step 2, transformation of the expression vector obtained in step 1 into a host cell or infection of the host cell with the viral vector obtained in step 1; Step 3, culture of the host cell in step 2; Step 4, collection of the cell culture supernatant and / or supernatant of cell lysate; Step 5, purification of the cell culture supernatant and / or supernatant of cell lysate to obtain a self-assembled gE nanoparticle solution.

[0020] Further, the expression vector in step 1 is a eukaryotic expression vector.

[0021] Further, the host cell transformed by the expression vector in step 2 includes but is not limited to CHO cells, yeast cells, HEK293 cells; further preferably, CHO cells.

[0022] Further, the CHO cells include but are not limited to CHO-S, CHO-K1, CHO-DG44 and the like cell lines.

[0023] Further, the yeast cells include but are not limited to Saccharomyces cerevisiae cells, Pichia pastoris cells, Hansenula cells and the like yeast cells.

[0024] Further, the viral vector in step 1 includes baculovirus vector, lentivirus vector, adenovirus vector, retrovirus vector, adeno-associated virus vector, herpes simplex virus vector, parainfluenza virus vector; further preferably, baculovirus vector.

[0025] Further, the host cell infected by the viral vector in step 2 includes but is not limited to insect cells, HEK293 cells; further preferably, insect cells.

[0026] Further, the insect cells include but are not limited to Sf9 cells, Sf21 cells, Hi5 cells.

[0027] Further, the viral vector for infecting the host cell in step 2 includes but is not limited to baculovirus vector, adenovirus vector, adeno-associated virus vector, lentivirus vector; further preferably, baculovirus vector.

[0028] Further, the host cell in step 3, if the host cell is transformed by the expression vector, the host cell can be either a polyclonal cell line or a monoclonal cell line, preferably a monoclonal cell line.

[0029] Further, the host cell of step 3, if the host cell is a host cell transformed with an expression vector, and the host cell is transformed into stable transformation (using a eukaryotic screening marker resistance gene and / or gene screening of the transformed host cell) or transformed into transient transformation (without using a eukaryotic screening marker resistance gene and / or gene screening of the transformed host cell), the host cell includes CHO cell, yeast cell, HEK293 cell; further, the host cell is preferably a CHO cell.

[0030] Further, the cell culture supernatant and / or supernatant of cell lysate of step 4 contains self-assembled gE nanoparticles, medium residual components and the like, and may contain host cell fragments, host cell proteins, host cell nucleic acids, host cell metabolites and the like; further, the supernatant of cell lysate is the supernatant obtained by lysing the host cell and centrifuging or the like.

[0031] Further, the purified cell culture supernatant and / or supernatant of cell lysate of step 5, the purification method includes but is not limited to any one of the following methods or a combination of multiple methods: precipitation method, chromatography method, dialysis method, ultrafiltration method, centrifugation method, nanofiltration method, microfiltration method; further, the chromatography method includes but is not limited to any one of the following chromatography methods or a combination of multiple chromatography methods: ion exchange chromatography, gel filtration chromatography, affinity chromatography, hydrophobic chromatography, reverse phase chromatography; further, the chromatography method also includes composite mode chromatography, the composite mode includes the composite mode of ion exchange chromatography and / or gel filtration chromatography and / or affinity chromatography and / or hydrophobic chromatography and / or reverse phase chromatography.

[0032] Further, the gE nanoparticle solution of step 5 has a purity (target protein; reducing SDS-PAGE method) of not less than 10.0% to not less than 99.9%.

[0033] Further, the gE nanoparticle solution of step 5 has a purity (nanoparticle) of not less than 10.0% to not less than 99.9%.

[0034] Further, the preparation of the immunopotentiator Quillaja saponin QS-21 solution of S2, the immunopotentiator Quillaja saponin QS-21 is derived from plant extraction or artificial synthesis.

[0035] Further, the immunopotentiator Quillaja saponin QS-21 is derived from natural plant extraction, and the raw material for plant extraction is derived from Quillaja saponaria (scientific name: Quillaja saponaria).

[0036] Further, S2 discloses a solution of the immunopotentiator Quillaja saponaria Molina saponin QS-21, wherein the concentration of Quillaja saponaria Molina saponin QS-21 is 50 μg-10000 μg / ml.

[0037] Further, S3 discloses a neutral liposome, which is composed of dioleoylphosphatidylcholine (DOPC) and cholesterol; further, the ratio of the dioleoylphosphatidylcholine (DOPC) and cholesterol is 2:1-8:1.

[0038] Further, S3 discloses a neutral liposome, which is composed of dioleoylphosphatidylcholine (DOPC) and cholesterol, wherein the concentration of the dioleoylphosphatidylcholine (DOPC) is 1000-16000 μg / ml, and the concentration of the cholesterol is 125-8000 μg / ml.

[0039] Further, S4 discloses a complex adjuvant, which contains the immunopotentiator Quillaja saponaria Molina saponin QS-21, the dioleoylphosphatidylcholine (DOPC) and the cholesterol.

[0040] Further, S5 discloses an immunogenic composition, which contains the self-assembled gE nanoparticle at a concentration of 5-400 μg / ml, the immunopotentiator Quillaja saponaria Molina saponin QS-21 at a concentration of 25-200 μg / ml, the dioleoylphosphatidylcholine (DOPC) at a concentration of 500-8000 μg / ml, and the cholesterol at a concentration of 62.5-4000 μg / ml.

[0041] In a third aspect, the present application provides an application of the immunogenic composition, which is applied to a VZV vaccine for preventing and / or improving varicella and / or herpes zoster and / or post-herpetic neuralgia.

[0042] In a fourth aspect, the present application provides a VZV vaccine, which contains the immunogenic composition. According to the VZV vaccine, other pharmaceutically acceptable excipients are further contained; further, the other excipients include, but are not limited to, a combination of any one or more of the following: polysorbate 80, polysorbate 20, sodium chloride, potassium chloride, sucrose, trehalose, glycine, histidine, histidine hydrochloride, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, acetic acid, sodium acetate, tris-hydroxymethyl aminomethane (Tris-HCl), tris-hydroxymethyl aminomethane hydrochloride (Tris-hydrochloric acid), sodium hydroxide, and hydrochloric acid.

[0043] Based on the above technical solutions, the present application has the following advantages and beneficial effects compared with the prior art: 1. The present application provides an immunogenic composition, which contains self-assembled gE nanoparticle, immunopotentiator Quillaja saponaria Molina saponin QS-21 and neutral liposome. The immunogenic composition can be applied to a VZV vaccine, which solves the technical problems of weak gE immunogenicity and severe side effects of the vaccine in the prior art.

[0044] 2. This invention provides a VZV vaccine, the immunogenicity of which is similar to that of Shingrix. ® Comparative: The serum gE-specific antibody levels in mice immunized twice with this vaccine were slightly lower than those in Shingrix. ® gE-specific CMI response (splenic cells) was slightly higher than that of Shingrix. ® This vaccine can induce not only gE-specific CMI responses and gE-specific antibodies, but also high levels of gI-specific CMI responses and significant gI-specific antibodies, which is beneficial for further improving the vaccine's effectiveness. 3. This invention provides a VZV vaccine, which is different from Shingrix. ® The immunogenicity of gE nanoparticles in this vaccine is higher than that of gE, thus reducing the need for immunostimulants in the vaccine (this vaccine contains only 50 μg of the immunostimulant saponin QS-21; Shingrix). ® It contains 50 μg of the immune enhancer saponin QS-21 and 50 μg of the immune enhancer 3D-MPLA, thereby reducing the inflammatory response induced by the immune enhancer and ultimately reducing the clinical side effects of the vaccine.

[0045] In summary, the immunogenic compositions and vaccines provided by this invention have good potential for clinical application. Attached Figure Description

[0046] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art are briefly introduced below. Obviously, the drawings described below are only some embodiments of this invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0047] Figure 1 This is a graph showing the SDS-PAGE detection results of gE or gE fusion protein in Example 1 of the present invention; in the graph, M / M1: protein marker; R: reducing SDS-PAGE; NR / NR: non-reducing SDS-PAGE; gE-gI2: gE-gI2 fusion protein; gE-gI: gE-gI fusion protein; NP-1: gE nanoparticle NP-1.

[0048] Figure 2 This is the SEC-HPLC detection chromatogram of gE nanoparticles NP-1 in Example 1 of this invention.

[0049] Figure 3 This is the particle size distribution (light intensity distribution) of gE nanoparticles NP-1 in Example 1 of the present invention.

[0050] Figure 4This is an electron microscope image (40,000x magnification) of gE nanoparticles NP-1 in Example 1 of this invention.

[0051] Figure 5 This is an electron microscope image (80,000x magnification) of gE nanoparticles NP-1 in Example 1 of this invention.

[0052] Figure 6 This is the SEC-MALS detection spectrum of gE nanoparticles NP-1 in Example 1 of this invention.

[0053] Figure 7 This is a graph showing the level of gE-specific antibody GMT in the serum of mice in each vaccine group after the second immunization in Example 3 of this invention.

[0054] Figure 8 This is a graph showing the proportion of gE-specific IL2+ and / or IFN-γ+ CD4+ T cells in the spleen of mice in each vaccine group after secondary immunization in Example 3 of this invention.

[0055] Figure 9 This is a graph showing the percentage of gI-specific IL2+ and / or IFN-γ+ CD4+ T cells in the spleen of mice in each vaccine group after secondary immunization in Example 3 of this invention. Detailed Implementation

[0056] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0057] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, regarding numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the numerical range is also specifically disclosed. Every smaller range between any stated numerical value or an intermediate value within a stated numerical range, and any other stated numerical value or an intermediate value within said numerical range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0058] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and / or materials have been described herein, any methods and / or materials similar or equivalent to those described herein may be used in the implementation and / or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe the methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the description herein shall prevail.

[0059] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope and / or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. The specification and embodiments of this invention are merely exemplary.

[0060] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0061] Example 1: Preparation of self-assembled gE nanoparticle solution Designing self-assembled gE nanoparticle monomers I. Extracellular sequences of VZV glycoprotein gE and gI The amino acid sequence information of VZV glycoproteins gE and gI is shown in Table 1 below.

[0062] Table 1. Extracellular sequence information of VZV glycoprotein gE and gI regions .

[0063] II. gI Th Tables (IEDB Table Database) The amino acid sequence information of the selected gI Th epitopes is shown in Table 2 below.

[0064] Table 2. Amino acid sequence information of the selected gI Th epitopes. .

[0065] Note: Th epitope 1, Th epitope 2 and Th epitope 3 have been confirmed as Th epitopes.

[0066] III. Design of gI polypeptide (PP) information (gIPP) containing gI Th epitopes. The amino acid sequence information of different gIPPs containing the gI Th epitope is shown in Table 3 below. gI2 has been disclosed in CN117100850A to enhance the immunogenicity of gE.

[0067] Table 3. Design of gI peptides containing gI Th epitopes .

[0068] IV. Expression of the experimental protein Step 1: Construct an expression vector containing a nucleic acid sequence encoding a self-assembled gE nanoparticle monomer or other experimental protein. (1) Design self-assembled gE nanoparticle monomers containing gI polypeptide sequences and other experimental proteins (as shown in Table 4 below). Table 4 Information on gE nanoparticle monomers or gE fusion proteins .

[0069] Note: N / A means not applicable. (2) Nucleic acid codon optimization and whole gene synthesis First, a DNA sequence encoding the signal peptide (MHSSALLCCLVLLTGVRA) was added to the 5' end (5' end of the positive strand) of the protein-coding DNA sequence, and a DNA sequence encoding the 6×His tag was added to the 3' end (3' end of the positive strand). Then, the DNA sequence was codon-optimized to improve translation efficiency. Next, a 5' UTR sequence and a stop codon (TGA) were added to the 5' end (5' end of the positive strand) and 3' end (3' end of the positive strand), respectively. Then, a Not I restriction enzyme cleavage site was added to the 5' end of the 5' UTR sequence, and an Xba I restriction enzyme cleavage site and a protective base were added to the 3' end of the stop codon (TGA). Finally, the entire DNA sequence was synthesized. The amino acid sequence of the NP-1 monomer containing the 6×His tag is shown in SEQ ID NO.5, and the DNA sequence is shown in SEQ ID NO.6.

[0070] (3) Construction of expression plasmids A synthetic DNA sequence containing the complete genome was ligated into the pCDNA3.4 vector. The cloning vector was transformed into competent bacteria, and the bacteria were then amplified in large quantities. The amplified bacteria were then spread onto solid culture medium in petri dishes and incubated for approximately 8 hours. Eight single colonies were then selected for cloning screening (PCR) and sequencing verification.

[0071] (4) Expression plasmid amplification After the positive clones were verified to be correct by sequencing, they were cultured and amplified in bacterial culture, and plasmids were extracted.

[0072] Step 2: Transform the expression vector obtained in Step 1 into host cells. (5) Transfection of CHO cells with expression vector (plasmid) Expression plasmids were transfected into CHO cells.

[0073] Step 3: Cultivate the host cells described in Step 2. (6) Cell culture and protein expression CHO cells with the transfection complex added were placed in an incubator and cultured for 3-7 days on a shaker at 36.5°C (±0.5°C) + 5% CO2, with appropriate feed added as needed.

[0074] Step 4: Collect cell culture supernatant (7) Collect cell culture supernatant When cell viability is below 50%, harvest the cell culture supernatant: centrifuge the harvested culture medium at 4500 rpm for 30 min, collect the cell culture supernatant, and filter it through a 0.22 μm filter.

[0075] Step 5: Purify the cell culture supernatant to obtain a solution of self-assembled gE nanoparticles or other protein solutions. (8) Protein purification Incubate the cell culture supernatant with AmMag™ Ni Magnetic Beads for at least 120 min, remove the supernatant, then equilibrate with 10 CV of 25 mM Tris-HCl + 300 mM NaCl (pH 7.0) buffer, and elute and collect with 25 mM Tris-HCl + 300 mM NaCl + 500 mM Imidazole (pH 7.0) buffer. Then, dialyze the collected protein three times using histidine buffer (10 mM histidine + 10% sucrose + 0.05% Tween 80; pH 6.5 ± 0.1) through a SNAKESKIN™ DIALYSIS TUBING™ dialysis bag, with each dialysis session lasting at least 2 hours.

[0076] (9) Protein purity detection Detection method (SDS-PAGE): Voltage 140~160V, electrophoresis for 45~60 minutes; staining and destaining are performed using the eStain™ L1 protein staining system. The difference between reduction electrophoresis and non-reduction electrophoresis is that a reducing agent (such as dithiothreitol) needs to be added to the electrophoresis system for reduction electrophoresis; no reducing agent is added for non-reduction electrophoresis.

[0077] Detection method (SEC-HPLC): The sample must be filtered through a 0.2 μm filter membrane before being injected into the chromatographic column. The chromatographic column is a TSKgel G3000SWxl, and the injection volume is approximately 20 μg. Relevant information is shown in Table 5 below.

[0078] Table 5. Information related to the SEC-HPLC method .

[0079] Test results: See Table 6 below. Figure 1 , Figure 2 As shown.

[0080] Table 6. Purity test results of gE nanoparticles or gE fusion proteins containing gIPP .

[0081] Note: 1. The reason why the nanoparticle band in the SDS-PAGE (non-reduction) purity (nanoparticle) spectrum is located in the sample well is: the molecular weight is extremely large, and the molecules have not moved by the end of electrophoresis, so the band is still located in the sample well.

[0082] 2. "#": When the purity (nanoparticles) of NP-1 is detected by SEC-HPLC, the software combines a small number of non-nanoparticle peaks and nanoparticle peaks into one peak.

[0083] Results analysis: Most of the monomer molecules in NP-1 self-assembled to form gE nanoparticles.

[0084] (10) Protein expression level detection Detection method: Protein concentration was detected using Nanodrop according to the UV-Vis spectrophotometry method in the General Rules of the Chinese Pharmacopoeia; then, the protein expression level was obtained by multiplying the protein concentration by the protein volume.

[0085] Test results are shown in Table 7 below.

[0086] Table 7. Concentration detection of designed gE nanoparticles or gE fusion proteins containing gIPP .

[0087] Results analysis: The protein expression levels in each experimental group were all higher than 8 mg.

[0088] (11) Particle size detection Detection method: NP-1 samples with significant differences in gIPP sequence length and high protein expression levels and purity (nanoparticles) were selected for particle size analysis. The samples were diluted to a protein concentration of 0.1~0.3 mg / ml and mixed thoroughly; particle size and PDI distribution were detected using a Malvern particle size analyzer (Zetasizer Advance).

[0089] Test results: See Table 8 below. Figure 3 As shown in (NP-1).

[0090] Table 8. Particle size determination of gE nanoparticles containing gIPP .

[0091] Results analysis: The average particle size of the self-assembled gE nanoparticles is 42 nm.

[0092] (12) Morphological (electron microscopy) detection Detection method: Microscopic morphology of NP-1 was analyzed. The sample was diluted to a protein concentration of 0.1~0.3 mg / ml; 10 μL of the diluent was dropped onto the surface of a 300-mesh carbon copper mesh and left to adhere for 10 min; then it was thoroughly rinsed with distilled water, followed by rapid staining twice with 2% sodium phosphotungstenate staining solution, 10 s each time; then the stained copper mesh was completely dried; finally, it was imaged and observed using a Hitachi HT7800 transmission electron microscope with an accelerating voltage of 80 kV.

[0093] Test results: such as Figure 4 and Figure 5 As shown.

[0094] Results analysis: Nanoparticles could be observed in NP-1 under both 40,000x and 80,000x magnification.

[0095] (13) Molecular weight (SEC-MALS) detection The molecular weight of NP-1 was determined using the SEC-MALS method.

[0096] Test results: See Table 9 below. Figure 6 As shown in (NP-1).

[0097] Table 9. Molecular weight determination of gE nanoparticles containing gIPP .

[0098] Results analysis: Most protein molecules in NP-1 exist in the form of hexadecimals.

[0099] Example 2: Preparation of vaccine (preparation of an immunogenic composition containing self-assembled gE nanoparticles and saponin QS-21) S1. Preparation of self-assembled gE virus-like nanoparticle solution using the method of Example 1. S2. Preparation of the immune enhancer saponin QS-21 solution (theoretical concentration 2 mg / ml) Accurately weigh 20 mg of saponin QS-21 (Desert King), then dissolve it completely in 5 ml of 5 mM histidine buffer solution (pH 6.5 ± 0.1), and then bring the volume up to 10 ml with the buffer solution and mix well to obtain the saponin QS-21 solution.

[0100] S3. Preparation of neutral liposomes (theoretical concentrations of dioleoylphosphatidylcholine and cholesterol are 5 mg / ml and 1.25 mg / ml, respectively). 1000 mg of dioleoylphosphatidylcholine (DOPC; Nippon Fine Chemicals Co., Ltd.) and 250 mg of cholesterol (Nippon Fine Chemicals Co., Ltd.) were accurately weighed into 20 ml volumetric flasks. DOPC and cholesterol were then dissolved in 10 ml of anhydrous ethanol, and the volume was adjusted to 20 ml. The mixture was thoroughly mixed to obtain the organic phase. 20 ml of the organic phase was injected into 180 ml of 10 mM histidine buffer solution (pH 6.5 ± 0.1; sucrose concentration 10%) to prepare the colostrum. The colostrum was then granulated using a liposome extruder to achieve a particle size of approximately 100 nm. The granulated liposomes were then ultrafiltered using a 30 kDa pore size membrane to remove residual ethanol. Finally, the liposomes were sterilized by filtration using a 0.22 μm sterilizing filter to obtain the final liposome product.

[0101] S4. Preparation of compound adjuvants Take 2 ml of liposomes, add 0.25 ml of saponin QS-21 solution, and stir well to obtain the compound adjuvant.

[0102] S5. Preparation of immunogenic compositions (vaccines) containing self-assembled gE nanoparticles. Vaccine preparation: 5ml volume; gE target concentration: 100μg / ml; saponin QS-21 target concentration: 100μg / ml; liposome components: dioleoylphosphatidylcholine and cholesterol target concentrations: 2mg / ml and 0.5mg / ml, respectively.

[0103] Add the calculated volume of gE nanoparticles, gE fusion protein, or gE solution to the adjuvant, then supplement the total volume to 5 ml with histidine solution (10 mM; pH 6.5 ± 0.1; 10% sucrose) and stir well. This is the gE nanoparticle, gE fusion protein, or gE adjuvant vaccine. The formula for calculating the target volume (ml) of added gE nanoparticles, gE fusion protein, or gE solution is: target mass (μg) of added gE nanoparticles, gE fusion protein, or gE solution / concentration (μg / ml) of gE nanoparticles, gE fusion protein, or gE solution. See Table 10 below for vaccine prescription information.

[0104] Table 10 Vaccine Prescription Information (Vaccine volume: 50 μl) .

[0105] Note: Shingrix is ​​a commercial vaccine that does not require preparation; its batch number is F7P54. " / " indicates that it is not applicable.

[0106] Example 3: Immunogenicity study of a vaccine (immunogenic composition containing self-assembled gE nanoparticles). I. Vaccination, Blood Collection, and Animal Culling Thirty-six female C57BL / 6 mice aged 6–8 weeks were randomly divided into 6 groups of 6 mice each. The total animal rearing time was approximately 100 days. The mice were then acclimatized for about one week. On day 35, each mouse was pre-immunized subcutaneously in the neck with one dose of attenuated varicella vaccine. On days 1 and 29, the mice were injected intramuscularly in the leg with the vaccine described in Example 2 (50 μl / mouse). On days 0 and 57, blood samples were collected and serum was separated. On day 57, the animals were sacrificed after blood collection, and the spleen was removed and spleen cells were separated.

[0107] II. Methods for Detecting Vaccine Immunogenicity (1) Detection method for gE-specific antibodies in serum The level of gE-specific antibody in the serum of all mice 28 days after secondary immunization was detected by indirect ELISA. Method: First, gE was coated into 96-well plates with carbonate buffer at a rate of 3 μg / well and incubated at 37°C for 60 min. Then, the plates were blocked with TPBS containing BSA and washed four times with TPBS. Next, all mouse serum was diluted at different dilutions (two-fold serial dilutions; with an initial dilution of 62,500, for a total of 8 dilutions) and added to each well at 100 μl. Samples, negative controls, and positive controls were replicated. The plates were incubated at 37°C for 1 hour, washed four times with TPBS, and then incubated with H2S ELISA. RP-labeled goat anti-mouse secondary antibody was incubated at 37°C for 1 hour, and the plate was washed 4 times with TPBS solution. Then, the plate was developed with TMB light-protected chromogenic solution for 15 minutes, and the reaction was terminated by adding 0.2M sulfuric acid. The OD value at 450 was read by an ELISA reader. The cut-off value was determined by taking 4 times the average OD value of the serum mixed sample prepared from blood collected on day 0, and the serum gE specific antibody level was calculated. The antibody GMT was also calculated (if the initial dilution of the serum sample in the vaccine group was negative in both wells, the vaccine group was assigned 1 / 4 of the minimum dilution).

[0108] (2) Detection method for gI-specific antibodies in serum Detection method: Except for the coating antigen being gI and the serum dilution factor (with 1250 times as the initial dilution factor, and a total of 8 dilutions) being different, all other methods are consistent with the "Detection Method of gE Specific Antibody in Serum".

[0109] (3) gE-specific cell-mediated immunoassay (CMI) detection method Twenty-eight days after the second immunization, the spleens of mice were harvested, and spleen single-cell suspensions were prepared. After adjusting the cell concentration, erythrocytes were lysed using erythrolysis buffer. Then, 10 μl of each cell suspension was added to trypan blue staining solution, mixed thoroughly, and counted. Based on the counting results, the cell concentration was diluted to 1×10⁻⁶ cells using T cell culture medium. 7Cells / ml; then add 100 μl to the corresponding wells of a U-shaped 96-well plate, and seal the remaining four wells with 250 μl of PBS each; stimulate cells with the peptide pool (gE peptide pool) to induce cytokine secretion; then add Containing Brefeldin A to block secretion; then, after cell live / dead staining, surface receptor FcR blocking, CD3, CD45, and CD4 surface staining, fixation and perforation, and intracellular staining of IL-2 and IFN-γ, the number of gE-specific IL2+ and / or IFN-γ+ CD4+ T cells in spleen CD4+ T cells is detected by flow cytometry.

[0110] (4) gI-specific cell-mediated immunoassay (CMI) detection method Except that the peptide pool is the gI peptide pool, the experimental method is consistent with the "gE-specific cell-mediated immunity (CMI) detection method".

[0111] III. Results of Vaccine Immunogenicity Testing (1) Detection results of gE-specific binding antibody in serum The results of the detection of gE-specific binding antibodies in serum are shown in Table 11 below. Figure 7 As shown.

[0112] Table 11 Serum gE-specific antibody levels .

[0113] Remark: # Both wells of the initial dilution of the serum sample in Group 1 (buffered) were negative, and the antibody level was labeled as "0 or <62500".

[0114] (2) Detection results of gI-specific binding antibody in serum The results of the detection of gI-specific binding antibodies in serum are shown in Table 12 below.

[0115] Table 12 Serum gI-specific antibody levels .

[0116] (3) Results of gE-specific CD4+ T cell levels (IL2+ and / or IFN-γ+) The results of gE-specific CD4+ T cell levels (IL2+ and / or IFN-γ+) detection are shown in Table 13 below. Figure 8 As shown.

[0117] Table 13 gE-specific CD4+ T cell levels (IL2+ and / or IFN-γ+) .

[0118] (4) Results of gI-specific CD4+ T cell levels (IL2+ and / or IFN-γ+) The results of gI-specific CD4+ T cell levels (IL2+ and / or IFN-γ+) detection are shown in Table 14 below. Figure 9 As shown.

[0119] Table 14 gI-specific CD4+ T cell levels (IL2+ and / or IFN-γ+) .

[0120] Finally, it should be noted that: The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An immunogenic composition, characterized in that, The immunogenic composition comprises: self-assembled gE nanoparticles, an immune enhancer, and neutral liposomes; The self-assembled gE nanoparticles are assembled by polymerization of several monomers; the monomers include the extracellular region of VZV glycoprotein E, a linker peptide, and a polypeptide containing a Th epitope in the extracellular region of VZV glycoprotein I.

2. The immunogenic composition according to claim 1, characterized in that, The amino acid sequence of the extracellular region of VZV glycoprotein E is shown in SEQ ID NO.1; the amino acid sequence of the linker peptide is SGS; the amino acid sequence of the extracellular region polypeptide of VZV glycoprotein I containing Th epitopes is shown in SEQ ID NO.3; and the amino acid sequence of the monomer is shown in SEQ ID NO.

4.

3. The immunogenic composition according to claim 2, characterized in that, The Th epitopes include at least Th epitope 1, Th epitope 2 and Th epitope 3; the amino acid sequence of Th epitope 1 is FCFRSVQVIRYDGCPRIRTS; the amino acid sequence of Th epitope 2 is RYDGCPRIRTSAFISCRYKH; and the amino acid sequence of Th epitope 3 is TSAFISCRYKHSWHYGNSTD.

4. The immunogenic composition according to claim 1, characterized in that, The immune enhancer is saponin QS-21; the neutral liposomes include dioleoylphosphatidylcholine and cholesterol; the ratio of dioleoylphosphatidylcholine to cholesterol is 2:1 to 8:

1.

5. The immunogenic composition according to claim 4, characterized in that, The concentration of self-assembled gE nanoparticles in the composition is 5–400 μg / ml, the concentration of the immune enhancer saponin QS-21 is 25–200 μg / ml, the concentration of dioleoylphosphatidylcholine is 500–8000 μg / ml, and the concentration of cholesterol is 62.5–4000 μg / ml.

6. A method for preparing the immunogenic composition according to any one of claims 1 to 5, characterized in that, Includes the following steps: S1. Preparation of self-assembled gE nanoparticle solution; S2. Prepare the immune enhancer saponin QS-21 solution; S3. Preparation of neutral liposomes; S4. Prepare a compound adjuvant by taking neutral liposomes and the immune enhancer saponin QS-21 solution; S5. Immunogenic compositions were prepared by taking the assembled gE nanoparticle solution and the composite adjuvant.

7. The method for preparing the immunogenic composition according to claim 6, characterized in that, The specific method for step S1 is as follows: Step 1: Construct an expression vector or viral vector, wherein the expression vector or viral vector contains a nucleic acid sequence encoding the monomer; Step 2: Transform the constructed expression vector into host cells or infect host cells with the constructed viral vector; Step 3, the cultured host cells; Step 4: Collect the cell culture supernatant and / or the supernatant of cell lysates; Step 5: Purify the cell culture supernatant and / or the supernatant of cell lysate to obtain a self-assembled gE nanoparticle solution.

8. The application of the immunogenic composition according to any one of claims 1 to 5 or the immunogenic composition prepared by the method according to claim 6 or 7, characterized in that, VZV vaccine is used to prevent and / or improve varicella and / or herpes zoster and / or postherpetic neuralgia.

9. A VZV vaccine, characterized in that, This includes the immunogenic composition according to any one of claims 1 to 5 or the immunogenic composition prepared by the method described in claim 6 or 7.

10. The VZV vaccine according to claim 9, characterized in that, It also contains other pharmaceutically acceptable excipients.

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