Immunogenic composition containing composite adjuvant as well as preparation method and application of immunogenic composition

Abstract

The invention discloses an immunogenic composition containing a composite adjuvant as well as a preparation method and application of the immunogenic composition. The immunogenic composition comprises a gE-gI fusion protein, an immunopotentiator, namely, saponin QS-21, and a neutral liposome, the gE-gI fusion protein comprises a VZV gE, a connecting peptide and a VZV gI. The immunogenic composition provided by the invention only contains one immunopotentiator, can be applied to VZV vaccines, and solves the problems of relatively weak gE immunogenicity and relatively large clinical side reaction of the vaccines prepared in the prior art. The immunogenicity of the vaccine is equivalent to that of the Xinanlii, but the vaccine contains fewer immunopotentiators and has lower clinical side effects; in addition, the vaccine can also induce a gI specific antibody and 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] This invention relates to the field of biomedical technology, specifically to an immunogenic composition containing a compound adjuvant, its preparation method, and its application. Background Technology

[0002] Varicella-zoster virus (VZV) is an alpha herpesvirus that infects humans, causing diseases such as varicella, herpes zoster, and postherpetic neuralgia (PHN). The VZV genome is approximately 125 kb, encoding 67 proteins, including envelope glycoproteins such as gB, gC, gE, gH, gI, and gL. Glycoprotein E is the most abundant and immunogenic glycoprotein on the VZV envelope and host cell membrane, containing numerous B-cell and T-cell epitopes. Glycoprotein I is also relatively abundant on the VZV envelope, exhibiting strong immunogenicity and containing confirmed Th (T helper) cell epitopes.

[0003] GlaxoSmithKline's recombinant protein shingles vaccine, Shingrix ® (Shingrix) ®This vaccine was approved by the FDA in 2017 for the prevention of shingles in adults aged 50 and older and immunocompromised adults aged 18 and older. It induces a higher gE-specific CMI (Cell-Mediated Immunity) response (inducing a higher number of gE-specific IL2+ and / or IFN-γ+CD4+ T cells) and gE-specific antibody levels in humans compared to live attenuated vaccines. The vaccine consists of the extracellular domain of glycoprotein E (gE) and the AS01B adjuvant system. The AS01B adjuvant system (compound adjuvant) contains 50 μg of the immunostimulant / adjuvant 3-O-deacylated-4'-monophospholipid A (3D-MPLA), 50 μg of the immunostimulant / adjuvant saponin QS-21, 1000 μg of dioleoylphosphatidylcholine (DOPC), and 250 μg of cholesterol. Saponin QS-21 can activate the NLRP3 (NLR family Pyrin domain protein 3) inflammasome, thereby releasing caspase-1-dependent cytokines IL-1β and IL-18, thus promoting Th17 cell maturation or driving INF-γ-mediated Th1 responses. 3D-MPLA, through interaction with TLR-4, activates MyD88 (myeloid differentiation factor 88) and TRIF (a TIR domain-inducing β-interferon adapter)-dependent signaling pathways, inducing the production of inflammatory cytokines and interferons, thereby enhancing the immune response. Compared to the live attenuated vaccines from Merck and Brno, GSK's Shingrix... ® It has a higher protection rate, but a higher rate of serious adverse reactions in clinical practice.

[0004] GSK announced Shingrix in 2017. ® A clinical review; the review discloses: Shingrix ® After injection into rabbits, it elicits high levels of the inflammatory marker C-reactive protein (CRP), up to 9 times higher in males and 5 times higher in females. High CRP levels indicate the initiation of an acute phase transition response and an increased systemic inflammatory response, which may be associated with clinical side effects such as lethargy, fatigue, and nausea. A 2025 study published in *Vaccine* indicated that vaccine side effects (tolerance) have become a major concern for Americans receiving Shingrix. ® The core obstacle is that the vaccination rate for this vaccine in people aged 50 and older (17%) is significantly lower than that of the influenza vaccine (47.4% in people aged 50-64; 71.3% in people aged 65 and older) and the pneumococcal vaccine (69% in people aged 65 and older). In my country, Shingrix... ® Side effects also have a significant negative impact on vaccination rates.

[0005] Shin An Li Shi ® The high incidence of clinical adverse reactions and serious adverse reactions (Grade 3 AEs) is mainly due to the use of two immune enhancers (saponin QS-21 and 3D-MPLA). The use of two immune enhancers / adjuvants may overactivate the immune system, leading to a large release of inflammatory factors and enhancing local or systemic inflammatory responses, thereby causing clinical adverse reaction symptoms such as fever, fatigue, and muscle aches. The nuvaxovid COVID-19 vaccine uses 50 μg of saponin adjuvant (QS-21 + QS-7) per dose. ® The clinical side effects were significantly lower with Shingrix using 50 μg of saponin adjuvant (QS-21) and 50 μg of 3D-MPLA. ® Detailed comparison information is shown in the table below.

[0006] Table 1. COVID-19 vaccine nuvaxovid ® Hexin Anglis ® Comparative information on clinical side effects

[0007] Current patents regarding the preparation of immunogenic compositions still have shortcomings: Patent CN112870344A improves the immunogenicity of gE by fusing expression of gE and the Fc terminus of human IgG, but carries the risk of inducing anti-Fc antibodies in humans; Patent CN114621356A greatly improves the immunogenicity of gE by fusing expression of gE with cytokine IL18 and the Fc terminus of human IgG, but carries the risk of inducing cytokine storm and anti-Fc antibodies; Patent WO2023 / 125976 A1 also provides a method for fusing expression of antigen and cytokine, the Fc terminus of human IgG, Padre universal Th epitope, and peptides binding to immune cell surface proteins, improving the immunogenicity of the antigen, but still carries the risk of inducing cytokine storm and anti-Fc antibodies; Patent 117003896A combines gE with Padre universal Th epitope, TT (tetanus toxin) P2... Th epitope fusion expression enhances the immunogenicity of gE, but it can only increase gE-specific CMI and cannot stimulate non-gE VZV-specific CMI. Patent CN117100850A enhances the immunogenicity of gE and VZV-specific CMI levels by fusing gE with multiple predicted Th epitopes of gB, gC, gH, gI, gK, gL, gM, and gN, but most of the predicted Th epitopes have not been confirmed by research, and the immune enhancement ability is weak.

[0008] Currently, clinical practice requires drugs that can induce high levels of VZV-specific CMI and gE-specific antibodies in the human body, and are also more effective than Shingrix. ®A new generation of vaccines with fewer side effects. Therefore, using only one immunostimulant (e.g., only 50 μg of QS-21 or 3D-MPLA) or significantly reducing the dosage of both immunostimulants (e.g., reducing both QS-21 and 3D-MPLA to 25 μg), while increasing Th epitopes in the antigen to improve VZV-specific CMI and gE-specific antibody levels, is an ideal technical route for developing next-generation vaccines. Th epitopes, such as Padre and / or P2, when fused with the antigen, have been shown to effectively activate Th cells (CD4+ T cells) and enhance the immunogenicity of the antigen.

[0009] Therefore, this application is hereby submitted. Summary of the Invention

[0010] The purpose of this invention is to provide an immunogenic composition containing a compound adjuvant, its preparation method, and its application, in order to solve the problem of significant clinical side effects in existing vaccines. To achieve the above objective, this invention provides the following technical solution: In a first aspect, the present invention provides an immunogenic composition containing a complex adjuvant, the immunogenic composition comprising: gE-gI fusion protein, the immune enhancer saponin QS-21, and neutral liposomes.

[0011] In some embodiments, the amino acid sequence of the gE-gI fusion protein is shown in SEQ ID NO.3; wherein the gE-gI fusion protein comprises VZV gE, a linker peptide, and VZV gI; the VZV gE is the extracellular region of VZV glycoprotein E, and its amino acid sequence is shown in SEQ ID NO.1; the VZV gI is the extracellular region of VZV glycoprotein I containing Th epitopes, and its amino acid sequence is shown in SEQ ID NO.2; the amino acid sequence of the linker peptide is SGS.

[0012] Furthermore, the aforementioned gE-gI fusion protein may contain various purification tags that facilitate protein purification; further, the purification tags include, but are not limited to, any one or more combinations 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 located at the N-terminus and / or C-terminus of the gE-gI fusion protein, preferably the C-terminus; further, when the purification tag is a 6XHis tag and located at the C-terminus of the gE-gI fusion protein, the amino acid sequence is as shown in SEQ ID NO.4, and the DNA sequence encoding the amino acid sequence of the gE-gI fusion protein includes, but is not limited to, SEQ ID NO.5.

[0013] The Th epitopes include at least Th epitope 1 (eptope ID: 839316), Th epitope 2 (eptope ID: 2224791), Th epitope 3 (eptope ID: 1597827), and Th epitope 4 (eptope ID: 840141); wherein the amino acid sequence of Th epitope 1 is FCFRSVQVIRYDGCPRIRTS; the amino acid sequence of Th epitope 2 is RYDGCPRIRTSAFISCRYKH; the amino acid sequence of Th epitope 3 is TSAFISCRYKHSWHYGNSTD; and further, the amino acid sequence of Th epitope 4 is PGINDAGVYVLLVRLDHSRS.

[0014] In this scheme, Th epitope 1 is located at positions AA82–AA101 in the VZV glycoprotein I (gI) sequence, and its amino acid sequence is FCFRSVQVIRYDGCPRIRTS. Furthermore, Th epitope 1 was confirmed by Ying Ying Kong in 2016 as a restriction epitope of HLA-DRB1*03:01, HLA-DRB1*13:01, HLA-DRB3*03:01, and HLA-DRB3*02:02, with IDs (IEDB IDs) of 2884824, 2884827, 2884826, and 2884825, respectively. Further, Th epitope 1 was confirmed by Ying Ying Kong in 2017 as an HLA-DRB3*01:01 and HLA-DR restriction epitope, with IDs (IEDB IDs) of 2884824, 2884827, 2884826, and 2884825, respectively. The IDs are 3497311 and 3497310, respectively; furthermore, the Th epitope 1 has been confirmed by Kerry J. Laing in 2019 as an HLA-DRB3*03:01 restrictive epitope, and its ID (IEDB ID) in the Immunotope Database (IEDB) is 5003302.

[0015] Th epitope 2 is located at positions AA91–AA110 in the VZV gI sequence, and its amino acid sequence is RYDGCPRIRTSAFISCRYKH. Furthermore, Th epitope 2 has been confirmed as a Th epitope by Kerry J. Laing in 2020, and its ID (IEDB ID) in the Immunotope Database (IEDB) is 8326833.

[0016] Furthermore, the Th epitope 3 is located at positions AA100 to AA119 in the VZV gI sequence, and its amino acid sequence is TSAFISCRYKHSWHYGNSTD; furthermore, the Th epitope 3 has been confirmed by Ying Ying Kong in 2017 as an HLA-DPB1*02:01 restrictive Th epitope, and its ID (IEDB ID) in the Immunotope Database (IEDB) is 3497348.

[0017] Further, the Th epitope 4 is located at positions AA136–AA155 in the VZV gI sequence, and its amino acid sequence is PGINDAGVYVLLVRLDHSRS; further, the Th epitope 4 was confirmed by Ying Ying Kong in 2016 as an HLA-DRB1*13:01 and HLA-DRB1*07:01-restricted Th epitope, with IDs (IEDB IDs) of 2884836 and 2884837 in the Immunotope Database (IEDB); further, the Th epitope 4 was confirmed by Ying Ying Kong in 2017 as an HLA-DRB1*11:01 and HLA-DRB4-restricted Th epitope, with IDs (IEDB IDs) of 3497330 and 3497331 in the Immunotope Database (IEDB); further, the Th epitope 4 has been confirmed by Kerry J. Laing was confirmed in 2019 as an HLA-DRB4*01:01 restricted Th epitope, with an ID (IEDB ID) of 5003315 in the Immunotope Database (IEDB).

[0018] In some embodiments, the neutral liposomes are composed of dioleoylphosphatidylcholine (DOPC) and cholesterol; wherein the ratio of DOPC to cholesterol is 2:1 to 8:1.

[0019] In some embodiments, the concentration of gE-gI fusion protein in the immunogenic composition of the compound adjuvant is 5-400 μg / ml, the concentration of the immunostimulant saponin QS-21 is 25-200 μg / ml, the concentration of dioleoylphosphatidylcholine (DOPC) is 500-8000 μg / ml, and the concentration of cholesterol is 62.5-4000 μg / ml.

[0020] Secondly, the present invention also provides a method for preparing an immunogenic composition containing a complex adjuvant, comprising the following steps: S1, prepare gE-gI fusion protein solution; S2, prepare the immune enhancer saponin QS-21 solution; S3, preparation of neutral liposomes; S4. Take a certain amount of the neutral liposomes prepared in step S3 and the saponin QS-21 solution prepared in step S2 and mix them evenly to prepare a composite adjuvant. S5, Prepare an immunogenic composition containing a compound adjuvant.

[0021] Further, the protein concentration in the gE-gI fusion protein solution in step S1 is 10–50,000 μg / ml; further, the gE-gI fusion protein solution contains a buffer solution; further, the buffer solution includes, but is not limited to, any one or more combinations of the following: phosphate buffer, histidine buffer, glycine buffer, citrate buffer, acetate buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, and 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer.

[0022] Further, the preparation of the gE-gI fusion protein solution described in S1 includes, but is not limited to, the following steps: Step 1 (constructing an expression vector or viral vector containing a nucleic acid sequence encoding the above-mentioned monomer); Step 2 (transforming the expression vector obtained in Step 1 into host cells or infecting host cells with the viral vector obtained in Step 1); Step 3 (culturing the host cells described in Step 2); Step 4 (collecting the cell culture supernatant and / or the supernatant of cell lysates); Step 5 (purifying the cell culture supernatant and / or the supernatant of cell lysates to obtain the gE-gI fusion protein solution).

[0023] Furthermore, the expression vector described in step 1 is a eukaryotic expression vector.

[0024] Further, the expression vector described in step 2 is transformed into host cells, and the host cells include, but are not limited to: CHO cells, yeast cells, and HEK293 cells; further, CHO cells are preferred.

[0025] Furthermore, the CHO cells include, but are not limited to, cell lines such as CHO-S, CHO-K1, and CHO-DG44.

[0026] Furthermore, the yeast cells include, but are not limited to, yeast cells such as Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha.

[0027] Further, the viral vector in step 1 includes baculovirus vector, lentivirus vector, adenovirus vector, retrovirus vector, adeno-associated virus vector, herpes simplex virus vector, and parainfluenza virus vector; further, baculovirus vector is preferred.

[0028] Furthermore, when the viral vector infects the host cell in step 2, the host cell includes, but is not limited to, insect cells and HEK293 cells; insect cells are further preferred.

[0029] Furthermore, the insect cells include, but are not limited to, Sf9 cells, Sf21 cells, and Hi5 cells.

[0030] Furthermore, the viral vector described in step 2 infects the host cell. The viral vector includes, but is not limited to, baculovirus vectors, adenovirus vectors, adeno-associated virus vectors, and lentivirus vectors. More preferably, baculovirus vectors are used.

[0031] Furthermore, in step 3, if the host cell is a host cell transformed by the expression vector, the host cell can be either a polyclonal cell line or a monoclonal cell line, with a monoclonal cell line being preferred.

[0032] Further, if the host cell in step 3 is a host cell transformed by the expression vector, and is transformed into a stable transgenic cell (using a resistance gene and / or gene with a eukaryotic selection marker to screen the transformed host cell) or a transient transgenic cell (not using a resistance gene and / or gene with a eukaryotic selection marker to screen the transformed host cell), the host cell includes CHO cells, yeast cells, and HEK293 cells; further, the host cell is preferably a CHO cell.

[0033] Furthermore, the cell culture supernatant and / or cell lysate supernatant in step 4 contain substances such as gE-gI fusion protein and residual culture medium components, and may contain substances such as host cell debris, host cell proteins, host cell nucleic acids, and host cell metabolites; furthermore, the cell lysate supernatant is obtained by lysing host cells and then centrifuging or other methods.

[0034] Further, the purification method for the cell culture supernatant and / or cell lysate supernatant in step 5 includes, but is not limited to, any one or a combination of the following methods: precipitation, chromatography, dialysis, ultrafiltration, centrifugation, nanofiltration, and microfiltration; further, the chromatography includes, but is not limited to, any one or a combination of the following chromatography methods: ion exchange chromatography, gel filtration chromatography, affinity chromatography, hydrophobic chromatography, and reversed-phase chromatography; further, the chromatography also includes composite mode chromatography, which includes a composite mode of ion exchange chromatography and / or gel filtration chromatography and / or affinity chromatography and / or hydrophobic chromatography and / or reversed-phase chromatography.

[0035] Further, the gE-gI fusion protein solution described in step 5 is characterized in that its purity (target protein; reducing SDS-PAGE method) is: not less than 30.0% to not less than 99.9%, such as not less than 30%, not less than 40%, not less than 50%, not less than 60%, not less than 70%, not less than 80%, not less than 90%, not less than 95%, not less than 96%, not less than 97%, not less than 98%, not less than 99%, or not less than 99.9%.

[0036] Furthermore, the immune enhancer saponin QS-21 solution prepared as described in S2 is derived from plant extracts or artificial synthesis.

[0037] Furthermore, the immune enhancer saponin QS-21 is derived from a natural plant extract, and the raw material used for the extraction comes from the South American soapberry tree (scientific name: Quillaja saponaria).

[0038] Further, in step S2, the concentration of the prepared immune enhancer saponin QS-21 solution is 50–10000 μg / ml.

[0039] Further, the neutral liposomes in step S3 are composed of dioleoylphosphatidylcholine (DOPC) and cholesterol; further, the ratio of dioleoylphosphatidylcholine (DOPC) to cholesterol is 2:1 to 8:1.

[0040] Further, the neutral liposomes described in step S3 have a dioleoylphosphatidylcholine (DOPC) concentration of 1000–16000 μg / ml and a cholesterol concentration of 125–8000 μg / ml.

[0041] Furthermore, the compound adjuvant described in step S4 contains the immune enhancer saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol.

[0042] Further, the immunogenic composition described in step S5 contains 5–400 μg / ml of gE-gI fusion protein, 25–200 μg / ml of the immune enhancer saponin QS-21, 500–8000 μg / ml of dioleoylphosphatidylcholine (DOPC) and 62.5–4000 μg / ml of cholesterol.

[0043] Thirdly, the present invention provides an application of an immunogenic composition containing a compound adjuvant for the prevention and / or improvement of varicella-zoster virus vaccines for varicella and / or herpes zoster and / or postherpetic neuralgia.

[0044] A varicella-zoster virus vaccine, comprising the above-mentioned immunogenic composition containing a compound adjuvant or the above-prepared immunogenic composition; the varicella-zoster virus vaccine further comprises other pharmaceutically acceptable excipients; further, the other excipients include, but are not limited to, any one or more combinations 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.

[0045] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects: This invention provides an immunogenic composition comprising a gE-gI fusion protein, the immune enhancer saponin QS-21, and neutral liposomes. This immunogenic composition can be applied to VZV vaccines, solving the technical problems of weak gE immunogenicity and severe vaccine side effects in existing vaccine preparation techniques.

[0046] The serum gE-specific antibody levels and gE-specific CMI response (splenic cells) in mice immunized twice with this vaccine were similar to those of Shingrix. ® (Prior technology) is comparable; the immunogenicity of the gE-gI fusion protein is higher than that of gE, thus reducing the types and dosages of immunostimulants in the vaccine (this vaccine contains only 50 μg of the immunostimulant saponin QS-21; Shingrix). ® Containing 50 μg of the immune enhancer saponin QS-21 and 50 μg of the immune enhancer 3D-MPLA, this vaccine can reduce the inflammatory response induced by the immune enhancer, thereby reducing clinical side effects. Furthermore, this vaccine can induce significant gI-specific antibody and gI-specific CMI responses, which is beneficial for further improving vaccine efficacy.

[0047] In short, with Xin An Li Shi ® Compared to other vaccines, the VZV vaccine provided by this invention has better clinical application potential. Therefore, the immunogenic composition provided by this invention has good clinical application potential! Attached Figure Description

[0048] 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.

[0049] 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.

[0050] Figure 2 This is the SEC-HPLC detection chromatogram of the gE-gI fusion protein in Example 1 of the present invention.

[0051] Figure 3 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.

[0052] Figure 4 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. Detailed Implementation

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] Example 1: Preparation of gE-gI fusion protein solution Design gE-gI fusion protein The amino acid sequence information of VZV gE and gI is shown in Table 2.

[0059] Table 2 VZV gE and gI sequence information

[0060] II. gI Th Tables (IEDB Table Database) The amino acid sequence information of the selected gI Th epitopes is shown in Table 3.

[0061] Table 3. Amino acid sequence information of the selected gI Th epitopes.

[0062] Note: Th epitopes 1, Th epitopes 2, Th epitopes 3 and Th epitopes 4 have been confirmed as Th epitopes.

[0063] III. Selected gI or gI polypeptides containing gI Th epitopes CN117100850A has disclosed that gI2 can enhance the immunogenicity of gE.

[0064] Table 4. Selected gI or gI peptides containing gI Th epitopes

[0065] IV. Expression of the experimental protein Step 1: Construct an expression vector containing a nucleic acid sequence encoding the gE-gI fusion protein or other experimental proteins. (1) Design gE fusion proteins containing gI or gI peptides and other experimental proteins. Relevant information is shown in Table 5. Table 5 Information on the gE-gI fusion protein

[0066] Note: N / A means not applicable.

[0067] (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 DNA sequence encoding the fusion protein, 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' and 3' ends (3' end of the positive strand), respectively, of the codon-optimized DNA sequence. 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 protective bases were added to the 3' end of the stop codon (TGA). Finally, the entire DNA sequence was synthesized. The amino acid sequence of the gE-gI fusion protein containing a 6×His tag (the 6X His tag is located at the C-terminus of the fusion protein) is shown in SEQ ID NO.4, and the DNA sequence is shown in SEQ ID NO.5.

[0068] (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).

[0069] (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.

[0070] Collect 45 ml of overnight (12-16 h) bacterial culture into a labeled 50 ml centrifuge tube; centrifuge at 8000 rpm for 4 min using an angle rotor centrifuge or at 4500 rpm for 15 min using a horizontal rotor centrifuge, then discard the supernatant and retain the bacterial cells.

[0071] Add 10 ml of Buffer P1 (containing RNase A) to the centrifuge tube and vortex for 6 minutes until no obvious clumps of bacteria are visible.

[0072] Add 10 ml of Buffer P2 to the centrifuge tube (if SDS precipitates, preheat in a 37℃~42℃ water bath until no reagent precipitates), then immediately rotate gently 6 times manually or mix for 60 seconds using a mixer.

[0073] Add 10 ml of Buffer P3 to the centrifuge tube and immediately invert it manually 5 to 15 times or mix it on a mixer for 90 seconds. After a white flocculent precipitate appears, centrifuge at 11,000 rpm for 4 minutes in an angle rotor centrifuge or at 4,500 rpm for 4 minutes in a horizontal rotor centrifuge.

[0074] Add 10 ml of ER Buffer to a 50 ml centrifuge tube, manually invert the tube 3-5 times to mix thoroughly, or place it on a mixer to mix for 10 seconds, then filter to remove the white precipitate and retain the liquid.

[0075] Before the filtration and loading step, add 25 ml of QBT (equilibration buffer; its components are: 10 mM Tris-HCl, 60% isopropanol, 1 mM EDTA) reagent to the chromatography column, which is placed on a centrifuge rack and filled with packing material, to equilibrate the packing material.

[0076] Add the ER buffer-treated liquid to the chromatography column; after the liquid has finished dripping, add 60 ml of QC (wash buffer; its components are: 0.85% sodium chloride, 60% isopropanol, 10 mM Tris-HCl and 1 mM EDTA) to the chromatography column and allow it to elute by gravity; after the QC has finished dripping, place the chromatography column into a labeled 50 ml centrifuge tube, add 10 ml of Elution Buffer, and allow it to elute by gravity; after elution, gently press the chromatography column against the centrifuge tube wall and then quickly remove it, add 7 ml of isopropanol to the collected filtrate, invert and mix 3-5 times, and centrifuge at 11000 rpm for 15 min in an angle rotor centrifuge or at 4500 rpm for 15 min in a horizontal centrifuge; after centrifugation, gently discard the supernatant, and add 5 ml of 75% ethanol to each centrifuge tube; after thoroughly rinsing the precipitate, centrifuge at 11000 rpm for 10 min in an angle rotor centrifuge or at 3700 rpm for 10 min in a horizontal centrifuge.

[0077] After centrifugation, gently discard the supernatant and invert the centrifuge tubes onto a piece of paper in ascending order of size. Add 50–500 μl of deionized water to the location of the plasmid and agitate 5–25 times to fully dissolve the plasmid in the water. Use a pipette to transfer the dissolved solution into the corresponding microporous filter column according to the tube number. Then, centrifuge at 14,000 rpm for 15–30 min in an angle rotor centrifuge. After centrifugation, transfer the plasmid into the corresponding EP tube according to the microporous filter column number.

[0078] Place 1.5ml EP tubes on a 96-well aliquot plate for plasmid aliquoting.

[0079] The plasmid concentration was detected using an enzyme-linked immunosorbent assay (ELISA) reader.

[0080] Step 2: Transform the expression vector obtained in Step 1 into host cells. (5) Transfection of CHO cells with expression vector (plasmid) The specific steps for transfecting CHO cells with the expression plasmid are as follows: Cell expansion: CHO cells were expanded at a rate of 0.2~0.4×10⁻⁶. 6 Cells were seeded at a density of 100 cells / ml in shake flasks containing serum-free medium and cultured at 36.5°C (±0.5°C) + 5% CO2 on a shaker in an incubator. Preparation of cells to be transfected: The day before transfection, CHO cells are seeded into Erlenmeyer flasks at an appropriate density for culture. Plasmid preparation: On the day of transfection, add an appropriate amount of expression vector (plasmid) to the buffer and mix well; Preparation of transfection complex: The transfection reagent is mixed with the plasmid in ③ according to the preferred ratio and incubated to form a transfection complex; Add transfection complex: After incubation, add the liquid from step ④ to the CHO cells to be transfected.

[0081] 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.

[0082] 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.

[0083] Step 5: Purify the cell culture supernatant to obtain gE fusion protein solution or other protein solution. (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.

[0084] (9) Protein purity detection Detection method (SDS-PAGE): Electrophoresis at 140-160V for 45-60 minutes. Stop electrophoresis after bromophenol blue reaches the bottom of the separating gel and remove the gel. Stain and destain using the eStain™ L1 protein staining system. The difference between reduction electrophoresis and non-reduction electrophoresis is that a reducing agent (e.g., dithiothreitol) needs to be added to the electrophoresis system for reduction electrophoresis; no reducing agent is added for non-reduction electrophoresis. Scan the gel and save the gel pattern.

[0085] 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. Information regarding the SEC-HPLC method is shown in Table 6.

[0086] Table 6. Information related to the SEC-HPLC method

[0087] Test results: as shown in Table 7. Figure 1 , Figure 2 As shown.

[0088] Table 7. Purity test results of gE-gI fusion protein or gE or gI

[0089] Note: The reason why the nanoparticle bands in the SDS-PAGE (non-reduction) purity (nanoparticle) spectrum are located in the sample wells is that the molecular weight is extremely large, and the molecules have not moved by the end of electrophoresis, so the bands are still located in the sample wells.

[0090] Results analysis: The vast majority of gE-gI fusion protein molecules exist in the form of monomers or oligomers.

[0091] (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.

[0092] Test results are shown in Table 8.

[0093] Table 8. Concentration detection of the designed gE fusion protein or gE or gI

[0094] Results analysis: The protein expression levels in each experimental group were all above 18 mg.

[0095] Example 2: Preparation of a vaccine (preparation of an immunogenic composition for the gE-gI fusion protein) S2, Prepare an immune enhancer solution of saponin QS-21 (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.

[0096] 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.

[0097] S4. Take a certain amount of the neutral liposomes prepared in step S3 and add them to the saponin QS-21 solution in step S2 to prepare a compound adjuvant; take 2 ml of neutral liposomes, add 0.25 ml of saponin QS-21 solution, and stir evenly to obtain the compound adjuvant.

[0098] S5, Prepare an immunogenic composition (vaccine) containing the gE-gI fusion protein. Vaccine preparation [5ml volume; target gE concentration of 100μg / ml; target concentration of saponin QS-21 of 100μg / ml; liposome components: target concentrations of dioleoylphosphatidylcholine and cholesterol of 2mg / ml and 0.5mg / ml, respectively] Add the calculated volume of gE-gI 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-gI fusion protein or gE adjuvant vaccine. The formula for calculating the target volume (ml) of gE-gI fusion protein or gE solution added is: target mass of gE-gI fusion protein or gE added (μg) / concentration of gE-gI fusion protein or gE solution (μg / ml). Vaccine prescription information is shown in Table 9.

[0099] Table 9 Vaccine Prescription Information (Vaccine volume: 50 μl)

[0100] Note: Xin An Li Shi ® This is a commercial vaccine, no preparation is required, batch number F7P54; " / " indicates not applicable.

[0101] Example 3: Immunogenicity study of the vaccine (immunogenic composition containing gE fusion protein) I. Vaccination, Blood Collection, and Animal Culling Thirty female C57BL / 6 mice aged 6–8 weeks were randomly divided into 5 groups of 6 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 day 1 and day 29, the mice were injected intramuscularly in the leg with the vaccine described in Case 2 (50 μl / mouse). On day 0 and day 57, blood samples were collected and serum was separated. On day 57, the animals were sacrificed after blood collection, the spleen was removed, and spleen cells were separated.

[0102] 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).

[0103] (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".

[0104] (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. 7 Cells / 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.

[0105] (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".

[0106] III. Results of Vaccine Immunogenicity Testing (1) Detection results of gE-specific binding antibody in serum Table 10 shows the results of the detection of gE-specific binding antibodies in serum. Figure 3 As shown.

[0107] Table 10 Serum gE-specific antibody levels

[0108] Note: " / " indicates that it was not detected; # 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".

[0109] (2) Detection results of gI-specific binding antibodies in serum, as shown in Table 11. Table 11 Serum gI-specific antibody levels

[0110] (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+) are shown in Table 12 and Figure 4 As shown.

[0111] Table 12 gE-specific CD4+ T cell levels (IL2+ and / or IFN-γ+)

[0112] (4) Results of gI-specific CD4+ T cell levels (IL2+ and / or IFN-γ+), as shown in Table 13. Table 13 gI-specific CD4+ T cell levels (IL2+ and / or IFN-γ+)

[0113] 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 containing a compound adjuvant, characterized in that, The immunogenic composition comprises: gE-gI fusion protein, the immune enhancer saponin QS-21, and neutral liposomes.

2. The immunogenic composition containing a compound adjuvant according to claim 1, characterized in that, The amino acid sequence of the gE-gI fusion protein is shown in SEQ ID NO.3; the gE-gI fusion protein includes VZV gE, a linker peptide, and VZV gI; the VZV gE is the extracellular region of VZV glycoprotein E, and its amino acid sequence is shown in SEQ ID NO.1; the VZV gI is the extracellular region of VZV glycoprotein I containing Th epitopes, and its amino acid sequence is shown in SEQ ID NO.2; the amino acid sequence of the linker peptide is SGS.

3. The immunogenic composition containing a compound adjuvant according to claim 2, characterized in that, The Th epitopes include at least Th epitope 1, Th epitope 2, Th epitope 3, and Th epitope 4; wherein the amino acid sequence of Th epitope 1 is FCFRSVQVIRYDGCPRIRTS; the amino acid sequence of Th epitope 2 is RYDGCPRIRTSAFISCRYKH; the amino acid sequence of Th epitope 3 is TSAFISCRYKHSWHYGNSTD; and the amino acid sequence of Th epitope 4 is PGINDAGVYVLLVRLDHSRS.

4. The immunogenic composition containing a compound adjuvant according to claim 1, characterized in that, The neutral liposomes are composed of dioleoylphosphatidylcholine and cholesterol; the ratio of dioleoylphosphatidylcholine to cholesterol is 2:1 to 8:

1.

5. The immunogenic composition containing a compound adjuvant according to claim 4, characterized in that, The concentration of the gE-gI fusion protein is 5–400 μg / ml, the concentration of the immune enhancer saponin QS-21 is 25–200 μg / ml, the concentration of the dioleoylphosphatidylcholine is 500–8000 μg / ml, and the concentration of the cholesterol is 62.5–4000 μg / ml.

6. A method for preparing an immunogenic composition containing a compound adjuvant, characterized in that, Includes the following steps: S1, Preparation of gE-gI fusion protein solution S2, prepare the immune enhancer saponin QS-21 solution; S3, preparation of neutral liposomes; S4. Take a certain amount of the neutral liposomes prepared in step S3 and the saponin QS-21 solution prepared in step S2 and mix them evenly to prepare a composite adjuvant. S5, Prepare an immunogenic composition containing a compound adjuvant.

7. The application of an immunogenic composition containing a compound adjuvant, characterized in that, A varicella-zoster virus vaccine used to prevent and / or improve varicella and / or shingles and / or postherpetic neuralgia.

8. A varicella-zoster virus vaccine, characterized in that, The varicella-zoster virus vaccine comprises the immunogenic composition containing a compound adjuvant as described in claims 1-5 or the immunogenic composition prepared in claim 6; the varicella-zoster virus vaccine further comprises other pharmaceutically acceptable excipients.

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