Adjuvant composition and application thereof in preparation of varicella vaccine

Abstract

The invention relates to an adjuvant composition and application thereof in preparation of a varicella vaccine, and provides a novel adjuvant composition which can induce an organism to generate a remarkable high-titer specific IgG antibody, enhance humoral immunity and induce strong cellular immunity, and particularly has a good application prospect for development of the varicella vaccine.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and in particular, to an adjuvant composition and its application in the preparation of varicella vaccine. Background Art

[0002] Adjuvants are non-specific immunomodulators, which refer to auxiliary substances that can enhance the body's immune response to antigens or change the type of immune response when injected into the body together with antigens or in advance. Adjuvants can be immunogenic or non-immunogenic. There are many types of adjuvants, and there is no unified classification method. The immunobiological effects of adjuvants are to enhance immunogenicity, increase the titer of antibodies, change the type of antibodies produced, and cause or enhance delayed hypersensitivity reactions, but the mechanism of action of adjuvants is not yet fully understood, and the mechanisms of action of different adjuvants are also different.

[0003] Aluminum adjuvants have been used as immune adjuvants for a long time, and their safety and immune-enhancing effects have long been proven. However, aluminum adjuvants lack or have only a weak adjuvant effect on some candidate vaccine antigens. Although they can enhance humoral immune responses, they have no strengthening effect on cellular immunity, and there is no obvious effect of enhancing immune responses in many human vaccines. Children's immune systems are still developing, and their responses to vaccines are different from those of adults, especially newborns and infants, whose kidneys have a weak ability to excrete aluminum. Therefore, with the emergence of more and more candidate vaccines, the demand for new clinically available adjuvants, especially vaccine adjuvants for children, is growing rapidly.

[0004] Chickenpox is an acute infectious disease caused by the varicella-zoster virus, which usually presents symptoms of systemic macules and papules. Children are more susceptible to infection. Most patients can form antibodies in their bodies after recovery to prevent re-infection, but the virus lurking in the body may cause herpes zoster under different conditions. The varicella virus can be transmitted through droplets, blister fluid, or objects contaminated by blister fluid. Usually there will be a high fever for 1 to 2 days, and the rash will be red macules and papules, which will turn into fluid-filled blisters after a few hours, and then into scabs after a few hours. People with positive antibodies can prevent reinfection, but there is a possibility that the virus will lurk in the ganglia, causing it to become herpes zoster in adulthood.

[0005] Therefore, it is necessary to develop a new adjuvant combination for vaccine development, especially for the preparation of varicella vaccine, to improve the immune response effect of the vaccine. Summary of the invention

[0006] In order to overcome the defects of the prior art, the present invention provides an adjuvant suitable for children's vaccines and its application, especially in varicella vaccine, specifically,

[0007] In a first aspect of the present invention, an adjuvant composition is provided, wherein the adjuvant composition comprises at least one of a STING agonist and / or a TLR4 receptor agonist.

[0008] Preferably, the adjuvant composition further comprises CpG.

[0009] Preferably, the STING agonist includes but is not limited to one or more of 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP or sting-22.

[0010] Preferably, the TLR4 receptor agonist includes but is not limited to MPLA, MF59 or MPL.

[0011] More preferably, the adjuvant composition comprises:

[0012] 1) STING agonists

[0013] 2) TLR4 receptor agonists

[0014] 3) STING agonists and TLR4 receptor agonists;

[0015] 4) STING agonists and CpG;

[0016] 5) TLR4 receptor agonist and CpG; or

[0017] 6) STING agonists, TLR4 receptor agonists and CpG.

[0018] Preferably, the ratio of the STING agonist to the TLR4 receptor agonist is (1-10):(1-20), for example (1, 2, 3, 4, 5, 6, 7, 8, 9, 10):(1, 2, 3, 4, 5, 10, 12, 15, 18, 20), more preferably, the ratio of the STING agonist to the TLR4 receptor agonist is 1:20, 2:20, 2:10, 2:5, 5:2, 10:2, 10:1 and the like.

[0019] Preferably, the ratio of the STING agonist to CpG is (1-20):(20-1), for example (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20):(20, 18, 15, 12, 10, 5, 4, 3, 2, 1), more preferably, the ratio of the STING agonist to CpG is 1:20, 2:20, 2:10, 2:5, 5:2, 10:2, 10:1, 20:1 and the like.

[0020] Preferably, the ratio of the TLR4 receptor agonist to CpG is (1-10):(4-120), for example (1, 2, 3, 4, 5, 6, 7, 8, 9, 10):(4, 5, 10, 15, 20, 25, 30, 40, 50, 80, 100, 120).

[0021] Preferably, the content of the STING agonist is 10-200 μg, which can be any range or value within the above range, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μg and the like.

[0022] The content of the TLR4 receptor agonist is 20-500 μg, which can be any range or value within the above range, for example, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 250, 260, 270, 280, 290, 300, 320, 350, 380, 400, 420, 450, 480, 500 μg and the like.

[0023] The CpG content is 1-50 μg, which can be any range or value within the above range, for example, 1, 1.5, 2, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 12, 15, 20, 25, 30, 35, 40, 45, 50 μg and the like.

[0024] In one embodiment, the adjuvant composition comprises:

[0025] 1) sting-22

[0026] 2) Sting-22 and MF59; or,

[0027] 3) MF59 and CpG.

[0028] Preferably, the adjuvant composition further comprises a buffer.

[0029] Preferably, the adjuvant does not contain aluminum adjuvant.

[0030] The second aspect of the present invention provides the use of the above-mentioned adjuvant composition in the preparation of varicella vaccine.

[0031] Preferably, the varicella vaccine comprises a fusion protein and an adjuvant composition, wherein the fusion protein comprises an antigenic protein of varicella-zoster virus. Further preferably, the fusion protein comprises glycoprotein E (VZV gE) of varicella-zoster virus (VZV) and an Fc fragment of IgG.

[0032] Preferably, the glycoprotein E of varicella-zoster virus (VZV) (VZV gE) comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; more preferably, the amino acid sequence of glycoprotein E of varicella-zoster virus (VZV) (VZV gE) is as shown in SEQ ID NO: 1;

[0033] Preferably, the IgG Fc fragment is the Fc fragment of human IgG;

[0034] More preferably, the Fc fragment of human IgG comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 2, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; more preferably, the amino acid sequence of the Fc fragment of human IgG is as shown in SEQ ID NO: 2.

[0035] Preferably, a linker is included between the glycoprotein E (VZVgE) of varicella-zoster virus (VZV) and the Fc fragment of IgG;

[0036] Preferably, the joint is a flexible joint;

[0037] Preferably, the flexible linker is selected from GGGGS (SEQ ID NO: 5), GGGGSGGGGS (SEQ ID NO: 6), GSGGGSGGGGSGGGGSA (SEQ ID NO: 7);

[0038] More preferably, the flexible polypeptide is GSGGGSGGGGSGGGGSA (SEQ ID NO: 7);

[0039] Preferably, the fusion protein comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 3, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; more preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 3.

[0040] Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO:3.

[0041] Preferably, the ratio of the fusion protein to the adjuvant is (1-8):(1-260).

[0042] Preferably, the ratio of the fusion protein to the TLR4 receptor agonist is (1-8):(10-240), for example (1, 2, 3, 4, 5, 6, 7, 8):(10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 220, 240).

[0043] Preferably, the ratio of the fusion protein to the STING agonist is (1-8):(2-50), for example (1, 2, 3, 4, 5, 6, 7, 8):(2, 3, 4, 5, 8, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50).

[0044] Preferably, the ratio of the fusion protein to CpG is (1-8):(2-20), for example (1, 2, 3, 4, 5, 6, 7, 8):(2, 3, 4, 5, 10, 15, 20).

[0045] The content of the fusion protein is 1-50 μg, which can be any range or value within the above range, for example, 1, 1.5, 2, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 12, 15, 20, 25, 30, 35, 40, 45, 50 μg and the like.

[0046] Preferably, the content of the STING agonist is 10-200 μg, which can be any range or value within the above range, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μg and the like.

[0047] The content of the TLR4 receptor agonist is 20-500 μg, which can be any range or value within the above range, for example, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 250, 260, 270, 280, 290, 300, 320, 350, 380, 400, 420, 450, 480, 500 μg and the like.

[0048] The CpG content is 1-50 μg, which can be any range or value within the above range, for example, 1, 1.5, 2, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 12, 15, 20, 25, 30, 35, 40, 45, 50 μg and the like.

[0049] Preferably, the varicella vaccine further comprises one or more pharmaceutically acceptable carriers.

[0050] The pharmaceutically acceptable carrier may be a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant or a lubricant but is not limited thereto.

[0051] The third aspect of the present invention provides a vaccine composition, which comprises a fusion protein and the above-mentioned adjuvant composition, wherein the fusion protein comprises an antigen protein of varicella-zoster virus.

[0052] Preferably, the fusion protein comprises a fusion protein of glycoprotein E of varicella-zoster virus (VZV) (VZV gE) and the Fc fragment of IgG.

[0053] Preferably, the glycoprotein E of varicella-zoster virus (VZV) (VZV gE) comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; more preferably, the amino acid sequence of glycoprotein E of varicella-zoster virus (VZV) (VZV gE) is as shown in SEQ ID NO: 1;

[0054] Preferably, the IgG Fc fragment is the Fc fragment of human IgG;

[0055] More preferably, the Fc fragment of human IgG comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 2, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; more preferably, the amino acid sequence of the Fc fragment of human IgG is as shown in SEQ ID NO: 2.

[0056] Preferably, a linker is included between the glycoprotein E (VZVgE) of varicella-zoster virus (VZV) and the Fc fragment of IgG;

[0057] Preferably, the joint is a flexible joint;

[0058] Preferably, the flexible linker is selected from GGGGS (SEQ ID NO: 5), GGGGSGGGGS (SEQ ID NO: 6), GSGGGSGGGGSGGGGSA (SEQ ID NO: 7);

[0059] More preferably, the flexible polypeptide is GSGGGSGGGGSGGGGSA (SEQ ID NO: 7);

[0060] Preferably, the fusion protein comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 3, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; more preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 3.

[0061] Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO:3.

[0062] Preferably, the ratio of the fusion protein to the adjuvant is (1-8):(1-260).

[0063] Preferably, the ratio of the fusion protein to the TLR4 receptor agonist is (1-8):(10-240), for example (1, 2, 3, 4, 5, 6, 7, 8):(10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 220, 240).

[0064] Preferably, the ratio of the fusion protein to the STING agonist is (1-8):(2-50), for example (1, 2, 3, 4, 5, 6, 7, 8):(2, 3, 4, 5, 8, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50).

[0065] Preferably, the ratio of the fusion protein to CpG is (1-8):(2-20), for example (1, 2, 3, 4, 5, 6, 7, 8):(2, 3, 4, 5, 10, 15, 20).

[0066] The content of the fusion protein is 1-50 μg, which can be any range or value within the above range, for example, 1, 1.5, 2, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 12, 15, 20, 25, 30, 35, 40, 45, 50 μg and the like.

[0067] Preferably, the content of the STING agonist is 10-200 μg, which can be any range or value within the above range, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μg and the like.

[0068] The content of the TLR4 receptor agonist is 20-500 μg, which can be any range or value within the above range, for example, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 250, 260, 270, 280, 290, 300, 320, 350, 380, 400, 420, 450, 480, 500 μg and the like.

[0069] The CpG content is 1-50 μg, which can be any range or value within the above range, for example, 1, 1.5, 2, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 12, 15, 20, 25, 30, 35, 40, 45, 50 μg and the like.

[0070] Preferably, the vaccine composition further comprises one or more pharmaceutically acceptable carriers.

[0071] The pharmaceutically acceptable carrier may be a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant or a lubricant but is not limited thereto.

[0072] The fourth aspect of the present invention provides a method for preparing the above-mentioned vaccine composition, the preparation method comprising the following steps:

[0073] The fusion protein involved in the second aspect is mixed with the above-mentioned adjuvant composition to obtain a varicella vaccine.

[0074] Preferably, the ratio of the fusion protein to the adjuvant is (1-8):(1-260).

[0075] Preferably, the ratio of the fusion protein to the TLR4 receptor agonist is (1-8):(10-240), for example (1, 2, 3, 4, 5, 6, 7, 8):(10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 220, 240).

[0076] Preferably, the ratio of the fusion protein to the STING agonist is (1-8):(2-50), for example (1, 2, 3, 4, 5, 6, 7, 8):(2, 3, 4, 5, 8, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50).

[0077] Preferably, the ratio of the fusion protein to CpG is (1-8):(2-20), for example (1, 2, 3, 4, 5, 6, 7, 8):(2, 3, 4, 5, 10, 15, 20).

[0078] The content of the fusion protein is 1-50 μg, which can be any range or value within the above range, for example, 1, 1.5, 2, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 12, 15, 20, 25, 30, 35, 40, 45, 50 μg and the like.

[0079] Preferably, the content of the STING agonist is 10-200 μg, which can be any range or value within the above range, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μg and the like.

[0080] The content of the TLR4 receptor agonist is 20-500 μg, which can be any range or value within the above range, for example, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 250, 260, 270, 280, 290, 300, 320, 350, 380, 400, 420, 450, 480, 500 μg and the like.

[0081] The CpG content is 1-50 μg, which can be any range or value within the above range, for example, 1, 1.5, 2, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 12, 15, 20, 25, 30, 35, 40, 45, 50 μg and the like.

[0082] A fifth aspect of the present invention provides a use of the above vaccine composition for preventing varicella.

[0083] Preferably, the use comprises preventing varicella in children.

[0084] The technical effect of the present invention is

[0085] The present invention provides a new adjuvant composition, Sting-22 and / or MF59; and various adjuvant combinations such as MF59 and CpG can induce the body to produce significant high-titer specific IgG antibodies, enhance humoral immunity; and can induce strong cellular immunity; and the combined adjuvants can show obvious synergistic effects.

[0086] The present invention replaces the aluminum adjuvant in the traditional vaccine by screening an adjuvant that is harmless to children, so that the vaccine composition of the present invention can achieve good humoral and cellular immune effects with a lower dose of antigen, making the vaccine composition of the present invention particularly suitable for children.

[0087] The above merely summarizes some aspects of the present invention and is not and should not be considered to limit the present invention in any aspect.

[0088] All patents and publications mentioned in this specification are incorporated herein by reference as a whole. Those skilled in the art will recognize that certain changes may be made to the present invention without departing from the concept or scope of the present invention. The following examples further illustrate the present invention in detail and should not be considered to limit the scope of the present invention or the specific methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0090] Figure 1 : VZV-gE-Fc purification results.

[0091] Figure 2 : Vaccination protocol for mice.

[0092] Figure 3 : Specific IgG antibody titers in the serum of mice on the 35th day after vaccination, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001.

[0093] Figure 4 : ELISPOT was used to detect the expression of IFN-γ in spleen lymphocytes of each group 14 days after the second immunization. *** represents P<0.001, ns represents non-significant difference. DETAILED DESCRIPTION

[0094] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0095] Example 1: Preparation of fusion protein VZV-gE-Fc

[0096] The recombinant herpes zoster subunit vaccine antigen target of this embodiment is VZV-gE. For details, please refer to CN116023510B, which is a part of this application and is cited as a whole in this application. When designing the antigen structure, a human IgG4 Fc fragment is added, and the VZV-gE protein and the human IgG Fc fragment are fused to form a VZV-gE-Fc stable dimer antigen structure to form a VZV gE-human IgG Fc fusion protein, whose amino acid sequence is shown in SEQ ID NO: 3. Two identical VZVgE-human IgG Fc fusion proteins are connected to form homodimers through disulfide bonds.

[0097] A VZV-gE-Fc fusion protein antigen gene sequence with high expression biased towards CHO-K1 cells was designed, and its nucleotide sequence is shown in SEQ ID NO: 4. The amino acid sequence of VZV-gE is shown in SEQ ID NO: 1, the amino acid sequence of IgG Fc is shown in SEQ ID NO: 2, the amino acid sequence of VZV gE-human IgG Fc fusion protein is shown in SEQ ID NO: 3, and the nucleic acid sequence of VZV gE-human IgG Fc fusion protein antigen is shown in SEQ ID NO: 4.

[0098] The gene fragment VZV-gE-Fc synthesized by GenScript Biotech Co., Ltd. was digested with HindIII and Not I, and then connected with the PKS001 empty vector (purchased from Zhongshan Kangsheng Biotechnology Co., Ltd., catalog number A13201). The successfully constructed vector was named PKS001gEFc. The recombinant expression vector PKS001gEFc was transformed into Escherichia coli top10 competent cells (purchased from Transgene, catalog number CD101), and the transformed bacteria were rejuvenated with LB liquid screening medium containing ampicillin and expanded. After shaking culture at 37°C overnight, the plasmid was extracted with an endotoxin-free plasmid extraction kit (enhanced) (Tiangen Biochemical Technology (Beijing) Co., Ltd., DP120-01), and electroporated to transform CHO-K1 cells (Zhongshan Kangtian Shenghe Biotechnology Co., Ltd., catalog number A14101). A high-expressing monoclonal strain with an expression level of 3 g / L was screened out through pressure screening, ELISA and Protein A HPLC, and cultured continuously. The cell density and cell viability were monitored every day for 12-14 days. When the cell viability dropped to 50-60%, the culture was stopped and the culture supernatant was harvested by centrifugation.

[0099] The culture supernatant was purified by affinity chromatography using a Protein A gel column, and then gradient eluted using DEAE Sepharose4FastFlow with different concentrations of NaCl solution, and then purified by Sephacryl S400 HR molecular sieve chromatography column ( Figure 1 ), the purity reached more than 97%, and the target product was collected for later use.

[0100] Example 2 Screening of related adjuvants and immunogenicity studies

[0101] In this example, the VZV gE-human IgG Fc (VZV-gE-Fc) fusion protein prepared in Example 1 was used as an immunogen, diluted with 20 mM histidine hydrochloride, 140 mM arginine hydrochloride, and 0.02% polysorbate 80 pH 6.0, and mixed evenly with MF59 (purchased from Invivogen, catalog number: vac-adx-10), CpG1018 adjuvant (purchased from Guangzhou Ruibo Biotechnology Co., Ltd.) or Sting-22 (purchased from MCE, Germany, 1 mg / 1 ml Cat No. HY-152955) at room temperature to prepare each adjuvant vaccine solution, and then placed at 2 to 8°C.

[0102] The above vaccine solution at 2-8°C is aseptically dispensed into 2 ml vials (or pre-filled glass syringes), 0.5 ml (or 1.0 ml) per vial, and stored at 2-8°C away from light after sealing.

[0103] The above vaccine solution was taken out, and immunogenicity study was carried out using C57BL / 6 mice (purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.) as animal model.

[0104] The specific method is as follows: the C57BL / 6 female mice in this example were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. 6-8 week old C57BL / 6 mice were randomly divided into groups, 5 mice in each group, and the above vaccine composition was injected intramuscularly, and different groups were set up: PBS control group, non-adjuvant vaccine group, various adjuvant vaccine groups (protein and adjuvant prepared in Example 1 (MF59 and / or CpG1018 adjuvant; MF59 and / or Sting-22 adjuvant)), attenuated varicella vaccine group, specific grouping refers to the immunization scheme of the specific experiment (Table 1), immunization at 0 and 3 weeks, blood collection at 3 and 5 weeks, spleen collection at 5 weeks, and the vaccination protocol of mice is as follows Figure 2 The ELISA method was used to detect the antibody titer (ie, total IgG) against VZV-gE-Fc protein in the serum.

[0105] ELISA method:

[0106] 1. Reagent Preparation

[0107] 1.1 Preparation of ELISA coating solution (1X): Take ELISA coating solution (10X) (purchased from Solebol, product number: C1055) and dilute it to 1X with sterile distilled water.

[0108] 1.2. PBS preparation: Take out PBS powder and dissolve each bag with 2L of sterile distilled water.

[0109] 1.3. Washing solution: PBST (PBS containing 0.05% Tween-20)

[0110] Measure 1L of filtered PBS into a blue-capped bottle, add 500μl Tween-20, mix thoroughly, and store at 2-8℃ for later use. Prepare the required volume of PBST according to the actual situation.

[0111] Note: PBST should be prepared fresh and used on the same day.

[0112] 1.4. Blocking solution and sample diluent (PBS containing 5% skim milk powder)

[0113] Weigh 5% skim milk powder of the volume of PBS (milk powder mass / g = PBS volume / ml x 5%), equilibrate the PBS taken out from 2-8°C to room temperature, measure the required volume of PBS solution into the centrifuge tube with milk powder added, and then fully dissolve it for use.

[0114] Note: The blocking solution and sample diluent should be prepared freshly and used on the same day.

[0115] 1.5. Dilute goat anti-mouse secondary antibody

[0116] Equilibrate 1 ml of secondary antibody (purchased from CST, item number 7076s) to room temperature, then aliquot and store at -20°C ± 5°C. Take out before each use and dilute at 1:4000, where the diluent is PBS.

[0117] Note: The diluted secondary antibody should be used on the same day.

[0118] 1.6. Stop solution (purchased from Solebro, product number C1058)

[0119] 2.ELISA to detect serum binding antibody titer:

[0120] 2.1 Coating: Take VZV-gE-his (prepared by Beijing Geneway Products Co., Ltd., with a concentration of 2 mg / ml) antigen stock solution and dilute it to 1000 ng / mL with ELISA coating solution (1X), coat the ELISA plate, 100 μl / well, and place it at 4°C overnight.

[0121] 2.2 Blocking: After taking out the coated plate from 2-8℃, wash the plate three times, each time with a washing volume of 300μl / well. If there is any washing solution left in the wells after washing, pat it dry on absorbent paper; then add the pre-prepared blocking solution to the coated wells, 300μl / well, cover with sealing film, and incubate at 37℃ for 60-90min.

[0122] 2.3 Serum dilution: Dilute the serum to be tested to an appropriate concentration using sample diluent in a centrifuge tube.

[0123] 2.4 Sample addition: Wash the blocked coated plate 3 times, with a washing volume of 300μl / well each time. If there is any washing solution left in the well after washing, pat it dry on absorbent paper. Add the diluted samples of various concentrations to the sample wells in sequence, 100μl / well; add 100μl of sample dilution as a blank control (Blk), set up 5 replicate wells, cover with sealing film, and incubate at 37℃ for 60min.

[0124] 2.5 Add secondary antibody: discard the sample, wash the plate 3 times, each time with a washing volume of 300μl / well. If there is any washing solution left in the well after washing, pat it dry on absorbent paper; add diluted secondary antibody, 100μl / well, cover with sealing film, and incubate at 37℃ for 60min.

[0125] 2.6 Color development: Wash the 96-well plate three times, with a washing volume of 300 μl / well each time. If there is any washing solution left in the wells after washing, pat it dry on absorbent paper, add single-component TMB color development solution 1 (taken out in advance and equilibrated to room temperature), 100 μl / well, and develop color at 25°C in the dark for 15 minutes.

[0126] 2.7 Termination: Add stop solution immediately after color development to terminate the reaction, 50 μl / well, and shake gently to mix.

[0127] 2.8 Detection: Place the ELISA plate into the ELISA reader and measure the absorbance at a wavelength of 450nm.

[0128] 2.9 Judgment: A value greater than 2.1 times the OD value of negative mice is considered positive.

[0129] Note: The following ELISA methods for detecting binding antibody titers are the same as above.

[0130] With reference to the screening doses of the previous experiments, the immunization scheme was designed according to Table 1 to immunize the animals, and the best adjuvant was screened by detecting the ELISA titers of groups ①-⑦ at 35 days after the second immunization.

[0131] Table 1 Immunization scheme for screening the best adjuvant

[0132]

[0133]

[0134] Table 2 Specific IgG antibody titers in the serum of mice on day 35 after vaccination

[0135]

[0136]

[0137] The results are shown in Table 2 and Figure 3 As shown, the conclusion of humoral immunity is:

[0138] 1. The results of ELISA antibody titer showed that ④>③>②>①, indicating that if only a single adjuvant is added, the effect of sting-22 is better than MF59.

[0139] 2. The ELISA antibody titer results showed that ⑥>⑤, indicating that if two adjuvants were added, the MF59+sting-22 adjuvant combination was better.

[0140] 3. The double adjuvant group was superior to the single adjuvant group.

[0141] 4. Both the single adjuvant group and the double adjuvant group were superior to the attenuated vaccine group.

[0142] Cellular Immunity Assays for Further Vaccine Screening:

[0143] Fourteen days after the second immunization, spleens were collected and spleen lymphocytes were isolated. The expression of IFN-γ in spleen lymphocytes of each group was detected by ELISPOT at fourteen days after the second immunization.

[0144] Table 3 ELISPOT results

[0145]

[0146]

[0147] The results are shown in Table 3 and Figure 4 As shown, the conclusion of cellular immunity is:

[0148] 1. ELISPOT results showed that among the single adjuvant groups, group ④ was the best, indicating that antigen alone plus sting-22 adjuvant can produce a higher level of cellular immunity.

[0149] 2. The double adjuvant group was significantly better than the single adjuvant group.

[0150] 3, ④, ⑤, ⑥ groups were significantly better than the attenuated vaccine group. The mean number of IFN-γ positive cells showed: ⑥>⑤>④, but there was no significant difference.

[0151] In summary, from the comprehensive perspective of humoral immunity and cellular immunity results: Sting-22 in the double adjuvant group and the single adjuvant group is a better adjuvant or adjuvant combination, which can improve both humoral immunity and cellular immunity.

[0152] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0153] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0154] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

[0155] Sequence Listing:

[0156] SEQ ID NO.1: Amino acid sequence 516aa of VZV-gE

[0157] SVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLE

[0158] NAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYG

[0159] DVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICL

[0160] KHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGV

[0161] LKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYH

[0162] SHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNS

[0163] GCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLY

[0164] VFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAW

[0165] TGGLA

[0166] SEQ ID NO.2: Amino acid sequence of IgG Fc, 228aa

[0167] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV

[0168] DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ

[0169] PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY

[0170] SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0171] SEQ ID NO.3: VZV gE-human IgG Fc fusion protein amino acid sequence, 761aa

[0172] SVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAGSGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0173] SEQ ID NO.4: Nucleic acid sequence of VZV gE-human IgG Fc fusion protein antigen, 2283 bp

[0174]

[0175] CTGTCTCTGAGCCTGGGA

[0176] SEQ ID NO: 5 Linker sequence

[0177] GGGGS

[0178] SEQ ID NO: 6 Linker sequence

[0179] GGGGSGGGGS

[0180] SEQ ID NO: 7 Linker sequence

[0181] GSGGGSGGGGSGGGGSA.

Claims

1. An adjuvant composition, characterized in that The adjuvant composition comprises at least one of a STING agonist and / or a TLR4 receptor agonist.

2. The adjuvant composition according to claim 1, characterized in that The adjuvant composition also includes CpG.

3. The adjuvant composition according to claim 1 or 2, characterized in that The STING agonist includes 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP or sting-22; The TLR4 receptor agonist includes MPLA, MPL or MF59.

4. Use of the adjuvant composition according to any one of claims 1 to 3 in the preparation of varicella vaccine.

5. The use according to claim 4, characterized in that: The varicella vaccine comprises a fusion protein and an adjuvant composition, wherein the fusion protein comprises an antigenic protein of varicella-zoster virus. Preferably, the fusion protein comprises glycoprotein E (VZV gE) of varicella-zoster virus (VZV) and an Fc fragment of IgG. Further preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID NO:

3.

6. A vaccine composition, characterized in that The vaccine composition comprises a fusion protein and the adjuvant composition according to any one of claims 1 to 3, wherein the fusion protein comprises an antigen protein of varicella-zoster virus.

7. The vaccine composition according to claim 6, characterized in that The fusion protein comprises a fusion protein of glycoprotein E ((VZV gE)) of varicella-zoster virus (VZV) and the Fc fragment of IgG. Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO:

3.

8. The vaccine composition according to any one of claims 6-7, characterized in that The ratio of the fusion protein to the adjuvant is (1-8):(1-260).

9. The vaccine composition according to any one of claims 6 to 8, characterized in that The ratio of the fusion protein to the TLR4 receptor agonist is (1-8):(10-240); The ratio of the fusion protein to the STING agonist is (1-8):(2-50); The ratio of the fusion protein to CpG is (1-8):(2-20).

10. The method for preparing the vaccine composition according to any one of claims 6 to 9, characterized in that: The preparation method comprises the following steps: Mixing the fusion protein with the adjuvant composition according to any one of claims 1 to 3 to obtain a varicella vaccine, wherein the fusion protein comprises an antigen protein of varicella-zoster virus; Preferably, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:3.

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