CN116212012A - Complex adjuvant and vaccine formulation comprising the same - Google Patents

Abstract

The invention relates to a composite adjuvant and a vaccine preparation containing it. The composite adjuvant comprises CpG oligodeoxynucleotide, QS-21 and liposome, wherein the CpG oligodeoxynucleotide is CpG7909. According to the present invention, the composite adjuvant can effectively enhance the antibody response and immune response of various vaccine preparations.

Description

Composite adjuvant and vaccine preparation containing it

technical field

The present invention relates to the field of vaccines. Specifically, the present invention relates to a composite adjuvant that can effectively enhance the antibody response and immune response of a vaccine preparation, and a vaccine preparation containing the composite adjuvant.

Background technique

Herpes zoster is an acute infectious skin disease caused by Varicella-Zoster Virus (VZV). Due to the neurotropic nature of the virus, it can remain dormant in the neurons of the dorsal root ganglia of the spinal cord for a long time after infection. When the resistance is low or tired, infected, or caught a cold, the virus can grow and multiply again, and move to the skin along the nerve fibers, causing intense inflammation to the affected nerves and skin.

Shingles presents clinically as vesicles on one side of the body, typically confined to a dermatome, often with radicular pain. Patients experience significant pain and discomfort that can last for weeks, months, or even years, reducing quality of life. The incidence rate increases significantly with age.

There are 9 types of VZV surface glycoproteins identified so far, namely gB, gC, gE, gH, gI, gK, gL, gM and gN. Among them, gE glycoprotein is the glycoprotein with the highest expression level of VZV, which plays a major role in the replication and assembly of the virus, and also mediates the spread of the virus between cells. In view of the strong immunogenicity of VZV gE, which can induce the body to produce an immune response against VZV, it has become one of the main candidate antigens for VZV subunit vaccines and DNA vaccines.

It is currently known that gE protein alone cannot induce a strong cellular immune response in animal models, and an adjuvant must be used to enhance the immune response of gE.

The new coronavirus (also known as the new coronavirus, or SARS-CoV-2) belongs to the B subgroup of the genus β, and is an enveloped single-stranded positive-sense RNA virus. The virus genus also includes severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), both of which interact with the host cell surface through the spike (Spike or S) protein on the surface of the virus particle. The receptor binds to infect the host. Specifically, the S protein on the surface of SARS-CoV-2 can recognize the angiotensin-converting enzyme 2 (Angiotensin-converting enzyme-2, ACE2) on the host cell through its receptor-binding domain (RBD). The virus invades the host cell. The neutralizing antibody induced by the SARS-CoV-2 S protein can significantly block the recognition pathway and reduce the infectivity of the virus. These studies fully illustrate the important role of S protein neutralizing antibodies in preventing virus invasion, and provide important guidance for the design of vaccines based on B cell humoral immunity. Meanwhile, T-cell immunity is crucial for clearing infected cells and viruses. Because once a small number of viruses escape the siege of neutralizing antibodies, the infected host cells will become a breeding ground for virus replication and even mutation, and at this time antigen-specific Tc cells can recognize the infected host cells and further clear the virus. Studies have found that severe COVID-19 patients have T cell exhaustion and reduced functional diversity, which reflects the importance of T cell immunity for disease control.

However, the currently commonly used aluminum adjuvant has a weak immune enhancement effect and mainly produces humoral immunity, which cannot meet the development needs of various vaccines including herpes zoster vaccine and new crown vaccine. Therefore, it is expected to obtain new compound adjuvants that can be used in various vaccines to enhance their effects.

Contents of the invention

In view of the above circumstances, an object of the present invention is to provide a composite adjuvant that can effectively enhance the antibody response and immune response of vaccine preparations.

In a first aspect, the present invention relates to a composite adjuvant comprising CpG oligodeoxynucleotide, QS-21 and liposome, wherein the CpG oligodeoxynucleotide is CpG7909.

In a second aspect, the present invention relates to a vaccine formulation comprising the above-mentioned composite adjuvant of the present invention.

According to the present invention, a compound adjuvant capable of effectively enhancing the antibody response and immune response of a vaccine preparation can be provided. In particular, the compound adjuvant of the present invention can specifically enhance T cell response and neutralizing antibody titer. Thus, the complex adjuvant of the present invention can reduce the amount of antigen necessary for vaccine formulations.

Description of drawings

Figure 1 shows the particle size test results of the compound adjuvant sample according to one embodiment of the present invention (Z-average: 113.5nm, PDI 0.196).

Fig. 2 shows the particle size test results of the compound adjuvant sample according to another embodiment of the present invention (Z-average: 96.7nm, PDI 0.164).

Figure 3 shows binding antibody responses in mice induced by different recombinant SARS-CoV-2 vaccine formulations according to one embodiment of the present invention.

Figure 4, according to one embodiment of the present invention, the pseudovirus neutralizing antibody response induced by different recombinant new coronavirus vaccine preparations in mice.

Figure 5 shows the secretion levels of IFN-γ, IL-2, TNF-α and IL-4 cytokines in lymphocytes produced by mice induced by different recombinant SARS-CoV-2 vaccine preparations according to one embodiment of the present invention.

Figure 6 According to one embodiment of the present invention, IFN-γ, IL-2, TNF-α and IL- 4 Percentage of positive cells.

Detailed ways

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document including definitions will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

All numerical ranges referred to in the present disclosure should be understood as disclosing all specific numerical values within the range, as well as subranges defined by any two numerical values within the range. For example, for 1%-10%, it should be understood as disclosing specific values such as 1%, 2%, 3%, 3.5%, 4.5%, 10%, and 1%-5%, 2%-6%, 3.5% -7.5% and other sub-ranges.

In one aspect, the present invention provides a complex adjuvant comprising CpG oligodeoxynucleotides, QS-21 and liposomes.

CpG oligodeoxynucleotide (CpG ODN) is a synthetic short single-stranded DNA molecule that contains unmethylated cytosine guanine dinucleotide sequence, which can mimic bacterial DNA binding and activate Toll in mammals including humans TLR9 directly activates B cells and monocytes, indirectly activates various immune effector cells such as NK cells and T cells, enhances their function and secretion of cytokines, and induces Th1 immune responses. The CpG ODN used in the present invention is CpG7909, also known as CpG2006, ODN7909 or ODN2006. CpG7909 has the following nucleotide sequence: TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID No. 1).

QS-21 is an active ingredient extracted from the bark of the South American soap tree (Quillaja Saponaria), which can induce mouse CD8+ cell immune response, produce IgG1 and IgG2a antibodies, and promote cytotoxic T lymphocytes (CTLs) to secrete Th1 cytokines , interleukin 2 and interferon gamma.

In a preferred embodiment, relative to the total weight of CpG oligodeoxynucleotides, QS-21 and liposomes, the content of CpG ODN is 10-30% by weight. In a more preferred embodiment, the content of CpG ODN is 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30% by weight.

In a preferred embodiment, relative to the total weight of CpG oligodeoxynucleotide, QS-21 and liposome, the content of QS-21 is 1-5% by weight. In a more preferred embodiment, the content of QS-21 is 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5% by weight.

In a preferred embodiment, the relative weight ratio of CpG ODN to QS-21 is 5:1 to 20:1. In a more preferred embodiment, the relative weight ratio of CpG ODN to QS-21 is 10:1.

In a preferred embodiment, the liposomes comprise phosphatidylcholine (PC) and cholesterol (CHOL). In a more preferred embodiment, the liposomes consist of phosphatidylcholine (PC) and cholesterol (CHOL).

Phosphatidylcholine (PC) is a structure in which choline is combined with two fatty acids through a phosphate radical to form an ester bond. The structural quaternary amines and hydroxyl groups on choline can be separated by multiple carbon atoms. The length of the two fatty acids is 12-18 carbon atoms, and there may be one or more unsaturated double bonds. Among them, dioleoylphosphatidylcholine (DOPC) having the structure shown below is preferable. The fatty acid of this structure is 18 carbons in length and contains 1 unsaturated double bond at the same time.

Cholesterol is a lipid widely present in the human body. It is an important component of cell membranes and can stabilize liposomes and reduce the permeability of lipid membranes. Cholesterol is composed of a steroidal part and a long side chain, which varies with different cholesterol derivatives. In one embodiment, cholesterol derivatives having the same steroidal moiety structure as in the following formula and a side chain length of 4-8 carbon chains are preferred. In another embodiment, cholesterol having the structure shown below is preferred.

In a preferred embodiment, relative to the total weight of CpG oligodeoxynucleotide, QS-21 and liposome, the content of phosphatidylcholine is 40-60% by weight. In a more preferred embodiment, the content of phosphatidylcholine is 40, 45, 50, 55 or 60% by weight.

In a preferred embodiment, the content of cholesterol is 10-15% by weight relative to the total weight of CpG oligodeoxynucleotide, QS-21 and liposome. In a more preferred embodiment, the content of cholesterol is 10, 10.5, 11, 12, 12.5, 13, 13.5, 14, 14.5 or 15% by weight.

Those skilled in the art will understand that although the above provides various examples for the content of each component such as CpG ODN, QS-21, phosphatidylcholine (PC) and cholesterol (CHOL) in the composite adjuvant of the present invention, They can be appropriately determined by those skilled in the art without being limited to the specific examples listed above.

In another aspect, the present invention provides a vaccine formulation comprising the complex adjuvant according to the present invention. In one embodiment, the vaccine formulation is a SARS-CoV-2 vaccine formulation. In a preferred embodiment, the vaccine formulation comprises the novel coronavirus spike protein (S protein). In a more preferred embodiment, the S protein has the following amino acid sequence: (SEQ ID No. 2), or has more than 90% thereof, such as 91%, 92%, 93%, 94%, 95%, 96% , 97%, 99%, and 99% identity amino acid sequences. In one embodiment, the nucleic acid encoding the S protein having the amino acid sequence of SEQ ID No. 2 has the nucleotide sequence shown in SEQ ID No. 3.

In yet another aspect, the present invention provides the use of the composite adjuvant according to the present invention in preparing a vaccine.

Example

Specific examples are provided below to further illustrate the present invention.

Example 1: Preparation of liposome complex adjuvant by ethanol injection method

Dioleoylphosphatidylcholine (DOPC) and cholesterol (CHOL) were purchased from Aveto (Shanghai) Pharmaceutical Technology Co., Ltd., QS-21 was purchased from Desert King International (USA), and CpG7909 was purchased from Guangzhou Ruibo Biotech Co., Ltd.

Buffer 1 preparation:

Weigh Na ₂ HPO ₄ 12H ₂ O 0.6446g, KH ₂ PO ₄ 1.1207g, and NaCl 1.1702g in a container, add 200ml of water for injection, stir to dissolve, and obtain 9mM Na ₂ HPO4, 41mM KH ₂ PO ₄ , 100mM NaCl , pH6.1 buffer. Labeled as Buffer 1. Sterilize and filter for use.

Buffer 2 preparation:

Weigh Na ₂ HPO ₄ 12H ₂ O 0.1432g, KH ₂ PO ₄ 0.2177g, and NaCl 1.755g in a container, add 200ml of water for injection, stir to dissolve, and obtain 2mM Na ₂ HPO ₄ , 8mM KH ₂ PO ₄ , 150mM NaCl, pH 6.1 buffer. Labeled as buffer 2. Sterilize and filter for use.

Organic phase solution preparation:

Accurately weigh 0.400g of DOPC and 0.100g of CHOL into an EP tube, add 20ml of absolute ethanol into it with a pipette, and ultrasonically dissolve to obtain a mixed solution of 20mg/ml DOPC and 5mg/ml CHOL.

QS-21 solution preparation:

Accurately weigh an appropriate amount of QS-21 plus dimethyl sulfoxide, dissolve it by ultrasonic, and prepare the concentration of QS-21 at 2 mg/ml.

CpG7909 solution preparation:

Accurately weigh 0.402g of CpG7909 freeze-dried powder, add 50ml of PBS to dissolve, and detect its concentration with an ultramicro spectrophotometer to be 5.242mg/ml.

Precisely measure 10ml of CpG7909 solution with a concentration of 5.242mg/ml, 5ml of QS-21 solution with a concentration of 2mg/ml, and 75ml of buffer solution 1 into a glass vial, stir and mix evenly, as the water phase.

Precisely measure 10ml of a mixed solution with a concentration of 20mg/ml DOPC and 5mg/ml CHOL, add it dropwise to the water phase, stir while adding, the stirring speed is 600rpm, and the temperature is room temperature. After all the dropwise addition was completed, the stirring was continued for 30 minutes. It is the intermediate product of liposome complex adjuvant.

Take 100ml of the liposome complex adjuvant intermediate product and use a high-pressure homogenizer to homogenize it at a pressure of 600 bar for 10 minutes and then collect the product.

The product obtained in the previous step was subjected to ultrafiltration and centrifugation using Amicon Ultra ultrafiltration tubes (molecular weight cut-off 30KD), and finally the solvent was changed to buffer 2.

Finally, the particle size of the sample was detected, as shown in Figure 1. A dynamic light scattering instrument (model Zetasizer nano) produced by Malvern was used for detection. Each sample was measured in triplicate.

Example 2: Preparation of liposome complex adjuvant by thin film hydration method

Organic phase solution preparation:

Accurately weigh 0.400g of DOPC and 0.100g of CHOL into an EP tube, add 20ml of absolute ethanol into it with a pipette, and ultrasonically dissolve to obtain a mixed solution of 20mg/ml DOPC and 5mg/ml CHOL.

QS-21 solution preparation:

Accurately weigh an appropriate amount of QS-21 plus dimethyl sulfoxide, dissolve it by ultrasonic, and prepare the concentration of QS-21 at 2 mg/ml.

CpG7909 solution preparation:

Accurately weigh 0.402g of CpG7909 freeze-dried powder, add 50ml of PBS to dissolve, and detect its concentration with an ultramicro spectrophotometer to be 5.242mg/ml.

Precisely measure 10ml of CpG7909 solution with a concentration of 5.242mg/ml, 5ml of QS-21 solution with a concentration of 2mg/ml, and 75ml of buffer solution 1 into a glass vial, stir and mix evenly, as the water phase.

Precisely measure 10ml of a mixed solution with a concentration of 20mg/ml DOPC and 5mg/ml CHOL, add it to a round bottom flask, use a rotary evaporator to remove the solvent, and then vacuum for 1 hour to remove the residual organic solvent to form a lipid film.

Slowly add the water phase into the flask for hydration at room temperature, continue to stir for 30 minutes and then sonicate for 5 minutes, which is the intermediate liposome complex adjuvant.

Take 100ml of the liposome complex adjuvant intermediate product and use a high-pressure homogenizer to homogenize it at a pressure of 600 bar for 10 minutes and then collect the product.

The product obtained in the previous step was subjected to ultrafiltration and centrifugation using Amicon Ultra ultrafiltration tubes (molecular weight cut-off 30KD), and finally the solvent was changed to buffer 2.

Finally, the particle size of the sample was detected, as shown in Figure 2.

Example 3. Vaccine prepared by mixing liposome complex adjuvant with novel coronavirus S protein antigen induces humoral immune response and cellular immune response

The amino acid sequence of the recombinant new coronavirus variant vaccine spike protein (Spike, hereinafter referred to as S protein) expressed by the Chinese hamster ovary cell line (CHO cell) expression system is shown in SEQ ID No. 2. Group 70 SPF-grade female BALB/c mice (6-8 weeks old). 10 mice in each group, 7 groups in total. The experimental mice in each group were immunized according to the table below. The immune site was the right hind leg muscle of the mouse. The immune volume was 0.1ml/mouse (0.05ml for each of the left and right hind legs). Each mouse was immunized twice. The immunization interval was 3 weeks. Blood was collected on the 13th day (D34) after the second immunization, and the blood sample was taken overnight at 4°C, centrifuged at 7000rpm for 10 minutes, and the serum was separated, inactivated at 56°C for 30 minutes, and then stored at -20°C. The neutralizing antibody titer in serum was detected by ELISA experiment method and pseudovirus neutralization experiment. Fourteen days after the second immunization (D35), 5 mice in each group were sacrificed, and the vaccine-induced cellular immune response was detected by ELISPOT and ICS.

Table 1. Animal experiment grouping information table

group antigen Adjuvant Negative control: PBS PBS none Experimental group 1: S+AH 5.0 μg S 50 μg Al(OH) ₃ Experimental group 2: S+AS01 _B 5.0 μg S AS01 _B , containing liposome+5μg MPLA+5μg QS-21 Experimental group 3: S+Lipo/CpG7909/QS21 5.0 μg S Liposome/50μg CpG7909/5.0μg QS-21 Experimental group 4: S+Lipo/MPLA/CpG7909 5.0 μg S Liposome/5.0μg MPLA/50μg CpG Experimental group 5: S+CpG7909 5.0 μg S 50 μg CpG7909 Experimental group 6: S+AH/CpG7909 5.0 μg S 50μg Al(OH) ₃ /50μg CpG7909

The detection of humoral immune response adopts enzyme-linked immunosorbent assay (Enzyme-Linked Immunosorbent Assay, ELISA) and pseudovirus neutralization assay.

Enzyme-linked immunosorbent assay method: take the used batch of antigen S protein and coat the enzyme plate, then add different dilutions of mouse serum, and then add Anti-Mouse IgG (H+L)-HRP Conjugate (purchased from Bio-rad Cat. No. 170-6516) secondary antibody was incubated at 37°C for 1 hour, and finally reacted with _{TMB substrate} (purchased from KPL, Cat. 2.1 times the group OD value of 0.05, less than 0.05 will be counted as 0.05) as the cut-off value.

The results of the binding antibody titers (GMT±95%CI) of each group showed (Figure 3): S+Lipo/CpG7909/QS21, S+Lipo/CpG7909/QS21 and S+AH/CpG7909 groups were all higher than the PBS group. It can induce a high-titer serum antibody response, and there is no significant difference in effect from the commercial adjuvant combined with S protein group (AS01 _B ).

Pseudovirus neutralization test method: The serum to be tested was diluted in multiples and mixed with 100 TCID ₅₀ pseudoviruses (using the mutant VSV virus genome as the backbone, the surface of the virus particles expresses the SASR-CoV-2 spike protein, and has no replication ability, prepared For details on the method, see Quantification of SARS-CoV-2 neutralizing antibody by a pseudotypedvirus-based assay. Nat Protoc. 2020 Nov; 15(11):3699-3715. doi: 10.1038/s41596-020-0394-5. Epub 2020 Sep 25 .PMID: 32978602.) Neutralize for 1 hour, add virus-serum mixture to BHK-21-hACE2-gluc cells in logarithmic growth phase (the cells were purchased from Wuhan University, 2×10 ⁴ cells per well), Place in a 37°C _CO2 incubator for 24 hours, add 50 μl of cell lysate (purchased from Promega, product number E1941) to each well for lysis for 30 minutes, and then add 50 μl of luciferase substrate (purchased from Perkin Elmer, product number 6066769), After 2 minutes of reaction at room temperature in the dark, the luminescence value was read, and the antibody neutralization titer when the serum inhibition rate was 50% was calculated according to the Reed-Muench rule.

The results of pseudovirus neutralizing titers (GMT±95%CI) in each group showed (Figure 4): the titers of pseudovirus neutralizing antibodies produced by mice induced by the S+Lipo/CpG7909/QS21 vaccine group were comparable to those of AS01 _B developed by GSK. The results of the pseudovirus neutralizing antibody titers produced by the vaccine induced by the adjuvant combined with S protein were comparable, and there was no significant difference between the two groups. And the pseudovirus neutralizing antibody titer of the S+Lipo/CpG7909/QS21 group was significantly higher than that of other groups with different adjuvant ratios. humoral immune response.

T cell reaction detection adopts intracellular cytokine staining method (Intracellular Cytokine Staining, ICS) and enzyme-linked immunospot detection method (Enzyme-linked immunospot, ELISPOT).

Intracellular cytokine staining method: separate mouse spleen lymphocytes, spread the cell suspension in a 96-well cell culture plate according to the number of 1×10 ⁶ cells per well. At the same time, a stimulating peptide library was added to each well at a final concentration of 1ug/ml (consisting of 169 polypeptides covering the full length of the S protein sequence, 18 amino acids in length and 11 amino acid overlaps between adjacent polypeptides, custom-synthesized by Genescript), 0.1% anti-CD28 antibody (purchased from BD, product number 553047) and 0.1% Golgi apparatus inhibitor (purchased from BD, product number 51-2301KZ), mix well and incubate in a cell culture incubator at 37°C for about 6 hours. Cells were collected by centrifugation, and stained with different fluorescein-labeled anti-CD3 monoclonal antibodies (purchased from Biolegend, catalog number 100214), anti-CD4 monoclonal antibodies (purchased from BD, catalog number 553047) and Aqua (purchased from Invitrogen, catalog number L34965). After treatment with cell membrane penetrating solution, use fluorescein-labeled anti-IL-2 monoclonal antibody (purchased from BD, catalog number 560547), anti-TNF-α monoclonal antibody (purchased from BD, catalog No. 557644), anti-IFN-γ monoclonal antibody (purchased from BD, Cat. No. 560660) and anti-IL-4 monoclonal antibody (purchased from BD, Cat. No. 554389) were used for intracellular staining to specifically detect CD4+ T cells and CD8+ T cells stimulated by S-polypeptide library in spleen cells of immunized mice The ability of T cells to secrete IFN-γ, IL-2, TNF-α and IL-4.

ICS experiment (Mean±SD) results showed (Figure 5): S+Lipo/CpG7909/QS21 group stimulated by S protein induced IFN-γ, IL -2. The proportion of TNF-α and IL-4 positive cells was significantly higher than that of all other groups. It shows that the vaccine prepared by liposome complex adjuvant "liposome+CpG+QS-21" combined with S antigen can induce T cell immune response significantly better than other groups.

ELISPOT method: isolate mouse spleen lymphocytes, spread the cell suspension in a 96-well cell culture plate according to the number of 5×10 ⁵ cells per well. At the same time, the stimulating polypeptide library and 0.1% anti-CD28 antibody were added to each well at a final concentration of 1 ug/ml, mixed evenly and incubated in a 37°C cell culture incubator for 48 hours. Add detection primary antibody R4-6A2-BAM (purchased from MABTECH, catalog number FS-4142-2), 5H4-biotin (purchased from MABTECH, catalog number FS-4142-2) and MT11B10-WASP (purchased from MABTECH, catalog number FSP-414245) -2), incubate at room temperature for 2 hours; then add detection secondary antibodies anti-BAM-490, SSA-550 and WASP-640 (secondary and primary antibodies are reagents provided in the same kit), incubate at room temperature for 1 hour in the dark , adding Fluorescence enhancer-II (purchased from MABTECH, product number FS-4142-10) at room temperature in the dark for 15 minutes to develop color, ELISPOT plate reader (model CTLAnalyzer) to scan and count.

The results of ELISPOT experiment (Mean±SD) showed (Figure 6): Lymphocytes in S+Lipo/CpG7909/QS21 and S+Lipo/MPL/QS21 groups could secrete strong IL-2 and IFN after being stimulated by S polypeptide -γ, TNF-α and IL-4 cytokines, among which S+Lipo/CpG7909/QS21 can significantly enhance the secretion of IL-4 cytokines, and the enhancement of IL-4 cytokines indicates that the vaccine induces a bias towards the Th2 pathway The immune response of IL-2, IFN-γ and TNF-α cytokines enhanced, indicating that the vaccine induced an immune response biased towards the Th1 pathway. The results showed that the S+Lipo/CpG7909/QS21 group could induce cytokine secretion that balances Th1 and Th2 pathways. The secretion levels of 4 cytokines in other adjuvant groups were lower than those in S+Lipo/CpG7909/QS21 group.

While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes and equivalents will now occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

sequence listing

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Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr

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Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn

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Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu

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Thr Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln

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Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn

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Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala

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Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr

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Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr

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Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala

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Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu

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Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln

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Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe

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Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Pro

980 985 990

Pro Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln

995 1000 1005

Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu

1010 1015 1020

Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys

1025 1030 1035

Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr

1040 1045 1050

His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe

1055 1060 1065

Leu His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr

1070 1075 1080

Ala Pro Ala Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu

1085 1090 1095

Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg

1100 1105 1110

Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val

1115 1120 1125

Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val

1130 1135 1140

Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu

1145 1150 1155

Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly

1160 1165 1170

Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu

1175 1180 1185

Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu

1190 1195 1200

Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp

1205 1210 1215

Pro Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala

1220 1225 1230

Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

1235 1240 1245

<210> 3

<211> 3750

<212>DNA

<213> Artificial sequence

<400> 3

atggagttcg gcctgagctg gctgttcctg gtggccatcc tgaagggcgt gcagtgtcag 60

tgcgtgaacc tgaccacaag gacccagctg ccacctgcct ataccaactc tttcacaagg 120

ggcgtgtact atcctgacaa ggtgtttcgg tccagcgtgc tgcactccac acaggatctg 180

tttctgccat tctttagcaa cgtgacctgg ttccacgcta tccacgtgtc cggcaccaat 240

ggcacaaaga gattcgacaa tcctgtgctg cccttcaacg atggcgtgta cttcgcctcc 300

accgagaaga gcaacatcat ccgcggctgg atctttggca ccaacactgga ctctaagaca 360

cagtccctgc tgatcgtgaa caatgctacc aacgtggtca tcaaggtgtg cgagttccag 420

ttttgtaatg atcccttcct gggcgtgtac tatcataaga acaataagag ctggatggag 480

tctgagtttc gggtgtattc ttccgccaac aattgtacat ttgagtacgt gtctcagcct 540

ttcctgatgg acctggaggg caagcagggc aatttcaaga acctgaggga gttcgtgttt 600

aagaatatcg atggctactt caagatctac agcaagcaca cccccatcaa cctggtgcgg 660

gacctgcctc agggcttctc tgccctggag cccctggtgg atctgcctat cggcatcaac 720

atcaccaggt ttcagacact gctggccctg catcggtcct acctgacacc cggcgacagc 780

tcttccggat ggaccgctgg agctgctgcc tactatgtgg gctatctgca gcctaggacc 840

ttcctgctga agtacaacga gaatggcacc atcacagacg ctgtggattg cgccctggac 900

cctctgagcg agaccaagtg tacactgaag tcttttaccg tggagaaggg catctatcag 960

acatctaatt tcagagtgca gccaaccgag tccatcgtgc gctttcctaa tatcacaaac 1020

ctgtgcccat ttggcgaggt gttcaacgct acccggttcg ccagcgtgta cgcttggaat 1080

aggaagcgga tcagcaactg cgtggccgac tattctgtgc tgtacaactc cgcctccttc 1140

tccaccttca agtgctatgg cgtgtccccc acaaagctga atgacctgtg ctttaccaac 1200

gtgtacgctg attcgttcgt gatcaggggc gacgaggtgc ggcagatcgc tcctggacag 1260

acaggcaaga tcgctgacta caattataag ctgccagacg acttcaccgg ctgcgtgatc 1320

gcctggaaca gcaacaatct ggattctaaa gtgggcggca actacaatta tctgtacaga 1380

ctgtttcgca agagcaatct gaagcccttc gagagggaca tctccacaga gatctaccag 1440

gccggcagca ccccttgcaa tggcgtggag ggctttaact gttatttccc actgcagtct 1500

tacggcttcc agcccaccaa cggcgtgggc tatcagcctt accgggtggt ggtgctgtct 1560

tttgagctgc tgcacgctcc agctacagtg tgcggaccca agaagtccac caatctggtg 1620

aagaacaagt gcgtgaactt caacttcaac ggactgaccg gaacaggcgt gctgaccgag 1680

agcaacaaga agttcctgcc atttcagcag ttcggcagag acatcgccga taccacagac 1740

gctgtgcgcg accccacagac cctggagatc ctggatatca caccctgctc cttcggcggc 1800

gtgagcgtga tcacaccagg aaccaataca tctaaccagg tggccgtgct gtatcaggac 1860

gtgaactgta ccgaggtgcc tgtggctatc cacgccgatc agctgacccc aacatggagg 1920

gtgtactcta ccggctccaa cgtgtttcag acaagggctg gatgtctgat cggagctgag 1980

catgtgaaca atagctatga gtgcgacatc ccaatcggcg ccggcatctg tgcttcctac 2040

cagacccaga caaacagccc aggaggaagc ggatctgtgg cttcccagag catcatcgct 2100

tataccatgt ccctgggcgc cgagaatagc gtggcttaca gcaacaatag catcgctatc 2160

ccaaccaact tcacaatctc cgtgaccaca gagatcctgc ccgtgtctat gaccaagaca 2220

tccgtggact gcacaatgta tatctgtggc gattccaccg agtgcagcaa cctgctgctg 2280

cagtacggct cgttttgtac ccagctgaat agagccctga caggcatcgc tgtggagcag 2340

gataagaaca cacaggaggt gttcgcccag gtgaagcaga tctacaagac cccacccatc 2400

aaggactttg gcggcttcaa tttttctcag atcctgccag atccctccaa gcccagcaag 2460

agatctttta tcgaggacct gctgttcaac aaggtgaccc tggctgatgc cggcttcatc 2520

aagcagtatg gcgattgcct gggcgacatc gctgctaggg acctgatctg tgcccagaag 2580

tttaatggcc tgaccgtgct gcctccactg ctgacagatg agatgatcgc tcagtacaca 2640

tccgccctgc tggccggcac catcacatct ggatggacct tcggcgctgg agctgccctg 2700

cagatccctt ttgccatgca gatggcttat cgcttcaacg gcatcggcgt gacccagaat 2760

gtgctgtacg agaaccagaa gctgatcgcc aatcagttta actccgctat cggcaagatc 2820

caggactctc tgagctctac agcctccgcc ctgggcaagc tgcaggatgt ggtgaatcag 2880

aacgctcagg ccctgaatac cctggtgaag cagctgtcca gcaacttcgg cgccatctct 2940

tccgtgctga atgatatcct gagcagactg gacccccctg aggctgaggt gcagatcgac 3000

agactgatca caggccgcct gcagagcctg cagacctacg tgacacagca gctgatcagg 3060

gctgccgaga tccgggcttc tgccaacctg gctgccacca agatgtctga gtgcgtgctg 3120

ggccagtcca agcgcgtgga cttttgtggc aagggctatc acctgatgtc tttccctcag 3180

tccgccccac acggcgtggt gtttctgcat gtgacctacg tgcccgctca ggagaagaac 3240

ttcaccacag ctcctgccat ctgccacgat ggcaaggccc attttccaag agagggcgtg 3300

ttcgtgtcta acggcaccca ttggtttgtg acacagcgca atttctacga gccccagatc 3360

atcaccacag acaatacctt cgtgtccggc aactgtgacg tggtcatcgg catcgtgaac 3420

aataccgtgt atgatcccct gcagcctgag ctggactctt ttaaggagga gctggataag 3480

tacttcaaga atcatacctc ccctgacgtg gatctgggcg acatcagcgg catcaatgcc 3540

tctgtggtga acatccagaa ggagatcgac aggctgaacg aggtggctaa gaatctgaac 3600

gagtccctga tcgatctgca ggagctgggc aagtatgagc agtacatcaa gtggccaggc 3660

agcggctata tcccagaggc tccaagagac ggacaggctt acgtgcgcaa ggatggcgag 3720

tgggtgctgc tgtctacctt cctgtgatga 3750

Claims (10)

1. A composite adjuvant comprising CpG oligodeoxynucleotides, QS-21 and liposomes, wherein the CpG oligodeoxynucleotides are CpG7909. 2. composite adjuvant according to claim 1, wherein, relative to the gross weight of CpG oligodeoxynucleotide, QS-21 and liposome, the content of CpG oligodeoxynucleotide is 10-30% by weight. 3. The composite adjuvant according to claim 1, wherein, relative to the total weight of CpG oligodeoxynucleotides, QS-21 and liposomes, the content of QS-21 is 1-5% by weight. 4. The composite adjuvant according to any one of claims 1-3, wherein the liposome comprises phosphatidylcholine and cholesterol. 5. The composite adjuvant according to claim 4, wherein, relative to the total weight of CpG oligodeoxynucleotides, QS-21 and liposomes, the content of the phosphatidylcholine is 40-60% by weight. 6. The composite adjuvant according to claim 4 or 5, wherein, relative to the total weight of CpG oligodeoxynucleotides, QS-21 and liposomes, the cholesterol content is 10-15% by weight. 7. The composite adjuvant according to claim 4, wherein the phosphatidylcholine is dioleoylphosphatidylcholine. 8. A vaccine formulation comprising the composite adjuvant according to any one of claims 1-6. 9. The vaccine formulation of claim 8, wherein the vaccine formulation is a novel coronavirus vaccine formulation. 10. The vaccine formulation according to claim 9, wherein the novel coronavirus vaccine formulation comprises the spike protein of the novel coronavirus with the amino acid sequence of SEQ ID No. 2.

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