CN113521275B

CN113521275B - Composite adjuvant and novel crown COVID-19 recombinant protein vaccine using same

Info

Publication number: CN113521275B
Application number: CN202010308118.6A
Authority: CN
Inventors: 郑泽宇; 王治伟; 范鑫; 张玮; 吕哲; 戈小琴; 李雅静; 高强
Original assignee: Sinovac Research & Development Co ltd
Current assignee: Sinovac Research & Development Co ltd
Priority date: 2020-04-17
Filing date: 2020-04-17
Publication date: 2024-06-25
Anticipated expiration: 2040-04-17
Also published as: CN113521275A

Abstract

The invention discloses a composite adjuvant containing CpG oligodeoxynucleotide and aluminum adjuvant, and a novel crown COVID-19 recombinant protein vaccine using the composite adjuvant. The CpG oligodeoxynucleotide can be used in combination with aluminum adjuvant to produce strong immunity enhancement effect. The composite adjuvant is applied to a novel crown COVID-19 recombinant protein vaccine, so that the residence time of the composite adjuvant in the body can be prolonged to a great extent, and the hydrolysis action of various hydrolases on the composite adjuvant is reduced. The novel coronavirus COVID-19 recombinant protein vaccine using the composite adjuvant provided by the invention has the advantages of simple preparation method, easy quality control, easy mass production, good safety and low toxic and side effects, and is particularly suitable for preventing and treating novel coronaviruses COVID-19 in various crowds including the crowds with low immune functions such as middle-aged and elderly people.

Description

Composite adjuvant and novel crown COVID-19 recombinant protein vaccine using same

Technical Field

The invention relates to the field of epidemic prevention and vaccine production, in particular to a composite adjuvant containing CpG oligodeoxynucleotide and aluminum adjuvant and a novel crown COVID-19 recombinant protein vaccine using the composite adjuvant.

Background

The novel coronavirus COVID-19 belongs to the coronavirus beta family, can be transmitted through the paths of respiratory tract spray transmission, contact, faeces-mouth transmission and the like, and is extremely easy to infect in the crowd. There is currently no specific treatment for the disease caused by the novel coronavirus.

The current vaccine for preventing virus infection applied to human body mainly comprises attenuated live vaccine prepared by attenuated virus, such as varicella attenuated live vaccine, measles attenuated live vaccine, etc.; and inactivated whole virus particle vaccines, such as enterovirus EV71 type inactivated vaccine, hepatitis A vaccine, and the like. However, attenuated vaccines take a long time to propagate the virus for many generations and then gradually attenuate most of the activity of the virus, making it difficult to meet the needs for prevention of acute infectious diseases. Inactivated vaccines have a number of problematic immunopathology problems, such as the possibility of some inactivated viruses entering humans exacerbating the disease during viral entry.

The recombinant protein vaccine is prepared through constructing the target antigen gene of virus on expression vector, converting to bacteria, yeast, mammal or insect cell, expressing great amount of antigen protein under certain inducing condition and purifying. The recombinant protein vaccine can induce organism to generate humoral immunity and cellular immunity, and can be rapidly prepared.

In the development of vaccines, the use of adjuvants must be considered in order to prepare a safe and effective vaccine. The adjuvant and the vaccine component are compatible, so that a stable, safe and immunogenic vaccine compound is formed. Thus, research into adjuvants for use in vaccines has been an important step in the research process of vaccines. Adjuvants as nonspecific immunopotentiators play a critical role in inducing an effective immune response after vaccination.

Conventional aluminum salt adjuvants, including aluminum hydroxide and aluminum phosphate and combinations thereof, are most widely used. Although the above adjuvant is widely approved for use in vaccine preparation, in practical application, the use of low-dose aluminum salt adjuvant alone has limited immunopotentiation effect on vaccine, while the use of increased dose aluminum salt adjuvant often causes side effects such as injection site swelling, granuloma, fever, pain, allergy, etc. Therefore, it is urgent to develop an ideal vaccine adjuvant that is broader, safer, more efficient, and at the same time, easier to produce and use.

Disclosure of Invention

A first object of the present invention is to provide a compound adjuvant. The composite adjuvant provided by the invention comprises CpG oligodeoxynucleotide and aluminum adjuvant. Preferably, the composite adjuvant consists of CpG oligodeoxynucleotides and an aluminum adjuvant.

CpG refers to dinucleotides composed of cytosine (C) and guanine (G) linked via phosphodiester bonds (phosphodiesterbonds, p), and CpG dinucleotides and two bases at the 5 'and 3' ends thereof form a CpG motif (CpG motifs). CpG motifs are also known as immunostimulatory sequences (immunostimulatory sequence, ISS), whereas CpG ODNs refer to oligodeoxynucleotides containing unmethylated CpG motifs. The immune system of vertebrates recognizes CpG DNA in the bacterial genome as a dangerous stimulatory signal through pattern recognition receptors (pattern recognition receptors, PRRs), thereby stimulating the body to produce an immunoprotection response.

The aluminium adjuvant according to the invention is selected from aluminium hydroxide and aluminium phosphate, preferably aluminium hydroxide. In terms of immune response mechanism, aluminum adjuvants' immune response is biased towards T helper cell type 2 (Th 2) responses, which are characterized by secretion of Th2 cytokines, such as IL-4 and IL-5, and production of IgG1 and IgE type antibodies. Studies have shown that aluminium hydroxide adjuvants can activate Th2 cells to secrete IL-4 and induce expression of MHC class II molecules and CD83, CD86, etc., ultimately inducing a humoral immune response, but a weak or absent single Cytotoxic T Lymphocyte (CTL) response. Whereas CpG ODN can promote maturation and activation of dendritic cells, macrophages and B cells, up-regulate expression of CD80, CD86, CD40 and MHC-II molecules, promote secretion of IL-6, IL-12, IFN-gamma and other Thl type cytokines, and induce the organism to generate Thl type immune response. In addition, cpG ODN is recognized by and bound to TLR9 on endosomes/lysosomes in cells after uptake into immune cells via endocytosis, resulting in dimerization of TLR9, ultimately causing related immune cells to secrete a range of cytokines and chemokines, triggering intracellular bactericidal mechanisms or inducing inflammatory responses, producing strong CTL induction.

The traditional aluminum adjuvant and CpG oligodeoxynucleotide are combined for use, so that complementation on an immune mechanism can be realized, and cell immunity and humoral immunity are stimulated at the same time, so that a stronger immune effect is generated. By balancing Th1 and Th2 immune responses, the risk of ADE is reduced.

As a preferred embodiment of the invention, the CpG oligodeoxynucleotide contains at least 2 CpG units, the chain length of which is at least 20bp, and all nucleotides in the CpG oligodeoxynucleotide are thio-modified.

As a preferred embodiment of the present invention, the CpG oligodeoxynucleotide sequence is selected from the group consisting of:

SEQ7:5'-TGACTGTGAACGTTCGAGATGA-3'; or (b)

SEQ8:5'-TCGACGTTCGTCGTTCGTCGTTC-3'; or (b)

SEQ9：5’TCGTCGTTTTGTCGTTTTGTCGTT-3’。

As a preferred scheme of the invention, the mass ratio of the CpG oligodeoxynucleotide to the aluminum adjuvant is 1: (0.01-1). Further preferably, the mass ratio of the CpG oligodeoxynucleotide to the aluminum adjuvant is 1: (0.06-0.09), 1: (0.1 to 0.15), 1: (0.2 to 0.5) or 1: (0.6-0.9). Specifically, the mass ratio of the CpG oligodeoxynucleotide to the aluminum adjuvant may be 1:0.06, 1:0.09, 1:0.1, 1:0.15, 1:0.2, 1:0.5, 1:0.6 or 1:0.9. according to the invention, the highest-efficiency compound adjuvant proportioning scheme is determined by researching the combination proportioning scheme of the CpG oligodeoxynucleotide and the aluminum adjuvant, so that the compound adjuvant can generate a strong immune enhancement effect.

A second object of the present invention is to provide the use of said complex adjuvant as an immunopotentiator or immunoadjuvant for a vaccine, or in increasing the immunogenicity of an antigen or vaccine of a vaccine.

As a preferred embodiment of the invention, the vaccine is a novel recombinant protein vaccine of COVID-19. The composite adjuvant is used as an adjuvant of the novel crown COVID-19 recombinant protein vaccine, can better excite cellular immunity and generates stronger immune effect. The preparation method is simple, the quality is easy to control, and the large-scale production is easy. Can be used for effectively preventing and treating pneumonia caused by infection of novel coronavirus COVID-19, even serious respiratory tract infection. Provides technical support for preventing and controlling diseases in China and even worldwide.

A third object of the present invention is to provide a vaccine using the composite adjuvant. The vaccine provided by the invention can achieve the effects of enhancing the immunity, prolonging the immunity time and reducing the antigen dosage due to the adoption of the composite adjuvant.

As a preferred embodiment of the present invention, in the vaccine, the dose of the compound adjuvant is 0.3 mg/dose to 6 mg/dose.

As a preferred embodiment of the present invention, the content of the antigen in the vaccine is 1. Mu.g/dose to 80. Mu.g/dose.

A fourth object of the present invention is to provide a novel corona COVID-19 recombinant protein vaccine using the composite adjuvant. The novel crown COVID-19 recombinant protein vaccine provided by the invention can achieve the effects of enhancing the immunity, prolonging the immunity time and reducing the antigen dosage due to the adoption of the composite adjuvant.

As a preferred embodiment of the present invention, the antigen of the novel crown COVID-19 recombinant protein vaccine comprises the receptor binding domain of the S protein of COVID-19 and the Fc fragment of IgG; or an ACE2 receptor binding domain and/or surface epitope comprising an S protein, which is the S protein of SARS or the S protein of COVID-19.

As a preferred embodiment of the present invention, the antigen of the vaccine has one or more amino acid sequences of SEQ1, SEQ2, SEQ3, SEQ4, SEQ5 and SEQ6, or an amino acid sequence having a similarity of 90% or more thereto. The preferred amino acid sequence of SEQ1 of the present invention encodes a fusion protein comprising the receptor binding domain of the S protein of CoVID-19 and the Fc fragment of IgG. The preferred amino acid sequences of SEQ2, SEQ3, SEQ4, SEQ5 and SEQ6 of the present invention are those which are highly conserved in the amino acid sequences of the S proteins of each of 2003Sars-CoV and CoVID-19. These sequences, although of varying length, cover the site of binding of the Spike protein to the ACE2 receptor and the major protein surface epitopes.

The amino acid sequences are shown in the sequence table of the invention, and are shown in the following table 1.

Table 1: amino acid sequence of interest

As a preferable scheme of the invention, in the novel crown COVID-19 recombinant protein vaccine, the dosage of the compound adjuvant is 0.3 mg/dose-6 mg/dose. Specifically, the content of the compound adjuvant in the recombinant protein vaccine may be 0.8 mg/dose, 0.95 mg/dose, 3.3 mg/dose, 3.45 mg/dose, 5.3 mg/dose, or 5.45 mg/dose.

As a preferable scheme of the invention, the novel crown COVID-19 recombinant protein vaccine contains 0.25-5 mg/dose of CpG oligodeoxynucleotide adjuvant. Specifically, the content of the CpG oligodeoxynucleotide adjuvant in the recombinant protein vaccine can be 0.25 mg/dose, 0.5 mg/dose, 1 mg/dose, 3 mg/dose, 2 mg/dose or 5 mg/dose.

As a preferable scheme of the invention, the novel crown COVID-19 recombinant protein vaccine contains 0.10-0.45 mg/dose of aluminum hydroxide adjuvant. Specifically, the content of the aluminum hydroxide adjuvant in the recombinant protein vaccine can be 0.15 mg/dose, 0.3 mg/dose or 0.45 mg/dose.

As a preferred embodiment of the present invention, the antigen content in the novel recombinant protein vaccine of the crown COVID-19 is 1. Mu.g/dose to 80. Mu.g/dose, preferably 20. Mu.g/dose to 40. Mu.g/dose. In particular, the antigen content of the recombinant vaccine may be 2.5 μg/dose, 10 μg/dose, 20 μg/dose, 40 μg/dose or 80 μg/dose.

As a preferred scheme of the invention, the novel crown COVID-19 recombinant protein vaccine is a liquid preparation and can be in various dosage forms. In particular, the recombinant protein vaccine may be an intramuscular liquid injection, an intravenous liquid injection, an intranasal liquid injection, an intradermal liquid injection or a subcutaneous liquid injection. In practical application, the preparation can be adjusted and selected according to clinical requirements such as transfection efficiency, local immunity monitoring and the like, for example, single dosage form is selected for injection immunization, or multiple mixed dosage forms are selected for injection immunization.

A fifth object of the present invention is to provide the use of the novel recombinant protein vaccine of crown COVID-19 using the composite adjuvant in the preparation of a medicament for preventing and/or treating diseases caused by COVID-19 infection. The disease is preferably pneumonia and syndrome, severe acute respiratory tract infection, intestinal tract disease, heart failure, renal failure or severe acute respiratory syndrome.

The novel crown COVID-19 recombinant protein vaccine using the composite adjuvant provided by the invention can effectively induce organisms to generate cellular immunity and humoral immunity, can be rapidly prepared, and is suitable for controlling epidemic situations of novel coronaviruses COVID-19.

Drawings

FIG. 1 is a schematic representation of serum IgG titers of COVID-19 recombinant vaccine immunized BALB/c mice with different CpG adjuvant and aluminum hydroxide adjuvant formulation schemes.

FIG. 2 is a schematic diagram showing the results of specific expression of S protein cytokines in serum.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

In each of the following examples and comparative example 2, the sequence of the CpG oligodeoxynucleotide (adjuvant) was 5'-TGACTGTGAACGTTCGAGATGA-3'.

In the following examples and comparative examples, the vaccine antigens have the sequences shown in SEQ 6.

Example 1

The embodiment provides a compound adjuvant composed of CpG oligodeoxynucleotide and aluminum hydroxide; wherein the mass ratio of the CpG oligodeoxynucleotide to the aluminum hydroxide is 1:0.6.

The present example further provides a combination of the compound adjuvant and a novel coronavirus COVID-19 recombinant vaccine.

The method comprises the following steps: the CpG oligodeoxynucleotide adjuvant and the aluminum hydroxide adjuvant are added into the vaccine stock solution filtered by a 0.22 mu m filter membrane, so that the concentration of the CpG adjuvant is 0.5 mg/dose, the content of the aluminum hydroxide adjuvant is 0.3 mg/dose, and the content of the vaccine antigen is 20 mu g/dose. The vaccine number obtained in this example is designated 20200401.

Example 2

The method comprises the following steps: the CpG oligodeoxynucleotide adjuvant and the aluminum hydroxide adjuvant are added into the vaccine stock solution filtered by a 0.22 mu m filter membrane, so that the concentration of the CpG adjuvant is 0.5 mg/dose, the content of the aluminum hydroxide adjuvant is 0.3 mg/dose, and the content of the vaccine antigen is 40 mu g/dose. The vaccine number obtained in this example is designated 20200402.

Example 3

The embodiment provides a compound adjuvant composed of CpG oligodeoxynucleotide and aluminum hydroxide; wherein the mass ratio of the CpG oligodeoxynucleotide to the aluminum hydroxide is 1:0.9.

The method comprises the following steps: the CpG oligodeoxynucleotide adjuvant and the aluminum hydroxide adjuvant are added into the vaccine stock solution filtered by a 0.22 mu m filter membrane, so that the concentration of the CpG adjuvant is 0.5 mg/dose, the content of the aluminum hydroxide adjuvant is 0.45 mg/dose, and the content of the vaccine antigen is 20 mu g/dose. The vaccine number obtained in this example is designated 20200403.

Example 4

The method comprises the following steps: the CpG oligodeoxynucleotide adjuvant and the aluminum hydroxide adjuvant are added into the vaccine stock solution filtered by a 0.22 mu m filter membrane, so that the concentration of the CpG adjuvant is 0.5 mg/dose, the content of the aluminum hydroxide adjuvant is 0.45 mg/dose, and the content of the vaccine antigen is 40 mu g/dose. The vaccine number obtained in this example is designated 20200404.

Example 5

The embodiment provides a compound adjuvant composed of CpG oligodeoxynucleotide and aluminum hydroxide; wherein the mass ratio of the CpG oligodeoxynucleotide to the aluminum hydroxide is 1:0.1.

The method comprises the following steps: the CpG oligodeoxynucleotide adjuvant and the aluminum hydroxide adjuvant are added into the vaccine stock solution filtered by a 0.22 mu m filter membrane, so that the concentration of the CpG adjuvant is 3 mg/dose, the content of the aluminum hydroxide adjuvant is 0.3 mg/dose, and the content of the vaccine antigen is 20 mu g/dose. The vaccine number obtained in this example is designated 20200405.

Example 6

The method comprises the following steps: the CpG oligodeoxynucleotide adjuvant and the aluminum hydroxide adjuvant are added into the vaccine stock solution filtered by a 0.22 mu m filter membrane, so that the concentration of the CpG adjuvant is 3 mg/dose, the content of the aluminum hydroxide adjuvant is 0.3 mg/dose, and the content of the vaccine antigen is 40 mu g/dose. The vaccine number obtained in this example is designated 20200406.

Example 7

The embodiment provides a compound adjuvant composed of CpG oligodeoxynucleotide and aluminum hydroxide; wherein the mass ratio of the CpG oligodeoxynucleotide to the aluminum hydroxide is 1:0.15.

The method comprises the following steps: the CpG oligodeoxynucleotide adjuvant and the aluminum hydroxide adjuvant are added into the vaccine stock solution filtered by a 0.22 mu m filter membrane, so that the concentration of the CpG adjuvant is 3 mg/dose, the content of the aluminum hydroxide adjuvant is 0.45 mg/dose, and the content of the vaccine antigen is 20 mu g/dose. The vaccine number obtained in this example is designated 20200407.

Example 8

The method comprises the following steps: the CpG oligodeoxynucleotide adjuvant and the aluminum hydroxide adjuvant are added into the vaccine stock solution filtered by a 0.22 mu m filter membrane, so that the concentration of the CpG adjuvant is 3 mg/dose, the content of the aluminum hydroxide adjuvant is 0.45 mg/dose, and the content of the vaccine antigen is 40 mu g/dose. The vaccine number obtained in this example is designated 20200408.

Example 9

The embodiment provides a compound adjuvant composed of CpG oligodeoxynucleotide and aluminum hydroxide; wherein the mass ratio of the CpG oligodeoxynucleotide to the aluminum hydroxide is 1:0.06.

The method comprises the following steps: the CpG oligodeoxynucleotide adjuvant and the aluminum hydroxide adjuvant are added into the vaccine stock solution filtered by a 0.22 mu m filter membrane, so that the concentration of the CpG adjuvant is 5 mg/dose, the content of the aluminum hydroxide adjuvant is 0.3 mg/dose, and the content of the vaccine antigen is 20 mu g/dose. The vaccine number obtained in this example is designated 20200409.

Example 10

The method comprises the following steps: the CpG oligodeoxynucleotide adjuvant and the aluminum hydroxide adjuvant are added into the vaccine stock solution filtered by a 0.22 mu m filter membrane, so that the concentration of the CpG adjuvant is 5 mg/dose, the content of the aluminum hydroxide adjuvant is 0.3 mg/dose, and the content of the vaccine antigen is 40 mu g/dose. The vaccine number obtained in this example is designated 20200410.

Example 11

The embodiment provides a compound adjuvant composed of CpG oligodeoxynucleotide and aluminum hydroxide; wherein the mass ratio of the CpG oligodeoxynucleotide to the aluminum hydroxide is 1:0.09.

The method comprises the following steps: the CpG oligodeoxynucleotide adjuvant and the aluminum hydroxide adjuvant are added into the vaccine stock solution filtered by a 0.22 mu m filter membrane, so that the concentration of the CpG adjuvant is 5 mg/dose, the content of the aluminum hydroxide adjuvant is 0.45 mg/dose, and the content of the vaccine antigen is 20 mu g/dose. The vaccine number obtained in this example is designated 20200411.

Example 12

The method comprises the following steps: the CpG oligodeoxynucleotide adjuvant and the aluminum hydroxide adjuvant are added into the vaccine stock solution filtered by a 0.22 mu m filter membrane, so that the concentration of the CpG adjuvant is 5 mg/dose, the content of the aluminum hydroxide adjuvant is 0.45 mg/dose, and the content of the vaccine antigen is 40 mu g/dose. The vaccine number obtained in this example is designated 20200412.

Comparative example 1

This comparative example provides a combination of an aluminum hydroxide adjuvant and a novel coronavirus COVID-19 recombinant vaccine.

The method comprises the following steps: the aluminium hydroxide adjuvant is added into the vaccine stock solution filtered by a 0.22 mu m filter membrane, so that the content of the aluminium hydroxide adjuvant is 0.45 mg/dose and the content of the vaccine antigen is 20 mu g/dose. The vaccine number obtained in this comparative example is designated 20200413.

Comparative example 2

This comparative example provides a combination of a CpG oligodeoxynucleotide adjuvant and a novel coronavirus COVID-19 recombinant vaccine.

The method comprises the following steps: the CpG oligodeoxynucleotide adjuvant is added into the vaccine stock solution filtered by a 0.22 mu m filter membrane, so that the concentration of the CpG adjuvant is 5 mg/dose, and the content of the vaccine antigen is 20 mu g/dose. The vaccine number obtained in this comparative example is designated 20200414.

Experimental example 1: immunological evaluation of adjuvant Effect on mouse model

1. Neutralizing antibody titer determination

The total 14 vaccine groups provided in each example and comparative example were diluted 1:4, 1:16, and 1:64 with physiological saline, and BALB/c mice were vaccinated using a 1mL syringe, 10 mice per group were immunized on day 0,7, one dose per abdominal cavity, and 4 weeks after the 1 st immunization were bled, and the neutralizing antibodies were detected. Neutralizing antibody titers GMT greater than 8 were considered positive and were considered protective. The results are shown in Table 2.

Table 2: neutralization titers of COVID-19 recombinant vaccine immunized BALB/c mice

The results show that COVID-19 vaccine immunization of BALB/c mice with different effective components and adjuvant contents can generate enough high neutralizing antibody titer. Vaccines with high adjuvant content are relatively more immunogenic than vaccines with low content of the same active ingredient. The invention provides a vaccine which can induce BALB/c mice to generate neutralizing antibodies with protective capability.

2. IgG antibody titer determination

A total of 14 vaccine groups as provided in each example and comparative example were subjected to IgG antibody titer determination. The method for detecting the titer of the IgG antibody is as follows:

Coating the new coronavirus on an ELISA plate according to the protein concentration of 1 mug/ml and 100 mug/hole, coating overnight at 2-8 ℃ or coating for more than 2 hours at 37 ℃, washing the plate and beating to dry, sealing by using 0.01M PBS containing 1% BSA or 10% calf serum, sealing by 200 mug/hole, sealing for 1-2 hours at 37 ℃, throwing out the liquid, and draining for standby. Serial dilution is carried out on the sample to be detected and negative serum control by adopting the sealing liquid, 100 mu l of each hole is added with a sealed ELISA plate, incubation is carried out for 60-70 minutes at 37 ℃, and the plate is washed and dried; adding HRP enzyme-labeled antibody corresponding to the anti-species, incubating for 45-60 minutes at 37 ℃, washing the plate and beating; after 50. Mu.l of each of the color-developing A/B solutions was added and the color development was performed at 37℃for 10-15 minutes, 2M H2SO4 was added to terminate. Analysis of results: the highest dilution factor when the OD value of the sample is more than or equal to 2.1 times of the OD value of the negative serum under the same dilution factor is the IgG antibody titer of the sample. If the negative control OD value is less than 0.05, it is calculated as 0.05.

The results are shown in FIG. 1. In the bar graph shown in fig. 1, the IgG antibody titer results for each vaccine group consisted of two left and right panels, with the left panel representing 7 days (7D) and the right panel representing 14 days (14D).

Experimental example 2: cytokine detection

The total 14 vaccine groups provided in each example and comparative example were immunized according to the procedures of 0, 7 days immunization and 28 days blood collection, and BALB/c mice were immunized respectively, and cell supernatants were collected for cell immunoassay. And detecting CD4+T cells which specifically express the S protein cytokines in serum by adopting an intracellular cytokine staining method and a flow cytometry method.

1. Separation of spleen lymphocytes

Mice were sacrificed by cervical dislocation and immersed in 70% alcohol for about 3min. The mice were aseptically removed from the spleens in a biosafety cabinet and placed on a 200 mesh cell sieve placed in a sterile dish. 10mL of RPMI1640 complete medium was added, the spleen was gently ground into single cells with a syringe plunger, and the cell sieve was rinsed with 10mL of RPMI1640 complete medium to obtain more spleen cells. The spleen cell suspension was transferred to a 50mL centrifuge tube and centrifuged at 500g level for 5min. The supernatant was discarded, the cells resuspended in 3m1×erythrocyte lysate, lysed at room temperature for 5min, added 27mL of RPM 1640 complete medium and centrifuged at 500g level for 5min. The supernatant was discarded, the cells were washed once with 20mL of complete medium of RPM 1640, resuspended in appropriate amount of medium, filtered through a 200 mesh cell sieve into 10mL tubes, and 50. Mu.L diluted 20-fold were counted for use.

2. In vitro stimulation of mouse spleen cells

The above isolated mouse spleen cells were diluted to 4X 10 ⁶ cells/mL in appropriate amounts and added to a 24-well plate at 0.5mL per well. Each mouse was provided with a specific CTL epitope stimulation well and no stimulation well, respectively. Specific epitope concentration was 2 μg/mL per peptide, and equivalent amount of DMSO was added without stimulation. As a positive control, wells were stimulated with PMA and ionomycin, where the PMA concentration was 100ng/mL and ionomycin concentration was 1. Mu.g/mL. While 1. Mu. L Brilliant Violet. Mu.421. TM. Anti-mouse CD107a was added to each well. After culturing the cells in a 5% CO2 cell incubator at 37℃for 1 hour, appropriate amounts of GolgiStop and/or GolgiPlug were added to each well as blockers of cytokine secretion. Staining of the relevant antigen was performed after a total of 6 hours of incubation for flow cytometry detection of intracellular cytokines.

3. Cell surface antigen and intracellular cytokine staining

After 6 hours of in vitro stimulation, splenocytes were transferred to a flow tube and centrifuged at 500g for 5 minutes at 4℃and the supernatant discarded. A suitable amount of the fluorescent-labeled antibodies PerCP/Cy5.5-conjugated anti-CD3 (clone 145-2c 11) and FITC conjugated anti-CD8 (clone 53-6.7) was diluted with PBS+2% FBS in the amounts recommended in the instructions, 50. Mu.L of each tube was added, gently mixed, and left at 4℃for 30 minutes. After 30 minutes, 3mL PBS+2% FBS was added to each tube, and 500g was centrifuged for 5 minutes at 4℃and the supernatant was discarded. mu.L Cytofix/CytopermTM Fixation and Permeabilizaiton Solution was added to each tube, and the cells were fixed and perforated by placing at 4℃for 20 minutes. After 20 minutes, 1mL of 1 XPerm/WashTM Buffer was added to each tube, centrifuged at 600g for 5 minutes at 4℃and the supernatant was discarded. A suitable amount of PE conjugated anti-IFN-gamma (clone XMG 1.2) antibody was diluted with 1 XPerm/WashTM Buffer in accordance with the instructions recommended to use, 50. Mu.L of each tube was added, gently mixed and left at 4℃for 30 minutes. Finally, each tube was washed once with 1mL of 1 XPerm/WashTM Buffer and 3mL of PBS, respectively, the supernatant was discarded and resuspended in 200. Mu.L of PBS and checked on the machine. To adjust the fluorescence compensation between the dyes at the time of detection, a non-staining tube, a single-staining PerCP/Cy5.5-conjugated anti-CD3 tube, a single-staining FITC conjugated anti-CD8 tube and a single-staining PEconjugated anti-IFN-gamma tube were set, wherein the PE conjugated anti-IFN-gamma single-staining tube used positively stimulated cells.

4. Flow cytometry detection

Flow cytometry detection was performed using BD FACS CantoTM. Firstly, regulating proper voltage of each channel, regulating fluorescence compensation among dyes by using a single fluorescence staining sample, then loading samples in sequence, and collecting data.

5. Intracellular cytokine staining flow cytometry detection results

The results of the intracellular cytokine staining flow cytometry are shown in figure 2. From the results of this experimental example, the novel coronavirus COVID-19 recombinant protein vaccine can induce to raise the serum antibody level. The novel crown vaccine using the composite adjuvant can generate high-level specific antibodies when being used for immunizing mice, and the immune effect is obviously stronger than that of a control vaccine using the aluminum hydroxide adjuvant and the CpG oligodeoxynucleotide independently. This experiment shows the good effect of the composite adjuvant in the novel coronavirus COVID-19 recombinant protein vaccine.

While the invention has been described in detail in the foregoing general description, embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

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<210> 3

<211> 345

<212> PRT

<213> Artificial sequence

<400> 3

Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala

1 5 10 15

Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp

20 25 30

Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr

35 40 45

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

50 55 60

Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro

65 70 75 80

Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp

85 90 95

Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys

100 105 110

Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn

115 120 125

Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly

130 135 140

Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu

145 150 155 160

Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr

165 170 175

Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val

180 185 190

Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn

195 200 205

Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn

210 215 220

Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr

225 230 235 240

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

245 250 255

Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr

260 265 270

Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val

275 280 285

Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr

290 295 300

Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly

305 310 315 320

Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala

325 330 335

Gly Ile Cys Ala Ser Tyr Gln Thr Gln

340 345

<210> 4

<211> 197

<212> PRT

<213> Artificial sequence

<400> 4

Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg

1 5 10 15

Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val

20 25 30

Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys

35 40 45

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

50 55 60

Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile

65 70 75 80

Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro

85 90 95

Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp

100 105 110

Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys

115 120 125

Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln

130 135 140

Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe

145 150 155 160

Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln

165 170 175

Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala

180 185 190

Thr Val Cys Gly Pro

195

<210> 5

<211> 261

<212> PRT

<213> Artificial sequence

<400> 5

Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala

1 5 10 15

Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp

20 25 30

Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr

35 40 45

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

50 55 60

Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro

65 70 75 80

Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp

85 90 95

Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys

100 105 110

Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn

115 120 125

Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly

130 135 140

Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu

145 150 155 160

Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr

165 170 175

Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val

180 185 190

Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn

195 200 205

Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn

210 215 220

Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr

225 230 235 240

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

245 250 255

Pro Cys Ser Phe Gly

260

<210> 6

<211> 1213

<212> PRT

<213> Artificial sequence

<400> 6

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

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

115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

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

405 410 415

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

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

465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Ala Ser Val Ala

675 680 685

Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

690 695 700

Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

705 710 715 720

Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

725 730 735

Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

755 760 765

Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

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

805 810 815

Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

820 825 830

Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

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

885 890 895

Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

900 905 910

Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

915 920 925

Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

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

945 950 955 960

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

965 970 975

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

980 985 990

Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val

995 1000 1005

Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn

1010 1015 1020

Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys

1025 1030 1035

Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val

1055 1060 1065

Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His

1070 1075 1080

Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn

1085 1090 1095

Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln

1100 1105 1110

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

1115 1120 1125

Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro

1130 1135 1140

Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn

1145 1150 1155

His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro

1205 1210

<210> 7

<211> 22

<212> DNA

<213> Artificial sequence

<400> 7

tgactgtgaa cgttcgagat ga 22

<210> 8

<211> 23

<212> DNA

<213> Artificial sequence

<400> 8

tcgacgttcg tcgttcgtcg ttc 23

<210> 9

<211> 24

<212> DNA

<213> Artificial sequence

<400> 9

tcgtcgtttt gtcgttttgt cgtt 24

Claims

1. The application of a composite adjuvant in preparing a vaccine is characterized in that the vaccine is a novel crown COVID-19 recombinant protein vaccine, the composite adjuvant consists of CpG oligodeoxynucleotide and an aluminum adjuvant, wherein the sequence of the CpG oligodeoxynucleotide is SEQ7, and all nucleotides in the CpG oligodeoxynucleotide are thio-modified;

the mass ratio of the CpG oligodeoxynucleotide to the aluminum adjuvant is 1: (0.06-0.15);

In the vaccine, the dosage of CpG oligodeoxynucleotide is 3mg-5 mg/dose, the dosage of aluminum adjuvant is 0.30mg-0.45 mg/dose, the content of recombinant protein of the novel crown COVID-19 is 20 mug/dose-40 mug/dose, and the amino acid sequence of antigen of the vaccine is shown as SEQ 6.

2. The use according to claim 1, characterized in that the mass ratio of CpG oligodeoxynucleotide to aluminium adjuvant is 1: (0.06-0.09).

3. The novel crown COVID-19 recombinant protein vaccine containing the composite adjuvant is characterized in that the composite adjuvant consists of CpG oligodeoxynucleotide and aluminum adjuvant, the sequence of the CpG oligodeoxynucleotide is SEQ7, and all nucleotides in the CpG oligodeoxynucleotide are thio-modified; the mass ratio of the CpG oligodeoxynucleotide to the aluminum adjuvant is 1: (0.06-0.15);

In the novel crown COVID-19 recombinant protein vaccine, the dosage of CpG oligodeoxynucleotide is 3mg-5 mg/dose, the dosage of aluminum adjuvant is 0.30mg-0.45 mg/dose, and the content of novel crown COVID-19 recombinant protein is 20 mug/dose-40 mug/dose;

the amino acid sequence of the antigen of the vaccine is shown in SEQ 6.

4. The novel crown COVID-19 recombinant protein vaccine according to claim 3, wherein the mass ratio of CpG oligodeoxynucleotide to aluminum adjuvant is 1: (0.06-0.09).

5. The novel corona-COVID-19 recombinant protein vaccine according to any one of claims 3 to 4 wherein said vaccine is a liquid formulation.

6. The novel crown COVID-19 recombinant protein vaccine of claim 5, wherein said vaccine is an intramuscular liquid injection, an intranasal liquid spray, an intradermal liquid injection or a subcutaneous liquid injection.

7. Use of the novel crown COVID-19 recombinant protein vaccine of any one of claims 3-6 in the manufacture of a medicament for the prevention of a disease caused by COVID-19 infection.