CN112494644B - Composite adjuvant, vaccine containing composite adjuvant and application - Google Patents
Composite adjuvant, vaccine containing composite adjuvant and application Download PDFInfo
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- CN112494644B CN112494644B CN202011578434.1A CN202011578434A CN112494644B CN 112494644 B CN112494644 B CN 112494644B CN 202011578434 A CN202011578434 A CN 202011578434A CN 112494644 B CN112494644 B CN 112494644B
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Classifications
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55505—Inorganic adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55516—Proteins; Peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention provides a compound adjuvant, a vaccine containing the compound adjuvant and application thereof, and belongs to the technical field of vaccine adjuvant preparation. The polypeptide adjuvant in the composite adjuvant can effectively promote the maturation of DC cells, has better presenting effect on antigen, and can further effectively improve the immune response level of T cells. The compound adjuvant provided by the invention can enhance humoral immunity and enhance the immune response of T cells, can stimulate organisms to generate higher neutralizing antibodies, and has good immunogenicity and protective effect. The immune adjuvant can be placed at normal temperature, can be transported in a short time, and is a safe, stable and effective vaccine adjuvant.
Description
Technical Field
The invention relates to the technical field of immune adjuvants, in particular to a composite adjuvant, a vaccine containing the composite adjuvant and application thereof.
Background
The immune adjuvant is abbreviated as adjuvant, namely non-specific immunoproliferative agent, which refers to auxiliary substances which can enhance the immune response capability of an organism to an antigen or change the type of immune response together with the antigen or injected into the organism in advance. Aluminum adjuvants are the most commonly used conventional adjuvants, but aluminum adjuvants can only enhance humoral immune responses and local (injection site) macrophage responses, and the immune enhancing effect is not balanced.
Disclosure of Invention
The invention aims to provide a composite adjuvant, a vaccine containing the composite adjuvant and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a compound adjuvant, which comprises the following components: polypeptide adjuvants and aluminum adjuvants;
the polypeptide adjuvant has a structure shown in a formula I;
preferably, the formulation of the compound adjuvant comprises an injection.
Preferably, the concentration of the polypeptide adjuvant in the injection is 0.05-50 mg/ml; the concentration of the aluminum adjuvant is 1.75mg/ml.
Preferably, the polypeptide adjuvant and aluminum adjuvant are packaged separately.
The invention also provides application of the composite adjuvant in preparing vaccines.
The invention also provides a vaccine containing the composite adjuvant according to the scheme, and the vaccine also contains an immunogen.
Preferably, the vaccine comprises a novel corona vaccine.
Preferably, the immunogen comprises a novel coronavirus surface S protein.
Preferably, the mass ratio of the aluminum adjuvant to the immunogen in the vaccine is (0.1-10): 1.
the invention also provides a preparation method of the vaccine according to the scheme, which comprises the following steps: and (3) adsorbing the immunogen by using an aluminum adjuvant, and then adding a polypeptide adjuvant to obtain the vaccine.
The invention provides a compound adjuvant, which comprises the following components: polypeptide adjuvants and aluminum adjuvants; the polypeptide adjuvant has a structure shown in a formula I;
the polypeptide adjuvant in the composite adjuvant can effectively promote the maturation of DC cells, has better presenting effect on antigen, and can further effectively improve the immune response level of T cells. The compound adjuvant provided by the invention can enhance humoral immunity and enhance the immune response of T cells, can stimulate organisms to generate higher neutralizing antibodies, and has good immunogenicity and protective effect. The immune adjuvant can be placed at normal temperature, can be transported in a short time, and is a safe, stable and effective vaccine adjuvant.
Drawings
FIG. 1 shows MS results for a polypeptide adjuvant of the present invention;
FIG. 2 shows the HPLC detection results of the polypeptide adjuvant of the present invention;
FIG. 3 is an electron microscope detection result, wherein A is an electron microscope image of a polypeptide adjuvant, B is an aluminum adjuvant electron microscope image, and C is a compound adjuvant electron microscope image;
FIG. 4 shows ELISA detection results;
FIG. 5 shows the results of detection of neutralizing antibodies;
FIG. 6 shows IFN-. Gamma.ELISPOT assay results;
FIG. 7 is a flow cytometer analysis of secretion levels of CD 137;
FIG. 8 is a flow cytometer analysis of IL-2 secretion levels.
Detailed Description
The invention provides a compound adjuvant, which comprises the following components: polypeptide adjuvants and aluminum adjuvants; the polypeptide adjuvant has a structure shown in a formula I;
in the present invention, the polypeptide adjuvant is preferably obtained synthetically by gill company.
In the present invention, the minimum gel forming concentration of the polypeptide adjuvant is 0.25mg/ml. The polypeptide adjuvant can be assembled into hydrogel when the concentration of the polypeptide adjuvant is 0.25mg/ml, and the polypeptide adjuvant has low gel forming concentration, small dosage and low cost.
In the present invention, the amino acid sequence of the polypeptide adjuvant is: f (phenylalanine) -E (glutamic acid) -Y (tyrosine); the Y is modified by Nap (2-naphthylpropylamine); the relative molecular mass of the polypeptide is 851.93. In the present invention, the preparation method of the polypeptide adjuvant is not particularly limited, and conventional methods in the art may be employed.
The phenylalanine and Nap in the polypeptide adjuvant provided by the invention contain aromatic rings, so that beta-sheet can be promoted, the polypeptide adjuvant provided by the invention drives a single peptide to form beta-sheet by utilizing aromatic interaction, polypeptide assembly is promoted, the hydrogel assembled by the polypeptide can encapsulate antigen, antigen slow release is promoted, immune response of an organism can be continuously caused, and the immune response effect of a vaccine is effectively enhanced.
In the present invention, the mass ratio of the polypeptide adjuvant to the aluminum adjuvant is preferably 100: (35 to 3500), more preferably 100: (100 to 1000), more preferably 100: (175-200).
In the examples of the present invention, the aluminum adjuvant is also referred to as an aluminum hydroxide adjuvant, which is purchased from General Chemical company in the united states.
In the present invention, the formulation of the compound adjuvant preferably includes an injection. In the present invention, the concentration of the polypeptide adjuvant in the injection is preferably 0.05 to 50mg/ml, more preferably 1 to 20mg/ml, still more preferably 5 to 10mg/ml; the concentration of the aluminum adjuvant is preferably 1.75mg/ml.
In the invention, the polypeptide adjuvant and the aluminum adjuvant in the compound adjuvant are preferably packaged independently and are matched with immunogen in situ during use.
The invention also provides application of the composite adjuvant in preparing vaccines.
The invention also provides a vaccine containing the composite adjuvant according to the scheme, and the vaccine also contains an immunogen.
In the present invention, the vaccine preferably comprises a novel coronal vaccine. In the present invention, the immunogen preferably comprises a novel coronavirus surface S protein. In the invention, the mass ratio of the aluminum adjuvant to the immunogen in the vaccine is preferably (0.1-10): 1, more preferably (1 to 5): 1, still more preferably 3.5:1. in the present invention, the dosage form of the vaccine preferably includes an injection.
In the present invention, alkaline phosphatase is preferably further included in the vaccine; the amount of alkaline phosphatase added is 0.5 to 1.5. Mu.L/mL of the vaccine, and more preferably 1. Mu.L/mL of the vaccine. Alkaline phosphatase-mediated dephosphorylation can significantly enhance the hydrophobic effect of peptides, promote the formation of β -sheets from individual peptides, and promote polypeptide assembly.
The invention also provides a preparation method of the vaccine according to the scheme, which comprises the following steps: and (3) adsorbing the immunogen by using an aluminum adjuvant, and then adding a polypeptide adjuvant to obtain the vaccine.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The materials and reagents used in the examples of the present invention were purchased from: IFN-. Gamma.ELISPOT was purchased from Abcam; all animal experiments were approved by the animal ethics committee of the medical biology institute of academy of sciences of china, operating strictly according to the laboratory animal ethics committee.
Example 1
1. The polypeptide adjuvant shown in the formula I is obtained by synthesis of Jier company. FFEY (glutamic acid and phosphoric acid form methyl ester group, carboxyl of tyrosine is exposed) is added into HATU and DIEA to activate on a solid-phase polypeptide synthesizer, then the mixture is subjected to condensation reaction with amino of 2-naphthylpropylamine, and then the mixture is hydrolyzed in 0.5M aqueous solution of aluminum hydroxide to remove methyl ester protecting group, so that a target product can be obtained.
2. MS detection and HPLC detection are carried out on the polypeptide adjuvant, the MS detection result is shown in figure 1, the HPLC detection result is shown in figure 2, the relative molecular mass of the polypeptide is 851.93 according to the MS detection result, the peak is started in 12.247min, the peak is reached in 12.642min, the peak is ended in 13.294min, and the purity can reach 95.5829%.
3. Electron microscopy of the polypeptide adjuvant:
weighing 2mg of polypeptide, adding 1ml of 0.9% physiological saline, adjusting pH to 9 with NaOH, and diluting to 1mg/ml with 0.9% physiological saline or PBS when in use; the aluminum adjuvant is 1.75mg/ml; the mass ratio of the aluminum adjuvant to the polypeptide adjuvant is 100:175, respectively mixing with a gun, wherein the detection concentration of the polypeptide adjuvant is 1 mg/ml; three samples pass through phosphotungstic acid negative dye, copper mesh 300 meshes, observe the structure of polypeptide under the electron microscope, show as fibrous structure under the electron microscope, can wrap up the immunogen after forming the fibre. As shown in figure 3, wherein A is an electron microscope image of the polypeptide adjuvant, B is an aluminum adjuvant electron microscope image, and C is a compound adjuvant electron microscope image.
Example 2
SPF-class Balb/c mice were kept in a sterile environment at 6-8 weeks of age. Comprising 3 groups of 6 mice each. A. Novel coronatine + aluminum adjuvant + polypeptide adjuvant group; B. novel coronatine + aluminum adjuvant group; C. a novel coronal S proteome; and (5) immunization after mixing.
The specific method comprises the following steps: A. the novel coronal S protein, aluminum adjuvant and polypeptide adjuvant group is immunized for 2 times, the mass ratio of S protein to aluminum adjuvant is 1, wherein the S protein is 50 mug per mouse: 3.5, the polypeptide adjuvant is 100 mug/mouse;
B. new coronal S protein+aluminum adjuvant group, S protein is 50 mug/mouse, mass ratio of S protein to aluminum adjuvant is 1:3.5;
C. new coronal S protein, S protein is 50 mug/mouse;
the immunization mode is intramuscular injection, the immunization interval is 0 and 3 weeks, blood is collected 10 days after the last immunization, spleen is taken, and relevant detection such as flow type detection, ELISPOT, ELISA detection, protective experiment detection, histology detection and the like are carried out.
ELISA detection
With dilution (0.012 mol/LNa 2 CO 3 ，0.038mol/LNaHCO 3 pH 9.6) separately diluting the novel coronal S protein packageWere incubated in 96-well plates at a final concentration of 5. Mu.g/ml, 4℃overnight. PBST plates were washed 3 times, blocked with PBS containing 2% bsa, incubated at 37 ℃ for 2h. The serum of the mice to be tested which is separated after the tail vein blood sampling is diluted by the sealing liquid, 100 mul/hole is diluted according to the dilution degree of 100 times, 500 times, 1000 times, 5000 times and 10000 times, and the mice are incubated for 1h at 37 ℃. PBST plates were washed 3 times, HRP-labeled goat anti-mouse IgG secondary antibody was added at a ratio of 1:8000 and incubated for 1h at 37 ℃. PBST plates were washed 3 times, TMB developing solution was added, 100. Mu.l/well, and developed for 5min. By 2M H 2 SO 4 The reaction was stopped, 50. Mu.l/well. OD values were measured with an ELISA detector at wavelengths of 450nm and 630 nm. The OD mean of 100-fold, 500-fold, 1000-fold, 5000-fold, 10000-fold serum dilutions of the protein vaccine + aluminum adjuvant + polypeptide group were respectively: 0.23, 0.19, 0.12, 0.09; OD average values of 100-fold, 500-fold, 1000-fold, 5000-fold and 10000-fold serum dilutions of the protein vaccine plus aluminum adjuvant group were respectively: 0.23, 0.16, 0.13, 0.07, 0.04; OD average values of 100 times, 500 times, 1000 times, 5000 times and 10000 times serum dilutions of the protein seedling group are respectively as follows: 0.26, 0.02, 0.006, 0.004, 0.003.
As a result of the detection, see fig. 4, the compound adjuvant can effectively enhance the antibody response caused by the novel crown vaccine in the mouse body. By testing multiple dilutions of serum antibodies, it can be seen that the composite adjuvant can effectively enhance IgG immune response levels.
Neutralizing antibody detection
In 96-well U bottom plate, 293TACE2 transgenic cells are added to make the number of cells per well be 2×10 4 Incubating at 37 ℃ per well and 100 μl/well, and growing cells into a monolayer; the serum to be tested isolated after the tail vein blood sampling of the mice was diluted 100. Mu.l/well at 20, 100, 500 times dilutions were added to a new 96-well U-bottom plate (final system: 50. Mu.l serum dilution+25. Mu.l medium (containing 32. Mu.g/ml, final concentration of polybrene) +25. Mu.l pseudovirus) and incubated in an incubator for 1h. After the incubation, the final infection solution was added to the cell culture plate, and after 24 hours, the fresh medium was changed, and after 48 hours, detection was performed. Before detection, 10ml LuciferaseAssay Buffer (-20deg.C) is preserved, and is dissolved in refrigerator at 4deg.C in advance), 1 bottle LuciferaseAssay Substrate (lyophilized) is added, and the temperature is balanced to 25deg.C; the Lucifer is added toddH for enzyme Cell C [ mu ] solution 5X Reagent (CCLR) 2 O was diluted to 1×, equilibrated to 25 ℃. The medium was discarded, 1 XCCLR 20 μl/well was added, and the plates were shaken to ensure complete coverage of the cells with CCLR. Before the culture plate is brought to the instrument, instrument parameters are adjusted, 100 mu l/hole of LuciferaseAssay System reagent is added, and data are detected and stored. The neutralization detection results of 20-fold, 100-fold and 500-fold serum dilutions of the protein vaccine, aluminum adjuvant and polypeptide group are respectively as follows: 90.47%, 83.04%, 64.25%; the neutralization detection results of 20 times, 100 times and 500 times serum dilutions of the protein vaccine plus aluminum adjuvant group are respectively as follows: 62.63%, 34.76%, 15.57%; the neutralization detection results of 20 times, 100 times and 500 times serum dilutions of the protein seedling group are respectively as follows: 38.15%, 29.94%, 18.76%; the neutralization detection results of the serum dilutions of 20 times, 100 times and 500 times of the control group are respectively as follows: 7.15%, 4.72% and 2.56%. The test results are shown in fig. 5, and the results in fig. 5 show that the compound adjuvant can effectively enhance the neutralizing antibody response caused by the novel crown vaccine in mice. By detecting the neutralizing antibodies of the serum of the mice, the compound adjuvant can be seen to effectively enhance the response level of the neutralizing antibodies of the mice relative to a control group, a protein vaccine group and a protein vaccine plus aluminum adjuvant group.
IFN-gamma ELISPOT detection
IFN-. Gamma.ELISPOT assay plates were added to 100. Mu.l/well and gently blotted dry on absorbent paper at 25℃for 10 min. Mu.l of sample/well was added, the sample comprising 50. Mu.l of stimulus diluent+50. Mu.l of cell diluent (5X 10) 5 Specific volumes of cells were counted and 1640 complete medium was added to 50 μl) with the stimulus added followed by cells. Covering with a cover, sealing, and sealing at 37deg.C, CO 2 And standing the incubator for 10-15 hours. Drying, adding PBST,300 μl/hole, washing the plate 3 times, and gently beating on absorbent paper. Add detectionAB, 100. Mu.l/well, cover 25℃for 1.5h, drain, add PBST, 300. Mu.l/well, wash the plate 3 times, gently shoot dry on absorbent paper. Add Diluted Streptavidin-AP Conjute, 100. Mu.l/well, cover 25℃for 1h, drain, add PBST, 300. Mu.l/well, wash the plate 3 times, gently shoot dry on absorbent paper. Adding ready-to-use BCIP/NBTbuffer, incubating at 25deg.C for 5-20min at 100 μl/well, developing, drying, and washing with distilled waterThe plates were gently patted dry on absorbent paper 3 times, the bottoms of the plates were gently peeled off, the excess water was wiped off, and after natural air drying, the plates were stored for a long period of time, and the reading points were detected with German AID ELISPOT READER (German AID ELISA). There were 184 spots on average in the protein vaccine + aluminum adjuvant + polypeptide group, 52 spots on average in the protein vaccine + aluminum adjuvant group, and 40 spots on average in the protein vaccine group. The results of fig. 6 show: the combination of the polypeptide adjuvant with the aluminum adjuvant can effectively enhance IFN-gamma (Th 1) immune response caused by the novel crown vaccine in mice compared with the aluminum adjuvant alone.
Flow cytometer analysis
Fresh spleen cells were extracted, washed twice with PBS, and the final concentration of the cell suspension was adjusted to 1X 10 6 And each ml. Adding 100 μl of stimulator and protein transport blocker, 37 ℃ and 5% CO 2 Incubate for 5h under conditions. CD3 BV510, CD4 FITC, CD8aAlexa with 4 anti-murine surface marker antibodies700. CD137PE, incubated at 4 ℃ for 30min in dark, and washed twice with PBS. 100 μl Fix/Perm solution was added, mixed well, incubated at 4deg.C in the dark for 20min, the supernatant was removed by centrifugation and washed 2 times with 1 XPerm/Wash. Staining was then performed with an antibody IL-2BV605 (eBioscience) against murine intracellular cytokines, incubated for 30min at 4 ℃. The cells were washed twice with PBS and immediately after resuspension were detected on-line using a Beckmann Coulter Cyto FLEX flow cytometer. Data analysis using flow Jo software, at least 10 samples per sample were obtained 5 Individual cells were subjected to scatter plot gates. Protein vaccine + aluminum adjuvant + polypeptide group CD137 + The average percentage of T cells was 4.92%; protein vaccine + aluminum adjuvant group CD137 + The average percentage of T cells was 1.18%; protein seedling group CD137 + The average percentage of T cells was 1.42%; protein vaccine, aluminum adjuvant and polypeptide group IL-2 + The average percentage of T cells was 6.19%; protein vaccine plus aluminum adjuvant group IL-2 + The average percentage of T cells was 1.08%; protein seedling group IL-2 + The average percentage of T cells was 1.07%. The results are shown in fig. 7 and 8. The results of fig. 7 and 8 show that: the compound adjuvant can effectively enhance T cell immune response caused by the novel crown vaccine in mice. By the detection of a plurality of cytokines,the results show that: the use of a polypeptide adjuvant in combination with an aluminum adjuvant as a novel crown vaccine adjuvant may be effective in enhancing the secretion levels of CD137 and IL-2 as compared to aluminum adjuvant alone.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A composite adjuvant comprising a polypeptide adjuvant and an aluminum adjuvant;
the polypeptide adjuvant has a structure shown in a formula I;
2. the compound adjuvant according to claim 1, wherein the dosage form of the compound adjuvant comprises an injection.
3. The compound adjuvant according to claim 2, wherein the concentration of the polypeptide adjuvant in the injection is 0.05-50 mg/ml; the concentration of the aluminum adjuvant is 1.75mg/ml.
4. A compound adjuvant according to any one of claims 1 to 3 wherein the polypeptide adjuvant and aluminium adjuvant are packaged separately.
5. Use of a compound adjuvant according to any one of claims 1 to 4 in the preparation of a vaccine.
6. A vaccine comprising a complex adjuvant according to any one of claims 1 to 4, said vaccine further comprising an immunogen.
7. The vaccine of claim 6, wherein the vaccine comprises a neocrown vaccine.
8. The vaccine of claim 7, wherein the immunogen comprises a novel coronavirus surface S protein.
9. Vaccine according to any one of claims 6 to 8, characterised in that the mass ratio of aluminium adjuvant to immunogen in the vaccine is (0.1 to 10): 1.
10. a method of preparing a vaccine according to any one of claims 6 to 9, comprising the steps of: and (3) adsorbing the immunogen by using an aluminum adjuvant, and then adding a polypeptide adjuvant to obtain the vaccine.
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