CN111939253A

CN111939253A - Phospholipid-coated aluminum nanoparticle vaccine adjuvant-delivery system carrying squal compound and SARS-CoV2 subunit vaccine

Info

Publication number: CN111939253A
Application number: CN202010814061.7A
Authority: CN
Inventors: 王理想; 许小锋; 白宗胜; 王旭之; 陈贤碧
Original assignee: Hefei Novel Gene Technology Service Co ltd
Current assignee: Anhui Lirui Investment Management Co.,Ltd.
Priority date: 2020-08-13
Filing date: 2020-08-13
Publication date: 2020-11-17
Anticipated expiration: 2040-08-13
Also published as: CN111939253B

Abstract

The invention belongs to the field of biological preparations, and discloses a phospholipid bilayer coated aluminum nanoparticle vaccine adjuvant-delivery system and a SARS-CoV2 subunit vaccine carrying a squal compound. The VADS takes aluminum nanoparticles as a carrier, phospholipid bilayers cover the surfaces of the aluminum nanoparticles, and a squal compound is clamped between the phospholipid bilayers; the invention also uses phospholipid carrying squal compound to coat the aluminum nanoparticle VADS and SARS-CoV2 antigen, to construct SARS-CoV2 subunit vaccine; the SARS-CoV2 subunit vaccine has high safety and strong immune induction effect, and can induce organism to generate high-level SCV2 antigen specific antibody and cytotoxic T cell by injection or mucosal inoculation.

Description

Phospholipid-coated aluminum nanoparticle vaccine adjuvant-delivery system carrying squal compound and SARS-CoV2 subunit vaccine

Technical Field

The invention relates to the technical field of biological medicines, in particular to a phospholipid-coated aluminum nanoparticle vaccine adjuvant-delivery system carrying a squal compound and a SARS-CoV2 subunit vaccine.

Background

Currently, SARS-CoV2(COVID-19, 2019, a novel coronavirus) infection pneumonia is spreading rapidly in the world and becoming a pandemic of infectious diseases seriously threatening human life. The economic loss caused by controlling the COVID-19 epidemic in the world cannot be estimated. Although researchers are developing vaccines capable of SARS-CoV2 overnight, there is no vaccine formulation available that can be widely vaccinated. Under the urgent situation, the SARS-CoV2 vaccine can be developed as soon as possible to protect susceptible people, and has important significance for ensuring human safety, maintaining social stability and recovering economic development.

The SARS-CoV2 vaccine currently under development mainly comprises the following types. 1) Inactivated SARS-CoV2 vaccine: it is a vaccine preparation directly used for killing SARS-CoV2 pathogen by physical or chemical method. 2) Attenuated SARS-CoV2 live vaccine: eliminating pathogenicity of pathogen by artificial directed mutagenesis method, or integrating SARS-CoV2 surface antigen Spike protein gene into non-pathogenic virus vector, such as adenovirus, etc. as vaccine preparation. The attenuated live vaccine has better immune induction effect, but has the risk of generating pathogenicity due to gene mutation and the immune negative effect generated by self induction of the vector virus. 3) Nucleic acid vaccine: the vaccine comprises RNA and DNA vaccines, and is characterized in that a protein antigen expression gene is transmitted to cells through a lipid nano-carrier, and an expression antigen stimulates an organism to generate immunity. 4) Subunit vaccine (pure antigen component vaccine): the SARS-CoV2 purified antigen was combined with a vaccine adjuvant-delivery system to prepare a vaccine formulation.

The SARS-CoV2 vaccines have the characteristics and advantages, but have the defects, and are summarized as the following two aspects. The development of a safe, potent, and easily constructed vaccine adjuvant-delivery system (VADS) remains the key to the preparation of SARS-CoV2 subunit vaccines.

Aluminum salt is a commonly used VADS and has been used clinically for over 90 years. Some of the main vaccine formulations in widespread use still contain aluminium salts VADS, such as the diphtheria, haemophilus influenzae, etc. However, the traditional aluminum adjuvant exists in a gel form due to particle aggregation, can activate Th2 cells to secrete IL-4, further promote Th2 type humoral immune response to generate antibodies, but is difficult to effectively induce cellular immune response, and cannot promote the body to generate cytotoxic T cells. In addition, the traditional aluminum adjuvant has the prominent weakness that the traditional aluminum adjuvant has strong local irritation and often causes inflammatory reaction at an inoculation position.

In conclusion, aluminum salts as vaccine adjuvants are to be further developed and optimized in terms of safety and immunostimulation efficacy.

Disclosure of Invention

In order to cope with the COVID-19 epidemic situation which is rapidly spreading in the world at present, the invention constructs the phospholipid-coated aluminum nanoparticle VADS carrying the squal compound and the SARS-CoV2 subunit vaccine based on the VADS. The specific technical scheme is summarized as follows:

a phospholipid-coated aluminum nanoparticle VADS carrying a squal compound is characterized in that: aluminum Nanoparticles (AN) are used as a carrier, Phospholipid (PL) bilayers cover the surfaces of the aluminum nanoparticles, and a squalane compound (sgualane) is sandwiched between the phospholipid bilayers.

Preferably, the squalane compound is squalene (squalene) or squalane (squalane).

Preferably, the phospholipid contains a main component consisting of one or more of Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylglycerol (PG), Phosphatidylserine (PS), and Sphingomyelin (SP);

preferably, the lipid material also contains a charged lipid material as an auxiliary component to adjust the charge property of the carrier;

preferably, the charged lipid material is Stearylamine (SA) or 1, 2-dioleoyl-3-trimethylammoniumpropane (DOTAP).

Further preferably, the phospholipid comprises phosphatidylcholine and the charged lipid material 1, 2-dioleoyl-3-trimethylammoniopropane.

Preferably, the aluminum nanoparticles are aluminum oxide, aluminum phosphate or aluminum hydroxide nanoparticles;

the particle diameter of the aluminum nanoparticles is preferably 100 nm or less.

The method for preparing VADS described in any of the above, characterized in that: the method comprises the following steps:

(1) putting a proper amount of phospholipid and a squal compound into a flask, adding an organic solvent for dissolving, removing the solvent through rotary evaporation, and forming a lipid film on the inner wall of a container;

preferably, the organic solvent is chloroform, and the mass ratio of the phospholipid to the squalane compound is 10-40: 1;

preferably, the mass ratio of the phospholipid to the squalane compound is 20-25: 1;

(2) adding the solution containing the aluminum nanoparticles into the flask, and fully stirring until lipid molecules are hydrated to form SPLAN;

preferably, the mass ratio of the aluminum nanoparticles to the phospholipid is 2-10: 1;

preferably, when the phase transition temperature of the phospholipid is higher than room temperature, the steps (1) and (2) are carried out at the temperature higher than the phase transition temperature of the phospholipid by more than 5 ℃; when the phase transition temperature of the phospholipid is lower than the room temperature, the steps (1) and (2) are carried out at the room temperature.

The invention also provides a subunit vaccine based on the phospholipid-coated aluminum nanoparticle vaccine adjuvant-delivery system with the squal compound, which is characterized in that: the carrier is any one of the vaccine adjuvant-delivery systems described above, and the vaccine antigen is linked or adsorbed on the carrier.

The invention also provides a SARS-CoV2 subunit vaccine based on the phospholipid coated aluminum nanoparticle vaccine adjuvant-delivery system of the sandwich squal compound, which is characterized in that: the carrier is any VADS, and SARS-CoV2 antigen is connected or adsorbed on the carrier.

The SARS-CoV2 antigen is SCV2 envelope protein SCV2-EP, or SARS-CoV2spike protein SCV2-SP, or SARS-CoV2spike protein receptor binding domain SCV 2-RBD;

preferably, SARS-CoV2spike protein receptor binding domain SCV 2-RBD.

The mass ratio of the aluminum nanoparticles to the SARS-CoV2 antigen is 5-40: 1;

preferably, the mass ratio of the aluminum nanoparticles to the SARS-CoV2 antigen is 10-20: 1;

preferably, the subunit vaccine is in the form of a liquid preparation or a lyophilized preparation obtained by freeze-drying.

The preparation method of any SARS-CoV2 subunit vaccine is characterized by comprising the following steps:

firstly, preparing SPLAN according to the method for preparing SPLAN, and mixing and stirring the prepared SPLAN and SARS-CoV2 antigen solution uniformly to obtain the product;

preferably, the SPLAN and SARS-CoV2 antigen solutions are HEPES aqueous solutions.

Squal compounds, such as squalene and squalane, are strongly lipophilic liquid oils at room temperature. The squalane compound can activate the immune system and induce the organism to generate TH1/TH2 mixed immune response.

Phospholipids are amphiphilic molecules constituting biological membranes and contain polar groups of phosphoric acid and nonpolar hydrophobic hydrocarbon chains, and thus can self-arrange to form a bilayer structure in aqueous solution due to hydrophobic interaction. The phospholipid has good biocompatibility, and can improve the biological safety of the preparation and promote the cellular uptake. And the lipophilic property in the middle of the phospholipid bilayer provides a loading space for the squalane compound.

Aluminum has strong phosphorus-philic properties and is capable of forming a strong binding support with phosphate group-containing molecules. The invention combines the adjuvant functions of aluminum salt and squal compound to develop better VADS-SPLAN; using SPLAN as a high-efficiency vaccine adjuvant-delivery system (VADS), SARS-CoV2 antigen is combined to construct SARS-CoV2 subunit vaccine (for example, squalene and SCV2-RBD, the structure is shown in FIG. 1).

The vaccine adjuvant-delivery system SPLAN constructed by the invention has high biocompatibility, the phospholipid bilayer has good biocompatibility and small local irritation, and is suitable for multi-path inoculation, for example, the inoculation can be carried out through the mucosa of the cavity, and the inoculation can also be carried out through subcutaneous, intradermal and intramuscular injection. The SARS-CoV2 subunit vaccine SPLAN-SCV2 based on SPLAN has high safety, no potential safety hazard caused by virus vector vaccine and no induced immune response deviating from target antigen. The SPLAN-SCV2 has improved aluminum salt adjuvant function, strong immune induction effect, and the aluminum nanoparticles activate immune system to generate humoral and cellular immunity; in particular, the SPLAN-SCV2 has simple preparation process, is suitable for large-scale production and is expected to cope with the COVID-19 epidemic situation.

The present application relates to the word abbreviations:

SCV2, SARS-CoV2, the segment acid respiratory syndrome coronavirus 2 (Severe respiratory syndrome coronaviruses-2);

SCV2-SP, SCV2spike protein (SCV 2spike protein);

SCV2-RBD, SCV2spike protein receptor-binding domain (SCV 2spike protein receptor binding domain);

COVID-19, coronavirus disease 2019(2019 coronavirus infection disease);

SPLAN, the sgualane-sandwiched phospholipid bilayer-coated aluminum nanoparticles (loaded with a phospholipid bilayer of squalane compound coated with aluminum nanoparticles);

VADS, vaccine adjuvant-delivery system;

SPLAN-SCV2, (SPLAN-based SCV2 subunit vaccine);

SPC, soy phosphatidyllcholine (stigmaphosphatidylcholine);

DOTAP,1, 2-dioleoyl-3-trimethyllamonium-propane (1, 2-dioleoyl-trimethylaminopropane).

Drawings

FIG. 1 is the structural diagram of SARS-CoV2 subunit vaccine constructed by the invention.

Figure 2 shows serum antibody IgG levels (n-5) in mice vaccinated with different prescribed vaccines.

Fig. 3 shows the relative levels of antigen-specific cytotoxic T cells (anti-Ag CTLs) (n-5) produced by mice vaccinated with the different vaccines.

Figure 4 shows that splenocytes of mice vaccinated with different prescriptions receive antigen stimulation again to produce IFN- γ levels (n-5).

Figure 5 shows serum IgG and lung lavage fluid (BALF) IgA levels (n-5) in mice vaccinated with different prescribed vaccines.

Fig. 6 shows the relative levels of antigen-specific cytotoxic T cells (anti-Ag CTLs) (n-5) generated by mice vaccinated with different vaccines.

Figure 7 shows that splenocytes of mice vaccinated with different prescriptions received antigen stimulation again to produce IFN- γ levels (n-5).

Detailed Description

The invention is further illustrated by the following examples, which are not intended to be limiting. The experimental procedures in the following examples are conventional unless otherwise specified.

And (3) reagent sources:

alumina nanoparticles: the aluminum oxidation method is used, as described in the literature references (M Changmai, J Primesh, M Purkait, Al2O3 nanoparticles synthesizing using vacuum oxidation agents: Defluoridation performance, J Sci: Adv Mater device 2017, Volume 2, Issue 4, December 2017, Pages 483-.

Aluminum hydroxide nanoparticles: made by an acid-base neutralization method, and concretely refers to a reference (Li X, Aldayel A, Cui Z.2014.aluminum hydroxide nanoparticles show a runner vacuum additive reaction product J Control Release 173: 148-.

Traditional aluminium hydroxide adjuvant

Purchased from InvivoGen (San Diego, Calif., USA), cat-alu-250.

Soy Phosphatidylcholine (SPC): purchased from ai Wei Tuo (Shanghai) pharmaceutical science and technology, Inc., CAS number 8030-76-0.

1, 2-dioleoyl-3-trimethylammoniopropane (1, 2-dioleoyl-3-trimethyllammonium-propane), (chloride), DOTAP, WM ═ 699): purchased from Avention (Shanghai) pharmaceutical science and technology, Inc., CAS number 132172-61-3.

Squalene (squalene), available from Merck (Shanghai, China) under code number S3626-100ML, CAS number 111-02-4.

Squalane (squalane), available from Merck (Shanghai, China) under item number S3626-100ML, CAS number 111-01-3.

In each embodiment of the invention, SARS-CoV2spike protein receptor binding domain SCV2-RBD (SARS-CoV2spike protein binding domain) is selected as a vaccine antigen purchased from InvivoGen (San Diego, CA, USA) under the cargo number his-SARS 2-RBD.

The amino acid sequence of SCV2-RBD-His (the complete sequence 319-541 of SCV2-SP, the end of which is linked with histidine) is shown in SEQ ID NO: 1:

RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF(GSGHHHHHH)

SCV2-RBD aqueous solution: the SCV2-RBD is dissolved in HEPES buffer solution to prepare the product.

The remaining reagents, if not indicated, were conventional in the art and were commercially available.

Example 1 construction of SPLAN and SCV2 subunit vaccines with SPC/DOTAP/Squalene and aluminum hydroxide nanoparticles

Lipid materials (SPC and DOTAP) and a proper amount of Squalene (the mass ratio of SPC/DOTAP/Squalene is 10:10:1) are placed in a pear-shaped flask, chloroform (the using amount is 3 times of the weight of the lipid materials) is added for dissolving, and the organic solvent is removed at room temperature and 90rpm (90 revolutions per minute) by rotary evaporation to form an SPC/DOTAP/Squalene film.

To the above flask, AN aqueous solution (10mM HEPES buffer, pH 7) containing aluminum hydroxide nanoparticles (AN, 1% w/v) having AN average particle size of 80nm was added according to AN/lipid material (SPC and DOTAP) at 10:1 (mass ratio), and rotated at 120rpm for 1 hour to sufficiently hydrate the SPC/DOTAP/Squalene thin film to form a SPLAN.

Subsequently, the flask was transferred to a constant temperature electromagnetic stirrer, AN electromagnetic stirrer was added, and 0.1% (w/v) of AN aqueous solution of SCV2-RBD antigen (Ag) (10mM HEPES buffer, pH 7) was slowly added dropwise under stirring at 200rpm at room temperature, in a mass ratio of AN/SCV2-RBD of 10:1, and stirring was continued for 30 minutes, thereby forming SCV 2-RBD-SPLAN.

The samples were characterized parametrically using the following method (n-3).

The particle size and zeta potential of the samples were measured by Dynamic Light Scattering (DLS) technique and Electrophoretic Light Scattering (ELS) technique using Zetasizer Nano ZS90(Malvern Panalytical) instrument at 25 ℃, aqueous medium and 90 degree angle.

Quantitative detection of squalenes by HPLC is described in the references (Novak A, et al, Development and validation of a simple high-performance liquid chromatography analytical method for a single chromatography determination of chromatography, chromatography and squalene in a partial chromatography evaluation biomed chromatography, 2018 Feb; 32(2) doi: 10.1002/bmc.4084).

The quantitative determination of SCV2-RBD was carried out by the micro-Bradford protocol method, which was described in the references (S Zuo, P Lundahl, A micro-Bradford membrane protein assay, Anal Biochem 284(1) (2000)162-4.), and the antigen-carrier binding efficiency (AE) was calculated by the following formula.

AE (%) - (Total Ag-free Ag)/Total Ag X100%

The results show that: aluminum hydroxide nanoparticles (AN) having AN average particle size of 80nm and zeta of-48 mV, the SPLAN prepared by the above method having AN average particle size of 93 nm and zeta of 35 mV; carrying the antigen to obtain SCV2-RBD-SPLAN with average particle diameter of 98 nm and zeta of 28 mV; 97% Squalene was entrapped in the SPLAN phospholipid bilayer, antigen AE 94%.

Animal vaccination experiment (n ═ 5): mice were vaccinated intramuscularly at 3 week intervals 2 times at a 2.5 μ g/50 μ L antigen dose. 3 control mice were also set and inoculated with the same dose and mode: (1) saline (Saline); (2) SCV2-RBD + commercial conventional aluminum hydroxide adjuvant (SCV 2-RBD-AM); (3) SCV2-RBD + aluminum hydroxide nanoparticles (SCV 2-RBD-AN).

After inoculation, the weight, feeding and activity behaviors of the mice of each group are not abnormal.

3 weeks after the second inoculation, the immune response of mice in the experimental and control groups (saline, SCV2-RBD-AM, SCV2-RBD-AN) was measured, including ELISA for measuring the level of mouse serum antigen-specific antibodies (IgG), and flow cytometry for detecting cytotoxic T cells (CTL, i.e., fluorescent marker SIINFEKLH-I)⁺CD8⁺T cell) levels, and ELISA detection of antigen restimulated immunized miceSpleen cells secrete IFN-gamma levels (the specific detection method of each parameter is described in the reference (Wang N, Zhen Y, Jin Y, Wang X, Li N, Jiang S, Wang T. binding differential types of multiple functional lipids loaded with ammonium bicarbonate to a fibrous microelectrode array as a variable microbial vaccine added with a reduced delivery system (DDS VAN). J Control Release.2017Jan 28; 246:12-29.) the detection results are shown in FIGS. 2, 3 and 4, wherein AM is a traditional aluminum hydroxide adjuvant (aluminum hydroxide) and AN is AN aluminum hydroxide nanoparticle.

Figure 2 shows the serum IgG levels (n-5) of mice vaccinated with the different prescribed vaccines, and the sera were diluted 1:3200 times (p <0.01) at the time of the assay.

Fig. 3 shows the relative levels of antigen-specific cytotoxic T cells (anti-Ag CTLs) (n ═ 5) (. x.p <0.01) generated by mice vaccinated with the different vaccines.

Figure 4 shows that splenocytes of mice vaccinated with different prescriptions received antigen stimulation again to produce IFN- γ levels (n-5) (. x.p < 0.01).

The results show that compared with SCV2-RB-AM and SCV2-RBD-AN control groups, the levels of IgG generated by inoculating SCV2-RBD-SPLAN mice are respectively improved by 2.2 times and 1.6 times, the levels of generated cytotoxic T Cells (CTL) are respectively improved by 8.1 times and 1.9 times, and the levels of IFN-gamma secretion stimulated by immune mouse spleen cell antigen are improved by 4.6 times and 2.5 times.

As can be seen, SPLAN is a potent VADS, and the SCV2 vaccine developed based on SPLAN can efficiently induce vaccinated mice to generate a Th1/Th2 mixed immune response, and is expected to become a vaccine preparation for preventing and controlling SCV 2.

Example 2 construction of SPLAN and SCV2 subunit vaccines with SPC/DOTAP/Squalane and alumina nanoparticles

Lipid materials (SPC and DOTAP) and a proper amount of Squalene (SPC/DOTAP/Squalane: 15:10:1, w/w) were placed in a pear-shaped flask, dissolved in chloroform (3 times the weight of the lipid materials), and rotary-evaporated at 90rpm (90 revolutions per minute) at room temperature to remove the organic solvent, thereby forming an SPC/DOTAP/Squalane film.

To the above flask, AN aqueous solution (10mM HEPES buffer, pH 7) containing alumina nanoparticles (AN, 1% w/v) having AN average particle size of 50nm was added according to AN/lipid material (SPC and DOTAP ═ 8:1 (mass ratio), and rotated at 120rpm for 1 hour to sufficiently hydrate the SPC/DOTAP/Squalane film to form AN SPLAN.

Then the flask is transferred to a constant temperature electromagnetic stirrer, AN electromagnetic stirring bar is added, 0.1% (w/v) of SCV2-RBD antigen aqueous solution (10mM HEPES buffer solution, pH 7) is slowly dripped under the stirring condition of room temperature and 200rpm according to the mass ratio of AN/SCV2-RBD being 20:1, and the stirring is continued for 30 minutes after the dripping is finished, thus forming SCV 2-RBD-SPLAN.

The sample was characterized for each parameter using the method described in example 1 (n-3). A method for quantitative detection of Squalane (Squalane) is described in the literature references (M Berekaa, A Steinbuchel, microbiological grade of the Multiply branched alkane 2,6,10,15,19,23-hexamethy et ratoascan (Squalane) by Mycobacterium fortuitum and Mycobacterium ratinebense, apple Environ Microbiol.2000Oct; 66(10):4462-7.doi: 10.1128/aem.66.10.4462-4467.2000).

The results show that: the average particle size of the Alumina Nanoparticles (AN) is 50nm, and the zeta is-45 mV, and the SPLAN prepared by the method has the average particle size of 59 nm and the zeta is 32 mV; carrying the antigen to obtain SCV2-RBD-SPLAN with average particle diameter of 66 nm and zeta of 23 mV; 96% Squalane was entrapped in the SPLAN phospholipid bilayer, with 91% antigen AE.

Animal vaccination experiment (n ═ 5): mice were vaccinated by inhalation into the lungs at a dose of 5 μ g/50 μ L antigen, 2 times at 3 week intervals. 3 control mice were also set and inoculated with the same dose: (1) sucking in an equal volume of Saline (Saline); (2) intramuscular injection of SCV2-RBD + commercial conventional aluminum hydroxide adjuvant (SCV2-RBD-AM) (inhalation of aluminum hydroxide gel resulted in asphyxiation death of mice); (3) the solution is inoculated with SCV2-RBD + alumina nanoparticles (SCV2-RBD-AN) by inhalation.

3 weeks after the second inoculation, the immune response of the mice in the experimental group and the control group was tested according to the method of example 1, and the test results are shown in FIGS. 5, 6 and 7, in which AM is commercial aluminum hydroxide adjuvant (aluminum microparticles); AN is alumina nanoparticles.

Figure 5 shows serum antibody IgG and lung lavage fluid (BALF) IgA levels (n-5) in mice vaccinated with different prescribed vaccines; serum IgG is diluted 1:3200 times during detection; lung wash IgA was tested at 1:400 fold dilutions (. p < 0.01).

Fig. 6 shows the relative levels of antigen-specific cytotoxic T cells (anti-Ag CTLs) (n ═ 5) (. x.p <0.01) generated by mice vaccinated with the different vaccines.

Figure 7 shows that splenocytes from mice vaccinated with different prescriptions were re-stimulated by antigen to produce IFN- γ levels (n-5) (. x.p < 0.01).

The results show that compared with SCV2-RB-AM and SCV2-RBD-AN control groups, the levels of IgG generated by serum antigen specific antibodies are respectively improved by 2.9 times and 1.9 times by inoculating SCV2-RBD-SPLAN mice, and the levels of IgA generated by lung flushing fluid antibodies are respectively improved by 6.9 times and 2.1 times; CTL generation is respectively improved by 5.7 times and 2.0 times; the spleen cells of the immunized mice are stimulated by the antigen again to generate IFN-gamma which is respectively improved by 6.0 times and 2.8 times. This indicates that the mice developed both TH1/TH2 mixed systemic immunity and strong mucosal immunity.

Therefore, the SCV2 vaccine prepared based on the SPLAN is expected to be inoculated through the lung mucosa, and the SCV2 infection invading the respiratory system is effectively prevented and treated.

SEQUENCE LISTING

<110> Hefeinuo Dai' er Gene science and technology services Co., Ltd

<120> phospholipid-coated aluminum nanoparticle vaccine adjuvant-delivery system carrying squal compound and SARS-CoV2 subunit vaccine

<130> P200514

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 232

<212> PRT

<213> Artificial Sequence

<220>

<223> SARS-CoV2spike protein receptor binding domain SCV2-RBD

<400> 1

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Ser Gly His His His His His His

225 230

Claims

1. A phospholipid-coated aluminum nanoparticle vaccine adjuvant-delivery system loaded with a squal compound, which is characterized in that: the preparation method is characterized in that aluminum nanoparticles are used as carriers, phospholipid bilayers cover the surfaces of the aluminum nanoparticles, and a squal compound is sandwiched between the phospholipid bilayers.

2. The vaccine adjuvant-delivery system of claim 1, wherein: the squalane compound is squalene or squalane.

3. The vaccine adjuvant-delivery system of claim 1, wherein: the phospholipid contains a main component consisting of one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine and sphingomyelin;

preferably also contains a charged lipid material as an auxiliary component to adjust the charge property of the carrier;

preferably the charged lipid material is stearylamine or 1, 2-dioleoyl-3-trimethylammoniumpropane.

4. The vaccine adjuvant-delivery system of claim 3, wherein: the phospholipid contains phosphatidylcholine and a charged lipid material 1, 2-dioleoyl-3-trimethylammoniumpropane.

5. The vaccine adjuvant-delivery system of claim 1, wherein: the aluminum nanoparticles are aluminum oxide, aluminum phosphate or aluminum hydroxide nanoparticles;

aluminum nanoparticles having a particle diameter of 100 nm or less are preferable.

6. The method of preparing a vaccine adjuvant-delivery system according to any of claims 1 to 5, comprising the steps of:

preferably, the mass ratio of the phospholipid to the squalane compound is 20-25: 1

(2) Adding the solution containing the aluminum nanoparticles into the flask, and fully stirring until lipid molecules are hydrated to form a vaccine adjuvant-delivery system SPLAN;

7. A subunit vaccine based on a phospholipid-coated aluminum nanoparticle vaccine adjuvant-delivery system with a squal compound entrapped therein, which is characterized in that: the carrier is a vaccine adjuvant-delivery system according to any one of claims 1 to 5, to which a vaccine antigen is attached or adsorbed.

8. A SARS-CoV2 subunit vaccine based on phospholipid coated aluminum nanoparticle vaccine adjuvant-delivery system carrying squal compound, which is characterized in that: the carrier is the vaccine adjuvant-delivery system of any one of claims 1 to 5, wherein the carrier is linked or adsorbed with SARS-CoV2 antigen.

9. The SARS-CoV2 subunit vaccine of claim 8, wherein: the SARS-CoV2 antigen is SCV2 envelope protein SCV2-EP, or SARS-CoV2spike protein SCV2-SP, or SARS-CoV2spike protein receptor binding domain SCV 2-RBD;

preferably, the SARS-CoV2 antigen is SCV 2-RBD;

preferably, the mass ratio of the aluminum nanoparticles to the SARS-CoV2 antigen is 5-40: 1;

further preferably, the mass ratio of the aluminum nanoparticles to the SARS-CoV2 antigen is 10-20: 1;

10. The method of preparing a SARS-CoV2 subunit vaccine as claimed in claim 8 or 9, wherein: preparing vaccine adjuvant-delivery system SPLAN according to the method of claim 6, mixing the prepared SPLAN with SARS-CoV2 antigen solution, and stirring;