An antigen-adaptable vaccine adjuvant system and its application in preparing new crown and monkeypox vaccines

Abstract

The application discloses an antigen-adaptable vaccine adjuvant system and application thereof in preparation of new crown and monkeypox vaccines, and belongs to the technical field of viral vaccines. The vaccine adjuvant system provided by the application comprises a liposome delivery system and a cyclic dinucleotide, the cyclic dinucleotide is wrapped by the liposome delivery system, the liposome delivery system comprises ionizable phospholipid, cationic lipid, auxiliary lipid and dimyristyl glycerol-polyethylene glycol 2000, and the molar proportion of the ionizable phospholipid in the liposome delivery system is 10-40% or 45-69%. By adjusting the proportion of ionizable lipids in the liposome, efficient adaptation to different pathogen antigens is realized, and a universal strategy for preparing efficient and broad-spectrum pathogen vaccines is provided.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of viral vaccines, and particularly relates to an antigen-adapted vaccine adjuvant system and application thereof in preparation of new crown and monkeypox vaccines. BACKGROUND

[0002] In the face of the continuous emergence of new pathogens, developing rapid and efficient vaccines has become an important direction and strategy for responding to the spread of various epidemics. The vaccine system based on viral recombinant protein as antigen is one of the mainstream technical routes for current vaccine development, such as the recombinant RBD vaccine of Zhifei Biological and the recombinant HPV capsid protein vaccine of Wantai Canghai. However, the recombinant protein antigen generally has the problems of low immunogenicity and weak induction of immune response. The widely used aluminum adjuvant is inefficient in inducing high-level antibody immune response on the one hand, and has an innate defect in stimulating antigen-specific T cell immunity on the other hand. Therefore, developing an adjuvant system with universal and efficient adjuvant activity for pathogen protein antigens has important strategic significance and technical reserve value. Different protein antigens have different immune characteristics. How to achieve the individualization of the same adjuvant system for different antigens and achieve the best immune effect still needs further research. SUMMARY

[0003] To solve the above technical problems, the application provides an antigen-adapted vaccine adjuvant system and application thereof in preparation of new crown and monkeypox vaccines, which realizes efficient adaptation to different pathogen antigens and induces optimal immune response characteristics by adjusting the ratio of ionizable lipids in the liposome.

[0004] To achieve the above-mentioned purposes, the application provides the following technical solutions:

[0005] On the one hand, the application provides an antigen-adapted vaccine adjuvant system, which comprises a liposome delivery system and a cyclic dinucleotide.

[0006] The liposome delivery system encapsulates the cyclic dinucleotide.

[0007] The liposome delivery system comprises ionizable phospholipids, cationic lipids, helper lipids and dimyristyl glycerol-polyethylene glycol 2000.

[0008] The ionizable phospholipids account for 10-40% or 45-69% in terms of molar ratio in the liposome delivery system.

[0009] Optionally, the molar ratio of the ionizable phospholipids, cationic lipids, helper lipids and dimyristyl glycerol-polyethylene glycol 2000 is:

[0010] 13-52: 28-77: 40: 0.4 or 59-90: 0-31: 40: 0.4.

[0011] Optionally, the ionizable phospholipid has the following structural formula:

[0012] .

[0013] Optionally, the preparation method of the ionizable phospholipid comprises the following steps:

[0014] Step 1) reacting n-nonyl alcohol, triethylamine and phosphorus oxychloride to obtain a reaction product P2;

[0015] Step 2) reacting n-bromo octane, ethanolamine, diisopropylethylamine and potassium iodide to obtain a reaction product P3;

[0016] Step 3) reacting the reaction product P2 with the reaction product P3 to obtain the ionizable phospholipid.

[0017] Optionally, the specific operation of step 1) is to mix n-nonyl alcohol, triethylamine and anhydrous tetrahydrofuran to obtain a mixture 1, mix phosphorus oxychloride and anhydrous tetrahydrofuran and cool to -25~-35℃ to obtain a mixture 2, and then mix the mixture 1 with the mixture 2 and react at -2~2℃ for 0.8~1.2h.

[0018] Optionally, after the reaction of step 1) is completed, the reaction product P2 is obtained by evaporation and concentration.

[0019] Optionally, the specific operation of step 2) is to dissolve n-bromo octane, ethanolamine, diisopropylethylamine and potassium iodide in ethanol, and heat to 85~95℃ and react for 14~16h.

[0020] Optionally, after the reaction of step 2) is completed, the reaction product P3 is obtained by evaporation and concentration, separation and purification in sequence.

[0021] Optionally, the specific operation of step 3) is to dissolve the reaction product P2 in anhydrous tetrahydrofuran and cool to -2~2℃ to obtain a P2 solution, and then dissolve the reaction product P3 and triethylamine in anhydrous tetrahydrofuran and react with the P2 solution for 3~5h.

[0022] Optionally, after the reaction of step 3) is completed, the ionizable phospholipid is obtained by evaporation and concentration, separation and purification in sequence.

[0023] Optionally, the cyclic dinucleotide has the following structural formula:

[0024] .

[0025] Optionally, the mass ratio of the auxiliary lipid to the cyclic dinucleotide is 0.5~5.

[0026] Optionally, the cationic lipid is selected from any one of ditetradecyl-dimethylammonium bromide, (2,3-dioleoyl-propyl)trimethylammonium chloride and dioleoylphosphatidylethanolamine.

[0027] Optionally, the helper lipid comprises cholesterol.

[0028] In the present application, the preparation method of the ionizable phospholipid has the advantages of no by-product and easy separation compared with the synthesis route of the ionizable phospholipid IP9 in the inventor's previous application CN115381938A.

[0029] In a second aspect, the present application provides a preparation method of the antigen-adapted vaccine adjuvant system, comprising the following steps:

[0030] Step 1) mixing the ionizable phospholipid, the cationic lipid, the helper lipid and dimyristyl glycerol-polyethylene glycol 2000 to obtain a mixture;

[0031] Step 2) mixing the mixture with the cyclic dinucleotide to obtain the antigen-adapted vaccine adjuvant system.

[0032] Optionally, in step 1), the ionizable phospholipid, the cationic lipid, the helper lipid and the ethanol solution of dimyristyl glycerol-polyethylene glycol 2000 are mixed to obtain a mixture.

[0033] Optionally, after the mixing in step 2) is completed, the mixture is diluted 3-5 times with phosphate buffered saline PBS to obtain the antigen-adapted vaccine adjuvant system.

[0034] In a third aspect, the present application provides the use of the antigen-adapted vaccine adjuvant system in the preparation of a vaccine.

[0035] In a fourth aspect, the present application provides a COVID-19 vaccine, which comprises the vaccine adjuvant system and a COVID-19 antigen.

[0036] Optionally, the dose of the cyclic dinucleotide in the COVID-19 vaccine is 0.25-1 mg / kg.

[0037] The dose of the COVID-19 antigen is 0.25-1 mg / kg.

[0038] In a fifth aspect, the present application provides a monkeypox virus vaccine, which comprises the vaccine adjuvant system and a monkeypox virus antigen.

[0039] Optionally, the dose of the cyclic dinucleotide in the monkeypox virus vaccine is 0.25-1 mg / kg.

[0040] The dose of the monkeypox virus antigen is 0.25-1 mg / kg.

[0041] Compared with the prior art, the present application has the following beneficial effects:

[0042] (1) The present application designs a liposome delivery system based on ionizable phospholipid IP9, which loads and delivers immunostimulant cyclic dinucleotide by adjusting the percentage of IP9, and constructs a new adjuvant system STINGsome.

[0043] (2) The present application selects cyclic dinucleotide CDG as a representative of the stimulant to construct the adjuvant system, and carries out delivery effect and vaccine adjuvant application experiments of different IP9 ratios. The vaccine antigen model is SARS-CoV-2 RBD protein antigen and Monkeypox virus A35R membrane protein antigen. Among them, the proportion of ionizable phospholipid IP9 is divided into 46% (named STINGsome60, abbreviated as STS60) and 23% (named STINGsome30, abbreviated as STS30). Cell and in vivo experiments show that the proportion of IP9 can significantly affect the cell uptake, interferon secretion, in situ sustained release and lymph node activation kinetics of STINGsomes.

[0044] (3) The in vivo vaccine immunization experiment of the present application further shows that the change of the proportion of ionizable phospholipid IP9 will affect the different antibody and T cell levels induced by STINGsomes against the same antigen. Based on this, the STINGsome adjuvant system constructed by adjusting the percentage of ionizable phospholipid IP9 in the present application can be adapted to different pathogen antigens, and also provides a universal strategy for preparing efficient and broad-spectrum pathogen vaccines. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 The present application STS60, STS30 Kinetics of uptake by macrophages (Note: A represents incubation for 4h; B represents incubation for 21h; Lipo represents commercial cationic liposome (Lipofectmine 3000); PFO represents perforin);

[0046] Figure 2 The present application STS60, STS30 Kinetics of induction of interferon secretion by macrophages (Note: A represents complete medium incubation; B represents basal medium incubation; Lipo represents commercial cationic liposome (Lipofectmine 3000); PFO represents perforin);

[0047] Figure 3 The present application STS60, STS30 Kinetics of in situ sustained release of CDG after subcutaneous injection at the root of the mouse tail (Note: Alum represents aluminum adjuvant as a positive control);

[0048] Figure 4Differences in immune activation characteristics of STS60 and STS30 in stimulating draining lymph nodes after subcutaneous injection at the base of the mouse tail;

[0049] Figure 5 After completion of vaccine immunization, the serum antibody titers of each group were determined based on ELISA, and STS30 induced higher levels of SARS-CoV-2 RBD antigen-specific antibody titers than STS60 (Note: * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001);

[0050] Figure 6 After re-stimulation with SARS-CoV-2 RBD protein antigen, the proportion of CD4 + T cell population statistics, where A is a scatter plot and B is a statistical plot, STS60 induced a higher proportion of SARS-CoV-2 RBD antigen-specific CD4 + T cells than STS30 (Note: * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001);

[0051] Figure 7 After completion of vaccine immunization, the serum antibody titers of each group were determined based on ELISA, and STS60 and STS30 induced monkeypox A35R antigen-specific antibody titers (Note: * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001);

[0052] Figure 8 After re-stimulation with monkeypox virus A35R protein antigen, the proportion of CD4 + T cell population statistics, where A is a flow scatter plot and B is a proportion statistical plot, STS30 induced a higher proportion of monkeypox A35R antigen-specific CD4 + T cells than STS60 (Note: * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001). DETAILED DESCRIPTION

[0053] The present application will be further described in conjunction with specific examples. The following description is only a few embodiments of the present application, and does not limit the present application in any form. Although the present application is disclosed as a preferred embodiment, it is not intended to limit the present application. Any skilled person in the art can make some changes or modifications to the disclosed technical content without departing from the scope of the technical solution, and equivalent embodiments are also included in the scope of the technical solution.

[0054] Unless otherwise specified, the raw materials in the examples of the present application are purchased through commercial channels and are used directly without any special treatment.

[0055] Unless otherwise specified, the analysis methods in the examples all use the conventional settings of instruments or devices and conventional analysis methods.

[0056] Example 1

[0057] Preparation of STINGsome adjuvant system:

[0058] The synthesis route of ionizable phospholipid IP9 is as follows:

[0059]

[0060] P2 synthesis:

[0061] Take n-nonyl alcohol 2 g (P1) and triethylamine 8.42 g and add to a round-bottom flask containing 60 mL of anhydrous tetrahydrofuran (THF); take 12.7 g of phosphorus oxychloride (POCl3) and dissolve in 60 mL of THF, and cool to -30°C; add P1 dropwise to the POCl3 solution, stir at 0°C for 1 h after the dropwise addition is complete, and concentrate the reaction crude product P2 by rotary evaporator after the reaction is complete;

[0062] P3 synthesis:

[0063] Take n-bromooctane (P3a) 3.8 g, ethanolamine (P3b) 30 g, diisopropylethylamine (DIEA) 20 g, and potassium iodide (KI) 1.03 g, dissolve in 300 mL of ethanol, and heat to 90°C, and react for 15 h. After the reaction is complete, concentrate the reaction product by rotary evaporator, and purify the product P3 by column chromatography;

[0064] IP9 synthesis:

[0065] Take P2 2.56 g and dissolve in 20 mL of THF, and cool to 0°C; take P3 2 g and triethylamine 0.708 g, dissolve in 20 mL of THF, and add dropwise to the P2 solution, and stir for 4 h. After the reaction is complete, concentrate the reaction product by rotary evaporator, and purify the target product IP9 by high-performance liquid chromatography (HPLC C 18 Column).

[0066] The cyclic dinucleotide CDG used in the present application has the following structural formula:

[0067] .

[0068] The compound is synthesized based on the route of the previous patent CN111592570B of the present inventor.

[0069] (2) Preparation of STINGsome:

[0070] Firstly, ethanol solutions of four compounds, IP9, DDAB (dodecyl dimethyl ammonium bromide), cholesterol and DMG-PEG2000 (dimyristyl glycerol-polyethylene glycol 2000) were mixed according to the molar ratio of 60:30:40:0.4 (STS60) and 30:60:40:0.4 (STS30) respectively. Then, CDG was added to the mixed solution of STS60 and STS30 according to the mass ratio of cholesterol:CDG=2.36 respectively, mixed uniformly, and then diluted 4 times with PBS to complete the preparation of STS60 and STS30.

[0071] Example 2

[0072] Evaluation of immune cell activation of STS60 and STS30:

[0073] Macrophage RAW-ISG was used for cell uptake and interferon secretion evaluation. After cell culture was completed, 500,000 cells / well were plated in a 24-well plate and cultured overnight. For cell uptake, carboxyfluorescein (FAM) and CDG were packaged into STS60 and STS30 at the same amount. The drug concentration was FAM 0.5 μg / mL and CDG 1 μg / mL. Cells were collected at 4h and 21h after administration for flow cytometry analysis (as shown in Figure 1 ). For interferon secretion, the stimulation concentration of STS60 and STS30 was CDG 1 μg / mL, and the volume per well was 500 μL. At different time points after administration, 70 μL of supernatant was taken, 10 μL of luciferase substrate was added, and the chemiluminescence intensity was measured using a microplate reader (as shown in Figure 2 ). Then the cells at the bottom of the well were collected and subjected to antibody staining (anti-CD86, Biolegend, 1 / 300 dilution) on ice. Commercially available cationic liposomes (Lipofectmine) and pore-forming protein (PFO) were used as controls in the above experiments. The experimental results showed that the proportion of IP9 could significantly affect the cell uptake and interferon secretion kinetics of STINGsomes (as shown in Figure 1 and Figure 2 ).

[0074] Example 3

[0075] Evaluation of in vivo in situ sustained release and lymph node activation of STS60 and STS30:

[0076] CDG and indocyanine green (ICG) were mixed and encapsulated into STS60, STS30 at the same time. BALB / c mice were injected subcutaneously at the tail with CDGSF (10 pg) + ICG (5 pg), STS60, STS30 and ICG (5 pg) + aluminum adjuvant (100 pg). The ICG fluorescence intensity was monitored regularly using IVIS (PerkinElmer) (as shown in Figure 3 PBS, TS60 and STS30 were injected subcutaneously at the bottom of the tail of BALB / c mice. 4h after injection, the proximal inguinal lymph nodes were collected and rapidly frozen with liquid nitrogen, and transcriptome sequencing was performed (as shown in Figure 4 The experimental results show that the percentage of IP9 can significantly affect the STS60, STS30 release curve, thereby leading to different lymph node activation characteristics. Compared with STS30, STS60 has a faster release speed and a stronger immune cell activation level (as shown in Figure 3 and Figure 4

[0077] Example 4

[0078] Preparation, immunization of COVID-19 and monkeypox virus vaccines with STS60, STS30 as adjuvant

[0079] Vaccine system: SARS-CoV-2 RBD protein or Monkeypox virus A35R membrane protein (10 pg per mouse; Beijing Yiqiao Shenzhou COVID-19 virus antigen RBD, item number 40592; Nanjing Oukai Biological Monkeypox virus antigen A35R, item number C1621) was mixed with STS60, STS30 (CDG 10 pg per mouse) prepared in advance in PBS buffer. The preparation process of the aluminum adjuvant group vaccine is described in the adjuvant instruction (Alhydrogel® adjuvant 2%). 6-8 week old female BALB / c mice were selected as vaccine immunization objects, 3-5 per group. Each mouse was injected with each group of samples by subcutaneous immunization at the root of the tail, and PBS was used as a blank control. The immunization process was carried out for a total of three times, with two weeks of interval injection, and the mice were processed one week after completing all immunization to collect spleen and serum samples.

[0080] Example 5

[0081] SARS-CoV-2 and Monkeypox virus specific IgG antibody titers

[0082] ​RBD protein and A35R were dissolved in 0.1M NaHCO3(pH=9.6) to prepare a 1 pg / mL solution, and added to a 96-well enzyme-labeled plate (Costar 3590) at a volume of 100 pL per well, and incubated at 4°C for 12 h. Then, PBST solution (0.05% Tween in PBS solution) was used for washing, and 0.25% gelatin PBS solution was added for blocking at room temperature for 3 h. After blocking was completed, PBS and PBST were used for washing multiple times and dried. Gradient-diluted serum of each group was added to the well plate at a volume of 100 pL per well, and incubated at 37°C for 1.5 h. PBS and PBST were used for washing multiple times and dried, and horseradish peroxidase (HRP)-modified rabbit anti-mouse IgG antibody (1 / 2000 dilution) was added at 100 pL, and incubated at 37°C for 1 h. After washing and drying again, 200 pL of 3,3',5,5'-tetramethylbenzidine (TMB) was added to each well, and after standing in the dark for 4 min, 2M sulfuric acid was added to terminate color development (50 pL). The absorbance value of each well was measured using an enzyme-labeled instrument (λ=450 nm). The definition of the antibody titer of the vaccine of the present application was the maximum dilution factor at which the OD 450 value was at least 0.1 higher than that of the blank group. The titer data showed that the STS60 and STS30 adjuvant systems could significantly improve the IgG antibody levels specific to the new crown RBD antigen (as shown in Figure 5 ) and the monkeypox A35R antigen (as shown in Figure 7 ). Among them, STS30 induced higher levels of RBD-specific IgG antibody titers than STS60; and in terms of A35R-specific IgG levels, STS60 and STS30 induced the same levels.

[0083] Example 6

[0084] Post-immunization spleen cell antigen restimulation flow analysis of mice:

[0085] Spleen tissues from mice immunized with RBD as antigen were first ground and dispersed, then filtered with 40 μm cell filter to obtain spleen cells. After red blood cells in the spleen cells were lysed with red blood cell lysate, cell counting was performed and added to 96-well plates (1 million). Antigen stimulation concentration: SARS-CoV-2 RBD protein 25 μg / mL, Monkeypox virus A35R protein 25 μg / mL. After incubation for 72 h, the cells were centrifuged, and αCD3 (17A2), αCD4 (RM4-4), αCD8α (53-6.7) antibodies were added for staining on ice for 1 h. For intracellular cytokine staining, after washing the cells, a membrane-penetrating reagent was added for treatment, and then intracellular cytokine (αIFN-γ (XMG1.2) and αTNF-α (MP6-XT22)) antibody incubation was performed, and finally flow analysis was performed. The flow results show that the STS60, STS30 adjuvant system can significantly improve the cell immune response intensity of the new crown RBD antigen (such as Figure 6 indicated) and the monkeypox A35R antigen (such as Figure 8 indicated). And STS60 induced higher levels of RBD antigen-specific T cells than STS30; while in terms of A35R, STS60 induced lower levels than STS30. This result proves that adjusting the ratio of IP9 can affect the immune bias of STINGsome adjuvant, and thus achieve optimal adaptation to different antigens.

[0086] The above is only a few embodiments of the present application, and does not limit the present application in any form. Although the preferred embodiments are disclosed as above, they are not intended to limit the present application. Any skilled person in the art can make some changes or modifications to the above disclosed technical content without departing from the scope of the technical solutions of the present application, which are equivalent to equivalent embodiments, and all belong to the scope of the technical solutions.

Claims

1. The application of an antigen-adaptive vaccine adjuvant system in the preparation of a COVID-19 vaccine, characterized in that, The vaccine adjuvant system includes a liposome delivery system and a cyclic dinucleotide; The liposome delivery system encapsulates the cyclic dinucleotide; The liposome delivery system contains ionizable phospholipids, cationic lipids, auxiliary lipids, and dimyristic glycerol-polyethylene glycol 2000; The ionizable phospholipids account for 23% of the molar percentage in the liposome delivery system. The ionizable phospholipid has the following structural formula: ； The cyclic dinucleotide has the following structural formula: ； The vaccine in question is a COVID-19 vaccine.

2. The application according to claim 1, characterized in that, The molar ratio of the ionizable phospholipid, cationic lipid, auxiliary lipid, and dimyristoylglycerol-polyethylene glycol 2000 is as follows: 13~52:28~77:40:0.4。 3. The application according to claim 1, characterized in that, The mass ratio of the auxiliary lipid to the cyclic dinucleotide is 0.5 to 5.

4. The application according to claim 1, characterized in that, The cationic lipid is selected from any one of bis(decyl)dimethylammonium bromide, (2,3-dioleoyl-propyl)trimethylammonium chloride, and dioleoylphosphatidylethanolamine.

5. The application according to claim 1, characterized in that, The auxiliary lipids include cholesterol.

6. The application according to claim 1, characterized in that, The preparation method of the antigen-adaptive vaccine adjuvant system includes the following steps: Step 1) Mix ionizable phospholipids, cationic lipids, auxiliary lipids and dimyristic glycerol-polyethylene glycol 2000 to obtain a mixture; Step 2) Mix the mixture with cyclic dinucleotides to obtain the antigen-adapted vaccine adjuvant system.

7. The application according to claim 6, characterized in that, In step 1), an ethanol solution of ionizable phospholipids, cationic lipids, auxiliary lipids, and dimyristic glycerol-polyethylene glycol 2000 is mixed to obtain a mixture.

8. The application according to claim 6, characterized in that, After mixing in step 2), dilute with phosphate-buffered saline (PBS) 3-5 times to obtain the antigen-adapted vaccine adjuvant system.

9. A COVID-19 vaccine, characterized in that, The COVID-19 vaccine comprises the vaccine adjuvant system as described in claim 1 and the COVID-19 antigen.

10. The COVID-19 vaccine according to claim 9, characterized in that, The dosage of cyclic dinucleotide in the COVID-19 vaccine is 0.25~1 mg / kg; The dosage of the SARS-CoV-2 antigen is 0.25~1 mg / kg.

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