Application of a small molecule adjuvant in vaccine preparation and vaccine prepared therefrom

Abstract

The present invention provides an application of a small molecule adjuvant in the preparation of a vaccine and a vaccine prepared therefrom, and belongs to the technical field of biological products. The present invention provides an application of S-4,12-dihydroxy[2.2]paracyclophane as an adjuvant in the preparation of a vaccine, and the vaccine prepared by S-4,12-dihydroxy[2.2]paracyclophane can not only induce a high level of humoral immune response but also enhance cellular immune response, and has good biosafety. It can be seen that the present invention enriches the types of current adjuvants and can be widely used in the development and research of vaccines.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological products, and specifically relates to the application of a small molecule adjuvant in the preparation of a vaccine and the prepared vaccine. Background Art

[0002] The emergence and large-scale outbreaks of infectious viruses, such as monkeypox and the novel coronavirus, have spurred a surge in the development of corresponding vaccines. However, the antigenic immunogenicity of inactivated vaccines, recombinant protein subunit vaccines, and nucleic acid vaccines is weak and insufficient to produce strong immune protection. Therefore, adjuvants are often added to enhance the protective efficacy of vaccines.

[0003] Currently, there are very few adjuvants approved for clinical use. On the one hand, new adjuvants have limited ability to enhance vaccine immunogenicity; on the other hand, many adjuvants have poor biosafety, limiting their widespread and large-scale use. Furthermore, the adjuvants approved for clinical use induce a single type of immune response. For example, while common aluminum adjuvants can induce a certain level of humoral immune response, they are less effective in inducing cellular and mucosal immunity. Therefore, there is an urgent need for adjuvants with superior biosafety, high immune response induction, and diverse immune response types.

[0004] S-4,12-dihydroxy[2.2]-p-cyclophane was discovered in the last century, and its physical and chemical properties have been extensively studied. Studies have shown that S-4,12-dihydroxy[2.2]-p-cyclophane possesses excellent optical properties, including the ability to absorb circularly polarized light with a specific rotational orientation. Furthermore, the molecule possesses a rigid structure and is amenable to chemical modification. Currently, S-4,12-dihydroxy[2.2]-p-cyclophane is primarily used in the preparation of upconversion materials, fluorescent and phosphorescent materials, and their optimized preparation through chemical modification. However, few biological applications have been discovered or reported. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a use of S-4,12-dihydroxy[2.2]paracyclophane as an adjuvant in the preparation of vaccines. The vaccine prepared with S-4,12-dihydroxy[2.2]paracyclophane can not only induce a high level of humoral immune response but also enhance the cellular immune response and has good biosafety.

[0006] The present invention also aims to provide a vaccine with good immunogenicity using S-4,12-dihydroxy[2.2]-p-cyclophane as an adjuvant.

[0007] The present invention provides the use of S-4,12-dihydroxy[2.2]paracyclophane of the structural formula I as an adjuvant in the preparation of vaccines.

[0008]

[0009] Preferably, the vaccine comprises an immunogen; the immunogen is a protein.

[0010] Preferably, the mass ratio of the protein to S-4,12-dihydroxy[2.2]paracyclophane is 10:3-3.5.

[0011] Preferably, the protein is a new coronavirus immune protein.

[0012] Preferably, the novel coronavirus immune protein is the novel coronavirus RBD protein;

[0013] The amino acid sequence of the SARS-CoV-2 RBD protein is shown in SEQ ID NO: 1.

[0014] The present invention provides a vaccine comprising S-4,12-dihydroxy[2.2]p-cyclophane of the structural formula I and an immunogen.

[0015] Preferably, the mass ratio of the immunogen to S-4,12-dihydroxy[2.2]paracyclophane is 10:3-3.5.

[0016] Preferably, in the vaccine, the final concentration of the S-4,12-dihydroxy[2.2]paracyclophane is 0.6-0.7 mg / ml.

[0017] Preferably, the immunogen comprises a viral protein;

[0018] The viral protein is the new coronavirus RBD protein;

[0019] In the vaccine, the final concentration of the SARS-CoV-2 RBD protein is 2 mg / ml.

[0020] The invention provides a method for preparing the vaccine, which comprises dissolving S-4,12-dihydroxy[2.2]-p-cyclophane in PBS containing 4% to 6% DMSO by volume, and mixing the solution with a PBS solution containing an immunogen to obtain the vaccine.

[0021] The present invention provides the use of S-4,12-dihydroxy[2.2]-p-cyclophane as an adjuvant in the preparation of vaccines. S-4,12-dihydroxy[2.2]-p-cyclophane and aluminum adjuvant were used as adjuvants to prepare protein subunit vaccines with the new coronavirus RBD protein. After immunization of mice, the new adjuvant S-4,12-dihydroxy[2.2]-p-cyclophane induced a level of humoral immunity comparable to that of aluminum hydroxide adjuvant in the new coronavirus protein subunit vaccine model, producing specific neutralizing antibodies. ELISpot experiments showed that S-4,12-dihydroxy[2.2]-p-cyclophane could significantly enhance the activation of immune cells compared to aluminum adjuvant in the new coronavirus protein subunit vaccine model, inducing a higher level of cellular immune response. At the same time, MTT cytotoxicity experiments showed that the adjuvant S-4,12-dihydroxy[2.2]-p-cyclophane had good biosafety. Therefore, S-4,12-dihydroxy[2.2]paracyclophane has great potential for preparing vaccines for clinical application, enriching the currently lackluster field of adjuvants. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The results of HPLC separation and purification of S-4,12-dihydroxy[2.2]-p-cyclophane are shown;

[0023] Figure 2 This is the H NMR spectrum identification result of S-4,12-dihydroxy[2.2]-p-cyclophane;

[0024] Figure 3 This is the result of ELISA test;

[0025] Figure 4 This is the result of IFN-gamma ELISpot assay;

[0026] Figure 5 This is the result of MTT assay. DETAILED DESCRIPTION

[0027] The present invention provides the use of S-4,12-dihydroxy[2.2]paracyclophane of the structural formula I as an adjuvant in the preparation of vaccines.

[0028]

[0029] In the present invention, the chemical formula of the S-4,12-dihydroxy[2.2]-cyclophane is C 16 H 16O2, with a relative molecular mass of 240.12, is slightly soluble in water and easily soluble in organic solvents such as DMF, DMSO, and EA. It has the appearance of a light yellow powder. In the present embodiment, the S-4,12-dihydroxy[2.2]-p-cyclophane was purchased from Daicel Corporation. S-4,12-dihydroxy[2.2]-p-cyclophane was separated and purified by HPLC. The results are shown in FIG. Figure 1 The purified S-4,12-dihydroxy[2.2]-paracyclophane was identified by H NMR spectrum. Figure 2 .

[0030] In the present invention, the vaccine preferably comprises an immunogen, which is preferably a protein. The protein is preferably a COVID-19 immune protein. The novel coronavirus immune protein is preferably a novel coronavirus RBD protein; the amino acid sequence of the novel coronavirus RBD protein is preferably as shown in SEQ ID NO: 1 (VQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN F), specifically refer to the prior art (Fitzgerald GA, Komarov A, Kaznadzey A, Mazo I, Kireeva ML. Expression of SARS-CoV-2 surface glycoprotein fragment319-640 in E. coli, and its refolding and purification. Protein Expr Purif. 2021 Jul; 183: 105861.) The mass ratio of the protein to S-4,12-dihydroxy[2.2]paracyclophane is preferably 10:3-3.5, more preferably 10:3.18.

[0031] The present invention provides a vaccine comprising S-4,12-dihydroxy[2.2]p-cyclophane of the structural formula I and an immunogen.

[0032] In the present invention, the mass ratio of the S-4,12-dihydroxy[2.2]-paracyclophane to the immunogen is preferably 3-3.5:10. In the vaccine, the final concentration of the S-4,12-dihydroxy[2.2]-paracyclophane is 0.6-0.7 mg / ml, more preferably 0.6058 mg / ml. The immunogen preferably comprises a viral protein; the viral protein is preferably the SARS-CoV-2 RBD protein. In the vaccine, the final concentration of the SARS-CoV-2 RBD protein is preferably 2 mg / ml.

[0033] The present invention provides a method for preparing the vaccine, which comprises dissolving S-4,12-dihydroxy[2.2]-p-cyclophane in DMSO and mixing the solution with a PBS solution containing an immunogen to obtain the vaccine.

[0034] The application of a small molecule adjuvant provided by the present invention in preparing a vaccine and the prepared vaccine are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] Method for preparing novel coronavirus immune protein vaccine as adjuvant using S-4,12-dihydroxy[2.2]-p-cyclophane

[0037] The S-4,12-dihydroxy[2.2]-p-cyclophane adjuvant was first dissolved in PBS containing 5% DMSO by volume and then mixed with a PBS solution containing the SARS-CoV-2 RBD protein at a mass ratio of 10:3.18, resulting in a final concentration of 0.21 mg / ml for the S-4,12-dihydroxy[2.2]-p-cyclophane and 0.67 mg / ml for the SARS-CoV-2 RBD protein. This was designated the novel adjuvant vaccine.

[0038] Comparative Example 1

[0039] The SARS-CoV-2 RBD protein was dissolved in PBS solution, and then mixed with aluminum hydroxide aqueous solution at a mass ratio of 2:7. The mixture was emulsified at 3000 rpm for 12 hours to obtain a final concentration of 0.67 mg / ml of SARS-CoV-2 RBD protein and a final aluminum hydroxide concentration of 2.35 mg / ml. The vaccine was recorded as aluminum adjuvant vaccine.

[0040] Example 2

[0041] (1) Immunization of Balb / c mice with novel adjuvant vaccine or aluminum adjuvant vaccine

[0042] Specific animal immunization method: The SARS-CoV-2 RBD protein was selected as the SARS-CoV-2 vaccine and mixed with the adjuvants S-4,12-dihydroxy[2.2]-paracyclophane and aluminum hydroxide to immunize SPF-grade, 6-8 week old female BALB / c mice. The specific animal experimental groups are as follows:

[0043] Group 1: Each mouse was injected with 10 μg of the novel adjuvant containing the SARS-CoV-2 RBD protein, designated as the novel adjuvant group;

[0044] Group 2: Each mouse was injected with 10 μg of SARS-CoV-2 RBD protein-containing aluminum adjuvant vaccine, recorded as the aluminum adjuvant group;

[0045] Group 3: Each mouse was injected with 10 μg of SARS-CoV-2 RBD protein, designated as the RBD group;

[0046] Group 4: Each mouse was injected with an equal volume of normal saline, which was recorded as the blank group.

[0047] Each group consisted of 6 mice. The drug was injected intramuscularly into the hind legs, with half the dose injected into the left and right hind legs.

[0048] Immunization timeline: After purchase, the mice were raised for one week to allow them to acclimate to the new environment. They were then immunized three times on the 8th, 22nd, and 36th days. Seven days after the last immunization, blood was collected from the eyeballs for ELISA testing, and spleen cells were harvested for ELISpot experiments.

[0049] All animal experiments were approved by the Animal Experiment Ethics Committee of the Institute of Medical Biology, Chinese Academy of Medical Sciences and performed in strict accordance with the regulations of the Yunnan Provincial Laboratory Animal Welfare and Ethics Committee.

[0050] (II) ELISA test to detect the level of specific IgG antibodies produced by mice induced by vaccines and adjuvants

[0051] The SARS-CoV-2 RBD protein subunit vaccine induces the body to produce specific antibodies (primarily IgG) to neutralize the SARS-CoV-2, thus providing immune protection. Therefore, it is of great significance to detect the level of specific IgG antibodies induced by the vaccine and adjuvant.

[0052] The specific implementation steps are as follows:

[0053] 1. Dilute the SARS-CoV-2 RBD antigen protein to a concentration of 5 μg / mL with an alkaline buffer solution containing 0.012 mol / L Na2CO3 and 0.03 mol / L NaHCO3 and coat a 96-well plate (100 μL per well). Incubate at 37°C for 2 h.

[0054] 2. Discard the antigen coating solution and add blocking solution (PBS containing 2% BSA) to the 96-well plate, add 100 μL to each well, and incubate at 37°C for 2 hours;

[0055] 3. Discard the blocking solution, add 300 μL PBST to each well and wash three times. Then, dilute the mouse serum (from the different treatment groups prepared above) to different concentrations in PBS containing 2% BSA, add 100 μL to each well, and incubate at 37°C for 1 hour.

[0056] 4. Discard the mouse serum, add 300 μL PBST to each well and wash three times, dilute HRP-labeled goat anti-mouse secondary antibody at 1:5000, add 100 μL of the above enzyme-labeled secondary antibody dilution solution to each well, and incubate at 37°C for 1 hour;

[0057] 5. Discard the secondary antibody diluent, add 300 μL PBST to each well, wash three times and control to dryness, then add TMB colorimetric solution, 100 μL / well, add 2M H2SO4 to each well after 15 minutes to stop color development, and then detect the OD value using an enzyme-linked immunosorbent assay at wavelengths of 450 nm and 630 nm.

[0058] The results of ELISA experiments showed that the ability of the novel adjuvant group to induce specific IgG antibodies was comparable to that of the aluminum adjuvant group (no significant difference between the two groups), and was significantly better than that of the RBD group (see Figure 3 ). This indicates that the new adjuvant S-4,12-dihydroxy[2.2]cyclopentane induces a level of humoral immunity comparable to that of aluminum adjuvant in the new coronavirus protein subunit vaccine model.

[0059] Example 3

[0060] IFN-gamma ELISpot assay to detect T cell activation levels in mice induced by vaccines and adjuvants

[0061] In addition to inducing high levels of humoral immune responses and producing specific neutralizing antibodies, the ability of vaccines and adjuvants to induce cellular immunity is currently receiving increasing attention. The ELISpot assay can detect the number of cytokines secreted by splenic immune cells, reflecting the level of splenic immune cell activation.

[0062] The specific implementation steps are as follows:

[0063] 1. Incubate the IFN-gamma ELISpot plate with 1640 medium containing 10% fetal bovine serum (200 μL per well) for 30 minutes at room temperature.

[0064] 2. Discard the culture medium and add spleen cell suspension (about 2.5×10 5 Cells, spleen cells derived from mice in each treatment group in Example 2) and stimulants (new coronavirus RBD protein), incubated at 37 ° C, 5% CO2, and a humidified environment for 24 hours;

[0065] 3. Discard the cells and wash the ELISpot plate five times by adding 200 μL PBS to each well. Dilute the detection antibody (R4-6A2-biotin) to a concentration of 1 μg / mL in PBS containing 0.5% fetal bovine serum. Add 100 μL of the detection antibody dilution to each well and incubate at room temperature for 2 h.

[0066] 4. Discard the detection antibody dilution solution, add 200 μL PBS to each well, wash the ELISpot plate 5 times, dilute streptavidin-labeled alkaline phosphatase (Streptavidin-ALP) at 1:1000, add 100 μL of the above dilution solution to each well, and incubate at room temperature for 1 hour;

[0067] 5. Discard the detection antibody diluent, add 200 μL PBS to each well to wash the ELISpot plate 5 times, filter the substrate solution (BCIP / NBT-plus) through a 0.45 μm filter, add 100 μL to each well and incubate for about 15 minutes;

[0068] 6. Wash the plate with tap water to stop color development, air-dry the ELISpot plate at room temperature, and then read the plate under a reader and record the number of spots.

[0069] The results of IFN-gammaELISpot experiments showed that the number of spots in the novel adjuvant group was significantly higher than that in the aluminum adjuvant group and the RBD group (see Figure 4 ). This shows that the new adjuvant S-4,12-dihydroxy[2.2]paracyclophane discovered in the present invention can significantly enhance the activation of immune cells and induce a higher level of cellular immune response in the new coronavirus protein subunit vaccine model compared to aluminum adjuvant.

[0070] Example 4

[0071] Evaluation of the biological safety of adjuvant S-4,12-dihydroxy[2.2]-p-cyclophane by MTT cytotoxicity assay

[0072] In addition to effectiveness, biosafety of adjuvants is also an important evaluation indicator. The present invention evaluated the biosafety of the novel adjuvant S-4,12-dihydroxy[2.2]paracyclophane by in vitro cell stimulation and MTT cell assay.

[0073] The specific implementation steps are as follows:

[0074] 1. Use a cell scraper to scrape the adherent DC cells cultured in a T75 culture flask and transfer them to a centrifuge tube. Centrifuge at 1800 rpm for 5 minutes, discard the supernatant, and resuspend the cells in DMEM medium to prepare a single-cell suspension. Count the cells using a cell counting plate.

[0075] 2. Inoculate the single cell suspension into a 96-well plate with a cell number of approximately 6×10 3 / well, cultured in a 37°C incubator containing 5% CO2 for 4 h;

[0076] 3. Set up four groups, namely the novel adjuvant group, aluminum adjuvant group, RBD group, and blank group, with three replicates in each group. Add 1 μg of SARS-CoV-2 RBD protein and 0.318 μg of S-4,12-dihydroxy[2.2]cyclopentane adjuvant to each well in the novel adjuvant group, 1 μg of SARS-CoV-2 RBD protein and 3.5 μg of aluminum hydroxide adjuvant to each well in the aluminum adjuvant group, 1 μg of SARS-CoV-2 RBD protein to each well in the RBD group, and an equal volume of normal saline to the blank group. Incubate in a 37°C incubator with 5% CO2 for 12 hours;

[0077] 4. Discard the culture medium, add 10 μL of MTT solution and 100 μL of fresh DMEM medium to each well, and culture in a 37°C incubator with 5% CO2 for 4 hours. Then discard the liquid in the wells, add 100 μL of Fomazan to each well, shake in the dark on a shaker for 15 minutes, and finally read the absorbance of each well at 570 nm using a spectrophotometer.

[0078] The results of MTT experiments showed that the cell survival rates of the novel adjuvant group were comparable to those of the aluminum adjuvant group, RBD group, and blank group, and there was no significant difference between them (see Figure 5 ). This indicates that the novel adjuvant S-4,12-dihydroxy[2.2]-paracyclophane discovered in the present invention has good biosafety at the cellular level.

[0079] It can be seen from the above examples that the adjuvant effect of S-4,12-dihydroxy[2.2]paracyclophane in vaccines provided by the present invention is evaluated in different dimensions. The present invention evaluates the adjuvant effect of S-4,12-dihydroxy[2.2]paracyclophane on the new crown protein subunit vaccine model and finds that: S-4,12-dihydroxy[2.2]paracyclophane can induce a comparable level of humoral immune response (producing comparable amounts of specific antibodies IgG) compared to traditional superior aluminum adjuvants, significantly enhances cellular immune response (activates more immune cells, produces more cytokines IFN-gamma), and has good biosafety (equivalent cell survival rates in in vitro cell experiments). It enriches the current types of adjuvants and provides a new means for conducting primate research and even vaccines approved for clinical use.

[0080] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of S-4,12-dihydroxy[2.2]paracyclophane of formula I as an adjuvant in the preparation of a vaccine, wherein the immunogen in the vaccine is a novel coronavirus RBD protein; Formula I; The mass ratio of the new coronavirus RBD protein and S-4,12-dihydroxy[2.2]paracyclophane is 10:3~3.

5.

2. The application according to claim 1, characterized in that: The amino acid sequence of the SARS-CoV-2 RBD protein is shown in SEQ ID NO:

1.

3. A vaccine, characterized in that Comprising S-4,12-dihydroxy[2.2]paracyclophane of formula I and an immunogen; Formula I; The immunogen is the SARS-CoV-2 RBD protein; The mass ratio of the immunogen to S-4,12-dihydroxy[2.2]paracyclophane is 10:3-3.

5.

4. The vaccine according to claim 3, characterized in that In the vaccine, the final concentration of the S-4,12-dihydroxy[2.2]paracyclophane is 0.6-0.7 mg / ml.

5. The vaccine according to claim 3 or 4, characterized in that In the vaccine, the final concentration of the SARS-CoV-2 RBD protein is 2 mg / ml.

6. The method for preparing the vaccine according to any one of claims 3 to 5, characterized in that: The S-4,12-dihydroxy[2.2]paracyclophane is dissolved in PBS containing 4% to 6% DMSO by volume, and then mixed with a PBS solution containing an immunogen to obtain the obtained product.

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