Novel immunologic adjuvant

Abstract

The invention discloses a novel immunologic adjuvant, a composition containing the immunologic adjuvant and a pharmaceutical composition. The immunologic adjuvant is picrasidine S. The immunologic adjuvant composition comprises picrasidine S and optionally one or more other types of immunologic adjuvants, such as an aluminum adjuvant. The pharmaceutical composition comprises an antigen and the immunologic adjuvant composition. Experiments prove that the picrasidine S can effectively stimulate dendritic cells to generate cell factors IFN-beta and IL-6, and has PS dose dependence; picrasidine S plays an important role in enhancing specific humoral immune response of different types of antigens, and has a stronger effect than an aluminum adjuvant; the picrasidine S immunity can enhance the anti-tumor immune response in different tumor models. According to the invention, a new candidate drug is provided for research and development of a new generation of vaccine adjuvants, and meanwhile, the medical application of the picrasidine S is also widened.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biology, specifically relating to a novel immune adjuvant, a composition containing the immune adjuvant, and a pharmaceutical composition. Background Technology

[0002] Vaccination protects humanity from numerous devastating diseases, making a tremendous contribution to global public health. It is estimated that vaccines save approximately 3 million lives each year. The application of vaccines has enabled the control and even complete eradication of many infectious diseases, such as smallpox and rinderpest. Existing disease prevention strategies include screening and treatment, drug therapy, and vaccination. Among these measures, vaccination is considered the most effective and safest method for reducing disease morbidity and mortality.

[0003] Traditional vaccines, including live attenuated vaccines and inactivated vaccines, are prepared by weakening or inactivating pathogens. Such vaccines retain most of their immunogenicity, mimicking the natural infection process well and inducing a long-lasting immune response. However, traditional vaccines also have significant drawbacks, such as the risk of autoimmune reactions, reactive and allergic side effects, as well as production risks and difficulties, and stringent requirements for cold chain transportation. These shortcomings of traditional vaccines spurred the development of subunit vaccines. Subunit vaccines use purified, immunogenic pathogen fragments as antigens; their components can be proteins, toxoids, polysaccharides, etc., offering advantages such as low adverse reactions, ease of large-scale production, and lower requirements for transportation and storage. However, subunit vaccines often have poor immunogenicity, therefore they usually need to be used in conjunction with adjuvants to achieve better immunization effects.

[0004] Adjuvants are non-immunogenic substances typically added to vaccines as immunostimulants or antigen delivery systems to stimulate and enhance the strength and duration of the immune response, reducing the amount of antigen used and the number of vaccinations. To date, hundreds of materials have been attempted as adjuvants, including bacterial metabolites, mineral oils / surfactants, microparticles, nucleic acids, liposomes, and polysaccharides. Currently, the most widely used and earliest adjuvant in vaccines worldwide is the aluminum adjuvant, including aluminum hydroxide, aluminum phosphate, and alum. Common vaccines for hepatitis A virus, hepatitis B virus, hepatitis E virus, and human papillomavirus are all aluminum adjuvant formulations. Aluminum adjuvants are very effective in inducing humoral immune responses, producing high titers of antibodies, but they rarely or never induce cellular immune responses. Cellular immunity is essential for fighting intracellular pathogens (such as Mycobacterium tuberculosis). Furthermore, vaccines that strongly induce T-cell immune responses are needed for diseases such as cancer, AIDS, tuberculosis, and malaria. In addition to insufficient cellular immune response, aluminum adjuvants are prone to inducing inflammatory responses and allergy-related immunoglobulin E (IgE) responses at the injection site. Therefore, there is an urgent clinical need for successful new vaccine adjuvants that can promote strong and sustained immune responses while overcoming the main limitations of traditional vaccine adjuvants.

[0005] This invention focuses on developing adjuvants that enhance dendritic cell function, thereby strengthening cellular immune responses. Dendritic cells are currently the most potent antigen-presenting cells and are the initiators of the body's adaptive T-cell immune response. Enhancing their antigen-presenting capacity and T-cell activation ability helps induce effective and sustained cellular immune responses. This invention aims to develop novel adjuvants that balance humoral and cellular immunity, helping to expand the application range of adjuvants and providing new theoretical basis and treatment strategies for the prevention and treatment of more diseases. Summary of the Invention

[0006] A key characteristic of adjuvants that induce cellular immune responses is their ability to effectively activate dendritic cells. Generally, the maturation of dendritic cells enhances their antigen-presenting capacity and their ability to activate T cells, a prerequisite for inducing effective and durable immune responses. To discover novel adjuvants that can induce cellular immune responses, the applicant previously screened approximately 7,000 small molecules in mouse bone marrow-derived dendritic cells, including 3,000 natural products and 4,000 small molecules in clinical trials or already marketed. They discovered that the plant-derived natural small molecule Picrasidine S (PS) can effectively stimulate bone marrow-derived dendritic cells to express the cytokine interferon β (IFN-β). Figure 1 ), induces the production of high levels of antibodies against the antigen in mice ( Figure 2-3 It also has significant anti-tumor effects. Figure 4).

[0007] The inventors focused on the function of quassoxidine S in antigen immunity and disease models, and evaluated its potential as a novel vaccine adjuvant by comparing it with traditional aluminum adjuvant, providing new theoretical basis and candidate drugs for the development of next-generation vaccine adjuvants.

[0008] The above-mentioned objective of the present invention is achieved through the following technical solution: In a first aspect, the present invention provides the use of quassin S as and / or in the preparation of an immune adjuvant.

[0009] The molecular formula of the quassinolide S is C 30 H 29 N4O4 + The structural formula is shown in equation (I):

[0010] Formula (I) There are no reports in existing studies on the use of small molecule quassoxidine S as an immune adjuvant.

[0011] Furthermore, the immune adjuvant is an adjuvant that can induce humoral and / or cellular immune responses.

[0012] In a second aspect, the present invention provides an immune adjuvant composition.

[0013] The immune adjuvant composition includes quassin S as described in the first aspect of the present invention.

[0014] Furthermore, the immune adjuvant composition may optionally include one or more other types of immune adjuvants, such as aluminum adjuvants, specifically aluminum hydroxide.

[0015] Further, in the immune adjuvant composition, the effective amount of quassinolide S can be 0.01-1 mg / dose, preferably 0.1 mg / dose. Optionally, the effective amount of other types of immune adjuvants (such as aluminum adjuvants) can be 0.01-1 mg / dose. The doses mentioned here refer to the dosage per administration.

[0016] Thirdly, the present invention provides the use of the immune adjuvant composition described in the second aspect of the present invention in the preparation of vaccines.

[0017] Furthermore, the vaccine may be an anti-tumor vaccine, such as an anti-melanoma vaccine or an anti-T-lymphoma vaccine.

[0018] Fourthly, the present invention provides a pharmaceutical composition.

[0019] The pharmaceutical composition includes an antigen and the immune adjuvant composition described in the second aspect of the present invention.

[0020] Furthermore, in the pharmaceutical composition, the content of the antigen is 0.01-1 mg per dose. The dose referred to here is the dose administered per administration.

[0021] Furthermore, in the pharmaceutical composition, the ratio of the antigen to the adjuvant composition can be determined by those skilled in the art through routine experiments for different antigen types.

[0022] Furthermore, the pharmaceutical composition may be an anti-tumor vaccine, such as an anti-melanoma vaccine and an anti-T-lymphoma vaccine.

[0023] According to one embodiment of the present invention, the antigen is NP-OVA antigen, the immune adjuvant is quassinolide S, and the mass ratio of NP-OVA antigen to quassinolide S is 50 μg:100 μg.

[0024] According to one embodiment of the present invention, the antigen is NP-KLH antigen, the immune adjuvant is quassinolide S, and the mass ratio of NP-KLH antigen to quassinolide S is 50 μg: 150 μg.

[0025] Fifthly, the present invention provides a method for inducing immunity.

[0026] The method for inducing immunity provided by the present invention includes administering the pharmaceutical composition described in the fourth aspect of the present invention to a subject; the immunity may be one or both of cellular immunity and humoral immunity, and the administration may be administering a therapeutically effective amount of the pharmaceutical composition.

[0027] This invention experimentally demonstrates that quassin S can effectively stimulate dendritic cells to produce cytokines IFN-β and IL-6 in a dose-dependent manner. Quassin S plays an important role in enhancing specific humoral immune responses to different antigens, and its effect is stronger than that of aluminum adjuvants. Quassin S immunization can enhance anti-tumor immune responses in different tumor models. This invention provides a new candidate drug for the development of next-generation vaccine adjuvants and also broadens the pharmaceutical applications of quassin S. Attached Figure Description

[0028] Figure 1To induce IFN-β expression in mouse bone marrow-derived dendritic cells (BMDCs) with quassinolide S (PS); (AB) BMDCs were cultured in vitro and stimulated with 10 μM PS for 8 h. Quantitative PCR was used to identify the cytokines IFN-β (A) and IL-6 (B); (C) BMDCs were stimulated with 10 μM PS for 8 h, and IFN-β in the culture medium was detected by ELISA; (DE) BMDCs were stimulated with 10 μM PS for different time periods, and quantitative PCR was used to identify the cytokines IFN-β (D) and IL-6 (E); (FG) BMDCs were stimulated with different concentrations of PS for 8 h, and quantitative PCR was used to identify the cytokines IFN-β (F) and IL-6 (G).

[0029] Figure 2 To enhance the humoral immune response to NP-OVA antigen with quassinolide S (PS), wild-type mice were subcutaneously immunized with PS and aluminum adjuvant (Al) in combination with NP-OVA antigen. Blood samples were collected from the eyeballs 14 days later, and the level of NP antigen-specific antibodies in the serum was detected by ELISA.

[0030] Figure 3 To enhance the humoral immune response to NP-KLH antigen with quassinolide S (PS), wild-type mice were subcutaneously immunized with PS and aluminum adjuvant (Al) in combination with NP-KLH antigen. Blood samples were collected from the eyeballs at 2, 4 and 6 weeks later, and the level of NP antigen-specific antibodies in the serum was detected by ELISA.

[0031] Figure 4 To enhance the anti-tumor immune response with quassinolide S (PS); (AB) wild-type mice were subcutaneously immunized with PS and aluminum adjuvant (Al) in combination with NP-OVA antigen, respectively, and E.G7 cells were inoculated 38 days later, and tumor size was monitored; (C) wild-type mice were subcutaneously immunized with PS and aluminum adjuvant (Al) in combination with NP-OVA antigen, respectively, and B16-OVA cells were inoculated 23 days later, and tumor incidence was monitored.

[0032] Figure 5 To evaluate the toxicity and pharmacokinetics of the quassinosteroid S (PS)-mediated vaccine; (A) Subcutaneous immunization of wild-type mice with PS in combination with NP-OVA antigen; (BC) Measurement of mouse body weight (B) and mortality (C) within 7 days post-immunization, monitored every two days; (DF) On day 7, organs were removed and their weights recorded (D and E) and H&E staining was performed (F); (G) Pharmacokinetics of PS in mice; T 1 / 2 Half-life; C max Maximum plasma concentration; AUC, area under the curve. Detailed Implementation

[0033] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0035] Definitions of terms in this invention The compounds in this article The compounds mentioned herein, or the compounds described herein, refer to compounds represented by formula (I) below.

[0036] (I) Subjects The term "subject" as used in this invention refers to an animal, including but not limited to primates (e.g., humans), cattle, sheep, goats, horses, dogs, pigs, cats, rabbits, rats, mice, fish, birds, and poultry such as chickens, ducks, geese, etc. Preferably, the animal is a mammal. In a preferred embodiment, the subject is a human.

[0037] immune cells In this invention, immune cells refer to all cells involved in and related to the immune response, as well as their precursor cells. Immune cells include T cells (e.g., CD4+ cells, CD8+ cells, and various other T cell subclasses), B cells (e.g., CD19+ cells), natural killer cells (NK cells), macrophages, monocytes, dendritic cells, and neutrophils.

[0038] Specific T lymphocytes and specific B lymphocytes expressing specific antigen receptors participate in mediating the adaptive immune response. B lymphocytes, upon receiving specific antigen stimulation, can be activated, proliferate, and differentiate into plasma cells, producing specific antibodies and mediating the humoral immune response. T lymphocytes, upon receiving specific antigen stimulation, can be activated, proliferate, and differentiate into effector T cells, mediating cellular immune responses and assisting humoral immune responses. Furthermore, during the initiation phase of the adaptive immune response, professional antigen-presenting cells (APCs) such as dendritic cells and monocytes / macrophages participate, presenting antigens to activate T cells. During the effector phase of the adaptive immune response, monocytes / macrophages, NK cells, and other cells participate, working synergistically with T cells and antibodies to clear antigens.

[0039] Cells involved in the innate immune response mainly include monocytes / macrophages, granulocytes, dendritic cells, NK cells, endothelial cells, mast cells, erythrocytes, platelets, and a few T and B lymphocyte subsets. Among them, NK cells are the third type of lymphocyte, possessing non-specific cytotoxic activity, and play an important role in the innate immune response against viral infections and tumors. Monocytes / macrophages and granulocytes have strong phagocytic and killing functions and participate in the inflammatory response by releasing large amounts of active products.

[0040] vaccine The "vaccine" of this invention refers to vaccines well-known to those skilled in the art, broadly encompassing any biological product that, after administration via injection or mucosal route, can induce the body to produce specific antibodies and / or cellular immunity against a specific pathogen, thereby providing the body with the ability to protect against or eliminate the pathogen. This includes proteins, polysaccharides, nucleic acids, live vectors, or infectious agents. A vaccine is an immunizing agent used to prevent infectious diseases, prepared by artificially attenuating, inactivating, or using genetic engineering methods from pathogenic microorganisms (such as bacteria, rickettsiae, viruses, etc.) and their metabolites. Vaccines retain the characteristic of pathogens stimulating the animal's immune system. When an animal comes into contact with this harmless pathogen, the immune system produces certain protective substances, such as immune hormones, active physiological substances, and specific antibodies. When the animal is exposed to the pathogen again, its immune system, following its previous memory, produces more protective substances to prevent harm from the pathogen.

[0041] The term "vaccine" as used herein refers to a preparation designed to induce an immune response against an antigen. Vaccines can be therapeutic, administered during treatment to enhance the immune response or drive a response in a specific direction, or they can be prophylactic, administered before or shortly after the onset of a disease. Vaccines can be both therapeutic and prophylactic in treating existing diseases and preventing future recurrences. Vaccines can be administered to subjects using methods of administration commonly used in the art. The terms "administration" or "application" as used herein include all suitable ways of providing a substance to a patient. Common routes of administration include oral, sublingual, transmucosal, transdermal, rectal, vaginal, subcutaneous, intramuscular, intravenous, intra-arterial, intrathecal, via catheter, via implant, etc.

[0042] The antigen of the present invention can be used to prepare drugs for inducing an immune response against the antigen in a subject. In one embodiment, the antigen of the present invention can be used to prepare a melanoma vaccine and a T-lymphoma vaccine.

[0043] The "vaccine adjuvant" or "adjuvant" of this invention is a vaccine adjuvant well known to those skilled in the art. The word adjuvant originates from the Latin word "Aduvare," meaning to assist or enhance. A vaccine adjuvant is an additive to a vaccine that, when injected into the body before or mixed with the antigen, can enhance the body's immune response to the antigen or alter the type of immune response. It is a non-specific immune enhancer and has no antigenicity itself.

[0044] Currently, there is no unified international standard for the classification of adjuvants. Commonly used adjuvants mainly include insoluble aluminum salt colloids, oil-water emulsions, microorganisms and their metabolites, nucleic acids and their analogues, cytokines, immunostimulatory complexes, propolis, liposomes, and ODN. The compounds of this invention (such as quassin S) can also be used as vaccine adjuvants and can exert excellent adjuvant functions. In this invention, quassin S can be used alone as a vaccine adjuvant or in combination with other commonly used adjuvants. The compound quassin S and these commonly used adjuvants can exert additive or synergistic effects, improving the immunogenicity of the antigen, thereby reducing the vaccine dosage or improving vaccine efficacy (e.g., reducing the dosage or number of vaccine administrations).

[0045] In one embodiment, the present invention also provides the use of the compound described herein, quassinine S, in the preparation of a vaccine adjuvant, preferably a melanoma vaccine and a T-lymphoma vaccine. In one embodiment, the vaccine adjuvant described herein further comprises one or more other substances that synergistically act as adjuvants with the compound. The effective amount of the compound described herein can be determined by those skilled in the art through conventional experiments; for example, the effective amount can be 0.01-1 mg / dose, including any value within the range of 0.01-1 mg, such as 0.01 mg / dose, 0.02 mg / dose, 0.03 mg / dose, 0.04 mg / dose, 0.05 mg / dose, 0.06 mg / dose, 0.07 mg / dose, 0.08 mg / dose, 0.09 mg / dose, 0.1 mg / dose, 0.2 mg / dose, 0.3 mg / dose, 0.4 mg / dose, 0.5 mg / dose, 0.6 mg / dose, 0.7 mg / dose, 0.8 mg / dose, 0.9 mg / dose, and 1 mg / dose vaccine.

[0046] The vaccine may contain another adjuvant, such as an aluminum adjuvant, specifically aluminum hydroxide adjuvant. The effective amount of the other adjuvant, such as aluminum adjuvant or aluminum hydroxide adjuvant, can be determined by those skilled in the art through routine experiments. For example, the effective amount may be 0.01-1 mg / dose, such as 0.01 mg / dose, 0.02 mg / dose, 0.03 mg / dose, 0.04 mg / dose, 0.05 mg / dose, 0.06 mg / dose, 0.07 mg / dose, 0.08 mg / dose, 0.09 mg / dose, and 0.1 mg / dose, 0.2 mg / dose, 0.3 mg / dose, 0.4 mg / dose, 0.5 mg / dose, 0.6 mg / dose, 0.7 mg / dose, 0.8 mg / dose, 0.9 mg / dose, and 1 mg / dose vaccine.

[0047] The effective amount of antigen in the vaccine can be determined by those skilled in the art through routine experiments. For example, the effective amount can be 0.01-1 mg / dose, such as 0.01 mg / dose, 0.02 mg / dose, 0.03 mg / dose, 0.04 mg / dose, 0.05 mg / dose, 0.06 mg / dose, 0.07 mg / dose, 0.08 mg / dose, 0.09 mg / dose, and 0.1 mg / dose, 0.2 mg / dose, 0.3 mg / dose, 0.4 mg / dose, 0.5 mg / dose, 0.6 mg / dose, 0.7 mg / dose, 0.8 mg / dose, 0.9 mg / dose, and 1 mg / dose vaccine.

[0048] drug The term "medicine" or "medicinal formulation" refers to a formulation in such a form as to allow the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional components that would cause unacceptable toxicity to a subject to be administered the formulation. In one embodiment, this document relates to the use of the compounds or pharmaceutical compositions described herein in the preparation of a medicament for the prevention or treatment of a subject's tumor, microbial infection, or allergy. The subject may be a human or animal, such as a mouse, rat, livestock, such as a dog, pig, cattle, or horse; or poultry, such as a chicken, duck, or goose. Those skilled in the art can determine the effective amount of the compound in the medicament or pharmaceutical formulation according to conventional methods, and determine the method of administration of the medicament according to conventional methods.

[0049] The experimental materials and methods used in the following embodiments are as follows: 1. Experimental materials: 1.1 Laboratory Animals All mouse-related experiments and procedures in this embodiment were conducted in accordance with the protocols approved by the Institute of Laboratory Animal Management and Use (IACUC) of Tsinghua University. The experimental mice were housed in the SPF (specific pathogen-free) animal housing platform at the Tsinghua University Laboratory Animal Center, with a 12-hour / 12-hour light cycle and a housing temperature of 23 degrees Celsius. The mice were cared for by professional breeders and had free access to water and food during their rearing period. All experimental mice were wild-type C57BL / 6 mice, 8-12 weeks old. 1.2 Main Reagents The main reagents and antibodies used in this embodiment are shown in Table 1.

[0050] Table 1. Main Reagents and Their Sources

[0051] 2. Experimental Methods: 2.1. RNA extraction using the Trizol method 1) Centrifuge the cells, discard the supernatant, add 1 mL of Trizol, mix well, and store at -80 ℃. 2) Remove the sample and thaw it at room temperature. Add 200 µL of chloroform, tighten the cap, and place it on a vortex mixer to mix thoroughly. 3) Centrifuge at 10,000 rpm for 5 min, remove the tube, transfer 400 µL of supernatant to a new centrifuge tube, add 800 µL of anhydrous ethanol and mix well, then freeze at -20 ℃ for 30 min. 4) Take out the sample and put it into a centrifuge. Centrifuge at 15,000 rpm for 10 min. Discard the supernatant and add 600 µL of 75% ethanol to wash the precipitate. 5) Centrifuge the sample at 15,000 rpm for 3 min, discard the supernatant, centrifuge again for 1 min, and remove the remaining supernatant with a pipette. 6) Air dry the sample at room temperature, add 30 µL of RNase-free water to dissolve the RNA, and store in a -80 ℃ refrigerator.

[0052] 2.2. RNA reverse transcription The RNA reverse transcription kit used in this study consisted of two steps: the first step was for removing the genomic DNA template, with the reaction system prepared as shown in Table 2, and the reaction conditions were 42 ℃ for 2 min and 4 ℃ for 2 min. The second step was the RNA reverse transcription reaction, with the reaction system prepared as shown in Table 3, and the reaction conditions were 37 ℃ for 15 min, 85 ℃ for 15 s, and held at 4 ℃.

[0053] After the two-step reaction is completed, the sample is stored in a -20 ℃ refrigerator for later use.

[0054] Table 2 RNA reverse transcription reaction system (step 1)

[0055] Table 3. RNA reverse transcription reaction system (step 2)

[0056] 2.3. Real-time quantitative PCR The expression level of the target gene was detected using cDNA obtained by reverse transcription of RNA as a template. The preparation of the qPCR reaction system is shown in Table 4, and the reaction conditions are shown in Table 5. After the reactants were mixed evenly, they were placed in a real-time PCR instrument for reaction.

[0057] After the qPCR reaction, a uniform linear interval was selected from the amplification curve to determine the CT value. The CT value of the Hprt gene was used as an internal reference, and 2... -ΔΔCt The method involves relative quantitative analysis of target genes in the test samples. The qPCR primer sequences used in this embodiment are shown in Table 6.

[0058] Table 4 qPCR reaction system

[0059] Table 5 qPCR reaction conditions

[0060] Table 6 qPCR primer sequences

[0061] 2.4. In vitro induction of bone marrow-derived dendritic cells 1) After euthanizing the mice by dislocation of the neck, disinfect them by soaking them in alcohol, and completely remove the tibia and femur. Take 4 bones from each mouse and remove the muscles attached to the bones. 2) Wash the bone three times with sterile PBS, then cut both ends of the bone with sterilized scissors; 3) Using a syringe equipped with a 25 G needle, draw RPMI-1640 medium to rinse all bone marrow into a sterile bacterial culture dish, collect the bone marrow cell suspension in the culture dish, and centrifuge at 1500 rpm × 10 min; 4) Discard the supernatant, add 1 mL of sterile 1 × ACK solution to suspend the cells and lyse the red blood cells, let stand at room temperature for 1 min to lyse the red blood cells, add 10 mL of RPMI-1640 culture medium to stop the process, and centrifuge at 1500 rpm × 5 min. 5) Discard the supernatant, resuspend the cells in RPMI-1640 complete medium containing rmGM-CSF 10 ng / mL and IL-4 10 ng / mL, divide into 6-well culture plates, 4 mL in each well, and incubate in a cell culture incubator at 37 ℃ containing 5% CO2. 6) After 48 h, gently blow the cells away and aspirate the suspended cells along with the culture medium, leaving only the adherent cells. 7) Add fresh complete culture medium and the same concentration of rmGM-CSF and IL-4 cytokines, and continue culturing; 8) On the 5th day, change the medium semi-quantitatively and replenish cytokines, and retain the suspension cells for continued culture; 9) On day 7, after gently blowing on the cells, collect all suspended cells, which are the enriched mouse bone marrow-derived dendritic cells (BMDC).

[0062] 2.5. Enzyme-linked immunosorbent assay (ELISA) 1) To detect the level of specific antibodies produced by mice immunized with NP-KLH or NP-OVA, 100 μL / well of 5 μg / mL NP-BSA was plated on a 96-well ELISA plate and incubated overnight at 4 °C; 2) Wash three times with washing buffer, add 200 μL of 3% gelatin blocking solution to each well, and block at room temperature for 2 hours; 3) Wash once with washing buffer, add serially diluted serum from different mice after immunization to the blocked ELISA plate, and incubate at room temperature for 2 h; 4) Wash 3-5 times with washing buffer, add 1:5000 diluted HRP-conjugated anti-mouse IgG, IgG1, IgG2b, IgG2c and IgG3 antibodies, and incubate at room temperature in the dark for 1 h. 5) Wash 5-7 times with washing buffer, add 100 μL of 1× TMB solution, and incubate for 15 min; 6) Add 50 μL of 1 M H3PO4 to stop the reaction. Read the plate at 450 nm and 570 nm using a plate reader. Subtract the 570 nm value from the 450 nm value before data analysis. All wash buffers used in the ELISA process were PBS buffer containing 0.05% Tween-20.

[0063] 2.6. Hematoxylin and eosin (H&E) staining 1) Skin tissue was fixed with 4% paraformaldehyde at 4 °C for 24 h, embedded in paraffin, and then sectioned. 2) Place the slices in an oven at 50-60 ℃ for 60 min, dewax them twice in xylene for 10 min each time, twice in 100% alcohol for 5 min each time, once in 95% and once in 75% graded alcohol for 5 min each time, and wash them twice in distilled water for 5 min each time. 3) Stain with hematoxylin solution for 5-10 min, rinse with tap water, differentiate with differentiation solution for 5 s, blue with blue solution for 1 min, and rinse twice with tap water for 5 min each time; 4) Stain in eosin solution for 2 min, then rinse with tap water for 10 s; 5) Place in 95% alcohol for 1 min, then place in 100% alcohol twice and xylene twice in sequence, each time for 5 min to dehydrate and clear. Remove the sections, air dry them, mount them with neutral resin, and observe them under a microscope.

[0064] Example 1: Determination of IFN-β expression in mouse bone marrow-derived dendritic cells induced by quassinolide S. Mice bone marrow-derived dendritic cells were stimulated for 8 h with 10 μM quasine S dissolved in DMSO. An equal volume of DMSO was added to the negative control (NC). RNA was extracted using the Trizol method, and quantitative PCR was performed after reverse transcription. The results showed that quasine S effectively stimulated dendritic cells to produce the cytokines IFN-β and IL-6. Figure 1 IFN-β expression is a key step in the activation of innate immunity and the maturation of dendritic cells, thereby inducing adaptive immunity. IL-6 is an important factor in inflammatory responses. ELISA was used to detect the cytokine IFN-β in the culture medium. Figure 1 C). BMDCs were stimulated with 10 μM quassinolide S for different time periods, and quantitative PCR was used to identify the cytokines IFN-β and IL-6. The production of IFN-β and IL-6 increased with increasing PS stimulation time. Figure 1 DE). BMDCs were stimulated with different concentrations of quassinolide S for 8 h, and the cytokines IFN-β and IL-6 were identified by quantitative PCR. The production of IFN-β and IL-6 was PS dose-dependent. Figure 1 These results indicate that quassinosteroids S has a regulatory function on dendritic cells.

[0065] Example 2: Determination of the effect of quassinosteroid S on enhancing antigen-specific humoral immune response In this embodiment, wild-type B6 mice were subcutaneously immunized with quassinolide S in conjunction with NP-OVA antigen. The injection solution was PBS buffer containing 50 μg NP-OVA antigen and 100 μg quassinolide S, with a total volume of 100 μL, and 50 μL was injected subcutaneously into each lateral arm. Quassinolide S was a 40 mg / mL solution pre-dissolved in DMSO. The vector control group consisted of PBS buffer containing 50 μg NP-OVA antigen and an equal volume of DMSO. We also compared this to aluminum adjuvant (Aluminum, A1), the most widely used adjuvant in human vaccines. The aluminum adjuvant group consisted of PBS buffer containing 50 μg NP-OVA antigen and 25 μL of aluminum adjuvant.

[0066] The mice in each of the above-mentioned quassinolide S group, carrier control group, and aluminum adjuvant group were 10.

[0067] Blood was collected from the eyeballs 14 days after immunization, and the level of NP antigen-specific antibodies in the serum was detected by ELISA. The level of NP antigen-specific antibodies in the serum of mice injected with quassoxidine S was significantly higher than that in the vector control group and also higher than that in the aluminum adjuvant group, indicating that quassoxidine S plays an important role in the specific humoral immune response to antigens and is more effective than aluminum adjuvant. Figure 2 ).

[0068] We also investigated the function of quassinolide S in immunization against another antigen, NP-KLH, and compared it with aluminum adjuvant. The injection consisted of PBS buffer containing 50 μg NP-KLH antigen and 150 μg quassinolide S, with a total volume of 100 μL, administered subcutaneously to both sides (50 μL each). Quassinolide S was a 40 mg / mL solution pre-dissolved in DMSO. The control group received PBS buffer containing 50 μg NP-KLH antigen and an equal volume of DMSO. The aluminum adjuvant group received PBS buffer containing 50 μg NP-KLH antigen and 25 μL of aluminum adjuvant.

[0069] The mice in each of the above-mentioned quassinolide S group, carrier control group, and aluminum adjuvant group were 6.

[0070] Blood samples were collected from the eyes 14, 28, and 42 days after immunization, and serum NP antigen-specific antibody levels were detected by ELISA. Results were observed at 2, 4, and 6 weeks post-immunization. Figure 3 The levels of NP antigen-specific antibodies in the serum of mice injected with quassoxidine S were significantly increased, and higher than those in the aluminum adjuvant group. Figure 3 The above demonstrates that quassin S plays an important role in enhancing specific humoral immune responses to different types of antigens, and its effect is stronger than that of aluminum adjuvants.

[0071] Example 3: Determination of the immune-enhancing and antitumor immune response of quassinosteroid S. The results of Example 2 showed that quassin S enhances antigen-specific humoral immune responses. To assess its protective function as a vaccine adjuvant, we examined the regulatory effect of quassin S as a vaccine adjuvant on disease progression in different types of tumor models. Wild-type mice were subcutaneously immunized with quassin S or an aluminum adjuvant in conjunction with NP-OVA antigen. The injection solution was PBS buffer containing 50 μg NP-OVA antigen and 100 μg quassin S, with a total volume of 100 μL, injected bilaterally in each eye (50 μL). Quassin S was a 40 mg / mL solution pre-dissolved in DMSO. The control group received PBS buffer containing 50 μg NP-OVA antigen and an equal volume of DMSO. The aluminum adjuvant group received PBS buffer containing 50 μg NP-OVA antigen and 25 μL aluminum adjuvant.

[0072] Mice 38 days post-immunization were subcutaneously inoculated with mouse T-lymphoma E.G7 cells expressing OVA antigen at a dose of 5 × 10⁻⁶. 6 / each, to monitor tumor growth ( Figure 4 A). The quassinolide S group, the carrier control group, and the aluminum adjuvant group each contained 10 mice. Compared with the control group, the tumor volume in the quassinolide S group mice was significantly smaller ( Figure 4 B).

[0073] In addition, mice were subcutaneously inoculated 23 days after immunization with B16 melanoma cells expressing the OVA antigen (B16-OVA) at a dose of 2 × 10⁻⁶. 6 / mouse / , to monitor tumor growth. The above-mentioned quassinolide S group, carrier control group, and aluminum adjuvant group each contained 6 mice. It can be seen that compared with the control group, the tumor incidence rate in the quassinolide S group was significantly lower, and lower than that in the aluminum adjuvant group ( Figure 4 C). The above results indicate that quassinolide S-immunization can enhance anti-tumor immune responses in different tumor models.

[0074] Example 4: Determination of the toxic side effects of quassoxidine S on the body To determine whether quassinolide S had toxic side effects on the body, we immunized mice with a PBS buffer solution containing 50 μg NP-OVA antigen and 100 μg quassinolide S, totaling 100 μL, administered subcutaneously to each side (50 μL). Quassinolide S was a 40 mg / mL solution pre-dissolved in DMSO. The control group received a PBS buffer solution containing 50 μg NP-OVA antigen. Figure 5 A). Subsequently, we measured the mice's weight every two days ( Figure 5 B) and survival rate Figure 5 C) Observation was conducted, and the spleen was collected 7 days later. Figure 5 D) and kidneys ( Figure 5 E) Observe and weigh, and perform H&E staining at the same time ( Figure 5 F) and quasicillin S showed no significant abnormalities. The results indicate that quasicillin S has no significant toxic side effects on the body. We evaluated its plasma concentration and stability in mice by measuring the pharmacokinetic parameters of quasicillin S using LC-MS / MS after a single intravenous administration. The results showed that quasicillin S has good pharmacokinetic characteristics, including a reasonable half-life (5.5 h), an ideal maximum plasma concentration (8423 ng / mL), and an area under the curve (AUC, 3656 ng / mL*h). Figure 5 F).

[0075] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The use of quassinolide S as and / or in the preparation of immune adjuvants; The molecular formula of the quassinolide S is C 30 H 29 N4O4 + The structural formula is shown in equation (I): Formula (I).

2. The application according to claim 1, characterized in that: The immune adjuvant is an adjuvant that can induce humoral and / or cellular immune responses.

3. An immune adjuvant composition comprising quassin S as described in claim 1.

4. The immunoadjuvant composition according to claim 3, characterized in that: The immune adjuvant composition may also optionally include one or more other types of immune adjuvants, such as aluminum adjuvants.

5. The immunoadjuvant composition according to claim 4, characterized in that: In the immune adjuvant composition, the effective amount of quassinolide S is 0.01-1 mg / dose; the effective amount of the other types of immune adjuvants is 0.01-1 mg / dose.

6. The use of the immune adjuvant composition according to claim 4 or 5 in the preparation of a vaccine.

7. The application according to claim 6, characterized in that: The vaccine in question is an anti-tumor vaccine.

8. A pharmaceutical composition comprising an antigen and an immune adjuvant composition according to any one of claims 3-5.

9. The pharmaceutical composition according to claim 8, characterized in that: In the pharmaceutical composition, the content of the antigen is 0.01-1 mg / dose.

10. The pharmaceutical composition according to claim 8 or 9, characterized in that: The pharmaceutical composition is an anti-tumor vaccine.

11. A method for inducing immunity, comprising administering to a subject the pharmaceutical composition of any one of claims 8-11; wherein the immunity is one or both of cellular immunity and humoral immunity, and the administration is the administration of a therapeutically effective amount of the pharmaceutical composition.

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