A novel vaccine adjuvant and uses thereof

Abstract

The present application discloses the use of compounds as shown in formula (I), in particular chloroiodohydroxyquin in the preparation of immunological adjuvants, wherein R1 and R2 are independently selected from F, Cl, Br, I; R3 is selected from nitro (-NO2), cyano (-CN), amino (-NH2), hydroxyl (-OH) and thiol (-SH). The present application also discloses immunological adjuvant compositions comprising said compounds and pharmaceutical compositions comprising said immunological adjuvant compositions and antigens.

Description

Technical Field

[0001] This invention relates to the field of immunology. More specifically, this invention relates to a novel vaccine adjuvant and its use. Background Technology

[0002] Vaccination protects humans from numerous devastating diseases and has made a significant 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 infectious diseases, such as smallpox and rinderpest. Existing disease prevention strategies include screening and treatment, drug therapy, and vaccination (1). Among these measures, vaccination is considered the most effective and safest method for reducing disease morbidity and mortality (2,3).

[0003] Adjuvants are an important component of modern vaccine development. Over the past few decades, hundreds of materials have been tried as adjuvants, but only a few have been approved for use in human vaccines. Among them, aluminum adjuvants, including aluminum hydroxide, aluminum phosphate, and alum (4), are the most widely used. Aluminum adjuvants can effectively induce Th2 cell-mediated humoral immune responses and produce high-titer antibodies, but the induced cellular immune responses are relatively weak (5), thus limiting their application in vaccines.

[0004] Since Edward Jenner developed the first successful smallpox vaccine in 1796, research on disease vaccines has made tremendous progress, and vaccination has made a huge contribution to global public health. Smallpox and rinderpest, two major infectious diseases, have been eradicated. Currently, vaccines targeting infectious agents can be divided into live attenuated vaccines, subunit vaccines, toxoid vaccines, and conjugate vaccines (6). The active ingredient in most vaccines is pathogen-derived antigens, which can induce a series of specific immune responses and build antigenic immune memory in the body.

[0005] Early vaccine formulations were not pure and often suffered from contamination by unrelated antigens, affecting their immunogenicity. With the advent of recombinant deoxyribonucleic acid (DNA) technology and synthetic chemistry, it became possible to manufacture high-purity antigens to induce more specific immune responses. However, pure antigen preparations have a major drawback: they often have weak immunogenicity. Therefore, such antigen preparations require the addition of adjuvants to achieve protective immunity, such as diphtheria-tetanus-pertussis (DTP), hepatitis A, and hepatitis B vaccines. These vaccines require the addition of exogenous adjuvants to enhance the immune response to the antigen (7). A seemingly simple strategy is to increase the antigen load in the vaccine to obtain the expected immune response, but this often leads to adverse reactions. Treanor et al. studied the safety and immunogenicity of different doses of inactivated subviral influenza A (H5N1) vaccines (8). The results showed that the incidence of injection site pain and tenderness was higher in vaccine recipients than in the placebo group, and the severity was significantly antigen dose-dependent (P<0.001). For this type of antigen, adding adjuvants can reduce the antigen content without compromising the immunogenicity of the vaccine.

[0006] Adjuvants are generally non-immunogenic materials that enhance the specific immune response of vaccines by physically or chemically binding to antigens. Over the past few decades, adjuvants have played a key role in more than 300 vaccines that combat more than 80 pathogens, cancers, and allergies (9). To date, hundreds of materials have been attempted as adjuvants, such as bacterial metabolites (10,11), mineral oils / surfactants (12), microparticles (13,14), nucleic acids (15), liposomes (16,17), and polysaccharides (18). However, only aluminum adjuvants are widely used globally, including aluminum hydroxide, aluminum phosphate, and alum (4).

[0007] Common vaccines against hepatitis A virus, hepatitis B virus, hepatitis E virus, and human papillomavirus are all aluminum adjuvant formulations. These prophylactic vaccines provide protective immunity primarily because aluminum adjuvants can effectively induce Th2 cell-mediated humoral immune responses, producing high-titer antibodies. Neutralizing antibodies are crucial for protection against viral or bacterial infections, but aluminum adjuvants rarely or never stimulate T helper 1 cell (Th1) immune responses (19), which are essential for combating intracellular pathogens such as Mycobacterium tuberculosis (20). Furthermore, vaccines that strongly induce T lymphocyte immune responses are needed for diseases such as cancer, AIDS, tuberculosis, and malaria (21-25). In addition to insufficient cellular immune responses, aluminum adjuvants are prone to inducing inflammatory responses and allergy-associated immunoglobulin E (IgE) responses at the injection site (26). Therefore, there is an urgent clinical need for successful novel vaccine adjuvants that can promote strong and sustained immune responses while overcoming the main limitations of traditional vaccine adjuvants.

[0008] This application 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 application aims to develop novel adjuvants that balance humoral and cellular immunity, which will help expand the application scope of adjuvants and provide new theoretical basis and treatment strategies for the prevention and treatment of more diseases. Summary of the Invention

[0009] 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 new adjuvants that can induce cellular immune responses, the inventors screened approximately 7,000 small molecules from mouse bone marrow-derived differentiated dendritic cells, including 3,000 natural products and 4,000 small molecules in clinical trials or already on the market. The inventors identified 12 small molecules that can effectively stimulate dendritic cells to express the cytokine interferon-β (IFN-β). IFN-β expression is a crucial step in innate immune activation and dendritic cell maturation, thereby inducing adaptive immunity. The inventors subcutaneously immunized wild-type mice with these small molecules and specific antigens, finding that clioquinol effectively induced high-titer antibodies against the specific antigens in mice and possessed anti-tumor function.

[0010] Chloroquine is a component of the prescription drug Vioform, used for topical antifungal treatment and the treatment of inflammatory skin diseases (27). Chloroquine is also known as chloroquinoline, 5-chloro-7-iodo-8-hydroxyquinoline, or 5-chloro-8-hydroxy-7-iodoquinoline. Its molecular formula is C9H5ClINO, and its structural formula is as follows:

[0011]

[0012] In addition, some articles have reported that cloiodril has antimalarial activity (28,29). No studies have reported on cloiodril as an immune adjuvant. The inventors focused on the function of cloiodril in antigen-mediated immunity and disease models, and evaluated its potential as a novel vaccine adjuvant by comparing it with the traditional adjuvant aluminum adjuvant, providing new theoretical basis and candidate drugs for the development of next-generation vaccine adjuvants.

[0013] Specifically, the present invention solves the problems existing in this field through the following technical solutions:

[0014] 1. The use of the compound represented by formula (I) in the preparation of immune adjuvants,

[0015]

[0016] R1 and R2 are independently selected from F, Cl, Br, and I;

[0017] R3 is selected from nitro (-NO2), cyano (-CN), amino (-NH2), hydroxyl (-OH), and thiohydroxyl (-SH).

[0018] 2. The use described in Implementation Scheme 1, wherein R3 is a hydroxyl group, and / or R1 is chlorine, and / or R2 is iodine.

[0019] 3. The use described in any one of embodiments 1-2, wherein the compound is chloroiodohydroxyquine.

[0020] 4. An immune adjuvant composition comprising the compound described in any one of embodiments 1-2, and optionally one or more other types of immune adjuvants, such as aluminum adjuvants, like aluminum hydroxide.

[0021] 5. The immunoadjuvant composition according to embodiment 4, wherein the compound is chloroiodohydroxyquine.

[0022] 6. The immune adjuvant composition according to any one of embodiments 4-5, wherein the effective amount of said compound is 0.01-1 mg / dose, preferably 0.4 mg / dose, and optionally, the effective amount of said other type of immune adjuvant, such as aluminum adjuvant, is 0.01-0.1 mg / dose.

[0023] 7. Use of the immune adjuvant composition according to any one of embodiments 4-6 in the preparation of a vaccine.

[0024] 8. The use described in Implementation Scheme 7, wherein the vaccine is an anti-tumor vaccine, such as an anti-melanoma and anti-colon cancer vaccine.

[0025] 9. A pharmaceutical composition comprising an antigen and an immune adjuvant composition according to any one of embodiments 4-6.

[0026] 10. The pharmaceutical composition described in Embodiment 9, wherein the content of the antigen is 0.01-0.1 mg / dose.

[0027] 11. The pharmaceutical composition according to any one of embodiments 9-10, which is an anti-tumor vaccine, such as an anti-melanoma and anti-colon cancer vaccine. Attached Figure Description

[0028] Figure 1 This indicates that chlorhexidine hydroxyquine induces the expression of IFN-β in differentiated dendritic cells derived from mouse bone marrow. (A) Chemical structural formula of chlorhexidine hydroxyquine. (B, C) Differentiated dendritic cells derived from mouse bone marrow were cultured in vitro, stimulated with chlorhexidine hydroxyquine for 8 hours, and the expression of cytokines IFN-β (B) and IL-6 (C) was identified by quantitative PCR.

[0029] Figure 2 This indicates that cloiodine hydroxyquine enhances the NP-KLH antigen-specific humoral immune response. (AE) Wild-type mice were subcutaneously immunized with cloiodine hydroxyquine in conjunction with the NP-KLH antigen. Blood was 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 This indicates that cloiodohydroxyquine enhances the NP-OVAL antigen-specific humoral immune response. (AE) Wild-type mice were subcutaneously immunized with cloiodohydroxyquine or aluminum adjuvant in conjunction with the NP-OVAL antigen. Blood was collected from the eyeballs 14 days later, and the level of NP antigen-specific antibodies in the serum was detected by ELISA.

[0031] Figure 4 This indicates that cloiodohydroxyquine enhances the NP-OVAL antigen-specific humoral immune response. (AE) Wild-type mice were subcutaneously immunized with cloiodohydroxyquine or aluminum adjuvant in conjunction with the NP-OVAL antigen. Blood was collected from the eyeballs 28 days later, and the level of NP antigen-specific antibodies in the serum was detected by ELISA.

[0032] Figure 5This indicates that cloiodohydroxyquine enhances the NP-OVAL antigen-specific humoral immune response. (AE) Wild-type mice were subcutaneously immunized with different doses of cloiodohydroxyquine or aluminum adjuvant in conjunction with the NP-OVAL antigen. Blood samples were collected from the eyeballs 14 days later, and the serum NP antigen-specific antibody levels were detected by ELISA.

[0033] Figure 6 This indicates that cloiodohydroxyquine enhances the NP-OVAL antigen-specific humoral immune response. (AE) Wild-type mice were subcutaneously immunized with different doses of cloiodohydroxyquine or aluminum adjuvant in conjunction with the NP-OVAL antigen. Blood samples were collected from the eyeballs 28 days later, and the level of NP antigen-specific antibodies in the serum was detected by ELISA.

[0034] Figure 7 This indicates that cloiodrial hydroxyquine enhances the antitumor immune response. (A) Wild-type mice were subcutaneously immunized with cloiodrial hydroxyquine or aluminum adjuvant in combination with NP-OVAL antigen, and B16-OVA cells were inoculated 36 days later. Tumor incidence was monitored. (B) Wild-type mice were subcutaneously immunized with cloiodrial hydroxyquine or aluminum adjuvant in combination with NP-OVAL antigen, and MC38-OVA cells were inoculated 46 days later. Tumor incidence was monitored.

[0035] Figure 8 This indicates that cloiodohydroxyquine has no obvious toxic side effects on the body. (A) Body weight of wild-type mice immunized with different doses of cloiodohydroxyquine or aluminum adjuvant synergistic with NP-OVAL antigen one day before, 7 days after, and 14 days after immunization. (B) HE staining results of skin at the injection sites of mice in the control group, cloiodohydroxyquine immunization group, and aluminum adjuvant immunization group. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0037] Terminology Explanation

[0038] The compounds in this article

[0039] The compounds referred to herein, or the compounds described herein, are quinoline compounds represented by the general formula (I) below.

[0040]

[0041] R1 and R2 are independently selected from F, Cl, Br, and I;

[0042] R3 is selected from nitro (-NO2), cyano (-CN), amino (-NH2), hydroxyl (-OH), and thiohydroxyl (-SH). In a preferred embodiment, R3 is hydroxyl. In a further preferred embodiment, R1 is chlorine. In a further preferred embodiment, R2 is iodine. In the most preferred embodiment, R1 is chlorine, R2 is iodine, and R3 is hydroxyl; this compound is also called chloroiodohydroxyquine. The structural formula of chloroiodohydroxyquine is as follows:

[0043]

[0044] Subjects

[0045] As used in this invention, the term "subject" 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, such as poultry, such as chickens, ducks, geese, etc. Preferably, the animal is a mammal. In a preferred embodiment, the subject is a human.

[0046] immune cells

[0047] 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), natural killer cells (NK cells), macrophages, monocytes, dendritic cells, and neutrophils.

[0048] 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.

[0049] 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.

[0050] The synergistic effect of the antigen and CpG ODN of the present invention induces both humoral and cellular immune responses, enhances the immune function of Th1 T cells, and greatly enhances the T cell immune response.

[0051] vaccine

[0052] 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 enabling the body to protect against or eliminate the pathogen. This includes proteins, polysaccharides, nucleic acids, live vectors, or infectious agents. A vaccine is an autoimmune preparation for preventing infectious diseases, made from pathogenic microorganisms (such as bacteria, rickettsiae, viruses, etc.) and their metabolites through artificial attenuation, inactivation, or genetic engineering. 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.

[0053] 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.

[0054] 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 colon cancer vaccine.

[0055] 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.

[0056] 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 cloiodrial hydroxyquine) can also be used as vaccine adjuvants and can exert excellent adjuvant functions. The compounds of this invention can be used alone as vaccine adjuvants or in combination with other commonly used adjuvants. The compounds and these commonly used adjuvants can exert additive or synergistic effects to enhance the immunogenicity of antigens, thereby reducing vaccine dosage or improving vaccine efficacy (e.g., reducing the dosage or number of administrations). Therefore, in one embodiment, this invention also provides the use of the compounds described herein in the preparation of vaccine adjuvants, preferably, the vaccines being melanoma vaccines and colon cancer vaccines. In one embodiment, the vaccine adjuvants described herein also include one or more other substances that work synergistically with the compounds to exert adjuvant effects. The effective amount of the compounds described herein can be determined by those skilled in the art through routine 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.

[0057] The vaccine may contain another adjuvant, such as an aluminum adjuvant or aluminum hydroxide adjuvant. The effective amount of the other adjuvant, such as an 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 0.01-0.1 mg / dose, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1 mg / dose.

[0058] 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 0.01-0.1 mg / dose, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 and 0.1 mg / dose.

[0059] drug

[0060] 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.

[0061] Example

[0062] 1. Experimental materials:

[0063] 1.1 Laboratory Animals

[0064] All mouse-related experiments and procedures in this study were conducted in accordance with the protocols approved by the Institute of Laboratory Animal Management and Use (IACUC) of Tsinghua University. 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. 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, aged 8-12 weeks.

[0065] 1.2 Main Reagents

[0066] The main reagents and antibodies used in this study are listed in Table 1.

[0067] Table 1. Main Reagents and Their Sources

[0068]

[0069] 2. Experimental Methods:

[0070] 1. RNA extraction using the Trizol method

[0071] 1) Centrifuge the cells, discard the supernatant, add 1 ml of Trizol, mix well, and store at -80°C.

[0072] 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.

[0073] 3) Centrifuge at 10,000 rpm for 5 minutes, then 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°C for 30 minutes.

[0074] 4) Take out the sample and put it into a centrifuge. Centrifuge at 15,000 rpm for 10 minutes. Discard the supernatant and add 600 μl of 75% ethanol to wash the precipitate.

[0075] 5) Place the sample in a centrifuge and centrifuge at 15,000 rpm for 3 minutes. Discard the supernatant and centrifuge for another minute. Use a pipette to remove the remaining supernatant.

[0076] 6) Air dry the sample at room temperature, add 30 μl of RNase-free water to dissolve the RNA, and store in a -80℃ freezer.

[0077] 2. RNA reverse transcription

[0078] The kit used for RNA reverse transcription in this study consisted of two steps: the first step was for removing the genomic DNA template, and the reaction system was prepared as shown in Table 2, with reaction conditions of 42℃ for 2 minutes followed by 4℃ for 2 minutes. The second step was the RNA reverse transcription reaction, and the reaction system was prepared as shown in Table 3, with reaction conditions of 37℃ for 15 minutes, 85℃ for 15 seconds, and constant temperature at 4℃.

[0079] After the two-step reaction is completed, the sample is stored in a -20°C refrigerator for later use.

[0080] Table 2 RNA reverse transcription reaction system (first step)

[0081]

[0082] Table 3. RNA Reverse Transcription Reaction System (Step 2)

[0083]

[0084] 3. Real-time quantitative PCR

[0085] 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.

[0086] 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 study are shown in Table 6.

[0087] Table 4 qPCR reaction system

[0088]

[0089] Table 5 qPCR reaction conditions

[0090]

[0091]

[0092] Table 6 qPCR primer sequences

[0093]

[0094] 4. In vitro induction of bone marrow-derived dendritic cells

[0095] 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.

[0096] 2) Wash the bone three times with sterile PBS, then cut both ends of the bone with sterilized scissors;

[0097] 3) Using a syringe equipped with a 25G needle, draw up RPMI-1640 culture 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 for 10 min.

[0098] 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 minute to lyse the red blood cells, add 10 ml of RPMI-1640 culture medium to stop, and centrifuge at 1500 rpm for 5 min.

[0099] 5) Discard the supernatant, suspend the cells in RPMI-1640 complete medium containing rmGM-CSF 10ng / ml and IL-4 10ng / ml, divide into 6-well culture plates, 4ml in each well, and incubate in a cell culture incubator at 37℃ with 5% CO2.

[0100] 6) After 48 hours, gently blow the cells away and aspirate the suspended cells along with the culture medium, leaving only the adherent cells.

[0101] 7) Add fresh complete culture medium and the same concentration of rmGM-CSF and IL4 cytokine, and continue culturing;

[0102] 8) On the 5th day, change the medium semi-quantitatively and replenish cytokines, and retain the suspension cells for continued culture;

[0103] 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).

[0104] 5. Enzyme-linked immunosorbent assay (ELISA)

[0105] 1) To detect the level of specific antibodies produced by mice immunized with NP-KLH or NP-OVAL, 100 μL / well of 5 μg / mL NP-BSA was plated on a 96-well ELISA plate and incubated overnight at 4°C.

[0106] 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;

[0107] 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 hours;

[0108] 4) Wash 3-5 times with washing buffer, add HRP-conjugated anti-mouse IgG, IgG1, IgG2b, IgG2c and IgG3 antibodies diluted 1:5000, and incubate at room temperature in the dark for 1 hour.

[0109] 5) Wash 5-7 times with washing buffer, add 100 μL of 1×TMB solution, and incubate for 15 min;

[0110] 6) Add 50 μL of 1M 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.

[0111] 6. Hematoxylin and eosin (H&E) staining

[0112] 1) Fix skin tissue with 4% paraformaldehyde at 4°C for 24 hours, embed it in paraffin, and then section it;

[0113] 2) Place the slices in an oven at 50-60℃ and bake for 60 minutes. Dewax them twice in xylene for 10 minutes each time, twice in 100% alcohol for 5 minutes each time, once in 95% and once in 75% graded alcohol for 5 minutes each time, and wash them twice in distilled water for 5 minutes each time.

[0114] 3) Stain with hematoxylin solution for 5-10 minutes, rinse with tap water, differentiate with differentiation solution for 5 seconds, blue solution for 1 minute, and rinse with tap water twice, 5 minutes each time;

[0115] 4) Stain in eosin solution for 2 minutes, then rinse with tap water for 10 seconds;

[0116] 5) Place the slide in 95% alcohol for 1 minute, then place it in 100% alcohol twice and xylene twice in sequence, for 5 minutes each time to dehydrate and clear it. Remove the slide, let it air dry, and then mount it with neutral resin for observation under a microscope.

[0117] Example 1: Determination of IFN-β expression in chloroiodohydroxyquine-induced differentiated dendritic cells derived from mouse bone marrow.

[0118] Mice bone marrow-derived dendritic cells were stimulated for 8 hours with 10 μM cloiodriquine dissolved in DMSO, while the control group received an equal amount of DMSO. RNA was extracted using the Trizol method, and quantitative PCR was performed after reverse transcription. The results showed that cloiodriquine effectively stimulated dendritic cells to produce the cytokine IFN-β (IFN-β). Figure 1 B), and does not induce excessively high levels of the pro-inflammatory cytokine IL-6 (B). Figure 1 C) It also does not affect cell growth status. IFN-β expression is a key step in the activation of innate immunity and the maturation of dendritic cells, thereby inducing adaptive immunity. This result shows that cloiodrial hydroxyquine has a regulatory function on dendritic cells.

[0119] Example 2: Determination of the effect of chloroiodine-hydroxyquine on enhancing antigen-specific humoral immune response

[0120] We subcutaneously immunized wild-type B6 mice with cloiocarbazin in combination with NP-KLH antigen. The injection solution was PBS buffer containing 50 μg NP-KLH antigen and 125 μg cloiocarbazin, with a total volume of 100 μl, administered bilaterally in 50 μl doses to each side. The cloiocarbazin solution was a 20 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. Blood samples were collected from the eyes 14 days after immunization, and the serum NP antigen-specific antibody levels were detected by ELISA. Compared with the control group, the serum NP antigen-specific antibody levels in mice injected with cloiocarbazin were significantly higher. Figure 2 ).

[0121] We also investigated the function of cloiodrial hydroxyquine in immunization against another antigen, NP-OVAL, and compared it with aluminum adjuvant, the most widely used adjuvant in human vaccines. The injection consisted of 100 μl of PBS buffer containing 50 μg NP-OVAL antigen and 350 μg cloiodrial hydroxyquine, administered subcutaneously to both sides (50 μl each). Cloiodrial hydroxyquine was a 50 mg / ml solution pre-dissolved in DMSO. The control group received PBS buffer containing 50 μg NP-OVAL antigen and an equal volume of DMSO. The aluminum adjuvant group received PBS buffer containing 50 μg NP-OVAL antigen and 25 μl of aluminum adjuvant. Blood samples were collected from the eyes 14 and 28 days after immunization, and serum NP antigen-specific antibody levels were detected by ELISA. The results showed that at 14 days post-immunization (… Figure 3 ) and 28 days Figure 4 The levels of NP antigen-specific antibodies in the serum of mice injected with cloiodrial hydroxyquine were significantly increased and higher than those in the aluminum adjuvant group, indicating that cloiodrial hydroxyquine plays an important role in enhancing specific humoral immune responses to different types of antigens and has a stronger effect than aluminum adjuvant.

[0122] To further identify the effective dose of cloiodrial hydroxyquine in regulating antigen-specific humoral immune responses, we immunized wild-type mice with different doses of cloiodrial hydroxyquine or aluminum adjuvant in combination with NP-OVAL antigen. The injection was a PBS buffer solution containing 50 μg NP-OVAL antigen and 100 μg / 200 μg / 400 μg cloiodrial hydroxyquine, with a total volume of 100 μl, administered subcutaneously to each side (50 μl). The cloiodrial hydroxyquine was a 50 mg / ml solution pre-dissolved in DMSO. The control group received PBS buffer solution containing 50 μg NP-OVAL antigen. The aluminum adjuvant group received PBS buffer solution containing 50 μg NP-OVAL antigen and 25 μl aluminum adjuvant. Blood samples were collected from the eyes 14 and 28 days after immunization, and serum NP antigen-specific antibody levels were detected by ELISA. It can be seen that at 14 days post-immunization (… Figure 5 ) and 28 days Figure 6The antibody levels in mouse serum increased with increasing doses of cloiodrial hydroxyquine, with the highest levels observed in the 400 μg group, which were also higher than those in the aluminum adjuvant group. This further demonstrates that cloiodrial hydroxyquine has a stronger function of enhancing antigen-specific humoral immune responses than aluminum adjuvant.

[0123] Example 3: Determination of the immune-enhancing and antitumor immune response of chloroiodine-hydroxyquine

[0124] The results of Example 2 showed that cloiodrial hydroxyquine enhances antigen-specific humoral immune responses. To assess its protective function as a vaccine adjuvant, we examined the regulatory effect of cloiodrial hydroxyquine as a vaccine adjuvant on disease progression in different tumor models. Wild-type mice were subcutaneously immunized with either cloiodrial hydroxyquine or aluminum adjuvant in conjunction with NP-OVAL antigen. The injection was a PBS buffer solution containing 50 μg NP-OVAL antigen and 350 μg cloiodrial hydroxyquine, with a total volume of 100 μl, injected bilaterally in each eye (50 μl). The cloiodrial hydroxyquine was a 50 mg / ml solution pre-dissolved in DMSO. The control group received PBS buffer solution containing 50 μg NP-OVAL antigen and an equal volume of DMSO. The aluminum adjuvant group received PBS buffer solution containing 50 μg NP-OVAL antigen and 25 μl aluminum adjuvant. Mice were subcutaneously inoculated 3 × 10⁶ B16 melanoma cells expressing OVA antigen (B16-OVA) 36 days post-immunization. 6 / mouse, monitoring tumor growth. It can be seen that, compared with the control group, the incidence of tumors in the cloiodrial-hydroxyquine group was significantly lower, and lower than that in the aluminum adjuvant group ( Figure 7 A).

[0125] In addition, we further validated this tumor model by inoculating mice immunized with cloiocarbazine or aluminum adjuvant in combination with NP-OVAL antigen with MC38 mouse colon cancer cells expressing OVA antigen (MC38-OVA). The immunization was performed using PBS buffer containing 50 μg NP-OVAL antigen and 400 μg cloiocarbazine, with a total volume of 100 μl, administered subcutaneously to each side (50 μl). Cloiocarbazine was a 50 mg / ml solution pre-dissolved in DMSO. The control group received PBS buffer containing 50 μg NP-OVAL antigen. The aluminum adjuvant group received PBS buffer containing 50 μg NP-OVAL antigen and 25 μl aluminum adjuvant. Forty-six days post-immunization, mice were subcutaneously inoculated with MC38 mouse colon cancer cells expressing OVA antigen (MC38-OVA), at a dose of 5 × 10⁻⁶. 6 / mouse, monitoring tumor growth. It can be seen that the tumor incidence rate in the cloiodrial-hydroxyquine group was also lower than that in the control group and the aluminum adjuvant group ( Figure 7 B) indicates that chloriodine-hydroxyquine immunization can enhance the anti-tumor immune response.

[0126] Example 4: Determination of the toxic side effects of cloiodrial hydroxyquine on the body

[0127] To investigate whether cloiodril-hydroxyquine caused toxic side effects, we weighed mice one day before and one / two weeks after subcutaneous immunization with different doses of cloiodril-hydroxyquine or aluminum adjuvant combined with NP-OVAL antigen. The immunization solution was PBS buffer containing 50 μg NP-OVAL antigen and 100 μg / 200 μg / 400 μg cloiodril-hydroxyquine, with a total volume of 100 μl, injected bilaterally (50 μl each). Cloiodril-hydroxyquine was a 50 mg / ml solution pre-dissolved in DMSO. The control group received PBS buffer containing 50 μg NP-OVAL antigen. The aluminum adjuvant group received PBS buffer containing 50 μg NP-OVAL antigen and 25 μl aluminum adjuvant. Compared with the control group, there was no significant change in mouse body weight after cloiodril-hydroxyquine immunization. Figure 8 A).

[0128] To detect whether skin lesions occurred at the injection sites in immunized mice, we collected skin samples from the injection sites of mice immunized two months after immunization with cloiocarbazine or aluminum adjuvant combined with NP-OVAL antigen and performed H&E staining. The injection solution was PBS buffer containing 50 μg NP-OVAL antigen and 350 μg cloiocarbazine, with a total volume of 100 μl, injected subcutaneously into each eye (50 μl on each side). The cloiocarbazine solution was a 50 mg / ml solution pre-dissolved in DMSO. The control group received PBS buffer containing 50 μg NP-OVAL antigen and an equal volume of DMSO. The aluminum adjuvant group received PBS buffer containing 50 μg NP-OVAL antigen and 25 μl aluminum adjuvant. It was observed that no obvious skin lesions occurred in the cloiocarbazine group mice. Figure 8 (B) We also examined the mice's kidneys, liver, and other internal organs, and found no obvious abnormalities. Based on the current results, cloiodrial hydroxyquine has no obvious toxic side effects on the body.

[0129] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

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Claims

1. The use of chloroiodohydroxyquine in the preparation of immune adjuvants.

2. The use of chloroiodohydroxyquine as an immune adjuvant in the preparation of vaccines that enhance antitumor immune responses.

3. The use according to claim 1 or 2, wherein, The effective dose of chloroiodohydroxyquine is 0.01-1 mg / dose.

4. The use according to claim 2, wherein the vaccine is an anti-tumor vaccine.

5. The use according to claim 2, wherein the vaccine is an anti-melanoma vaccine or an anti-colon cancer vaccine.

6. Use of chloroiodohydroxyquine as an immune adjuvant in the preparation of pharmaceutical compositions that enhance antitumor immune responses.

7. The use according to claim 6, wherein, The pharmaceutical composition also contains an antigen.

8. The use according to claim 7, wherein the content of said antigen is 0.01-0.1 mg / dose.

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