Application of grifola frondosa extract in preparation of vaccine composition

Abstract

The invention relates to application of grifola frondosa extract as a vaccine adjuvant and a vaccine composition containing the adjuvant. The invention finds that the grifola frondosa extract beta-glucan is used as the adjuvant in the vaccine composition, so that the immune effect of the vaccine is remarkably improved, the activation of antigen presenting cells is remarkably improved, and the grifola frondosa extract beta-glucan is an ideal vaccine adjuvant for potential preventive tumor vaccines and therapeutic tumor vaccines.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to the use of extracts of Grifola frondosa as adjuvants in preventive and / or therapeutic vaccines. Background Technology

[0002] Adjuvants are a key component of vaccines, also known as nonspecific immune enhancers. They are not antigenic themselves, but when injected into the body together with or beforehand, they can enhance the immunogenicity of the antigen or alter the type of immune response induced by the antigen. The functions of adjuvants include promoting the body's immune response and reducing vaccine dosage and production costs. Aluminum adjuvants remain the most commonly used adjuvants in humans, but because they only have a Th2-type effect and lack a Th1-type effect, this is a significant disadvantage for vaccines that rely on Th1-type cellular immunity.

[0003] Therefore, there is an urgent need to develop new adjuvants with significant practical application and demand value. Summary of the Invention

[0004] The purpose of this invention is to provide an adjuvant component that can simultaneously and efficiently enhance Th1 and Th2 immunity and its application.

[0005] In a first aspect of the invention, there is provided the use of an extract of Grifola frondosa for the preparation of an adjuvant; wherein the extract of Grifola frondosa is β-glucan or contains at least 80 wt%, preferably 85%, more preferably 90% β-glucan, based on the total weight of the extract of Grifola frondosa.

[0006] The β-glucan has the following structural units:

[0007]

[0008] in,

[0009] It is glucose;

[0010] n is an integer between 100 and 750, preferably between 400 and 500, and more preferably 450.

[0011] In another preferred embodiment, the average molecular weight of the β-glucan is 3-400 kDa, preferably 100-400 kDa.

[0012] In another preferred embodiment, the adjuvant is used for:

[0013] (1) Enhances the production of OVA-specific IgG;

[0014] (2) Enhance the expression of IgG1, IgG2a and IgG2b;

[0015] (3) Simultaneously enhances the immune effects of Th1 and Th2 types;

[0016] (4) Promotes the expression of CD86 in antigen-presenting cells.

[0017] In another preferred embodiment, the antigen-presenting cells are selected from the group consisting of: monocytes-phagocytes, dendritic cells, B cells, and Langerhans cells.

[0018] A second aspect of the present invention provides an adjuvant composition comprising:

[0019] (i) Extracts from Grifola frondosa;

[0020] (ii) A pharmaceutically acceptable carrier;

[0021] The extract of Grifola frondosa is β-glucan; the β-glucan has the following structural units:

[0022]

[0023] in,

[0024] It is glucose;

[0025] n is an integer between 100 and 750, preferably between 400 and 500, and more preferably 450.

[0026] In another preferred embodiment, the content of Grifola frondosa extract in the adjuvant composition is 0.01-99 wt%, more preferably 0.1-90 wt%, based on the total weight of the adjuvant composition.

[0027] In another preferred embodiment, the adjuvant composition is present in unit dose form.

[0028] In another preferred embodiment, each unit dose of the adjuvant composition contains 0.1-1000 μg, preferably 1-500 μg, more preferably 50-500 μg of Grifola frondosa extract, said unit dose being the clinically used reference dose for a single administration (injection).

[0029] A third aspect of the present invention provides a vaccine composition comprising:

[0030] (a) One or more antigens;

[0031] (b) Extracts from Grifola frondosa; and

[0032] (c) A pharmaceutically acceptable carrier;

[0033] The extract of Grifola frondosa is β-glucan; the β-glucan has the following structural units:

[0034]

[0035] in,

[0036] It is glucose;

[0037] n is an integer between 100 and 750, preferably between 400 and 500, and more preferably 450.

[0038] In another preferred embodiment, the mass ratio of the β-glucan to the antigen in the vaccine composition is (0.1-1000):1; preferably, (0.1-100):1; more preferably, (0.1-10):1; even more preferably, (0.5-5):1.

[0039] In another preferred embodiment, the vaccine composition is used to prevent and / or treat diseases associated with antigens or pathogens.

[0040] In another preferred embodiment, the vaccine composition is a therapeutic vaccine and / or a preventive vaccine.

[0041] In another preferred embodiment, the therapeutic vaccine is a therapeutic tumor vaccine.

[0042] In another preferred embodiment, the preventive vaccine is a preventive tumor vaccine.

[0043] In another preferred embodiment, the vaccine composition is used to treat or prevent tumors or cancers selected from the group consisting of: skin cancer, pancreatic cancer, liver cancer, brain cancer, stomach cancer, lung cancer, breast cancer, intestinal cancer, prostate cancer, ovarian cancer, cervical cancer, esophageal cancer, hematologic malignancies, and bone tumors.

[0044] In another preferred embodiment, the antigen is a protein, peptide, polysaccharide, lipid, lipopolysaccharide, or nucleic acid of a pathogen.

[0045] In another preferred embodiment, the pathogen is selected from viruses, bacteria, fungi, or combinations thereof.

[0046] In another preferred embodiment, the antigen is selected from tumor-specific antigens, tumor-associated antigens, or combinations thereof.

[0047] In another preferred embodiment, the virus is selected from human immunodeficiency virus, human papillomavirus, hepatitis A virus, hepatitis B virus, poliovirus, influenza virus, coronavirus, yellow fever virus, rotavirus, herpes simplex virus, herpes zoster virus, rubella virus, measles virus, poliovirus, or combinations thereof.

[0048] In another preferred embodiment, the bacteria are selected from Mycobacterium tuberculosis, Neisseria meningitidis, Haemophilus influenzae, Candida albicans, Salmonella, Shigella, Staphylococcus aureus, Streptococcus pneumoniae, or combinations thereof.

[0049] In another preferred embodiment, the fungus is selected from Candida, Aspergillus, Cryptococcus, Pneumocystis, Aspergillus, Zygomycetes, or combinations thereof.

[0050] In another preferred embodiment, the protein is selected from capsid proteins, outer shell proteins, secretory proteins, membrane proteins, cell wall proteins, nucleoproteins, or combinations thereof.

[0051] In another preferred embodiment, the vaccine composition is in the form of an injectable formulation, an inhalable formulation, or an oral formulation.

[0052] In another preferred embodiment, the dosage form of the vaccine composition is selected from the following: powder for injection, suspension, aqueous injection, spray, aerosol, powder inhaler, adhesive tablet, sublingual tablet or film.

[0053] A fourth aspect of the invention provides the use of a vaccine composition as described in the third aspect of the invention, said vaccine composition for stimulating an immune response in a subject.

[0054] In another preferred embodiment, the immune response includes the activation of B cells, the activation of T cells, the production of antibodies, and / or the release of cytokines.

[0055] A fifth aspect of the invention provides a method for treating and / or preventing a disease, comprising the step of administering the vaccine composition of the third aspect of the invention to a subject in need.

[0056] In another preferred embodiment, the disease includes diseases caused by pathogens.

[0057] In another preferred embodiment, the pathogen includes viruses, bacteria, and fungi.

[0058] In another preferred embodiment, the virus is selected from human immunodeficiency virus, human papillomavirus, hepatitis A virus, hepatitis B virus, poliovirus, influenza virus, coronavirus, yellow fever virus, rotavirus, herpes simplex virus, herpes zoster virus, rubella virus, measles virus, poliovirus, or combinations thereof.

[0059] In another preferred embodiment, the bacteria are selected from Mycobacterium tuberculosis, Neisseria meningitidis, Haemophilus influenzae, Candida albicans, Salmonella, Shigella, Staphylococcus aureus, Streptococcus pneumoniae, or combinations thereof.

[0060] In another preferred embodiment, the fungus is selected from Candida, Aspergillus, Cryptococcus, Pneumocystis, Aspergillus, Zygomycetes, or combinations thereof.

[0061] In another preferred embodiment, the disease includes related diseases such as AIDS, tuberculosis, influenza, and tumors.

[0062] In another preferred embodiment, the tumor is selected from the group consisting of: skin cancer, pancreatic cancer, liver cancer, brain cancer, stomach cancer, lung cancer, breast cancer, intestinal cancer, prostate cancer, ovarian cancer, cervical cancer, esophageal cancer, hematologic malignancy, and bone tumor.

[0063] In another preferred embodiment, the object includes a human or a non-human mammal.

[0064] In another preferred embodiment, the non-human mammals include non-human primates (such as monkeys).

[0065] A sixth aspect of the present invention provides a method for promoting the activation of antigen-presenting cells in vitro, comprising the steps of:

[0066] The antigen-presenting cells were contacted with an extract of Grifola frondosa to promote the activation of the antigen-presenting cells.

[0067] The extract of Grifola frondosa is β-glucan; the β-glucan has the following structural units:

[0068]

[0069] in,

[0070] It is glucose;

[0071] n is an integer between 100 and 750, preferably between 400 and 500, and more preferably 450.

[0072] In another preferred embodiment, the concentration of the β-glucan extract from the maitake mushroom is 0.1-1000 μg / mL; preferably, 1-1000 μg / mL; more preferably, 5-500 μg / mL; even more preferably, 5-100 μg / mL.

[0073] In another preferred embodiment, the activation is to increase the expression level of CD86.

[0074] In another preferred embodiment, the antigen-presenting cells are selected from the group consisting of: monocytes-phagocytes, dendritic cells, B cells, and Langerhans cells.

[0075] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0076] Figure 1 The absorbance of OVA-specific total IgG after immunizing mice with saline, ovalbumin (OVA) alone, OVA+GFPBW1 (50 μg), and OVA+GFPBW1 (300 μg) are shown.

[0077] Figure 2 The absorbance of OVA-specific IgG1 / 2a / 2b in mice after immunization with saline, ovalbumin (OVA) alone, OVA+GFPBW1 (10 μg), OVA+GFPBW1 (50 μg), and OVA+GFPBW1 (300 μg) is shown.

[0078] Figure 3 The study showed the tumor growth and survival rate of mice immunized with saline, ovalbumin (OVA) alone, OVA+GFPBW1 (10 μg), OVA+GFPBW1 (50 μg), and OVA+GFPBW1 (300 μg) before inoculation with B16-OVA melanoma cells.

[0079] Figure 4 This study showed the tumor growth and survival rate of mice after immunization with B16-OVA melanoma cells, using saline, ovalbumin (OVA) alone, OVA+GFPBW1 (10 μg), OVA+GFPBW1 (50 μg), and OVA+GFPBW1 (300 μg).

[0080] Figure 5 This shows the expression level of CD86 and the percentage of FITC-OVA phagocytosed after treatment of antigen-presenting cells with GFPBW1 and similar polysaccharides (glucan-1, glucan-2, glucan-3). Detailed Implementation

[0081] Through extensive and in-depth research, the inventors unexpectedly discovered that β-glucan, an extract of Grifola frondosa, can be used as an adjuvant in vaccine compositions, significantly improving the immunogenicity of vaccines and simultaneously enhancing both Th1 and Th2 immune responses. In particular, it effectively enhances the immunogenicity of both prophylactic and therapeutic tumor vaccines. Based on this discovery, the present invention was completed.

[0082] the term

[0083] In this invention, the term "adjuvant" is a key component of a vaccine, also known as a nonspecific immune enhancer. It is not antigenic itself, but when injected into the body together with or beforehand with an antigen, it can enhance the immunogenicity of the antigen or alter the type of antigen-induced immune response. The functions of adjuvants include promoting the body's immune response and reducing vaccine dosage and production costs.

[0084] In this invention, the term "antigen" refers to a molecule or combination of molecules that elicits an immune response in order to be recognized by an individual's immune system. This antigen may be foreign to the host seeking an immune response. In this case, the antigen may be a protein expressed by bacteria or a virus. Antigens can also be autoantigens, i.e., proteins expressed by host cells, such as tumor antigens.

[0085] In this invention, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to them at the doses and concentrations used.

[0086] In this invention, the term "comprising" generally means including, encompassing, containing, or including. In some cases, it also means "as" or "consisting of".

[0087] extract of maitake mushroom

[0088] Grifola frondosa (Fr.) SFGray (also known as Maitake mushroom) is a medicinal and edible fungus belonging to the Polyporaceae family, genus Grifola frondosa, class Agaricomycetes, subphylum Basidiomycota. Wild Grifola frondosa is mainly found in Japan. During feudal times, it was primarily used as a culinary ingredient in Japan due to its excellent flavor. Its medicinal value was only gradually discovered by Japanese scholars in the 1980s. Grifola frondosa extract has been shown to have therapeutic effects on AIDS (Acquired Immunodeficiency Syndrome), diabetes, and hypertension.

[0089] β-glucan (GFPBW1) is a β-glucan extracted from Grifola frondosa, with an average molecular weight of about 300 kDa.

[0090] Extracts and preparation methods

[0091] As used herein, the term "extract" includes water-soluble and / or fat-soluble extracts. There are no particular limitations on the methods that can be used to prepare the β-glucan extract of this invention. Water-soluble and / or fat-soluble extracts can be obtained using conventional methods from Grifola frondosa. These methods include solvent extraction, extraction, and / or chromatography.

[0092] In this invention, there are no particular limitations on the solvent used for solvent extraction. Representative examples include (but are not limited to): one or a mixture of several solvents selected from water, ethanol, methanol, acetone, and ethyl acetate. The extraction can be performed once or multiple times.

[0093] In this invention, there are no particular limitations on the solvent used for solvent extraction. Representative examples include (but are not limited to): one or more solvents selected from n-butanol, ethyl acetate, dichloromethane, chloroform, cyclohexane, and petroleum ether. The extraction can be performed once or multiple times.

[0094] In this invention, there are no particular limitations on column chromatography, and representative examples include (but are not limited to): activated carbon, silica gel, reversed-phase silica gel, macroporous resin, dextran gel, or a combination of several of these.

[0095] Pharmaceutical composition and administration method

[0096] The present invention also provides a pharmaceutical composition. The pharmaceutical composition of the present invention can be therapeutic or prophylactic (e.g., a vaccine). The pharmaceutical composition of the present invention comprises an effective amount of the adjuvant component of the present invention, an effective amount of one or more antigens, and optionally a pharmaceutically acceptable carrier, diluent, or excipient.

[0097] In this invention, these (vaccine) compositions comprise an immune antigen, the adjuvant of this invention, and are generally combined with a "pharmaceutically acceptable carrier," which includes any carrier that does not itself induce antibodies harmful to an individual receiving the composition. Examples of suitable carriers include (but are not limited to) proteins, lipid aggregates (such as oil droplets or liposomes), etc. These carriers are well known to those skilled in the art. Additionally, these carriers may optionally act as immunostimulants ("adjuvants").

[0098] In addition, the (vaccine) composition of the present invention may also contain additional adjuvants. Representative vaccine adjuvants include (but are not limited to) the following: inorganic adjuvants, such as aluminum hydroxide, alum, etc.; synthetic adjuvants, such as artificially synthesized double-stranded polynucleotides (double-stranded polyadenylate, uridine acid), levamisole, isoprotinin, etc.; oils, such as Freund's adjuvant, peanut oil emulsified adjuvant, mineral oil, vegetable oil, etc.

[0099] Typically, vaccine compositions or immunogenic compositions can be formulated as injectable preparations, such as liquid solutions or suspensions; they can also be formulated as solid forms suitable for reconstitution into solutions or suspensions or liquid excipients prior to injection. The formulation may also be emulsified or encapsulated in liposomes to enhance adjuvant effects.

[0100] The composition can be formulated into mono- or multi-component dosage forms. Each dosage form contains a predetermined amount of active substance calculated to produce the desired therapeutic effect, as well as suitable pharmaceutical excipients.

[0101] The prepared pharmaceutical composition can be administered via conventional routes, including (but not limited to): intravenous, intramuscular, intraperitoneal, subcutaneous, intradermal, oral, or local administration.

[0102] When using the (vaccine) composition, a safe and effective amount of the vaccine polypeptide or peptide group of the present invention is administered to a human, wherein the safe and effective amount is generally at least about 1 microgram peptide / kg body weight, and in most cases does not exceed about 8 milligram peptide / kg body weight, preferably about 1 microgram to 1 milligram peptide / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0103] The main advantages of this invention include:

[0104] (1) The extract of Grifola frondosa in this invention can effectively enhance the immunogenicity of vaccines as a vaccine adjuvant.

[0105] (2) The extract of Grifola frondosa of the present invention can simultaneously enhance the Th1 and Th2 immune effects.

[0106] (3) The extract of Grifola frondosa of the present invention can significantly improve the immunity of both preventive and therapeutic tumor vaccines.

[0107] (4) The extract of Grifola frondosa in this invention is derived from plant extracts, and is safe for oral administration with few side effects.

[0108] (5) Compared with similar polysaccharides, the extract of Grifola frondosa of the present invention has a better effect on promoting the activation / maturation of antigen-presenting cells.

[0109] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions (such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0110] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0111] Experimental materials:

[0112] The extraction and separation method of Grifola frondosa extract GFPBW1 is as follows: Grifola frondosa fruiting bodies were defatted for two weeks and then air-dried. The solid residue after boiling water extraction was soaked in 5% sodium hydroxide and reacted at 4°C for 4 hours. The extract was neutralized and precipitated with ethanol to obtain the alkaline-extracted crude polysaccharide GFPB. The latter was then subjected to DEAE-cellulose chromatography with distilled water as the eluent to obtain GFPBW1.

[0113] glucan-1, glucan-2, and glucan-3 are all plant-derived polysaccharides. Glucan-1 was obtained from *Globe Amaranth* through boiling water extraction and ethanol precipitation to obtain crude polysaccharide. The crude polysaccharide was then eluted using a DEAE-Fast Flow column with 0.05M NaCl as the eluent to obtain secondary crude polysaccharide. The secondary crude polysaccharide was then purified using a Sephacryl S-200 column with 0.2M NaCl to obtain a homogeneous fraction. Glucan-2 and glucan-3 were obtained from *Astragalus membranaceus* tubers through boiling water extraction and ethanol precipitation to obtain crude polysaccharide. The crude polysaccharide was then eluted using a DEAE-Fast Flow column with 0.05M NaCl to obtain secondary crude polysaccharide, which was subsequently purified using a Sephacryl S-200 column to obtain a homogeneous fraction.

[0114] Example 1: Grifola frondosa extract (GFPBW1) can enhance the immunogenicity of the model antigen OVA (ovalbumin).

[0115] 1.1 Immunized animals

[0116] Twenty adult C57 mice were randomly divided into four groups: saline group, ovalbumin (OVA) alone group, OVA+GFPBW1 (50 μg) group, and OVA+GFPBW1 (300 μg) group, with five mice in each group. Mice were immunized twice via intramuscular injection, 14 days apart. The first immunization was defined as day 0. Blood samples were collected via the orbital venous plexus on days 7, 14, 21, 28, and 35. Serum was collected by centrifugation and analyzed.

[0117] 1.2 ELISA detection of OVA-specific total IgG

[0118] Coat a 96-well plate overnight at 4°C with 100 μL of OVA solution (5 μg / ml). After blocking with 3% BSA, add serially diluted serum and incubate at 37°C for 1 hour. After washing, add 100 μL of enzyme-labeled goat anti-mouse IgG (dilution ratio 1:5000) to each well and incubate for 1 hour. After washing, add 100 μL of tetramethylbenzidine (TMB) substrate solution for 20 min, terminate the reaction with 2M H2SO4, and measure the absorbance at 450 nm using an Infinite F50 microplate reader (Tecan).

[0119] The results are as follows Figure 1 As shown, compared to the OVA-only immunization group, starting from day 7 after the second immunization, the OD values ​​of both OVA+GFPBW1 immunization groups were significantly higher. Figure 1 A, 1B, 1C) indicate that the OVA-specific total IgG content is higher, and the OVA+GFPBW1 (300μg) group is more significantly higher than the OVA+GFPBW1 (50μg) group.

[0120] Example 2: Grifola frondosa extract (GFPBW1) can simultaneously induce Th1 and Th2 immune responses.

[0121] 2.1 Immunized animals

[0122] Twenty-five adult C57 mice were randomly divided into five groups: saline group, ovalbumin (OVA) alone group, OVA+GFPBW1 (10 μg), OVA+GFPBW1 (50 μg), and OVA+GFPBW1 (300 μg), with five mice in each group. Mice were immunized twice via intramuscular injection, 14 days apart. Blood was collected via the orbital venous plexus on day 14 after the second immunization, and serum was collected by centrifugation for analysis.

[0123] 2.2 ELISA detection of OVA-specific IgG1 / 2a / 2b

[0124] Coat a 96-well plate overnight at 4°C with 100 μL of OVA solution (5 μg / ml). After blocking with 3% BSA, add serially diluted serum and incubate at 37°C for 1 hour. After washing, add 100 μL of enzyme-labeled goat anti-mouse IgG1 / IgG2a / IgG2b (dilution ratio 1:5000) to each well and incubate for 1 hour. After washing, add 100 μL of tetramethylbenzidine (TMB) substrate solution for 20 min, terminate the reaction with 2M H2SO4, and measure the absorbance at 450 nm using an Infinite F50 microplate reader (Tecan).

[0125] The results are as follows Figure 2As shown, compared with the OVA-only immunization group, the OVA+GFPBW1 immunization group had significantly higher levels of OVA-specific IgG1, IgG2a, and IgG2b. Figure 2 (A, 2B, 2C) For OVA-specific IgG1, all three immunization groups with OVA+GFPBW1 showed higher levels than the OVA-only group. For OVA-specific IgG2a, only the OVA+GFPBW1 (300 μg) group showed higher levels than the OVA-only group. For OVA-specific IgG2b, both the OVA+GFPBW1 (50 or 300 μg) groups showed higher levels than the OVA-only group. IgG1 and IgG2a / 2b represent Th1 and Th2 effects, respectively. In conclusion, GFPBW1 can simultaneously induce Th1 and Th2 immune effects.

[0126] Example 3: Inhibition of tumor growth by Grifola frondosa extract (GFPBW1) as an adjuvant in prophylactic tumor vaccines.

[0127] Experimental methods: Forty C57 mice were randomly divided into five groups: saline group, ovalbumin (OVA) alone group, OVA+GFPBW1 (10 μg), OVA+GFPBW1 (50 μg), and OVA+GFPBW1 (300 μg), with eight mice in each group. The mice were immunized twice by intramuscular injection (14 days apart). Six weeks later, B16-OVA melanoma cells were inoculated under the armpit. Starting from day 8, the length and width of the tumor were measured every other day. The tumor volume was calculated as 0.5 * length * width * width.

[0128] The results are as follows Figure 3 As shown, compared to the OVA-only immunization group, the tumors in the three immunization groups (OVA + GFPBW1) were significantly inhibited. Figure 3 (A, 3B) In particular, the 50 and 300 μg groups of OVA+GFPBW1 showed 100% tumor inhibition, and the same trend was observed in animal survival rates. The survival rate of mice in the OVA+GFPBW1 group was significantly higher than that in the OVA-only group, indicating that GFPBW1 can play a good adjuvant role in preventive tumor vaccines.

[0129] Example 4: Inhibition of tumor growth by Grifola frondosa extract (GFPBW1) as an adjuvant in therapeutic tumor vaccines.

[0130] Experimental methods: Forty C57 mice were randomly divided into five groups: saline group, ovalbumin (OVA) alone group, OVA+GFPBW1 (10 μg), OVA+GFPBW1 (50 μg), and OVA+GFPBW1 (300 μg), with eight mice in each group. After inoculating B16-OVA melanoma cells under the armpit, the mice were immunized twice by intramuscular injection on days 6 and 13. Starting from day 8, the length and width of the tumor were measured every other day. The tumor volume was calculated as 0.5 * length * width * width.

[0131] The results are attached. Figure 4 As shown, compared to the OVA-only immunization group, the tumors in the three immunization groups (OVA + GFPBW1) were significantly inhibited. Figure 4 A and Figure 4 B) The survival rate of mice in the OVA+GFPBW1 group was also higher than that in the OVA alone group, indicating that GFPBW1 can play a good adjuvant role in therapeutic tumor vaccines.

[0132] Example 5: Compared with similar polysaccharides, the extract of Grifola frondosa (GFPBW1) has a better ability to promote the activation of antigen-presenting cells.

[0133] Experimental methods:

[0134] 1) Single-cell suspensions were prepared from the spleens of C57 mice and nine groups were set up: saline group, two GFPBW1 concentration groups (5 and 50 μg / mL), two glucan-1 concentration groups (5 and 50 μg / mL), two glucan-2 concentration groups (5 and 50 μg / mL), and two glucan-3 concentration groups (5 and 50 μg / mL). The single-cell suspensions were added and the cells were stimulated for 48 h before the cells were collected. The activation index CD86 was detected by flow cytometry.

[0135] 2) Human monocyte-macrophage cell line THP-1 cells were induced to become macrophages by adding PMA for 24 h. Different concentrations of GFPBW1 (5 and 50 μg / mL), glucan-1 (5 and 50 μg / mL), glucan-2 (5 and 50 μg / mL), and glucan-3 (5 and 50 μg / mL) were added and the cells were stimulated for 24 h. Then, FITC-OVA was added and the cells were incubated in a 37°C incubator in the dark for 1.5 h. The cell suspension was collected, gently pipetted and mixed, and then analyzed by instrument.

[0136] The results are as follows Figure 5 As shown, compared to polysaccharides of the same class (glucan-1, glucan-2, glucan-3), the GFPBW1 group significantly promoted the increase of CD86. Figure 5 A, Figure 5 B and Figure 5 C), and its corresponding ability to absorb FITC-OVA is weaker. Figure 5 D) indicates that GFPBW1 has a better ability to promote the activation of antigen-presenting cells compared with similar polysaccharides, suggesting that the extract of Grifola frondosa, GFPBW1, is more suitable as an immune adjuvant.

[0137] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of an extract from Grifola frondosa, characterized in that, Used for preparing adjuvants; wherein, the extract of Grifola frondosa is β-glucan; the β-glucan has the following structural units: in, It is glucose; n is an integer between 100 and 750, preferably between 400 and 500, and more preferably 450.

2. The use as described in claim 1, characterized in that, The average molecular weight of the β-glucan is 3-400 kDa, preferably 100-400 kDa.

3. The use as described in claim 1, characterized in that, The adjuvant is used for: (1) Enhances the production of OVA-specific IgG; (2) Enhance the expression of IgG1, IgG2a and IgG2b; (3) Simultaneously enhances the immune effects of Th1 and Th2 types; (4) Promotes the expression of CD86 in antigen-presenting cells.

4. An adjuvant composition, characterized in that, The adjuvant composition comprises: (i) The extract of Grifola frondosa as described in claim 1; (ii) A pharmaceutically acceptable carrier; The extract of Grifola frondosa is β-glucan; the β-glucan has the following structural units: in, It is glucose; n is an integer between 100 and 750, preferably between 400 and 500, and more preferably 450.

5. The adjuvant composition of claim 4, wherein the adjuvant composition is present in unit dose form.

6. A vaccine composition, characterized in that, The vaccine composition comprises: (a) One or more antigens; (b) Extracts from Grifola frondosa; and (c) A pharmaceutically acceptable carrier; The extract of Grifola frondosa is β-glucan; the β-glucan has the following structural units: in, It is glucose; n is an integer between 100 and 750, preferably between 400 and 500, and more preferably 450.

7. The vaccine composition according to claim 6, characterized in that, The vaccine composition is in the form of an injectable formulation, an inhalable formulation, or an oral formulation.

8. The vaccine composition according to claim 6, characterized in that, The dosage form of the vaccine composition is selected from the following: powder for injection, suspension, aqueous injection, spray, aerosol, powder inhaler, adhesive tablet, sublingual tablet or film.

9. Use of the vaccine composition as described in claim 6, characterized in that, The vaccine composition is used to stimulate an immune response in a subject.

10. A method for promoting the activation of antigen-presenting cells in vitro, characterized in that, Including the following steps: Contacting antigen-presenting cells with an extract of Grifola frondosa promotes the activation of the antigen-presenting cells. The extract of Grifola frondosa is β-glucan; the β-glucan has the following structural units: in, It is glucose; n is an integer between 100 and 750, preferably between 400 and 500, and more preferably 450.

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