Application of ganoderma lucidum spore powder in oral vaccine adjuvant and product of ganoderma lucidum spore powder

Abstract

The invention provides application of ganoderma lucidum spore powder in an oral vaccine adjuvant and a product of the ganoderma lucidum spore powder, and belongs to the field of vaccine adjuvants. The invention provides application of ganoderma lucidum spore powder in oral vaccine adjuvants. As an oral vaccine adjuvant, the ganoderma lucidum spore powder provided by the invention can improve the concentration level of serum IFN-alpha and enhance the IFN-alpha mediated immune cell response, thereby assisting the vaccine in enhancing the immunity and providing a basis for clinical research.

Description

Technical Field

[0001] The invention belongs to the field of vaccine adjuvants, and specifically relates to the application of ganoderma lucidum spore powder in oral vaccine adjuvants and products thereof. Background Art

[0002] At present, a variety of vaccines have passed clinical trials and completed large-scale vaccination. However, about half a year after the injection, the antibody titer level dropped significantly. Five months after the injection, the effects of vaccines such as mRNA-1273, BNT162b2, and Ad26.COV2.S all dropped by at least 20% from the peak period. The problem of reduced vaccine efficacy is also common in vaccines for other infectious diseases, including vaccines for influenza, whooping cough, and mumps. Therefore, enhancing the effectiveness of vaccines and maintaining high levels of antibody titers for a long time are the key to the next step of vaccine development.

[0003] Appropriate vaccine adjuvants can enhance immune responses and prolong vaccine effects. Currently, common adjuvants such as aluminum hydroxide, MF59, Matrix-M, and AS03 have been used in the development of COVID-19 vaccines and have achieved good results. However, the number of vaccine adjuvants currently approved for clinical use is limited, and they have potential safety risks and limitations. Aluminum hydroxide, as the most commonly used immune adjuvant, mainly assists vaccines by activating innate immunity and promoting Th2 cell responses. Therefore, it is not suitable for vaccines that rely on CD8 + Aluminum hydroxide is difficult to work on vaccines that provide antiviral effects on T cells or Th1 cells. In addition, since aluminum salts are neurotoxic and may cause allergic reactions, doubts about the safety of aluminum hydroxide as a vaccine adjuvant have been raised in recent years. AS03 is an emulsified adjuvant developed by GlaxoSmithKline. It has been reported that H1N1 vaccines adjuvanted with AS03 are associated with an increased incidence of narcolepsy in children. Another emulsified adjuvant, MF59, can only work at the injection site and draining lymph nodes. Compared with vaccines without adjuvants, vaccines using MF59 adjuvant are more likely to cause local or systemic reactions. Based on the above considerations, it is necessary to develop adjuvants that can systemically activate immune responses and are safe and stable.

[0004] Traditional Chinese herbal extracts can meet the safety requirements of vaccine adjuvants and can be used as alternative vaccine adjuvants for clinical applications. At present, clinical studies have proven the feasibility of Chinese herbal extract adjuvants. Among them, Ganoderma lucidum polysaccharides, Ganoderma lucidum proteins and other ingredients can be used as adjuvants for tetanus vaccines, tumor vaccines and chicken Newcastle vaccine, effectively promoting dendritic cell maturation, activating Th1 response, and promoting cytokine secretion. In particular, Ganoderma lucidum spore powder is rich in Ganoderma lucidum active ingredients, which can be used for anti-tumor and immune regulation. However, there are currently no reports on Ganoderma lucidum ingredients as adjuvants for human epidemic vaccines, and related experiments are often conducted on animals. Therefore, the possibility of Ganoderma lucidum ingredients being used as adjuvants for a wide range of human vaccines remains to be explored. Summary of the invention

[0005] In order to solve the above problems, the present invention provides the application of Ganoderma lucidum spore powder in oral vaccine adjuvant and its products. The present invention provides the application of Ganoderma lucidum spore powder in oral vaccine adjuvant. The Ganoderma lucidum spore powder provided by the present invention is used as an oral vaccine adjuvant to increase the serum IFN-α concentration level and enhance the IFN-α-mediated immune cell response, thereby assisting the vaccine in enhancing immunity and providing a basis for clinical research.

[0006] Specific technical solutions include:

[0007] In one aspect, the present invention provides a use of Ganoderma lucidum spore powder in preparing an oral vaccine adjuvant.

[0008] Preferably, the Ganoderma lucidum spore powder comes from any one of Xianzhi No. 3, Xianzhi No. 4 and Xianzhi No. 5.

[0009] Preferably, the Ganoderma lucidum spore powder is wall-broken Ganoderma lucidum spore powder.

[0010] Preferably, the preparation of the Ganoderma lucidum spore powder includes breaking the spore walls of the Ganoderma lucidum spores and removing the spore walls after breaking.

[0011] Preferably, the oral vaccine adjuvant is Ganoderma lucidum spore powder.

[0012] On the other hand, the present invention provides a vaccine preparation, which includes a vaccine active ingredient and an oral vaccine adjuvant, and the oral vaccine adjuvant is the above-mentioned Ganoderma lucidum spore powder.

[0013] Preferably, the vaccine active ingredient is a new coronavirus vaccine.

[0014] Preferably, the new coronavirus vaccine is selected from any one of an adenovirus vector vaccine, an inactivated vaccine and a recombinant protein vaccine.

[0015] Preferably, the vaccine preparation comprises an oral vaccine, an intravenous vaccine, an arterial vaccine or a subcutaneous vaccine.

[0016] On the other hand, the present invention provides the use of the above-mentioned Ganoderma lucidum spore powder or the above-mentioned vaccine preparation in the preparation of immunomodulatory drugs.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] (1) The present invention uses Ganoderma lucidum spore powder as an oral vaccine adjuvant to assist the booster shot of the new crown vaccine. Compared with the unintervention control group, Ganoderma lucidum spore powder increased the antibody titer of the vaccine in subjects with moderate basic antiviral ability.

[0019] (2) The present invention provides the use of Ganoderma lucidum spore powder in enhancing IFN-α-mediated immune cell response. Compared with a simple booster injection, Ganoderma lucidum spore powder also increases the concentration level of serum IFN-α.

[0020] (3) The present invention provides a product that is safe, non-toxic and has no side effects within an effective dose. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Statistical chart of the differences in neutralizing antibody inhibition rates among groups with high immune levels at different time points; among them, there was no significant difference between the drug intervention group and the control group at each time point.

[0022] Figure 2 Statistical chart of the differences in neutralizing antibody inhibition rate in the medium immune level group at different time points; among them, there was no significant difference between the drug intervention group and the control group before injection, on the 10th day after injection, and on the 180th day after injection; on the 90th day after injection, the drug intervention group vs. the control group, *P<0.05.

[0023] Figure 3 Statistical chart of the differences in neutralizing antibody inhibition rates in low-immunity groups at different time points; among them, there was no significant difference between the drug intervention group and the control group at each time point.

[0024] Figure 4 Statistical diagram of the inter-group differences in the intensity of B cell / plasma cell response to IFN-α on the 90th day after injection; among them, drug intervention group vs control group, ****P<0.0001.

[0025] Figure 5 Statistical diagram of the inter-group differences in the intensity of myeloid cell response to IFN-α on the 90th day after injection; among them, drug intervention group vs control group, ****P<0.0001.

[0026] Figure 6 Statistical chart of the inter-group differences in the intensity of T cell response to IFN-α on the 90th day after injection; among them, drug intervention group vs control group, ****P<0.0001.

[0027] Figure 7 The difference in serum IFN-α concentration results 90 days after injection of the new crown booster shot. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0029] The sources of materials involved in the present invention are shown in Table 1:

[0030] Table 1 Source of materials

[0031] Reagents / Materials Manufacturer / Source Article number / Batch number Ganoderma lucidum spores Zhejiang Shouxiangu Pharmaceutical Co., Ltd. Lingzhi No.3 ELISA Kits Elabscience E-EL-E602 Serum IFN-α concentration detection kit Elabscience E-EL-H6125

[0032] In the following examples, “*” indicates that P<0.05 is significant, “**” indicates that P<0.01 is highly significant, “***” indicates that P<0.001 is extremely significant, and “****” indicates that P<0.0001.

[0033] Preparation Example: Preparation of Ganoderma Lucidum Spore Powder

[0034] (1) Weigh the raw material of Lingzhi spore No. 3 (from Zhejiang Shouxiangu Pharmaceutical Co., Ltd.), pass it through an 80-mesh sieve to remove larger gravel and sand;

[0035] (2) Place 2000 g of sieved spores into a mixing tank, add 16000 g of distilled water, stir for 20 minutes and then let stand for 15 minutes. Remove the deflated spores and other floating impurities from the feeding port, and drain the sludge and gravel that sink to the bottom of the water from the drain port at the bottom of the mixing tank. Allow the middle layer of spore suspension to overflow into an iron tank and pump it into a filter centrifuge to remove moisture;

[0036] (3) The wet spores after centrifugation are placed in a tray in a vacuum microwave dryer, evenly spread out, and dried at low temperature. The material thickness is ≤4cm, the temperature is ≤60°C, and it is dried to a moisture content of 6% to obtain 1400g of dry spores; (4) The dried spores are broken by a fluidized bed airflow mill to obtain 1400g of broken spore powder of Ganoderma lucidum; the broken spore powder is tested according to NYT1677-2008 standard, and the broken spore rate is 98%. The contents of chromium, nickel, and lead in the broken spore powder of Ganoderma lucidum are 0.65mg / kg, 0.14mg / kg, and 0.079mg / kg, respectively, which are not significantly increased compared with 0.5mg / kg, 0.17mg / kg, and 0.085mg / kg before the spores are broken, and meet the requirements of GB2762-2012 for safety limits in food;

[0037] (5) 1400 g of broken spore powder is put into an extraction tank, and 14000 g of 60% ethanol solution is added, and the mixture is soaked at 80° C. for 2 hours, soaked twice, and then ultrasonically soaked with 10000 g of distilled water at 60° C. for 1 hour each time. All soaking solutions are combined and filtered using a filtering centrifuge. The solution that passes through the filter screen is the wall-removed Ganoderma lucidum spore powder filtrate, and the solution on the filter screen is the spore powder wall shell; the filter bag of the filtering centrifuge has an aperture of 800 mesh, a frequency of 40 Hz, and a rotation speed of 1000 rpm;

[0038] (6) Concentrate the filtrate at 60°C until there is no alcohol taste and the specific gravity of the concentrate is 1.05;

[0039] (7) The concentrated solution was dried into solid powder by microwave drying to obtain 400 g of Ganoderma lucidum spore powder.

[0040] Example 1: Detection of the effect of Ganoderma lucidum spore powder on improving the neutralizing antibody inhibition rate in people with moderate immunity. Experimental materials: Ganoderma lucidum spore powder.

[0041] Test population: 195 subjects, all of whom have completed two doses of the new crown vaccine injection for more than 6 months. The new crown vaccine booster model is CoronaVac, produced by Sinovac Biotech Co., Ltd.

[0042] Experimental methods:

[0043] (1) The subjects were divided into a control group and a drug intervention group, and both were given a booster shot (third shot) of the COVID-19 vaccine. The drug intervention group took Ganoderma lucidum spore powder from the first day of injection, twice a day, one bag (2g) each time, and continued to take it for 6 months. The serum of the subjects was collected before injection (day 0), 10 days, 90 days and 180 days, and the serum neutralizing antibody inhibition rate was measured.

[0044] (2) The ELISA kit was used to detect the serum SARS-CoV-2S protein-specific IgG antibody levels of all subjects before injection. According to the serum SARS-CoV-2S protein-specific IgG antibody levels, the subjects were divided into groups with different basic antiviral immunity. Specifically, there were low-immunity groups with serum SARS-CoV-2S protein-specific IgG levels in the lowest 25% range, high-immunity groups with levels in the highest 25% range, and medium-immunity groups with levels in the remaining 50% range between the two. The differences in neutralizing antibody inhibition rates between the drug intervention group and the control group at different time points were measured for the three groups, and the rank sum test was used as the statistical method.

[0045] The results are as follows Figure 1-3 As shown in the figure, Day0 means before injection, Day10 means the 10th day after injection, Day90 means the 90th day after injection, and Day180 means the 180th day after injection; Control means the control group, and Drug means the drug intervention group. Figure 1 The figure is a statistical chart showing the differences in neutralizing antibody inhibition rates among groups with high immune levels at different time points; among them, there was no significant difference between the drug intervention group and the control group at each time point. Figure 2The figure is a statistical chart showing the differences in neutralizing antibody inhibition rates in the medium immune level group at different time points; among them, there was no significant difference between the drug intervention group and the control group before injection, on the 10th day after injection, and on the 180th day after injection; on the 90th day after injection, the drug intervention group vs. the control group, *P<0.05. Figure 3 The figure is a statistical chart showing the differences in neutralizing antibody inhibition rates in low-immunity groups at different time points; among them, there was no significant difference between the drug intervention group and the control group at each time point.

[0046] analyze Figure 1-3 It can be found that in the group with moderate immunity, 90 days after the injection of the new crown vaccine booster shot, the neutralizing antibody level in the drug intervention group was significantly higher than that in the control group (drug intervention group: 74.82±20.17%; control group: 67.67±19.26%, P<0.05). In the groups with low and high levels of basic immunity, there was no significant difference in the intervention of Ganoderma lucidum spore powder. This shows that in the group with moderate basic immunity, compared with the simple injection of booster shots, Ganoderma lucidum spore powder can significantly enhance the neutralizing antibody inhibition rate, and the 90th day after injection is the onset time of the drug.

[0047] Example 2: Detection of Ganoderma lucidum spore powder in enhancing IFN-α response level

[0048] According to the classification of different antiviral basic immunity groups in Example 1, based on the medium immunity level group, three male subjects and three female subjects were randomly selected from the control group and the drug intervention group, and PBMC samples were extracted before injection and on the 90th day after injection. The neutralizing antibody inhibition rates of the selected samples were close to the average neutralizing antibody inhibition rate in the group.

[0049] According to the 10X genomics workflow, single-cell transcriptome sequencing was performed on PBMC samples. The R language package Seurat was used to perform quality control, standardization, dimensionality reduction, and clustering of single-cell transcriptome data, and known markers were used to annotate cell populations. The "INTERFERON ALPHA RESPONSE" entry genes in the MSigDB HALLMARK database were selected as the IFN-α response-related gene set, and the AddModuleScore function was used to score the gene set to quantify the IFN-α response level of the cells. The IFN-α response scores of PBMCs between the drug intervention group and the control group were compared on day 90 after injection, and the rank sum test was used as the statistical method.

[0050] The results are as follows Figure 4-6 As shown, Day90_Control is the control group on the 90th day after injection, and Day90_Drug is the drug intervention group on the 90th day after injection. Figure 4The figure is a statistical chart showing the inter-group differences in the intensity of B cell / plasma cell response to IFN-α on the 90th day after injection; among them, drug intervention group vs control group, ****P<0.0001. Figure 5 The figure is a statistical chart showing the inter-group differences in the intensity of myeloid cell response to IFN-α on the 90th day after injection; among them, drug intervention group vs control group, ****P<0.0001. Figure 6 The figure is a statistical chart of the inter-group differences in the intensity of T cell response to IFN-α on the 90th day after injection; among them, drug intervention group vs control group, ****P<0.0001.

[0051] Depend on Figure 4-6 It can be seen that compared with the simple booster injection, the IFN-α response intensity of B cells / plasma cells, myeloid cells and T cells in the drug intervention group was significantly improved. This shows that Ganoderma lucidum spore powder has a positive effect on both innate and adaptive immune cells in enhancing the response to IFN-α.

[0052] Example 3: Detection of Ganoderma lucidum spore powder in increasing serum IFN-α levels

[0053] The serum IFN-α concentration on the 90th day after injection was measured by mixed sample using a serum IFN-α concentration detection kit, and a total of 6 repeated measurements were performed. The subjects of the serum samples were the same as the subjects of the sequencing samples in Example 2. The statistical method used was the t-test. The results are shown in Figure 7 As shown, drug intervention group vs control group, *P<0.05; compared with simple booster injection, Ganoderma lucidum spore powder can significantly increase serum IFN-α levels (drug intervention group: 51.81±15.50pg / ml; control group: 35.18±14.82pg / ml, P<0.05).

Claims

1. An application of Ganoderma lucidum spore powder in preparing an oral vaccine adjuvant.

2. The use according to claim 1, characterized in that: The ganoderma lucidum spore powder comes from any one of Xianzhi No. 3, Xianzhi No. 4 and Xianzhi No.

5.

3. The use according to claim 1, characterized in that: The ganoderma lucidum spore powder is wall-broken ganoderma lucidum spore powder.

4. The use according to claim 3, characterized in that: The preparation of the ganoderma spore powder includes breaking the wall of the ganoderma spores and removing the wall after the wall is broken.

5. The use according to any one of claims 1 to 4, characterized in that: The oral vaccine adjuvant is Ganoderma lucidum spore powder.

6. A vaccine preparation, characterized in that The vaccine preparation comprises a vaccine active ingredient and an oral vaccine adjuvant, and the oral vaccine adjuvant is ganoderma lucidum spore powder.

7. The vaccine preparation according to claim 6, characterized in that The active ingredient of the vaccine is the new coronavirus vaccine.

8. The vaccine preparation according to claim 7, characterized in that The new coronavirus vaccine is selected from any one of an adenovirus vector vaccine, an inactivated vaccine and a recombinant protein vaccine.

9. The vaccine preparation according to claim 6, characterized in that The vaccine preparations include oral vaccines, intravenous vaccines, arterial vaccines or subcutaneous vaccines.

10. Use of the Ganoderma lucidum spore powder according to any one of claims 1 to 5 or the vaccine preparation according to any one of claims 6 to 9 in the preparation of immunomodulatory drugs.

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