High-performance immunologic adjuvant and application thereof in shortening antibody production cycle

Abstract

The invention relates to the technical field of immunologic adjuvants, and discloses a high-performance immunologic adjuvant and application thereof in shortening the antibody production cycle, and a preparation method of the high-performance immunologic adjuvant comprises the following steps: combining plant polysaccharide with chitosan nanoparticles to obtain plant polysaccharide-chitosan nanoparticles; the plant polysaccharide-chitosan nano-particles are wrapped with lipidosome, and the high-performance immunologic adjuvant is obtained; wherein the plant polysaccharide is one or more of burdock polysaccharide, polygonatum odoratum polysaccharide and roxburgh rose polysaccharide. By applying the adjuvant combination, the antibody generation time can be effectively shortened, rapid generation of a high-titer antibody is promoted, the plant polysaccharide used in the adjuvant combination is natural, excellent biocompatibility and safety are shown, the side effect risk in immunoreaction is reduced, and the adjuvant combination is more suitable for clinical application.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoadjuvants, and more particularly, to a high-performance immunoadjuvant and its application in shortening the antibody production cycle. Background Art

[0002] Antibody production is an important branch in the field of modern biotechnology, playing a crucial role in disease treatment, diagnosis, and biological research. However, the traditional antibody production process is time-consuming and costly, which limits its clinical and commercial applications. Immunoadjuvants, as auxiliary substances to enhance the immune response, have potential application value in shortening the antibody production cycle, increasing antibody yield and quality.

[0003] Traditional immunoadjuvants such as Freund's adjuvant are effective but have certain toxicity and side effects, restricting their use in clinical applications. Therefore, the development of new high-performance immunoadjuvants that can effectively stimulate the immune response while reducing adverse reactions is a current research hotspot.

[0004] Plant polysaccharides are a class of natural macromolecular compounds widely present in plants, with various biological activities, including immunomodulatory effects. In recent years, studies have found that certain plant polysaccharides can act as immunoadjuvants, effectively enhancing the immune response of the body to vaccines or antigens, while having relatively low toxicity and side effects. For example, astragalus polysaccharide and ginseng polysaccharide have been proven to enhance the immune response, increase antibody levels, and have low toxicity to the body.

[0005] Therefore, the development of new plant polysaccharide-based immunoadjuvants is of great significance for improving the efficiency and safety of antibody production. By utilizing the immunomodulatory properties of plant polysaccharides, safer and more effective adjuvants can be designed, thereby promoting the rapid production of antibodies to meet clinical and commercial needs. In addition, plant polysaccharides are widely sourced and relatively inexpensive, which makes it possible for the large-scale and economical production of antibodies. Future research can further explore the immunoadjuvant effects of different plant polysaccharides, optimize the formulation, and evaluate their application potential in different types of antibody production. With the in-depth research, plant polysaccharide-based immunoadjuvants are expected to become an important force driving the development of the antibody production field. Summary of the Invention

[0006] In view of this, the present invention proposes a high-performance immunoadjuvant and its application in shortening the antibody production cycle, aiming to provide a plant polysaccharide-based immunoadjuvant that can effectively improve the antibody production efficiency, shorten the antibody production cycle, and has relatively low toxicity and side effects.

[0007] The present invention proposes a preparation method for a high-performance immunoadjuvant, including:

[0008] The plant polysaccharide is combined with chitosan nanoparticles to obtain plant polysaccharide-chitosan nanoparticles;

[0009] The plant polysaccharide-chitosan nanoparticles are encapsulated by liposomes to obtain the high-performance immune adjuvant;

[0010] Wherein, the plant polysaccharide is one or more of burdock polysaccharide, polygonatum odoratum polysaccharide and rosa roxburghii tratt polysaccharide.

[0011] Preferably, the preparation method of the chitosan nanoparticles includes:

[0012] The chitosan nanoparticles are prepared by dissolving chitosan in an acetic acid solution and using the ion gel method.

[0013] Preferably, when the plant polysaccharide is combined with chitosan nanoparticles to obtain plant polysaccharide-chitosan nanoparticles, it includes:

[0014] Under the action of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, the burdock polysaccharide is combined with chitosan nanoparticles through the reaction of amino group and carboxyl group to obtain the plant polysaccharide-chitosan nanoparticles.

[0015] The present invention also provides a high-performance immune adjuvant, which is prepared according to the preparation method of the above-mentioned high-performance immune adjuvant.

[0016] The present invention also provides an application of the above-mentioned high-performance immune adjuvant, and the application at least includes one of the following:

[0017] A) The application of the high-performance immune adjuvant in the preparation of antibodies;

[0018] A) The application of the high-performance immune adjuvant in shortening the antibody generation cycle.

[0019] The present invention also provides a product for shortening the antibody generation cycle, and the active ingredient of the product is the above-mentioned high-performance immune adjuvant.

[0020] The present invention also provides a vaccine composition, and the vaccine composition includes an antigen and the above-mentioned high-performance immune adjuvant.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1. High-performance adjuvant combination:

[0023] The polysaccharide combination (such as polygonatum odoratum polysaccharide, rosa roxburghii tratt polysaccharide and burdock polysaccharide) adopted by the present invention can synergistically enhance the immune response, significantly improve the antibody titer and cellular immune response compared with single polysaccharide or traditional adjuvant.

[0024] 2. Shorten the antibody generation cycle:

[0025] The experimental results show that the adjuvant combination of the present invention can effectively shorten the antibody generation time, promote the rapid production of high-titer antibodies, and exhibit a faster immune response speed compared with commercially available traditional adjuvants.

[0026] 3. Safety and biocompatibility:

[0027] The plant polysaccharides used in the present invention are derived from nature, exhibit excellent biocompatibility and safety, reduce the risk of side effects in immune responses, and are more suitable for clinical applications.

[0028] 4. Significantly enhance the cellular immune response:

[0029] The adjuvant combination of the present invention not only improves the antibody generation efficiency but also significantly enhances the proliferation and activation of CD4+ and CD8+ T cells, promotes the secretion of cytokines, and thus enhances the cellular immune capacity of the body.

[0030] 5. Wide adaptability:

[0031] This adjuvant combination is applicable to a variety of antigens, can be widely used in vaccine development and disease prevention, and improves the flexibility and effectiveness of antibody production.

[0032] 6. Economy:

[0033] Since the polysaccharides in the present invention are derived from common plant resources, the raw materials are easily accessible and the cost is low, so it has good economic benefits.

[0034] In summary, the immune adjuvant of this invention is superior to the prior art in terms of improving the immune effect, shortening the production cycle, and ensuring safety, showing broad application prospects. Brief Description of the Drawings

[0035] By reading the following detailed description of the preferred embodiments, various other advantages and benefits will become clear to those of ordinary skill in the art. The drawings are only for the purpose of showing the preferred embodiments and are not considered to be a limitation of the present invention. Moreover, throughout the drawings, the same reference numerals are used to represent the same components. In the drawings:

[0036] Figure 1 is the expression result of cell surface markers;

[0037] Figure 2 is the detection result of phagocytic function;

[0038] Figure 3 is one of the detection results of cellular immune responses

[0039] Figure 4This is the second result of the cellular immune response detection. Detailed implementation manners

[0040] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided so that the present disclosure can be more thoroughly understood and the scope of the present disclosure can be fully conveyed to those skilled in the art. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the drawings and in combination with the embodiments.

[0041] Example 1 In vitro screening of polysaccharides

[0042] I. Experimental materials

[0043] Cells: Mouse bone marrow-derived dendritic cells (BMDC), mouse macrophage cell line RAW264.7.

[0044] Polysaccharide samples: Polygonatum odoratum polysaccharide (POP), Arctium lappa polysaccharide (ALP), Rosa roxburghii polysaccharide (RRP).

[0045] Controls: Phosphate-buffered saline (PBS), lipopolysaccharide (LPS, positive control).

[0046] Culture medium: RPMI-1640 medium, 10% fetal bovine serum (FBS), penicillin-streptomycin mixture.

[0047] Antibodies: APC-labeled anti-CD11c, PE-labeled anti-MHC-II, FITC-labeled anti-CD86 (for BMDC); APC-labeled anti-F4 / 80, PE-labeled anti-CD80, FITC-labeled anti-CD40 (for RAW264.7).

[0048] ELISA kits: IL-12, TNF-α, IFN-γ, IL-6.

[0049] II. Experimental procedures

[0050] 1. Flow cytometry for detecting cell surface markers

[0051] 1.1 Preparation of bone marrow-derived dendritic cells (BMDC)

[0052] In this study, bone marrow cells were extracted from C57BL / 6 mice and treated with erythrocyte lysate. Subsequently, the cells were inoculated into RPMI-1640 complete medium containing 10 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF) and 10 ng / mL interleukin-4 (IL-4) and cultured continuously for 7 days. The fresh medium was changed on the 3rd day of culture, and the cells were collected on the 6th day for subsequent experiments.

[0053] 1.2 Culture of RAW264.7 cells

[0054] The RAW264.7 cell line was cultured in RPMI-1640 complete medium and placed in an incubator at 37 °C and 5% CO2. When the cells grew to approximately 80% confluence, cell passage and subsequent experiments were performed.

[0055] 1.3 Polysaccharide stimulation experiment

[0056] BMDC cells and RAW264.7 cells were respectively inoculated into 6-well plates, the cell density was adjusted to 50%, and 6 experimental groups were set up: PBS control group, lipopolysaccharide (LPS, 1 μg / mL), polysaccharide group (POP, 100 μg / mL), acidic polysaccharide group (ALP, 100 μg / mL), and recombinant polysaccharide group (RRP, 100 μg / mL). They were cultured under the conditions of 37 °C and 5% CO2 for 24 hours.

[0057] 1.4 Fluorescent staining of cell surface markers

[0058] After collecting the cells, they were washed twice with phosphate buffer saline (PBS). BMDC cells were fluorescently stained with CD11c, major histocompatibility complex II (MHC-II), and CD86 antibodies, while RAW264.7 cells were stained with F4 / 80, CD80, and CD40 antibodies. All operations were carried out under light-proof conditions, and the incubation time was 30 minutes.

[0059] 1.5 Flow cytometry detection

[0060] The proportion of marker-positive cells was detected by flow cytometry to analyze the maturation status of dendritic cells and macrophages.

[0061] 1.6 Experimental results

[0062] Figure 1 And Table 1 shows the experimental results of the expression of cell surface markers.

[0063] Table 1

[0064]

[0065]

[0066] According to Table 1 and Figure 1 it can be seen that, compared with the PBS group, the LPS group, ALP group, POP group and RRP group can all significantly increase the expression levels of maturation markers in BMDC and RAW264.7 cells. Among them, the LPS group, as the positive control, has the strongest ability to induce cell maturation. Among the polysaccharide groups in the experiment, the ALP group has the relatively optimal ability to induce the maturation of BMDC and RAW264.7 cells, followed by the POP group and RRP group, but both show certain immune-stimulating activities. Figure 1 Intuitively demonstrates the differences in the expression levels of cell surface markers among different groups, further confirming the reliability of the experimental results. These results provide important experimental basis for the subsequent evaluation of the application of high-performance immune adjuvants in shortening the antibody production cycle.

[0067] 2. Flow cytometry detection of cell phagocytosis function

[0068] 2.1 Cell seeding and polysaccharide stimulation

[0069] Inoculate bone marrow-derived dendritic cells (BMDC) and RAW264.7 cells into 6-well plates, adjust the cell density to 50%, and set up six groups of experiments, including PBS control group, lipopolysaccharide (LPS, 1 μg / mL), polysaccharide group (POP, 100 μg / mL), acidic polysaccharide group (ALP, 100 μg / mL), and recombinant polysaccharide group (RRP, 100 μg / mL), and then culture for 24 hours at 37°C and 5% CO2.

[0070] 2.2 Cell phagocytosis experiment

[0071] Add fluorescently labeled Escherichia coli particles (MOI = 10:1) to the cells and incubate at 37°C for 2 hours.

[0072] 2.3 Flow cytometry detection

[0073] Wash the cells three times with cold phosphate-buffered saline (PBS), and then collect cell samples for flow cytometry detection to measure the fluorescence intensity, and further evaluate the phagocytic ability of the cells.

[0074] 2.4 Experimental results

[0075] Figure 2 And Table 2 shows the detection results of cell phagocytosis function.

[0076] Table 2

[0077]

[0078]

[0079] According to Table 2 and Figure 2 it can be seen that compared with the PBS group, the LPS group, ALP group, POP group and RRP group can all significantly improve the phagocytic ability of BMDC and RAW264.7 cells. Among them, the LPS group, as the positive control, showed the strongest enhancement effect on phagocytic activity. Among the polysaccharide groups, the ALP group induced the highest phagocytic activity, and the phagocytic ability of BMDC and RAW264.7 cells increased by about 82% and 11 times that of the PBS group, respectively. The POP group and RRP group also showed a certain enhancement effect on phagocytic activity, but were slightly weaker than the ALP group. Figure 2 Intuitively demonstrates the differences in cell phagocytic ability among different groups, further verifying the accuracy of the experimental results. These results provide valuable experimental data for subsequent evaluation of the effect of high-performance immune adjuvants on cell phagocytic function, and contribute to a deeper understanding of their mechanism of action in the immune response.

[0080] Preparation of Immune Adjuvant in Example 2

[0081] I. Core Materials

[0082] Arctium lappa polysaccharide (ALP) as the main adjuvant component; chitosan nanoparticles (CSNPs) as the carrier; lipid modification (liposome encapsulation) to enhance the immune activation effect.

[0083] II. Preparation Method

[0084] Preparation of nanoparticles: Dissolve chitosan in acetic acid solution, and prepare chitosan nanoparticles by the ionotropic gelation method.

[0085] Modification of Arctium lappa polysaccharide: Combine Arctium lappa polysaccharide with chitosan nanoparticles through the reaction of amino group and carboxyl group to increase its stability and targeting. This step is promoted by using the activator 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC).

[0086] Liposome encapsulation: Coat a liposome layer composed of phospholipids outside the Arctium lappa polysaccharide-chitosan nanoparticles to simulate the cell membrane environment, improve the biocompatibility of the nanoparticles, and enhance the targeting effect on antigen-presenting cells.

[0087] Characterization and purification: Characterize the nanoparticles by transmission electron microscopy (TEM) and dynamic light scattering (DLS) to ensure that their particle size is between 100 - 200 nm. Purify the nanoparticles by ultrafiltration method.

[0088] Preparation of Immune Adjuvant in Example 3

[0089] The preparation method is the same as that of Example 2, and the only difference is that the polysaccharide is polygonatum odoratum polysaccharide.

[0090] Preparation of the adjuvant in Example 4

[0091] The preparation method is the same as that of Example 2, and the only difference is that the polysaccharide is rosa roxburghii tratt polysaccharide.

[0092] Preparation of the adjuvant in Example 5

[0093] The preparation method is the same as that of Example 2, and the only difference is that the polysaccharide is a 1:1 mixture of polygonatum odoratum polysaccharide and rosa roxburghii tratt polysaccharide.

[0094] Preparation of the adjuvant in Example 6

[0095] The preparation method is the same as that of Example 2, and the only difference is that the polysaccharide is a 1:1 mixture of arctium lappa polysaccharide and rosa roxburghii tratt polysaccharide.

[0096] Preparation of the adjuvant in Example 7

[0097] The preparation method is the same as that of Example 2, and the only difference is that the polysaccharide is a 1:1:1 mixture of polygonatum odoratum polysaccharide, rosa roxburghii tratt polysaccharide and arctium lappa polysaccharide.

[0098] Example 3

[0099] For the adjuvants prepared in Examples 2 - 7, combined with the conventional antibody preparation process, an animal immunization experiment design was used to verify their effects of shortening the antibody production cycle and improving the antibody titer. The following are the specific preparation steps and experimental data.

[0100] I. Experimental materials

[0101] 1. Experimental animals and materials

[0102] Experimental animals: 6 - 8 - week - old C57BL / 6 mice, 10 mice in each group.

[0103] Grouping: There are a total of 6 experimental groups and 1 commercial adjuvant control group. The adjuvants used in each group are as follows:

[0104] No - adjuvant group: Do not use any adjuvant;

[0105] Example 2 group: Polygonatum odoratum polysaccharide (POP);

[0106] Example 3 group: Rosa roxburghii tratt polysaccharide (RRP);

[0107] Example 4 group: Arctium lappa polysaccharide (ALP);

[0108] Example 5 group: Polygonatum odoratum polysaccharide + rosa roxburghii tratt polysaccharide (POP + RRP);

[0109] Example 6 group: Arctium lappa polysaccharide + Rosa roxburghii Tratt polysaccharide (ALP + RRP);

[0110] Example 7 group: Polygonatum odoratum polysaccharide + Rosa roxburghii Tratt polysaccharide + Arctium lappa polysaccharide (POP + RRP + ALP);

[0111] Commercially available adjuvant group: The commonly used Freund's Adjuvant on the market was used as the control group.

[0112] APC-labeled anti-CD3, FITC-labeled anti-CD4, PE-labeled anti-CD8.

[0113] 2. Antigen selection

[0114] Antigen: Ovalbumin (OVA) was used as the model antigen, and the target of the antibody was anti-OVA antibody.

[0115] 3. Primary immunization: Each immunoadjuvant was mixed with OVA antigen (50 μg / mouse) at a ratio of 1:1 and then subcutaneously injected into mice (100 μL / mouse).

[0116] Booster immunization: The first booster immunization was carried out on the 14th day after the primary immunization, and the dose was the same as that of the primary immunization.

[0117] Second booster immunization: The second booster immunization was carried out on the 28th day, and the dose was the same as that of the primary immunization.

[0118] Sample collection: Mouse serum was collected by orbital puncture on the 7th day after each immunization for subsequent analysis of antibody titer and immune response.

[0119] II. Experimental detection

[0120] 1. Detection of the contents of CD4+ and CD8+ in mouse spleen T cells by flow cytometry

[0121] 1.1 Preparation of mouse spleen cells

[0122] Spleen tissues were taken from experimental mice, ground using a cell filter, and then washed with 20 mL of RPMI-1640 medium. The washed cell suspension was centrifuged at 1500 rpm for 5 minutes in a centrifuge.

[0123] 1.2 Staining of cell surface markers

[0124] The cell pellet was collected and washed twice with PBS. Subsequently, the cell samples were stained with CD3, CD4, and CD8 antibodies and incubated for 30 minutes in the dark.

[0125] 1.3 Lysis treatment of red blood cells

[0126] Add 2 mL of red blood cell lysate to the cell suspension, react for 5 minutes in the dark at room temperature, then centrifuge at 1500 rpm for 5 minutes, and discard the supernatant. After adding 1 mL of PBS and mixing well, centrifuge again at 1500 rpm for 5 minutes and discard the supernatant. Finally, resuspend the cell pellet in 400 uL of PBS for flow cytometry detection.

[0127] 1.4 Flow cytometry detection

[0128] Use flow cytometry to detect the contents of CD4+ and CD8+ in mouse spleen T cells, and then analyze the antibody titer and immune response.

[0129] 1.5 Detection results

[0130] Figure 3 and Figure 4 are the detection results of cellular immune response. Table 3 shows the specific data of the detection results of cellular immune response.

[0131] Table 3

[0132] Group CD4+ T cells (%) CD8+ T cells (%) Adjuvant-free group 11.1±1.5 12.1±1.5 Example 2 (POP) 30.0±2.3 21.3±1.3 Example 3 (RRP) 30.9±2.2 19.1±0.7 Example 4 (ALP) 38.1±1.8 26.0±0.6 Example 5 (POP + RRP) 45.7±2.7 29.3±1.1 Example 6 (ALP + RRP) 47.0±1.6 32.9±1.6 Example 7 (POP + RRP + ALP) 49.8±2.3 40.4±2.0 Commercially available adjuvant control group 42.5±2.1 34.3±1.2

[0133] According to Table 3 and Figure 3 - 4 it can be seen that compared with the adjuvant-free group, all the example groups can significantly increase the contents of CD4+ T cells and CD8+ T cells in the mouse spleen. Among them, the example 7 group (POP+RRP+ALP) induced the highest contents of CD4+ T cells and CD8+ T cells, which were increased by about 353% and 234% respectively compared with the adjuvant-free group. The example 6 group (ALP+RRP) and the example 5 group (POP+RRP) also showed relatively high T cell contents, but were slightly lower than the example 7 group. Although the commercially available adjuvant control group could also increase the T cell content, the effect was not as good as that of the example 7 group, the example 6 group and the example 5 group. Figure 3 and Figure 4 intuitively show the differences in T cell contents among different groups, further confirming the reliability of the experimental results. These results provide important experimental basis for subsequent evaluation of the application of high-performance immune adjuvants in enhancing cellular immune response, indicating that the immune adjuvant combination of the present invention has significant immune enhancement effect and is expected to provide new strategies for vaccine research and development and therapeutic antibody production.

[0134] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the above examples, those of ordinary skill in the art should understand that: modifications or equivalent replacements can still be made to the specific embodiments of the present invention, and any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the protection scope of the claims of the present invention.

Claims

1. A preparation method of a high-efficiency immune adjuvant, characterized in that, Comprising: Combining plant polysaccharides with chitosan nanoparticles to obtain plant polysaccharide-chitosan nanoparticles; Using liposomes to encapsulate the plant polysaccharide-chitosan nanoparticles to obtain the high-performance immune adjuvant; Wherein, the plant polysaccharide is one or more of arctium lappa polysaccharide, polygonatum odoratum polysaccharide and rosa roxburghii tratt polysaccharide.

2. The preparation method of the high-performance immune adjuvant according to claim 1, wherein The preparation method of the chitosan nanoparticles comprises: Dissolving chitosan in an acetic acid solution and preparing the chitosan nanoparticles by an ionic gelation method.

3. The preparation method of the high-efficiency immune adjuvant according to claim 1, wherein When combining plant polysaccharides with chitosan nanoparticles to obtain plant polysaccharide-chitosan nanoparticles, it includes: Under the action of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, combining arctium lappa polysaccharide with chitosan nanoparticles through the reaction of amino group and carboxyl group to obtain the plant polysaccharide-chitosan nanoparticles.

4. A high-performance immune adjuvant, characterized in that, Prepared according to the preparation method of the high-performance immune adjuvant described in any one of claims 1-3.

5. Use of the high-efficiency immune adjuvant according to claim 4, characterized in that, The application at least includes one of the following: A) The application of the high-performance immune adjuvant in the preparation of antibodies; A) The application of the high-performance immune adjuvant in shortening the antibody generation cycle.

6. A product for shortening the antibody generation cycle, characterized in that, The active ingredient of the product is the high-performance immune adjuvant described in claim 4.

7. A vaccine composition, characterized in that, The vaccine composition comprises an antigen and the high-performance immune adjuvant described in claim 4.

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