Application of Macranthoidin B in Vaccine Adjuvants

Abstract

The application of Macranthoidin B in vaccine adjuvant relates to the application of Macranthoidin B and belongs to the technical field of biological medicine. The primary purpose of the application is to provide a novel use of Macranthoidin B, i.e. the application in the preparation of vaccine adjuvant. The application of Macranthoidin B in the preparation of vaccine adjuvant is provided. The Macranthoidin B is used as an active ingredient of a vaccine adjuvant in a vaccine composition. When used in combination with a protein antigen (ovalbumin), Macranthoidin B can induce an adaptive immune response which is significantly superior to benchmark adjuvants QS-21 and R848, including higher levels of antigen-specific IgG, more antigen-specific memory B cells and stronger antigen-specific T cell responses. Meanwhile, Macranthoidin B does not cause significant liver toxicity at an effective dose, and has better safety. The application provides a new candidate compound for the development of efficient and safe vaccine adjuvants.

Description

Technical Field

[0001] This invention relates to the application of Macronthoidin B and belongs to the field of biomedical technology. Background Technology

[0002] Vaccines are one of the most effective means of preventing and controlling infectious diseases. Adjuvants are a key component of vaccines, used to enhance and modulate the immune response against antigens, thereby reducing the amount of antigen used, improving immunization efficacy, and prolonging the duration of protection. Currently, the types of adjuvants approved for human vaccines are limited, mainly including aluminum adjuvants, MF59, the AS01 series (including QS-21), and AS04. Among them, saponin QS-21, derived from the soap tree, is used in blockbuster products such as the shingles vaccine Shingrix and the malaria vaccine Mosquirix due to its ability to strongly stimulate cellular and humoral immunity.

[0003] However, QS-21 has significant limitations. First, it possesses potential hemolytic activity and injection site reactions. Second, studies have shown that even a single low-dose injection (e.g., 5 μg) can cause a significant increase in serum transaminase (ALT / AST) levels within a short period, indicating a risk of hepatotoxicity (see experimental data in this invention), which limits its wider application. To overcome the toxicity of QS-21, it is usually necessary to combine it with other components (such as liposomes and MPL) to form a complex adjuvant system (such as AS01), which increases the complexity and cost of the process. Therefore, developing novel saponin adjuvants that combine potent immunostimulatory activity with good safety has become an important research direction in the vaccine field. Summary of the Invention

[0004] The primary objective of this invention is to provide a novel use for Macronthoidin B, namely, its application in the preparation of vaccine adjuvants. An application of Macronthoidin B in the preparation of vaccine adjuvants is provided.

[0005] This invention is expected to address the hepatotoxicity risk associated with saponin QS-21.

[0006] Another object of the present invention is to provide a safe and effective vaccine composition comprising Macronthoidin B.

[0007] The use of Macranthoidin B in vaccine adjuvants, wherein Macranthoidin B is the active ingredient in the vaccine composition as a vaccine adjuvant.

[0008] The macronthoidin B is used to enhance the innate and / or adaptive immune responses elicited by the vaccine.

[0009] The enhanced innate immune response is manifested as the aggregation and / or activation of dendritic cells, monocytes and / or neutrophils in draining lymph nodes.

[0010] The activation is manifested by the upregulation of one or more of the cell surface co-stimulatory molecules CD40, CD80, CD86, and major histocompatibility complex class II molecules.

[0011] The major histocompatibility complex II molecule is an MHC-II molecule.

[0012] The Macronthoidin B, when used, exhibits a lower risk of hepatotoxicity than QS-21.

[0013] The enhanced adaptive immune response manifests itself in one or more of the following ways:

[0014] 1) Significantly increases the level of antigen-specific IgG antibodies;

[0015] 2) Induces the production of memory B cells with higher frequency of antigen specificity and class switching;

[0016] 3) Activate a greater number of antigen-specific T cells and promote their secretion of IL-2 cytokine.

[0017] The active ingredient of Macronthoidin B, used as a vaccine adjuvant, is formulated together with the protein antigen to form a vaccine composition.

[0018] The protein antigen is ovalbumin.

[0019] The vaccine composition comprises an immunologically effective amount of 1 μg of protein antigen and an immunologically effective amount of 1 μg of Macranthoidin B as a vaccine adjuvant.

[0020] The vaccine composition is administered via intramuscular injection.

[0021] This invention provides the first systematic evaluation of the immunoadjuvant effects of macronthoidin B. When used in combination with a protein antigen (ovalbumin), macronthoidin B induces an adaptive immune response significantly superior to the baseline adjuvants QS-21 and R848, including higher levels of antigen-specific IgG, more antigen-specific memory B cells, and a stronger antigen-specific T cell response. Simultaneously, it exhibits a lower risk of hepatotoxicity than QS-21. It not only possesses immunostimulatory capabilities superior to or equivalent to QS-21 but also demonstrates significant advantages in terms of safety.

[0022] Compared with the prior art, the present invention has the following significant advantages and positive effects:

[0023] 1. Powerful innate immune activation: Macranthoidin B can effectively mobilize and activate dendritic cells, monocytes and neutrophils, especially significantly upregulating CD40, CD80, CD86 and MHC-II molecules on the surface of these cells, laying a solid foundation for the subsequent initiation of a strong adaptive immune response.

[0024] 2. Comprehensive and powerful adaptive immune induction capacity: In the OVA protein vaccine model, the level of antigen-specific IgG, the frequency of memory B cells, and the number of IL-2 secreting T cells induced by the Macronthoidin B adjuvant group were significantly higher than those induced by the QS-21 and TLR7 / 8 agonist R848 adjuvant groups, indicating that it can induce balanced and powerful humoral and cellular immunity.

[0025] 3. Excellent safety profile: A single injection of Macronthoidin B did not cause a significant increase in serum ALT and AST, while the same dose of QS-21 led to a significant increase in transaminase levels. This indicates that Macronthoidin B has a superior safety window compared to QS-21, providing important assurance for its clinical application.

[0026] 4. A potential alternative to QS-21 is provided: Macronthoidin B, as a novel saponin adjuvant, has shown advantages in both efficacy and safety, and is expected to replace or partially replace QS-21 for the development of a new generation of highly effective and safe vaccines, especially those that require a strong T-cell immune response (such as antiviral and antitumor vaccines). Attached Figure Description

[0027] Figure 1 This is a frequency analysis diagram of innate immune cell subsets in the drainage lymph nodes at the injection site in Example 1. In the diagram, A represents the flow cytometry gating strategy for dendritic cells, monocytes, and neutrophils; B represents the frequency of dendritic cells in the drainage lymph nodes of each group of mice; C represents the frequency of monocytes in the drainage lymph nodes of each group of mice; and D represents the frequency of neutrophils in the drainage lymph nodes of each group of mice.

[0028] Figure 2This is a comparison chart of the innate immunity assessment of the vaccine adjuvant Macronthoidin B and the baseline adjuvant in Example 1. In the figure, A represents the expression levels of CD40, CD80, CD86 and MHC-II molecules on the surface of dendritic cells in the draining lymph nodes as detected by flow cytometry (expressed as mean fluorescence intensity, MFI); B represents the expression levels of CD40, CD80, CD86 and MHC-II molecules on the surface of draining monocytes as detected by flow cytometry (expressed as mean fluorescence intensity, MFI); and C represents the expression levels of CD40, CD80, CD86 and MHC-II molecules on the surface of draining neutrophils as detected by flow cytometry (expressed as mean fluorescence intensity, MFI).

[0029] Figure 3 This is a comparison chart of the hepatotoxicity assessment of the vaccine adjuvant Macronthoidin B and the baseline adjuvant in Example 2. In the figure, A represents the level of ALT (A) in the serum of mice 12 hours after injection, and B represents the level of AST (B) in the serum of mice 12 hours after injection.

[0030] Figure 4 This is a comparison chart of the humoral immune response elicited by the vaccine adjuvant Macronthoidin B and the baseline adjuvant in OVA vaccine in Example 2. In the figure, A represents the dynamic change of anti-OVA specific IgG in serum after three immunizations, B represents the flow cytometry gating strategy of OVA specific memory B cells, and C represents the frequency of OVA specific memory B cells in the spleen.

[0031] Figure 5 A comparative evaluation chart of antigen-specific T-cell immune responses induced by the vaccine adjuvant Macronthoidin B and the baseline adjuvant in OVA vaccines. In the chart, A represents a dot plot of the number of OVA-specific IL-2 secreting T cells in the spleen detected by ELISpot, and B represents a statistical plot of the number of OVA-specific IL-2 secreting T cells in the spleen detected by ELISpot. Detailed Implementation

[0032] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0033] Specific Implementation Method 1: The application of Macranthoidin B in vaccine adjuvants in this implementation method, wherein Macranthoidin B is used as the active ingredient of vaccine adjuvant in the vaccine composition.

[0034] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that the Macronthoidin B is used to enhance the innate and / or adaptive immune responses elicited by the vaccine. Everything else is the same as in Specific Implementation Method One.

[0035] Specific Implementation Method Three: This implementation method differs from Specific Implementation Method One or Two in that the enhanced innate immune response manifests as the aggregation and / or activation of dendritic cells, monocytes, and / or neutrophils in the draining lymph nodes. Everything else is the same as in Specific Implementation Method One or Two.

[0036] Specific Implementation Method Four: This implementation method differs from Specific Implementation Methods One to Three in that the activation is manifested by the upregulation of one or more of the cell surface co-stimulatory molecules CD40, CD80, CD86, and major histocompatibility complex II molecules. Everything else is the same as in Specific Implementation Methods One to Three.

[0037] Specific Implementation Method Five: This implementation method differs from Specific Implementation Methods One to Four in that the major histocompatibility complex II molecule is an MHC-II molecule. Everything else is the same as in Specific Implementation Methods One to Four.

[0038] Specific Implementation Method Six: This implementation method differs from one of Specific Implementation Methods One to Five in that the enhancement of the adaptive immune response manifests in one or more of the following ways:

[0039] (a) Increased titer of antigen-specific antibodies;

[0040] (b) An increase in the number of antigen-specific memory B cells;

[0041] (c) An increase in the number of antigen-specific T cells and / or an enhanced capacity for cytokine secretion. Other aspects are the same as in any one of embodiments one through five.

[0042] Specific Implementation Method Seven: This implementation method differs from one of Specific Implementation Methods One to Six in that the enhancement of the adaptive immune response manifests in one or more of the following ways:

[0043] 1) Significantly increases the level of antigen-specific IgG antibodies;

[0044] 2) Induces the production of memory B cells with higher frequency of antigen specificity and class switching;

[0045] 3) Activate a greater number of antigen-specific T cells and promote their secretion of IL-2 cytokine. Other aspects are the same as in any of the specific implementation methods one through six.

[0046] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Methods One to Seven in that the active ingredient of Macronthoidin B as a vaccine adjuvant is formulated together with the protein antigen to form a vaccine composition. Everything else is the same as in Specific Implementation Methods One to Seven.

[0047] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Methods One to Eight in that the protein antigen is ovalbumin. Everything else is the same as in Specific Implementation Methods One to Eight.

[0048] Specific Embodiment Ten: This embodiment differs from Specific Embodiments One to Nine in that the vaccine composition contains an immunologically effective amount of 1 μg of protein antigen and an effective amount of 1 μg of macronthoidin B as a vaccine adjuvant. Everything else is the same as in Specific Embodiments One to Nine.

[0049] The effects of the present invention were verified using the following embodiments:

[0050] Example 1: Activation effect of Macronthoidin B on the innate immune system

[0051] 1. Experimental Materials

[0052] C57BL / 6 mice (6-8 weeks old, female, purchased from GemPharmatech); Dipsacoside B, Macranthoidin A, Macranthoidin B (all with a purity >98%); QS-21 (purchased from AbMole, 141256-04-4); TLR7 / 8 agonist R848 (purchased from InvivoGen, 144875-48-9); PBS buffer (purchased from Biosharp, BL2218); flow cytometry antibodies are listed in Table 1; ALT / AST detection ELISA kit (Nanjing Jiancheng, C010-2-1 / C009-2-1).

[0053] Table 1. Antibodies used in flow cytometry experiments

[0054] Antibody name Cloned ID brand IgM RMM-1 Biolegend IgD 11-26c.2a Biolegend CD19 6D5 Biolegend CD45R / B220 RA3-6B2 Biolegend MHC-II M5-114.15.2 Ebioscience CD11b M1 / 70 Ebioscience CD11c N418 Ebioscience Ly6C HK1.4 Biolegend Ly6G IA8 Ebioscience CD40 3 / 23 BD CD80 16-10A1 Ebioscience CD86 GL1 Ebioscience

[0055] 2. Experimental Methods

[0056] (1) Animal grouping and treatment: Mice were randomly divided into several groups (n=5) and injected intramuscularly with PBS, 1 μg or 5 μg of Dipsacoside B, Macronthoidin A, Macronthoidin B, 1 μg of QS-21, or 25 μg of R848, respectively. Serum and draining lymph nodes (dLNs) at the injection site were collected 12 hours after injection.

[0057] (2) Hepatotoxicity assessment: The concentrations of ALT and AST in serum were detected using a commercially available ELISA kit. The procedure is as follows:

[0058] 1) Preparation of standard curve: Add phosphate buffer, sodium pyruvate standard solution, matrix solution, DNPH solution and sodium hydroxide solution respectively according to the table below (Table 2).

[0059] Table 2

[0060] Kong Hao 0 1 2 3 4 5 0.1 mol / L phosphate buffer (μL) 5 5 5 5 5 5 2 μmol / mL sodium pyruvate standard solution (μL) 0 2 4 6 8 10 Matrix buffer (μL) 20 18 16 14 12 10 2,4-Dinitrophenylhydrazine solution (μL) 20 20 20 20 20 20 Mix well and react at 37°C for 20 minutes. 0.4 mol / L NaOH (μL) 200 200 200 200 200 200

[0061] After reacting at 37℃, the mixture was allowed to stand at room temperature for 15 minutes, and the OD value (510 nm) of each well was measured.

[0062] A standard curve was fitted with the absolute OD value (minus the zero OD value) as the x-axis and the KAMN units as the y-axis.

[0063] 2) Sample determination

[0064] Follow the sample addition sequence below (unit: μL), mix well, let stand at room temperature for 15 minutes, and measure the 510nm OD value.

[0065] Table 3

[0066] step Measurement well (μL) Control well (μL) Matrix solution (preheated to 37℃) 20 20 Serum to be tested 5 — Gently shake to mix, react at 37°C for 30 minutes. 2,4-Dinitrophenylhydrazine solution 20 20 Serum to be tested — 5 Gently shake to mix, react at 37°C for 20 minutes. 0.4 mol / L NaOH 200 200

[0067] Read the OD value of each well and calculate the absolute OD value (OD value of the measured well - OD value of the control well).

[0068] 3) Plot the standard curve and calculate the absolute OD value of each standard hole (subtract the OD value of the 0 hole).

[0069] The absolute OD value is used as the x-axis, and the corresponding Kármán units are used as the y-axis (the corresponding values ​​of the ALT / AST standard curve are shown in the appendix of the instruction manual).

[0070] The calculation formula was obtained by performing polynomial fitting (R² ≥ 0.99) using Excel.

[0071] 4) Result Calculation

[0072] 1. Find the corresponding Kamen unit from the standard curve based on the absolute OD value of the sample.

[0073] 2. Formula for calculating serum ALT / AST activity:

[0074] ALT / AST activity (U / L) = Kamen's units × 0.482 × N

[0075] in:

[0076] 0.482: Conversion factor between Kamen units and International Units (U / L)

[0077] N: Sample dilution factor (1 if undiluted)

[0078] (3) Flow cytometry analysis: Drained lymph node tissue was gently ground and filtered through a 70-micron sterile cell filter. Cells were resuspended in PBS and centrifuged at 400 g for 10 minutes to obtain isolated monocytes. Cells were then resuspended in PBS for subsequent in vitro experiments. Dead cells were removed by staining with Fixed Viability Dye eFluor™ 506, followed by Fc receptor blocking and staining with a mixture of antibodies targeting different cell surface markers. Data were acquired using a BD LSRFortessa flow cytometer and analyzed using FlowJo V.10.1 software. Dendritic cells, monocytes, and neutrophils were identified using a gating strategy, and the frequencies and mean fluorescence intensities of their surface CD40, CD80, CD86, and MHC-II molecules were analyzed.

[0079] 3. Experimental Results

[0080] (1) Cell recruitment: All adjuvants caused a dose-dependent increase in the frequency of dendritic cells and monocytes in dLNs ( Figure 1 (BD). Among them, QS-21 and R848 showed the strongest efficacy in inducing cell recruitment.

[0081] (2) Cell activation: Although slightly inferior to QS-21 and R848 in cell recruitment, Macronthoidin B excels in activating antigen-presenting cells. For example... Figure 2 As shown in AC, the expression levels of CD40, CD80, CD86, and MHC-II molecules (MFI) in the macronoidin B treatment group (especially the 1 μg dose group) were significantly higher than or comparable to those in the QS-21 group in dendritic cells, monocytes, and neutrophils. In particular, macronoidin B showed a significant activating effect on monocytes and neutrophils. These data indicate that macronoidin B is a highly potent innate immunostimulant capable of creating an immune microenvironment favorable for T cell initiation.

[0082] (3) Safety: such as Figure 1As shown in Figure B, compared with the PBS control group, mice injected with 5 μg QS-21 showed significantly elevated serum ALT and AST levels, while mice injected with 1 μg or 5 μg Macranthoidin B, Macranthoidin A, Dipsacoside B, or R848 showed no significant changes in serum transaminase levels. This indicates that Macranthoidin B did not exhibit significant hepatotoxicity at the experimental dose.

[0083] Example 2: The effect of Macronthoidin B in inducing adaptive immune responses in protein vaccines

[0084] 1. Experimental Materials

[0085] Ovalbumin (purchased from Sigma, A5503), IgG antibody (purchased from Proteintech, SA00001-1); ELISpot kit (purchased from MabTech, 3441-4APW), erythrocyte lysis buffer (Solarbio, R1010), and other materials were the same as in Example 1.

[0086] 2. Experimental Methods

[0087] (1) Vaccine preparation and immunization: Ovalbumin (OVA protein) (1 μg) was mixed with Macronthoidin B (1 μg), QS-21 (1 μg) or R848 (25 μg) to prepare vaccine formulations. C57BL / 6 mice (n=5 / group) were immunized by intramuscular injection for a total of three times, with each immunization two weeks apart.

[0088] (2) Sample collection: Serum was collected 7 days after the first immunization (Day 7), 7 days after the first booster immunization (Day 21), and 14 days after the last immunization (Day 35). 14 days after the last immunization, mice were sacrificed and spleens were collected.

[0089] (3) Antibody reaction detection: The level of anti-OVA specific IgG antibody in serum was detected by ELISA.

[0090] (4) Memory B cell detection: Spleen tissue was gently ground and filtered through a 70 μm sterile cell filter. Cells were resuspended in PBS and centrifuged at 400 g for 10 minutes. The cell pellet was resuspended in erythrocyte lysis buffer and incubated at 4°C for 5 minutes to lyse erythrocytes. The lysis process was then terminated with 1×PBS, followed by washing the cells at 400 g for 5 minutes to separate splenic monocytes. Cells were then resuspended in PBS for subsequent in vitro experiments. OVA-specific B cells were labeled using a fluorescent probe prepared from biotinylated OVA and streptavidin-BV421 / APC. By flow cytometry, class-switched B cells gated to IgD-IgM-CD19+ were further screened, and cells that were double-negative for OVA-BV421 and OVA-APC (excluding non-specific binding) but positive for either channel were defined as OVA-specific memory B cells.

[0091] (5) T cell response detection: Splenic cells were isolated (specific method as above), and analyzed at 1×10⁻⁶ cells. 5 Cells were seeded in wells and stimulated with OVA peptide (10 μg / ml) for 20 h. IL-2 secreting cells were detected using a commercial ELISpot kit, and results are expressed as the number of spot-forming cells per million cells.

[0092] 3. Experimental Results

[0093] (1) Antibody reaction: such as Figure 4 As shown in Figure A, low levels of anti-OVA IgG were detectable in the Macronthoidin B and R848 adjuvant groups 7 days after the first booster immunization (Day 21), while no detectable antibodies were induced in the QS-21 group at this time. After three immunizations (Day 35), the antibody levels induced by the Macronthoidin B adjuvant group were significantly higher than those in the QS-21 and R848 groups, indicating that it can induce a stronger and potentially faster humoral immune response.

[0094] (2) Memory B cell response: such as Figure 4 As shown in Figure C, the frequency of OVA-specific memory B cells in the spleen of mice in the OVA-Macranthoidin B vaccine group was the highest, higher than that in the OVA-QS-21 and OVA-R848 groups. This suggests that Macronthoidin B can induce more durable humoral immunity, providing a basis for long-term protection.

[0095] (3) T cell response: ELISpot results showed ( Figure 5In mice in the OVA-Macranthoidin B group (AB), the number of OVA-specific T cells secreting IL-2 was the highest, significantly higher than in the other two groups, especially the R848 group. This indicates that Macronthoidin B can effectively stimulate strong T-cell immunity, which is crucial for clearing intracellular pathogens and tumor cells.

[0096] in conclusion

[0097] This invention demonstrates through systematic in vivo experiments that the natural saponin compound Macronthoidin B is a highly effective and safe vaccine adjuvant candidate. It powerfully activates the innate immune system and induces a comprehensive, balanced, and robust adaptive immune response, including high levels of specific antibodies, a large number of memory B cells, and activated T cells. Furthermore, it exhibits superior safety compared to QS-21 under experimental conditions. Therefore, Macronthoidin B has significant potential for the development of novel preventative or therapeutic vaccines.

Claims

1. The application of Macronthoidin B alone as an adjuvant for low-hepatotoxic protein vaccines, characterized in that... Macronthoidin B is the active ingredient in the vaccine adjuvant of the low hepatotoxicity protein vaccine composition.

2. The application of Macronthoidin B as a low-hepatotoxicity protein vaccine adjuvant according to claim 1, characterized in that... The macronthoidin B is used to enhance the innate and / or adaptive immune responses elicited by the vaccine.

3. The application of Macronthoidin B as a low-hepatotoxicity protein vaccine adjuvant according to claim 2, characterized in that... The enhanced innate immune response is manifested as the aggregation and / or activation of dendritic cells, monocytes and / or neutrophils in draining lymph nodes.

4. The application of Macronthoidin B as a single adjuvant for a low-hepatotoxicity protein vaccine according to claim 3, characterized in that... The activation is manifested by the upregulation of one or more of the cell surface co-stimulatory molecules CD40, CD80, CD86, and major histocompatibility complex class II molecules.

5. The application of Macronthoidin B as a single adjuvant for a low-hepatotoxicity protein vaccine according to claim 4, characterized in that... The major histocompatibility complex II molecule is an MHC-II molecule.

6. The application of Macronthoidin B as a low-hepatotoxicity protein vaccine adjuvant according to claim 2, characterized in that... The enhanced adaptive immune response manifests itself in one or more of the following ways: (a) Increase in antigen-specific antibody titers; (b) An increase in the number of antigen-specific memory B cells; (c) An increase in the number of antigen-specific T cells and / or an enhanced ability to secrete cytokines.

7. The application of Macronthoidin B as a low-hepatotoxicity protein vaccine adjuvant according to claim 2, characterized in that... The enhanced adaptive immune response manifests itself in one or more of the following ways: 1) Significantly increases the level of antigen-specific IgG antibodies; 2) Induces the production of memory B cells with higher frequency of antigen specificity and class switching; 3) Activate a greater number of antigen-specific T cells and promote their secretion of IL-2 cytokine.

8. The application of Macronthoidin B as a low-hepatotoxicity protein vaccine adjuvant according to claim 1, characterized in that... The active ingredient of Macronthoidin B, used as a vaccine adjuvant, is formulated together with the protein antigen to form a vaccine composition.

9. The application of Macronthoidin B as a low-hepatotoxicity protein vaccine adjuvant according to claim 8, characterized in that... The protein antigen is ovalbumin.

10. The application of Macronthoidin B as a single adjuvant for a low-hepatotoxicity protein vaccine according to claim 8, characterized in that... The vaccine composition comprises an immunologically effective amount of 1 μg of protein antigen and an immunologically effective amount of 1 μg of Macranthoidin B as a vaccine adjuvant.

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