Immunologic adjuvant composition and application thereof

Abstract

The invention relates to the technical field of immunologic adjuvants, in particular to an immunologic adjuvant composition and application thereof. The immunologic adjuvant composition disclosed by the invention comprises a TLR9 (toll-like receptor 9) agonist CpG-ODN and simvastatin. The invention finds that under the condition that simvastatin is combined with CpG-ODN, the immune intensity can be improved under the condition that the immune quality of the CpG-ODN is not changed, simvastatin can enhance innate immune activation caused by the CpG-ODN, but other mevalonic acid inhibitors (such as mevastatin and atorvastatin) do not have the effect.

Description

Technical Field

[0001] The present invention relates to the technical field of immune adjuvants, and in particular to an immune adjuvant composition and its application, to an adjuvant activity enhancer for improving the immune strength of existing TLR adjuvants and an immune adjuvant composition containing the adjuvant activity enhancer, and in particular to the application of the immune adjuvant composition in preparing vaccines. Background Art

[0002] CpG is a characteristic sequence in DNA, which refers to a specific arrangement of cytosine (C) and guanine (G) connected by phosphodiester bonds in DNA. CpG sequences are usually found in "enriched regions" in the genome and play important biological functions in many organisms. At the same time, it is a TLR9 agonist that activates the immune system and promotes the production of cytokines. This makes CpG sequences a focus of attention in vaccine development and immunotherapy. TLR3 agonist Poly(I:C), TLR7 / 8 agonist R848, and TLR1 / 2 agonist Pam3 are also worth studying in immunology.

[0003] Simvastatin is a commonly used lipid-lowering drug that mainly reduces low-density lipoprotein cholesterol (LDL-C) and total cholesterol in the blood by inhibiting the activity of hydroxymethylglutaryl CoA reductase (HMG-CoA reductase) in the liver to reduce the risk of cardiovascular diseases such as heart disease and stroke.

[0004] Previous studies have shown that the mevalonate pathway can enhance the adjuvant effect, but found that it is not related to inflammatory factors. Patients with gene deletions in the mevalonate pathway have severe inflammatory factors, and studies have shown that in mice with mevalonate pathway defects, the TLR4 agonist LPS causes higher inflammatory factors, but different Toll-like receptors initiate immune responses by recognizing different types of pathogen-associated molecular patterns, and their downstream signal transduction and activated transcription factors often differ. TLR4 focuses on bacterial infections, while TLR9 plays an important role in viral infections. At the same time, there is no research on the combination of the mevalonate pathway inhibitor simvastatin and the TLR9 agonist as a vaccine adjuvant. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide an immune adjuvant composition and its application in view of the deficiencies in the prior art.

[0006] In order to solve the above technical problems, the present invention discloses an immune adjuvant composition and its application. The specific technical scheme is as follows:

[0007] An immune adjuvant composition comprises a TLRs agonist and simvastatin.

[0008] Wherein, the TLRs agonist is any one of a TLR1 agonist, a TLR2 agonist, a TLR7 agonist, a TLR8 agonist or a TLR9 agonist, preferably a TLR9 agonist.

[0009] Wherein, the TLR9 agonist is CpG-ODN, preferably CpG-ODN1826.

[0010] Wherein, the molar ratio of the TLRs agonist to the simvastatin is 1-30:20. Preferably, the molar ratio of the TLR9 agonist to the simvastatin is 1-30:20.

[0011] Preferably, the immune adjuvant composition further comprises a solvent, and the solvent is physiological saline.

[0012] Further preferably, the immune adjuvant composition is prepared according to the following method: dissolving simvastatin with DMSO to obtain a simvastatin solution, dissolving CpG-ODN with PBS to obtain a CpG solution, and mixing the simvastatin solution and the CpG solution in physiological saline according to a ratio.

[0013] In a second aspect, the present invention provides use of the immune adjuvant composition described in the first aspect in preparing a vaccine.

[0014] Among them, the vaccine is the new crown vaccine.

[0015] Among them, the antigen of the new coronavirus vaccine is RBD.

[0016] In a third aspect, the present invention provides a use of the immune adjuvant composition described in the first aspect in the preparation of a drug for treating an immune disease, wherein the immune disease is preferably a pulmonary immune disease.

[0017] Beneficial effects:

[0018] The present invention combines a variety of TLRs agonists with the mevalonate pathway inhibitor simvastatin (or mevastatin, or atorvastatin) to study whether the adjuvant activity of the TLRs agonists can be enhanced, and speculates that inhibiting mevalonate can promote the activation of TLRs and enhance the adjuvant activity of TLRs, which provides a certain theoretical basis for high antibodies and cytokines in genetic patients and provides a basis for the combined application of statin adjuvants and TLRs adjuvants.

[0019] The present invention utilizes TLR9 agonist and simvastatin to form an immune adjuvant composition, which can enhance the innate immunity of the lungs, have higher antibody titers, increase the content of pro-inflammatory cytokines, enhance the activation of lymph node germinal centers, enhance the activation of immune cells such as T cells and dendritic cells, and have the ability to reside, thereby having long-lasting immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0021] Figure 1 This is a diagram showing the expression of pro-inflammatory factors after different Toll-like receptor agonists combined with simvastatin. Figure 1 A in the figure is the expression of pro-inflammatory factor IL-6. Figure 1 B in the figure shows the expression of pro-inflammatory factor TNF. **p<0.01, ***p<0.001.

[0022] Figure 2 The effects of different concentrations of CpG combined with different statins on IL-6 in MH-S cells. Figure 2 A in is simvastatin, Figure 2 B in is mevastatin, Figure 2 C in the figure represents atorvastatin, compared with the control group, **p<0.01, ***p<0.001.

[0023] Figure 3 To investigate the effects of simvastatin combined with CPG on proinflammatory factors in MH-S cells. Figure 3 The A in the expression is the pro-inflammatory factor IL-6. Figure 3 B in the expression is IL-1β. Figure 3 C in the figure represents TNF-α, compared with the control group, *p<0.05, **p<0.01, ***p<0.001.

[0024] Figure 4 The effect of simvastatin combined with CPG on chemokines of MH-S cells. Figure 4 A in the figure is the chemokine CCL3. Figure 4 B in the figure is the chemokine CCL5. Figure 4 C in the figure is chemokine Cxcl10. Compared with the control group, **p<0.01, ***p<0.001.

[0025] Figure 5 Analysis of transcriptome sequencing results of MH-S cells. Figure 5 A in is PCA analysis, Figure 5 B in the figure is the volcano plot comparison result between the control group and the simvastatin group. Figure 5 C in the figure is the GO enrichment analysis result of the control group and the simvastatin group. Figure 5 D in the figure is the KEGG enrichment analysis result of the control group and the simvastatin group. Figure 5 E in the figure is the comparison result of PI3K-AKT signaling pathway genes between the control group and the simvastatin group. Figure 5F in the figure is the comparison result of Toll-like receptor signaling pathway genes between the CpG group and the simvastatin + CpG group.

[0026] Figure 6 It is the content of immunoglobulin in bronchoalveolar lavage fluid and serum. Figure 6 A in the figure is IgA in bronchoalveolar lavage fluid after the first immunization. Figure 6 B in the figure is the IgG in the bronchoalveolar lavage fluid of the first immunization. Figure 6 C in the figure is IgG2a in the first immune serum. Figure 6 D in the figure is the IgG in the first immune serum. Figure 6 E in the figure is IgA in bronchoalveolar lavage fluid after the second immunization. Figure 6 F in the figure is IgG in bronchoalveolar lavage fluid after the second immunization. Figure 6 G in the figure is the comparison of IgA in bronchoalveolar lavage fluid after the second immunization with that in the first immunization. Figure 6 H in the figure is the comparison of IgG in bronchoalveolar lavage fluid after the second immunization with that in the first immunization. *p<0.05, **p<0.01, ***p<0.001.

[0027] Figure 7 Immunofluorescence sections and flow cytometry results of cervical lymph nodes. Figure 7 A in the figure is the immunofluorescence section result of cervical lymph nodes. Figure 7 B in the figure is the flow cytometry result of cells expressing B220 and FAS. ***p<0.001.

[0028] Figure 8 The results of flow cytometry are shown in Figure 2. Figure 8 A in the figure represents the expression of CD8 + 、CD44 + 、CD45 + 、CD103 + cells, Figure 8 B in the figure expresses CD45 + 、CD4 + and CD103 + cells, Figure 8 C in the figure represents the expression of CD45 + 、CD103 + and MHCII + of dendritic cells.

[0029] Fig. 9 The mRNA expression levels of inflammatory factors in bronchoalveolar lavage fluid after the first immunization. **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0030] The mouse alveolar macrophages (MH-S) described in the following examples were obtained from the Traditional Chinese Veterinary Laboratory of the College of Veterinary Medicine of Nanjing Agricultural University; the ICR mice were purchased from Yangzhou University and were raised in an animal room with suitable temperature, humidity and lighting, with free access to food and water, in accordance with ethical requirements and respecting animal welfare.

[0031] The reagents used in the following examples and their sources are as follows: fetal bovine serum (Sigma, USA); 1640 culture medium (Gibco, USA); penicillin-streptomycin dual antibody (Sigma, USA); simvastatin (MCE); CpG (the CpG described in the following examples is CpG-ODN1826, purchased from Sangon Biotech); IgG2A (Invitrogen), IgA, IgG, IgM, CD4, CD8, CD44, CD45, CD103, MHCII (eBioscience, Thermo Fisher Scientific);

[0032] The instruments and their sources used in the following examples are as follows: a constant temperature CO2 incubator (Thermo Corporation, USA); a centrifuge (Thermo Corporation, USA); an ELISA reader (Shanghai Thermo Fisher Scientific Inc.); and a flow cytometer (BD FACSCalibur).

[0033] In the following examples, the simvastatin described was used in the form of a simvastatin solution for cell or mouse experiments, and the solvent of the simvastatin solution was DMSO; the CpG described was used in the form of a CpG solution for cell or mouse experiments, and the solvent of the CpG solution was PBS (154 mM, pH = 7.4).

[0034] Example 1 Cell culture

[0035] 1. Cell recovery

[0036] Before the experiment, the clean bench was sterilized by ultraviolet irradiation for 30 minutes, and the required fetal bovine serum (FBS) was inactivated at 56°C in advance. 1640 culture medium (containing 2% v / v double antibody, the double antibody is 100 μg / mL penicillin and 100 μg / mL streptomycin) was added at a ratio of 10% v / v, and preheated at 37°C before use. After the MH-S cells were quickly dissolved, they were transferred to a centrifuge tube containing 5 mL of fresh culture medium, and centrifuged at 800r / min for 5 minutes after being blown evenly. The supernatant was discarded after centrifugation, and an appropriate amount of fresh culture medium was added to blow and mix the cells evenly. 1 mL of cells was placed in a culture bottle containing 4 mL of 1640 culture medium, and then evenly shaken, placed in a constant temperature incubator, and the culture conditions were set to 37°C and 5% CO2 for culture.

[0037] 2. Cell passaging

[0038] When the cells adhere to the wall and grow to a density of about 80%, observe whether the cells are differentiated. If not, blow the cells off the culture bottle, collect them in a centrifuge tube, centrifuge at 1000r / min for 5min, discard the supernatant, add appropriate amount of culture medium to mix the cells, put 1mL of cells into a culture bottle containing 4mL of 1640 culture medium, shake evenly, place in a constant temperature incubator, set the culture conditions to 37℃, 5% CO2 for 24h.

[0039] 3. Cell cryopreservation

[0040] When the cells adhere to the wall and grow to a cell density that reaches 80-90% of the culture dish area, observe under a microscope whether the cells are differentiated. If not, gently blow off the cells and collect them in a centrifuge tube. Centrifuge at 1000r / min for 5min, discard the supernatant, add the prepared freezing solution (serum:DMSO v:v=9:1), then blow the cells to resuspend them and put them into a freezing tube. Place them in a freezing box and freeze them in a -80℃ refrigerator, and finally transfer them to a liquid nitrogen tank for storage.

[0041] Example 2 Effects of different Toll-like receptor agonists combined with simvastatin

[0042] CpG is an agonist of Toll-like receptor 9 (TLR9). In this example, simvastatin was used in combination with CpG or other Toll-like signaling pathway receptor agonists (including TLR3 agonist Poly(I:C), TLR7 / 8 agonist R848, and TLR1 / 2 agonist Pam3) on MH-S cell lines in vitro. The specific method is as follows: MH-S macrophages in good condition and undifferentiated were cultured at 8×10 3 The cells were evenly inoculated into 96-well plates and cultured in a 37°C, 5% CO2 incubator for 24 hours. Simvastatin (added at a concentration of 2 μmol / L) was added for 12 hours, and then a Toll-like signaling pathway receptor agonist (added at a concentration of 0.1 μmol / L) was added. After 24 hours of culture, the levels of IL-6 and TNF after administration were determined by Q-PCR. Figure 1 As shown in the figure, compared with the control group (only culture medium was added), the use of TLRs agonists alone can significantly increase the content of proinflammatory factors IL-6 and TNF, but when combined with simvastatin, CpG has a more significant effect than other Toll-like receptor agonists. Interestingly, after Poly(I:C) was combined with simvastatin, both proinflammatory factors showed a downward trend.

[0043] Example 3 Effects of different statins combined with CpG on the secretion of proinflammatory factor IL-6

[0044] This example detects the effect of different statins (simvastatin, mevastatin and atorvastatin) combined with CpG on IL-6 secretion. When MH-S cells were stimulated with three statins at a concentration of 2 μmol / L combined with different concentrations of CpG (0.1 μmol / L, 0.3 μmol / L, 0.7 μmol / L, 1.5 μmol / L and 3.0 μmol / L), the specific method was the same as in Example 2. When mevastatin was used in combination with atorvastatin and CpG, a trend of decreasing IL-6 content was produced in each concentration group, as shown in FIG. Figure 2 B and Figure 2 This may be related to their strong natural immunosuppressive effect. Studies have shown that mevastatin and atorvastatin can play an anti-inflammatory role by reducing the levels of inflammatory cytokines (such as IL-6, TNF-α, etc.). This characteristic has attracted attention in some studies of chronic inflammation and autoimmune diseases. When different concentrations of CpG are used with 2μmol / L simvastatin, except for the 3μmol / L group, all of them show an upward trend, such as Figure 2 As shown in A, when the concentration of CpG was 0.1 μmol / L, the IL-6 content was significantly increased after combined use compared with the use of CpG alone (P<0.01), while when other concentrations were used, no significant increase was achieved compared with the use of CpG alone. Therefore, we chose 0.1 μmol / L as the CpG concentration for subsequent experiments.

[0045] Example 4 Determination of inflammatory factors and chemokines in MH-S cells

[0046] Take MH-S macrophages in the logarithmic growth phase, which are in good condition and undifferentiated, and evenly inoculate the cells into 24-well plates with 1 mL of the system, with 2 × 10 cells per well. 5 After culturing for 24 hours in an incubator at 37°C and 5% CO2, a blank group, a simvastatin group, a CpG group, and a simvastatin combined with CpG group were set. The concentration of simvastatin added was 2 μmol / L, and the concentration of CpG added was 0.1 μmol / L. As described in Example 2, simvastatin was added for 12 hours, and then CpG was added for 24 hours. The supernatant culture medium was collected and the content of inflammatory factors and chemokines was determined by q-PCR.

[0047] In the inflammatory response, macrophages are key inflammatory cells involved in the initiation of the process, and promote a variety of inflammatory-related diseases by secreting a large number of related pro-inflammatory mediators. In this example, simvastatin at a concentration of 2 μmol / L combined with 0.1 μmol / L CpG was used to stimulate alveolar macrophages MH-S, and the levels of pro-inflammatory factors in the cells were detected by Q-PCR. Figure 3As shown in the results, compared with the control group, simvastatin combined with CpG significantly increased the levels of proinflammatory factors IL-6 (p<0.01), IL-1β, and TNF-α (p<0.001). Therefore, the results show that simvastatin can enhance the proinflammatory effect of CpG.

[0048] When the human body is defending and clearing invading pathogens and other foreign bodies, it has a function that directs the chemotaxis of immune cells. The substance that can cause this function is called chemokine. Some chemokines are considered to be pro-inflammatory cytokines, which can induce cells of the immune system to enter the infection site during the immune response. CCL3, CCL5, and Cxcl10 are all chemokines secreted by Th1 cells and can reflect the immune response mediated by Th1 cells. The levels of these three chemokines in cells were detected by Q-PCR. Figure 4 As shown in the figure, compared with the control group, CpG significantly increased the content of CCL3, but there was no significant difference in the content of CCL5 and Cxcl10. Compared with CpG alone, CpG combined with simvastatin significantly increased the content of the three chemokines, indicating that simvastatin can promote the Th1 immune response induced by CpG as an adjuvant.

[0049] Example 5: Changes in CpG transcriptome genes in combination with simvastatin

[0050] In this example, transcriptome sequencing was performed on the cells obtained by the method described in Example 4. Figure 5 As shown in the figure, first of all, from the PCA diagram, the explained variance of PC1 and PC2 is 48.68% and 20.69% respectively. These two principal components together explain 69.37% of the data variation, which is enough to show that there are differences between samples. Figure 5 The four groups were clearly separated, indicating that the gene expression patterns of these four groups were significantly different (e.g. Figure 5 ). Figure 5 Figure B shows that compared with the control group, a large number of genes were up-regulated or down-regulated after the addition of simvastatin. GO enrichment analysis and KEGG enrichment analysis showed that the genes that changed in the statin group were mainly related to pro-inflammatory signaling pathways such as Toll-like receptors, NF-κB, TNF-α, and TH-17 (e.g. Figure 5 Combined with previous studies, we know that statins inhibit the mevalonate pathway and are linked to the PI3K-AKT pathway, such as Figure 5Figure E shows that the statin group did have changes in PI3K-AKT expression compared with the control group, with both PI3K isoforms Pik3r5 / 6 upregulated and NF-κB substrate Rela upregulated, both of which are consistent with pro-inflammatory trends. KEGG gene enrichment analysis showed that it was also related to many pro-inflammatory signaling pathways; at the Toll-like receptor signaling pathway gene level, such as Figure 5 As shown in F, compared with the use of CpG alone, simvastatin combined with CpG upregulated the expression of inflammatory factors such as TNF, IL-1β, IL-6, and chemokines such as CCL3 and CCL5, which is consistent with the previous results. This shows that simvastatin can enhance the adjuvant activity of CpG.

[0051] Example 6 Animal Grouping and Treatment

[0052] Six-week-old ICR male mice were divided into five groups: control group, RBD group (new crown vaccine antigen, purchased from OkayBio), simvastatin group, CpG group, and simvastatin + CpG group. They were placed in the animal room of the experimental animal center for a week to familiarize themselves with the environment. After a week of familiarization with the environment, the first nasal immunization was performed on the first day, and the second nasal immunization was performed two weeks later (day 14). The process of each nasal immunization experiment: First, the ICR mice were anesthetized by intraperitoneal injection of 2.5% Avertin working solution. After the mice lost their autonomous activity, the restrained mice were grabbed with the left hand, and the pipette was held in the right hand to slowly drip 200μL of the drug into the mouse's nostrils. After the mouse inhaled into the nasal cavity, the above operation was repeated until all 200μL were inhaled. The grouping is as follows:

[0053] Control group: add 200 μL of normal saline;

[0054] RBD group: 5.12 μL RBD and 194.88 μL saline were added;

[0055] Simvastatin group: 1 μL simvastatin, 5.12 μL RBD, and 193.88 μL normal saline were added;

[0056] CpG group: 5 μL CpG, 5.12 μL RBD and 189.88 μL saline were added;

[0057] Simvastatin + CpG group: 5 μL CpG, 1 μL simvastatin, 5.12 μL RBD and 188.88 μL normal saline were added.

[0058] The RBD described above is an RBD solution, the solvent of which is physiological saline, and the concentration is 3 mg / mL; the CpG described above is a CpG solution, the solvent of which is PBS (154 mM, pH=7.4), and the concentration of which is 0.02 mg / mL; the simvastatin described above is a simvastatin solution, the solvent of which is DMSO, and the concentration of which is 0.02 mg / mL.

[0059] After the nasal drip is completed, the mouse is kept in the nasal drip posture for a period of time to prevent the drug solution from leaking out and affecting the experimental data.

[0060] 48 hours after administration, 10% chloral hydrate was prepared and mice were anesthetized by intraperitoneal injection. Blood was first collected and placed in a 37°C incubator for 1 hour, and centrifuged at 4000 r / min, 4°C for 30 minutes to separate serum. Subsequently, bronchoalveolar lavage fluid was collected for antibody ELISA determination, and cervical lymph nodes were used for immunofluorescence sectioning and flow cytometry detection.

[0061] 1. Determination of immunoglobulin content in serum and bronchoalveolar lavage fluid

[0062] ELISA was used to measure the amount of immunoglobulin binding in serum and bronchoalveolar lavage fluid. The specific steps are as follows:

[0063] (1) Draw the standard curve: set up the standard wells, and add 50 μL of the existing standard of different concentrations to each well. (2) Add the sample to be tested: set up blank wells (control wells without adding samples and enzyme-labeled reagents, and the rest of the steps are the same) and the sample wells to be tested. First, add 40 μL of the sample diluent to the enzyme-labeled plate in the sample well to be tested, then add 10 μL of the sample to be tested, and gently shake to mix.

[0064] (3) Add enzyme: Add 100 μL of enzyme-labeled reagent to each well except the blank well.

[0065] (4) Incubation: Seal the ELISA-coated plate with a sealing film and incubate in a 37°C incubator for 60 min.

[0066] (5) Liquid preparation: Calculate the required amount of detergent and dilute the concentrated detergent 20 times with distilled water for later use.

[0067] (6) Washing: Carefully peel off the sealing film, discard the liquid, spin dry and fill each well with washing solution. Let it stand for 30 seconds and then discard it. Pat dry on absorbent paper. Repeat this process 5 times.

[0068] (7) Color development: First add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, seal the enzyme-labeled plate with a sealing film, and place it in a 37°C incubator away from light for 15 minutes for color development.

[0069] (8) Termination: Add 50 μL of stop solution to each well to terminate the reaction.

[0070] (9) Determination: Within 15 minutes after adding the stop solution, measure the absorbance (OD value) of each well at a wavelength of 450 nm using an ELISA reader and adjust the value to zero using a blank well.

[0071] 2. Determination of immunofluorescence sections of cervical lymph nodes

[0072] Cervical lymph node samples were fixed with 4% paraformaldehyde and permeabilized (with 0.1% Triton X-100). Blocked with PBS (154mM, pH=7.4) containing 5% BSA to prevent nonspecific binding. Samples were treated with specific primary antibodies at 4°C overnight. Unbound primary antibodies were washed away with PBS (154mM, pH=7.4). Samples were incubated with fluorescently labeled secondary antibodies, usually for 1-2 hours at room temperature. Unbound secondary antibodies were washed away with PBS (154mM, pH=7.4). Sections were blocked with sealing fluid. Observed and images were taken using a fluorescence microscope.

[0073] 3. Determination of dendritic cell activation and memory T cell retention

[0074] Place the cervical lymph node tissue in a medium containing enzymes, gently mince, digest for 30-60 minutes, and prepare a single cell suspension. Filter through a cell sieve to remove tissue clumps and cell clusters. Centrifuge at an appropriate centrifugal rate and remove the supernatant. Resuspend the cells in an appropriate medium and add antibodies for labeling. Detect the activation and retention of dendritic cells and the retention of memory T cells in the cervical lymph nodes by flow cytometry.

[0075] Example 7 Simvastatin combined with CpG increases the content of specific antibodies

[0076] The above cell experiment related examples found that simvastatin can enhance the activity of CpG as an adjuvant. This example further evaluates whether simvastatin can enhance the activity of CpG adjuvant in the long term. The bronchoalveolar lavage fluid and serum were collected on the 3rd and 16th days, and the antibody levels of specific antibodies and their subtypes in the bronchoalveolar lavage fluid and serum were determined by ELISA. In the immune response, enhanced Th1 immunity can produce higher levels of IgG and its subtypes, and IgA can also assist the Th1 immune response on the mucosa, jointly protecting the body from infection.

[0077] like Figure 6As shown, after the first immunization, simvastatin combined with CpG significantly enhanced the antibody titer levels of IgA and IgG in bronchoalveolar lavage fluid (p<0.05) and IgG (p<0.01) and IgG2a (p<0.001) in serum. However, after the second immunization, the antibody titer did not increase significantly compared with the first immunization, but was still significantly higher than that of the CpG group, indicating that simvastatin can enhance the activity of CpG as an adjuvant in a long-term manner.

[0078] Example 8 Simvastatin combined with CpG increases the content of cells expressing B220 and FAS

[0079] Immunofluorescence sections of cervical lymph node germinal centers and flow cytometric analysis Figure 7 As shown, Figure 7 B in the figure shows that the number of cells expressing B220 and FAS in the germinal center increased significantly (p<0.001), indicating an increase in the content of B cells and an increase in cell apoptosis.

[0080] Example 9 Simvastatin combined with CpG enhances the activation and retention of dendritic cells and memory T cells

[0081] CD103 is highly expressed on dendritic cells resident in the lungs and is also a marker highly expressed on tissue-resident memory T cells. Figure 8 Compared with the CpG group, the results showed that simvastatin combined with CpG increased the expression of resident dendritic cells and CD4 + 、CD8 + The increase in T lymphocytes indicated that simvastatin combined with CpG had a long-term immunomodulatory effect.

[0082] Example 10 Simvastatin combined with CpG enhances innate immunity in the lungs

[0083] The bronchoalveolar lavage fluid was collected from the immunized mice after the first immunization, and the inflammatory cytokines in the bronchoalveolar lavage fluid were determined by Q-PCR. Fig. 9 The results showed that compared with the control group, CpG stimulation can significantly increase the expression of some cytokines that mediate humoral immunity, such as IL-6 and IL-10 (p<0.001), and the co-stimulatory cytokine TNF-α also increased significantly. In acute and chronic inflammatory responses, chemokine levels usually increase, promoting the aggregation of inflammatory cells, including CC chemokines and CXC chemokines; CpG stimulation can increase the expression of these factors. When simvastatin was added in advance to inhibit the mevalonate pathway, compared with CpG stimulation alone as an adjuvant, the combination of drugs as an adjuvant can more significantly stimulate the production of inflammatory factors (p<0.001), and simvastatin enhances the adjuvant activity of CpG.

[0084] The present invention provides an immune adjuvant composition and its application ideas and methods. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be considered as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. An immune adjuvant composition, characterized in that: Including TLRs agonists and simvastatin.

2. The immunoadjuvant composition according to claim 1, characterized in that The TLRs agonist is any one of a TLR1 agonist, a TLR2 agonist, a TLR7 agonist, a TLR8 agonist or a TLR9 agonist.

3. The immunoadjuvant composition according to claim 2, characterized in that: The TLRs agonist is a TLR9 agonist.

4. The immune adjuvant composition according to claim 3, characterized in that The TLR9 agonist is CpG-ODN.

5. The immune adjuvant composition according to claim 1, characterized in that: The molar ratio of the TLRs agonist to the simvastatin is 1-30:

20.

6. The immune adjuvant composition according to claim 5, characterized in that The molar ratio of the TLRs agonist to the simvastatin is 1:

20.

7. Use of the immune adjuvant composition according to any one of claims 1 to 6 in the preparation of a vaccine.

8. The use according to claim 7, characterized in that: The vaccine described is the new coronavirus vaccine.

9. The use according to claim 8, characterized in that: The antigen of the new coronavirus vaccine is RBD.

10. Use of the immune adjuvant composition according to any one of claims 1 to 6 in the preparation of a drug for treating immune diseases, wherein the immune disease is preferably a pulmonary immune disease.

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