An immunological adjuvant composition and uses thereof

Abstract

The present application relates to the technical field of immunological adjuvant, and particularly relates to an immunological adjuvant composition and application thereof.The immunological adjuvant composition comprises a TLR9 agonist CpG-ODN and simvastatin.It is found that, in the case of combination of simvastatin and CpG-ODN, the intensity of immunization can be improved without changing the immunization quality of CpG-ODN, and simvastatin can enhance the natural immune activation caused by CpG-ODN, but other mevalonate inhibitors (such as mevastatin and atorvastatin) do not have this effect.

Description

Technical Field

[0001] This invention relates to the field of immune adjuvant technology, specifically to an immune adjuvant composition and its application, to an adjuvant activity enhancer that improves the immune efficacy of existing TLR adjuvants and an immune adjuvant composition containing the adjuvant activity enhancer, and further to the application of the immune adjuvant composition in vaccine preparation. Background Technology

[0002] CpG is a characteristic sequence in DNA, referring to a specific arrangement in DNA where cytosine (C) and guanine (G) are linked by phosphodiester bonds. CpG sequences are typically found in "enriched regions" of the genome and play important biological functions in many organisms. They are also TLR9 agonists, activating the immune system and promoting cytokine production. This has made CpG sequences a focus of attention in vaccine development and immunotherapy. Other CpG sequences, including the TLR3 agonist Poly(I:C), the TLR7 / 8 agonist R848, and the TLR1 / 2 agonist Pam3, also have research value in immunology.

[0003] Simvastatin is a commonly used lipid-lowering drug that primarily works by inhibiting the activity of hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase) in the liver, thereby reducing the risk of cardiovascular diseases such as heart disease and stroke.

[0004] Previous studies have demonstrated that the mevalonate pathway can enhance adjuvant efficacy, but these studies have found no correlation with inflammatory factors. Patients with gene deletions in the mevalonate pathway exhibit severe inflammatory factor activity. Furthermore, research has shown that in mevalonate pathway-deficient mice, the TLR4 agonist LPS induces higher levels of inflammatory factors. However, 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 infection, while TLR9 plays a crucial role in viral infection. Currently, there are no studies on the combined use of the mevalonate pathway inhibitor simvastatin and a 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, which addresses the shortcomings of the prior art.

[0006] To address the aforementioned technical problems, this invention discloses an immune adjuvant composition and its application. The specific technical solution is as follows:

[0007] An immune adjuvant composition comprising a TLR agonist and simvastatin.

[0008] 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] The TLR9 agonist is CpG-ODN, preferably CpG-ODN1826.

[0010] 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 includes a solvent, wherein the solvent is physiological saline.

[0012] More preferably, the immune adjuvant composition is prepared by the following method: simvastatin is dissolved in DMSO to obtain a simvastatin solution, CpG-ODN is dissolved in PBS to obtain a CpG solution, and the simvastatin solution and CpG solution are mixed in physiological saline according to the specified ratio.

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

[0014] The vaccine mentioned is a COVID-19 vaccine.

[0015] The COVID-19 vaccine in question uses RBD as its antigen.

[0016] Thirdly, the present invention provides the use of the immune adjuvant composition described in the first aspect in the preparation of a medicament for treating immune diseases. The immune diseases are preferably pulmonary immune diseases.

[0017] Beneficial effects:

[0018] This invention investigates whether combining various TLRs agonists with the mevalonate pathway inhibitor simvastatin (or mevastatin, or atorvastatin) can enhance the adjuvant activity of TLRs agonists. It also hypothesizes that inhibiting mevalonate can promote TLR activation and enhance TLRs adjuvant activity. This provides a theoretical basis for the high levels of antibodies and cytokines in genetic patients and lays the foundation for the combined application of statin adjuvants and TLRs adjuvants.

[0019] This invention utilizes a 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 the germinal centers of lymph nodes, enhance the activation of immune cells such as T cells and dendritic cells and enable them to reside, thus providing long-lasting immunity. Attached Figure Description

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

[0021] Figure 1 This diagram shows the expression of pro-inflammatory factors after combining different Toll-like receptor agonists with simvastatin. Figure 1 In the diagram, A represents the expression of the pro-inflammatory factor IL-6. Figure 1 Figure B in the graph represents the expression of the 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 the text refers to simvastatin. Figure 2 B in the formula is mevastatin. Figure 2 C in the figure represents atorvastatin. Compared with the control group, **p<0.01, ***p<0.001.

[0023] Figure 3 The effect of simvastatin combined with CPG on pro-inflammatory factors in MH-S cells. Figure 3 A in the text represents the pro-inflammatory factor IL-6. Figure 3 B in the text stands for IL-1β. Figure 3 C in the study was 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 MH-S cell chemokines. Figure 4 In this context, A represents the chemokine CCL3. Figure 4 B in the equation represents the chemokine CCL5. Figure 4 C in the figure represents the chemokine Cxcl10. Compared with the control group, **p<0.01, ***p<0.001.

[0025] Figure 5 Transcriptome sequencing results of MH-S cells were analyzed. Figure 5 In this context, A represents PCA analysis. Figure 5 B in the figure represents the comparison of volcano maps between the control group and the simvastatin group. Figure 5 C in the figure represents the GO enrichment analysis results of the control group and the simvastatin group. Figure 5 D in the figure represents the KEGG enrichment analysis results of the control group and the simvastatin group. Figure 5 E in the figure represents the comparison results of PI3K-AKT signaling pathway genes between the control group and the simvastatin group. Figure 5F in the figure represents the comparison of Toll-like receptor signaling pathway genes between the CpG group and the simvastatin + CpG group.

[0026] Figure 6 This refers to the levels of immunoglobulins in bronchoalveolar lavage fluid and serum. Figure 6 The A in the text refers to IgA in the bronchoalveolar lavage fluid after the first immunization. Figure 6 In this context, B represents IgG from the bronchoalveolar lavage fluid during the first immunization. Figure 6 In this context, C represents IgG2a from the first immunized serum. Figure 6 In this context, D represents IgG from the first immunization serum. Figure 6 The E in the text refers to IgA in the bronchoalveolar lavage fluid after the second immunization. Figure 6 The F in the text represents IgG in the bronchoalveolar lavage fluid after the second immunization. Figure 6 In the figure, G represents the comparison of IgA in the bronchoalveolar lavage fluid after the second immunization with that after the first immunization. Figure 6 In the figure, H represents the comparison of IgG in bronchoalveolar lavage fluid after the second immunization with that after the first immunization. *p<0.05, **p<0.01, ***p<0.001.

[0027] Figure 7 The results are from immunofluorescence sections and flow cytometry analysis of cervical lymph nodes. Figure 7 In the diagram, A represents the immunofluorescence section result of the cervical lymph nodes. Figure 7 In the figure, B represents the flow cytometry results of cells expressing B220 and FAS. ***p<0.001.

[0028] Figure 8 The results are from flow cytometry. Figure 8 A in the expression CD8 + CD44 + CD45 + CD103 + cells, Figure 8 B in the text represents CD45. + CD4 + and CD103 + cells, Figure 8 C in the text represents CD45. + CD103 + and MHCII + Dendritic cells.

[0029] Figure 9 The values ​​represent the mRNA expression levels of inflammatory factors in bronchoalveolar lavage fluid after the first immunization. **p<0.01, ***p<0.001. Detailed Implementation

[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, Nanjing Agricultural University; the ICR mice were purchased from Yangzhou University and housed in an animal room with suitable temperature, humidity and light, with free access to food and water, in accordance with ethical requirements and respect for animal welfare.

[0031] The reagents used in the following examples and their sources are as follows: fetal bovine serum (Sigma-Aldrich, USA); 1640 medium (Gibco, USA); penicillin-streptomycin antibiotics (Sigma-Aldrich, USA); simvastatin (MCE); CpG (the CpG mentioned 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 used in the following examples and their sources are as follows: constant temperature CO2 incubator (Thermo Fisher Scientific, USA); centrifuge (Thermo Fisher Scientific, USA); microplate reader (Shanghai Thermo Fisher Scientific Co., Ltd.); flow cytometer (BD FACSCalibur).

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

[0034] Example 1 Cell Culture

[0035] 1. Cell resuscitation

[0036] Before the experiment, the clean bench was sterilized by UV irradiation for 30 min, and the required fetal bovine serum (FBS) was inactivated at 56℃. 1640 medium (containing 2% v / v antibiotics, namely 100 μg / mL penicillin and 100 μg / mL streptomycin) was added at a ratio of 10% v / v and preheated to 37℃ before use. After the MH-S cells rapidly lysed, they were transferred to centrifuge tubes containing 5 mL of fresh medium, mixed thoroughly by pipetting, and centrifuged at 800 rpm for 5 min. The supernatant was discarded after centrifugation, and an appropriate amount of fresh medium was added to mix the cells. 1 mL of cells was then transferred to a culture flask containing 4 mL of 1640 medium, shaken thoroughly, and placed in a constant temperature incubator at 37℃ with 5% CO2 for incubation.

[0037] 2. Cell passage

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

[0039] 3. Cell cryopreservation

[0040] When the cells adhere to the culture dish and grow to a density of 80-90% of the dish area, observe under a microscope whether the cells have differentiated. If they have not differentiated, gently blow off the cells and collect them in a centrifuge tube. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add the prepared cryopreservation solution (serum:DMSO v:v = 9:1), resuspend the cells by pipetting, and then place them in cryovials. Place them in a cryopreservation box and freeze them at -80°C. 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 embodiment, simvastatin is used in combination with CpG or other Toll-like signaling pathway receptor agonists (including the TLR3 agonist Poly(I:C), the TLR7 / 8 agonist R848, and the TLR1 / 2 agonist Pam3) in vitro on the MH-S cell line. The specific method is as follows: well-conditioned and undifferentiated MH-S macrophages are used at a ratio of 8 × 10⁶ cells / year. 3 Cells / well were evenly seeded into 96-well plates and incubated at 37°C with 5% CO2 for 24 h. Simvastatin (2 μmol / L) was added for 12 h, followed by the addition of a Toll-like signaling pathway receptor agonist (0.1 μmol / L). After 24 h of incubation, the levels of IL-6 and TNF were measured using Q-PCR. Figure 1 As shown, compared to the control group (culture medium only), the use of TLR agonists alone significantly increased the levels of pro-inflammatory cytokines IL-6 and TNF. However, when combined with simvastatin, CpG showed a more significant effect compared to other Toll-like receptor agonists. Interestingly, the combination of Poly(I:C) and simvastatin resulted in a decrease in both pro-inflammatory cytokines.

[0043] Example 3: Effects of different statins combined with CpG on the secretion of the pro-inflammatory cytokine IL-6

[0044] This example investigated the effect of different statins (simvastatin, mevastatin, and atorvastatin) combined with CpG on IL-6 secretion. When MH-S cells were stimulated with the 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 decreasing trend in IL-6 levels was observed in all the set concentration groups. Figure 2 B and Figure 2 As shown in C. This may be related to their strong innate immunosuppressive effects. Studies have shown that mevastatin and atorvastatin can exert anti-inflammatory effects by reducing the levels of inflammatory cytokines (such as IL-6, TNF-α, etc.), a characteristic that has attracted attention in some studies of chronic inflammation and autoimmune diseases. When different concentrations of CpG are combined with 2 μmol / L simvastatin, except for the 3 μmol / L group, all show an increasing trend, such as Figure 2 As shown in A, when the concentration of CpG is 0.1 μmol / L, the combined use of CpG significantly increases the IL-6 content compared with CpG alone (P<0.01). However, other concentrations do not show a significant increase compared with CpG alone. Therefore, we selected 0.1 μmol / L for CpG concentration in subsequent experiments.

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

[0046] Log-phase MH-S macrophages, in good condition and undifferentiated, were seeded evenly into 24-well plates at a concentration of 1 mL, with 2 × 10⁶ cells per well. 5 After incubation at 37°C and 5% CO2 for 24 hours, control groups, simvastatin group, CpG group, and simvastatin combined with CpG group were established. The concentration of simvastatin was 2 μmol / L, and the concentration of CpG was 0.1 μmol / L. The experimental method described in Example 2 was followed by adding simvastatin for 12 hours, then adding CpG for 24 hours. The supernatant culture medium was collected, and the contents of inflammatory factors and chemokines were determined by q-PCR.

[0047] In inflammatory responses, macrophages are key inflammatory cells involved in initiating the process, promoting various inflammation-related diseases by secreting large amounts of related pro-inflammatory mediators. In this example, alveolar macrophages (MH-S) were stimulated with 2 μmol / L simvastatin combined with 0.1 μmol / L CpG, and the levels of pro-inflammatory factors in the cells were detected using Q-PCR. Figure 3As shown, compared with the control group, simvastatin combined with CpG significantly increased the levels of pro-inflammatory factors IL-6 (p<0.01), IL-1β, and TNF-α (p<0.001). Therefore, the results indicate that simvastatin can enhance the pro-inflammatory effect of CpG.

[0048] When the human body defends against and eliminates invading pathogens and other foreign substances, it has a function that directs immune cells to chemotaxis. Substances that induce this function are called chemokines. Some chemokines are considered pro-inflammatory cytokines, which can induce immune system cells to enter the site of infection during the immune response. CCL3, CCL5, and Cxcl10 are all chemokines secreted by Th1 cells and can reflect Th1 cell-mediated immune responses. The levels of these three chemokines in cells are detected using Q-PCR. Figure 4 As shown, compared with the control group, CpG significantly increased the level of CCL3, but there was no significant difference in the levels of CCL5 and Cxcl10. However, the combination of CpG and simvastatin significantly increased the levels of the three chemokines compared with CpG alone. This indicates 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] This embodiment performs transcriptome sequencing on the cells obtained by the method described in Example 4. For example... Figure 5 As shown in the PCA plot, PC1 and PC2 explained 48.68% and 20.69% of the variance, respectively. These two principal components together explained 69.37% of the data variation, which is sufficient to indicate differences between samples. The samples from the control, simvastatin, CpG, and simvastatin + CpG groups... Figure 5 The four groups were clearly separated, indicating that their gene expression patterns differed significantly (e.g., Figure 5 (As shown in A). Figure 5 B in the study showed that, compared to the control group, the addition of simvastatin resulted in the upregulation or downregulation of a large number of genes. GO enrichment analysis and KEGG enrichment analysis revealed that the genes altered 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 (As shown in C and D in the diagram); based on previous research, it is understood that statin inhibition of the mevalonate pathway is linked to the PI3K-AKT pathway, such as... Figure 5The E-values ​​in the study showed that the statin group did indeed exhibit changes in the PI3K-AKT pathway compared to the control group. Both PI3K gene isoforms, Pik3r5 / 6, were upregulated, as was the NF-κB substrate Rela, both consistent with a pro-inflammatory trend. KEGG gene enrichment analysis also revealed involvement in numerous pro-inflammatory signaling pathways; at the Toll-like receptor signaling pathway gene level, such as... Figure 5 As shown in F, compared to CpG alone, simvastatin combined with CpG upregulated the expression of inflammatory factors such as TNF, IL-1β, and IL-6, as well as chemokines such as CCL3 and CCL5, consistent with previous results. This indicates that simvastatin can enhance the adjuvant activity of CpG.

[0051] Example 6: Animal grouping and treatment

[0052] Six-week-old male ICR mice were randomly divided into five groups: a control group, an RBD group (COVID-19 vaccine antigen, purchased from OkayBio), a simvastatin group, a CpG group, and a simvastatin + CpG group. They were placed in the animal facility of the experimental animal center for one week to acclimatize to the environment. After one week of acclimatization, the first intranasal immunization was administered on day one, and the second intranasal immunization was administered two weeks later (day 14). The procedure for each intranasal immunization was as follows: ICR mice were first anesthetized by intraperitoneal injection of 2.5% Avertin working solution. After the mice lost spontaneous movement, the mice were restrained with the left hand, and 200 μL of the drug was slowly dripped into the nostrils of the mice using a pipette in the right hand. The process was repeated until all 200 μL was inhaled. The groupings are as follows:

[0053] Control group: 200 μL of physiological saline was added;

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

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

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

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

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

[0059] After administering the medication via nasal drops, the mice were kept in the nasal drop position for a period of time to prevent the medication from leaking out and affecting the experimental data.

[0060] Forty-eight hours after drug administration, a 10% chloral hydrate solution was prepared and the mice were anesthetized by intraperitoneal injection. Blood was first collected and incubated at 37°C for 1 hour. Then, the mixture was centrifuged at 4000 rpm and 4°C for 30 minutes to obtain serum. Subsequently, bronchoalveolar lavage fluid was collected for antibody ELISA assay, 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] The amount of immunoglobulins bound in serum and bronchoalveolar lavage fluid was measured using ELISA. The specific procedures are as follows:

[0063] (1) Plotting the standard curve: Set up standard wells, adding 50 μL of existing standards of different concentrations to each well. (2) Adding test samples: Set up blank wells (control wells without sample and enzyme-labeled reagent, the rest of the operation is the same) and test sample wells. First, add 40 μL of sample dilution to the enzyme-labeled plate of the test sample wells, and then add 10 μL of test sample, and gently shake to mix.

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

[0065] (4) Incubation: After sealing the enzyme-labeled plate with the sealing film, incubate it in a 37°C incubator for 60 minutes.

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

[0067] (6) Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each hole with washing liquid, let stand for 30 seconds and then discard, pat dry on absorbent paper, repeat this 5 times.

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

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

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

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

[0072] Neck lymph node samples were fixed and infiltrated with 4% paraformaldehyde (using 0.1% Triton X-100). Blocked with PBS (154 mM, pH 7.4) containing 5% BSA to prevent nonspecific binding. Samples were treated with specific primary antibodies overnight at 4°C. Unbound primary antibodies were washed away with PBS (154 mM, pH 7.4). Samples were incubated with fluorescently labeled secondary antibodies, typically for 1–2 hours at room temperature. Unbound secondary antibodies were washed away with PBS (154 mM, pH 7.4). Sections were blocked with mounting medium. Observation and imaging were performed using a fluorescence microscope.

[0073] 3. Measurement of dendritic cell activation and memory T cell residence

[0074] Cervical lymph node tissue was placed in an enzyme-containing culture medium, gently chopped, and digested for 30-60 minutes to prepare a single-cell suspension. Tissue blocks and cell clusters were removed by filtration through a cell sieve. The cells were centrifuged at an appropriate rate to remove the supernatant. The cells were resuspended in a suitable culture medium and labeled with antibodies. Flow cytometry was used to detect the activation and residence of dendritic cells and memory T cells in the cervical lymph nodes.

[0075] Example 7: Simvastatin combined with CpG increased the content of specific antibodies.

[0076] The aforementioned cell-related experiments revealed that simvastatin can enhance the activity of CpG as an adjuvant. This embodiment further evaluates whether simvastatin can enhance the activity of CpG adjuvant in the long term. Bronchoalveolar lavage fluid and serum were collected on days 3 and 16, and the levels of specific antibodies and their subtypes in the collected bronchoalveolar lavage fluid and serum were measured by ELISA. In the immune response, enhanced Th1 immunity can produce higher levels of IgG and its subtypes, while IgA on the mucosa can also assist the Th1 immune response, jointly protecting the body from infection.

[0077] like Figure 6As shown, after the first immunization, simvastatin combined with CpG significantly enhanced antibody titers in bronchoalveolar lavage fluid (p<0.05) and serum (p<0.01) for IgA and IgG2a (p<0.001). However, after the second immunization, the antibody titers did not significantly increase compared to the first immunization, but were still significantly higher than those in 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 increased the content of cells expressing B220 and FAS.

[0079] Immunofluorescence sections and flow cytometry analysis of cervical lymph node germinal centers, such as Figure 7 As shown, Figure 7 The B cells in the germinal centers showed a significant increase in the amount of cells expressing B220 and FAS (p<0.001), indicating an increase in B cell content and apoptosis.

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

[0081] CD103 is highly expressed on resident dendritic cells in the lungs and is also a marker of high expression on tissue-resident memory T cells. For example... Figure 8 The results showed that, compared to the CpG group, simvastatin combined with CpG reduced the number of resident dendritic cells and CD4+. + CD8 + Increased T lymphocytes indicate that simvastatin combined with CpG has a long-lasting immunomodulatory effect.

[0082] Example 10: Simvastatin combined with CpG enhances the innate immunity of the lungs.

[0083] Bronchoalveolar lavage fluid was collected from immunized mice after the first immunization, and inflammatory cytokines were measured using Q-PCR. Figure 9 The results showed that, compared with the control group, CpG stimulation significantly increased the expression of some cytokines mediating humoral immunity, such as IL-6 and IL-10 (p<0.001), and the co-stimulatory cytokine TNF-α was also significantly increased. In acute and chronic inflammatory responses, chemokine levels are usually elevated, promoting the aggregation of inflammatory cells, including CC chemokine and CXC chemokine; CpG stimulation can increase the expression of these factors. Furthermore, when simvastatin was pre-added to inhibit the mevalonate pathway, the combined use of simvastatin as an adjuvant more significantly stimulated the production of inflammatory factors than CpG stimulation alone (p<0.001), indicating that simvastatin enhanced the adjuvant activity of CpG.

[0084] This invention provides an immune adjuvant composition and its application, along with a method. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. An immune adjuvant composition, characterized in that, The formulation includes a TLRs agonist and simvastatin, wherein the TLRs agonist is a TLR9 agonist, and the TLR9 agonist is CpG-ODN1826; the molar ratio of the TLRs agonist to the simvastatin is 1-30:

20.

2. The immunoadjuvant composition according to claim 1, characterized in that, The molar ratio of the TLRs agonist to the simvastatin is 1:

20.

3. The use of the immune adjuvant composition according to any one of claims 1-2 in the preparation of a vaccine.

4. The application according to claim 3, characterized in that, The vaccine mentioned is a COVID-19 vaccine.

5. The application according to claim 4, characterized in that, The COVID-19 vaccine in question uses RBD as its antigen.

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