A calcium-doped manganese carbonate multimodal vaccine delivery system and its preparation method and application

Abstract

The present invention discloses a calcium-doped manganese carbonate multimodal vaccine delivery system, its preparation method, and application. The vaccine delivery system is obtained by loading listeriolysin O with calcium-doped manganese carbonate microspheres, wherein the calcium-doped manganese carbonate microspheres are prepared by the following method: (1) stirring and mixing a CaCl2 aqueous solution and a MnCl2 aqueous solution, and then adding sodium polystyrene sulfonate to obtain a mixed solution of CaCl2 and MnCl2 containing PSS; (2) adding sodium polystyrene sulfonate to a Na2CO3 aqueous solution, mixing uniformly, and then adding the mixed solution dropwise to the mixed solution of CaCl2 and MnCl2 containing PSS, stirring and reacting, to obtain calcium-doped manganese carbonate microspheres. The multimodal tumor vaccine delivery system constructed by the present invention can conceal the toxicity of LLO and improve cellular immune response, and has important application value in the field of tumor vaccines.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and in particular relates to a calcium-doped manganese carbonate multimodal vaccine delivery system and a preparation method and application thereof. Background Art

[0002] With the success of tumor immunotherapy in clinical practice, tumor vaccines have received increasing attention. Vaccination with tumor vaccines can induce the body to produce tumor antigen-specific cellular immune responses, ultimately producing cytotoxic T lymphocytes (CTLs) that directly and specifically kill tumor cells. However, it is difficult to achieve this goal using only tumor antigens. The induction of cellular immune responses of tumor vaccines mainly relies on vaccine adjuvants to directly stimulate the immune system, or relies on intelligent delivery systems to intelligently trigger lysosomal escape and cross-presentation of tumor antigens. Therefore, the construction of a multimodal vaccine delivery system has become the first choice, which can not only act as a vaccine adjuvant, but also deliver tumor antigens in the desired manner, but there are currently few materials that meet the multimodal requirements.

[0003] Listeriolysin O (LLO) is not only an excellent vaccine adjuvant, but also can promote cellular immune responses to tumor vaccines. LLO is a virulence protein secreted by Listeria that destroys cell membranes by binding to cholesterol molecules. This property gives LLO the potential to escape lysosomes and deliver bioactive substances to the cytoplasm. Studies have shown that liposomes containing LLO can promote the escape of tumor antigen OVA into the cytoplasm, induce cellular immune responses, and significantly increase OVA-specific CTL activity. Therefore, LLO, which has multiple functions, is suitable for constructing multimodal vaccine delivery systems, promoting antigen cross-presentation, and inducing cellular immune responses. However, LLO has certain cytotoxicity, which limits its application in biological vaccines.

[0004] Multifunctional MnCO3 microspheres are ideal carrier materials with ideal properties for constructing multimodal tumor vaccine delivery systems. 2+ It was recently discovered for the first time that it plays a key role in the body's natural immune process against viral infection and specifically increases the sensitivity of animals to DNA viruses. 2+ It is released into the cytoplasm, triggering signal transduction and inducing anti-DNA virus response. 2+ It has a good tumor immune surveillance effect and can promote CD8 + The proliferation of T cells and tumor infiltration, killing tumor cells. The findings of these studies have stimulated people's strong interest in using manganese-containing materials as vaccine adjuvants. In addition, Mn 2+ It is also widely used in MRI imaging in cancer diagnosis. Overall, pH-sensitive, biodegradable MnCO3 microspheres appear to be a promising material for constructing a multimodal vaccine delivery system that can not only provide Mn2+ MnCO3 microspheres can be used as adjuvants and can also deliver antigens to the cytoplasm of APCs. However, we found that MnCO3 microspheres are fragile and have a smooth surface, which limits their antigen loading capacity. To date, there are no reports of MnCO3 being used as a vaccine adjuvant or carrier. Therefore, it is of great significance to improve the stability and antigen loading capacity of MnCO3 microspheres and utilize them to construct multimodal vaccine delivery systems. Summary of the Invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a calcium-doped manganese carbonate multimodal vaccine delivery system.

[0006] Another object of the present invention is to provide an application of the calcium-doped manganese carbonate multimodal vaccine delivery system in the preparation of vaccine immune adjuvants.

[0007] Another object of the present invention is to provide the use of the calcium-doped manganese carbonate multimodal vaccine delivery system as a vaccine immune adjuvant in the preparation of vaccines.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A calcium-doped manganese carbonate multimodal vaccine delivery system is obtained by loading listeriolysin O (LLO) onto calcium-doped manganese carbonate microspheres (Ca@MnCO3); wherein the calcium-doped manganese carbonate microspheres (Ca@MnCO3) are prepared by the following method:

[0010] (1) stirring and mixing a CaCl2 aqueous solution and a MnCl2 aqueous solution to obtain a mixed solution of CaCl2 and MnCl2; then adding sodium polystyrene sulfonate (PSS) to obtain a mixed solution of CaCl2 and MnCl2 containing PSS;

[0011] (2) Sodium polystyrene sulfonate (PSS) is added to a Na2CO3 aqueous solution and mixed evenly, and then added dropwise to the mixed solution of CaCl2 and MnCl2 containing PSS obtained in step (1) and stirred for reaction, centrifuged, washed, and freeze-dried to obtain calcium-doped manganese carbonate microspheres (Ca@MnCO3).

[0012] The mass ratio of the calcium-doped manganese carbonate microspheres (Ca@MnCO3) to listeriolysin O (LLO) is 250 to 500:1, preferably 500:1.

[0013] The CaCl2 and MnCl2 mixture in step (1) 2+ and Mn 2+ The molar ratio is 1:1.

[0014] The concentration of the CaCl2 aqueous solution described in step (1) is preferably 0.6 mol / L.

[0015] The concentration of the MnCl2 aqueous solution described in step (1) is preferably 0.6 mol / L.

[0016] The volume ratio of the CaCl2 aqueous solution to the MnCl2 aqueous solution in step (1) is 1:1.

[0017] The amount of sodium polystyrene sulfonate (PSS) added in step (1) is calculated based on the ratio of 0.2 mg sodium polystyrene sulfonate per milliliter (mL) of the mixed solution of CaCl2 and MnCl2.

[0018] The amount of sodium polystyrene sulfonate (PSS) added in step (2) is calculated based on 0.5 mg of sodium polystyrene sulfonate per milliliter (mL) of Na2CO3 solution.

[0019] The concentration of the Na2CO3 aqueous solution described in step (2) is preferably 0.6 mol / L.

[0020] The molar ratio of CaCl2, MnCl2 and Na2CO3 in the reaction system described in step (2) is 5:5:4 (i.e., the molar ratio of the total amount of CaCl2 and MnCl2 to Na2CO3 is 5:2).

[0021] The volume ratio of the mixed solution of CaCl2 and MnCl2 containing PSS to the Na2CO3 aqueous solution described in step (2) is 5:2.

[0022] The stirring reaction conditions in step (2) are: stirring at 600 rpm for 30 min.

[0023] The centrifugal conditions described in step (2) are: centrifugation at 4000 rpm for 5 minutes.

[0024] The washing in step (2) is performed by alternating washing with anhydrous ethanol and deionized water; preferably, the washing is performed by alternating washing with anhydrous ethanol and deionized water four times.

[0025] Application of the calcium-doped manganese carbonate multimodal vaccine delivery system in the preparation of vaccine immune adjuvants.

[0026] The vaccine is a tumor vaccine.

[0027] The calcium-doped manganese carbonate multimodal vaccine delivery system is used as a vaccine immune adjuvant in the preparation of vaccine preparations.

[0028] The vaccine preparation comprises the calcium-doped manganese carbonate multimodal vaccine delivery system and an antigen.

[0029] The antigen is preferably ovalbumin (OVA).

[0030] The calcium-doped manganese carbonate multimodal vaccine delivery system is used as a vaccine immune adjuvant in the preparation of a vaccine preparation, which comprises adding an antigen and listeriolysin O (LLO) to a solvent, then adding calcium-doped manganese carbonate microspheres (Ca@MnCO3), and incubating with stirring at room temperature for more than 24 hours to obtain the vaccine preparation.

[0031] The mass ratio of the calcium-doped manganese carbonate microspheres to the antigen is 50 to 100:3, preferably 100:3.

[0032] The mass ratio of the calcium-doped manganese carbonate microspheres to listeriolysin O (LLO) is 250 to 500:1, preferably 500:1.

[0033] The solvent is physiological saline.

[0034] The stirring speed is preferably 100 rpm.

[0035] The vaccine preparation is administered by subcutaneous injection, preferably in the groin.

[0036] The vaccine preparation was stored at 4°C.

[0037] The present invention has the following advantages and effects compared to the prior art:

[0038] 1. MnCO3 is acid-sensitive and can release Mn 2+ , which can enhance the immune stimulation effect, but it is fragile and has weak antigen loading capacity. Calcium doping forms a rough surface, which greatly increases the specific surface area and stability of the microspheres. In order to overcome the brittleness and low loading capacity of manganese carbonate, the present invention takes advantage of the pH sensitivity, good stability and high loading capacity of calcium carbonate, and constructs Ca-doped MnCO3 microspheres (Ca@MnCO3) through a one-pot method to improve the stability and antigen loading capacity of MnCO3 microspheres.

[0039] 2. LLO can promote antigen presentation, but its toxicity limits its application. In order to overcome the defects of existing immune adjuvants such as weak immunity induced by LLO and high toxicity, the present invention constructs a multimodal tumor vaccine delivery system (Ca@MnCO3 / LLO) by physically adsorbing LLO on Ca@MnCO3 microspheres. This porous carrier material can hide the toxicity of LLO and improve cellular immune response.

[0040] 3. The Ca@MnCO3 in the present invention has good biocompatibility, simple synthesis, low cost, economical and environmentally friendly; at the same time, the Ca@MnCO3 in the present invention has good dispersibility in water and physiological saline, large specific surface area, and is convenient for preparing vaccine preparations.

[0041] 4. The present invention uses Ca@MnCO3 / LLO as an immune delivery system for the first time, which can promote the uptake of antigens by DC2.4 cells, and use LLO to promote the lysosomal escape of antigens, promote the cross-presentation of antigens, promote the entry of antigens into peripheral immune organs, promote the proliferation of spleen cells, and induce higher levels of antigen-specific IgG antibody titers, IFN-γ, IL-4 and IL-10, achieving a more excellent cellular immune response. Therefore, it has important application value in the field of vaccine therapy.

[0042] 5. The multimodal tumor vaccine delivery system (Ca@MnCO3 / LLO) constructed by the present invention has the following advantages as an immune adjuvant for improving cellular immune response: (1) The material sources for synthesizing Ca@MnCO3 are abundant, the preparation method is simple, and the biosafety is good; (2) Ca@MnCO3 has good dispersibility in water and physiological saline, which is convenient for the production and preparation of immune vaccine preparations; (3) Ca@MnCO3 has a large specific surface area and a rough surface, which can efficiently load antigens; (4) Ca@MnCO3 can adhere to the cell surface, so that the antigens can be more effectively recognized and taken up by antigen presenting cells (APCs), thereby inducing the body's immune response; (5) Ca@MnCO3 microspheres can effectively protect the antigen OVA and the adjuvant LLO from damage by acids, alkalis, proteases, etc. in the body, thereby improving the utilization rate of the antigen; (6) LLO can promote the escape of antigens from lysosomes and improve the cellular immune response; (7) Ca@MnCO3 microspheres can hide the toxicity of LLO. The constructed multimodal tumor vaccine delivery system has good biosafety, and the system itself can act as an adjuvant (LLO and Mn 2+ ), and can also intelligently co-deliver antigen OVA and adjuvant LLO through various mechanisms; (8) The multimodal vaccine delivery system can promote the entry of antigens into peripheral immune organs and induce higher levels of antigen-specific IgG antibody titers, IFN-γ, IL-4 and IL-10, so it has important application value in the field of tumor immunotherapy.

[0043] 6. The present invention found that when the Ca@MnCO3 / LLO / antigen vaccine preparation is inoculated into the body, the vaccine delivery system can slowly release the antigen, slowly dissolve in the acidic lysosome and release the adjuvant and antigen, promoting the antigen OVA and adjuvant Mn 2+The vaccine delivery system can release the antigen slowly, allowing the body to continuously receive immune stimulation and induce a long-term and effective immune response. Therefore, this research work provides a new perspective for the development of new tumor vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is the SEM image of Ca@MnCO3 and MnCO3 microspheres.

[0045] Figure 2 This is the N2 adsorption and desorption curve of Ca@MnCO3 and MnCO3 microspheres.

[0046] Figure 3 It is an evaluation diagram of the antigen OVA loading capacity of Ca@MnCO3; among them, A is the elemental analysis diagram of Ca@MnCO3 and Ca@MnCO3 after loading the antigen OVA; B is the antigen loading result diagram of MnCO3 and Ca@MnCO3.

[0047] Figure 4 It is a graph showing the hemolysis results after incubation of erythrocytes with LLO, Ca@MnCO3 / OVA / LLO, ii-Ca@MnCO3 / OVA / LLO and H2O.

[0048] Figure 5 It is the distribution diagram of the vaccine preparation in DC2.4 cells; A is the result of flow cytometry detection; B is the average fluorescence intensity of the vaccine preparation.

[0049] Figure 6 The figure is the result of immunohistochemical staining.

[0050] Figure 7 This is a graph showing the results of IgG antibody titer determination.

[0051] Figure 8 This is a diagram showing the results of an IFN-γ secretion experiment.

[0052] Figure 9 This is a diagram showing the results of an IL-4 secretion experiment.

[0053] Figure 10 This is a diagram showing the results of an IL-10 secretion experiment. DETAILED DESCRIPTION

[0054] The present invention will be described in further detail below in conjunction with the examples, but embodiments of the present invention are not limited thereto. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually based on conventional experimental conditions or the experimental conditions recommended by the manufacturer. Unless otherwise stated, the reagents and raw materials used in the present invention can be obtained commercially.

[0055] Ovalbumin (OVA) involved in the examples of the present invention was purchased from Sigma, and Listeriolysin O (LLO) was purchased from Prospec.

[0056] Example 1 Preparation of Ca@MnCO3 microspheres

[0057] 1. Ca@MnCO3 microspheres are prepared by the following steps:

[0058] (1) First, prepare aqueous solutions of CaCl2, MnCl2, and Na2CO3 with a concentration of 0.6 M.

[0059] (2) 5 mL of CaCl2 aqueous solution and 5 mL of MnCl2 aqueous solution were mixed and stirred to obtain a mixed solution of CaCl2 and MnCl2, and then 2 mg of sodium polystyrene sulfonate (PSS) was added to obtain a mixed solution of CaCl2 and MnCl2 containing PSS.

[0060] (3) Take 4 mL of Na2CO3 solution, add 2 mg of PSS, mix well, and then add it dropwise to the mixture of CaCl2 and MnCl2 containing PSS, and stir at 600 rpm for 30 min.

[0061] (4) The particles obtained by the reaction were collected and centrifuged (4000 rpm, 5 min), washed four times with anhydrous ethanol and deionized water alternately, and freeze-dried to obtain Ca@MnCO3 microspheres, which were sealed and stored at room temperature.

[0062] 2. MnCO3 microspheres are prepared by the following steps:

[0063] (1) Mix 4 mg of NH4HCO3 and 0.1 mg of MnCl2 in 20 mL of deionized water and stir for 30 min to prepare a nanoseed solution.

[0064] (2) The nanoseed solution was mixed with 1 L of MnCl2 aqueous solution (6 mM, containing 0.5% isopropanol) and stirred uniformly. At 50°C, 1 L of NH4HCO3 aqueous solution (0.06 M, containing 0.5% (v / v) isopropanol) was added and rapidly stirred (2000 rpm) for 30 min. Subsequently, the mixed solution was cooled to room temperature and neutralized with HCl to pH = 7.

[0065] (3) The obtained particles were washed three times with deionized water and freeze-dried for 24 h.

[0066] The morphology of MnCO3 and Ca@MnCO3 microspheres was observed by SEM. Figure 1As shown in the figure: During the antigen loading process, pure MnCO3 microspheres are unstable and will be destroyed under 100rpm stirring. After calcium doping, the surface of the prepared Ca@MnCO3 microspheres is rough, showing good dispersion and stability. Subsequently, the specific surface area of ​​the microspheres was analyzed using a fully automatic specific surface and porosity analyzer. The results are shown in the figure. Figure 2 As shown: After Ca doping, the specific surface area of ​​MnCO3 microspheres is greatly increased.

[0067] Example 2 Construction of a multimodal vaccine delivery system and its application as a vaccine immune adjuvant

[0068] 1. Antigen loading of Ca@MnCO3 microspheres

[0069] (1) 5 mg of Ca@MnCO3 microspheres prepared in Example 1 were added to 1 mL of OVA solution (300 μg / mL, prepared with physiological saline), stirred at room temperature for 24 h (100 rpm), and the obtained Ca@MnCO3 / OVA microspheres were stored at 4°C. The antigen-loaded MnCO3 microspheres prepared in Example 1 were used as a control. At different time points (0, 6, 12, 24, 48, 72 h), the suspension was centrifuged and the supernatant was taken. The OVA concentration in the supernatant was detected using a BCA kit, and the elements of the microspheres were analyzed using an energy dispersive X-ray spectrometer (EDS) attached to the SEM. The results are shown in Figure 2. Figure 3 As shown in A: After Ca@MnCO3 was loaded with the antigen OVA, nitrogen was found in the Ca@MnCO3 / OVA microspheres, indicating that the Ca@MnCO3 microspheres were successfully loaded with OVA.

[0070] (2) Subsequently, the OVA loading capacity of different doses of MnCO3 and Ca@MnCO3 microspheres (the dosage of microspheres was 5 mg and 10 mg, and the preparation method was the same as Example 1) was tested and compared. Figure 3 As shown in Figure 2B: The level of OVA loaded on MnCO3 microspheres was very low, while 5mg Ca@MnCO3 microspheres could load 300μg OVA within 24h and maintain the loading within 72h, indicating that calcium doping increased the ability and stability of MnCO3 microspheres to load OVA.

[0071] 2. Preparation of vaccine preparations

[0072] Using ovalbumin (OVA) as the model antigen, a 1 mL mixed solution of OVA (containing 300 μg) and LLO (containing 20 μg) was prepared in saline. This mixed solution was added to 5 mg or 10 mg of Ca@MnCO3 microspheres and stirred at room temperature (100 rpm) for 24 h to produce the Ca@MnCO3 / OVA / LLO and ii-Ca@MnCO3 / OVA / LLO vaccine formulations, respectively. Using the same protocol, a Ca@MnCO3 / OVA vaccine formulation without LLO was prepared. A vaccine formulation prepared with aluminum adjuvant and OVA served as a positive control (without Ca@MnCO3 and LLO), a blank control with saline, and a 300 μg / mL OVA solution (prepared in saline) as a negative control. Vaccine formulations were stored at 4°C. Vaccines with different formulations were prepared according to Table 1.

[0073] Table 1 Vaccine formulations for subcutaneous injection

[0074]

[0075] 3. Hemolysis test

[0076] Fresh blood was drawn from a healthy individual and centrifuged at 1000×g for 5 minutes. The lower layer of red blood cells was removed and washed three times with normal saline. The washed red blood cells were diluted with normal saline to a suspension concentration of 16% (v / v). 100 μL of the test solution (deionized water (H2O), LLO solution containing 2 μg of LLO, Ca@MnCO3 / OVA / LLO and ii-Ca@MnCO3 / OVA / LLO vaccine preparations prepared according to the formula in Table 1) was taken, 200 μL of the above red blood cell suspension was added, and incubated at 37°C for 15 minutes. Subsequently, centrifugation was performed for 5 minutes (1000×g), and 100 μL of the supernatant was taken and its absorbance (OD) at 540 nm was measured using a microplate reader (Multiskan MK3, Thermo Fisher, USA). The results were repeated three times.

[0077] The results are as follows Figure 4 As shown in the figure, after the reaction with pure LLO, the hemolysis rate of erythrocytes was 98.4%, while after the reaction with Ca@MnCO3 / OVA / LLO and ii-Ca@MnCO3 / OVA / LLO microspheres, the hemolysis rates of erythrocytes were only 3.97% and 2.27%, respectively. This shows that LLO can be completely adsorbed by Ca@MnCO3 microspheres, thus masking its toxicity.

[0078] IV. OVA Uptake by DC2.4 Cells

[0079] DC2.4 cells (Huiying Biotechnology) were seeded in 24-well culture plates (1×10 5cells / well) and incubate in a 37°C incubator for 24 hours. Wash twice with PBS buffer, and add PBS buffer, Cy5.5-OVA, Ca@MnCO3 / Cy5.5-OVA, Ca@MnCO3 / Cy5.5-OVA / LLO, and ii-Ca@MnCO3 / Cy5.5-OVA / LLO (first use Cy5.5 fluorescently labeled OVA (Cy5.5-OVA), then prepare according to the recipe in Table 1 above) to test solution, in triplicate, and continue incubation at 37°C for 6 hours. Finally, the culture medium was aspirated, and the cells were washed with PBS buffer to remove free particles. The cells were resuspended and analyzed by flow cytometry.

[0080] The results are as follows Figure 5 As shown, compared with Cy5.5-OVA alone, the OVA internalization rate was significantly higher in all Ca@MnCO3-containing groups, with the highest OVA internalization rate in the ii-Ca@MnCO3 / OVA / LLO group. This indicates that the introduction of Ca@MnCO3 and LLO greatly increased antigen uptake, which facilitated further antigen presentation.

[0081] 5. Mouse Immunization Scheme and Determination of Various Immune Indicators

[0082] C57BL / 6 female mice (4-6 weeks old, Beijing Huafukang) were randomly divided into 5 groups (n=5) and subcutaneously inoculated with 100 μL of OVA, Ca@MnCO3 / OVA, Ca@MnCO3 / OVA / LLO, ii-Ca@MnCO3 / OVA / LLO, and Alum / OVA test solutions prepared according to the formula in Table 1. A total of two inoculations were performed, with an interval of 7 days. On the 7th day after the second inoculation, serum was separated from the blood of the experimental mice and stored at -20°C for later use. Subsequently, the mice were sacrificed by cervical dislocation, and splenocytes were isolated from their spleens, and splenocyte suspensions of different concentrations were prepared for later use.

[0083] 1. Immunohistochemistry

[0084] C57BL / 6 female mice (4-6 weeks old) were randomly divided into five groups (n=5) and subcutaneously inoculated with 100 μL of OVA, Ca@MnCO3 / OVA, Ca@MnCO3 / OVA / LLO, ii-Ca@MnCO3 / OVA / LLO, and Alum / OVA test solutions (30 μg OVA / mouse). On days 2 and 7, spleens were harvested and fixed with 4% paraformaldehyde for immunohistochemical analysis. Finally, the distribution of the OVA antigen in the spleen was observed using a microscope (Leica DMI6000, Germany).

[0085] The results are as follows Figure 6As shown, on day 2, OVA levels in the spleens of all mice were low. However, on day 7, OVA levels in the Ca@MnCO3-containing groups increased significantly compared to OVA alone. The OVA level in the ii-Ca@MnCO3 / OVA / LLO group was the highest on day 7. This suggests that longer retention of the antigen at the injection site and increased internalization facilitated antigen migration to the spleen, promoting antigen presentation.

[0086] 2. Determination of IgG Antibody Titer

[0087] Enzyme-linked immunosorbent assay (ELISA) was used to measure the titer of OVA-specific antibodies in mouse serum. First, an OVA antigen solution (10 μg / mL) was prepared in carbonate buffer (0.1 M, pH 9.6) and added to a 96-well plate (100 μL / well). The plate was coated overnight at 4°C. The next day, the plate was washed three times with PBS containing 0.05% (v / v) Tween-20 (PBST). A blocking buffer (2% (v / v) bovine serum albumin in PBST, 200 μL / well) was added and the plate was incubated in a microplate shaker incubator for 1 hour (37°C, 400 rpm). Subsequently, the plate was washed three times with PBST, and serially diluted serum samples (100 μL / well) were added in triplicate, and incubation continued for 2 hours. The plate was then washed three times with PBST and tapped until no residual liquid remained. Next, horseradish peroxidase-conjugated goat anti-mouse antibody HRP-IgG (BioLegend) was added to the plate (100 μL / well, diluted 1:2000 v / v with serum diluent) and incubated for another 1 hour. Subsequently, the plate was washed four times with PBST and tapped until no residual liquid remained in the plate. 3,3',5,5'-tetramethylbenzidine (TMB) substrate (100 μL / well) was added in the dark and incubated for 15 minutes. Finally, H2SO4 stop solution (100 μL / well) was added to terminate the color reaction, and the absorbance (ODs, 450 nm) was measured using a microplate reader.

[0088] The results are as follows Figure 7 As shown, compared with the OVA alone and Alum / OVA groups, the antibody levels in the Ca@MnCO3-containing groups were significantly increased. Among them, the ii-Ca@MnCO3 / OVA / LLO group had the highest antibody level. This indicates that Ca@MnCO3 and LLO exhibit powerful vaccine adjuvant function and significantly enhance the immune response.

[0089] 3. ELISA determination of cytokine levels secreted by splenocytes

[0090] The spleen cell suspension was inoculated into 12-well plates (5 × 10 5cells / well), and the antigen OVA solution (final concentration: 25 μg / mL) was added, and the spleen cells were stimulated for another 60 hours. Subsequently, the cell supernatant was collected, and the secretion levels of cytokines IFN-γ, IL-4, and IL-10 in the supernatant were detected using ELISA kits. Briefly, CaptureAntibody solution (Thermo Fisher Scientific) was added to a 96-well plate (100 μL / well) and coated overnight at 4°C. The next day, the plate was washed four times with PBST, 200 μL of Assay Diluent A solution was added, and incubated for 1 hour. Subsequently, the plate was washed four times with PBST, and the spleen cell supernatant (100 μL / well) was added and incubated for 2 hours. Subsequently, it was washed four times with PBST and the plate was tapped until there was no residual liquid in the wells. Then, 100 μL of HRP-conjugated avidin (Avidin-HRP) solution was added to each well, incubated at 37°C for 1 hour, and the plate was washed four times with PBST. Next, 100 μL of TMB colorimetric solution was added to each well. After color development for 15 min, 100 μL of H2SO4 solution was added per well to terminate the colorimetric reaction. Finally, the OD value at 450 nm was measured using a microplate reader.

[0091] The results are as follows Figures 8-10 As shown, compared with OVA alone, the levels of IFN-γ, IL-4, and IL-10 were higher in the Ca@MnCO3-containing group. Among them, the introduction of Ca@MnCO3 and LLO induced higher IFN-γ secretion, and the ii-Ca@MnCO3 / OVA / LLO group had the highest levels of IFN-γ, IL-4, and IL-10. This indicates that the introduction of Ca@MnCO3 can trigger stronger humoral immunity, while the introduction of LLO triggers stronger cellular immunity.

[0092] In summary, the Ca@MnCO3 prepared in the present invention has good dispersibility, can improve the fragility and antigen loading capacity of MnCO3, and hide the toxicity of LLO. Different vaccine formulations were prepared with OVA and then injected subcutaneously into mice. The results showed that the Ca@MnCO3-based formulation can significantly enhance the uptake of antigens by DC2.4 cells, spleen cell proliferation, IgG levels and cytokine secretion (IFN-γ, IL-4 and IL-10). This is mainly due to the protection and sustained release ability of Ca@MnCO3 for antigens, which is conducive to DCs to take up and present more antigens. Ca@MnCO3 can slowly dissolve in acidic lysosomes and release adjuvants and antigens, promote the escape of antigens and adjuvants to the cytoplasm, and achieve cross-presentation of antigens and cellular immunity. In summary, the multimodal Ca@MnCO3 / LLO vaccine delivery system has the potential to be applied to vaccine delivery systems for subcutaneous immunization.

[0093] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A calcium-doped manganese carbonate multimodal vaccine delivery system, characterized in that: The calcium-doped manganese carbonate microspheres are loaded with listeriolysin O; wherein the calcium-doped manganese carbonate microspheres are prepared by the following method: (1) stirring and mixing a CaCl2 aqueous solution and a MnCl2 aqueous solution to obtain a mixed solution of CaCl2 and MnCl2; then adding sodium polystyrene sulfonate to obtain a mixed solution of CaCl2 and MnCl2 containing PSS; (2) adding sodium polystyrene sulfonate to a Na2CO3 aqueous solution, mixing uniformly, and then adding dropwise to the mixed solution of CaCl2 and MnCl2 containing PSS obtained in step (1) to stir the reaction, centrifuge, wash, and freeze-dry to obtain calcium-doped manganese carbonate microspheres; The mass ratio of the calcium-doped manganese carbonate microspheres to listeriolysin O is 250-500:1; The concentration of the CaCl2 aqueous solution described in step (1) is 0.6 mol / L; The concentration of the MnCl2 aqueous solution described in step (1) is 0.6 mol / L; The CaCl2 and MnCl2 mixture in step (1) 2+ and Mn 2+ The molar ratio is 1:1; The amount of sodium polystyrene sulfonate added in step (1) is calculated based on the ratio of 0.2 mg sodium polystyrene sulfonate per milliliter of the mixed solution of CaCl2 and MnCl2; The concentration of the Na2CO3 aqueous solution described in step (2) is 0.6 mol / L; The molar ratio of CaCl2, MnCl2 and Na2CO3 in the reaction system described in step (2) is 5:5:4; The amount of sodium polystyrene sulfonate added in step (2) is calculated based on the ratio of 0.5 mg sodium polystyrene sulfonate per milliliter of Na2CO3 solution; The volume ratio of the mixed solution of CaCl2 and MnCl2 containing PSS to the Na2CO3 aqueous solution described in step (2) is 5:2; The stirring reaction conditions in step (2) are: stirring at 600 rpm for 30 min.

2. The calcium-doped manganese carbonate multimodal vaccine delivery system according to claim 1, characterized in that: The mass ratio of the calcium-doped manganese carbonate microspheres to listeriolysin O is 500:

1.

3. The calcium-doped manganese carbonate multimodal vaccine delivery system according to claim 1, characterized in that: The centrifugation conditions in step (2) are: centrifugation at 4000 rpm for 5 min; The washing in step (2) is performed by alternating washing with anhydrous ethanol and deionized water.

4. Use of the calcium-doped manganese carbonate multimodal vaccine delivery system according to any one of claims 1 to 3 in the preparation of vaccine immune adjuvants.

5. Use of the calcium-doped manganese carbonate multimodal vaccine delivery system according to any one of claims 1 to 3 as a vaccine immune adjuvant in the preparation of a vaccine preparation.

6. The use according to claim 5, characterized in that: The vaccine formulation includes a calcium-doped manganese carbonate multimodal vaccine delivery system and an antigen; The antigen is ovalbumin; The mass ratio of the calcium-doped manganese carbonate microspheres to the antigen is 50-100:

3.

7. The use according to claim 5, characterized in that: Adding the antigen and listeriolysin O to a solvent, then adding calcium-doped manganese carbonate microspheres, and incubating with stirring at room temperature for more than 24 hours to obtain the vaccine preparation; The mass ratio of the calcium-doped manganese carbonate microspheres to listeriolysin O is 250-500:1; The solvent is physiological saline.

8. The use according to claim 7, characterized in that: The vaccination route of the vaccine preparation is subcutaneous injection.

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