Manganese salt adjuvant system based on dextran particle capsule shrinkage as well as preparation method and application of manganese salt adjuvant system

Abstract

The invention belongs to the technical field of immunology, and discloses a manganese salt adjuvant system based on dextran particle capsule shrinkage and a preparation method and application thereof.The manganese salt adjuvant system comprises beta-dextran particles GPs of a hollow spherical shell structure from yeast and manganese hydroxide colloid encapsulated in the dextran particles, the preparation method comprises the following steps: soaking the GPs in a MnCl2 solution, after the inner cavity of the GPs fully absorbs the MnCl2 solution, separating the GPs adsorbing MnCl2, then adding the GPs into a NaOH solution, forming stable manganese hydroxide colloid by manganese ions in the inner cavity of the GPs, and washing after separation to obtain the dextran particle encapsulated manganese salt adjuvant system. The GPMnOH particles carry various antigens at the same time in various modes such as electrostatic adsorption, chemical coupling or hydrophobic interaction, and an ideal carrier is provided for co-delivery immunization of the antigens and adjuvants.

Description

Technical Field

[0001] This invention relates to the field of immunology, and in particular to a manganese salt adjuvant system based on dextran particle encapsulation, its preparation method, and its application. Background Technology

[0002] Manganese adjuvants, as innovative adjuvants, can efficiently activate cellular immunity, promote humoral immunity, activate mucosal immune responses, and induce secretory IgA production, thus serving as mucosal immune adjuvants. Compared to aluminum adjuvants, manganese adjuvants possess stronger immune activation and cellular immune activation functions, thus exhibiting significant clinical development potential and application value. However, manganese adjuvants still face significant limitations in practical applications. Traditional soluble manganese adjuvants suffer from poor targeting of immune organs, easy diffusion into non-target tissues, and the need for high doses to achieve effective immune activation during in vivo application, which limits their maximum adjuvant effect potential. Furthermore, although manganese is a necessary trace element for the human body, high doses can cause a dramatic increase in blood manganese levels, leading to high toxicity. How to enhance the adjuvant effect of manganese salts, thereby correspondingly reducing their dosage and minimizing toxic side effects on normal tissues, has become a significant challenge in the clinical application of manganese salt adjuvants. Summary of the Invention

[0003] The purpose of this invention is to address the technical deficiencies in existing technologies by providing a manganese salt adjuvant system based on dextran particle encapsulation, its preparation method, and its applications. Manganese ions are precisely encapsulated within dextran particles to form manganese hydroxide, and the immune activation capacity of this adjuvant system is then evaluated in detail. Experimental results confirm that, compared to traditional manganese adjuvant strategies, this adjuvant system can induce highly efficient humoral immunity, cellular immunity, and long-lasting immune memory through subcutaneous administration, achieve synergistic activation of "local pulmonary mucosal immunity - systemic immunity" through nasal administration, and exhibit specific immunomodulatory effects against respiratory viruses (such as SARS-CoV-2).

[0004] The technical solution adopted to achieve the purpose of this invention is: A manganese salt adjuvant system based on dextran particle encapsulation includes yeast-derived hollow spherical shell-like β-glucan particles (GPs) and manganese hydroxide colloid encapsulated within the dextran particles.

[0005] In the above technical solution, the particle size of the β-glucan particles (GPs) is 2~4 μm.

[0006] In the above technical solution, the mass percentage of manganese in the manganese salt adjuvant system is 2.28~6.42wt%.

[0007] Another aspect of the present invention includes a method for preparing the manganese salt adjuvant system of dextran particle encapsulation, comprising the following steps: β-glucan particles (GPs) were immersed in a MnCl2 solution. After the inner cavity of the β-glucan particles (GPs) fully absorbed the MnCl2 solution, the β-glucan particles (GPs) adsorbed with MnCl2 were separated. Then, NaOH solution was added, and manganese ions in the inner cavity of the β-glucan particles (GPs) formed a stable manganese hydroxide colloid. The loosely bound Mn(OH)2 on the surface of the β-glucan particles (GPs) was removed by ultrasonication. After separation and washing, a manganese salt adjuvant system of condensed dextran particles was obtained.

[0008] In the above technical solution, the concentration of MnCl2 solution is 50~100 mM, and the concentration of NaOH solution is 2~14 mM.

[0009] Another aspect of the present invention includes the application of the manganese salt adjuvant system of the dextran particles in vaccine preparation.

[0010] Another aspect of the invention includes a vaccine comprising a manganese salt adjuvant system encapsulated in dextran particles and an antigen loaded onto the manganese salt adjuvant system via electrostatic adsorption, chemical coupling, or hydrophobic interaction.

[0011] In the above technical solution, the antigen includes OVA protein or Spike protein.

[0012] Another aspect of the present invention includes a method for preparing the vaccine as follows: The manganese salt adjuvant system encapsulated in the dextran particles is mixed with the antigen and incubated in physiological saline to obtain the vaccine.

[0013] In the above technical solution, the vaccine is administered via intramuscular, percutaneous, subcutaneous, or nasal immunization routes.

[0014] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention develops a novel manganese salt adjuvant system, GPMnOH particles, based on β-glucan particles. Compared with conventional manganese salt adjuvant systems, GPMnOH particles significantly enhance the activation ability of manganese adjuvants on the immune system, resulting in comprehensive activation and enhancement of dendritic cells, and consequently leading to a significant improvement in the body's immune response.

[0015] 2. GPMnOH particles induce strong humoral and cellular immune responses against multiple antigens, such as ovalbumin (OVA) and the Spike protein of SARS-CoV-2, and provide highly efficient neutralizing protection against the novel coronavirus through co-encapsulation and co-delivery with antigen proteins. At the same time, when administered via nasal inhalation, the system also induces highly efficient mucosal responses against multiple antigens, such as OVA and the Spike protein of SARS-CoV-2.

[0016] 3. The manganese salt adjuvant system based on dextran particle encapsulation created in this invention significantly enhances the adjuvant effect of manganese salt, reduces the amount of manganese salt adjuvant required, and induces a wide range of enhanced immune responses, including systemic immunity and mucosal immunity. At the same time, this adjuvant system can simultaneously carry various antigens through electrostatic adsorption, chemical coupling, or hydrophobic interactions, providing an ideal carrier for the co-delivery of antigens and adjuvants for immunity. Attached Figure Description

[0017] Figure 1 The preparation and characterization of GPMnOH particles; Figure 2 GPMnOH particles induce BMDC activation in vitro; Figure 3 GPMnOH particles, when administered subcutaneously, induce antigen-specific humoral immune responses in vivo. Figure 4 It refers to the titer of antigen-specific neutralizing antibodies induced in vivo by subcutaneous administration of GPMnOH particles; Figure 5 It represents the number of CTLs that induce specific secretion of IFN-γ in vivo after subcutaneous administration of GPMnOH particles; Figure 6 It is the maintenance of immune memory and function mediated by subcutaneous administration of GPMnOH particles; Figure 7 GPMnOH granules are administered via nasal drops to enhance pulmonary mucosal immunity; Figure 8 Intranasal inoculation of GPMnOH mediates an increase in pulmonary plasma cells and memory B cells; Figure 9 GPMnOH induces an increase in resident memory T cells (TRM) in lung mucosal tissue; Figure 10 GPMnOH administered intranasally induces systemic immunity. Figure 11 Intranasal inoculation with GPMnOH induces SARS-CoV-2 antigen-specific mucosal and systemic immune responses. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0019] The following abbreviations are used in the following examples: GP: β-glucan particles; APC: antigen-presenting cells; rpm: rotation speed per minute; BMDC: bone marrow-derived dendritic cells; GM-CSF: granulocyte-macrophage colony-stimulating factor; IL: interleukin; IFN-γ: interferon-γ; TNF-α: tumor necrosis factor-α; MHC: major histocompatibility complex; CD: leukocyte differentiation antigen; PBMC: peripheral blood mononuclear cells; ELISA: enzyme-linked immunosorbent assay; ELISPOT: enzyme-linked immunospot assay; CTL: cytotoxic T lymphocytes.

[0020] Example 1 1.1 A method for preparing a manganese salt adjuvant system (GPMnOH particles) based on dextran particle encapsulation includes the following steps: Step 1: Preparation of yeast-derived β-glucan particles (GPs) β-glucan particles (GPs) were extracted from *Saccharomyces cerevisiae*. The simplified steps are as follows: First, 40 g of *Saccharomyces cerevisiae* was resuspended in double-distilled water (ddH2O), washed three times with ddH2O, and centrifuged at 2000 rpm for 5 min each time. The precipitate was then resuspended in 1M NaOH solution, stirred at 80°C for 1 h, and washed three times with ddH2O, centrifuged at 2000 rpm for 5 min each time. The pH was adjusted to 4-5 with 1M hydrochloric acid, stirred at 55°C for 1 h, centrifuged at 3500 rpm for 5 min, washed three times with ddH2O, and centrifuged at 3500 rpm for 5 min each time. The precipitate was resuspended in isopropanol five times, washed five times with isopropanol, and centrifuged at 3500 rpm for 2 min each time. The precipitate was resuspended in acetone, washed twice with acetone, and centrifuged at 3500 rpm for 2 min each time. Finally, the obtained β-glucan particles (GPs) were naturally dried in a fume hood, dispersed in a mortar, and dried and stored at room temperature.

[0021] Step 2: Preparation of a manganese salt adjuvant system based on dextran particle encapsulation (GPMnOH particles) The steps for preparing GPMnOH are as follows ( Figure 1 (AB): Take an appropriate amount of β-glucan granules (GPs), first soak them in MnCl2 solution, then completely remove excess solution by centrifugation. Under stirring conditions, add the remaining granules to an appropriate amount of NaOH solution, and continue stirring at room temperature for a suitable time to allow the MnCl2 to settle. 2+In-situ precipitation formed manganese hydroxide (Mn(OH)2) gel. Subsequently, the sample was sonicated in an ice-water bath to remove loosely bound Mn(OH)2 on the particle surface. The particles were collected by centrifugation and washed twice with physiological saline to obtain GPMnOH particles. The monodispersity and flocculation state of the GPMnOH particles were observed using transmission electron microscopy (TEM). Figure 1 (C); The content of manganese salt in particles was quantitatively detected using inductively coupled plasma mass spectrometry (ICP-MS). Figure 1 (Medium D), specifically 2.28~6.42wt%. 1.2 A vaccine (GPMnOH-OVA) is prepared by the following method: To load the antigen, 1 mg of prepared GPMnOH particles were mixed with 10 μg of OVA in 1.0 mL of physiological saline and incubated overnight at 4°C to achieve antigen binding, thus obtaining antigen particles, denoted as GPMnOH-OVA.

[0023] A vaccine (GPMnOH-Spike) is prepared by the following method: The prepared GPMnOH particles (1 mg) were mixed with Spike (2.5 μg) in 1.0 mL of physiological saline and incubated overnight at 4°C to achieve antigen binding, resulting in antigen particles, denoted as GPMnOH-Spike. Example 2 Detection of GPMnOH particle-induced BMDC activation I. Cultivation of BMDC Primary bone marrow mononuclear cells were isolated from the femur and tibia of C57BL / 6N mice. The cells were cultured for 6 days in RPMI 1640 medium containing inducing factors (5% inactivated FBS, 1% penicillin-streptomycin, 40 ng / mL GM-CSF, 20 ng / mL IL-4 and 50 μM β-mercaptoethanol) at 37°C and 5% CO2 to generate bone marrow-derived dendritic cells (BMDCs).

[0024] II. Activation Detection of BMDC To assess the adjuvant-induced activation effect, PBS and Mn were used. 2+ +OVA(Mn) 2+BMDCs were incubated overnight with OVA (mixed in free form), MnOH-OVA (MnOH-adsorbed protein antigen OVA), and GPMnOH-OVA (GPMnOH-adsorbed protein antigen OVA), respectively. Cells and supernatants were then collected to assess cell activation status and cytokine secretion levels. For cell activation analysis, non-specific binding was blocked with TruStain FcX™ PLUS (anti-mouse CD16 / 32) antibody, followed by staining with FITC or PerCP / Cyanine 5.5-labeled anti-mouse CD11c antibody, and additional staining for surface markers (including CD80, CD86, CD40, and IA / IE (MHCII)) at 4°C for 30 min, followed by flow cytometry analysis. Simultaneously, the concentrations of IFN-γ, TNF-α, IL-6, and IL-12p70 in the culture supernatant were detected using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions.

[0025] Given that the expression levels of co-stimulatory molecules reflect the degree of activation and immune response of BMDCs, we used flow cytometry to detect the expression levels of co-stimulatory molecules (CD80, CD86, CD40) and major histocompatibility complex II (MHC II) molecules on the surface of BMDCs. The results showed that, compared with the MnOH-OVA treatment group, the GPMnOH-OVA treatment group showed significantly enhanced surface activation characteristics, including the expression levels of CD80, CD86, MHC II, and CD40. Figure 2 GPMnOH-OVA promotes the activation of BMDCs and amplifies the expression of various inflammatory cytokines, which may lead to a significant enhancement of the immune response.

[0026] Example 3 Detection of antigen-specific immune response induced by subcutaneous administration of GPMnOH particles: I. GPMnOH particles induce antigen-specific humoral immune responses in vivo. To investigate the ability of vaccine particles to induce a humoral immune response, we selected the model antigen OVA and the SARS-CoV-2-related antigen Spike as antigens loaded onto the vaccine particles. BaLB / c mice were randomly assigned to groups and treated with PBS and Mn... 2+ +OVA, MnOH-OVA, and GPMnOH-OVA (all containing 20 μg manganese salt adjuvant, 10 μg OVA / 2.5 μg Spike antigen) were administered to mice in each group subcutaneously in the groin area, once every two weeks, for a total of three immunizations. Figure 3(A). After immunization, serum was collected from immunized mice on day 7, and the titers of OVA-specific IgG, IgG1, and IgG2a in the serum were detected by ELISA. In summary, 100 μL of 1 μg / mL LOVA / 200 ng / mL Spike solution was coated into each well of a 96-well plate and incubated overnight at 4°C. The plates were washed four times with PBST (phosphate-buffered saline containing Tween-20) and blocked with 5% skim milk for 2 hours. Serum from each immunization group was diluted initially at a 1:50 ratio, followed by five-fold serial dilutions for a total of eight dilutions. The diluted serum was added to the pre-coated antigen wells and incubated at room temperature for 2 hours. After washing four times with PBST, horseradish peroxidase (HRP)-conjugated anti-mouse IgG or IgG1 and IgG2a was added and incubated at room temperature for 1 hour. After washing four times with PBST, 100 μL of TMB single-component chromogenic buffer was added and incubated at room temperature for 2 minutes. The chromogenic process was terminated by adding 100 μL of 2N H2SO4. OD values ​​were measured at 450 nm using a multi-mode microplate reader. Positive wells were defined as those with an OD value greater than twice the average OD value of the negative serum group.

[0027] Experimental Results and Conclusions: Compared with MnOH-OVA, GPMnOH-OVA induced higher levels of OVA-specific IgG and the highest titer of IgG2a (Th1-biased antibody); this indicates that GPMnOH-OVA can enhance humoral immunity and has the advantage of shifting the immune response towards the Th1 type. Figure 3 (B). Similarly, in immunization against SARS-CoV-2-related antigens, the GPMnOH-Spike immunization group showed higher antigen-specific antibody titers compared to MnOH-Spike, along with a significant increase in IgG2a titers (Th1 bias). Figure 3 (C)

[0028] To investigate whether GPMnOH particles could enhance the neutralizing antibody response against Spike protein, a vaccine was prepared by combining them with Spike protein. After three doses of immunization, mouse serum was separated for neutralizing antibody titer detection based on pseudovirus. The simplified steps for producing SARS-CoV-2 pseudovirus carrying the luciferase reporter gene are as follows: Packaging plasmid psPAX2, luciferase reporter plasmid pMD2.G-Luciferase, and spike protein-expressing plasmid pcDNA3.1-SARS-CoV-2 (strain WA1 / 2020) were co-transfected into human embryonic kidney (HEK) 293T cells using PEI transfection reagent. After 48 hours, the culture supernatant was collected and filtered through a 0.45 μm filter to obtain the pseudovirus. Heat-inactivated serum samples were serially diluted and mixed with an equal volume of pseudovirus, then incubated at 37°C for 1 hour. The mixture was then added to 96-well plates (2 × 10⁶ cells per well) pre-coated with 293T cells expressing the hACE2 receptor.4 (100 cells), and continue culturing at 37°C and 5% CO2 for 48 h. Neutralizing antibody titers were determined using a luciferase reporter gene assay kit according to the manufacturer's instructions. Titer was defined as the highest serum dilution that reduced the relative optical units (RLU) value by 50% compared to the average value of the negative serum control group.

[0029] Neutralizing antibody titer assays based on pseudoviruses showed that GPMnOH-Spike (GPMnOH adsorbed the protein antigen Spike, containing 20 μg Mn) was effective. 2+ The neutralizing antibody titer induced by the immunization group containing 2.5 μg Spike was significantly increased, which is related to the fact that MnOH-Spike (MnOH adsorbs the protein antigen Spike, containing 20 μg Mn) significantly increased the titer of neutralizing antibodies. 2+ The neutralizing antibody titer in the serum of the immunized group (2.5 μg Spike) was 2.1 times that of Mn 2+ +Spike(Mn 2+ Mixed with Spike in free form, containing 20 μg Mn 2+ The antibody response to the SARS-CoV-2 Spike protein was 9.4 times that of the group immunized with 2.5 μg Spike. This indicates that GPMnOH particles can significantly enhance the body's neutralizing antibody response against the SARS-CoV-2 Spike protein. Figure 4 ).

[0030] II. Detection of the number of CTLs that specifically secrete IFN-γ induced by GPMnOH particles in vivo To detect the number of cell-mediated cytokines (CTLs) capable of generating specific antigen responses in the body after vaccine immunization, we used ELISPOT to detect the number of CTLs that could secrete IFN-γ after in vitro antigen restimulation of immune cells. Immunized C57BL / 6N mice were randomly divided into groups and treated with PBS and Mn... 2+ +OVA, MnOH-OVA, and GPMnOH-OVA vaccine particles were administered to mice subcutaneously in the groin area, with three immunizations every two weeks. Seven days after the end of immunization, peripheral blood was collected from each group of mice, and peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation. The cells were counted and their density adjusted to 5 × 10⁻⁶. 6Cells / mL were added to pre-activated plates coated with IFN-γ antibody, 100 μL per well, along with RPMI 1640 complete medium (5% heat-sterilized FBS, 1% penicillin-streptomycin bispecific antibiotic, and 50 μM β-mercaptoethanol) containing OVA (10 μg / mL), and incubated at 37°C for 48 h. After incubation, the supernatant was discarded, and the cells were washed four times with PBS. Biotin-labeled detection antibody R4-6A2 was added to a final concentration of 1.0 μg / mL, 100 μL per well, and incubated at room temperature for 2 h. After washing four times with PBS, 100 μL of streptavidin-conjugated alkaline phosphatase was added to each well, and incubated at room temperature for 1 h. 100 μL of NBT / BCIP chromogenic reagent was added to each well. After complete spot development, 100 μL of chromogenic stop solution was added to each well to stop the development. The cells were washed four times with ddH2O to detect IFN-γ spot-forming cells. The number of single-celled cells (SFCs) was measured. The methods for detecting the levels of cytokines IFN-γ and IL-4 in the culture supernatant were consistent with those described above for detecting BMDCs in the culture supernatant.

[0031] Experimental Results and Conclusions: Compared with the MnOH-OVA immunization group, the GPMnOH-OVA immunization group mice produced a significant increase in IFN-γ SFCs in their PBMCs, approximately 1.6 times and 2.8 times that of the former. Figure 5 (AB). Compared with the MnOH-Spike immunization group, the number of IFN-γ secreting speckled cells (SFCs) in mice in the GPMnOH-Spike immunization group was significantly increased, approximately 1.7 times and 4.4 times higher, respectively. Figure 5 The results (CD) indicate that GPMnOH particles can significantly enhance antigen-specific cellular immune responses.

[0032] III. GPMnOH particle-mediated immune memory Flow cytometry was further used to detect phenotypic changes in T cell subsets in the peripheral blood of immunized mice, in order to investigate the ability of GPMnOH particles to induce long-acting memory T cell responses and effector functions. Similar to the humoral immunity assay, PBS and MnO2 were selected on day seven after three immunizations. 2+For mice in the +OVA, MnOH-OVA, and GPMnOH-OVA groups, 0.5 mL of peripheral blood was collected from each mouse. The blood was slowly injected into a centrifuge tube containing EDTA-K2 anticoagulant, and gently inverted to mix to prevent blood clotting. Three times the volume of erythrocyte lysis buffer was added to the anticoagulated peripheral blood, and the mixture was incubated at room temperature for 5 min, with gentle shaking twice during the incubation period to accelerate erythrocyte lysis. Subsequently, the mixture was centrifuged at 300×g for 5 min, the supernatant was discarded, and 50 μL of the adjusted cell suspension was added to a flow cytometry tube. Diluted antibodies were added and mixed, and the mixture was incubated at 4℃ in the dark for 30 min. After incubation, the mixture was centrifuged at 300×g for 5 min, the supernatant was discarded, and flow cytometry was performed.

[0033] Experimental Results and Conclusions: Compared with the MnOH-Spike immunization group and Mn alone... 2+ Compared to the Spike immune group, peripheral blood CD4 levels + T cells and CD8 + After T cells are stimulated by in vitro antigens, the levels of Th1-type cytokines (IFN-γ and TNF-α) secreted by T cells also increase significantly. Figure 6 (Middle A). Compared with MnOH-Spike immunization group and Mn alone. 2+ Compared to the Spike immunization group, GPMnOH-Spike immunized mice showed increased CD8 activity. + T cells and CD4 + The proportion of CD44hi and CD62Llo cells in T cells increased significantly. Figure 6 (B). These data indicate that GPMnOH particles promote the differentiation of naive T cells into effector memory phenotypes and help maintain the effector function of T cells, which is crucial for maintaining T cell-mediated antiviral immunity.

[0034] Example 4 Detection of antigen-specific immune response induced by GPMnOH granule nasal drops: I. Intranasal administration of GPMnOH particles to induce pulmonary mucosal immunity in an OVA model With PBS, Mn 2+ +OVA, MnOH-OVA and GPMnOH-OVA (20μg Mn 2+ Mice were immunized by intranasal administration of 10 μg OVA (n=5) on days 0, 14 and 28, with an administration volume of 50 μL. Serum and bronchoalveolar lavage fluid (Balf) of immunized mice were collected on the seventh day after the second and third immunizations (day 21, day 35). The titers of OVA-specific IgG, IgG1 and IgG2a in the serum were detected by ELISA.

[0035] The mice were euthanized, and a 1-2 cm longitudinal incision was made along the midline of the neck to separate the subcutaneous tissue and muscle, exposing the trachea. The trachea was gently lifted with hemostats, and an incision was made in the middle of the trachea. A pipette was inserted and secured with silk sutures to prevent it from falling off. Sterile saline was slowly injected, 1 mL per mouse, and left to stand for 30 seconds to allow the saline to fully contact the alveoli. The bronchoalveolar lavage fluid was then gently aspirated with a syringe.

[0036] The results showed that compared with the MnOH-OVA immunization group and Mn alone, 2+ Compared to the OVA immunization group, GPMnOH-OVA immunization induced higher levels of OVA-specific IgG and IgGA, and was able to produce antibodies more rapidly. Figure 7 Higher IgA levels indicate that GPMnOH elicited a strong mucosal immune response in the lungs, while the simultaneous increase in IgG suggests that it also triggered an effective systemic immune response.

[0037] To examine changes in the lymphocyte population, mice were euthanized via cervical dislocation after immunization, and their intact lungs were dissected. The lung tissue was then cut into 1-2 mm pieces using surgical scissors. 3 Small tissue fragments were collected and 5 mL of 0.125% trypsin working solution preheated to 37°C was added. The fragments were gently ground for 5-8 minutes, avoiding excessive force that could damage the cells. The dispersion of the tissue fragments was observed every 2 minutes until the fragments became significantly smaller and the grinding solution became turbid. A 70 μm cell sieve was placed on a 15 mL sterile centrifuge tube, and the ground tissue suspension was slowly poured into the sieve to remove cell clumps and impurities. The collected filtrate was the preliminary single-cell suspension, which was then analyzed by flow cytometry after lysing.

[0038] Data showed that the MnOH-OVA immunization group and Mn alone... 2+ Compared with the +OVA immunization group, GPMnOH-OVA immunization significantly increased the proportion of IgG-specific plasma cells and IgA-specific plasma cells in lung tissue. Figure 8 AD); and induced the production of more memory B cells ( Figure 8 (EF). T lymphocyte detection results showed that GPMn-OVA immune-induced a significant increase in the proportion of resident memory T cells (TRM) in lung mucosa tissue, including CD4+. + TRM and CD8 + TRM cells all showed a synchronous upward trend ( Figure 9 These results indicate that GPMnOH can synergistically induce efficient B cell responses and TRM cell-mediated cellular immunity at the respiratory mucosa site, establishing a multi-layered and durable local immune protection against respiratory pathogens.

[0039] II. Intranasal administration of GPMnOH induces systemic immunity in an OVA model. To investigate whether the adjuvant could still induce a systemic immune response after intranasal administration, serum from immunized mice was collected, and the titers of OVA-specific IgG and IgA in the serum were detected by ELISA. Similar to the results of antibody titer detection in Balf, the serum levels of OVA-specific IgG and IgA in GPMnOH-OVA-immunized mice were significantly increased. Figure 10 (AB), and these results indicate that the GPMnOH-OVA vaccine successfully elicited a robust systemic immune response. High levels of IgG provided the body with core protection against the systemic spread of pathogens, indicating strong neutralizing activity, long-lasting immune memory, and activation of cellular immunity; while the synchronous increase in serum IgA further linked systemic immunity with mucosal defense, enhancing immune support for systemic mucosal tissues.

[0040] II. Immunostimulation of SARS-CoV-2 antigen mediated by intranasal administration of GPMnOH particles To evaluate the immunomodulatory effect of GPMnOH particles on a wider range of antigens, the SARS-CoV-2 virus spike protein was selected as the antigen and co-administered with GPMnOH particles for animal immunization. The preparation of GPMnOH-Spike vaccine particles and subsequent animal immunization procedures were consistent with those described earlier for OVA-based antigens. After three doses of immunization, mouse serum and bronchoalveolar lavage fluid were separated for antigen-specific antibody titer detection using ELISA. Additionally, lymphocytes from the mouse spleen and lungs were isolated for ELISPOT cellular immune response evaluation.

[0041] The results of the antigen-specific antibody titer assay showed that, in bronchoalveolar lavage fluid, the titer of antibodies against MnOH-Spike immunoassay and MnOH alone was significantly higher than that against MnOH-Spike immunoassay. 2+ Compared to the +Spike immunization group, the GPMnOH-Spike immunization group showed higher antigen-specific IgG and IgA antibody titers. Figure 11 (AB). Similar results were observed in serum titer testing; the Spike-specific IgG titer in the serum of the GPMnOH-Spike immunized group was 5.0 times that of the MnOH-Spike immunized group, and was significantly higher than that of the MnOH-Spike immunized group. 2+ +6.8 times that of the Spike immunization group; Spike-specific IgA titers were 3.0 times and 4.1 times higher than the former ( Figure 11 Medium CD).

[0042] ELISPOT experimental results showed that, compared with the MnOH-Spike immunization group and Mn alone, 2+Compared to the Spike immunization group, the number of IFN-γ-secreting speckled cells (SFCs) in the spleen of mice immunized with GPMnOH-Spike was significantly increased, approximately 1.8 times and 2.7 times that of the former. Figure 11 (MnOH-Spike immunized mice). Simultaneously, the number of Spike-specific SFCs in the lungs of mice in the GPMnOH-Spike immunized group was also significantly increased, reaching 5.4 times that of the MnOH-Spike immunized group and significantly higher than that of the MnOH-Spike immunized group. 2+ +1.9 times that of the Spike immune group ( Figure 11 (GPMnOH). These results indicate that nasal administration of GPMnOH particles can not only induce specific respiratory mucosal immune responses, but also significantly induce systemic cellular immune responses, providing a three-dimensional and comprehensive immune barrier for viral prevention.

[0043] In summary, this invention successfully constructed a novel manganese adjuvant system (GPMnOH) with highly homogeneous structure by encapsulating manganese hydroxide colloid within the cavity of dextran particles, significantly enhancing its synergistic activation effect on the immune system. Compared with manganese hydroxide, the GPMnOH vaccine can synergistically induce a highly efficient and multi-layered local lymphocyte response in the lungs. Regarding mucosal immunity, GPMnOH immunization significantly increased the proportion of antigen-specific IgG and IgA plasma cells in lung tissue and induced the production of more memory B cells. Regarding cellular immunity, GPMnOH particularly significantly induced a substantial increase in the proportion of resident memory T cells (TRM) in lung mucosal tissue, including CD4+. + With CD8 + The TRM subgroup establishes a durable local cellular immune barrier against respiratory pathogens. This strategy not only provides a new approach to defending against SARS-CoV-2, but its "low-dose, high-efficiency" adjuvant delivery concept also has important implications for the future development of inhaled mucosal vaccines against various respiratory pathogens.

[0044] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A manganese salt adjuvant system based on dextran particle encapsulation, characterized in that, It includes yeast-derived hollow spherical shell-like β-glucan particles (GPs) and manganese hydroxide colloids encapsulated within the glucan particles.

2. The manganese salt adjuvant system based on dextran particle encapsulation as described in claim 1, characterized in that, The β-glucan particles (GPs) have a particle size of 2-4 μm.

3. The manganese salt adjuvant system based on dextran particle encapsulation as described in claim 1, characterized in that, The manganese salt adjuvant system contains 2.28 to 6.42 wt% manganese by mass.

4. The method for preparing the manganese salt adjuvant system of dextran particle encapsulation as described in claim 1, characterized in that, Includes the following steps: β-glucan particles (GPs) were immersed in a MnCl2 solution. After the inner cavity of the β-glucan particles (GPs) fully absorbed the MnCl2 solution, the β-glucan particles (GPs) adsorbed with MnCl2 were separated. Then, NaOH solution was added, and manganese ions in the inner cavity of the β-glucan particles (GPs) formed a stable manganese hydroxide colloid. The loosely bound Mn(OH)2 on the surface of the β-glucan particles (GPs) was removed by ultrasonication. After separation and washing, a manganese salt adjuvant system of condensed dextran particles was obtained.

5. The preparation method according to claim 4, characterized in that, The concentration of MnCl2 solution is 50~100 mM, and the concentration of NaOH solution is 2~14 mM.

6. The application of the manganese salt adjuvant system of dextran particle encapsulation as described in any one of claims 1 to 3 in vaccine preparation.

7. A vaccine, characterized in that, Manganese salt adjuvant systems comprising dextran particles encapsulated and antigens loaded onto the manganese salt adjuvant systems via electrostatic adsorption, chemical coupling, or hydrophobic interactions.

8. The vaccine as described in claim 7, characterized in that, The antigen includes OVA protein or Spike protein.

9. The vaccine as described in claim 8, characterized in that, The method for preparing the vaccine is as follows: The manganese salt adjuvant system encapsulated in the dextran particles is mixed with the antigen and incubated in physiological saline to obtain the vaccine.

10. The vaccine as described in claim 8, characterized in that, The vaccine is administered via intramuscular, percutaneous, subcutaneous, or nasal immunization routes.

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