Preparation method and application of vesicle vaccine based on chitosan antigen delivery system

Abstract

The invention discloses a preparation method and application of a vesicle vaccine based on a chitosan antigen delivery system, the vesicle vaccine stimulates cells to secrete the vesicle vaccine with an antigen presentation function, and the method related to the system specifically comprises the following steps: step 1, mixing antigen protein with a chitosan aqueous solution and a sodium tripolyphosphate aqueous solution, and carrying out electrostatic interaction to obtain a vesicle vaccine solution; chitosan nanoparticles loaded with the antigen protein are prepared; and 2, treating immune cells by using the antigen-loaded chitosan nanoparticles, collecting cell culture supernatant, and collecting and preparing vesicles by adopting a method of combining multi-step differential centrifugation with ultrafiltration. After the antigen delivery system based on chitosan is co-incubated with cells, antigen protein can be effectively delivered into immune cells, meanwhile, the immune cells are promoted to generate a large number of antigen-related vesicles with efficient immunogenicity through the adjuvant effect and autophagy regulation effect of chitosan, and the immunogenicity of the antigen-related vesicles is improved. The application potential of the artificial antigen presenting cell is realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to a preparation method and application of a vesicle vaccine based on a chitosan antigen delivery system. BACKGROUND

[0002] Vaccines based on extracellular vesicles (such as exosome vaccines) have shown great potential in the fields of cancer treatment and infectious disease prevention due to their natural immune activation ability, targeting ability and excellent biocompatibility. However, although the vesicle vaccine is safer than the traditional vaccine and has fewer side effects in clinical application, the immunogenicity of naturally secreted vesicles is usually insufficient to induce a strong and effective immune response. By enhancing the immunogenicity and functionality of the vesicles, it is expected to overcome the application bottleneck of the existing vesicle vaccines.

[0003] To address the aforementioned problems, this invention designs a multifunctional antigen delivery vesicle system that enhances the uptake, processing, and presentation of antigens by immune cells, promoting the secretion of highly immunogenic vesicles with antigen-presenting functions. To this end, we utilize the natural adjuvant chitosan to synthesize chitosan-based ovalbumin (OVA) loading nanoparticles (NP), abbreviated as CS-OVA-NP, and utilize their antigen delivery and simultaneous immune activation stimulation capabilities to prepare vaccine vesicles. These nanoparticles not only effectively load antigen proteins but also enhance macrophage adhesion through electrostatic interactions, significantly increasing the macrophage uptake rate of antigen proteins. Furthermore, chitosan not only acts as an adjuvant to activate antigen presentation and immune activation in macrophages but also enhances vesicle secretion, which may be related to its ability to promote autophagy. We isolated and purified macrophage-derived vesicles using ultrafiltration. We discovered that the vesicle surface received immune activation signals from source cells, directly activating the in vitro proliferation of specific T cells without relying on other adjuvants or antigen-presenting cells. This indicates that the vesicles can deliver antigen peptide-major histocompatibility complex (MHC) formed after antigen protein treatment. This antigen presentation mode significantly improved the efficiency of immune activation, demonstrating the potential of vesicles as artificial nanoantigen delivery carriers. Further in vivo evaluation of the vesicle's immunogenicity revealed that the vesicle vaccine rapidly activated the body's immune response. Vesicles secreted by cells regulated by CS-OVA-NP showed significantly increased antibody levels as early as the seventh day post-immunization. This rapid response may be due to the direct delivery of pretreated antigen peptide-MHC by the vesicle vaccine, omitting the in vivo antigen processing process and thus accelerating the initiation of the immune response. Furthermore, by directly stimulating T cells, the vesicle vaccine significantly enhanced cellular immunity while activating humoral immunity. Therefore, we can prepare antigen-presenting vesicles by directly loading viral antigens onto chitosan particles. This significantly improved the survival rate of mice under lethal doses of influenza virus challenge. In summary, this study presents a simple, universal, and effective strategy for preparing in vitro vesicle vaccines. This method eliminates the need for genetic engineering of the vaccine; it simply involves delivering viral antigens into immune cells to prepare vesicle vaccines, providing a new approach for the development of future universal vaccines. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes a method for preparing and applying a vesicle vaccine based on a chitosan antigen delivery system. The method involves mixing an antigen protein with an aqueous chitosan solution and an aqueous sodium tripolyphosphate solution, and then preparing chitosan nanoparticles loaded with the antigen protein through electrostatic interactions. These nanoparticles not only effectively load the antigen protein but also enhance adhesion to macrophages through electrostatic interactions, significantly improving the uptake rate of the antigen protein by macrophages. Furthermore, chitosan not only acts as an adjuvant to activate antigen presentation and immune activation in macrophages but also enhances vesicle secretion, which may be related to its ability to promote autophagy. We isolated and purified macrophage-derived vesicles using ultrafiltration. The surface of these vesicles received immune activation signals from the source cells, directly activating the in vitro proliferation of specific T cells without relying on other adjuvants or antigen-presenting cells, indicating that these vesicles can deliver the antigen peptide-major histocompatibility complex formed after antigen protein treatment. In in vivo experiments, this vesicular vaccine directly delivered pretreated antigenic peptides-major histocompatibility complexes, omitting the in vivo antigen processing process, directly stimulating T cells, and rapidly initiating the body's immune response. Significant antibody levels were detectable as early as seven days post-injection. This vesicular vaccine significantly enhanced cellular immunity while activating humoral immunity. Since the synthesis principle of chitosan nanoparticles is based on electrostatic interactions, this synthesis strategy has a certain degree of versatility and is expected to be applicable to the loading of other antigenic proteins (as long as the reaction solution (pH≈5) is negatively charged), making it suitable for developing vaccines against different pathogens. In summary, the chitosan-based vesicular vaccine of this invention can achieve the purpose of stimulating the body's immunity and preventing and controlling infectious diseases.

[0005] A method for preparing and applying a vesicle vaccine based on a chitosan antigen delivery system includes the following steps:

[0006] Step 1: Mix the antigen protein with chitosan aqueous solution and sodium tripolyphosphate aqueous solution, and prepare chitosan nanoparticles loaded with antigen protein through electrostatic interaction;

[0007] Step 2: After co-incubating immune cells with the aforementioned antigen-loaded chitosan nanoparticles, the cell supernatant is collected, and a vesicle solution is prepared by multi-step differential centrifugation combined with ultrafiltration, which is the vesicle vaccine based on the chitosan delivery system.

[0008] The chitosan-based antigen delivery system of this invention, after co-incubation with cells, can effectively deliver antigen proteins into immune cells. Simultaneously, through the adjuvant and autophagy-regulating effects of chitosan itself, it promotes the production of a large number of highly immunogenic antigen-associated vesicles by immune cells. These vesicles are collected using a simplified ultrafiltration method. These vesicles can directly stimulate the proliferation of antigen-specific T cells in vitro, showing potential for application as artificial antigen-presenting cells. In vivo, it can stimulate highly effective antigen-specific immune protection, making it a highly efficient vesicle vaccine system.

[0009] Step one involves preparing chitosan nanoparticles loaded with antigen proteins, specifically including:

[0010] Prepare a chitosan solution using ultrapure water, adjust the pH to 4.5-5.0, filter the solution, add the antigen protein to a final concentration of 0.02-0.2 mg / mL, then add an aqueous solution of sodium tripolyphosphate under gentle magnetic stirring. Continue stirring the reaction solution for 15-45 min, centrifuge the reaction solution, discard the supernatant, and obtain chitosan nanoparticles loaded with antigen protein.

[0011] In step one, adjust the pH to 4.7-4.8. Add the antigen protein to a final concentration of 0.05-0.15 mg / mL. Continue stirring the reaction solution for 25-35 minutes.

[0012] In step two, immune cells are prepared into vesicle solutions using a multi-step differential centrifugation combined with ultrafiltration, specifically including:

[0013] After culturing the immune cells, the cells were washed with PBS, and then the culture medium was replaced with complete culture medium containing chitosan nanoparticles loaded with antigen proteins. The cells were incubated for 6–10 h. After incubation, the cells were washed with PBS, replaced with serum-free culture medium, and cultured for another 16–22 h. During the vesicle collection stage, the vesicle solution was obtained by centrifugation.

[0014] In step two, incubate for 7–9 hours. Continue culturing for another 18–20 hours. The centrifugation process specifically includes:

[0015] All centrifugation operations were performed at 3–5°C. First, the culture medium was collected and centrifuged sequentially at 250–350g for 8–12 min, 1800–2200g for 8–12 min, and 9500–10500rpm for 25–35 min. The supernatant was collected after each centrifugation step. Then, the medium was centrifuged at 3500–4500g for 25–35 min using a 100kDa (Molecular weight cut-off, MWCO) ultrafiltration tube. The filtrate in the centrifuge tube was discarded, PBS was added, and the medium was centrifuged at 3500–4500g for 10–20 min as a washing step. The collected liquid was the vesicle solution.

[0016] A further preferred embodiment is a method for preparing a vesicle vaccine based on a chitosan delivery system, the method comprising the following steps:

[0017] Step 1: Prepare 5-10 mL of chitosan solution with a concentration of 3-5 mg / mL and a degree of deacetylation greater than 80% using ultrapure water. Adjust the pH to 4.7-4.8 with 1M NaOH aqueous solution. Filter the solution through a 0.22 μm filter membrane. Add the antigen protein; for example, add ovalbumin (OVA) to a final concentration of 0.1 mg / mL. Then, add 2-4 mL of sodium tripolyphosphate (TPP) aqueous solution with a concentration of 1.6-1.9 mg / mL under gentle magnetic stirring. Continue stirring the reaction solution at room temperature for 30 min. Transfer the reaction solution to a 15 mL conical centrifuge tube and centrifuge at 4°C and 10,000 rpm for 30 min. Discard the supernatant to obtain chitosan nanoparticles loaded with the antigen protein (CS-OVA-NP).

[0018] Step Two: Vesicles derived from immune cells were prepared using a low-speed, multi-step differential centrifugation combined with ultrafiltration. The specific procedure is as follows: RAW264.7 cells were cultured to 80% confluence, washed with PBS, and then replaced with complete medium containing CS-OVA-NP (concentration of 1.43 mg / mL based on chitosan content), and incubated for 8 hours. After incubation, the cells were thoroughly washed with PBS, replaced with serum-free medium, and cultured for another 19 hours. During the vesicle collection phase, all centrifugation operations were performed at 4°C. First, the medium was collected by sequentially centrifuging at 300g for 10 min, 2000g for 10 min, and 10000 rpm for 30 min, collecting the supernatant at each centrifugation step. Subsequently, the cells were centrifuged at 4000g for 30 min using a 100 kDa MWCO ultrafiltration tube, the filtrate in the centrifuge tube was discarded, 15 mL of PBS was added, and the cells were centrifuged at 4000g for 15 min as a wash. The liquid collected from the filter device was the prepared vesicle solution.

[0019] The pH of the solution was adjusted to 4.7-4.8 because chitosan nanoparticles have the smallest particle size and relatively high antigen protein loading rate within this pH range (as measured experimentally).

[0020] Further, in step one, the chitosan solution concentration was 4 mg / mL, and the volume was 5 mL; the sodium tripolyphosphate concentration was 1.8 mg / mL, and the volume was 2 mL; the final concentration of ovalbumin in the reaction solution was 0.1 mg / mL. In step two, the concentration of CS-OVA-NP co-incubated with RAW264.7 cells was 1.43 mg / mL, and the co-incubation time was 8 h. The serum-free culture time was 19 h. Vesicles were collected by ultrafiltration at 4000 g for 30 min. They were then washed with PBS and centrifuged at 4000 g for 15 min.

[0021] Furthermore, the stirring is magnetic stirring at a speed of 700 rpm.

[0022] A chitosan nanoparticle and vesicle vaccine prepared by the above method.

[0023] Application of chitosan nanoparticles prepared by the above method in in vitro antigen delivery to cells.

[0024] Application of a vesicle vaccine prepared by the above method in artificial antigen-presenting cells ( / in vitro T cell proliferation).

[0025] Application of a vesicle vaccine prepared by the above method in cancer immunotherapy and infectious disease prevention and control.

[0026] The beneficial effects of this invention are as follows:

[0027] (1) Preparation of vesicular vaccines with antigen-presenting capabilities via material methods: This invention constructs a multifunctional antigen delivery system based on chitosan. Chitosan-antigen protein nanoparticles not only effectively load antigen proteins but also enhance adhesion to macrophages through electrostatic interactions, significantly improving the uptake efficiency of antigen proteins by macrophages. Furthermore, chitosan acts as an adjuvant to activate antigen presentation and immune responses in macrophages and may enhance vesicle secretion by promoting autophagy. Macrophages treated with this material are able to secrete vesicles with stronger immunogenicity.

[0028] (2) Vesicle vaccines possess antigen-presenting capabilities that can directly activate T cells: The vesicles prepared in this invention carry MHC-I and MHC-II molecules from the source cells on their surface and are effectively loaded with processed antigenic peptides, enabling them to deliver antigen information to T cells. Notably, these vesicles can directly activate antigen-specific T cells in vitro without the need for exogenous adjuvants or other immune cells. This indicates that the vesicles carry the necessary signals for T cell activation and can function as antigen-presenting cells in vitro. This characteristic facilitates the rapid induction of potent antigen-specific cellular immune responses in vivo.

[0029] (3) After co-incubation with cells, the CS-OVA-NP of this invention can effectively deliver antigen proteins into immune cells. Simultaneously, through the adjuvant and autophagy-regulating effects of chitosan itself, it promotes the release of more and more immunogenic vesicles from immune cells. Vesicles are collected using a simplified ultrafiltration method. These vesicles can directly stimulate the proliferation of antigen-specific T cells in vitro, demonstrating the potential for application as artificial antigen-presenting cells. Furthermore, these vesicles not only possess the advantages common to vesicle vaccines, such as high biocompatibility, high safety, and the ability to be stored for a long period without losing immunotherapeutic activity, but also activate strong antigen-specific cellular immunity and relatively durable antigen-specific humoral immunity in vivo, significantly improving the body's protective ability against high-titer viral environments. The vesicle vaccine preparation strategy proposed in this invention is simple and universal, requiring no genetic engineering modification of the vaccine. By directly delivering viral antigens into immune cells, vesicle vaccines can be prepared, providing a new approach for the development of future universal vaccines, especially with broad application potential in dealing with different viruses.

[0030] (4) Developing a material-based universal viral vaccine preparation strategy: The vesicle vaccine preparation strategy proposed in this invention is simple and universal, requiring no genetic engineering modification of the vaccine. Vesicle vaccines can be prepared by directly delivering viral antigens into immune cells, providing a new approach for the development of universal vaccines in the future, especially with broad application potential in dealing with different viruses. Attached Figure Description

[0031] Figure 1 Transmission electron microscopy (TEM) images of ovalbumin (OVA) loaded (right) and unloaded (left) chitosan particles, scale bar: 100 nm;

[0032] Figure 2 Cytotoxicity of CS-NP (Chitosan nanoparticle), OVA, and CS-OVA-NP at various concentrations;

[0033] Figure 3 Confocal electron microscopy image of RAW 264.7 cells taking up CS-OVA-NP, where chitosan (CS) is labeled with SF555, the cytoskeleton is labeled with AF488 phalloidin, and the cell nuclei are labeled with Hoechst. Scale bar: 20 μm.

[0034] Figure 4 The uptake of CS-NP, OVA, and CS-OVA-NP by RAW264.7 cells over time;

[0035] Figure 5TEM image of a vesicle, scale bar: 100 nm;

[0036] Figure 6 Protein encapsulation efficiency of CS-OVA-NP prepared at different TPP concentrations;

[0037] Figure 7 The abundance of CD63, TSG101 and Calnexin proteins in vesicles of each group;

[0038] Figure 8 The abundance of LC3-I and LC3-II proteins in vesicles of each group;

[0039] Figure 9 The abundance of MHC I and MHC II proteins in vesicles of each group;

[0040] Figure 10 The proliferation of OVA-specific T cells under vesicle stimulation in each group;

[0041] Figure 11 Immunization schedule for CS-OVA vesicle (CS-OVA-EV) vaccine (n=5);

[0042] Figure 12 The serum anti-OVA IgG titers in mice at different time points after immunization;

[0043] Figure 13 The results of ELISPOT assay on day 21 post-immunization show the speckled image (left) and speckled count (right) of interferon-γ (IFN-γ) SFCs. *P represents <0.05, **P represents <0.01, ***P represents <0.001.

[0044] Figure 14 Immunization schedule for extracellular vesicle (CS-H1N1-EV) vaccine treated with chitosan nanoparticles loaded with H1N1 protein (n=7);

[0045] Figure 15 The titers of anti-H1N1 IgG in mouse serum at different time points after immunization;

[0046] Figure 16 Survival curves (left) and weight changes (right) of mice after challenge. Detailed Implementation

[0047] The present invention will now be described in detail with reference to the accompanying drawings and preferred embodiments. The objectives and effects of the present invention will become clearer from this description. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, all percentages mentioned in the following embodiments are mass percentages.

[0048] Example 1

[0049] Prepare 5 mL of a chitosan solution with a 4 mg / mL concentration and a degree of deacetylation greater than 80% using ultrapure water. Adjust the pH to 4.7-4.8 with 1 M NaOH aqueous solution. Filter the solution through a 0.22 μm filter membrane. Add the antigen protein; for example, ovalbumin (OVA) is added to a final concentration of 0.1 mg / mL. Then, add 2 mL of a 1.8 mg / mL sodium tripolyphosphate (TPP) aqueous solution with gentle magnetic stirring. Continue stirring the reaction solution at room temperature for 30 min. Transfer the reaction solution to a 15 mL conical centrifuge tube and centrifuge at 4 °C and 10,000 rpm for 30 min. Discard the supernatant to obtain chitosan nanoparticles loaded with the antigen protein (CS-OVA-NP).

[0050] Immunocyte-derived vesicles were prepared using a combination of low-speed multi-step differential centrifugation and ultrafiltration. The specific procedure was as follows: RAW264.7 cells were cultured to 80% confluence, washed with PBS, and then replaced with complete medium containing CS-OVA-NP (gibco DMEM medium with +10% FBS (fetal bovine serum) +1% P / S (penicillin-streptomycin)) (CS concentration 1.43 mg / mL), and incubated for 8 h. After incubation, the cells were thoroughly washed with PBS, replaced with serum-free medium (gibco DMEM medium with +1% P / S (penicillin-streptomycin), and cultured for another 19 h. During vesicle collection, all centrifugation was performed at 4°C. First, the medium was collected by sequential centrifugation at 300g for 10 min, 2000g for 10 min, and 10000 rpm for 30 min, collecting the supernatant at each centrifugation step. Subsequently, the solution was centrifuged at 4000g for 30 min using a 100kDa MWCO ultrafiltration tube. The filtrate in the centrifuge tube was discarded, 15mL of PBS was added, and the solution was centrifuged at 4000g for 15 min as a wash. The liquid in the Filter Device was collected, which is the prepared vesicle solution (CS-OVA-EV).

[0051] Example 2

[0052] Prepare 10 mL of a chitosan solution with a concentration of 4 mg / mL and a degree of deacetylation greater than 80% using ultrapure water. Adjust the pH to 4.7-4.8 with 1M NaOH aqueous solution. Filter the solution through a 0.22 μm filter membrane. Add the antigen protein; for example, ovalbumin (OVA) is added to a final concentration of 0.1 mg / mL. Then, add 4 mL of a 1.8 mg / mL sodium tripolyphosphate (TPP) aqueous solution with gentle magnetic stirring. Continue stirring the reaction solution at room temperature for 30 min. Transfer the reaction solution to a 15 mL conical centrifuge tube and centrifuge at 4 °C and 10,000 rpm for 30 min. Discard the supernatant to obtain chitosan nanoparticles loaded with the antigen protein (CS-OVA-NP).

[0053] Immunocyte-derived vesicles were prepared using a combination of low-speed multi-step differential centrifugation and ultrafiltration. The specific procedure was as follows: RAW264.7 cells were cultured to 80% confluence, washed with PBS, and then replaced with complete medium containing CS-OVA-NP (CS concentration 1.43 mg / mL), and incubated for 8 hours. After incubation, the cells were thoroughly washed with PBS, replaced with serum-free medium, and cultured for another 19 hours. During vesicle collection, all centrifugation was performed at 4°C. First, the medium was collected by sequential centrifugation at 300g for 10 min, 2000g for 10 min, and 10000 rpm for 30 min, collecting the supernatant at each centrifugation step. Then, the cells were centrifuged at 4000g for 30 min using a 100 kDa MWCO ultrafiltration tube, the filtrate was discarded, 15 mL of PBS was added, and the cells were washed by centrifugation at 4000g for 15 min. The liquid collected from the filter device was the prepared vesicle solution (CS-OVA-EV).

[0054] Example 3

[0055] We obtained CS-H1N1-NP loaded with H1N1 virus lysis buffer using a method similar to that in Example 1. The specific procedure for UV inactivation of H1N1 was as follows: after the virus solution was thawed, it was spread in a petri dish and irradiated with a UV lamp for 30 minutes. 1×10 7 PFU mL -1H1N1 virus (WSN) was concentrated in a 50kDa MWCO ultrafiltration tube, and 10% Triton X-100 was added to a final concentration of 1%, followed by lysis at room temperature for 90 minutes. The viral lysate was purified using the HiPPR Detergent Removal Spin Column Kit. The specific steps are as follows: (1) Place an empty centrifuge column in a 2mL centrifuge tube. Gently shake with HiPPR detergent to remove the resin. Using a 5mL pipette tip, add an appropriate volume of resin suspension to the centrifuge column. (2) Centrifuge at 1500g for 1 minute to remove the storage buffer and discard the separated liquid. (3) Add an appropriate volume of PBS (PBS:resin = 1:1 (v:v)), centrifuge at 1500g for 1 minute, and discard the separated liquid. Repeat this step twice. (4) Insert the bottom plug and place the column into a new collection tube. (5) Slowly add the sample directly to the top of the compacted resin bed. (6) Cover the column and gently vortex to mix the sample with the resin. Incubate at room temperature for 10 minutes. (7) Loosen the cap slightly, remove the bottom stopper, and place the column into the collection tube. Centrifuge at 1500g for 2 minutes and collect the sample without detergent. Discard the column used.

[0056] Based on the cytotoxicity results, purification twice was recommended for safety. The cytotoxicity mainly originated from residual Trithon X-100. Pure free H1N1 lysis buffer or CS-H1N1-NP showed no cytotoxicity (no significant cell death was observed after 8 hours of co-culturing).

[0057] The purified H1N1 lysis buffer (1 mL of virus concentrated to 100 μL, with any shortfall made up with PBS) was premixed with 5 mL of 4 mg / mL CS solution. Under gentle stirring, 2 mL of 1.8 mg / mL TPP aqueous solution was added. Stirring continued for 30 min. The resulting solution was centrifuged at 10,000 rpm for 30 min at 4 °C. The supernatant was discarded, and the solution was resuspended in ultrapure water to obtain CS-H1N1-NP.

[0058] Preparation of CS-H1N1-EV: The experimental steps are the same as in Example 1, except that CS-OVA-NP in Example 1 is replaced with CS-H1N1-NP (the concentration based on CS remains unchanged), and the other steps are the same.

[0059] Specifically, vesicles derived from immune cells were prepared using a combination of low-speed, multi-step differential centrifugation and ultrafiltration. The specific procedure was as follows: RAW264.7 cells were cultured to 80% confluence, washed with PBS, and then replaced with complete medium containing CS-H1N1-NP (CS concentration 1.43 mg / mL) for 8 hours. After incubation, the cells were thoroughly washed with PBS, replaced with serum-free medium, and cultured for another 19 hours. During vesicle collection, all centrifugation was performed at 4°C. First, the medium was collected, followed by sequential centrifugation at 300g for 10 min, 2000g for 10 min, and 10000 rpm for 30 min, collecting the supernatant at each centrifugation step. Subsequently, the solution was centrifuged at 4000g for 30 min using a 100kDa MWCO ultrafiltration tube. The filtrate in the centrifuge tube was discarded, 15mL of PBS was added, and the solution was centrifuged at 4000g for 15 min as a wash. The liquid in the Filter Device was collected, which is the prepared vesicle solution (CS-H1N1-EV).

[0060] Morphology characterization of chitosan nanoparticles: TEM

[0061] Transmission electron microscopy (TEM) characterization of chitosan nanoparticles

[0062] After centrifugation, the reaction solution was resuspended in ultrapure water at an appropriate concentration, dropped onto a carbon support membrane, and excess liquid was absorbed with filter paper after 1 minute. The solution was then dried overnight. The instrument's accelerating voltage was 100 kV.

[0063] The presence of (using transmission electron microscopy) Figure 1 (Right) or no load ( Figure 1 (Left) Morphological characterization of chitosan nanoparticles containing OVA protein. From... Figure 1 It can be observed that the chitosan nanoparticles obtained under these synthesis conditions exhibit an irregular, near-spherical structure. Since the electron microscope samples need to be dehydrated before loading, and the nanoparticle size potentiometer measures the hydrated particle size, the former's particle size is usually smaller than the latter's.

[0064] Cytotoxicity characterization of chitosan nanoparticles: CCK8

[0065] RAW264.7 cells were cultured in DMEM medium (hereinafter referred to as complete medium) containing 10% fetal bovine serum, 100 U / mL penicillin, and streptomycin. The cell culture conditions were a humidified incubator at 37°C and 5% CO2. Unless otherwise specified, the culture conditions for RAW264.7 cells were the same and will not be repeated hereafter.

[0066] With 5×10 4RAW264.7 cells were seeded in 96-well plates at a density of 10 cells / well. Cell adhesion was observed overnight. The cells were washed twice with PBS, and the culture medium was replaced. CS nanoparticles (CS group), free OVA protein (OVA group), and CS-OVA-NP (CS-OVA group) were dispersed in ultrapure water and mixed with an equal volume of DMEM (2×) medium containing 20% ​​FBS, 200 U / mL penicillin, and streptomycin (hereinafter referred to as DMEM (2×) complete medium). The mixture was then added to the medium at a specific concentration. After co-incubating the materials with the cells for 24 h, the cells were washed twice with PBS, and the culture medium was replaced. 10 μL of CCK8 solution was added to each well, and after incubation for 2 h, the absorbance at 450 nm was measured using a microplate reader.

[0067] Before the formal cell experiments began, the cytotoxicity of different concentrations of CS-NP, OVA, and CS-OVA-NP on RAW264.7 cells was tested using the CCK-8 assay kit. The results are as follows: Figure 2 As shown in the image, the CCK-8 kit assesses cell viability by detecting the activity of mitochondrial enzymes within cells. When the water-soluble tetrazolium dye in the reagent enters the cells, dehydrogenases in the mitochondria of living cells reduce it to a yellow formazan compound. The amount of formazan generated is directly proportional to the number of viable cells. After incubation for a certain period, the cell proliferation or survival can be quantitatively determined by measuring the absorbance at 450 nm.

[0068] from Figure 2 As can be seen, CS-OVA-NP did not exhibit significant toxicity to RAW264.7 cells at concentrations of 1.43 mg / mL or less. At low concentrations, cell viability data for all groups exceeded 100%, which may be because CS-NP, OVA, and CS-OVA-NP promoted RAW264.7 cell proliferation or enhanced mitochondrial activity at low concentrations.

[0069] Characterization of chitosan particle delivery efficiency: laser confocal microscopy & flow cytometry

[0070] Laser confocal microscopy characterizes cellular uptake:

[0071] CS-SF555 staining protocol: Dissolve 10 mg / mL Solar Fluor 555 and SE in DMSO. Mix the two solutions at a ratio of CS:Solar Fluor 555,SE = 20 mg:1 μL. After reacting in the dark for 10 minutes, transfer to a dialysis bag with an MWCO of 3500 and dialyze using 100 times the volume of ultrapure water for 2 days, changing the solution every 24 hours. Adjust the pH of the dialyzed chitosan aqueous solution to 4.7-4.8 with 5% acetic acid aqueous solution, filter through a 0.22 μm filter membrane to obtain the CS-SF555 solution, and store it in the dark at 4°C for later use.

[0072] Staining protocol for cytoskeleton-AF488: After co-incubation of materials and cells, wash three times with PBS, fix with 1% paraformaldehyde at room temperature for 15 min. Wash twice with PBS. Treat with strong immunostaining permeabilization buffer for 15 min. Wash twice with PBS. Use QuickBlock staining solution. TM Incubate with immunostaining blocking buffer for 10 min. Wash twice with PBS. Add Alexa Fluor. TM Incubate with 488g Phalloidin at room temperature for 45 minutes. Wash three times with PBS.

[0073] Nuclear-Hoechst staining protocol: After co-incubation of materials and cells, add Hoechst 33342 live cell staining solution (1×) and stain for 4 min. Wash 3 times with PBS.

[0074] Laser confocalization experiment: with 1.5 × 10 6 RAW264.7 cells were seeded in confocal culture dishes at a density of 1 cell / dish. Cells adhered overnight. The cells were washed twice with PBS, and the culture medium was replaced. Following the staining protocol, stained chitosan nanoparticles were synthesized using method 2.3.1 and mixed with an equal volume of DMEM (2×) complete culture medium, then added to the medium at a specific concentration. The material was co-incubated with the cells for 4 hours before subsequent staining. After adding strong anti-fluorescence quenching mounting medium, the uptake of the material by the cells was observed using a confocal microscope.

[0075] To better observe the specific uptake of materials by cells, we synthesized chitosan nanoparticles using SF555 and SE-stained CS and unlabeled OVA protein, and employed Alexa Fluor... TM Phalloidin was used to stain actin in RAW264.7 cells, and the staining was observed at a high magnification. The results are as follows: Figure 3 As shown.

[0076] Alexa Fluor TM 488 Phalloidin can selectively bind to F-actin, revealing the cytoskeleton and thus outlining the cell contours. From Figure 3 It can be seen that CS-OVA-NP can be effectively taken up by cells. Figure 3 For example, among 55 cells that showed complete nuclear outlines, 50 cells took up CS-OVA-NP to varying degrees, and the cell uptake rate of the material exceeded 90%.

[0077] Flow cytometry characterizes cellular uptake

[0078] With 1×10 6RAW264.7 cells were seeded in 12-well plates at a density of 10 cells / well. Cells adhered overnight. The cells were washed twice with PBS, and the culture medium was changed. Synthetic chitosan nanoparticles loaded with OVA-FITC (CS-OVA-FITC-NP) were used. CS-NP, free OVA-FITC protein, and CS-OVA-FITC-NP were dispersed in ultrapure water and mixed with an equal volume of DMEM (2×) complete culture medium, then added to the medium at a specific concentration. After co-incubation with the cells for a certain period, the cells were thoroughly washed with PBS. The green fluorescence of the cells was observed using flow cytometry.

[0079] To more accurately quantify cellular uptake of the material and explore the optimal co-incubation time, chitosan nanoparticles were synthesized using unstained CS and OVA-FITC. Cells were collected after 8 h, 12 h, and 24 h of co-incubation, and the green fluorescence of the cells was detected using flow cytometry. Figure 4 Flow cytometry uses lasers to detect intracellular fluorescence signals. When cells engulf FITC-labeled particles, they are irradiated with a laser, causing FITC to emit green fluorescence. The instrument detects and records the fluorescence intensity, thereby assessing the proportion of cells that have taken up FITC-labeled particles and the strength of the fluorescence signal.

[0080] In other flow cytometry experiments, CS exhibited a certain degree of autofluorescence. Therefore, the CS group was specifically analyzed in this experiment to eliminate the influence of CS autofluorescence on the results. Figure 4 As the co-incubation time increased, the uptake of CS-OVA-NP by RAW264.7 cells showed a continuous upward trend, while the uptake of free OVA protein remained unchanged. Furthermore, at the 8-hour incubation point, the uptake of CS-OVA-NP by RAW264.7 cells significantly exceeded the uptake of free OVA protein. These results indicate that chitosan, as a carrier, significantly improves the efficiency of OVA protein uptake by cells, and that time is a crucial factor affecting the uptake amount.

[0081] Vesicle morphology characterization: TEM

[0082] The obtained EV solution was diluted appropriately and dropped onto a copper grid. After negative staining with 4% uranium acetate, it was observed using a transmission electron microscope with an accelerating voltage of 100 kV.

[0083] The morphology of the prepared vesicles was characterized by transmission electron microscopy, and the results are as follows: Figure 5As shown, vesicles exhibit a typical cup-shaped structure. Extracellular vesicles are typically nearly spherical in vivo, encapsulated by a phospholipid bilayer. During TEM sample preparation, the central portion of the vesicle collapses due to dehydration. Simultaneously, the negative staining agent uranium acetate primarily surrounds the outer structure of the vesicle, rarely penetrating its interior, further emphasizing the cup-shaped appearance of the vesicle.

[0084] Chitosan nanoparticles effectively load antigen proteins

[0085] Characterization of encapsulation efficiency of chitosan nanoparticles

[0086] The encapsulation efficiency of chitosan nanoparticles for OVA protein was determined using the Bicinchoninic Acid Assay (BCA). The specific method was as follows: after centrifugation of the reaction solution, the supernatant was collected, mixed, and the free protein concentration was determined using a BCA protein concentration assay kit. A standard curve was plotted based on OVA aqueous solutions of known concentrations. The standard curve (R) was used... 2 The concentration of free protein in the supernatant was calculated (>0.99), and the encapsulation efficiency of chitosan for OVA protein was then determined. The specific formula is as follows:

[0087]

[0088] To determine the efficiency of chitosan particles in encapsulating proteins, we used the BCA method to measure the protein encapsulation efficiency of CS-OVA-NP prepared at different TPP concentrations.

[0089] from Figure 6 As can be seen, the encapsulation efficiency of chitosan particles for the target protein increases with increasing TPP concentration. Specifically, when the TPP concentration increases from 1.7 mg / mL to 1.8 mg / mL, the protein encapsulation efficiency rises significantly from 23.2% to 48.7%; however, when the TPP concentration increases from 1.8 mg / mL to 1.9 mg / mL, the encapsulation efficiency only increases from 48.7% to 53.9%, a smaller increase. The selected TPP concentrations all resulted in solutions with a pH close to 9.3. Increasing the TPP concentration raises the overall pH of the reaction system, which is beneficial for chitosan precipitation. Simultaneously, because the isoelectric point of OVA protein is 4.8, increasing the pH allows OVA to carry more negative charges, resulting in stronger electrostatic attraction with the positively charged chitosan. Furthermore, as a small molecule anion, higher concentrations of TPP promote better cross-linking of chitosan, thereby encapsulating more target protein. Further increasing the TPP concentration resulted in the rapid precipitation of a flocculent white precipitate in the reaction solution, suggesting that the maximum TPP concentration in this synthesis system is around 1.9 mg / mL. Figure 6Based on this trend, we speculate that the protein encapsulation limit for OVA under this synthetic system is around 53.9%. In summary, considering the average particle size, PDI, Zeta potential, and protein encapsulation efficiency, we ultimately selected a TPP concentration of 1.8 mg / mL as the subsequent synthetic conditions.

[0090] Materials regulate the release of antigen-presenting vesicles from immune cells.

[0091] Surface protein characterization of vesicles

[0092] Western blot characterization of vesicle surface proteins was performed using the following steps: An equal volume of lysis buffer was added to each EV sample. 1.5 mL of lysis buffer was added to each 15 cm dish of cells, and lysis was carried out on ice for 15 min. The lysis buffer was collected by pipetting. The lysis buffer was then centrifuged at 14,000 rpm for 15 min at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA kit. 10 μg of protein was mixed with loading buffer and incubated at 100°C for 10 min. The sample was loaded onto a 12% SDS-PAGE gel for electrophoresis and then transferred to a PVDF membrane. After washing three times with TBST, the membrane was blocked with 5% skim milk at room temperature for 1 h. After washing three times with TBST, the membrane was incubated with primary antibody overnight at 4°C. After washing three times with TBST, the membrane was incubated with secondary antibody (Goat anti-Rabbit IgG(H+L)-HRP) at room temperature for 2 h. After washing three times with TBST, exposure buffer was evenly added to the membrane, and banding was photographed.

[0093] In Western blotting experiments, the proteins involved in identifying vesicle marker proteins are mainly classified into five categories: (1) transmembrane proteins associated with the plasma membrane and / or endosomes; (2) cytoplasmic proteins in EVs; (3) non-EV components separated together with EVs; (4) transmembrane proteins, lipid-binding proteins, and soluble proteins associated with intracellular compartments outside the plasma membrane and endosomes; and (5) secretory proteins recovered from EVs. [1] We selected CD63 from the Tetraspanins family (class 1), TSG101 from class 2, and Calnexin from class 4 as markers for our study of vesicles. CD63 is a protein associated with transmembrane transport. TSG101 is a key component of the ESCRT (Endosomal Sorting Complex Required for Transport) complex, involved in the formation of intracellular multivesicles and the sorting of vesicle contents. Calnexin is a chaperone protein residing in the endoplasmic reticulum membrane, mainly involved in protein folding and quality control processes.

[0094] from Figure 7It can be observed that the surface of CS-OVA-EVs is rich in CD63 protein, contains a small amount of TSG101 protein, and a very small amount of Calnexin. The presence of CD63 and TSG101 indicates that the prepared CS-OVA-EVs have typical vesicle characteristics. The presence of Calnexin protein, which is absent in exosomes, suggests that the vesicles prepared by this method contain some proteins derived from the endoplasmic reticulum, possibly indicating that some vesicles originate from the endoplasmic reticulum. The endoplasmic reticulum may contain antigen-presenting related proteins.

[0095] Furthermore, studies have found that chitosan can promote autophagy. Therefore, we further investigated whether CS-OVA-EV is related to autophagosomes. A significant characteristic of autophagosomes is the decreased ratio of LC3-I to LC3-II on their surface. To further investigate the obtained vesicles, we examined the ratio of LC3-I to LC3-II on the surface of CS-OVA-EV.

[0096] from Figure 8 As can be seen, the ratio of LC3-I to LC3-II on the surface of CS-OVA-EV did not change significantly, and its level was not much different from that of the control group (PBS). This indicates that the vesicles we prepared were not derived from autophagosomes, or at least not mainly composed of autophagosomes.

[0097] Whether CS-OVA-EV possesses high immunogenicity is related to the abundance of antigen-presenting proteins expressed on its surface. Therefore, we selected MHC, the first signal required for antigen-presenting cells to activate T cells, for our study. To better analyze the differences in protein expression between vesicles and their source cells, in this Western blotting experiment, we collected the source cells of the vesicles, lysed them, and then loaded them onto the sample.

[0098] from Figure 9 The results showed that the abundance of MHC-I and MHC-II in RAW264.7 cells was slightly increased after material treatment, indicating that the material can activate RAW264.7 cells. Further analysis of MHC expression on the surface of CS-OVA-EV revealed that it was consistent with the MHC-I and MHC-II expression in RAW264.7 cells, indicating that CS-OVA-EV acquired MHC from the source cells. This suggests that the vesicles have the potential to stimulate T cell proliferation and can be considered as an artificial nano-APC.

[0099] Research on the immune function of vesicles

[0100] This study investigated the immune activation function of EVs by evaluating their ability to stimulate the proliferation of antigen-specific T cells in vitro. OT-1 mouse T cells express TCRs that specifically recognize the OVA 257-264 peptide, thus exhibiting natural OVA specificity. CFSE-labeled cells exhibit uniform and stable fluorescence, with the fluorescence intensity halving with each cell division, allowing for the detection of cell proliferation. The materials were used to stimulate CFSE-labeled OT-1 mouse T cells, and the fluorescence signal was detected by flow cytometry to assess the proliferation of antigen-specific T cells. Specific experimental procedures were as follows: Spleen cells from OT-1 mice were obtained, passed through a 70μm cell sieve, and the resulting cells were processed using EasySep. TM CD3 was isolated using a mouse T-cell isolation kit. + T cells. Viable cell count was determined using trypan blue. CFSE working solution was added, and the cells were incubated at room temperature in the dark for 30 min. Cells were washed twice with PBS, resuspended in complete culture medium, and cultured at 5 × 10⁶ cells / mL. 5 Cells per well were seeded in 48-well plates. 10 μL of EV was added to each well (each 10 μL of EV is converted from 1 × 10⁻⁶ cells / well). 7 (Preparation of individual cells), mix well. After incubation for 3 days, collect cells, wash with PBS, and detect fluorescence signals using flow cytometry.

[0101] To more precisely investigate the immune activation function of vesicles and their potential as vaccines, we employed a T-cell proliferation assay to study whether vesicles could stimulate the proliferation of antigen-specific T cells. Whether CS-OVA-EVs generated from RAW264.7 cells treated with CS-OVA-NP have the ability to activate OVA-specific T cells would demonstrate whether CS-OVA-EV vesicles can carry antigenic peptides, which is crucial for their potential as a vaccine.

[0102] APCs deliver antigenic information to T cells via MHC molecules loaded with antigenic peptides, activating naive T cells and promoting their proliferation and differentiation into CTLs. CTLs then specifically kill infected cells or pathogens by recognizing the pMHC presented by APCs; this process is the core mechanism of adaptive cellular immunity. We isolated CD3+ cells from the spleen of OT-1 mice using the EasySep™ mouse T cell isolation kit. + T cells, by evaluating CS-OVA-EV for OVA-specific CD3 +The activation of T cells was investigated to verify whether the MHC molecules on the EV effectively load antigen peptides and transmit information, while also observing its overall activation effect on T cell proliferation. pMHC signaling alone is insufficient to fully activate T cells. If CS-OVA-EV promotes antigen-specific T cell proliferation in the absence of exogenous adjuvants or other immune cells, it indicates that it possesses all the key signals required to drive T cell proliferation and can effectively activate the body's specific cellular immunity as a vaccine.

[0103] During each cell division, the CFSE fluorescence is evenly distributed to the two daughter cells, causing the fluorescence signal peak to split into a sub-peak with half the fluorescence intensity. As the cell proliferates multiple times, the fluorescence intensity gradually decreases. Figure 10 Visually, CS-OVA-EV can induce at least four divisions of antigen-specific T cells, while no significant T cell proliferation was observed in other control groups. Flow jo analysis was used to analyze the proliferation data, calculating the percentage of divided cells (the ratio of proliferating cells to the total number of cells before stimulation). The percentages of divided cells in the PBS, CS-EV, OVA-EV, and CS-OVA-EV groups were 4.3%, 10.4%, 6.0%, and 36.3%, respectively. The percentage of divided cells in the CS-OVA-EV group was 6.05 times that of the OVA-EV group, indicating that the chitosan-antigen complex delivery system is more effective than free antigen proteins in promoting the secretion of antigen-presenting vesicles by immune cells. The comparison with the CS-EV group shows that the promoting effect of CS-OVA-EV on antigen-specific T cell proliferation is not solely induced by the chitosan material, ruling out the possibility of false positives. In summary, CS-OVA-EV can directly activate OVA-specific T cells and significantly promote their proliferation in vitro without the participation of exogenous adjuvants or other immune cells, possessing all the key signals required for antigen presentation and performing the function of antigen-presenting cells.

[0104] Vesicle vaccines rapidly initiate the body's immune response and activate balanced humoral immunity and powerful cellular immunity.

[0105] While vesicle-based vaccines offer advantages such as high biocompatibility and safety, they suffer from low immunogenicity. To enhance their immunogenicity, we introduced a natural adjuvant, chitosan, to synthesize chitosan-antigen-protein composite nanoparticles. This multifunctional antigen delivery system not only improves the uptake of antigens by macrophages but also directly activates macrophages as an adjuvant and promotes EV secretion by regulating autophagy. Since the synthesis of chitosan-antigen-protein composite nanoparticles is based on electrostatic interactions and the reaction conditions are mild, it is expected to be widely applicable to loading different types of antigen proteins.

[0106] In our experiments, we successfully prepared vesicles capable of activating the proliferation of specific T cells in vitro. These vesicles not only carry antigen information but also possess key signals for stimulating T cell activation, holding promise for vaccine development. In this chapter, we first used OVA as a model antigen, synthesized CS-OVA-NP, stimulated RAW264.7 cells, collected the secreted CS-OVA-EV, and then injected it subcutaneously into mice to examine the in vivo immunogenicity of CS-OVA-EV. In this animal experiment, we controlled the same number of source cells and examined the in vivo immunogenicity of vesicles secreted by the same number of RAW264.7 cells after different treatments. We assessed the strength of the antigen-specific humoral immune response induced by the vaccine by detecting the level of OVA-specific antibodies in mouse serum using enzyme-linked immunosorbent assay (ELISA); and assessed the strength of the antigen-specific cellular immune response induced by the vaccine by detecting the level of IFN-γ secreted by mouse spleen cells using enzyme-linked immunospot (ELISPOT) assay.

[0107] To further validate the universality of this EV vaccine preparation strategy and evaluate its protective efficacy against specific viruses, we used a lysed H1N1 influenza virus surrogate antigen (OVA) in our second animal experiment. Influenza viruses are highly variable, with different variant strains emerging each epidemic season. Therefore, it is necessary to develop vaccines that can be rapidly prepared and target specific strains to effectively address these variations. Meanwhile, existing influenza vaccines primarily focus on inducing humoral immunity, with less emphasis on strongly stimulating cellular immunity. For individuals who cannot produce sufficient protective antibody levels (such as the elderly), enhancing cellular immune responses is particularly crucial. Our prepared vesicles can directly activate specific T cell proliferation, and are expected to have a significant effect on enhancing cellular immune responses. Furthermore, the vesicle vaccine preparation strategy exhibits a certain degree of universality. Based on this, we chose an influenza virus model to evaluate the universal immunoprotective efficacy of the CS-antigen-EV vaccine.

[0108] We prepared the CS-H1N1-EV influenza vaccine using a method similar to that used for the CS-OVA-EV vaccine. Under conditions of consistent vesicle source cell numbers, we found that the concentration of vesicle proteins secreted by RAW264.7 cells treated with CS-NP or free OVA was significantly lower than that of cells treated with CS-OVA-NP (i.e., the experimental group). Therefore, in the second animal experiment, we appropriately diluted the vesicles in the experimental group to ensure similar protein concentrations across all groups, thereby evaluating the immunoprotective efficacy of different vesicle vaccines under the same protein concentration conditions.

[0109] In an influenza virus model, we also subcutaneously injected mice with the EV vaccine. To assess the difference in immunoprotective effect between the EV vaccine and the traditional nanoparticle vaccine, we established a group of mice that were subcutaneously injected with CS-H1N1-NP. A booster immunization was administered four weeks after the primary immunization, and blood samples were collected from mice two weeks after each immunization. Serum H1N1-specific antibody levels were detected by ELISA to assess the intensity of the antigen-specific humoral immune response. On day 45 after the primary immunization, the mice were challenged with H1N1 influenza virus to observe the protective effect of the CS-H1N1-EV vaccine against high-titer influenza virus.

[0110] Vesicle vaccine based on model antigen OVA (CS-OVA-EV)

[0111] Vesicle vaccine immunization regimen using OVA as a model protein

[0112] Female BALB / c mice aged 6-8 weeks were randomly divided into four groups of 5 mice each. Following method 3.3.1, 100 μL of vesicles secreted by RAW264.7 cells treated with CS-NP (CS-EV group), free OVA (OVA-EV group), and CS-OVA-NP (CS-OVA-EV group) were collected from each group. The vesicles were then further divided according to the same cell source (10T). 8 The mice were injected subcutaneously with cells per 100 μL, while the PBS group received 100 μL of PBS. The day of injection was recorded as day 0. Blood samples were collected from the retroorbital vein on days 7, 14, and 21 post-injection. Mice were euthanized by cervical dislocation on day 21, and spleen cells were collected for in vitro culture.

[0113] Group Description

[0114]

[0115] Determination of OVA-specific IgG titers induced by CS-OVA-EV vaccine

[0116] Whole blood was collected on days 7, 14, and 21 post-immunization, and serum was obtained by centrifugation. The level of OVA-specific antibodies in mouse serum was detected by ELISA. The specific steps were as follows: 100 μL of 5 μg / ml OVA protein was added to a high-binding 96-well plate and incubated overnight at 4°C. The plate was washed three times with washing buffer (PBS + 0.3% Tween 20) and blotted dry. 100 μL of blocking dilution buffer (PBS + 0.3% Tween 20 + 5% BSA) was added to each well and the plate was blocked at 37°C for 1 hour and blotted dry. Mouse serum was serially diluted with blocking dilution buffer, 100 μL per well, and incubated at 37°C for 1 hour. The plate was washed six times with washing buffer, and 100 μL of 1:10000 diluted Goat anti-Mouse IgG (H+L) Secondary Antibody (HRP) was added to each well and incubated at 37°C for 1 hour. Wash six times with washing buffer, add 100 μL of TMB chromogenic solution to each well, incubate at room temperature for a period of time, and then add 50 μL of 2 mol / L H2SO4 stop solution. Detect the OD value (Optical Density) at 450 nm using a microplate reader. The second strongest OD value in the PBS group mouse serum is used as the positive cutoff; wells with OD values ​​greater than this value are considered positive.

[0117] Assay for CS-OVA-EV vaccine-induced cellular immunity

[0118] Mice were euthanized by cervical dislocation on day 21 post-immunization, and spleen cells were harvested for in vitro culture. Cellular immunity levels in mouse spleen cells were detected using ELISPOT. The specific steps were as follows: Plates were pre-wetted with 70% ethanol, washed three times with PBS, and then coated with Capture antibody and incubated overnight at 4°C. After washing once with Blocking Solution, the plates were incubated at room temperature for 2 hours. Equal amounts of OVA protein and OVA peptide were mixed and prepared into a 200 μg / mL stimulation solution using complete culture medium, with 100 μL added to each well. Mouse spleen cells were collected, ground, and passed through a 70 μm sieve, with an appropriate amount of viable cells added to each well. The plates were incubated at 37°C in a humidified incubator with 5% CO2 for 16 hours. The cell suspension was aspirated. The plates were washed twice with deionized water. The plates were washed three times with Wash Buffer I. 100 μL of Detection antibody dilution was added to each well, and the plates were incubated at room temperature for 2 hours. The solution was discarded, and the plates were washed three times with Wash Buffer I. Add 100 μL of Enzyme Conjugate diluent to each well. Incubate at room temperature for 1 hour. Wash the plate four times with Wash Buffer I. Wash the plate three times with Wash Buffer II. Add 100 μL of Final Substrate Solution to each well. Observe the spot development. Wash the plate with deionized water to terminate the reaction. Pat and air dry the plate at room temperature until completely dry. Count the spots automatically using an ELISA reader.

[0119] Antigen-specific humoral immune response induced by CS-OVA-EV vaccine

[0120] Antigen-specific humoral immunity is an immune response primarily mediated by B cells and antibodies. Its main function is to recognize and eliminate pathogens in body fluids, ensuring the body is protected from infection, and it is an important component of adaptive immunity. When pathogens enter the body, antigen-specific cells (APCs) process and present antigens, which are then displayed to helper T cells via the antigen peptide-MHC II complex. Helper T cells activate B cells, promoting their proliferation and differentiation into plasma cells that produce and release large quantities of antibodies. Antibodies can specifically bind to pathogens, inactivating them or promoting clearance by phagocytes. The strength of antigen-specific humoral immunity can be reflected by detecting the level of antigen-specific antibodies in serum.

[0121] like Figure 11 As shown, in the in vivo experiment using OVA as a model antigen, mice were subcutaneously injected with EV vaccine, and blood samples were collected on days 7, 14, and 21 post-immunization to measure the serum OVA-specific IgG levels. Figure 12As shown, on day 7 post-immunization, the antibody levels in the serum of mice in the CS-OVA-EV group were significantly increased, reaching 34 times that of the PBS group, 11 times that of the CS-EV group, and 3 times that of the OVA-EV group, respectively. The results on day 7 indicate that CS-OVA-EV can rapidly induce a specific humoral immune response. On day 14 post-immunization, the level of OVA-specific antibodies in the serum of mice in the CS-OVA-EV group further increased significantly, reaching 88 times that of the PBS, CS-EV, and OVA-EV groups. Notably, at this point, the antibody levels in the CS-EV and OVA-EV groups were close to those in the PBS group. While the OVA-EV group showed a significant increase in antibody levels on day 7 post-immunization, there was no significant difference between it and the PBS group on day 14, indicating that although vesicles stimulated by free antigen proteins can induce specific humoral immunity in the short term, they fail to trigger a durable protective immune response. On day 21 post-immunization, although the specific antibody levels in the CS-OVA-EV group decreased somewhat, they remained at a high level, 58 times that of the PBS group, indicating that it still has a certain protective effect. In contrast, the antibody levels (OD values ​​in the ELISA experiment) in the CS-EV and OVA-EV groups were lower than those in the PBS group, indicating that these two groups had lost the protective effect of humoral immunity at this point. These data suggest that CS-OVA-EV can rapidly induce durable protective specific humoral immunity. This rapid response may stem from the fact that CS-OVA-EV can directly activate T cells, bypassing the antigen processing and handling by APCs in vivo, thereby accelerating the initiation of specific humoral immunity. Simultaneously, OVA-EV also showed a certain rapid initiation trend, further supporting the hypothesis that the initiation speed may be related to the vesicle structure derived from immune cells. The CS-OVA-EV group exhibited durable antibody levels, indicating that it effectively activated the body's overall specific humoral immunity. OVA-EV, however, failed to achieve this effect, further highlighting the necessity of constructing a chitosan-antigen complex delivery system.

[0122] Antigen-specific cellular immune response induced by CS-OVA-EV vaccine

[0123] Cellular immunity is primarily mediated by T cells, whose function is to specifically recognize and eliminate pathogens and abnormal cells, constituting an important component of adaptive immunity. The spleen, as a vital immune organ, contains immune cells such as T cells, B cells, macrophages, and dendritic cells (DCs). IFN-γ is a key antiviral cytokine secreted by various immune cells, including innate lymphocyte populations such as natural killer (NK) cells and innate lymphocytes, as well as adaptive immune cells such as helper T cell 1 (Th1) cells and cytotoxic T lymphocytes (CTLs). IFN-γ can induce macrophage polarization, enhancing their killing ability and antigen-presenting function, and plays an important role in local antiviral responses. In vaccine-induced immune memory, IFN-γ derived from memory T cells is crucial, directing monocytes, DCs, NK cells, and NKT cells to strongly express effector cytokines and guide microbial killing pathways during infection.

[76] Elevated IFN-γ levels typically indicate an active cellular immune response. Therefore, detecting the level of IFN-γ secreted by spleen cells can reflect the strength of the body's cellular immunity.

[0124] ELISPOT is a highly sensitive sandwich ELISA technique based on enzyme-linked immunosorbent assay (ELISA), widely used for the in vitro detection and counting of single cells secreting specific cytokines. In an ELISPOT assay, capture antibodies are pre-coated onto the bottom of wells, specifically binding to cytokines secreted by immune cells. Subsequently, through enzyme signal amplification and colorimetric reaction, visual spots are formed on the wells, each spot precisely corresponding to a cell secreting a specific cytokine, known as spot-forming cells (SFCs). An ELISA reader can then accurately count the number of cells secreting specific cytokines.

[0125] Both NK cells and T cells (mainly Th1 cells and CTLs) in the spleen can secrete IFN-γ. In this experiment, we co-cultured spleen cells with a stimulation medium containing a mixture of OVA protein and OVA peptide. As a specific antigen, OVA can selectively activate T cells that have recognized and remembered the antigen, thereby triggering a specific immune response. Therefore, the ELISPOT results here primarily assess the degree of activation of specific cellular immunity. After stopping the ELISPOT staining, images of the spots on the wells were taken using an ELISA reader, and the number of spots in the wells was counted. Figure 13It can be seen that CS-OVA-EV significantly increased the proportion of SFCs in mouse spleen cells, which was 17.37 times that of the PBS group, 2.24 times that of the CS-EV group, and 1.43 times that of the OVA-EV group. This indicates that CS-OVA-EV can more effectively activate adaptive immune cells and promote their secretion of IFN-γ, meaning that CS-OVA-EV is also excellent in inducing protective antigen-specific cellular immunity. Simultaneously, by enhancing IFN-γ secretion, CS-OVA-EV further improved the body's antiviral capacity.

[0126] Immunization regimen of vesicle vaccine (CS-H1N1-EV) in influenza animal models

[0127] We obtained CS-H1N1-NP using a similar, but not exactly the same, method. First, we performed UV inactivation of 1×10⁻⁶ ppm. 7 PFU mL-1 H1N1 virus (WSN). After concentration in a 50kDa MWCO ultrafiltration tube, 10% Triton X-100 was added to a final concentration of 1%, and the mixture was lysed at room temperature for 90 min. The viral lysate was purified using the HiPPR Detergent Removal Spin Column Kit. The specific steps are as follows: (1) Place an empty centrifuge column in a 2mL centrifuge tube. Gently shake with HiPPR detergent to remove the resin. Using a 5mL pipette tip, add an appropriate volume of resin suspension to the centrifuge column. (2) Centrifuge at 1500g for 1 min to remove the storage buffer and discard the liquid. (3) Add an appropriate volume of PBS (PBS:resin = 1:1 (v:v)), centrifuge at 1500g for 1 min, and discard the liquid. Repeat this step twice. (4) Insert the bottom plug and place the column into a new collection tube. (5) Slowly add the sample directly to the top of the compacted resin bed. Cover the column and gently vortex to mix the sample with the resin. Incubate at room temperature for 10 minutes. (6) Slightly loosen the cap, remove the bottom stopper, and place the column into the collection tube. Centrifuge at 1500g for 2 minutes and collect the sample without detergent. Discard the column used. Premix the purified H1N1 lysis buffer with CS solution, and add TPP aqueous solution under gentle stirring. Continue stirring for 30 minutes. Centrifuge the resulting solution at 10000rpm for 30 minutes at 4℃, discard the supernatant, and resuspend in ultrapure water to obtain CS-H1N1-NP.

[0128] Preparation of CS-H1N1-EV: The experimental steps are the same as in Example 1, except that CS-OVA-NP in Example 1 is replaced with CS-H1N1-NP (the concentration based on CS remains unchanged), and the other steps are the same.

[0129] In an animal model experiment of H1N1 influenza virus infection, 6-8 week old female BALB / c mice were randomly divided into six groups of seven each. The prepared vesicular vaccine was diluted with PBS to a similar protein concentration (measured by the BCA method) and injected subcutaneously into the mice (100 μL). The PBS group received 100 μL of PBS (both PBS control groups received the same treatment during the immunization phase, differing only in subsequent challenge procedures). The CS-H1N1-NP group received 100 μL of CS-H1N1-NP diluted with ultrapure water. The day of the initial immunization was recorded as day 0. Blood was collected from the retroorbital vein two weeks after the initial immunization. A booster immunization was performed four weeks after the initial immunization. Blood was collected from the retroorbital vein two weeks after the booster immunization. Nasal challenge was performed three days later. Only the PBS-N group did not receive challenge and served as a full survival control. Weight changes in mice were monitored after challenge; mice with a weight below 75% were euthanized. Fifteen days after the viral challenge, all surviving mice were euthanized.

[0130] Group Description

[0131]

[0132]

[0133] Assay for H1N1-specific IgG titers induced by CS-H1N1-EV vaccine

[0134] Whole blood was collected on days 14 and 42 after the initial immunization, and serum was obtained by centrifugation. The level of H1N1-specific antibodies in mouse serum was detected by ELISA. The antigen coating procedure involved adding 100 μL of 1×10⁻⁶ H1N1-specific antibody to a high-binding 96-well plate. 6 PFU / mL UV-inactivated virus solution, coated overnight at 4°C. IgG samples were obtained from serum samples in this animal experiment. The remaining steps were described in the section "Determination of OVA-specific IgG titers induced by CS-OVA-EV vaccine".

[0135] The protective effect of CS-H1N1-EV vaccine against influenza

[0136] H1N1 nasal challenge was administered 45 days after the initial immunization. To test the protective efficacy of the CS-H1N1-EV vaccine in a high viral titer environment, our challenge dose was 50 times the LD50 (Lethal Dose 50, determined experimentally and calculated using the Reed-Muench method), specifically 6.3 × 10⁻⁶. 2 (PFU / 50μL). Monitor mouse weight changes after challenge and plot mouse survival curves and weight change graphs.

[0137] In animal experiments using OVA as a model antigen, the CS-OVA-EV vaccine demonstrated advantages such as a universal and rapid preparation protocol, rapid initiation of the body's immune response, and activation of strong cellular immunity. Rapid initiation of cellular immunity means that in the early stages of viral invasion, T cells can be activated to clear virus-infected cells, thereby preventing further viral replication and spread. We believe this characteristic has potential applications in environments with high viral titers. We used lysed H1N1 virus to replace the model antigen OVA in the synthesis of CS-H1N1-NP to verify the practicality and universality of this EV vaccine preparation strategy. Because the principle of chitosan-antigen composite nanoparticle synthesis is based on electrostatic interaction and has certain universality, this substitution is effective. Subsequently, we stimulated RAW264.7 cells with CS-H1N1-NP and collected the secreted CS-H1N1-EV by ultrafiltration. We appropriately diluted the CS-H1N1-EV vesicles (approximately three-fold) to ensure similar protein concentrations across the groups, thereby enabling the evaluation of the immunoprotective efficacy of different vesicle vaccine groups under conditions of similar protein concentrations. Figure 14 As shown, in this influenza model animal experiment, mice were initially immunized with EV vaccine subcutaneously, designated as day 0. A booster immunization was administered four weeks after the initial immunization. Blood samples were collected two weeks after each immunization. Serum H1N1-specific antibody levels were detected using ELISA in blood samples collected on days 14 and 42. Figure 15 As can be seen, on day 14, the H1N1-EV, CS-H1N1-EV, and CS-H1N1-NP groups all showed certain antibody levels. The antibody level in the CS-EV group was comparable to that in the PBS group, mainly because CS-EV does not carry any antigenic information and serves only as a control group to exclude the influence of chitosan. On day 42, the H1N1-specific antibody level in the CS-H1N1-NP group increased significantly, reaching 4.36 times that of the PBS group, while the antibody level in the CS-H1N1-EV group was close to that of the CS-H1N1-NP group, reaching 3.03 times that of the PBS group. This indicates that the CS-H1N1-EV vaccine has a similar humoral immune protection effect to traditional nanoparticle vaccines. The antibody levels in the H1N1-EV and CS-EV groups were comparable to those in the PBS group.

[0138] The immune protection effect of CS-H1N1-EV vaccine against influenza infection

[0139] Influenza virus challenge was performed on day 45, three days after the second blood draw. To evaluate the immunoprotective effect of the CS-H1N1-EV vaccine in a high viral titer environment, a challenge dose of 50 times the LD50 was selected. In the two PBS groups, PBS-N was not challenged and served as a normal weight control, while PBS-Y underwent challenge treatment to verify the lethality of the H1N1 influenza virus. After influenza virus infection, the mice's body weight gradually decreased, reaching its lowest point between days 6 and 10 post-infection. If no deaths occurred, the mice's body weight gradually recovered as the disease subsided. Figure 16 As can be seen, compared with the control groups, the CS-H1N1-EV vaccine still showed good protective efficacy against the deadly influenza virus with an LD50 up to 50 times higher, with a survival rate of 57.1%. Although the antigen-specific antibody levels induced by traditional nanoparticle vaccines were comparable to those of the CS-H1N1-EV vaccine, their protective efficacy was poor, with a survival rate of only 14.3%. This result indicates that the antigen-specific cellular immunity activated by the CS-H1N1-EV vaccine played an important role under high-dose influenza virus attack, which is consistent with our design intent.

Claims

1. A method for preparing a vesicle vaccine based on a chitosan antigen delivery system, characterized in that, Includes the following steps: Step 1: Mix the antigen protein with chitosan aqueous solution and sodium tripolyphosphate aqueous solution, and prepare chitosan nanoparticles loaded with antigen protein through electrostatic interaction; Step 2: After co-incubating immune cells with chitosan nanoparticles loaded with antigen proteins, the cell supernatant is collected, and a vesicle vaccine solution is prepared by multi-step differential centrifugation combined with ultrafiltration, which is the vesicle vaccine based on the chitosan antigen delivery system.

2. The method for preparing a vesicle vaccine based on a chitosan antigen delivery system according to claim 1, characterized in that, Step one involves preparing chitosan nanoparticles loaded with antigen proteins, specifically including: Prepare a chitosan solution using ultrapure water, adjust the pH to 4.5-5.0, filter the solution, add the antigen protein to a final concentration of 0.02-0.2 mg / mL, then add an aqueous solution of sodium tripolyphosphate under gentle magnetic stirring. Continue stirring the reaction solution for 15-45 min, centrifuge the reaction solution, discard the supernatant, and obtain chitosan nanoparticles loaded with antigen protein.

3. The method for preparing a vesicle vaccine based on a chitosan antigen delivery system according to claim 2, characterized in that, In step one, adjust the pH to 4.7-4.

8.

4. The method for preparing a vesicle vaccine based on a chitosan delivery system according to claim 2, characterized in that, In step one, the antigen protein is added to a final concentration of 0.05–0.15 mg / mL.

5. The method for preparing a vesicle vaccine based on a chitosan antigen delivery system according to claim 2, characterized in that, In step one, the reaction solution is stirred for another 25–35 minutes.

6. The method for preparing a vesicle vaccine based on a chitosan delivery system according to claim 1, characterized in that, In step two, after co-incubating immune cells with chitosan nanoparticles loaded with antigen proteins, the cell supernatant is collected, and a vesicle vaccine solution is prepared using a multi-step differential centrifugation combined with ultrafiltration method, specifically including: After culturing the immune cells, the cells were washed with PBS, and then the culture medium was replaced with complete culture medium containing chitosan nanoparticles loaded with antigen proteins. The cells were incubated for 6–10 h. After incubation, the cells were washed with PBS, replaced with serum-free culture medium, and cultured for another 16–22 h. During the vesicle collection stage, a vesicle solution was prepared by multi-step differential centrifugation combined with ultrafiltration.

7. The method for preparing a vesicle vaccine based on a chitosan delivery system according to claim 6, characterized in that, In step two, incubate for 7–9 hours.

8. The method for preparing a vesicle vaccine based on a chitosan delivery system according to claim 6, characterized in that, In step two, continue culturing for another 18–20 hours.

9. The method for preparing a vesicle vaccine based on a chitosan delivery system according to claim 6, characterized in that, Step two, which employs a multi-step differential centrifugation combined with ultrafiltration method, specifically includes: All centrifugation operations were performed at 3–5°C. First, the culture medium was collected and centrifuged sequentially at 250–350g for 8–12 min, 1800–2200g for 8–12 min, and 9500–10500rpm for 25–35 min. The supernatant was collected after each centrifugation step. Then, the medium was centrifuged at 3500–4500g for 25–35 min using a 100kDa MWCO ultrafiltration tube. The filtrate in the centrifuge tube was discarded, PBS was added, and the medium was centrifuged at 3500–4500g for 10–20 min as a washing step. The collected liquid was the vesicle solution.

10. The use of the vesicle solution prepared by the preparation method according to any one of claims 1 to 9 in the preparation of vaccines for cancer immunotherapy and infectious disease prevention.

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