Manganese-based platelet carrier vaccine based on biomimetic mineralization technology and its application in immunotherapy

Abstract

The present application belongs to the technical field of vaccines, and particularly relates to a manganese-based platelet carrier vaccine based on biomimetic mineralization technology and application thereof in immunotherapy. A manganese-based shell is deposited on the surface of a platelet, and the platelet surface expresses immune active molecules such as CD40L and PF4, which can activate dendritic cells (DCs) through direct contact or secretion of cytokines and promote antigen presentation; and the deposited manganese ions can activate the cGAS-STING pathway and simultaneously up-regulate the expression of CD40 / CD80 / CD86 / MHC-II costimulatory molecules on the surface of DCs. The vaccine adjuvant provided by the present application can load antigens to form a vaccine complex of "three-in-one" of antigens (loaded on the platelet membrane), platelets and manganese shells, which not only retains the natural targeting ability of platelets, but also enhances DC cross-presentation through sustained release of manganese ions, and ultimately synergistically activates CD8 + T cells and antibody responses, and enhances the body's anti-viral immune response.

Description

Technical Field

[0001] This invention belongs to the field of vaccine technology, specifically relating to a manganese-based platelet vector vaccine based on biomimetic mineralization technology and its application in immunotherapy. Background Technology

[0002] The rise of immunotherapy marks a paradigm shift in disease management, its core lying in the precise regulation of the immune system to stimulate or suppress specific immune responses. Within this framework, prophylactic vaccines constitute the forefront of adaptive immunization, their efficacy relying on the successful activation of innate immunity and the subsequent induction of antigen-specific T-cell and B-cell memory. As one of the most effective disease prevention methods in modern medicine, vaccines have evolved from traditional inactivated, attenuated, and vector vaccines to novel nucleic acid and subunit vaccines. With advancements in molecular biology and immunology in recent years, new vaccine antigens have been continuously identified. However, due to limitations such as insufficient immunogenicity and short duration of immune response in most highly purified recombinant antigens, novel vaccines typically rely on adjuvants to enhance the level and duration of vaccine-induced protection.

[0003] Currently, the mainstream adjuvants approved in clinical practice are still primarily traditional aluminum adjuvants. While widely used, traditional adjuvants mainly induce Th2 immune responses, with insufficient activation of Th1 immune responses (e.g., IFN-γ). + CD8 + T-cell responses are weak, making them ill-equipped to combat intracellular viruses, thus prompting researchers to continuously develop novel adjuvant systems. In recent years, the rise of metal immunotherapy, which utilizes metal ions to enhance immunotherapy, has provided entirely new avenues for adjuvant development. Among these, manganese ions (Mn)... 2+ Manganese ions can target and activate the unique cGAS-STING pathway, promoting the secretion of type I interferon, which is key to enhancing cytotoxic CD8+ T cell responses. Therefore, manganese ions are a highly promising candidate novel adjuvant. However, although manganese ions can address the insufficient ability of vaccines to initiate immune effector cell responses, their clinical application still faces many limitations, including a short in vivo half-life (<2h), rapid in vivo metabolism (low utilization), inefficient antigen loading methods, and low co-delivery efficiency. These issues urgently require efficient integration through vector engineering strategies. Summary of the Invention

[0004] The purpose of this invention is to develop a manganese-based platelet vector vaccine based on biomimetic mineralization technology and its application in immunotherapy, which successfully targets immune effector sites and induces Th1 and Th2 immune responses, solving the problem that traditional vectors cannot simultaneously achieve both targeting and sustained immune memory.

[0005] The present invention provides a vaccine adjuvant comprising platelets and manganese ions deposited on the surface of the platelets.

[0006] The present invention provides a method for preparing the vaccine adjuvant described in the above technical solution, comprising the following steps: mixing platelets, manganese chloride, sodium hydroxide and physiological saline, incubating at 20~25℃ for 0.5~1h to obtain the incubated product; mixing the incubated product with prostaglandin E1, separating the solid and liquid, collecting the precipitate to obtain the vaccine adjuvant.

[0007] Preferably, the concentration ratio of platelets to manganese chloride in the mixture of platelets, manganese chloride, sodium hydroxide, and physiological saline is 1×10⁻⁶. 7 ~2×10 8 Cells / mL: 0.5~1mM; The concentration ratio of platelets to sodium hydroxide in the mixture of platelets, manganese chloride, sodium hydroxide, and physiological saline is 1×10⁻⁶. 7 ~2×10 8 Cells / mL: 50~200μM.

[0008] Preferably, the oscillation frequency of the incubation is 50~100 rpm.

[0009] Preferably, the concentration of prostaglandin E1 in the mixture of the incubated product and prostaglandin E1 is 1~5 μM.

[0010] Preferably, the solid-liquid separation method includes centrifugation; the centrifugation temperature is 20~25℃, the centrifugal force is 800~1000×g, and the time is 10~15min.

[0011] This invention provides the application of the vaccine adjuvant described in the above technical solution or the vaccine adjuvant prepared by the preparation method described in the above technical solution in the preparation of vaccines.

[0012] Preferably, the vaccine includes an antiviral therapeutic vaccine.

[0013] This invention provides a manganese-based platelet vector vaccine, comprising an antigen and a vaccine adjuvant; the vaccine adjuvant is the vaccine adjuvant described in the above technical solution or a vaccine adjuvant prepared using the preparation method described in the above technical solution.

[0014] The present invention provides a method for preparing the manganese-based platelet vector vaccine described in the above technical solution, comprising the following steps: mixing the vaccine adjuvant and antigen, incubating at 20~25℃ for 0.5~1h to obtain the incubated product; mixing the incubated product with prostaglandin E1, separating the solid and liquid, collecting the precipitate to obtain the manganese-based platelet vector vaccine.

[0015] Beneficial effects: This invention provides a vaccine adjuvant comprising platelets and manganese ions deposited on the platelet surface. The invention deposits a manganese-based shell on the platelet surface, which expresses immunologically active molecules such as CD40L and PF4. These molecules can activate dendritic cells (DCs) through direct contact or secretion of cytokines and promote antigen presentation. Furthermore, the deposited manganese ions can activate the cGAS-STING pathway and upregulate the expression of co-stimulatory molecules CD40 / CD80 / CD86 / MHC-II on the DC surface. The vaccine adjuvant provided by this invention can load antigens, forming a "three-in-one" vaccine complex of antigen (loaded on the platelet membrane), platelets, and the manganese shell. This not only retains the natural targeting ability of platelets but also enhances DC cross-presentation through the continuous release of manganese ions, ultimately synergistically activating CD8+. + T cells react with antibodies, enhancing the body's antiviral immune response. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The images show the characterization results of the manganese-based platelet vector vaccine; where a represents the scanning electron microscope (SEM) results, b represents a representative image of the energy dispersive spectroscopy (EDS) results, and * represents... p <0.05; c represents the relative quantitative result of EDS elements; d represents the detection result of inductively coupled plasma mass spectrometry (ICP-MS); Figure 2 The results represent the quality control findings for the manganese-based platelet vector vaccine; where a represents the flow cytometry results of Erythrina lectin (ECA) and ricin agglutinin (RCA); b represents the flow cytometry results of Annexin V; *** represents... p <0.001; Figure 3 The results of the in vivo distribution study of the manganese-based platelet vector vaccine are shown below; where a is the adsorption efficiency; b is the recovery rate; c is the representative images of platelet distribution in vivo; d is the representative images of platelet distribution in organs 24 hours after platelet reinfusion; e is the localization results in the spleen, liver, and lungs; * indicates... p <0.05; Figure 4 The results of the humoral immune response evaluation of the manganese-based platelet vector vaccine are as follows: a) is the titer of anti-RBD IgG antibody; b) is the neutralization response to SARS-CoV-2 pseudovirus. Figure 5 The results represent the evaluation of the cellular immunogenicity of the manganese-based platelet vector vaccine; among which, *** represents... p <0.001. Detailed Implementation

[0018] The present invention provides a vaccine adjuvant comprising platelets and manganese ions deposited on the surface of the platelets.

[0019] The present invention provides a method for preparing the vaccine adjuvant described in the above technical solution, comprising the following steps: mixing platelets, manganese chloride, sodium hydroxide and physiological saline, incubating at 20~25℃ for 0.5~1h to obtain the incubated product; mixing the incubated product with prostaglandin E1, separating the solid and liquid, collecting the precipitate to obtain the vaccine adjuvant.

[0020] This invention involves mixing platelets, manganese chloride, sodium hydroxide, and physiological saline, and incubating at 20-25°C for 0.5-1 hour to obtain the incubated product.

[0021] In one embodiment, the incubation temperature of the present invention is 22°C. In one embodiment, the incubation time of the present invention is 1 hour. In one embodiment, the oscillation frequency of the incubation of the present invention is 50~100 rpm; in another embodiment, the oscillation frequency of the incubation of the present invention is 80 rpm.

[0022] In one embodiment, the concentration ratio of platelets to manganese chloride in the mixture of platelets, manganese chloride, sodium hydroxide, and physiological saline described in this invention is 1×10⁻⁶. 7 ~2×10 8 Platelets / mL: 0.5~1mM; As another embodiment, the concentration ratio of platelets to manganese chloride in the mixture of platelets, manganese chloride, sodium hydroxide and physiological saline of the present invention is 2×10⁻⁶. 8 Platelets / mL: 1 mM. As one embodiment, the concentration ratio of platelets to sodium hydroxide in the mixture of platelets, manganese chloride, sodium hydroxide, and physiological saline described in this invention is 1 × 10⁻⁶. 7 ~2×10 8 Platelets / mL: 50~200μM; As another embodiment, the concentration ratio of platelets to sodium hydroxide in the mixture of platelets, manganese chloride, sodium hydroxide and physiological saline of the present invention is 2×10⁻⁶. 8 Platelets / mL: 100 μM. This invention limits the concentration ratio of platelets, manganese chloride, and sodium hydroxide, which has the advantages of product stability, ease of quality control, and preservation of platelet functional activity.

[0023] After obtaining the incubated product, the present invention mixes the incubated product with prostaglandin E1, separates the solid and liquid components, collects the precipitate, and obtains the vaccine adjuvant.

[0024] In one embodiment, the concentration of prostaglandin E1 in the mixture of the incubated product and prostaglandin E1 described in this invention is 1-5 μM; in another embodiment, the concentration of prostaglandin E1 in the mixture of the incubated product and prostaglandin E1 described in this invention is 2 μM. Prostaglandin E1 can prevent excessive platelet activation caused by shear force during solid-liquid separation.

[0025] In one embodiment, the solid-liquid separation method of the present invention includes centrifugation. In one embodiment, the centrifugation temperature is 20-25°C; in another embodiment, the centrifugation temperature is 22°C. In one embodiment, the centrifugal force is 800-1000 × g; in another embodiment, the centrifugal force is 1000 × g. In one embodiment, the centrifugation time is 10-15 min; in another embodiment, the centrifugation time is 10 min.

[0026] In one embodiment, the present invention resuspends the vaccine adjuvant in physiological saline to obtain a vaccine adjuvant suspension.

[0027] This invention deposits manganese ions on the surface of platelets. Compared with platelets without deposited manganese ions, manganese mineralization increases platelet desialylation, alters their in vivo circulation time and distribution pattern, and improves the targeted accumulation rate in the spleen. This, in turn, forms immune complexes with dendritic cells (DCs), enabling antigen-Mn binding. 2+ Common targeted delivery.

[0028] In view of the advantages of the vaccine adjuvant provided by the present invention, the application of the vaccine adjuvant described in the above technical solution or the vaccine adjuvant prepared by the preparation method described in the above technical solution in the preparation of vaccines also falls within the protection scope of the present invention.

[0029] In one embodiment, the vaccine of the present invention includes an antiviral therapeutic vaccine. In another embodiment, the vaccine of the present invention includes an antitumor therapeutic vaccine that targets immune effector sites and induces Th1 and Th2 immune responses.

[0030] This invention provides a manganese-based platelet vector vaccine, comprising an antigen and a vaccine adjuvant; the vaccine adjuvant is the vaccine adjuvant described in the above technical solution or a vaccine adjuvant prepared using the preparation method described in the above technical solution.

[0031] In one embodiment, the antigen of this invention comprises the receptor-binding domain of the anti-mouse SARS-CoV-2 spike protein and / or chicken ovalbumin. This invention is illustrated using the receptor-binding domain of the anti-mouse SARS-CoV-2 spike protein and chicken ovalbumin as examples, but these should not be construed as representing the entire scope of protection of this invention. In one embodiment, when the antigen of this invention is chicken ovalbumin, the concentration of the vaccine adjuvant and the antigen is 1 × 10⁻⁶. 7 ~2×10 8 The vaccine adjuvant is measured in platelet counts at 100 μg / mL. As one embodiment, when the antigen is the receptor-binding region of the mouse SARS-CoV-2 spike protein, the concentration of the vaccine adjuvant and antigen is 1 × 10⁻⁶. 7 ~2×10 8 The vaccine adjuvant is 20 μg / mL, and the adjuvant is expressed as a number of platelets.

[0032] The present invention provides a method for preparing the manganese-based platelet vector vaccine described in the above technical solution, comprising the following steps: mixing the vaccine adjuvant and antigen, incubating at 20~25℃ for 0.5~1h to obtain the incubated product; mixing the incubated product with prostaglandin E1, separating the solid and liquid, collecting the precipitate to obtain the manganese-based platelet vector vaccine.

[0033] In this invention, the vaccine adjuvant and antigen are mixed and incubated at 20-25°C for 0.5-1 hour to obtain the incubated product.

[0034] In one embodiment, the incubation temperature of the present invention is 22°C. In one embodiment, the incubation time of the present invention is 30 minutes. In one embodiment, the oscillation frequency of the incubation of the present invention is 50~100 rpm; in another embodiment, the oscillation frequency of the incubation of the present invention is 80 rpm.

[0035] After obtaining the incubated product, the present invention mixes the incubated product with prostaglandin E1, separates the solid and liquid components, collects the precipitate, and obtains the manganese-based platelet vector vaccine.

[0036] In one embodiment, the concentration of prostaglandin E1 in the mixture of the incubated product and prostaglandin E1 described in this invention is 1-5 μM; in another embodiment, the concentration of prostaglandin E1 in the mixture of the incubated product and prostaglandin E1 described in this invention is 2 μM. Prostaglandin E1 can prevent excessive platelet activation caused by shear force during solid-liquid separation.

[0037] In one embodiment, the solid-liquid separation method of the present invention includes centrifugation. In one embodiment, the centrifugation temperature is 20-25°C; in another embodiment, the centrifugation temperature is 22°C. In one embodiment, the centrifugal force is 800-1000 × g; in another embodiment, the centrifugal force is 1000 × g. In one embodiment, the centrifugation time is 10-15 min; in another embodiment, the centrifugation time is 10 min.

[0038] In one embodiment, the present invention utilizes physiological saline to resuspend the precipitate to obtain a suspension of manganese-based platelet carrier vaccine.

[0039] This invention utilizes the manganese-based platelet vector vaccine for humoral immunization. Compared with traditional vaccines obtained with aluminum adjuvants, the manganese-based platelet vector vaccine of this invention, after intramuscular injection, significantly increases the titer of IgG antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein in mice; and the maximum inhibitory concentration (IC50) of neutralizing antibodies against SARS-CoV-2 pseudoviruses is also significantly increased. 50 This invention utilizes the manganese-based platelet vector vaccine for cellular immunization. In a mouse hepatotropic adenovirus infection model, the hilar lymph nodes in the manganese-based platelet vector vaccine immunization group were significantly larger than those in the traditional vaccine group obtained with aluminum adjuvant, and the antigen-specific CD8+ was significantly higher. + T cell infiltration, activation, and release of antiviral-related cytokines were all significantly increased. The manganese-based platelet vector vaccine provided by this invention can respond to viral infection and has antiviral therapeutic effects.

[0040] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes the manganese-based platelet vector vaccine based on biomimetic mineralization technology and its application in immunotherapy, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0041] The materials and their sources used in the embodiments of this invention are as follows: manganese chloride (Sigma); ethylenediaminetetraacetic acid (EDTA, Sigma); prostaglandin E1 (PGE1, MCE); ovalbumin (OVA, Sigma); receptor-binding domain of mouse SARS-CoV-2 spike protein (SARS-CoV-2 Spike RBD, Novizan); flow cytometry antibodies against ECA, RCA, Annexin V, CD8a, CD25, CD69, CD44, CD107α, Granzyme B, IFNγ, and TNF-α were purchased from Biolegend; flow cytometry antibody against Tetramer was purchased from MBL; recombinant primary antibody against CD3 was purchased from Abcam; recombinant primary antibodies against CD45R and CD11c were purchased from Invitrogen.

[0042] Example 1 Preparation of manganese mineralized platelets 1. Place mouse whole blood anticoagulated with EDTA into a low-speed centrifuge, set the temperature to 22℃, the centrifugal force to 150×g, and centrifuge for 20 min to obtain the upper platelet-rich plasma.

[0043] 2. Add 2 μM of prostaglandin E1 (PGE1) to platelet-rich plasma, mix well, and place in a low-speed centrifuge. Set the temperature to 22℃ and the centrifugal force to 1000×g, and centrifuge for 10 min to obtain purified platelets.

[0044] 3. Adjust the platelet concentration to 2 × 10 using normal saline. 8 Platelets per mL were added, along with 1 mM manganese chloride and 100 μM sodium hydroxide, and incubated in a platelet constant temperature shaking incubator for 1 h. The incubation temperature was 22 °C and the shaking frequency was 800 rpm to obtain manganese mineralized platelets (PLT@MnOx), which were used as vaccine adjuvants.

[0045] Example 2 Preparation of manganese-based platelet vector vaccine 1. Add 2 μM of PGE1 to the manganese mineralized purified platelets obtained in Example 1, then place them in a low-speed centrifuge. Set the temperature to 22°C and the centrifugal force to 1000×g. After centrifugation for 10 min, discard the supernatant and resuspend in physiological saline.

[0046] 2. Repeat step 1.

[0047] 3. Adjust the manganese-mineralized platelet concentration to 2×10 using normal saline. 8 Add 100 μg / mL of ovalbumin (OVA) to each platelet, mix well, and incubate in a platelet constant temperature shaking incubator for 30 min; the incubation temperature is 22℃ and the shaking frequency is 800 rpm.

[0048] 4. After adding 2 μM of PGE1, place the mixture in a low-speed centrifuge, set the temperature to 22℃ and the centrifugal force to 1000×g, centrifuge for 10 min, discard the supernatant, and resuspend the mixture in physiological saline to obtain the suspension of the manganese-based platelet vector vaccine.

[0049] Comparative Example 1 Preparation of platelet vector vaccines 1. Add 2 μM of PGE1 to the purified platelets obtained in step 2 of Example 1, and then place them in a low-speed centrifuge. Set the temperature to 22°C and the centrifugal force to 1000×g. After centrifugation for 10 min, discard the supernatant and resuspend in physiological saline.

[0050] 2. Repeat step 1.

[0051] 3. Adjust the platelet concentration to 2 × 10 using normal saline. 8 Add 100 μg / mL of ovalbumin (OVA), mix well, and incubate in a platelet constant temperature shaking incubator for 30 min.

[0052] 4. After adding 2 μM of PGE1, place the mixture in a low-speed centrifuge, set the temperature to 22℃ and the centrifugal force to 1000×g, centrifuge for 10 min, discard the supernatant, and resuspend the mixture in physiological saline to obtain the platelet vector vaccine suspension.

[0053] Test Example 1 Characterization of manganese-based platelet vector vaccines The manganese-based platelet vector vaccine (PLT@MnOx) obtained in Example 2 and the platelet vector vaccine (PLT) obtained in Comparative Example 1 were resuspended in 4% paraformaldehyde. After incubation at room temperature for 15 min, the plates were centrifuged at 22°C and 1000×g for 10 min. The supernatant was discarded, and different concentration gradients (30%, 50%, 70%, 90%, 100%) of ethanol were added for gradient dehydration. Finally, the platelets were resuspended in deionized water and detected by scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) and inductively coupled plasma mass spectrometry (ICP-MS). The detection results are as follows: Figure 1 As shown.

[0054] according to Figure 1 It can be seen that, compared with the PLT constructed in Comparative Example 1, the surface of PLT@MnOx constructed in Example 1 becomes smoother and is accompanied by an increase in particle size. Figure 1 (a) Mn 2+ Deposits on platelets ( Figure 1 (bd). The example successfully constructed PLT@MnOx.

[0055] Test Example 2 Quality control of manganese-based platelet vector vaccines The average fluorescence intensity of Erythrina lectin (ECA) and ricin agglutinin (RCA) reflected changes in platelet desialylation levels, while the expression level of Annexin V reflected changes in the proportion of platelets undergoing apoptosis. Manganese-based platelet vector vaccine (PLT@MnOx) obtained in Example 2 and platelet vector vaccine (PLT) obtained in Comparative Example 1 were collected. The expression levels of ECA, RCA, and Annexin V were detected by flow cytometry surface staining. The results are as follows: Figure 2 As shown.

[0056] according to Figure 2 It can be seen that the desialylation level of PLT@MnOx obtained in Example 2 is significantly upregulated compared to that of PLT obtained in Comparative Example 1. Figure 2 (a), but no obvious apoptosis was observed ( Figure 2 (b) Compared to unmineralized platelets, manganese mineralization increased platelet desialylation, suggesting that it may alter platelet circulation time and distribution patterns in vivo. Furthermore, manganese mineralization is a mild modification method that does not affect platelet activity.

[0057] Example 3 Preparation of manganese-based platelet vector vaccine labeled with fluorescent dye Same as Example 2, except that in step 3, ovalbumin (OVA) is replaced with OVA labeled with near-infrared fluorescent dye CY5.5.

[0058] Comparative Example 2 Preparation of platelet vector vaccines labeled with fluorescent dyes Similar to Comparative Example 1, the only difference is that in step 3, ovalbumin (OVA) is replaced with OVA labeled with the near-infrared fluorescent dye CY5.5.

[0059] Test Example 3 In vivo distribution patterns of manganese-based platelet vector vaccines The fluorescently labeled manganese-based platelet vector vaccine (PLT@MnOx) obtained in Example 3 and the fluorescently labeled platelet vector vaccine (PLT) obtained in Comparative Example 2 were collected separately by centrifugation. The antigen loading efficiency was calculated directly by the change in fluorescence signal between platelets and the remaining DiD in the solution. The platelets were then intravenously infused into 8-week-old male C57BL / 6 mice. Fluorescence signals were detected at different time points using small animal in vivo imaging. Mice were euthanized 24 hours later, and samples were collected for multicolor immunofluorescence experiments. The detection results are as follows: Figure 3 As shown.

[0060] according to Figure 3It can be seen that manganese mineralization does not affect the platelet's ability to adsorb antigens. Figure 3 (a) From 4 hours post-infusion, the recovery rate of PLT@MnOx was significantly lower than that of the control group in terms of platelet count ( Figure 3 (b) At the same time, it is more enriched in the spleen of mice, and the spleen still has signals after 48 hours, which prolongs the half-life and improves the utilization rate. Figure 3 (cd). Samples were collected 24 hours after infusion, and multicolor immunofluorescence staining was performed simultaneously on the liver and lungs, which also showed high concentrations of PLT@MnOx. The results showed that PLT@MnOx was more localized in the germinal centers of the spleen, and enhanced infiltration of B cells, T cells, and dendritic cells (DCs) was also observed in the liver and lungs. Figure 3 (e). Manganese mineralization alters the in vivo distribution of platelets, enhancing their targeting ability and causing them to accumulate more in secondary lymphoid organs. It may also overcome the shortcomings of traditional vaccines in terms of poor targeting, delivering the antigen and adjuvant in the "three-in-one" design to the immune effector site, thereby systematically enhancing the body's immune response.

[0061] Example 4 Same as Example 2, except that in step 3, 100 μg / mL of ovalbumin (OVA) is replaced with 20 μg / mL of the receptor-binding domain (RBD) of the mouse SARS-CoV-2 spike protein.

[0062] Comparative Example 3 The receptor-binding domain (RBD) of the mouse SARS-CoV-2 spike protein was mixed with aluminum adjuvant (Alu) at a mass ratio of 1:1, and after vortexing, it was allowed to stand at 25°C for 15 min to obtain the aluminum adjuvant + RBD vaccine.

[0063] Test Example 4 Evaluation of humoral immune efficacy of manganese-based platelet vector vaccine Male C57BL / 6 mice aged 6-8 weeks were randomly divided into four treatment groups, designated MOCK, RBD, Alu, and PLT@MnOx, with five mice in each group. The following treatments were administered: MOCK: No treatment or intervention is performed on the mice; RBD: The receptor-binding domain of the mouse SARS-CoV-2 spike protein was resuspended in physiological saline and injected intramuscularly for a total of 3 times, with an interval of 3 weeks between each injection, and the dose of each injection was 50 μg. Alu: The aluminum adjuvant + RBD vaccine obtained in Comparative Example 3 was resuspended in physiological saline and administered intramuscularly for a total of 3 injections, with an interval of 3 weeks between each injection. The dosage of each injection was 50 μg (antigen dose). PLT@MnOx: The manganese-based platelet vector vaccine (PLT@MnOx) obtained in Example 4 was resuspended in physiological saline and administered intramuscularly for a total of 3 injections, with an interval of 3 weeks between each injection. The dosage of each injection was 50 μg (antigen dose).

[0064] Week 0 was defined as the first injection. Serum from mice in each treatment group was collected at week 8 for neutralizing antibody titers and pseudovirus neutralization tests. Results are as follows: Figure 4 As shown.

[0065] according to Figure 4 It can be seen that, compared with the Alu treatment group, the PLT@MnOx treatment group induced the production of higher titers of anti-RBD IgG antibodies. Figure 4 (a) Meanwhile, the PLT@MnOx treatment group induced a more effective neutralizing response to SARS-CoV-2 pseudoviruses compared to the Alu treatment group, with the maximum inhibitory concentration (IC50) of the anti-SARS-CoV-2 pseudovirus neutralizing antibody being higher. 50 Significantly superior to conventional vaccines obtained with aluminum adjuvants ( Figure 4 (b). PLT@MnOx is superior to traditional adjuvants in inducing antigen-specific humoral immune responses in mice.

[0066] Comparative Example 4 Chicken ovalbumin (OVA) of 100 μg / mL was mixed with aluminum adjuvant (Alu) at a mass ratio of 1:1, vortexed, and then allowed to stand at 25°C for 15 min to obtain aluminum adjuvant + OVA vaccine.

[0067] Comparative Example 5 100 μg / mL of ovalbumin (OVA) was mixed with 50 μg / mL of MnCl2, vortexed, and then allowed to stand at 25°C for 15 min to obtain the MnCl2+OVA vaccine.

[0068] Test Example 5 Evaluation of the cellular immunogenicity of manganese-based platelet vector vaccine Male C57BL / 6 mice aged 6-8 weeks were randomly divided into 7 treatment groups, designated as MOCK, PBS, OVA, Alu, MnCl2, PLT, and PLT@MnOx, respectively. Each treatment group consisted of 5 mice, and the following treatments were administered: MOCK: No treatment or intervention is performed on the mice; PBS: PBS buffer was administered intramuscularly in three injections, with each injection spaced three weeks apart. Each injection consisted of an equal volume of PBS without the antigen. OVA: Chicken ovalbumin was resuspended in physiological saline and injected intramuscularly for a total of 3 times, with an interval of 3 weeks between each injection. The dosage of each injection was 50 μg. Alu: The aluminum adjuvant + OVA vaccine obtained in Comparative Example 4 was resuspended in physiological saline and administered intramuscularly for a total of 3 injections, with an interval of 3 weeks between each injection. The dosage of each injection was 50 μg (antigen dose). MnCl2: The MnCl2+OVA vaccine obtained in Comparative Example 5 was resuspended in physiological saline and administered intramuscularly for a total of 3 injections, with an interval of 3 weeks between each injection. The dosage of each injection was 50 μg (antigen dose). PLT: The platelet vector vaccine obtained in Comparative Example 1 was resuspended in physiological saline and administered intramuscularly for a total of 3 injections, with an interval of 3 weeks between each injection. The dosage of each injection was 50 μg (antigen dose).

[0069] PLT@MnOx: The manganese-based platelet vector vaccine obtained in Example 2 was resuspended in physiological saline and administered intramuscularly for a total of 3 injections, with an interval of 3 weeks between each injection. The dosage of each injection was 50 μg (antigen dose).

[0070] Week 0 is the first injection. After two immunizations, 4×10⁴ mmol / L is administered via tail vein in week 8. 8 PFU / mouse of hepatophilic adenovirus expressing OVA. Mice were euthanized 48 h post-infection, and hilar lymph nodes from each treatment group were collected. Surface staining for Tetramer indicator antigen-specific CD8 was detected by flow cytometry. + T cell changes; detection of CD25 and CD69 expression levels indicates CD8+ + Changes in T cell activation levels; detection of granzyme B, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) to indicate changes in the secretion levels of CD8+ T cell antiviral killing-related cytokines, results as follows: Figure 5 As shown.

[0071] according to Figure 5 It can be seen that, compared with the MnCl2 treatment group and the Alu treatment group, the PLT@MnOx treatment group showed higher levels of antigen-specific CD8+ in the hilar lymph nodes of mice. + T cell count, CD8 + T cell activation level and CD8 + The secretion levels of T cell antiviral killing-related cytokines were significantly increased. PLT@MnOx was superior to aluminum adjuvant manganese chloride alone in inducing antigen-specific cellular immune responses in mice, overcoming the deficiency of traditional adjuvants in insufficient cellular immune activation.

[0072] As can be seen from the above, the vaccine adjuvant provided by this invention can load antigens to form a "three-in-one" vaccine complex consisting of antigen (loaded on the platelet membrane), platelet, and manganese shell. This not only retains the natural targeting ability of platelets but also enhances DC cross-presentation through the continuous release of manganese ions, ultimately synergistically activating CD8. +T cells react with antibodies, enhancing the body's antiviral immune response.

[0073] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a vaccine adjuvant in the manufacture of a vaccine, characterised in that, The vaccine adjuvant includes platelets and manganese ions deposited on the surface of the platelets.

2. Use according to claim 1, characterized in that, The preparation method of the vaccine adjuvant includes the following steps: platelets, manganese chloride, sodium hydroxide and physiological saline are mixed and incubated at 20~25℃ for 0.5~1h to obtain the incubated product; the incubated product is mixed with prostaglandin E1, solid-liquid separation is performed, and the precipitate is collected to obtain the vaccine adjuvant.

3. Use according to claim 2, characterized in that, The concentration ratio of the platelet and the manganese chloride in the mixed solution of the platelet, the manganese chloride, the sodium hydroxide and the physiological saline is 1 x 10 7 ~2 x 10 8 0.5~1mM; The concentration ratio of the platelet and sodium hydroxide in the mixed solution of the platelet, manganese chloride, sodium hydroxide and normal saline is 1 x 10 7 ~2 x 10 8 ~200 μM.

4. The application according to claim 2, characterized in that, The oscillation frequency of the incubation is 50~100 rpm.

5. The application according to claim 2, characterized in that, The concentration of prostaglandin E1 in the mixture of the incubated product and prostaglandin E1 is 1~5 μM.

6. The application according to claim 2, characterized in that, The solid-liquid separation method includes centrifugation; the centrifugation temperature is 20~25℃, the centrifugal force is 800~1000×g, and the time is 10~15min.

7. The application according to claim 1, characterized in that, The vaccines include antiviral therapeutic vaccines.

8. A manganese-based platelet vector vaccine, characterized in that, It includes antigens and vaccine adjuvants; the vaccine adjuvant is the vaccine adjuvant used in any one of claims 1 to 7.

9. The method for preparing the manganese-based platelet vector vaccine according to claim 8, characterized in that, The process includes the following steps: mixing the vaccine adjuvant and antigen, incubating at 20-25°C for 0.5-1 h to obtain the incubated product; mixing the incubated product with prostaglandin E1, separating the solid and liquid components, collecting the precipitate, and obtaining the manganese-based platelet vector vaccine.

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