Application of cerium oxide nano material in vaccine adjuvant and / or vaccine inactivator

Abstract

The invention relates to an application of a cerium oxide nano-material in a vaccine adjuvant and / or a vaccine inactivator, in particular to a vaccine, when the cerium oxide nano-material is the vaccine adjuvant, the vaccine comprises the vaccine adjuvant and an immunogen; or when the cerium oxide nano material is a vaccine inactivating agent, the vaccine is a composite vaccine, the composite vaccine comprises the vaccine inactivating agent and a virus, and the virus is embedded in the cerium oxide nano material; the preparation method of the composite vaccine comprises the step of mineralizing and inactivating viruses by using a vaccine inactivator, wherein the vaccine inactivator is cerium salt. The cerium oxide can be used as a vaccine adjuvant and also can be used as a virus inactivator. The maturation of dendritic cells and macrophages can be promoted by means of regulating the level of active oxygen in immune cells and the like, and the antigen presentation efficiency is improved, so that the vaccine-induced body fluid and cellular immune response is enhanced; meanwhile, cerium salt can directly act on virus surface protein for mineralization, efficient and safe virus inactivation is achieved, and preparation of inactivated vaccines is facilitated.

Description

Technical Field

[0001] This invention relates to the field of vaccine formulation technology, and in particular to the application of cerium oxide nanomaterials in vaccine adjuvants and / or vaccine inactivators. Background Technology

[0002] Vaccine adjuvants are key excipients in vaccine formulations, enhancing the immunogenicity of antigens and promoting the strength and duration of the immune response, thereby improving the protective effect of vaccines. Traditional aluminum salt adjuvants have been the most widely used type of adjuvant in clinical practice since the last century due to their good safety profile. However, their immune enhancement mechanism mainly relies on the activation of the inflammatory microenvironment, inducing Th2 immune responses, and is insufficient in responding to complex viral, bacterial, and tumor antigens (Aluminum Adjuvants-'Back to the Future'. Pharmaceutics. 2023;15(7):1884). While some novel adjuvants, such as emulsion adjuvants (MF59, AS03) and adjuvants based on pattern recognition receptors (such as TLR agonists), have made some breakthroughs, the number of clinically approved types is extremely limited. They face problems such as scarce raw materials, high preparation costs, significant potential toxic side effects, and patent and technology barriers (Emerging concepts in the science of vaccine adjuvants. Nature reviews drug discovery, 2021, 20(6), 454–475; From Sharks to Yeasts: Squalene in the Development of Vaccine Adjuvants. Pharmaceuticals (Basel). 2022;15(3):265.). Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides an application of cerium oxide nanomaterials in vaccine adjuvants and / or vaccine inactivators, the technical solution of which is as follows: A vaccine, characterized in that, when cerium oxide nanomaterials are used as vaccine adjuvants, the vaccine comprises a vaccine adjuvant and an immunogen; When cerium oxide nanomaterials are used as vaccine inactivators, the vaccine is a composite vaccine, which includes a vaccine inactivator and a virus, with the virus encapsulated within the cerium oxide nanomaterials; the preparation method of the composite vaccine includes: mineralizing the vaccine inactivator to inactivate the virus, wherein the vaccine inactivator is a cerium salt.

[0004] Cerium oxide (CeO2) is an important rare earth oxide, and its nanostructured form has attracted widespread attention in the biomedical field in recent years. Nanostructured cerium oxide possesses unique CeO2 content. 3+ / Ce 4+ Its reversible mutational capacity and abundant surface oxygen vacancies enable it to exhibit a variety of enzyme activities, including superoxide dismutase (SOD), catalase (CAT), oxidase (OXD), and peroxidase (POD). This characteristic allows it to effectively regulate reactive oxygen species (ROS) levels, demonstrating excellent dual-regulation of antioxidant and pro-oxidative effects. Increased ROS levels in dendritic cells and macrophages help promote their maturation and antigen presentation functions, thereby enhancing CD4+. + T cells, B cells, and CD8 + T-cell activation significantly enhances the body's immune response. Compared to traditional adjuvants, cerium oxide not only serves as an antigen delivery carrier but also possesses immune-activating capabilities, further improving vaccine efficacy.

[0005] It is worth emphasizing that cerium oxide not only functions as an adjuvant but also as a novel virus inactivating agent. Because cerium salts bind to proteins on the surface of viruses, they mineralize in an alkaline environment to form cerium oxide that encapsulates the virus, achieving mineralization and inactivation of virus particles, thus preparing safe and effective inactivated vaccines. This "dual function" allows cerium oxide to participate in vaccine preparation (virus inactivation) and also act as an adjuvant in the formulation to enhance immunity, demonstrating unique technological advantages.

[0006] In one embodiment, when the cerium oxide nanomaterial is a vaccine adjuvant, the volume ratio of the cerium oxide nanomaterial to the immunogen is 1:1 to 1:10.

[0007] In one embodiment, when the cerium oxide nanomaterial is used as a vaccine inactivator, the volume ratio of the cerium oxide nanomaterial to the virus is 1:1 to 1:10.

[0008] In one embodiment, the cerium salt includes at least one of cerium nitrate, cerium chloride, or cerium acetate.

[0009] In one embodiment, the type of vaccine includes inactivated vaccines, live attenuated vaccines, subunit vaccines, nucleic acid vaccines, or recombinant protein vaccines.

[0010] In one embodiment, the vaccine includes an animal vaccine or a human vaccine; the animal includes fish, poultry, or mammals.

[0011] This invention also provides a method for preparing the above-mentioned vaccine, wherein when cerium oxide nanomaterials are used as vaccine adjuvants, the method for preparing the vaccine includes the following steps: Preparation of cerium oxide nanomaterials: Dissolve cerium salt, add sodium hydroxide and hydrogen peroxide, stir to react and generate precursor precipitate, collect the precipitate by centrifugation, wash, dry and calcine to obtain cerium oxide nanomaterials; Vaccine preparation: Cerium oxide nanomaterials are dissolved in water or PBS to form a vaccine adjuvant with a final concentration of 0.01~50 mg / mL. The vaccine adjuvant is then mixed with the immunogen to obtain the vaccine.

[0012] In one embodiment, the molar concentration ratio of the cerium salt: the sodium hydroxide: the hydrogen peroxide is 1:(1~10):(0.5~5). The cerium salt includes at least one of cerium nitrate, cerium chloride, or cerium acetate.

[0013] In one embodiment, the cerium oxide nanomaterial has an average particle size of 2-1000 nm and a morphology including one or more of the following: spherical, cubic, polyhedral, sheet-like, or rod-like.

[0014] In one embodiment, in the step of preparing cerium oxide nanomaterials, the stirring reaction temperature is 20~200℃ and the reaction time is 0.5~72h.

[0015] In one embodiment, in the step of preparing cerium oxide nanomaterials, the washing solution is anhydrous ethanol and water.

[0016] In one embodiment, in the step of preparing cerium oxide nanomaterials, the drying is performed overnight at 25~100°C.

[0017] In one embodiment, in the step of preparing cerium oxide nanomaterials, the calcination temperature is 100~800℃ and the calcination time is 4~8h.

[0018] When cerium oxide nanomaterials are used as vaccine inactivators, the preparation method of the composite vaccine includes the following steps: dissolving cerium salt in water, adding live virus, stirring and reacting at 3~35℃ for 0.5~2h to generate precursor cerium ions and virus complex, adding sodium hydroxide, stirring and reacting at 3~35℃ to generate precipitate, centrifuging to collect the precipitate, and washing to obtain the composite vaccine.

[0019] In one embodiment, the volume ratio of the cerium salt: the live virus: the sodium hydroxide is 1:(1~10):(0.5~5). The initial concentration of the cerium salt is 100 mM, and the initial concentration of the sodium hydroxide is 1 M.

[0020] In one embodiment, the reaction time for adding sodium hydroxide and stirring is 70-75 hours.

[0021] The cerium oxide nanomaterials described in this invention exhibit significant advantages in vaccine adjuvant applications due to their unique physicochemical properties. Cerium oxide possesses tunable surface oxygen vacancies and reversible Ce2O3. 3+ / Ce 4+ Cerium oxide exhibits redox activity, thus displaying various enzyme-like activities such as oxidases and peroxidases. It can effectively regulate reactive oxygen species (ROS) levels, thereby promoting the activation and maturation of dendritic cells and macrophages, and enhancing humoral and cellular immune responses. Simultaneously, cerium oxide nanomaterials possess high specific surface area, good dispersibility, and excellent biocompatibility. Their preparation process is simple, low-cost, and suitable for large-scale production, providing a reliable guarantee for their application in animal and human vaccines.

[0022] Based on the aforementioned characteristics, cerium oxide has significant advantages in its application as a vaccine adjuvant. Firstly, cerium oxide possesses multi-mechanism immunomodulatory capabilities, enhancing immunity through ROS homeostasis regulation and alleviating inflammation-related toxicity. Secondly, it combines the functions of virus inactivation and adjuvant, improving the overall protective efficacy of inactivated vaccines. Thirdly, it exhibits good biocompatibility and safety, and does not easily accumulate toxic products in vivo. Fourthly, its synthesis methods are mature, inexpensive, and easy to scale up. Fifthly, it is not dependent on scarce raw materials or patent restrictions, possessing the potential for independent and controllable industrialization. Therefore, cerium oxide nanomaterials can not only compensate for the shortcomings of aluminum salt adjuvants and some novel adjuvants, but may also become a new direction for breakthroughs in vaccine adjuvant research and development. In conclusion, applying cerium oxide nanomaterials to both vaccine inactivation and adjuvant processes has significant scientific value and application prospects, providing solid technical support for promoting the development of new vaccines and achieving equitable global access to vaccines.

[0023] Compared with the prior art, the present invention has the following beneficial effects: 1. Dual function: It can be used as a virus inactivator in vaccine preparation, and as an adjuvant to enhance the immune response.

[0024] 2. Multi-mechanism immunomodulatory capabilities: Cerium oxide nanomaterials, through pathways such as regulating ROS levels, not only promote the maturation of dendritic cells and macrophages, but also enhance CD4+. + T cells, CD8 + The activation of T cells and B cells significantly enhances the immune response to the vaccine.

[0025] 3. Tunable enzyme-like activity: By controlling the particle size, morphology, and surface oxygen vacancy concentration of cerium oxide nanoparticles, the Ce activity can be precisely adjusted. 3+ / Ce 4+ The ratio enhances the activity of oxidase-like enzymes (OXD) and peroxidase-like enzymes (POD), enabling controllable regulation of ROS levels in immune cells.

[0026] 4. Good biocompatibility: Cerium oxide nanomaterials exhibit low toxicity and excellent biocompatibility in vivo, and can be safely used as adjuvants for animal and human vaccines without destroying the integrity of the vaccine immunogen.

[0027] 5. Simple preparation and controllable cost: Cerium oxide nanomaterials can be prepared through alkaline precipitation, hydrogen peroxide oxidation, calcination and other processes. The operation is simple and the conditions are mild, which is suitable for large-scale production and ensures stable and consistent material performance.

[0028] 6. Wide applicability: Cerium oxide nanomaterials can be applied to various types of vaccines, such as inactivated vaccines, live attenuated vaccines, subunit vaccines, nucleic acid vaccines, or recombinant protein vaccines. They can also be used in vaccines for fish, poultry, mammals, and humans, combining versatility and flexibility.

[0029] 7. Enhancing vaccine efficacy: Experiments have shown that cerium oxide, as a vaccine adjuvant, can significantly improve vaccine protection rates; and vaccines prepared through mineralization inactivation achieve a 100% protection rate. Cerium oxide enhances the body's ability to protect against pathogens, and has significant practical application value and potential for widespread application. Attached Figure Description

[0030] Figure 1 A schematic diagram of the preparation process of cerium oxide nanomaterials; Figure 2 Here is an electron microscopy characterization image of the cerium oxide nanomaterials in Example 1; Figure 3 Here is an electron microscopy characterization image of cerium oxide nanomaterials in Comparative Example 1; Figure 4 Here are electron micrographs of cerium oxide nanomaterials in Comparative Example 2; Figure 5 Here are electron micrographs of the comparative example cerium oxide nanomaterials; Figure 6 Characterization diagrams of oxidase-like enzymes (A) and peroxidase-like enzymes (B) in cerium oxide nanomaterials; Figure 7 Characterization diagram of ROS levels in mouse dendritic cells (A) and macrophages (B) enhanced by cerium oxide nanomaterials; Figure 8 Immunological effect diagram comparing cerium oxide nano-adjuvant vaccine and unadjuvanted vaccine; Figure 9 Figure showing the effect of cerium oxide nanoparticles on the immunological efficacy of vaccines; Figure 10 A schematic diagram of the preparation process for a live virus mineralized inactivated vaccine; Figure 11 Electron micrograph of a live virus mineralized inactivated vaccine; Figure 12This is an illustration of the immunological effects of a mineralized inactivated vaccine. Detailed Implementation

[0031] This invention addresses the limitations of existing vaccine adjuvants, such as their limited variety, singular mechanisms of action, and the toxicity or technical barriers of some novel adjuvants. It provides a novel application of cerium oxide nanomaterials in vaccine adjuvants, serving both as a vaccine adjuvant and a virus inactivating agent. The aim is to promote the maturation of dendritic cells and macrophages by regulating intracellular reactive oxygen species (ROS) levels, thereby improving antigen presentation efficiency and enhancing vaccine-induced humoral and cellular immune responses. Simultaneously, cerium salts can directly act on viral surface proteins for mineralization, achieving efficient and safe virus inactivation and facilitating the preparation of inactivated vaccines.

[0032] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0034] The cerium acetate (Ce(C2H3O2)3) and sodium hydroxide used in this invention were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; PBS (Phosphate Buffered Saline) was purchased from Wuhan Saiweier Biotechnology Co., Ltd.; the frog iridovirus was the largemouth bass frog virus (LMBV), which was isolated, identified and preserved by the inventors' team at the Pearl River Fisheries Research Institute of the Chinese Academy of Fishery Sciences, as referenced in "Molecular Epidemiology and Histopathological Analysis of Largemouth Bass Frog Virus".

[0035] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all test methods are conventional test methods in this field.

[0036] Example 1 1. This invention provides a method for preparing cerium oxide nanomaterials. The specific steps are as follows: S1: Weigh a certain amount of cerium acetate (Ce(C2H3O2)3) and dissolve it in deionized water to adjust the concentration to 0.1M; S2: Add appropriate amounts of sodium hydroxide (final concentration 0.333M) and hydrogen peroxide (final concentration 0.1M) to the solution, stir and react at 40°C for 6 hours to generate a precursor precipitate; in this embodiment, the molar concentration ratio of cerium salt: sodium hydroxide: hydrogen peroxide is 1:3.33:1; S3: Collect the precipitate by centrifugation, and wash it three times with anhydrous ethanol and deionized water respectively to remove impurities; S4: Place the precipitate in an oven and dry it overnight at 80°C; S5: Finally, calcination was carried out at 200℃ for 6 hours in air atmosphere to obtain cerium oxide nanomaterials with good dispersibility and crystallinity.

[0037] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that no hydrogen peroxide was added during the preparation process, and no drying or calcination was performed.

[0038] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that no drying or calcination was performed during the preparation process.

[0039] Comparative Example 3 The only difference between Comparative Example 3 and Example 1 is that calcination was not performed during the preparation process.

[0040] Implementation effect evaluation 1. Electron microscopy characterization of cerium oxide nanomaterials The cerium oxide nanomaterials prepared in Example 1 were dispersed in deionized water, ultrasonicated to homogenize, and then dropped onto a copper mesh carrier. After natural drying, they were characterized by transmission electron microscopy (TEM).

[0041] The results are as follows Figure 2 As shown, the cerium oxide nanomaterials are nearly spherical or cubic particles with an average particle size of about 5~10 nm and good dispersibility, indicating that cerium oxide has been successfully prepared.

[0042] The difference between Comparative Example 1 and Example 1 is that no hydrogen peroxide was added during the preparation process, and no drying or calcination was performed.

[0043] The results are as follows Figure 3 As shown, cerium oxide nanomaterials are nearly spherical or rod-shaped particles with an average particle size of about 5~10 nm and poor dispersibility.

[0044] The difference between Comparative Example 2 and Example 1 is that no drying or calcination was performed during the preparation process.

[0045] The results are as follows Figure 4 As shown, cerium oxide nanomaterials are nearly spherical with an average particle size of about 5~10 nm. They exhibit severe aggregation and poor dispersibility, making them unsuitable as adjuvants.

[0046] The only difference between Comparative Example 3 and Example 1 is that calcination was not performed during the preparation process.

[0047] The results are as follows Figure 5 As shown, cerium oxide nanomaterials are nearly spherical with an average particle size of about 5~10 nm. They exhibit severe aggregation and poor dispersibility, making them unsuitable as adjuvants.

[0048] 2. Enzyme activity detection of cerium oxide nanomaterials To investigate the biological functions of cerium oxide nanomaterials, colorimetric methods were used to detect their oxidase-like and peroxidase-like activities.

[0049] Oxidase assay: S1: Add cerium oxide nanomaterials to a reaction system with TMB (3,3',5,5'-tetramethylbenzidine) as the substrate; S2: Incubate the above mixture at room temperature; S3: Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance of the above reaction products at 300-700 nm.

[0050] Peroxidase assay: S1: Add cerium oxide nanomaterials to a reaction system containing TMB and H2O2; S2: Incubate the above mixture at room temperature; S3: Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance of the above reaction products at 300-700 nm.

[0051] Experimental results are as follows Figure 6 As shown, cerium oxide nanomaterials possess both oxidase-like and peroxidase-like activities.

[0052] Compared with the oxidase-like and peroxidase-like activities of Comparative Examples 1-3 and Example 1, the enzyme activities of both were low, and due to the low activity, no valid data could be detected.

[0053] 3. Regulation of ROS levels in mouse dendritic cells and macrophages by cerium oxide nanomaterials S1: Mouse-derived macrophages (Ana-1) and dendritic cells (DC2.4) were seeded in 96-well plates and allowed to adhere for 24 hours. S2: Cerium oxide nanomaterials (final concentration 50 μg / mL) were incubated separately to stimulate the control group, while PBS was added. S3: Intracellular reactive oxygen species (ROS) levels were detected using the DCF-DA fluorescent probe, and fluorescence intensity was detected using a multi-functional microplate reader.

[0054] The results are as follows Figure 7 As shown, compared with the control group, the dendritic cells in the cerium oxide-treated group ( Figure 7 A) and macrophages ( Figure 7 B) The internal ROS level increased significantly, suggesting that cerium oxide can enhance the activity of immune cells by regulating redox reactions.

[0055] 4. Evaluation of the immunization effect of cerium oxide nano-adjuvant vaccines To verify the immune-enhancing effect of cerium oxide nanomaterials as vaccine adjuvants, an inactivated frog iridovirus (LMBV) vaccine was selected as a model vaccine for protection rate experiments.

[0056] In the experiment, the preparation method of cerium oxide nanoadjuvant vaccine was as follows: cerium oxide nanomaterials were mixed with immunogens to obtain cerium oxide nanoadjuvant vaccine.

[0057] The specific method is as follows: S1: Healthy largemouth bass (Micropterus salmoides) fry were selected and randomly divided into three groups: PBS group, injected with PBS only; LMBV group, injected with inactivated LMBV only; LMBV+CeO2 group, injected with inactivated virus and cerium oxide (immunogen and cerium oxide volume mixing ratio of 1:1, final cerium oxide concentration of 1 mg / mL), and injected intraperitoneally. S2: 21 days after immunization, the three groups of fish were challenged with LMBV virus and observed for 15 days. The cumulative mortality rate and survival rate were recorded and calculated.

[0058] Experimental results are as follows Figure 8 As shown, the survival rate of the control group with PBS inactivated vaccine was only 12.9%, and the survival rate of the LMBV inactivated vaccine group was only 56.7%, while the survival rate of the experimental group with added cerium oxide nanomaterial adjuvant was significantly improved, reaching approximately 86.7%. These results indicate that cerium oxide nanomaterials can significantly enhance the immunoprotective effect of the LMBV inactivated vaccine.

[0059] The difference between Comparative Example 1 and Example 1 is that the cerium oxide nanomaterials were not prepared with hydrogen peroxide and were not subjected to any drying or calcination. The results are as follows: Figure 9 As shown, the survival rate of the cerium oxide (cerium oxide-1) experimental group was 13% higher than that of the LMBV inactivation group.

[0060] The difference between Comparative Example 2 and Example 1 is that the cerium oxide nanomaterials prepared in Comparative Example 2 did not undergo any drying or calcination. The results are as follows... Figure 9 As shown, the survival rate of the cerium oxide (cerium oxide-2) experimental group was not improved compared to the survival rate of the LMBV inactivated group.

[0061] Example 2 This embodiment provides a method for preparing a live virus mineralized inactivated vaccine. Figure 10 The specific steps are as follows: S1: Weigh a certain amount of cerium acetate (Ce(C2H3O2)3) and dissolve it in deionized water to adjust the concentration to 0.1M; S2: Add an equal volume of live LMBV virus to the solution and stir at 4°C for 1 hour to generate a precursor cerium ion complex with the virus. S3: Add an equal volume of sodium hydroxide (final concentration 0.333M) to the solution, stir and react at 4°C for 72 hours to form a precipitate; in this example, the volume ratio of cerium salt: live virus: sodium hydroxide is 1:1:1; S4: The obtained precipitate was collected by centrifugation, washed three times with deionized water to remove impurities and obtain the composite vaccine. The vaccine was then subjected to five blind passages of cells, and the results showed that the cells were free of lesions.

[0062] Implementation effect evaluation 1. Electron microscopy characterization The nanomaterials obtained in Example 2 were dispersed in deionized water, ultrasonicated to homogenize, and then dropped onto a copper mesh carrier. After natural drying, they were characterized by transmission electron microscopy (TEM).

[0063] The results are as follows Figure 11 As shown, cerium oxide forms around the virus, and the virus is embedded in cerium oxide, indicating that the virus@cerium oxide composite vaccine has been successfully prepared.

[0064] 2. Evaluation of the immunization efficacy of live virus mineralization vaccine To verify the immunoprotective effect of cerium oxide nanomaterials as vaccine adjuvants and inactivators, live frog iridovirus (LMBV) was selected as a model for mineralization vaccine preparation, and protection rate experiments were conducted. The specific methods are as follows: S1: Healthy largemouth bass (Micropterus salmoides) fry were selected and randomly divided into three groups: PBS group, which was injected with PBS only; and LMBV@CeO2 group, which was injected with mineralized inactivated virus and cerium oxide (final concentration of cerium oxide was 1 mg / mL) via intraperitoneal injection.

[0065] S2: 21 days after immunization, the three groups of fish were challenged with LMBV virus and observed for 15 days. The cumulative mortality rate and survival rate were recorded and calculated.

[0066] Experimental results are as follows Figure 12 As shown, the survival rate of the PBS inactivated vaccine in the control group was only 0%, while the survival rate of the LMBV inactivated vaccine group was 100%. The results indicate that mineralized vaccines can significantly enhance the immunoprotective effect of LMBV inactivated vaccines.

[0067] The results shown in the embodiments of this invention demonstrate that cerium oxide nanomaterials have a good immune-enhancing effect as a vaccine adjuvant. Those skilled in the art will understand that the cerium oxide adjuvant is not only applicable to LMBV vaccines, but can also be applied to other fish virus vaccines (such as mandarin fish rhabdovirus, iridovirus, etc.), as well as animal vaccines for poultry, mammals, and even human vaccines, including but not limited to inactivated vaccines, live attenuated vaccines, subunit vaccines, nucleic acid vaccines, or recombinant protein vaccines.

[0068] Cerium oxide possesses a high specific surface area, enzyme-like activity, and good biocompatibility. Its surface oxygen vacancy formation energy is low, making it prone to forming Ce. 3+ / Ce 4+ The coexisting structure exhibits both oxidase-like (OXD) and peroxidase-like (POD) activities. By modulating the enzyme-like activity of CeO2 nanoparticles, the levels of reactive oxygen species (ROS) in dendritic cells (DCs) and macrophages can be regulated. Increased ROS can promote the maturation and antigen presentation of DCs and macrophages, and enhance CD4+. + T cells, B cells, and CD8 + T-cell activation significantly improves the efficiency of vaccine immune response. Furthermore, this invention is the first to propose using cerium oxide as a virus inactivating agent. Mineralization inactivation is achieved through the interaction of cerium salts with live viruses, simultaneously endowing the vaccine with natural adjuvant function and avoiding the toxic side effects of traditional chemical inactivating agents. This invention is applicable to various vaccine formulations, including animal vaccines (fish, poultry, mammals, etc.) or human vaccines.

[0069] To achieve the above objectives, the technical solution adopted by the present invention is as follows: 1. The preparation process of cerium oxide nanomaterials is as follows: it is prepared by alkaline precipitation, hydrogen peroxide oxidation, calcination and other processes, using cerium nitrate, cerium chloride or cerium acetate as precursors, supplemented with appropriate amounts of oxidant or surfactant, and reacted at 20~200℃ for 0.5~72h. After centrifugation, washing and calcination at 100~800℃, cerium oxide nanomaterials with an average particle size of 2~1000nm are obtained. The morphologies include spheres, cubes, polyhedra or rods.

[0070] 2. Structural Regulation and Functional Characterization: By controlling the surface oxygen vacancy concentration, particle size, and doping elements of cerium oxide nanomaterials, the structure of Ce can be adjusted. 3+ / Ce 4+ The ratio was adjusted to enhance the activity of oxidase-like enzymes (OXD) and peroxidase-like enzymes (POD). The prepared material exhibited significant ROS-regulating ability in in vitro experiments, promoting the activation and maturation of dendritic cells and macrophages, and enhancing CD4+. + T cells, CD8 + The immune activation effect of T cells and B cells.

[0071] 3. Virus Inactivation Application: Utilizing the interaction properties of cerium salts with proteins containing carboxyl groups, virus particles can be directly treated and mineralized to prepare inactivated vaccines. Experiments have shown that inactivated vaccines prepared using cerium salt-mineralized live viruses achieve 100% protection in fish model experiments, significantly outperforming conventional chemical inactivation methods.

[0072] 4. Construction of adjuvant formulation: Cerium oxide nanomaterials were dissolved in sterile water or PBS (Phosphate Buffered Saline) to prepare a 0.01~50 mg / mL solution, which was used as an adjuvant component of the vaccine formulation.

[0073] 5. Preparation and application of vaccines: Cerium oxide nanomaterials are mixed with immunogens at a mass ratio of 1:1 to 1:10, or live viruses are directly mineralized with cerium salts to prepare finished vaccines, which are then used in animal vaccines (fish, poultry, mammals, etc.) or human vaccines, including inactivated vaccines, live attenuated vaccines, subunit vaccines, nucleic acid vaccines, or recombinant protein vaccines.

[0074] Experimental results show that in mouse dendritic cell and macrophage experiments, cerium oxide significantly increases ROS levels and promotes immune cell maturation. In fish model experiments, the combined use of cerium oxide and inactivated vaccines, compared to unadjuvanted vaccines, increases the protection rate of frog iridovirus (LMBV) vaccines by 30%. Furthermore, cerium oxide-based vaccines prepared by directly mineralizing live viruses with cerium salts achieve 100% protection. Therefore, this invention provides a novel, safe, and efficient vaccine preparation and immune enhancement technology that combines the functions of an immune adjuvant and virus inactivation.

[0075] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0076] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A vaccine, characterized in that, When cerium oxide nanomaterials are used as vaccine adjuvants, the vaccine comprises both the vaccine adjuvant and the immunogen. When cerium oxide nanomaterials are used as vaccine inactivators, the vaccine is a composite vaccine, which includes a vaccine inactivator and a virus, with the virus encapsulated within the cerium oxide nanomaterials; the preparation method of the composite vaccine includes: mineralizing the vaccine inactivator to inactivate the virus, wherein the vaccine inactivator is a cerium salt.

2. The vaccine according to claim 1, characterized in that, When the cerium oxide nanomaterial is used as a vaccine adjuvant, the volume ratio of the cerium oxide nanomaterial to the immunogen is 1:1 to 1:

10.

3. The vaccine according to claim 1, characterized in that, When the cerium oxide nanomaterial is used as a vaccine inactivator, the volume ratio of the cerium oxide nanomaterial to the virus is 1:1 to 1:

10.

4. The vaccine according to claim 3, characterized in that, The cerium salt includes at least one of cerium nitrate, cerium chloride, or cerium acetate.

5. The vaccine according to any one of claims 1-4, characterized in that, The types of vaccines include inactivated vaccines, live attenuated vaccines, subunit vaccines, nucleic acid vaccines, or recombinant protein vaccines.

6. The vaccine according to any one of claims 1-4, characterized in that, The vaccine may be an animal vaccine or a human vaccine; the animal may be a fish, poultry or a mammal.

7. The method for preparing the vaccine according to any one of claims 1-6, characterized in that, When cerium oxide nanomaterials are used as vaccine adjuvants, the vaccine preparation method includes the following steps: Preparation of cerium oxide nanomaterials: Dissolve cerium salt, add sodium hydroxide and hydrogen peroxide, stir to react and generate precursor precipitate, collect the precipitate by centrifugation, wash, dry and calcine to obtain cerium oxide nanomaterials; Vaccine preparation: Cerium oxide nanomaterials are dissolved in water or PBS to form a vaccine adjuvant with a final concentration of 0.01~50 mg / mL. The vaccine adjuvant is then mixed with the immunogen to obtain the vaccine.

8. The preparation method according to claim 7, characterized in that, According to the molar concentration ratio, the cerium salt: the sodium hydroxide: the hydrogen peroxide is 1:(1~10):(0.5~5). The cerium salt includes at least one of cerium nitrate, cerium chloride, or cerium acetate.

9. A method for preparing the vaccine according to any one of claims 1-6, characterized in that, When cerium oxide nanomaterials are used as vaccine inactivators, the preparation method of the composite vaccine includes the following steps: dissolving cerium salt in water, adding live virus, stirring and reacting at 3~35℃ for 0.5~2h to generate precursor cerium ions and virus complex, adding sodium hydroxide, stirring and reacting at 3~35℃ to generate precipitate, centrifuging to collect the precipitate, and washing to obtain the composite vaccine.

10. The preparation method according to claim 9, characterized in that, According to the volume ratio, the cerium salt: the live virus: the sodium hydroxide is 1:(1~10):(0.5~5). The initial concentration of the cerium salt is 100 mM, and the initial concentration of the sodium hydroxide is 1 M.

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