A composite nano-adjuvant preparation based on zirconium metal organic framework and its application in animal vaccines

Abstract

The application provides a kind of composite nano adjuvant preparation based on zirconium metal organic framework and its application in animal vaccine.The nano adjuvant preparation includes zirconium metal organic framework material and one or more polymers, and the mass ratio of the porous zirconium metal organic framework material to the polymer is 1:1-3.The composite nano adjuvant preparation based on zirconium metal organic framework provided by the application has good stability and safety, can effectively enhance immune response and attack protection effect, and can be widely used in the preparation of animal inactivated vaccine, attenuated vaccine, recombinant protein vaccine, polypeptide vaccine and nucleic acid vaccine.The composite adjuvant preparation has low cost, simple preparation process and easy scale production.

Description

Technical Field

[0001] This invention belongs to the field of immunomodulatory technology, specifically relating to a composite nanoadjuvant formulation based on a zirconium metal-organic framework and its application in animal vaccines. Background Technology

[0002] In animal disease control, vaccines are the most economical and effective tool. Traditional live attenuated vaccines have high immunogenicity, but their development cycle is long and poses biosafety risks. Inactivated vaccines and subunit vaccines are relatively safe and are a research hotspot in the development of new vaccines. However, inactivated and subunit vaccines have limited immunogenicity and require adjuvants to achieve long-term effective protection. Adjuvants are substances that can bind specifically or non-specifically to antigens, inducing an effective and long-lasting immune response in the body, thus playing a supporting role. Adjuvants are of great significance in vaccine production. They can act on both the body and the antigen simultaneously. The appropriate use of adjuvants can greatly enhance the body's immune response to antigens and produce durable immune protection, reduce the amount of immunogen required, and lower vaccine production costs.

[0003] Ideal vaccine adjuvants need to be safe, effective, targeted, and economical. Currently, aluminum salt adjuvants, oil emulsion adjuvants, and natural active substances (such as propolis and chitosan) are commonly used adjuvants in animal vaccines, but they still have shortcomings in terms of functionality, targeting, safety, preparation processes, and universality. Therefore, developing safe and effective novel adjuvants is crucial for vaccine development. Summary of the Invention

[0004] To address the problems in the prior art, this invention provides a composite nano-adjuvant formulation based on zirconium metal-organic frameworks and its application in animal vaccines. The composite formulation, composed of zirconium metal-organic framework materials, immune agonists, and polymers, can be used as an adjuvant to combine with multiple antigen components to obtain a complete vaccine. This vaccine exhibits good stability and safety, and effectively enhances the immune effect.

[0005] The technical problem solved by this invention is achieved by the following technical solution:

[0006] The first objective of this invention is to provide a composite nanoadjuvant formulation based on a zirconium metal-organic framework, characterized in that it comprises a porous zirconium metal-organic framework material and one or more polymers; wherein the mass ratio of the porous zirconium metal-organic framework material to the polymer is 1:1 to 3.

[0007] Furthermore, it also includes an immune agonist, which is loaded in the nanopores of the porous zirconium metal-organic framework material or adsorbed on the surface of the material.

[0008] Furthermore, the mass ratio of the porous zirconium metal-organic framework material, polymer, and immune agonist is 1:1 to 3:0.1 to 1.

[0009] Furthermore, the porous zirconium metal-organic framework material is unmodified UIO-66 or a zirconium metal-organic framework material modified with a polycationic / anionic polymer.

[0010] Furthermore, the porous zirconium metal-organic framework material has a diameter of 50–300 nm, a zeta potential of -50–+50 mV, and a concentration of 0.2–2.0 mg / mL.

[0011] Furthermore, the immune agonist is one or more of Toll-like receptor agonists and interferon gene stimulating factor (STING) agonists.

[0012] Furthermore, the polymer is carbomer, or a mixture of carbomer and one or more hydrophilic polymers selected from dextran, carboxymethyl chitosan, sodium alginate, hyaluronic acid, and carboxymethyl cellulose.

[0013] Furthermore, when there are multiple polymers, the carbomer accounts for 50-80% of the total polymer mass.

[0014] Furthermore, the carbomer concentration is 1.5–6.0 mg / mL.

[0015] Furthermore, the preparation method of UIO-66 is as follows: adding a tetravalent zirconium salt to a polar organic solvent and stirring until completely dissolved to obtain a tetravalent zirconium salt solution; adding an organic ligand to a polar organic solvent and stirring until completely dissolved to obtain an organic ligand solution; thoroughly mixing the tetravalent zirconium salt solution and the organic ligand solution, adding a regulator, stirring and mixing, and then carrying out a solvothermal reaction at 100-200°C; cooling after the reaction to obtain the reaction product.

[0016] The reaction product was separated into solid and liquid phases to obtain a precipitate, which was then washed to obtain zirconium metal-organic framework material UIO-66.

[0017] Furthermore, the tetravalent zirconium salt solution contains at least one of zirconium chloride, zirconium oxychloride, zirconium sulfate, zirconium carbonate, zirconium silicate, zirconium acetate, zirconium n-propoxide, zirconium n-butoxide, and zirconium acrylate.

[0018] Furthermore, the polar organic solvent is at least one of dimethylformamide, formamide, dimethyl sulfoxide, and tetrahydrofuran; the organic ligand is at least one of terephthalic acid, 2-aminoterephthalic acid, and isophthalic acid.

[0019] Furthermore, the molar ratio of the tetravalent zirconium salt to the organic ligand is 1:5 to 5:1.

[0020] Furthermore, the regulator is at least one selected from formic acid, acetic acid, propionic acid, benzoic acid, trifluoroacetic acid, isoamyl alcohol, and hydrochloric acid.

[0021] Furthermore, the solvothermal reaction time is 6 to 36 hours.

[0022] Furthermore, the zirconium metal-organic framework material modification process is as follows: the obtained zirconium metal-organic framework material UIO-66 is dispersed in deionized water, a polycationic / anionic polymer is added, and after stirring, the precipitate is collected by centrifugation to obtain the zirconium metal-organic framework material modified by the polycationic / anionic polymer.

[0023] Furthermore, the polycationic / anionic polymer is a water-soluble polycationic / anionic polymer, including but not limited to polyethyleneimine, poly-L-lysine, polypropyleneamine hydrochloride, polydiallyldimethylammonium chloride, polyacrylic acid, sodium polystyrene sulfonate, or sodium alginate.

[0024] The second objective of this invention is to provide a method for preparing a composite nanoadjuvant formulation based on a zirconium metal-organic framework, characterized by comprising the following steps:

[0025] Zirconium metal-organic framework materials are dispersed in physiological saline or citrate buffer solution, and then autoclaved to obtain a dispersion of zirconium metal-organic framework materials.

[0026] An immune agonist solution is prepared by dispersing an immune agonist in physiological saline or citrate buffer solution and then sterilizing it by filtration.

[0027] The polymer is dispersed in physiological saline or citrate buffer solution, and then the polymer solution is obtained by filtration or autoclaving.

[0028] The zirconium metal-organic framework material dispersion is thoroughly mixed with the polymer solution, or the above three solutions, under sterile conditions to obtain a nanocomposite adjuvant formulation based on zirconium metal-organic framework material.

[0029] Furthermore, the zirconium metal-organic framework material dispersion is first mixed with an immune agonist solution and incubated at room temperature to allow the immune agonist to be loaded into the nanopores of the porous zirconium metal-organic framework material or adsorbed onto the material surface, thereby obtaining a sterile dispersion solution; then, it is thoroughly mixed with the polymer solution at a volume ratio of 1:1 to obtain a nanocomposite adjuvant formulation based on zirconium metal-organic framework material.

[0030] Furthermore, before autoclaving, NaOH is added to adjust the pH of the polymer solution to approximately 7.0.

[0031] The third objective of this invention is to provide an application of a zirconium metal-organic framework-based composite nanoadjuvant formulation in animal vaccines.

[0032] Furthermore, the composite nanoadjuvant formulation is combined with an immunogen for use in animal vaccines.

[0033] Furthermore, the vaccine is prepared by mixing a sterile zirconium metal-organic framework nanocomposite adjuvant with an animal immunogen at a volume ratio of 1:5 under sterile conditions to obtain a complete animal vaccine.

[0034] Furthermore, the vaccine is administered to the animal via intraperitoneal, subcutaneous, or intramuscular injection to complete the immunization.

[0035] Furthermore, the immunoantigen includes at least one of inactivated virus, attenuated virus, recombinant protein antigen, polypeptide, and nucleic acid.

[0036] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0037] This invention combines a zirconium metal-organic framework (ZMOR) nanocomposite adjuvant with an immunogenic antigen, effectively improving vaccine performance and enhancing immune protection. The ZMOR nanocomposite adjuvant is compatible with a variety of antigenic components, exhibiting broad applicability. The vaccine prepared by this invention demonstrates good stability and safety, and does not cause adverse reactions in animals during injection. The vaccine based on this nanocomposite adjuvant induces the body to produce rapid, high-titer antibodies. The composite adjuvant formulation of this invention is inexpensive, has a simple preparation process, and is easily mass-produced.

[0038] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and in order to make the above and other objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0039] Figure 1 Appearance images of the aqueous dispersions of UIO-66, UIO-66-PAA, and UIO-66-PEI nanoparticles prepared for this invention (A in the figures is the aqueous dispersion of UIO-66, B is the aqueous dispersion of UIO-66-PAA, and C is the aqueous dispersion of UIO-66-PEI).

[0040] Figure 2 Scanning electron microscope (SEM) images of UIO-66, UIO-66-PAA, and UIO-66-PEI nanoparticles prepared in this invention (A and B are UIO-66 nanoparticles, C and D are UIO-66-PAA nanoparticles, and E and F are UIO-66-PEI nanoparticles).

[0041] Figure 3 X-ray crystal diffraction (XRD) patterns of the UIO-66, UIO-66-PAA, and UIO-66-PEI nanoparticles prepared in this invention;

[0042] Figure 4 Hydration particle size distribution and Zeta potential of UIO-66, UIO-66-PAA, and UIO-66-PEI nanoparticles prepared in this invention;

[0043] Figure 5 The cytotoxicity results of the UIO-66, UIO-66-PAA, and UIO-66-PEI nanoparticles prepared in this invention are shown in the figure.

[0044] Figure 6 The results of loading model proteins and nucleic acids onto UIO-66, UIO-66-PAA, and UIO-66-PEI nanoparticles prepared in this invention are shown in the figure.

[0045] Figure 7 The figure shows the results of the determination of IL-2, IL-4, IL-6, IFN-γ and TNF-α in the serum of mice after immunization with pseudorabies vaccine 1. Detailed Implementation

[0046] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0047] In addition, unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing from the market or prepared by existing methods.

[0048] Example 1: Preparation of zirconium metal-organic framework material UIO-66

[0049] Add 160 mg (0.69 mmol) of zirconium chloride to 15 mL of dimethylformamide and stir magnetically at 300 rpm for 30 min to fully dissolve the zirconium chloride into a zirconium salt solution for later use.

[0050] Add 115 mg (0.69 mmol) of terephthalic acid to 15 mL of dimethylformamide and stir magnetically at 300 rpm for 30 min to fully dissolve the terephthalic acid into an organic ligand solution for later use.

[0051] The zirconium salt solution and the organic ligand solution were thoroughly mixed, and 2 mL of acetic acid was added as a regulator. After stirring for 15 min, a mixed solution was obtained. The mixed solution was transferred to a stainless steel reactor and then placed in a constant temperature oven at 120℃ for 24 h to carry out a solvothermal reaction. After the reaction was completed, the stainless steel reactor was removed and allowed to cool naturally to room temperature to obtain the reaction product.

[0052] The reaction product was centrifuged at 10,000 rpm for 5 min to obtain a precipitate. The precipitate was first suspended in dimethylformamide, and then centrifuged at 6,000–12,000 rpm for 5–10 min to collect the first precipitate. The first precipitate was then suspended in ethanol, and then centrifuged at 6,000–12,000 rpm for 5–10 min to collect the second precipitate. The second precipitate was then suspended in water, and then centrifuged at 6,000–12,000 rpm for 5–10 min to collect the third precipitate, thus obtaining the zirconium metal-organic framework material UIO-66.

[0053] Example 2: Preparation of zirconium metal-organic framework materials modified with polycationic / anionic polymers

[0054] The zirconium metal-organic framework material UIO-66 obtained in Example 1 was dispersed in deionized water to obtain a 10 mg / mL UIO-66 aqueous dispersion. Polyacrylic acid (PAA, with a final concentration of 50 mg / mL) was added to the UIO-66 aqueous dispersion. After stirring continuously for 1 h, the precipitate was collected by centrifugation at 10,000 rpm for 5 min. The precipitate was washed twice with water (10,000 rpm, 5 min) and finally dispersed in water to obtain a PAA-modified zirconium metal-organic framework material UIO-66-PAA aqueous dispersion.

[0055] The zirconium metal-organic framework material UIO-66 obtained in Example 1 was dispersed in deionized water to obtain a 10 mg / mL UIO-66 aqueous dispersion. Polyethyleneimine (PEI, with a final concentration of 50 mg / mL) was added to the UIO-66 aqueous dispersion. After stirring continuously for 1 h, the precipitate was collected by centrifugation at 10,000 rpm for 5 min. The precipitate was washed twice with water (10,000 rpm, 5 min) and finally dispersed in water to obtain a PEI-modified zirconium metal-organic framework material UIO-66-PEI aqueous dispersion.

[0056] See appendix Figure 1 , 2 3. After dispersing the UIO-66 obtained in Examples 1 and 2 in deionized water, their appearance, electron microscopy scan images, and X-ray crystal diffraction patterns were observed with the aqueous dispersions of UIO-66-PAA and UIO-66-PEI nanoparticles, respectively. It can be seen that:

[0057] The UIO-66 nanoparticles of this invention can be stably dispersed in water to form a milky white suspension. The aqueous dispersions of UIO-66-PAA and UIO-66-PEI nanoparticles are also stable milky white suspensions.

[0058] The SEM images show that the UIO-66 nanoparticles are octahedral particles with a diameter of 150-200 nm, exhibiting monodispersity and uniform particle size. Figure 2 A, 2B); SEM images of the dried aqueous dispersions of UIO-66-PAA and UIO-66-PEI nanoparticles are respectively... Figure 2 C, 2D, 2E, and 2F show that UIO-66-PAA and UIO-66-PEI are octahedral particles with a diameter of 150nm-200nm and uniform particle size.

[0059] The XRD pattern shows that UIO-66 has good crystallinity, and the modification with polyacrylic acid and polyethyleneimine did not damage the crystal structure of UIO-66.

[0060] Figure 4 The hydration particle size distribution and Zeta potential diagrams of UIO-66, UIO-66-PAA, and UIO-66-PEI nanoparticles are shown. It can be seen that the hydration particle size of UIO-66 is mainly distributed between 100-300 nm, which is consistent with the SEM results. The Zeta potential of UIO-66 is approximately +35 mV, indicating a positive charge. The hydration particle sizes of UIO-66-PAA and UIO-66-PEI are also mainly distributed between 100-300 nm, which is basically consistent with the results of UIO-66. The Zeta potentials of UIO-66-PAA and UIO-66-PEI are -20 mV and +48 mV, respectively, indicating that polyacrylic acid and polyethyleneimine were successfully modified onto the surface of UIO-66, resulting in UIO-66-PAA nanoparticles with a negative surface charge and UIO-66-PEI nanoparticles with a positive surface charge.

[0061] Figure 5 The figures show the cytotoxicity assays of UIO-66, UIO-66-PAA, and UIO-66-PEI nanoparticles. It can be seen that UIO-66 showed no significant cytotoxicity at a concentration as high as 0.2 mg / mL, and UIO-66-PAA and UIO-66-PEI also showed no significant cytotoxicity at concentrations as high as 0.2 mg / mL.

[0062] Figure 6The image shows the results of loading model proteins and nucleic acids onto the UIO-66, UIO-66-PAA, and UIO-66-PEI nanoparticles prepared in this invention. DNA-Cy3 nucleic acid and HRP enzyme were added to the aqueous dispersions of the three nanoparticles (UIO-66, UIO-66-PAA, and UIO-66-PEI) prepared in Example 2, respectively, and incubated at room temperature for 2 hours, followed by centrifugation. The changes before and after centrifugation were observed, showing that all three nanoparticles could efficiently load protein and nucleic acid molecules, confirming their potential as inactivated virus (containing protein, lipids, and nucleic acid) vaccines, recombinant protein vaccines, peptide vaccines, and nucleic acid vaccines.

[0063] Example 3: Preparation of UIO-66-PAA / carbomer nanocomposite adjuvant formulation

[0064] The UIO-66-PAA prepared in Example 2 was diluted with physiological saline to 6 mg / mL and then sterilized by autoclaving for later use.

[0065] Weigh 600 mg of carbomer and add it to a round-bottom flask containing 90 mL of physiological saline. Fix the round-bottom flask on a magnetic stirrer and stir continuously for 2 hours. Add NaOH to the above solution to adjust the pH of the carbomer solution to about 7.0, and then add physiological saline to make up the volume to 100 mL to obtain a carbomer solution with a concentration of 6 mg / mL. Then, obtain a sterile carbomer solution by filtration or high-temperature sterilization for later use.

[0066] The above-mentioned UIO-66-PAA sterile dispersion and carbomer solution were thoroughly mixed under sterile conditions at a volume ratio of 1:1 to obtain the UIO-66-PAA / carbomer nanocomposite adjuvant, wherein the final concentrations of both UIO-66-PAA and carbomer were 3 mg / mL.

[0067] Example 4: Preparation of UIO-66-PAA / carbomer / carboxymethyl cellulose nanocomposite adjuvant formulation

[0068] The UIO-66-PAA prepared in Example 2 was diluted with physiological saline to 6 mg / mL and then sterilized by autoclaving for later use.

[0069] Weigh 400 mg of carbomer into a round-bottom flask containing 80 mL of physiological saline. Fix the flask on a magnetic stirrer and stir continuously for 2 hours. Add NaOH to the above solution to adjust the pH of the carbomer solution to about 7.0, then add 200 mg of carboxymethyl cellulose and continue stirring for 30 minutes. Then, bring the volume to 100 mL with physiological saline. The final concentrations of carbomer and carboxymethyl cellulose are 4 mg / mL and 2 mg / mL, respectively. Then, obtain a sterile carbomer / carboxymethyl cellulose solution by filtration or high-temperature sterilization for later use.

[0070] The above-mentioned UIO-66-PAA sterile dispersion was thoroughly mixed with carbomer / carboxymethyl cellulose solution at a volume ratio of 1:1 under sterile conditions to obtain UIO-66-PAA / carbomer / carboxymethyl cellulose nanocomposite adjuvant, wherein the final concentrations of UIO-66-PAA, carbomer, and carboxymethyl cellulose were 3 mg / mL, 2 mg / mL, and 1 mg / mL, respectively.

[0071] Example 5: Preparation of UIO-66-PAA / Poly(I:C) / Carbomer Nanocomposite Adjuvant Formulation

[0072] The UIO-66-PAA prepared in Example 2 was diluted with physiological saline to 10 mg / mL and then sterilized by autoclaving for later use.

[0073] 150 mg of the Toll-like receptor 3 (TLR3) agonist Poly(I:C) was dissolved in 10 mL of physiological saline to obtain a Poly(I:C) solution with a concentration of 15 mg / mL. The solution was then sterilized by filtration to obtain a sterile Poly(I:C) solution for later use.

[0074] Sterile UIO-66-PAA solution and sterile Poly(I:C) solution were mixed at a volume ratio of 3:2 and incubated at room temperature for 2 hours to allow Poly(I:C) to be adsorbed into the pores of UIO-66-PAA, thus obtaining a sterile dispersion of UIO-66-PAA / Poly(I:C).

[0075] Weigh 600 mg of carbomer and add it to a round-bottom flask containing 90 mL of physiological saline. Fix the round-bottom flask on a magnetic stirrer and stir continuously for 2 hours. Add NaOH to the above solution to adjust the pH of the carbomer solution to about 7.0, and then add physiological saline to make up the volume to 100 mL to obtain a carbomer solution with a concentration of 6 mg / mL. Then, obtain a sterile carbomer solution by filtration or high-temperature sterilization for later use.

[0076] The above-mentioned UIO-66-PAA / Poly(I:C) sterile dispersion and carbomer solution were thoroughly mixed at a volume ratio of 1:1 to obtain the UIO-66-PAA / Poly(I:C) carbomer nanocomposite adjuvant formulation.

[0077] Example 6: Preparation of a porcine pseudorabies virus vaccine (pseudorabies vaccine 1) based on UIO-66-PAA / carbomer nanocomposite adjuvant

[0078] The UIO-66-PAA / carbomer nanocomposite adjuvant prepared in Example 3 was thoroughly mixed with the prepared porcine pseudorabies virus (C1201 strain / ΔgE) inactivated antigen at a volume ratio of 1:5 to prepare a pseudorabies inactivated vaccine (the viral titer before inactivation was 1×10⁻⁶). 6.0 TCID 50 / ml), named pseudorabies vaccine 1.

[0079] The prepared complete vaccine was placed at 4°C to observe its stability. The vaccine compositions prepared in this example were all homogeneous suspensions with good stability, and could be stably stored at 4°C for at least 1 year.

[0080] Example 7: Preparation of a porcine pseudorabies virus vaccine (pseudorabies vaccine 2) based on UIO-66-PAA / carbomer / carboxymethyl cellulose nanocomposite adjuvant.

[0081] The UIO-66-PAA / carbomer / carboxymethyl cellulose nanocomposite adjuvant prepared in Example 4 was thoroughly mixed with the prepared porcine pseudorabies virus (C1201 strain / ΔgE) inactivated antigen at a volume ratio of 1:5 to prepare a pseudorabies inactivated vaccine (the viral titer before inactivation was 1×10⁻⁶). 6.0 TCID 50 / ml), named pseudorabies vaccine 2.

[0082] The prepared complete vaccine was placed at 4°C to observe its stability. The vaccine compositions prepared in this example were all homogeneous suspensions with good stability, and could be stably stored at 4°C for at least 1 year.

[0083] Example 8: Preparation of a porcine pseudorabies virus vaccine (pseudorabies vaccine 3) based on UIO-66-PAA / Poly(I:C) / carbomer nanocomposite adjuvant.

[0084] The UIO-66-PAA / Poly(I:C) / carbomer nanocomposite adjuvant prepared in Example 5 was thoroughly mixed with the prepared porcine pseudorabies virus (C1201 strain / ΔgE) inactivated antigen at a volume ratio of 1:5 to prepare a pseudorabies inactivated vaccine (the viral titer before inactivation was 1×10⁻⁶). 6.0 TCID 50 / ml), named pseudorabies vaccine 3.

[0085] The prepared complete vaccine was placed at 4°C to observe its stability. The vaccine compositions prepared in this example were all homogeneous suspensions with good stability, and could be stably stored at 4°C for at least 1 year.

[0086] Experiment 1: Detection of the immunoprotective effect of UIO-66-PAA / carbomer nanocomposite adjuvant on mice

[0087] 1. Evaluation of serological immune enhancement effect

[0088] Example 6 prepared pseudorabies vaccine 1, and simultaneously prepared control vaccine A using the same antigen and commercial aluminum gel adjuvant. Five Kunming mice were injected intraperitoneally with each vaccine, 0.1 mL / mouse, while the blank control group received only the same volume of physiological saline. Immunization was repeated every two weeks for a total of two immunizations. Blood samples were collected at 14, 21, and 28 days after the first immunization, and serum was separated for later use. All mice in each group showed normal results after immunization, with no adverse reactions.

[0089] (1) Serum gB antibody level

[0090] gB protein is a conserved structural protein of pseudorabies virus and its most important protective antigen protein, playing a crucial role in inducing an immune response. Detecting gB antibody levels after immunization can evaluate the effectiveness of the vaccine.

[0091] Antibody titers were detected in serum samples taken 14, 21, and 28 days post-immunization using a commercial gB antibody ELISA kit. The results (see Table 1) showed that antibodies in the experimental group mice began to turn positive approximately 2 weeks after vaccination, and antibody levels gradually increased over time, reaching their highest level approximately 4 weeks post-immunization.

[0092] Table 1. gB antibody levels in each experimental group at different time points

[0093]

[0094] Table 1 shows that the antibodies of pseudorabies vaccine 1 turned positive after two weeks of immunization, and the antibody production rate was faster. The antibody level produced was almost 29.9 times that of the control vaccine A, indicating that the vaccine formulated with the vaccine adjuvant of the present invention has a stronger immunizing effect.

[0095] (2) Serum levels of cytokines IL-2, IL-4, IL-6, IFN-γ, and TNF-α

[0096] IL-2, IL-4, IL-6, IFN-γ, and TNF-α are important immunomodulatory factors with various biological activities. Among them, IFN-γ, TNF-α, and IL-2 are Th1-type cytokines mainly produced by activated T cells and NK cells, primarily involved in cellular immune responses and inflammatory reactions, and can induce the body's immune response to shift towards the Th1 type. IL-4 and IL-6, produced by activated Th2-type cells, can enhance the interaction between B cells and T cells, promote humoral immune responses, and induce CD4+. + The directed differentiation of Th immature precursor cells, and the levels of IL-2, IL-4, IL-6, IFN-γ and TNF-α can evaluate the early immune status of the body.

[0097] The levels of IL-2, IL-4, IL-6, IFN-γ, and TNF-α in serum 28 days post-immunization were measured using commercially available kits. A standard curve was plotted with the measured concentrations of standards on the x-axis and their absorbance values ​​on the y-axis. The concentration of the test sample could be determined from its absorbance value, and multiplied by the sample dilution factor to obtain the specific levels of IL-2, IL-4, IL-6, IFN-γ, and TNF-α in the test sample. The results are as follows: Figure 7 As shown, 28 days after immunization, the secretion levels of IL-2, IFN-γ, and TNF-α in pseudorabies vaccine 1 were significantly higher than those in the control vaccine; the secretion levels of IL-4 and IL-6 were also higher than those in the control vaccine 28 days after immunization. This indicates that the vaccine formulated with the adjuvant of the present invention can stimulate humoral and cellular immunity more quickly and strongly, further demonstrating its better immune effect.

[0098] 2. Mouse challenge protection test

[0099] Fifty female Kunming mice weighing 18–22 g were randomly divided into three groups: an experimental group (n=20) and a control group (n=10). Each group was immunized with a separate vaccine via intraperitoneal injection (0.1 mL / mouse). The blank control group received only the same volume of physiological saline. Immunization was repeated every two weeks for a total of two immunizations. Twenty-eight days after the initial immunization, a challenge test was conducted. Mice in all inactivated vaccine groups and half of the blank control group were intraperitoneally injected with 0.1 ml of porcine pseudorabies virus strain WH-13 (containing 500 LD50). Clinical manifestations of the mice were observed for 10 consecutive days after challenge, and mortality rates in the challenge control group and each experimental group were recorded. Mice in the negative control group developed pseudorabies symptoms on day 4 after challenge, exhibiting restlessness, ruffled and disheveled fur, and mostly died 16–24 hours after symptom onset. The protection rates of the inactivated vaccine groups varied within 10 days, as shown in Table 2.

[0100] Table 2 Survival status of mice after virus challenge

[0101] Group Number of survivors / Total number of mice in the experimental group Protection rate Pseudorabies vaccine 1 18 / 20 90％ Comparison with vaccine A 7 / 20 35％ Challenge to control 0 / 10 0.0％

[0102] The results of the challenge protection test showed that the pseudorabies vaccine 1 could produce a protection rate of over 90% after immunization, with a good immunization effect, which was significantly higher than that of the control vaccine, indicating that the vaccine formulated with the vaccine adjuvant of the present invention has a good protective effect against pseudorabies virus.

[0103] Experiment 2: Detection of the immunoprotective effect of UIO-66-PAA / carbomer nanocomposite adjuvant on pigs

[0104] 1. Protection experiment of this genus of animals against viral challenge in pigs.

[0105] Piglets were randomly divided into four groups: two immunization groups (pseudorabies vaccine 1 and control vaccine A), one challenge control group (no immunization, challenged), and one blank control group (no immunization, challenged), with five piglets in each group. One group of piglets was immunized with pseudorabies vaccine 1 prepared in Example 6 and control vaccine A, respectively, by intramuscular injection of 2 mL of vaccine into the left side of the neck of each piglet. The piglets were observed for 30–60 minutes after immunization, and the occurrence of stress reactions caused by vaccination (depression, frothing at the mouth, vomiting, collapsing, difficulty breathing, cyanosis, etc.) was observed and recorded. The blank control group was given only the same volume of physiological saline, without immunization or challenge. Immunization was repeated every two weeks, for a total of two immunizations.

[0106] Post-challenge observation, temperature measurement, and disease severity assessment: Body temperature was measured once on the morning of the day before (-1 day), the day of challenge (0 day), and for 21 consecutive days after challenge. Appetite, mental state, and any clinical symptoms were observed daily in the experimental pigs. If any pig in the immunized group or challenge control group developed a temperature of 40.8℃ or 40.9℃, the temperature was measured again in the afternoon, and the data from the two measurements were collected. Disease severity and protection were assessed according to the draft quality standards for porcine pseudorabies gene-deleted inactivated vaccine (C1201 / △gE strain).

[0107] Table 3 Clinical manifestations of piglets after immune challenge

[0108]

[0109]

[0110] *Note: A—Depression, loss of appetite; B—Difficulty breathing (including coughing, wheezing, open-mouth breathing, abdominal breathing); C—Runny nose, diarrhea, vomiting; D—Neurological symptoms (ataxia, paralysis, limb flailing, opisthotonus, head tilt, dog sitting, etc.); E—Death; "None"—No clinical symptoms. In clinical manifestations, "+" indicates mild or transient clinical symptoms; "++" indicates significant clinical symptoms; "+++" indicates severe clinical symptoms.

[0111] The results of the pig challenge protection experiment showed that: none of the pseudorabies vaccine group 1 developed the disease, with only slight clinical symptoms; 3 / 5 of the control vaccine group A developed the disease and exhibited obvious clinical symptoms, mainly depression, loss of appetite, and difficulty breathing; 4 / 5 of the challenge control group developed the disease, with severe clinical symptoms, including frothing at the mouth and near death; the blank control group had normal body temperature (38.8℃~39.8℃), normal mental state and appetite, and no abnormal clinical symptoms. The results indicate that pseudorabies vaccine 1 achieves a 100% protection rate after immunization, significantly higher than the 40% protection rate of the control vaccine, further demonstrating in animal studies that the vaccine adjuvant of this invention does indeed have excellent protective effects against pseudorabies virus. Furthermore, the vaccine adjuvant of this invention has good safety in pigs, producing no adverse reactions after immunization.

[0112] Experiment 3: Detection of the protective effect of UIO-66-PAA / carbomer / carboxymethyl cellulose nanocomposite adjuvant against viral challenge in mice.

[0113] Forty female Kunming mice weighing 18–22 g were randomly divided into three groups: an experimental group (n=15) and a control group (n=10). The experimental group mice were immunized intraperitoneally with pseudorabies vaccine 2 prepared in Example 7 and control vaccine A, respectively, at a dose of 0.1 mL per mouse. The blank control group was given the same volume of physiological saline. Immunization was repeated every two weeks for a total of two immunizations. Twenty-eight days after the initial immunization, a challenge test was conducted. Mice in all inactivated vaccine groups and half of the blank control group were intraperitoneally injected with 0.1 ml of porcine pseudorabies virus strain WH-13 (containing 500 LD50). Clinical manifestations of the mice were observed for 10 consecutive days after challenge, and mortality rates in the challenge control group and each test group were recorded. Mice in the negative control group developed pseudorabies symptoms on day 4 after challenge, including restlessness, ruffled and disheveled fur, and mostly died 16–24 hours after the onset of symptoms. The protection rates of the inactivated vaccine groups varied within 10 days, as shown in Table 4.

[0114] Table 4. Survival status of mice after virus challenge

[0115] Group Number of survivors / Total number of mice in the experimental group Protection rate Pseudorabies vaccine 2 13 / 15 87％ Comparison with vaccine A 5 / 15 33％ Challenge to control 0 / 10 0.0％

[0116] The results of the challenge protection test showed that the pseudorabies vaccine 2 could produce a protection rate of nearly 90% after immunization, with a better immunization effect and significantly higher than the control vaccine, indicating that the vaccine formulated with the vaccine adjuvant of the present invention has a good protective effect against pseudorabies virus.

[0117] Experiment 4: Detection of the immunoprotective effect of UIO-66-PAA / Poly(I:C) / carbomer nanocomposite adjuvant on mice.

[0118] 1. Evaluation of serological immune enhancement effect

[0119] Example 8 prepared pseudorabies vaccine 3, and simultaneously prepared control vaccine A using the same antigen and commercial aluminum gel adjuvant. Five Kunming mice were injected intraperitoneally with each vaccine, 0.1 mL / mouse, while the blank control group received only the same volume of physiological saline. Immunization was performed twice, every two weeks. Blood samples were collected at 14, 21, and 28 days after the first immunization, and serum was separated for later use. All mice in each group showed normal results after immunization, with no adverse reactions.

[0120] (1) Serum gB antibody level

[0121] Antibody titers were detected in serum samples taken 14, 21, and 28 days post-immunization using a commercial gB antibody ELISA kit. The results (see Table 5) showed that antibodies in the experimental group mice began to turn positive approximately 2 weeks after vaccination, and antibody levels gradually increased over time, reaching their highest level approximately 4 weeks post-immunization.

[0122] Table 5. gB antibody levels in each experimental group at different time points

[0123]

[0124]

[0125] Table 5 shows that the antibodies of pseudorabies vaccine 3 turned positive after two weeks of immunization, and the rate of antibody production was significantly higher than that of control vaccine A. Moreover, the antibody level produced after 28 days was almost 23.7 times that of control vaccine A, indicating that the compound adjuvant vaccine formulated with the vaccine adjuvant of the present invention has a stronger immunizing effect.

[0126] 2. Mouse challenge protection test

[0127] Forty female Kunming mice weighing 18–22 g were randomly divided into three groups: experimental groups (n=15 per group) and challenge control groups (n=10 per group). Each group was immunized with a separate vaccine via intraperitoneal injection (0.1 ml / mouse). The blank control group received only the same volume of physiological saline. Immunization was repeated every two weeks for a total of two immunizations. Twenty-eight days after the initial immunization, a challenge test was conducted. Mice in all inactivated vaccine groups and half of the blank control groups were intraperitoneally injected with 0.1 mL of porcine pseudorabies virus strain WH-13 (containing 500 LD50). Clinical manifestations of the mice were observed for 10 consecutive days after challenge, and mortality rates in the challenge control group and each experimental group were recorded. Mice in the negative control group developed pseudorabies symptoms on day 4 after challenge, exhibiting restlessness, ruffled and disheveled fur, and mostly died 16–24 hours after symptom onset. The protection rates of the inactivated vaccine groups varied within 10 days, as shown in Table 6.

[0128] Table 6 Survival status of mice after virus challenge

[0129] Group Number of survivors / Total number of mice in the experimental group Protection rate Pseudorabies vaccine 3 14 / 15 93.4％ Comparison with vaccine A 5 / 15 33％ negative control 0 / 5 0.0％

[0130] The results of the challenge protection test showed that the pseudorabies vaccine 3 could produce a protection rate of more than 90% after immunization, with a better immunization effect, which was significantly higher than that of the control vaccine A, indicating that the compound adjuvant vaccine formulated with the vaccine adjuvant of the present invention has a good protective effect on mice.

[0131] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0132] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A composite nano-adjuvant formulation based on a zirconium metal-organic framework, characterized in that, Including porous zirconium metal-organic framework materials and one or more polymers, The porous zirconium metal-organic framework material is a zirconium metal-organic framework material modified with polyacrylic acid. The polymer is carbomer, or a mixture of carbomer and one or more hydrophilic polymers selected from dextran, carboxymethyl chitosan, sodium alginate, hyaluronic acid, and carboxymethyl cellulose. The mass ratio of the porous zirconium metal-organic framework material to the polymer is 1:

1.

2. The composite nanoadjuvant formulation based on a zirconium metal-organic framework as described in claim 1, characterized in that: It also includes immune agonists, which are loaded in the nanopores of the porous zirconium metal-organic framework material or adsorbed on the surface of the material.

3. The composite nanoadjuvant formulation based on a zirconium metal-organic framework as described in claim 2, characterized in that: The mass ratio of the porous zirconium metal-organic framework material, polymer, and immune agonist added is 1:1:

1.

4. The composite nanoadjuvant formulation based on a zirconium metal-organic framework as described in claim 1, characterized in that: The porous zirconium metal-organic framework material has a diameter of 50~300nm, a zeta potential of -20mV, and a concentration of 0.2~2.0mg / mL.

5. A composite nanoadjuvant formulation based on a zirconium metal-organic framework as described in any one of claims 1-4, characterized in that: The method for preparing the zirconium metal-organic framework material is as follows: adding a tetravalent zirconium salt to a polar organic solvent and stirring until completely dissolved to obtain a tetravalent zirconium salt solution; adding an organic ligand to a polar organic solvent and stirring until completely dissolved to obtain an organic ligand solution; thoroughly mixing the tetravalent zirconium salt solution and the organic ligand solution, adding a regulator, stirring and mixing, and then carrying out a solvothermal reaction at 100~200℃; cooling after the reaction to obtain the reaction product. The reaction product was separated into solid and liquid phases to obtain a precipitate, which was then washed to obtain zirconium metal-organic framework material UIO-66.

6. The composite nanoadjuvant formulation based on a zirconium metal-organic framework as described in claim 5, characterized in that: The modification process of the zirconium metal-organic framework material is as follows: the obtained zirconium metal-organic framework material UIO-66 is dispersed in deionized water, a polyanionic polymer is added, and after stirring, the precipitate is collected by centrifugation to obtain the zirconium metal-organic framework material modified by the polyanionic polymer.

7. A method for preparing a composite nano-adjuvant formulation based on a zirconium metal-organic framework as described in any one of claims 1-6, characterized in that, Includes the following steps: Zirconium metal-organic framework materials are dispersed in physiological saline or citrate buffer solution, and then autoclaved to obtain a dispersion of zirconium metal-organic framework materials. An immune agonist solution is prepared by dispersing an immune agonist in physiological saline or citrate buffer solution and then sterilizing it by filtration. The polymer is dispersed in physiological saline or citrate buffer solution, and then the polymer solution is obtained by filtration or autoclaving. The zirconium metal-organic framework material dispersion and polymer solution, or the zirconium metal-organic framework material dispersion, immune agonist solution and polymer solution, are thoroughly mixed under sterile conditions to obtain a nanocomposite adjuvant formulation based on zirconium metal-organic framework material.

8. The method for preparing a composite nano-adjuvant formulation based on a zirconium metal-organic framework as described in claim 7, characterized in that: First, the zirconium metal-organic framework material dispersion is mixed with an immune agonist solution and incubated at room temperature to allow the immune agonist to be loaded into the nanopores of the porous zirconium metal-organic framework material or adsorbed onto the surface of the material, thereby obtaining a sterile dispersion solution; then, it is thoroughly mixed with the polymer solution at a volume ratio of 1:1 to obtain a nanocomposite adjuvant formulation based on zirconium metal-organic framework material.

9. The application of a zirconium metal-organic framework-based composite nanoadjuvant preparation obtained by any one of the preparation methods in claims 7-8 in the preparation of animal vaccines, wherein the composite nanoadjuvant preparation is mixed with an immunogenic antigen for use in animal vaccines.

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