Veterinary composite water-soluble adjuvant and method for its preparation

Abstract

The application discloses a veterinary compound water-soluble adjuvant and a preparation method thereof, and belongs to the technical field of vaccine adjuvants. The application provides a veterinary compound water-soluble adjuvant to solve the problem of poor biocompatibility of traditional adjuvants in the prior art and to provide a stable and efficient novel carbomer adjuvant. The veterinary compound water-soluble adjuvant is composed of 10%-30% crosslinked polymers, 1%-6% solution stabilizers, 2.5%-7.5% polysaccharide immunopotentiators and 56.5%-86.5% solvents. The four components are mutually restrained and mutually enhanced, physical slow release-chemical activation-local low stimulation integration is realized, and it is proved that each component is indispensable in a limited proportion, so that a better adjuvant selection is provided for the market.

Description

Technical Field

[0001] This invention belongs to the field of vaccine adjuvant technology, and in particular relates to a veterinary compound water-soluble adjuvant and its preparation method. Background Technology

[0002] Adjuvants are substances that non-specifically enhance the body's immune response to antigens. They are substances added to vaccines to enhance the immunogenicity of antigens. While not antigenic themselves, adjuvants can synergistically enhance the immune effect with immunogenic antigens, for example, increasing the protection rate of weak immunogenic antigens from <40% to >90% (e.g., avian influenza H5 subtype vaccine). They can also save antigen dosage, reducing antigen usage by 4-10 times (e.g., foot-and-mouth disease vaccine). Furthermore, they have the effect of prolonging immunity, extending antibody duration by 2-3 times (e.g., rabies vaccine). The essence of adjuvant science lies in altering the antigen presentation process, overcoming immune tolerance, and achieving a transformation from "low antigen dosage to high protective efficacy," making it of core application value in veterinary vaccine development. Its core functions include the following aspects: (1) Enhancing immunogenicity: promoting the activation of antigen-presenting cells (APCs) and enhancing antibody titer and immune persistence; (2) Reducing antigen dosage: reducing the unit dose of antigen requirement through sustained release effect or immune signal amplification; (3) Directional regulation of immune response type: such as enhancing Th1 / Th2 balance or mucosal immunity to adapt to the clearance mechanism of different pathogens.

[0003] Vaccination is the core means of preventing and controlling animal infectious diseases. More than 35% of important diseases worldwide still lack effective vaccines (such as ASF and PRDC). The core bottlenecks are in the following three aspects: (1) Weak immunogenic antigens: Protein antigens with a molecular weight of <20 kDa (such as PCV2 Cap) cannot activate sufficient immune memory, and the seroconversion rate of neutralizing antibodies is <40%, which leads to insufficient immune protection; (2) Insufficient efficacy of traditional adjuvants. Traditional aluminum adjuvants only stimulate Th2 humoral immunity, and the protection rate against intracellular pathogens that require cell immunity (such as Salmonella) is ≤35%; while oil emulsion adjuvants (such as Montanide™ ISA 206) cause injection site granulomas (incidence rate >25%), and the high viscosity (>200 mPa·s) easily hinders on-site injection; (3) New adjuvants face economic barriers. For example, the cost of TLR agonists (such as Poly I:C) is as high as $180-220 / g, which cannot meet the needs of large-scale application in animal husbandry. In the existing technology, traditional adjuvants face serious biocompatibility challenges. For example, aluminum adjuvants have a greater than 15% probability of causing persistent granulomas at the injection site, while mineral oil adjuvants have an abscess incidence rate as high as 20%-30%.

[0004] Carbomer ( CarbomerAs an anionic polyacrylic acid polymer, carbomer adjuvant forms a pH-neutral (6.5-7.4) sustained-release environment in muscle tissue through its unique biodegradable gel network structure. The carboxyl groups (-COOH) on its molecular chain partially ionize under physiological conditions, forming a hydrated gel layer that significantly reduces macrophage overactivation. The three-dimensional cross-linked network of carbomer adjuvant possesses intelligent antigen-controlled release capabilities. When antigen molecules are embedded in the gel pores (average pore size 50-200 nm), they gradually swell under the influence of tissue fluid osmotic pressure through a remodeled immune response kinetics-sustained-release effect-gel mechanism. The antigen is released through a dual mechanism of diffusion and erosion, extending its half-life compared to traditional adjuvants, thereby ensuring a sustained capture window for antigen-presenting cells (APCs).

[0005] Carbomer adjuvants possess excellent engineerability, low-shear processing compatibility, good freeze-thaw stability, and bacterial tolerance. Therefore, developing carbomer-based adjuvants is a strategic necessity for upgrading veterinary vaccines. Its necessity is rooted in the three core logics of safety, immunity, and industry, aiming to overcome the delivery challenges of weakly immunogenic antigens while reducing tissue side effects and lowering preparation costs. Summary of the Invention

[0006] To address the problem of poor biocompatibility of traditional adjuvants in the prior art, and to provide a stable and efficient novel carbomer adjuvant, this invention provides a veterinary composite water-soluble adjuvant and its preparation method.

[0007] One of the objectives of this invention is to provide a veterinary compound water-soluble adjuvant, which comprises the following components: 10%-30% crosslinked polymer, 1%-6% solution stabilizer, 2.5%-7.5% polysaccharide immune enhancer, and 56.5%-86.5% solvent.

[0008] In a preferred embodiment of the present invention, the crosslinking polymer is polyacrylamide and carbomer 974P.

[0009] In a preferred embodiment of the present invention, the polyacrylamide has a particle size of 0.1 micrometers.

[0010] In a preferred embodiment of the present invention, the solution stabilizer is poloxamer or mannitol.

[0011] In a preferred embodiment of the present invention, the polysaccharide immune enhancer is one or more of the following: American ginseng root polysaccharide, Acanthopanax senticosus polysaccharide, glucan, and trehalose.

[0012] In a preferred embodiment of the present invention, the solvent is an aqueous solution containing a salt ion buffer.

[0013] In a preferred embodiment of the present invention, the aqueous solution containing the salt ion buffer is a phosphate buffer solution with a pH of 6.5.

[0014] The second objective of this invention is to provide the application of the above-mentioned veterinary compound water-soluble adjuvant in the preparation of veterinary vaccines.

[0015] A third objective of this invention is to provide a veterinary vaccine comprising the aforementioned veterinary compound water-soluble adjuvant.

[0016] The fourth objective of this invention is to provide a method for preparing the above-mentioned veterinary compound water-soluble adjuvant, the method comprising the following steps:

[0017] S1: Dissolve 10%-30% crosslinking polymer and 1%-6% solution stabilizer in 56.5%-86.5% solvent, and stir continuously at 60-80℃ and 1200-1800 rpm for 40-60 min to obtain a dispersion;

[0018] S2: Dissolve 2.5%-7.5% of polysaccharide immune enhancer in the dispersion obtained in S1, and stir continuously for 30-60 min at 40-55℃ and 600-800 rpm to obtain a veterinary compound water-soluble adjuvant.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] This invention achieves a synergistic effect of step-by-step locking between components through a quaternary confinement ratio of "10%-30% crosslinking polymer, 1%-6% solution stabilizer, 2.5%-7.5% polysaccharide immune enhancer, and 56.5%-86.5% solvent," specifically:

[0021] (1) Skeleton structure: Under high shear conditions of 60-80℃, the specific weight parts of carbomer / polyacrylamide (0.1 µm particle size) of this invention first swell into a cross-linked three-dimensional gel network, providing anchoring space for subsequent molecules; if the weight parts of carbomer / polyacrylamide are too low, sufficient gel structure cannot be formed to effectively encapsulate the antigen and provide a continuous "antigen warehouse effect"; if the weight parts of carbomer / polyacrylamide exceed 30%, the gel will become extremely viscous, making it difficult to inject through the needle, resulting in a poor user experience and clogging of the syringe.

[0022] (2) Stabilizer immediate embedding: Poloxamer reduces interfacial tension through amphiphilic blocks, preventing excessive aggregation of the backbone; Mannitol replaces water molecules with hydrogen bonds, simultaneously completing freeze-drying and reconstitution protection, so that the network maintains uniform pore size in freeze-drying-injection-body temperature cycle; The minimum effective weight of stabilizer in the specific weight parts of this invention can provide basic protection (such as anti-oxidation, inhibition of microorganisms, and prevention of freeze-drying damage) for the entire system (including antigens, polysaccharides, etc.); However, excessively high weight parts of stabilizer will increase the osmotic pressure or viscosity of the adjuvant, and trigger unexpected chemical side reactions.

[0023] (3) Immunostimulants precisely occupy the 2.5%-7.5% window: Polysaccharide immunostimulants need to reach a certain local concentration to be effectively captured by pattern recognition receptors (PRRs) of immune cells (such as macrophages and dendritic cells); In the specific weight parts of the polysaccharide immunostimulants of this invention, the TLR2 / 4 and CLR ligands are physically captured by the network pores, and the local concentration instantly reaches the PRR recognition threshold, releasing a "danger signal" to activate the immune system (NF-κB / MAPK), leading to the activation and maturation of APCs, and the secretion of various cytokines (such as IL-2, IL-6, IL-12, TNF-α or IFN-γ) and chemokines, thereby strongly activating Th1 (cellular immunity) and / or Th2 (humoral immunity) responses; and combined with the physical presentation of carbomer, it provides a powerful immune stimulation signal, achieving the dual enhancement of "physical presentation + chemical stimulation".

[0024] Too low a weight percentage of polysaccharide immune enhancers will result in insignificant immune-enhancing effects; while too high a weight percentage of polysaccharide immune enhancers will overactivate the immune system, triggering systemic inflammatory responses (such as fever and fatigue) and severe local adverse reactions (redness, swelling, heat, pain, and even ulcers), thus reducing safety.

[0025] (4) The solvent is the dependent variable. The pH=6.5 phosphate buffer not only neutralizes the negative charge of the carboxyl group of carbomer, so that the system can be quickly converted to a neutral microenvironment of pH=7.0-7.2 at the injection site to avoid alkaline damage of aluminum adjuvant >8.5, but also adjusts the osmotic pressure through ionic strength, and together with poloxamer, reduces the viscosity to ensure that it can be injected at room temperature and gelled at body temperature.

[0026] Therefore, the veterinary compound water-soluble adjuvant provided by this invention is stable and highly effective, forming an antigen reservoir at the injection site and continuously releasing antigens for more than 72 hours. Carbomer adjuvant creates a neutral (7.0-7.2) microenvironment at the injection site, avoiding the alkaline damage of aluminum adjuvants (pH>8.5) and the foreign body reaction of oil adjuvants. It can significantly reduce the incidence of granulomas (compared to mineral oil adjuvants). It can significantly increase the neutralizing antibody titer of subunit vaccines, induce a balanced Th1 / Th2 response through a dual-signal activation pathway, and induce IFN-γ secretion of CD4. + The frequency of T cells is higher than that of traditional adjuvants, and the specific CD8 cells are more abundant. + Higher T-cell amplification values ​​provide a better adjuvant option on the market.

[0027] The scaffold's "physical reservoir" continuously releases antigens for over 72 hours, stabilizers maintain network integrity and lyophilization recovery, polysaccharides continuously provide chemical stimulation within the "warehouse," and solvents regulate pH and osmotic pressure throughout the process. These four elements mutually restrain and enhance each other, ultimately reducing the incidence of granulomas, significantly increasing neutralizing antibody titers, and inducing IFN-γ secretion of CD4. + T cell frequency is higher than with traditional adjuvants, and specific CD8 + The T-cell amplification value is higher, achieving an integrated synergistic effect of "physical sustained release - chemical activation - local low stimulation", and proving that each component is indispensable within the specified ratio, providing a better adjuvant option for the market. Attached Figure Description

[0028] Figure 1 The images show the appearance results of the stability test of the veterinary compound water-soluble adjuvants prepared in Comparative Example 1 and Example 1 after storage at 25°C for 7 days; from left to right, they are Comparative Example 1 and Example 1.

[0029] Figure 2 The stability test results of the veterinary compound water-soluble adjuvant prepared in Comparative Example 2 after storage at 4℃ and 25℃ for 7 days are shown in the figure; from left to right, the storage conditions are 4℃ and 25℃ respectively.

[0030] Figure 3 The images show the appearance results of the stability test of the veterinary compound water-soluble adjuvants prepared in Examples 1, 2, and 3 after storage at 25°C for 17 days; from left to right, they are Example 1, Example 2, and Comparative Example 3.

[0031] Figure 4 The particle size distribution of the veterinary composite water-soluble adjuvant prepared in Example 1 is shown in the graph.

[0032] Figure 5 The particle size distribution of the veterinary composite water-soluble adjuvant prepared in Example 2 is shown in the graph.

[0033] Figure 6 The particle size distribution of the veterinary composite water-soluble adjuvant prepared in Comparative Example 1 is shown in the graph.

[0034] Figure 7 The particle size distribution of the veterinary composite water-soluble adjuvant prepared in Comparative Example 2 is shown in the graph.

[0035] Figure 8 The particle size distribution of the veterinary composite water-soluble adjuvant prepared in Comparative Example 3 is shown in the graph.

[0036] Figure 9 The image shows the results of the HI antibody potency test.

[0037] Figure 10 The graph shows the results of the BVDV neutralizing antibody potency test. Detailed Implementation

[0038] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention.

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0040] All chemical reagents used in the following examples were of analytical grade and purchased from Sinopharm Group.

[0041] The recombinant avian influenza H5N1 virus antigen solution, bovine viral diarrhea virus (BVM) NMG strain, inactivated BVM NMG strain virus solution, and MDBK cells used in the following examples were all provided by the College of Veterinary Medicine, China Agricultural University. They were dissolved in sterile physiological saline according to the amounts indicated on the bottle labels. The HA titer was determined using the hemagglutination assay (96-well microplate method), and the titer should be no less than 1:256. The BVM NMG strain virus solution and inactivated antigen solution used should have a concentration ≥10. 5 TCID 50 .

[0042] The phosphate buffer solution prepared in the following examples is prepared as follows: 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 are weighed and dissolved in 1000 mL of distilled water. After mixing, the phosphate buffer solution is obtained.

[0043] Example 1:

[0044] S1: Dissolve 30% crosslinking polymer and 5% solution stabilizer in 62% solvent (phosphate buffer, pH 6.5), and stir continuously at 60°C and 1200 rpm for 60 min to obtain a dispersion; the 30% crosslinking polymer is composed of 25% polyacrylamide (particle size 0.1 μm) and 5% carbomer 974P; the 5% solution stabilizer is composed of 4% poloxamer and 1% mannitol;

[0045] S2: Dissolve 3% polysaccharide immune enhancer (the 3% polysaccharide immune enhancer is composed of 1% American ginseng root polysaccharide, 1% dextran and 1% trehalose) in the dispersion obtained in S1; stir continuously for 30 min at 40℃ and 600 rpm to obtain veterinary compound water-soluble adjuvant 1.

[0046] Example 2:

[0047] S1: Dissolve 10% crosslinking polymer and 4% solution stabilizer in 85% solvent (phosphate buffer, pH 6.5), and stir continuously at 60°C and 1200 rpm for 60 min to obtain a dispersion; the 10% crosslinking polymer is composed of 8% polyacrylamide (particle size 0.1 μm) and 2% carbomer 974P; the 4% solution stabilizer is composed of 2% poloxamer and 2% mannitol;

[0048] S2: Dissolve 5% polysaccharide immune enhancer (the 1% polysaccharide immune enhancer is composed of 2% American ginseng root polysaccharide, 1% Acanthopanax senticosus polysaccharide, 1% glucan and 1% trehalose) in the dispersion obtained in S1; stir continuously for 30 min at 40℃ and 600 rpm to obtain veterinary compound water-soluble adjuvant 2.

[0049] Comparative Example 1:

[0050] S1: Dissolve 40% crosslinking polymer and 5% solution stabilizer in 54.5% solvent (phosphate buffer, pH 6.5), and stir continuously at 60°C and 1200 rpm for 60 min to obtain a dispersion; the 40% crosslinking polymer is composed of 20% polyacrylamide (particle size 0.1 μm) and 20% carbomer 974P; the 5% solution stabilizer is composed of 4% poloxamer and 1% mannitol;

[0051] S2: Dissolve 3% polysaccharide immune enhancer (the 3% polysaccharide immune enhancer is composed of 1% American ginseng root polysaccharide, 1% dextran and 1% trehalose) in the dispersion obtained in S1; stir continuously for 30 min at 40℃ and 600 rpm to obtain veterinary compound water-soluble adjuvant 3.

[0052] Comparative Example 2:

[0053] S1: Dissolve 30% crosslinking polymer and 15% solution stabilizer in 52% solvent (phosphate buffer, pH 6.5), and stir continuously at 60°C and 1200 rpm for 60 min to obtain a dispersion; the 40% crosslinking polymer is composed of 25% polyacrylamide (particle size 0.1 μm) and 5% carbomer 974P; the 5% solution stabilizer is composed of 10% poloxamer and 5% mannitol;

[0054] S2: Dissolve 3% polysaccharide immune enhancer (the 3% polysaccharide immune enhancer is composed of 1% American ginseng root polysaccharide, 1% dextran and 1% trehalose) in the dispersion obtained in S1; stir continuously for 30 min at 40℃ and 600 rpm to obtain veterinary compound water-soluble adjuvant 4.

[0055] Comparative Example 3:

[0056] S1: Dissolve 30% crosslinking polymer and 5% solution stabilizer in 55% solvent (phosphate buffer, pH 6.5), and stir continuously at 60°C and 1200 rpm for 60 min to obtain a dispersion; the 30% crosslinking polymer is composed of 25% polyacrylamide (particle size 0.1 μm) and 5% carbomer 974P; the 5% solution stabilizer is composed of 4% poloxamer and 1% mannitol;

[0057] S2: Dissolve 10% polysaccharide immune enhancer (the 10% polysaccharide immune enhancer is composed of 3% American ginseng root polysaccharide, 3% Acanthopanax senticosus polysaccharide, 3% glucan and 1% trehalose) in the dispersion obtained in S1; stir continuously for 30 min at 40℃ and 600 rpm to obtain veterinary compound water-soluble adjuvant 5.

[0058] Effect Experiment:

[0059] 1. Stability testing

[0060] The veterinary compound water-soluble adjuvants 1-2 prepared in Examples 1-2 and 3-5 prepared in Comparative Examples 1-3 were placed at 4℃, 25℃, and 37℃ respectively to observe their morphological stability. Observations were made daily for 7 days; weekly for 7 days to one month; and monthly thereafter. The observation period was 1 year at 4℃, 6 months at 25℃, and 6 months at 37℃, with photographic stability test results recorded.

[0061] As shown in Tables 1-3, the veterinary compound water-soluble adjuvants 3-5 prepared in Comparative Examples 1-3 exhibited varying degrees of stability issues under different temperature conditions (4℃, 25℃, and 37℃), such as viscosity, layering, or precipitation. Viscosity, layering, or precipitation all indicate poor adjuvant stability. Viscosity affects the adjuvant's performance and the animal's vaccination experience; layering indicates poor component dispersion, affecting vaccine uniformity and efficacy; precipitation affects vaccine homogeneity and immunization effect.

[0062] The veterinary compound water-soluble adjuvants 1-2 prepared in Examples 1-2 all exhibited good long-term stability under different temperature conditions (4℃, 25℃ and 37℃), with no obvious changes in physical properties (such as precipitation, layering, viscosity, etc.), making them suitable for long-term storage and use, and even more suitable for use as water-soluble adjuvants for veterinary vaccines.

[0063] Table 1

[0064]

[0065] Table 2

[0066]

[0067] Table 3

[0068]

[0069] like Figure 1 As shown, the veterinary composite water-soluble adjuvant 3 prepared in Comparative Example 1 exhibited a viscous paste-like texture after being stored at 25°C for 7 days, while the veterinary composite water-soluble adjuvant 1 prepared in Example 1 exhibited a flowing liquid texture under the same conditions. This indicates that the excessively high content of highly cross-linked polymers in the veterinary composite water-soluble adjuvant provided by this invention leads to the adjuvant becoming viscous, thereby reducing its stability.

[0070] like Figure 2As shown, the veterinary compound water-soluble adjuvant 4 prepared in Comparative Example 2 exhibited obvious solution stratification after being stored at 4℃ and 25℃ for 7 days. This indicates that the excessively high content of solution stabilizer in the veterinary compound water-soluble adjuvant provided by this invention can lead to stratification and thus reduce the stability of the adjuvant.

[0071] like Figure 3 As shown, the veterinary compound water-soluble adjuvants 1-2 prepared in Examples 1-2 exhibited a flowing liquid texture after being stored at 25°C for 14 days; while the veterinary compound water-soluble adjuvant 5 prepared in Comparative Example 3 showed a precipitation phenomenon after being stored at 25°C for 14 days. This indicates that the excessively high content of polysaccharide immune enhancers in the veterinary compound water-soluble adjuvants provided by this invention leads to significant precipitation and suspension phenomena, thereby reducing the stability of the adjuvant.

[0072] The veterinary composite water-soluble adjuvant provided by this invention achieves the formation of a uniform gel network within the synergistic range of four components—crosslinked polymer, solution stabilizer, polysaccharide immunostimulant, and solvent—by confining each component to a specific range. Specifically, polyacrylamide or carbomer 974P swells fully under high-speed shear to establish a three-dimensional framework; poloxamer reduces interfacial tension and imparts thermally reversible gel properties through amphiphilic blocks; mannitol replaces hydrogen bond positions of water molecules to inhibit ice crystal damage during freeze-drying and low-temperature storage; plant polysaccharides provide immune signals via PRR agonism within a safe concentration range of 2.5%-7.5% without excessive precipitation; and finally, a phosphate buffer system with pH=6.5 locks the osmotic pressure and ionic strength within a range compatible with tissue fluid, preventing salting out or pH drift.

[0073] Stability testing results for Comparative Examples 1-3 (veterinary compound water-soluble adjuvants 3-5) showed that a lack of sufficient poloxamer / mannitol stabilizer, or the selection of excessively high content of highly cross-linked polymers as the base material, both led to continuous water absorption and swelling of the carbomer skeleton during storage, resulting in an exponential increase in viscosity and a "viscous" phenomenon. A lack of sufficient polysaccharide concentration and buffer regulation caused ionic strength instability, leading to charge shielding of the carbomer network and phase separation, resulting in a "layering" phenomenon. Excessively high polysaccharide concentrations caused polysaccharides to self-aggregate and complex with salt ions, resulting in precipitation and a "precipitation" phenomenon. Therefore, this invention achieves the dual standards of "homogeneous injectability" and "long-term storage stability" through the mutual restraint and complementarity within a quaternary ratio window of skeleton-stabilizer-immunostimulant-solvent.

[0074] 2. Particle size detection

[0075] The particle size of the veterinary compound water-soluble adjuvants 1-2 prepared in Examples 1-2 and the veterinary compound water-soluble adjuvants 3-5 prepared in Comparative Examples 1-3 was determined using a Master sizer laser particle size analyzer, and the results were statistically analyzed and plotted.

[0076] like Figure 4-5 As shown, the particle size distribution of veterinary compound water-soluble adjuvants 1-2 (Examples 1-2) is unimodal and concentrated, with the main peak located around 0.1 µm, consistent with the "polyacrylamide particle size of 0.1 micrometers" described in this invention. Figure 6-8 As shown, the particle size distributions of the veterinary composite water-soluble adjuvants 3-5 prepared in Comparative Examples 1, 2, and 3 all showed significant right tailing, with the main peak shifting towards 1-10 µm and accompanied by multiple peaks, indicating that they exhibited significant aggregation.

[0077] As can be seen, this invention first forms a 0.1µm-scale three-dimensional framework by limiting the cross-linked polymer to 10%-30% and shearing it at 60-80℃; then, by limiting the solution stabilizer to 1%-6%, poloxamer reduces interfacial tension and inhibits particle aggregation through amphiphilic blocks, and mannitol maintains the original particle size by replacing water molecules through hydrogen bonds during freeze-drying and low-temperature storage; at the same time, pH=6.5 phosphate buffer maintains the stability of the particle surface charge and prevents salting-out aggregation. The particle size detection results of Comparative Examples 1-3 (veterinary compound water-soluble adjuvant 3-5) show that in Comparative Examples 1-2, due to the lack of sufficient poloxamer / mannitol, the carbomer could not be dispersed in time after swelling, and the hydrophobic interaction and charge shielding between particles were enhanced, resulting in increased particle size and the appearance of multiple peaks; in Comparative Example 3, the amount of polysaccharide added exceeded 7.5%, which aggravated aggregation by bridging the particles through hydrogen bonds. Therefore, the veterinary composite water-soluble adjuvant provided by the present invention maintains a narrow single-peak distribution at the 0.1 µm level under the synergistic effect of four mechanisms: skeleton formation, interface stabilization, freeze-drying protection, and charge shielding, and has better colloidal stability and injectability.

[0078] 3. Immune organ index detection and splenic lymphocyte proliferation activity detection

[0079] (1) Six- to eight-week-old SPF-grade Kunming mice (purchased from the National Institutes for Food and Drug Control) were randomly divided into the following six groups: blank control group, Example 1 group, Example 2 group, Comparative Example 1 group, Comparative Example 2 group and Comparative Example 3 group, with five mice in each group. The mice in each group were injected intraperitoneally with 0.2 mL of the corresponding adjuvant, while the blank control group was injected intraperitoneally with an equal volume of physiological saline. After being cultured under the same conditions for 14 days, the mice were euthanized by cervical dislocation, and the thymus and spleen were dissected, the surface blood was wiped dry, the wet mass was weighed using an electronic balance, and the thymus and spleen indices were calculated.

[0080] As shown in Table 4, after 14 days of culture following intraperitoneal injection, the thymus index of mice in Examples 1-2 reached 2.64±0.30 mg / g and 2.97±0.12 mg / g, respectively, and the spleen index reached 8.33±0.24 mg / g and 8.12±0.22 mg / g, respectively, both significantly higher than those of the blank control group (1.99±0.21 mg / g; 5.68±0.11 mg / g), and significantly higher than those of mice in Comparative Examples 1-3. This demonstrates that the veterinary compound water-soluble adjuvant provided by this invention can effectively amplify the central and peripheral immune organs.

[0081] Table 4

[0082]

[0083] (2) Under aseptic conditions, the spleens of mice were harvested. The spleen cells were washed with 3 mL of pre-cooled PBS buffer containing 1% penicillin and antibiotics until the spleen turned white. The washing solution was collected in 5 mL centrifuge tubes and centrifuged at 1500 r / min for 10 min. The supernatant was discarded, and 2 mL of red blood cell lysis buffer was added to each tube to resuspend the cells. The cells were allowed to stand for 3 min and then centrifuged at 1500 r / min for 10 min. The cells were washed twice with 1% penicillin and antibiotic-PBS and resuspended in 1640 buffer containing 1% penicillin and antibiotics to adjust the cell concentration to 500,000 cells / mL. 6 wells of a 96-well plate were selected and 200 μL of PBS was added as blank control. 100 μL of cell suspension was added to the remaining wells. 100 μL of the corresponding veterinary compound water-soluble adjuvant 1-5 was added to the intervention groups (Example 1, Example 2, Comparative Example 1, Comparative Example 2, and Comparative Example 3). 100 μL of culture medium was added to the blank control group. 3 replicates were made for each group. The cells were incubated at 37℃ for 72 h. 4. h, add 5 mg / mL MTT solution to each well of the culture plate, 10 μL / well, incubate at 37℃ for 4 h, centrifuge at 1500 r / min for 10 min, discard the supernatant, add 150 μL LDMSO to each well, dissolve for 10 min, and measure the absorbance (OD) value at 490 nm using a microplate reader at 30 min.

[0084] As shown in Table 5, the splenic lymphocyte OD of groups 1-2 in Examples 490 The values ​​were 0.89±0.18 and 0.77±0.15, respectively, significantly higher than the blank control group (0.51±0.21), and significantly higher than comparative group 1 (0.70±0.19), comparative group 2 (0.53±0.10), and comparative group 3 (0.31±0.04). This demonstrates that the veterinary compound water-soluble adjuvant provided by this invention can more effectively drive T cell proliferation.

[0085] Table 5

[0086]

[0087] In summary, this invention utilizes a 0.1 µm-scale three-dimensional network formed by a defined cross-linked polymer. This structure can fully encapsulate the antigen and maintain a neutral microenvironment of pH 7.0-7.2 at the injection site, avoiding the alkaline damage associated with traditional adjuvants (aluminum adjuvants). By limiting the content of solution stabilizers, particle aggregation is prevented. Simultaneously, its thermally reversible gel properties synergize with carbomer at body temperature to form an "antigen reservoir," continuously releasing antigens for more than 72 hours, thereby continuously stimulating the thymus and spleen. By defining polysaccharide immune enhancers as PRR agonists, they are recognized by TLR2 / 4 and CLRs on the surface of macrophages and dendritic cells, activating the NF-κB / MAPK pathway, inducing the secretion of IL-2, IL-12, and IFN-γ, promoting the Th1 / Th2 balanced response, and thus amplifying the migration and proliferation of thymocytes to the peripheral spleen. In contrast, Comparative Examples 1 and 2 lacked sufficient poloxamer / mannitol, leading to easy aggregation of the carbomer network in the body, short antigen release, and weak immune stimulation. In Comparative Example 3, the polysaccharide content exceeded the upper limit of 7.5%, and local inflammation inhibited cell proliferation, resulting in limited improvement in thymus and spleen indices. Excessive inflammation inhibited cell proliferation, leading to a decrease in OD value.

[0088] Therefore, this invention achieves a dual effect of "physical sustained release + chemical activation" through a quaternary ratio window of skeleton-stabilizer-immunostimulant-solvent, directly demonstrating superior immune amplification ability in organ index and spleen lymphocyte proliferation activity, making it more suitable as a water-soluble adjuvant for veterinary vaccines.

[0089] 4. Safety Testing

[0090] Six- to eight-week-old SPF-grade Kunming mice were randomly divided into the following eight groups: A1, B1, C1, D1, E1, H5N1 antigen solution group, control group, with 15 mice in each group; and a blank group with 5 mice.

[0091] Under aseptic conditions, the veterinary compound water-soluble adjuvants 1-2 prepared in Examples 1-2 and the veterinary compound water-soluble adjuvants 3-5 prepared in Comparative Examples 1-3 were heated to 30°C. The above-mentioned veterinary compound water-soluble adjuvants were weighed with recombinant avian influenza H5N1 virus antigen solution at a volume ratio of 1:9 and added to beakers. Using a rod-shaped homogenizer, the mixture was emulsified at 500 r / min for 25 min to obtain vaccines containing the corresponding adjuvants. In each group of 15 mice, 5 mice were subcutaneously injected with 1 mL of adjuvant, 5 mice were subcutaneously injected with 2 mL of vaccine containing the corresponding adjuvant, and 5 mice were intraperitoneally injected with 1 mL of vaccine containing the corresponding adjuvant.

[0092] Under aseptic conditions, Gel 01PR adjuvant (manufactured by SEPPIC, France) was heated to 30°C. The Gel 01PR adjuvant and recombinant avian influenza H5N1 virus antigen solution were weighed separately at a volume ratio of 1:9 and added to beakers. Using a rod-shaped homogenizer, the mixture was emulsified at 500 r / min for 25 min to obtain a vaccine containing Gel 01PR adjuvant, designated as the control group. In the control group, 5 mice were subcutaneously injected with 1 mL of Gel 01PR adjuvant, 5 mice were subcutaneously injected with 2 mL of the vaccine containing Gel 01PR adjuvant, and 5 mice were intraperitoneally injected with 1 mL of the vaccine containing Gel 01PR adjuvant.

[0093] In the H5N1 antigen solution group, 5 mice were subcutaneously injected with 1 mL of H5N1 virus antigen solution, 5 mice were subcutaneously injected with 2 mL of H5N1 virus antigen solution, and 5 mice were intraperitoneally injected with 1 mL of H5N1 virus antigen solution. In the blank group, 5 mice were selected and no treatment was given. After immunization, the mice in each group were observed to see if there were any abnormalities in their organs. The abnormal reactions or deaths of the animals were observed and recorded.

[0094] As shown in Tables 6-7, in groups A1 (Example 1) and B1 (Example 2), regardless of whether the mice were injected subcutaneously or intraperitoneally with adjuvants or vaccines containing the corresponding adjuvants, the survival rate of all mice was 100%. No hair loss, hardening, or ulceration occurred at the injection site, and the mice in both groups had normal appetite and mental state. However, mice in groups C1 (Comparative Example 1), D1 (Comparative Example 2), and E1 (Comparative Example 3) all experienced varying degrees of mortality. When 2 mL of vaccine was injected subcutaneously, the survival rate of mice in groups C1 and E1 dropped to 60%, accompanied by hair loss and hardening at the injection site, and decreased appetite and mental state. In group E1, ulceration was even observed at the injection site of 1 mL of intraperitoneal vaccine. In contrast, the H5N1 simple antigen solution, the control group, and the blank group showed no abnormalities throughout the process. Therefore, the veterinary compound water-soluble adjuvant provided by this invention has high safety.

[0095] Table 6

[0096]

[0097] Table 7

[0098]

[0099] Note: + indicates existence, - indicates non-existence.

[0100] 5. HI antibody potency test

[0101] Healthy, susceptible SPF chickens aged 4-5 weeks (purchased from Beijing Nongxiao Poultry Co., Ltd., and tested negative for H5N1 antibody) were randomly divided into the following 5 groups: Group A1 (Example 1), Group B1 (Example 2), H5N1 antigen solution group, blank group, and control group, with 5 chickens in each group.

[0102] Under aseptic conditions, the veterinary compound water-soluble adjuvants 1-2 prepared in Examples 1-2 were heated to 30°C. The veterinary compound water-soluble adjuvants were weighed with H5N1 inactivated virus antigen solution at a volume ratio of 1:9 and added to beakers. The mixtures were emulsified for 25 min at 500 r / min using a rod homogenizer. Each chicken in each group was intramuscularly injected with 0.3 mL of the vaccine containing the corresponding adjuvant, and the groups were named A1 and B1, respectively.

[0103] Under aseptic conditions, Gel 01PR adjuvant produced by SEPPIC (France) was heated to 30°C. The Gel 01PR adjuvant and recombinant avian influenza H5N1 virus antigen solution were weighed separately at a volume ratio of 1:9 and added to beakers. Using a rod-shaped homogenizer, the mixture was emulsified at 500 r / min for 25 min to obtain a vaccine containing Gel 01PR adjuvant. 0.3 mL of the vaccine containing the corresponding adjuvant was injected intramuscularly into each chicken in the control group.

[0104] In the H5N1 antigen solution group, each chicken was injected intramuscularly with 0.3 mL of H5N1 inactivated virus antigen solution, while the five chickens in the blank group were not vaccinated. Blood samples were collected from each group of chickens on days 7, 14, 21, and 28 post-immunization, and serum was separated. HI antibodies were measured using avian influenza virus H5 subtype antigen.

[0105] like Figure 9 As shown, from 7 to 28 days post-immunization, the H5N1 HI antibody titers in groups 1 and 2 of Examples 1 and 2 continuously increased over time, reaching a peak on day 28, significantly higher than the H5N1 antigen solution group and also superior to the commercially available Gel 01PR control group. No antibodies were produced in the blank group. It is evident that the veterinary compound water-soluble adjuvant provided by this invention significantly increases HI antibody levels in poultry models, rapidly and persistently enhancing humoral immunity in birds.

[0106] 6. BVDV neutralizing antibody potency test

[0107] Healthy yellow cattle (purchased from Jinyu Baoling Biotechnology Co., Ltd., and tested negative for BVDV antibody) were randomly divided into the following 5 groups: Group A1 (Example 1), Group B1 (Example 2), BVDV antigen solution group, blank group, and control group, with 5 cattle in each group.

[0108] Under aseptic conditions, the veterinary compound water-soluble adjuvants 1-2 prepared in Examples 1-2 were heated to 30°C. The veterinary compound water-soluble adjuvants were weighed with BVDV inactivated virus antigen solution at a volume ratio of 1:9 and added to beakers. Using a rod homogenizer, the mixture was emulsified at 500 r / min for 25 min. Each cattle in each group was intramuscularly injected with 1 mL of the vaccine containing the corresponding adjuvant, and the groups were named A1 and B1, respectively.

[0109] Under aseptic conditions, the ISA 206 adjuvant produced by SEPPIC (France) was heated to 34°C. The ISA 206 adjuvant and BVDV inactivated virus antigen solution were weighed separately at a volume ratio of 5:5 and added to beakers. Using a rod homogenizer, the mixture was emulsified at 500 r / min for 25 min. 1 mL of the vaccine containing the ISA 206 adjuvant was injected intramuscularly into each cattle, and this was designated as the control group.

[0110] In the BVDV antigen solution group, each head of cattle was injected with 1 mL of BVDV inactivated virus antigen solution. In the blank group, 5 cattle were not vaccinated. Blood samples were collected and serum was separated on days 7, 14, 21, and 28 after immunization. A booster immunization was performed on day 28 after the first immunization. Blood samples were collected on days 14 and 21 after the second immunization, and serum was separated. Cell neutralization experiments were conducted using bovine viral diarrhea virus (NMG) strain and MDBK cells.

[0111] like Figure 10 As shown, from 7-28 days after the first immunization and from 14-21 days after the second immunization, the neutralizing antibody titers of bovine serum against the BVDV NMG strain in groups 1 and 2 of Examples continuously increased over time, reaching a peak on day 21 after the second immunization, significantly higher than those in the BVDV antigen solution group and the ISA206 oil adjuvant control group. The blank group showed no neutralizing activity throughout the entire process. Therefore, the veterinary composite water-soluble adjuvant provided by this invention significantly increases the BVDV neutralizing antibody titer in the bovine model and can still rapidly, persistently, and efficiently induce neutralizing antibodies in large ruminants.

[0112] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A veterinary composite water-soluble adjuvant, characterized in that, The veterinary compound water-soluble adjuvant component is as follows: 30% cross-linked polymer, 5% solution stabilizer, 3% polysaccharide immune enhancer and 62% solvent; The cross-linked polymer is polyacryl, carbomer 974P; The particle size of the polyacryl is 0.1 microns; The polysaccharide immune enhancer is composed of American ginseng root polysaccharide, wujia polysaccharide, dextran, trehalose or composed of American ginseng root polysaccharide, dextran, trehalose; The solution stabilizer is poloxamer, mannitol; The solvent is an aqueous solution containing a salt ion buffer; The aqueous solution containing a salt ion buffer is a phosphate buffer with pH=6.

5.

2. Use of the veterinary compound water-soluble adjuvant of claim 1 in the preparation of a veterinary vaccine.

3. A veterinary vaccine comprising, The veterinary vaccine comprises the veterinary compound water-soluble adjuvant of claim 1.

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