Water-soluble immune composite adjuvant, vaccine and preparation method of water-soluble immune composite adjuvant

Abstract

The invention belongs to the technical field of biology, mainly relates to a preparation method of a composite adjuvant, and particularly relates to a water-soluble immune composite adjuvant, a vaccine and a preparation method of the vaccine. According to the compound adjuvant, every 300 mL of the compound adjuvant is prepared from 30 mL to 50 mL of carbomer stock solution, 30 mL to 37.5 mL of polyinosinic stock solution, 800 mg to 900 mg of triterpenoid saponin, 1.2 g of chitosan oligosaccharide and 20 mL of chitosan solution. Wherein the concentration of the carbomer stock solution is 30 mg / mL, the concentration of the polyinosinic-polycytidylic acid stock solution is 50 mg / mL, and the concentration of the chitosan solution is 600 mg / mL. The water-soluble immune composite adjuvant disclosed by the invention does not contain mineral oil components, granuloma of an injection part caused by viscous oil adjuvant vaccines is avoided, the water-soluble immune composite adjuvant is conveniently mixed with antigens in use, the economic benefits and the use convenience of farmers are effectively improved, and the water-soluble immune composite adjuvant is easy to industrially produce. And meanwhile, the gleditsia sinensis saponin enzymatic hydrolysate and the chitosan oligosaccharide enzymatic hydrolysate contained in the adjuvant have a synergistic effect, so that a relatively strong immune effect is achieved, humoral immune response and cellular immune response can be stimulated, and the immune persistence can be enhanced.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and mainly relates to a method for preparing a compound adjuvant, specifically a water-soluble immune compound adjuvant, a vaccine, and a method for preparing the same. Background Technology

[0002] Adjuvants, also known as immunomodulators or immune enhancers, are additives used in vaccines to enhance the body's immune response to antigens or alter the type of immune response. Currently, oil-emulsion adjuvants are widely used in veterinary biological products, especially in animal vaccines. They can promote the production of high-titer antibodies against various antigens, reducing the dosage and frequency of antigen administration. Common oil-emulsion adjuvants include Freund's adjuvant, white oil Span adjuvant, MF-59, ISA 206, ISA720, Adjuvant-65, and SAF. Oil-emulsion adjuvants are storage-type adjuvants, typically composed of mineral oil (such as white oil), emulsifiers (such as Span-85 or Tween-80), and stabilizers (such as aluminum stearate). These components are mixed to form a water-in-oil (W / O) or oil-in-water (O / W) emulsion. However, while oil-emulsion adjuvants function effectively in the body, they also have several drawbacks and limitations. First, oil adjuvants are viscous, making injection difficult and prone to forming granulomas at the injection site, posing health risks. Second, oil adjuvants have limited response types, primarily stimulating humoral immunity with weak stimulation of cellular immunity, making them ineffective against complex pathogen infections. Third, oil adjuvants have poor safety profiles; subcutaneous injection can easily cause severe local inflammation or even ulcers, and residual fluid may threaten environmental and biosafety. Furthermore, oil adjuvants can damage bacterial cell membranes, making them unsuitable for live vaccine development and limiting their applicability. To address these issues, this invention employs a water-soluble adjuvant, which not only stimulates humoral immune responses but also effectively triggers cellular immune responses. It offers advantages such as safety, no residue, and ease of injection, successfully avoiding the injection site granuloma problem caused by the viscosity of oil adjuvant vaccines, thereby effectively improving the economic benefits for farmers. More importantly, the adjuvant of this invention—a water-soluble immune complex adjuvant—has low skin irritation, is easily absorbed, and possesses strong immune effects and long-lasting immunity, demonstrating broader application prospects in the prevention and treatment of diseases. Summary of the Invention

[0003] In view of this, the present invention provides a water-soluble immune adjuvant, a vaccine, and a method for preparing the same.

[0004] Firstly, this invention provides a water-soluble immune adjuvant, wherein each 300 mL of the adjuvant formulation contains: 30-50 mL of carbomer stock solution, 30-37.5 mL of polyinosinic-polycytidylic acid (poly I:C) stock solution, 800-900 mg of triterpenoid saponins, 1.2 g of chitosan oligosaccharide, and 20 mL of chitosan solution; wherein the concentration of the carbomer stock solution is 30 mg / mL, the concentration of the poly I:C stock solution is 50 mg / mL, and the concentration of the chitosan solution is 600 mg / mL.

[0005] Preferably, the chitosan oligosaccharide in the composite adjuvant is enzymatically hydrolyzed by a combination of chitosanase and chitinase.

[0006] Preferably, the triterpenoid saponins in the compound adjuvant are hydrolyzed by a combination of α-glucosidase and galactosidase.

[0007] Preferably, the composite adjuvant is prepared using PBS buffer, which has the following formulation: per 5000 mL, it contains 612.5 mL of 0.2 M disodium hydrogen phosphate solution, 637.5 mL of 0.2 M disodium hydrogen phosphate solution, 45 g NaCl, 3.72 g KCl, 25 mL Tween 80, and the remainder is distilled water, with a pH of 6.80.

[0008] Secondly, this invention provides a method for preparing a composite water-soluble adjuvant and a vaccine, the specific steps of which are as follows: S1. Preparation of PBS buffer: Weigh 71.6g of disodium hydrogen phosphate dodecahydrate and 27.6g of sodium dihydrogen phosphate monohydrate, and dissolve each in 1L of purified water to prepare a 0.2M solution; measure 612.5mL of disodium hydrogen phosphate solution and 637.5mL of sodium dihydrogen phosphate solution, add 45g of NaCl, 3.72g of KCl and 25mL of Tween 80, stir and mix well, adjust the pH to 6.80, and finally make up to 5000mL with distilled water for later use; S2. Preparation of Poly I:C stock solution: Weigh 5g of Poly I:C, dissolve in distilled water and bring the volume to 100mL to prepare a 50mg / mL Poly I:C stock solution for later use. S3. Preparation of Carbomer Stock Solution: Weigh 30g of carbomer, add 500mL of distilled water, stir at 60-70℃ and 500-1200rpm, add 1.0mol / L NaOH during stirring until a gel is formed, then add distilled water to make up to 1000mL to prepare a 30 mg / mL carbomer stock solution, which is then autoclaved for later use. S4. Preparation of triterpenoid saponin hydrolysate: Weigh 900 mg of triterpenoid saponin and add it to 100 mL of PBS buffer from step S1. Stir and mix at 200-800 rpm, adjust the pH to 5.5, add 0.4 g of α-glucosidase at 35-40℃ for 3 h, then adjust the pH to 6.70, add 0.3 g of galactosidase at 35-40℃ for 3 h, and finally inactivate the enzyme at high temperature to obtain the triterpenoid saponin hydrolysate for later use. S5. Preparation of chitosan oligosaccharide solution: Add 1.2g of chitosan oligosaccharide to 250-300mL of distilled water, mix well, adjust the pH to 5.3, add 0.6g of chitosanase at 50-55℃ for 4h of enzymatic hydrolysis; then adjust the pH to 5.5, add 0.2g of chitinase at 45-50℃ for 3h of enzymatic hydrolysis, inactivate the enzyme at high temperature after the hydrolysis is completed, and concentrate to 1 / 10 of the original volume of the enzymatic hydrolysis solution to obtain chitosan oligosaccharide hydrolysate for later use; S6. Preparation of water-soluble immunomodulatory adjuvant: Measure 30-50 mL of carbomer stock solution, and slowly add 30-37.5 mL of polyinosinic-polycytidylic acid stock solution while stirring at 300-400 rpm. After mixing, adjust the pH to 6.80 to form a basic gel matrix. Subsequently, slowly add all the triterpenoid saponin hydrolysate and chitosan oligosaccharide hydrolysate in sequence, and adjust the pH to 6.80 again to obtain a mixture of the basic gel matrix and the two hydrolysates. Weigh 1.2 g of chitosan and dissolve it in 20 mL of 0.8%-1.0% (v / v) acetic acid aqueous solution, and stir until completely dissolved to prepare a chitosan solution. Under shear stirring at 600-1500 rpm, quickly pour the chitosan solution into the mixture of the basic gel matrix and the two hydrolysates, and continue shearing for 10-15 min to complete cross-linking. Adjust the volume to 300 mL with PBS buffer to obtain the water-soluble immunomodulatory adjuvant, and store at 2-8℃. S7. Preparation of compound aqueous adjuvant vaccine: The antigen is slowly added to the adjuvant solution while stirring at 300-400 rpm. The mixture is stirred continuously at 20-25℃ for 20-30 minutes until homogeneous, thus obtaining the compound aqueous adjuvant vaccine.

[0009] Preferably, the triterpenoid saponins (triterpenoid saponin compounds) of the present invention may be selected from one or more of the following: saikosaponin, ginsenoside, astragaloside, phytolaccaside, saponin, and platycodonside.

[0010] More preferably, the triterpenoid saponins (triterpenoid saponin compounds) described in this invention are saponins from saponins.

[0011] Preferably, the saponin from the saponin tree described in this invention is a triterpenoid saponin extracted from the bark of the South American saponin tree (Quillaja Saponaria). The process involves mechanically crushing the saponin bark, extracting it with water, filtering and clarifying it, refining it through chromatography, and then freeze-drying or spray-drying it. The saponin material contains 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% saponins.

[0012] More preferably, the content of saponins from the soap tree is 90%; Preferably, the carbomer in this invention is carbomer 974P.

[0013] Preferably, the concentration of the α-glucosidase of the present invention is 5000 U / g; and the concentration of the galactosidase is 4000 U / g.

[0014] Preferably, the concentration of chitosanase in this invention is 3000 U / g; the concentration of chitinase is 5000 U / g.

[0015] Preferably, the antigen described in this invention is selected from one or more of the following: classical swine fever virus E2 protein, porcine circovirus Cap protein, porcine epidemic diarrhea virus S protein, porcine reproductive and respiratory syndrome virus GP5 protein, and foot-and-mouth disease virus VPI protein.

[0016] More preferably: the antigen described in this invention is porcine circovirus Cap protein, purchased from Shanghai Jingfeng Biotechnology Co., Ltd., with a concentration of 1 mg / ml.

[0017] Preferably, the mass ratio of the antigen to the adjuvant in this invention is 2:1.

[0018] The present invention has the following beneficial effects: 1. The water-soluble immune adjuvant of this invention does not contain mineral oil, thus avoiding granulomas at the injection site caused by the viscosity of oil adjuvant vaccines. It is easy to mix with the antigen, effectively improving the economic benefits and ease of use for farmers, and is easy to industrialize.

[0019] 2. In this invention, the synergistic effect of saponin hydrolysate, chitosan and chitosan oligosaccharide hydrolysate has a strong immune effect, which can stimulate humoral immune response and cellular immune response.

[0020] 3. The water-soluble immune adjuvant of this invention is safe and non-toxic, with minimal skin irritation and easy absorption.

[0021] 4. In this invention, the saponin hydrolysate and chitosan oligosaccharide hydrolysate work synergistically to enhance the persistence of immunity. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The images show the skin reaction at the injection sites of each group of guinea pigs; the blue area on the left is the injection site of Group 1 of Example 1, and the red area on the right is the injection site of the positive control group.

[0024] Figure 2 This is a comparison chart of porcine circovirus immune antibodies in different groups of piglets.

[0025] Figure 3 This is a product image of the adjuvant in Example 3 of the present invention. Detailed Implementation

[0026] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.

[0027] Galactosidase was purchased from Anhui Datang Bioengineering Co., Ltd.; α-glucosidase was purchased from Anhui Cool Bioengineering Co., Ltd.; chitosanase was purchased from Jiangsu Jinheyuan Bioengineering Co., Ltd.; chitinase was purchased from Jiangsu Rongchuang Bioengineering Co., Ltd.; chitosan and chitosan oligosaccharide were purchased from Hefei Youzhiyuan Food Ingredients Co., Ltd.

[0028] Example 1 This embodiment demonstrates that the water-soluble immune adjuvant of the present invention is safe and non-toxic, has low skin irritation, and is easily absorbed.

[0029] This embodiment provides a water-soluble immune adjuvant, the specific steps of which are as follows: S1. Preparation of PBS buffer: Weigh 71.6g of disodium hydrogen phosphate dodecahydrate and 27.6g of sodium dihydrogen phosphate monohydrate, and dissolve each in 1L of purified water to prepare a 0.2M solution; measure 612.5mL of disodium hydrogen phosphate solution and 637.5mL of sodium dihydrogen phosphate solution, add 45g of NaCl, 3.72g of KCl and 25mL of Tween 80, stir and mix well, adjust the pH to 6.80, and finally make up to 5000mL for later use; S2. Preparation of Poly I:C stock solution: Weigh 5g of Poly I:C, dissolve in distilled water and bring the volume up to 100mL to prepare Poly I:C stock solution for later use. S3. Carbomer stock solution preparation: Weigh 30g of carbomer, add 500mL of distilled water, stir at 60℃ and 500rpm, add 1.0mol / L NaOH during the stirring process until a gel is formed, then add distilled water to make up to 1000mL to obtain carbomer stock solution, which is then autoclaved for later use. S4. Preparation of saponin hydrolysate: Weigh 800 mg of saponin and add it to 100 mL of PBS buffer. Stir and mix at 200 rpm. Adjust the pH to 5.5. Add 0.4 g of α-glucosidase (5000 U / g) at 35°C and hydrolyze for 3 h. Then adjust the pH to 6.70. Add 0.3 g of galactosidase (4000 U / g) at 35°C and hydrolyze for 3 h. After the hydrolysis is complete, inactivate the enzyme at high temperature to obtain saponin hydrolysate for later use. S5. Preparation of chitosan oligosaccharide solution: Add 1.2g of chitosan oligosaccharide to 250mL of distilled water, mix well, adjust the pH to 5.3, add 0.6g of chitosanase with a concentration of 3000U / g at 50℃ and hydrolyze for 4h; then adjust the pH to 5.5, add 0.2g of chitinase with a concentration of 5000U / g at 45℃ and hydrolyze for 3h. After the hydrolysis is completed, inactivate the enzyme at high temperature and concentrate to 1 / 10 of the original volume to obtain chitosan oligosaccharide hydrolysate for later use. S6. Preparation of water-soluble immune adjuvant: Measure 30 mL of carbomer stock solution, and slowly add 30 mL of polyinosinic-polycytidylic acid stock solution while stirring at 300 rpm. After mixing, adjust the pH to 6.80 to form a basic gel matrix. Subsequently, slowly add all the triterpenoid saponin hydrolysate and chitosan oligosaccharide hydrolysate in sequence, and adjust the pH to 6.80 again to obtain a mixture of basic gel matrix and two hydrolysates. Weigh 1.2 g of chitosan and dissolve it in 20 mL of 0.8% (v / v) acetic acid aqueous solution, and stir until completely dissolved to prepare a chitosan solution. Under shear stirring at 600 rpm, quickly pour the chitosan solution into the mixture of basic gel matrix and two hydrolysates, and continue shearing for 10 min to complete cross-linking. Adjust the volume to 300 mL with PBS buffer to obtain the water-soluble immune adjuvant, and store at 2℃. S7. Preparation of composite aqueous adjuvant vaccine: Porcine circovirus Cap protein antigen is slowly added to the adjuvant solution, wherein the ratio of antigen to adjuvant is 2:1. While adding, the mixture is stirred at 300 rpm and stirred continuously at 20°C for 20 minutes until it is uniformly mixed to obtain composite aqueous adjuvant vaccine.

[0030] Research on safety performance Experiment 1: Safety test of water-soluble compound adjuvant in mice Animals: SPF-grade BALB / c mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., were 6 weeks old and weighed 17-22g. The mice were housed in an environment with 12-hour light-dark cycles, a constant temperature of 25±2℃ and humidity of 50%±10%, with free access to water and food, and were acclimatized for 7 days.

[0031] Grouping: 20 mice were randomly divided into two groups: a control group and an Example 1 group, with 10 mice in each group.

[0032] Experiment: In Example 1, mice were subcutaneously injected with 100 μL of compound water adjuvant vaccine, while the control group was subcutaneously injected with 100 μL of physiological saline. The health and survival of the mice were observed and their weight was measured daily for 7 consecutive days. The experimental results are shown in Tables 1 and 2.

[0033]

[0034]

[0035] (1) Analysis of mouse survival Table 1 shows the survival rate of mice in the safety experiment. As can be seen from the table, the number of deaths in both the control group and the Example 1 group was 0. This indicates that during this experimental observation period, no mice died due to vaccine injection in either the control group (receiving saline injection) or the Example 1 group (receiving the compound aqueous adjuvant vaccine), and both groups of mice remained healthy. This demonstrates that, in terms of its impact on mouse survival, the compound aqueous adjuvant vaccine injection did not produce a negative effect.

[0036] (2) Analysis of weight data Table 2 shows the weight data of the mice in the safety experiment. As can be seen from the table, the average weight of the control group before immunization was 18.6g. After immunization, the weight gradually increased from 18.9g on day 1 to 20.6g on day 7. This indicates that under normal feeding conditions and without any special adverse factors, the mice grew well and their weight increased normally over time. In Example 1, the average weight before immunization was 18.8g. The weight remained at 18.8g on day 1 after immunization, and then also increased, reaching 20.4g on day 7. Although the weight did not increase on day 1 after immunization, the subsequent growth trend was similar to that of the control group, and the overall growth rate and trend were quite close to those of the control group. This indicates that the compound aqueous adjuvant vaccine administered to the Example 1 group did not cause adverse reactions to the growth and development of the mice.

[0037] In summary, in this mouse safety experiment, the combined aqueous adjuvant vaccine injection in Example 1 group showed similar effects on mouse survival and weight changes as the control group, indicating that the combined aqueous adjuvant vaccine of the present invention has no adverse effects on mice and has good safety.

[0038] Experiment 2: Safety test of compound water-soluble adjuvant on piglets Animals: 12 healthy piglets aged 3-4 weeks were tested by PCR and ELISA and found to be free of porcine circovirus type 2 (PCV2) virus and antibodies.

[0039] Grouping: The animals were randomly divided into two groups of six each: the Example 1 group and the control group.

[0040] Experiment: In Example 1, piglets were injected intramuscularly with 4 mL of compound water adjuvant vaccine in the neck, while the control group was injected with 4 mL of physiological saline in the neck. The health and survival status of the piglets were observed and recorded daily. The temperature was measured every 6 hours after injection for 5 consecutive days. The experimental results are shown in Tables 3 and 4.

[0041]

[0042]

[0043] Conclusion Analysis (1) Analysis of piglet survival Table 3 shows the survival rate of piglets in the safety experiment. As can be seen from the table, 7 days after injection of the compound aqueous adjuvant vaccine, there were no deaths among piglets in either the control group or the Example 1 group, and all piglets remained healthy. This indicates that the injection of the compound aqueous adjuvant vaccine did not have a negative effect on piglet survival.

[0044] (2) Analysis of average daily body temperature of piglets Table 4 shows the average daily body temperature of piglets in the safety experiment. As can be seen from the table, the average body temperature of the control group on the day of injection was 38.6℃, and the average temperature fluctuated between 39.0-39.6℃ for 1-5 days after injection. The average body temperature of the Example 1 group on the day of injection was 39.3℃, and the average temperature fluctuated between 38.7-39.9℃ for 1-5 days after injection. Although the body temperature fluctuated in both groups, the range was similar, and no abnormally large fluctuations were observed, indicating that the injection of the compound aqueous adjuvant vaccine in Example 1 group did not have an adverse effect on the body temperature of piglets.

[0045] In summary, in this safety experiment on piglets, the piglet survival and body temperature changes in the group injected with the compound water adjuvant vaccine in Example 1 were similar to those in the control group, indicating that the compound adjuvant of the present invention has no adverse effects on piglets and is safe and non-toxic.

[0046] Studies on skin irritation Experiment 3: Skin irritation test of compound water-soluble adjuvant Animals: SPF-grade female guinea pigs, 300-350g, purchased from Beijing Vital River Laboratory Animal Co., Ltd.

[0047] Test: Guinea pigs were shaved on their backs. After shaving, the corresponding adjuvants were injected intradermally into the shaved areas. In Example 1, 0.1 mL of a compound water-soluble adjuvant was injected, while the positive control group received 0.1 mL of NAS04 double adjuvant (purchased from Shanghai Anyikang Biotechnology Co., Ltd.). Six hours after injection, the skin reaction at the injection site was observed and recorded, with particular attention to redness, swelling, and induration. The experimental results are as follows: Figure 1 As shown.

[0048] Conclusion Analysis Figure 1 The figures show the skin reactions at the injection sites of guinea pigs in each group. As can be seen from the figures, the positive control group showed significant redness and swelling at the injection sites, forming indurations of approximately 8 × 10 mm, indicating significant skin irritation. In contrast, no redness or induration was observed at the injection sites of the Example 1 group. Therefore, the composite water-soluble adjuvant in Example 1 exhibits good biocompatibility and significantly lower irritation compared to similar commercially available products.

[0049] In summary, the composite water-soluble adjuvant of the present invention is safe and non-toxic, has low skin irritation, and is easily absorbed.

[0050] Example 2 This embodiment demonstrates that the synergistic effect of saponin hydrolysate, chitosan, and chitosan oligosaccharide hydrolysate has a strong immune effect.

[0051] This embodiment provides a water-soluble immune adjuvant, the specific steps of which are as follows: S1. Preparation of PBS buffer: Weigh 71.6g of disodium hydrogen phosphate dodecahydrate and 27.6g of sodium dihydrogen phosphate monohydrate, and dissolve each in 1L of purified water to prepare a 0.2M solution; measure 612.5mL of disodium hydrogen phosphate solution and 637.5mL of sodium dihydrogen phosphate solution, add 45g of NaCl, 3.72g of KCl and 25mL of Tween 80, stir and mix well, adjust the pH to 6.80, and finally make up to 5000mL for later use; S2. Preparation of Poly I:C stock solution: Weigh 5g of Poly I:C, dissolve in distilled water and bring the volume up to 100mL to prepare Poly I:C stock solution for later use. S3. Carbomer stock solution preparation: Weigh 30g of carbomer, add 500mL of distilled water, stir at 800rpm at 65℃, add 1.0mol / L NaOH during stirring until a gel is formed, then add distilled water to make up to 1000mL to obtain carbomer stock solution, which is then autoclaved for later use. S4. Preparation of saponin hydrolysate: Weigh 850 mg of saponin and add it to 100 mL of PBS buffer. Stir and mix at 500 rpm. Adjust the pH to 5.5. Add 0.4 g of α-glucosidase (5000 U / g) at 37°C and hydrolyze for 3 h. Then adjust the pH to 6.70. Add 0.3 g of galactosidase (4000 U / g) at 37°C and hydrolyze for 3 h. After the hydrolysis is complete, inactivate the enzyme at high temperature to obtain saponin hydrolysate for later use. S5. Preparation of chitosan oligosaccharide solution: Add 1.2g of chitosan oligosaccharide to 270mL of distilled water, mix well, adjust the pH to 5.3, add 0.6g of chitosanase with a concentration of 3000U / g at 52℃ and enzymatically hydrolyze for 4h; then adjust the pH to 5.5, add 0.2g of chitinase with a concentration of 5000U / g at 47℃ and enzymatically hydrolyze for 3h. After the enzymatic hydrolysis is completed, inactivate the enzyme at high temperature and concentrate to 1 / 10 of the original volume to obtain chitosan oligosaccharide hydrolysate for later use. S6. Preparation of water-soluble immune adjuvant: Measure 40 mL of carbomer stock solution, and slowly add 34 mL of polyinosinic-polycytidylic acid stock solution while stirring at 350 rpm. After mixing, adjust the pH to 6.80 to form a basic gel matrix. Subsequently, slowly add all the triterpenoid saponin hydrolysate and chitosan oligosaccharide hydrolysate in sequence, and adjust the pH to 6.80 again to obtain a mixture of basic gel matrix and two hydrolysates. Weigh 1.2 g of chitosan and dissolve it in 20 mL of 0.9% (v / v) acetic acid aqueous solution, and stir until completely dissolved to prepare a chitosan solution. Under shear stirring at 1000 rpm, quickly pour the chitosan solution into the mixture of the basic gel matrix and two hydrolysates, and continue shearing for 12 min to complete cross-linking. Adjust the volume to 300 mL with PBS buffer to obtain a water-soluble immune adjuvant. Store at 5℃. S7. Preparation of composite aqueous adjuvant vaccine: Porcine circovirus Cap protein antigen is slowly added to the adjuvant solution, wherein the ratio of antigen to adjuvant is 2:1. While adding, the mixture is stirred at 350 rpm and stirred continuously at 22°C for 25 minutes until it is uniformly mixed to obtain composite aqueous adjuvant vaccine.

[0052] Comparative Example 1: In step S6, the formed basic gel matrix was directly adjusted to 300 mL with PBS buffer to obtain a water-soluble immune complex adjuvant, that is, the complex adjuvant did not contain saponin hydrolysate, chitosan oligosaccharide hydrolysate and chitosan solution, and the remaining steps were the same as in Example 2.

[0053] Comparative Example 2: Except for step S6, which does not involve the addition of chitosan, the remaining steps are the same as in Example 2.

[0054] Comparative Example 3: Except for step S6, which does not involve adding chitosan oligosaccharide hydrolysate, the remaining steps are the same as in Example 2.

[0055] Comparative Example 4: Except for step S6, in which soap tree saponin hydrolysate is not added, the other steps are the same as in Example 2.

[0056] Comparative Example 5: Except for step S4, in which saponins are not enzymatically hydrolyzed, and step S5, in which chitosan oligosaccharides are not enzymatically hydrolyzed, the remaining steps are the same as in Example 2.

[0057] Comparative Example 6: Except for step S4, which does not involve enzymatic hydrolysis of saponins, the other steps are the same as in Example 2.

[0058] Comparative Example 7: Except for step S5, in which the chitosan oligosaccharide is not enzymatically hydrolyzed, the other steps are the same as in Example 2.

[0059] Immunization experiment in mice Effects of water-soluble compound immune adjuvants on cellular and humoral immunity in mice Animals: SPF-grade BALB / c mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., were 6 weeks old and weighed 18-22g. The mice were housed in an environment with 12-hour light-dark cycles, a constant temperature of 25±2℃ and humidity of 50%±10%, with free access to water and food, and were acclimatized for 7 days.

[0060] Grouping: 60 mice were randomly divided into 10 groups: blank control group, Example 2 group, comparative examples 1-7 groups, and positive control group, with 6 mice in each group.

[0061] Experiment: Mice in Example 2 group and Comparative Examples 1-7 were subcutaneously injected with 100 μL of the compound aqueous adjuvant vaccine; mice in the positive control group were subcutaneously injected with 100 μL of ISA206 adjuvant vaccine (purchased from Shanghai Yubo Biotechnology Co., Ltd.); mice in the blank control group were subcutaneously injected with 100 μL of physiological saline. Spleens were dissected and harvested on day 14 post-immunization. Splenic cells were analyzed using the following methods, the specific methods of which are as follows: Detection method: After rinsing the mouse spleen tissue in a 60mm glass petri dish containing 2mL of pre-cooled PBS, cut it into small pieces with ophthalmic scissors, gently grind the tissue, collect the cell suspension, filter it through a 200-mesh cell sieve, centrifuge at 300g for 5min, discard the supernatant, wash twice with PBS, centrifuge at 300g for 5min, discard the supernatant, add 3 times the volume of red blood cell lysis buffer, lyse at room temperature for 5min, wash twice with PBS, centrifuge and collect the cell pellet for later use.

[0062] Resuspend cells in 1 mL of 1640 medium, add BrefeldinA (1 μL / case) and PMA / TPA (1 μL / case) to each tube, incubate at 37°C for 4 h, centrifuge and discard the supernatant, wash with PBS, centrifuge and discard the supernatant, resuspend cells in 100 μL PBS, and count cells. After adjusting the cell concentration, the live and dead dyes were prepared at a ratio of 1:1000. The cells were resuspended in 1000 μL and incubated at room temperature in the dark for 10 min. After centrifugation, the supernatant was discarded. The cells were washed with PBS, centrifuged again, and the supernatant was discarded. The cells were resuspended in 50 μL of PBS. FcBlock (0.5 μL / case) was added to each tube and incubated at room temperature in the dark for 10 min. The cells were resuspended in 50 μL of PBS. CD3 (2.5 μL / case), CD4 (0.5 μL / case), and CD8 (1.25 μL / case) were added to each tube and incubated at room temperature in the dark for 30 min. Fixation Buffer (500 μL) was added to each tube and incubated at room temperature for 20 min. The cells were then centrifuged and the supernatant was discarded. Each tube was washed twice with 250 μL of Permeabilization Buffer, centrifuged, and the cells were resuspended in 100 μL of Permeabilization Buffer. TNF-α (2 μL / case), IL-2 (2 μL / case), and IFN-γ (2 μL / case) were added to each tube, and the cells were incubated overnight at 4°C. After centrifugation at 350g for 5 min, the supernatant was discarded. Each tube was washed with 250 μL of Permeabilization Buffer, centrifuged at 350g for 5 min, and the supernatant was discarded. The cells were resuspended in 300 μL of PBS and analyzed. The experimental results are shown in Tables 5 and 6.

[0063]

[0064]

[0065] 1. Cell viability Survival+ (%): refers to the percentage of surviving cells out of the total number of cells during preparation and testing.

[0066] The survival rate of each group remained at a high level, and the difference between groups was small. This fully demonstrates that the experimental operation had little effect on cell viability, the cells were in excellent condition, and could effectively provide a reliable basis for the subsequent analysis of immune indicators.

[0067] 2. T lymphocyte ratio CD3+(%): refers to the percentage of T lymphocytes in the total lymphocytes (or the detected monocytes). The CD3+(%) in Group 2 was 43.07±3.85, the highest among all groups. This highlights that the proportion of mature T lymphocytes in this group was significantly higher than that in other groups, laying a solid foundation for cellular immunity.

[0068] CD3+CD4+(%): This refers to the percentage of helper T cells among all T cells (CD3+ cells). The CD3+CD4+(%) in Group 2 reached 64.96±1.68, the highest compared to other groups. This indicates that Group 2 had an abundant number of helper T cells, enabling them to more efficiently assist various immune cells in their functions.

[0069] CD3+CD8+(%): This refers to the percentage of cytotoxic T cells among all T cells (CD3+ cells). The CD3+CD8+(%) in Example 2 group was 31.42±0.64, slightly higher than other groups. This indicates that the proportion of cytotoxic T cells in Example 2 group was slightly more prominent, and its killing ability against pathogen-infected cells and tumor cells was relatively optimal.

[0070] 3. Cytokine secretion CD3+CD4+IL-2+(%): This refers to the percentage of CD4+ T cells capable of producing interleukin-2 (IL-2). The CD3+CD4+IL-2+(%) in Example 2 group was 2.29±0.97, the highest compared to Comparative Examples 1-7. This indicates that the helper T cells in Example 2 have a strong ability to secrete IL-2, playing a positive role in promoting the proliferation and differentiation of T and B cells.

[0071] CD3+CD4+TNF-α+(%): This refers to the percentage of CD4+ T cells capable of producing tumor necrosis factor-α (TNF-α). The CD3+CD4+TNF-α+(%) in Example 2 group was 3.40±1.93, the highest compared to Comparative Examples 1-7. This indicates that Example 2 group has a superior ability to participate in immune regulation and inflammatory responses.

[0072] CD3+CD4+IFN-γ+(%): This refers to the percentage of CD4+ T cells capable of producing interferon-γ (IFN-γ). The CD3+CD4+IFN-γ+(%) in Example 2 group was 2.62±0.26, the highest compared to Comparative Examples 1-7. This indicates that Example 2 group can effectively activate immune cells such as macrophages, further enhancing the body's immune defense function.

[0073] Based on the above indicators, the Example 2 group showed outstanding advantages in terms of the proportion of mature T lymphocytes, helper T cells, cytotoxic T cells, and related cytokine secretion, which strongly proves that the compound aqueous adjuvant vaccine of Example 2 group has the most outstanding effect in promoting cellular immunity.

[0074] Example 3 This embodiment demonstrates that the synergistic effect of saponin hydrolysate and chitosan oligosaccharide hydrolysate enhances the persistence of immunity.

[0075] This embodiment provides a water-soluble immune adjuvant, the specific steps of which are as follows: S1. Preparation of PBS buffer: Weigh 71.6g of disodium hydrogen phosphate dodecahydrate and 27.6g of sodium dihydrogen phosphate monohydrate, and dissolve each in 1L of purified water to prepare a 0.2M solution; measure 612.5mL of disodium hydrogen phosphate solution and 637.5mL of sodium dihydrogen phosphate solution, add 45g of NaCl, 3.72g of KCl and 25mL of Tween 80, stir and mix well, adjust the pH to 6.80, and finally make up to 5000mL for later use; S2. Preparation of Poly I:C stock solution: Weigh 5g of Poly I:C, dissolve in distilled water and bring the volume up to 100mL to prepare Poly I:C stock solution for later use. S3. Carbomer stock solution preparation: Weigh 30g of carbomer, add 500mL of distilled water, stir at 70℃ and 1200rpm, add 1.0mol / L NaOH during stirring until a gel is formed, then add distilled water to make up to 1000mL to obtain carbomer stock solution, which is then autoclaved for later use. S4. Preparation of saponin hydrolysate: Weigh 900 mg of saponin and add it to 100 mL of PBS buffer. Stir and mix at 800 rpm. Adjust the pH to 5.5. Add 0.4 g of α-glucosidase (5000 U / g) at 40 °C and hydrolyze for 3 h. Then adjust the pH to 6.70. Add 0.3 g of galactosidase (4000 U / g) at 40 °C and hydrolyze for 3 h. After the hydrolysis is complete, inactivate the enzyme at high temperature to obtain saponin hydrolysate for later use. S5. Preparation of chitosan oligosaccharide solution: Add 1.2g of chitosan oligosaccharide to 300mL of distilled water, mix well, adjust the pH to 5.3, add 0.6g of chitosan enzyme with a concentration of 3000U / g at 55℃ and hydrolyze for 4h; then adjust the pH to 5.5, add 0.2g of chitinase with a concentration of 5000U / g at 50℃ and hydrolyze for 3h. After the hydrolysis is completed, inactivate the enzyme at high temperature and concentrate to 1 / 10 of the original volume to obtain chitosan oligosaccharide hydrolysate for later use. S6. Preparation of water-soluble immunomodulatory adjuvant: 50 mL of carbomer stock solution was weighed and slowly added to 37.5 mL of polyinosinic-polycytidylic acid stock solution under stirring at 400 rpm. After mixing, the pH was adjusted to 6.80 to form a basic gel matrix. Subsequently, all the triterpenoid saponin hydrolysate and chitosan oligosaccharide hydrolysate were added sequentially and slowly, and the pH was adjusted to 6.80 again to obtain a mixture of the basic gel matrix and the two hydrolysates. 1.2 g of chitosan was weighed and dissolved in 20 mL of 1.0% (v / v) acetic acid aqueous solution and stirred until completely dissolved to prepare a chitosan solution. Under shear stirring at 1500 rpm, the chitosan solution was quickly poured into the mixture of the basic gel matrix and the two hydrolysates. Shearing was continued for 15 min to complete cross-linking. The volume was adjusted to 300 mL with PBS buffer to obtain the water-soluble immunomodulatory adjuvant, which was stored at 8°C. The actual product image of the adjuvant in this embodiment is shown below. Figure 3 As shown; S7. Preparation of composite aqueous adjuvant vaccine: Porcine circovirus Cap protein antigen is slowly added to the adjuvant solution, wherein the ratio of antigen to adjuvant is 2:1. While adding, the mixture is stirred at 400 rpm and stirred continuously at 25°C for 30 minutes until it is homogeneous, thus obtaining the composite aqueous adjuvant vaccine.

[0076] Animals: SPF-grade KM mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., were 6 weeks old and weighed 18-22g. The mice were housed in an environment with 12-hour light-dark cycles, a constant temperature of 25±2℃ and humidity of 50%±10%, with free access to water and food, and were acclimatized for 7 days.

[0077] Comparative Example 8: Except for step S4, in which saponins are not enzymatically hydrolyzed, and step S5, in which chitosan oligosaccharides are not added for enzymatic hydrolysis, the remaining steps are the same as in Example 3.

[0078] Comparative Example 9: Except for step S4, in which the saponins of the soap tree are not enzymatically hydrolyzed, the other steps are the same as in Example 3.

[0079] Comparative Example 10: Except for step S5, which does not involve the addition of chitosan oligosaccharide for enzymatic hydrolysis, the remaining steps are the same as in Example 3.

[0080] Experiment on the persistence of immunity in piglets Animals: 36 healthy piglets aged 3-4 weeks were tested by PCR and ELISA and found to be free of porcine circovirus type 2 (PCV2) virus and antibodies.

[0081] Grouping: 36 piglets were randomly divided into 6 groups: blank control group, Example 3 group, comparative examples 8-10 group and positive control group (ISA206 group), with 6 piglets in each group.

[0082] experiment: After grouping, the Example 3 group and Comparative Examples 8-10 groups were subcutaneously injected with 2 mL of the corresponding compound aqueous adjuvant vaccine; the positive control group was subcutaneously injected with 2 mL of ISA206 adjuvant vaccine; and the blank control group was subcutaneously injected with 2 mL of physiological saline. This continued for 90 days. Blood samples were collected from the anterior vena cava at 7, 14, 21, 28, 42, 56, and 90 days post-immunization. Serum was separated, and antibody levels were detected using ELISA. The antibody titers in each group were measured to determine the changes in antibody levels. The results are shown in Table 7 and... Figure 2 As shown.

[0083]

[0084] Table 7 and Figure 2 This is a comparison chart of porcine circovirus immune antibodies in piglets. It shows that at key immunization time points of 7, 14, 21, 28, 42, 56, and 90 days post-immunization, the antibody levels in the compound aqueous adjuvant group (Example 3) were higher than those in the ISA206 group (Comparative Examples 8-10), demonstrating its stronger ability to stimulate antibody production and a significant increase in antibody levels. Furthermore, over a longer period from 21 to 90 days post-immunization, the Example 3 group maintained a consistently high antibody level, exhibiting a significant advantage in immune persistence compared to the Comparative Examples 8-10 and the ISA206 group, confirming the strong immune persistence effect of the compound aqueous adjuvant vaccine.

[0085] Analysis of the mechanism by which saponin hydrolysate and chitosan oligosaccharide hydrolysate synergistically enhance immune persistence: After being acted upon by α-glucosidase and galactosidase, saponin hydrolysate may generate small molecule active substances that are more easily recognized by immune cells. These substances can enhance the efficiency of the initial immune response by activating the phagocytic capacity and cytokine secretion of antigen-presenting cells (such as macrophages and dendritic cells). Meanwhile, the oligosaccharide fragments produced by the degradation of chitosan and chitinase in chitosan oligosaccharide hydrolysate can not only promote the proliferation and differentiation of B lymphocytes to increase antibody production, but also maintain the long-term activity of immune memory cells by regulating the balance of T lymphocyte subsets. When the two work synergistically, the antigen presentation process initiated by the saponin hydrolysate provides sufficient antigen stimulation for the specific immune cell proliferation induced by the chitosan oligosaccharide hydrolysate. At the same time, the immune microenvironment created by the chitosan oligosaccharide hydrolysate can prolong the activation time of immune cells by the saponin hydrolysate, forming a continuous immune regulatory chain of "initiation-amplification-maintenance", thereby significantly enhancing antibody production capacity and prolonging its duration, ultimately achieving an improvement in immune persistence.

[0086] The above-described embodiments are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of protection of the present invention. It should be noted that for those skilled in the art, any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the scope of protection of the present invention.

Claims

1. A water-soluble immune adjuvant, characterized in that: Each 300ml of the compound adjuvant formulation contains: 30-50mL of carbomer stock solution, 30-37.5mL of polyinosinic-polycytidylic acid (poly I:C) stock solution, 800-900mg of triterpenoid saponins, 1.2g of chitosan oligosaccharide, and 20mL of chitosan solution; wherein the concentration of the carbomer stock solution is 30 mg / mL, the concentration of the poly I:C stock solution is 50mg / mL, and the concentration of the chitosan solution is 600mg / mL.

2. The water-soluble immune adjuvant according to claim 1, characterized in that: In the compound adjuvant, the chitosan oligosaccharide is hydrolyzed by a combination of chitosanase and chitinase.

3. The water-soluble immune adjuvant according to claim 2, characterized in that: The triterpenoid saponins in the compound adjuvant are hydrolyzed by a combination of α-glucosidase and galactosidase.

4. The method for preparing a water-soluble immune adjuvant according to claim 3, characterized in that: The specific preparation method of the product is as follows: S1. Preparation of PBS buffer: Weigh 71.6g of disodium hydrogen phosphate dodecahydrate and 27.6g of sodium dihydrogen phosphate monohydrate, and dissolve each in 1L of purified water to prepare a 0.2M solution; measure 612.5mL of disodium hydrogen phosphate solution and 637.5mL of sodium dihydrogen phosphate solution, add 45g of NaCl, 3.72g of KCl and 25mL of Tween 80, stir and mix well, adjust the pH to 6.80, and make up to 5000mL for later use; S2. Preparation of Poly I:C stock solution: Weigh 5g of Poly I:C, dissolve in distilled water and bring the volume up to 100mL to prepare Poly I:C stock solution for later use. S3. Carbomer stock solution preparation: Weigh 30g of carbomer, add 500mL of distilled water, stir at 60-70℃ and 500-1200rpm, add 1.0mol / L NaOH during stirring until a gel is formed, then add distilled water to make up to 1000mL to obtain carbomer stock solution, autoclave and set aside for later use. S4. Preparation of triterpenoid saponin hydrolysate: Weigh 900 mg of triterpenoid saponin and add it to 100 mL of PBS buffer from step S1. Stir and mix at 200-800 rpm, adjust the pH to 5.5, add 0.4 g of α-glucosidase at 35-40℃ for 3 h, then adjust the pH to 6.70, add 0.3 g of galactosidase at 35-40℃ for 3 h, and finally inactivate the enzyme at high temperature to obtain the triterpenoid saponin hydrolysate for later use. S5. Preparation of chitosan oligosaccharide solution: Add 1.2g of chitosan oligosaccharide to 250-300mL of distilled water, mix well, adjust the pH to 5.3, add 0.6g of chitosanase at 50-55℃ for 4h of enzymatic hydrolysis; then adjust the pH to 5.5, add 0.2g of chitinase at 45-50℃ for 3h of enzymatic hydrolysis, inactivate the enzyme at high temperature after the hydrolysis is completed, and concentrate to 1 / 10 of the original volume of the enzymatic hydrolysis solution to obtain chitosan oligosaccharide hydrolysate for later use; S6. Preparation of water-soluble immunomodulatory adjuvant: Measure 30-50 mL of carbomer stock solution, and slowly add 30-37.5 mL of polyinosinic-polycytidylic acid stock solution while stirring at 300-400 rpm. After mixing, adjust the pH to 6.80 to form a basic gel matrix. Subsequently, slowly add all the triterpenoid saponin hydrolysate and chitosan oligosaccharide hydrolysate in sequence, and adjust the pH to 6.80 again to obtain a mixture of the basic gel matrix and the two hydrolysates. Weigh 1.2 g of chitosan and dissolve it in 20 mL of 0.8%-1.0% (v / v) acetic acid aqueous solution, and stir until completely dissolved to prepare a chitosan solution. Under shear stirring at 600-1500 rpm, quickly pour the chitosan solution into the mixture of the basic gel matrix and the two hydrolysates, and continue shearing for 10-15 min to complete cross-linking. Adjust the volume to 300 mL with PBS buffer to obtain the water-soluble immunomodulatory adjuvant, and store at 2-8℃.

5. The method for preparing a water-soluble immune adjuvant according to claim 4, characterized in that: The triterpenoid saponins are selected from one or more of the following: bupleurum saponins, ginsenosides, astragalus saponins, phytolacca saponins, saponins, and platycodon saponins.

6. The method for preparing a water-soluble immune adjuvant according to claim 4, characterized in that: The concentration of the α-glucosidase is 5000 U / g; the concentration of the galactosidase is 4000 U / g.

7. The method for preparing a water-soluble immune adjuvant according to claim 4, characterized in that: The concentration of the chitosanase is 3000 U / g; the concentration of the chitinase is 5000 U / g.

8. A water-soluble immune adjuvant vaccine, characterized in that: The vaccine is a mixture of the water-soluble immune adjuvant and antigen as described in any one of claims 1-3; The preparation method of the compound aqueous adjuvant vaccine is as follows: the antigen is slowly added to the adjuvant solution while stirring at a speed of 300-400 rpm to mix evenly. The mixture is then stirred continuously at 20-25℃ for 20-30 minutes until it is homogeneous, thus obtaining the compound aqueous adjuvant vaccine.

9. The water-soluble immune adjuvant vaccine according to claim 8, characterized in that: The antigen is porcine circovirus Cap protein, and the antigen concentration is 1 mg / mL.

10. A water-soluble immune adjuvant vaccine according to claim 8, characterized in that: The mass ratio of the antigen to the adjuvant is 2:1.

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