Water-soluble complex immunological adjuvant, immunological water adjuvant vaccine, and preparation method and application thereof

Abstract

The application discloses a water-soluble composite immunoadjuvant, an immunological water adjuvant vaccine and a preparation method and application thereof. The water-soluble composite immunoadjuvant comprises carbomer, poloxamer and an immunopotentiator; in the water-soluble composite immunoadjuvant, the concentration of the carbomer is 1.0-20.0 mg / mL, the concentration of the poloxamer is 0.1-2.0 mg / mL, and the concentration of the immunopotentiator is 1.0-40.0 mg / mL. The water-soluble composite immunoadjuvant does not contain mineral oil components, has a simple synthesis process, takes the carbomer as an antigen slow-release agent, takes cimetidine or fucoidan as the immunopotentiator and has the ability to enhance cellular immunity, takes the poloxamer as an antigen stabilizer, can effectively produce an immunopotentiating effect, effectively avoids obvious local side effects caused by oily adjuvants stimulating the body, is easy to absorb in an animal body, is high in safety and can effectively improve the economic benefits of breeders.

Description

Technical Field

[0001] This invention relates to the field of bioimmunotechnology, specifically to a water-soluble composite immune adjuvant, an immune water adjuvant vaccine, its preparation method, and its application. Background Technology

[0002] Large-scale treatment with traditional antibiotics easily leads to drug resistance, highlighting the crucial role of highly effective vaccines in infectious disease prevention. Currently, live attenuated and inactivated vaccines against bacteria are widely used. However, live attenuated vaccines carry the risk of virulence reversion, while inactivated vaccines suffer from low immunoprotective efficacy. Therefore, developing subunit vaccines that can elicit highly effective immunogenicity and possess high safety is a preferred approach. However, current methods for immunizing with subunit vaccine antigens alone have limitations; subunit vaccine antigens only elicit strong immunity when combined with adjuvants.

[0003] Vaccine adjuvants are preparations formulated with antigens to enhance the immune response. Vaccine adjuvants are divided into immunostimulants and delivery agents. Immunostimulants primarily enhance the immune effect by increasing the recruitment of immune cells and the secretion of cytokines, such as saponins, Toll-like receptor agonists, and cytokine preparations. Antigen delivery agents primarily enhance the immune effect by presenting antigens to antigen-presenting cells and slowly releasing antigens, such as emulsion adjuvants, microparticle adjuvants, and mineral salt adjuvants. Currently, commonly used oil-based adjuvants have strong immune effects, but they are prone to causing significant stress to the body and have considerable side effects on animals. Mineral oil adjuvants, in particular, cause significant damage and have strong toxic side effects in animals. Oil-emulsion adjuvants mainly include Freund's adjuvant, white oil adjuvant, ISA201 adjuvant, and ISA206 adjuvant. Therefore, researching an adjuvant that can reduce animal stress and side effects while simultaneously stimulating both cellular and humoral immunity is a current research direction. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a water-soluble composite immune adjuvant, an aqueous adjuvant vaccine, its preparation method, and its application. Compared with conventional oil-emulsion adjuvants, the water-soluble composite immune adjuvant of this invention reduces irritation to the animal body, enhances safety, and reduces residue problems. Furthermore, it incorporates an immune enhancer that can induce cellular immune responses, building upon the sustained-release properties of water-soluble adjuvants, aiming to simultaneously enhance the cellular and humoral immune capabilities of the vaccine adjuvant.

[0005] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides a water-soluble composite immune adjuvant, which includes carbomer, poloxamer, and an immune enhancer; In the water-soluble compound immunoadjuvant, the concentration of carbomer is 1.0~20.0 mg / mL, the concentration of poloxamer is 0.1~2.0 mg / mL, and the concentration of the immunostimulant is 1.0~40.0 mg / mL. The carbomer is any one of carbomer 974, carbomer 940, carbomer 971, carbomer 934, carbomer 980 and carbomer 941; The poloxamer is either poloxamer 188 or poloxamer 407; The immune enhancer is either cimetidine or fucoidan.

[0006] Furthermore, in the water-soluble compound immune adjuvant, the concentration of carbomer is 5.0~10.0 mg / mL, the concentration of poloxamer is 1.0 mg / mL, and the concentration of the immune enhancer is 10.0~20.0 mg / mL.

[0007] Furthermore, in the water-soluble compound immunoadjuvant, the concentration of carbomer 974 is 5.0 mg / mL, the concentration of poloxamer 188 is 1.0 mg / mL, and the concentration of fucoidan is 20.0 mg / mL.

[0008] The present invention also provides a method for preparing the aforementioned water-soluble composite immune adjuvant, comprising the following steps: (1) Dissolve carbomer in water, adjust the pH to 6.8-7.0, and sterilize to obtain a carbomer solution; (2) Dissolve the immune enhancer in a solvent to prepare an immune enhancer solution; (3) Dissolve poloxamer in water to obtain a poloxamer solution; (4) Mix carbomer solution, immune enhancer solution and poloxamer solution evenly to obtain water-soluble compound immune adjuvant.

[0009] Furthermore, in step (1), the concentration of the carbomer solution is 10.0~20.0 mg / mL; In step (2), the concentration of the immune enhancer solution is 12.5~50.0 mg / mL; In step (3), the concentration of the poloxamer solution is 10.0~20.0 mg / mL.

[0010] Furthermore, when the immune enhancer is cimetidine, the solvent is a 0.1-0.3 M hydrochloric acid solution, and the pH of the immune enhancer solution is adjusted to 6.8-7.0; When the immune enhancer is fucoidan, the solvent is water.

[0011] The present invention also provides the application of the aforementioned water-soluble composite immune adjuvant in the preparation of water-adjuvanted immune vaccines.

[0012] The present invention also provides an aqueous adjuvant vaccine, the aqueous adjuvant vaccine comprising the aforementioned water-soluble complex adjuvant and antigen; The antigen is Pasteurella multocida PlpE protein; The nucleotide sequence of the protein PlpE is shown in SEQ ID NO: 1; In the aforementioned aqueous adjuvant vaccine, the concentration of the antigen is 400-600 μg / mL.

[0013] Furthermore, in the aforementioned aqueous adjuvant vaccine, the concentration of the antigen is 500 μg / mL.

[0014] The present invention also provides a method for preparing the aforementioned aqueous adjuvant vaccine, the specific steps of which are: adding the antigen to the water-soluble composite adjuvant at a speed of 400~600 r / min and stirring for 5~8 min to obtain the aqueous adjuvant vaccine.

[0015] Furthermore, the speed is 500 r / min and the stirring time is 5 min.

[0016] The principle of this invention: The reasons for selecting the raw materials in this invention are as follows: (1) Carbomer is commonly used in the pharmaceutical industry as an emulsifier, gelling agent, suspension stabilizer, and tablet binder. Various types of carbomer do not produce significant toxic effects after injection into experimental animals. Carbomer-based acrylic polymers have been evaluated as adjuvants for veterinary vaccines and are used in combination with various other types of adjuvants for swine veterinary vaccines. Carbomer has a low application cost and is suitable for veterinary vaccination. Carbomer has been used as an adjuvant for various pathogens, such as equine influenza virus, porcine parvovirus, porcine circovirus, and Staphylococcus aureus mastitis. The carbomer-based adjuvant ADJ, when used in combination with Toll-like immune agonists GLA and CpG, can significantly enhance T cell immune responses and induce the proliferation of a large number of antigen-specific CD8+ and CD4+ T cells. Studies have shown that carbomer, whether used alone with antigens, or in combination with the oil-in-water adjuvant MF59, or in combination with lecithin, has been proven to be an adjuvant with good safety and tolerability, demonstrating excellent biocompatibility and biodegradability.

[0017] (2) Currently, common immunostimulants that can enhance immunity include small molecule Toll-like receptor agonists, polysaccharides, and chemically synthesized agents. However, some small molecule agonists are often expensive and costly. Polysaccharide or chemical adjuvants are more suitable for practical application. Polysaccharide adjuvants are widely used in vaccine adjuvant research due to their strong intrinsic immunomodulatory function, biocompatibility, and biodegradability. Chitosan, dextran, mannan, and traditional Chinese medicine polysaccharides have been approved as human vaccine adjuvants. Naturally derived polysaccharides with modified groups have stronger immunomodulatory effects. Animals immunized with sulfated polysaccharides showed stronger expression of Th1 and Th2 cytokines. Fucoidan is a natural sulfated polysaccharide that does not require chemical modification. Sulfated polysaccharides have good biocompatibility and fewer side effects, and they also have immunostimulatory effects. They can improve the phagocytic activity of macrophages and promote the secretion of nitric oxide, reactive oxygen species, and various cytokines.

[0018] (3) Chemically synthesized immunostimulants include levamisole, cimetidine, isoprostine, praziquantel, ranitidine, and famotidine. Among them, levamisole and cimetidine are commonly used. Cimetidine, as an H2R antagonist, can reverse histamine-mediated immunosuppression and has significant immunomodulatory capabilities. Studies have shown that cimetidine can activate dendritic cells (DCs) and promote the maturation of immature dendritic cells (imDCs), further enhancing their antigen presentation capabilities. Cimetidine may activate imDCs by targeting TLR / MAPK signaling. Some studies have used cimetidine and levamisole as adjuvants in combination with hepatitis B surface antigen, and the results show that cimetidine and levamisole have comparable levels of humoral immunity.

[0019] (4) Adding antigen stabilizers to subunit vaccines can effectively ensure that antigens do not precipitate or fold. Poloxamer 188 is an aqueous formulation stabilizer that can enhance the suspension stability of the solution and enhance the solubility of the aqueous solution, thus ensuring good structural stability of the antigen protein. Studies have shown that poloxamer 188 also has the ability to guide the antigen to the lymphatic vessels, that is, it has the ability to enhance the immune response.

[0020] The beneficial effects of this invention are: The acrylic polymer carbomer in the composition of this invention exhibits good stability and a certain viscosity. Upon injection into an animal, it slowly releases antigens, delaying the antigenic immune response and increasing the duration of the immune response. Cimetidine, an immunostimulant and H2R antagonist, enhances antigen presentation by activating dendritic cells and promoting the maturation of immature dendritic cells. Fucoidan, a natural sulfated polysaccharide immunostimulant, stimulates the production of natural killer cells and promotes dendritic cell maturation, inducing strong cell-mediated and humoral immune responses, and possessing anti-inflammatory, antiviral, antioxidant, and antitumor activities. Poloxamer 188, a surfactant, stabilizes proteins in vaccine adjuvants, preventing protein self-folding, and also acts as a stabilizer in aqueous formulations. This water-soluble composite immunostimulant of the present invention does not contain mineral oil, thus avoiding significant irritation to the animal body and effectively preventing damage to the injection site caused by oily components. It also offers the advantages of sustained release, immunostimulation, and good stability. Attached Figure Description

[0021] Figure 1 Antibody level graph for screening water-adjuvanted vaccines in mouse experiments; Figure 2 A graph showing the change in body weight in mice for screening immunized water-adjuvant vaccines. Figure 3 Local reaction at the injection site for screening water-adjuvanted vaccines in mouse experiments; In the diagram, 1, 2, 3, 4, 5 and 6 represent, respectively, water-adjuvanted vaccine 1, water-adjuvanted vaccine 2, water-adjuvanted vaccine 3, water-adjuvanted vaccine 4, adjuvanted ISA206 vaccine and PBS vaccine; Figure 4 The diagram shows the physical properties of a water-soluble compound immunoadjuvant. In the figure, A is water-soluble compound immune adjuvant 5, and B is water-soluble compound immune adjuvant 3; Figure 5 Antibody levels in mice after immunization with a water-adjuvanted vaccine; Figure 6 This is a graph showing the proliferation level of mouse spleen lymphocytes. Figure 7 This is a graph showing the antibody subtype level determination. Figure 8 This is a graph showing the IFN-γ levels in mice after immunization. Figure 9 Figure showing the weight change in mice after immunization with a water-adjuvanted vaccine; Figure 10 This graph shows the changes in biochemical indicators in mice after immunization. Figure 11HE staining images of tissue and organ sections from mice after immunization; Figure 12 A diagram showing the local reaction of mice after immunization with a reduced concentration of the immune enhancer. Detailed Implementation

[0022] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0023] Experimental materials description: Carbomer 974, Poloxamer 188: Beijing Fengli Precision Medicine Co., Ltd.; Cimetidine (99%), Fucoidan (≥98%): Shanghai Aladdin Biochemical Technology Co., Ltd.; ISA206 adjuvant: Seppic, France; The nucleotide sequence of protein PlpE is shown in SEQ ID NO: 1.

[0024] Example 1 Preparation of water-soluble complex immune adjuvant 1 1. Carbomer 974 solution: Place 0.25 g of carbomer 974 powder in 25 mL of ultrapure water and let it swell overnight (12~16 h). After stirring and mixing, adjust the pH to 7.0 with 10.0 M sodium hydroxide solution, autoclave at 121℃ for 20 min to prepare a 10.0 mg / mL carbomer solution.

[0025] 2. Cimetidine solution: Weigh 0.5 g of cimetidine powder and dissolve it in 14 mL of 0.15 M hydrochloric acid solution. Adjust the pH to 6.8-7.0 with 1.0 M sodium hydroxide solution. Dilute with ultrapure water to obtain 20.0 mL of cimetidine solution. Filter the solution through a 0.22 μm filter for later use.

[0026] 3. Poloxamer 188 solution: Weigh 0.05 g of poloxamer 188 powder, dissolve it in 5 mL of ultrapure water to prepare a 10.0 mg / mL poloxamer 188 solution, and filter it through a 0.22 μm filter for later use.

[0027] 4. Preparation of water-soluble compound immune adjuvant: Mix 25 mL of carbomer 974 solution, 20 mL of cimetidine solution and 5 mL of poloxamer 188 solution evenly to obtain 50 mL of water-soluble compound immune adjuvant 1 (0.5% carbomer + 10.0 mg / mL cimetidine + 0.1% poloxamer).

[0028] Example 2 Preparation of water-soluble complex immune adjuvant 2 1. Carbomer 974 solution: Place 0.5 g of carbomer 974 powder in 25 mL of ultrapure water and let it swell overnight (12~16 h). After stirring and mixing, adjust the pH to 7.0 with 10.0 M sodium hydroxide solution. Autoclave at 121℃ for 20 min to prepare a 20.0 mg / mL carbomer solution.

[0029] 2. Cimetidine solution: Weigh 0.5 g of cimetidine powder and dissolve it in 7 mL of 0.15 M hydrochloric acid solution. Adjust the pH to 6.8-7.0 with 1.0 M sodium hydroxide solution. Dilute with ultrapure water to obtain 20 mL of cimetidine solution. Filter through a 0.22 μm filter for later use.

[0030] 3. Poloxamer 188 solution: Weigh 0.05 g of poloxamer 188 powder, dissolve it in 5 mL of ultrapure water to prepare a 10.0 mg / mL poloxamer 188 solution, and filter it through a 0.22 μm filter for later use.

[0031] 4. Preparation of water-soluble compound immune adjuvant: Mix 25 mL of carbomer 974 solution, 20 mL of cimetidine solution and 5 mL of poloxamer 188 solution evenly to obtain 50 mL of water-soluble compound immune adjuvant 2 (1% carbomer + 10.0 mg / mL cimetidine + 0.1% poloxamer).

[0032] Example 3 Preparation of water-soluble complex immune adjuvant 3 1. Carbomer 974 solution: Place 0.25 g of carbomer 974 powder in 25 mL of ultrapure water and let it swell overnight (12~16 h). After stirring and mixing, adjust the pH to 7.0 with 10.0 M sodium hydroxide solution, autoclave at 121℃ for 20 min to prepare a 10.0 mg / mL carbomer solution.

[0033] 2. Fucoidan solution: Weigh 1.0 g of fucoidan powder and dissolve it in 20 mL of ultrapure water to prepare a 50.0 mg / mL fucoidan stock solution. Filter the solution using a 0.22 μm filter for later use.

[0034] 3. Poloxamer 188 solution: Weigh 0.05 g of poloxamer 188 powder, dissolve it in 5 mL of ultrapure water to prepare a 10.0 mg / mL poloxamer 188 solution, and filter it through a 0.22 μm filter for later use.

[0035] 4. Preparation of water-soluble compound immune adjuvant: Mix 25 mL of carbomer 974 solution, 20 mL of fucoidan solution and 5 mL of poloxamer 188 solution evenly to obtain 50 mL of water-soluble compound immune adjuvant 3 (0.5% carbomer + 20.0 mg / mL fucoidan + 0.1% poloxamer).

[0036] Example 4 Preparation of water-soluble complex immune adjuvant 4 1. Carbomer 974 solution: Place 0.5 g of carbomer 974 powder in 25 mL of ultrapure water and let it swell overnight (12~16 h). After stirring and mixing, adjust the pH to 7.0 with 10.0 M sodium hydroxide solution. Autoclave at 121℃ for 20 min to prepare a 20.0 mg / mL carbomer solution.

[0037] 2. Fucoidan solution: Weigh 1.0 g of fucoidan powder and dissolve it in 20 mL of ultrapure water to prepare a 50.0 mg / mL fucoidan stock solution. Filter the solution using a 0.22 μm filter for later use.

[0038] 3. Poloxamer 188 solution: Weigh 0.05 g of poloxamer 188 powder, dissolve it in 5 mL of ultrapure water to prepare a 10.0 mg / mL poloxamer 188 solution, and filter it through a 0.22 μm filter for later use.

[0039] 4. Preparation of water-soluble compound immune adjuvant: Mix 25 mL of carbomer 974 solution, 20 mL of fucoidan solution and 5 mL of poloxamer 188 solution evenly to obtain 50 mL of water-soluble compound immune adjuvant 4 (1% carbomer + 20.0 mg / mL fucoidan + 0.1% poloxamer).

[0040] Example 5 Preparation of water-soluble complex immune adjuvant 5 1. Carbomer 974 solution: Place 0.25 g of carbomer 974 powder in 25 mL of ultrapure water and let it swell overnight (12~16 h). After stirring and mixing, adjust the pH to 7.0 with 10.0 M sodium hydroxide solution, autoclave at 121℃ for 20 min to prepare a 10.0 mg / mL carbomer solution.

[0041] 2. Fucoidan solution: Weigh 0.5 g of fucoidan powder and dissolve it in 20 mL of ultrapure water to prepare a 25.0 mg / mL fucoidan stock solution. Filter the solution using a 0.22 μm filter for later use.

[0042] 3. Poloxamer 188 solution: Weigh 0.05 g of poloxamer 188 powder, dissolve it in 5 mL of ultrapure water to prepare a 10.0 mg / mL poloxamer 188 solution, and filter it through a 0.22 μm filter for later use.

[0043] 4. Preparation of water-soluble compound immune adjuvant: Mix 25 mL of carbomer 974 solution, 20 mL of fucoidan solution and 5 mL of poloxamer 188 solution evenly to obtain 50 mL of water-soluble compound immune adjuvant 5 (0.5% carbomer + 10.0 mg / mL fucoidan + 0.1% poloxamer).

[0044] Example 6 Preparation of Water-Adjuvanted Immunization Vaccine 1 Add the water-soluble compound adjuvant 1 from Example 1 to a sterilized beaker. Under the action of a magnetic stirrer, slowly add the antigen protein PlpE to the beaker at a speed of 500 r / min and stir continuously for 5 min to obtain the water-adjuvanted immune vaccine 1 (rPlpE-0.5% kcal + 10.0 mg / mL succinate + 0.1% povidone-iodine). The final concentration of PlpE protein is 500.0 μg / mL.

[0045] Example 7 Preparation of Water-Adjuvanted Immunization Vaccine 2 Add the water-soluble compound adjuvant 2 from Example 2 to a sterilized beaker. Under the action of a magnetic stirrer, slowly add the antigen protein PlpE to the beaker at a speed of 500 r / min and continue stirring for 5 min to obtain the water-adjuvanted immune vaccine 2 (rPlpE-1% carbohydrate + 10.0 mg / mL succinate + 0.1% povidone-iodine). The final concentration of PlpE protein is 500.0 μg / mL.

[0046] Example 8 Preparation of Water-Adjuvanted Immunization Vaccine 3 Add the water-soluble compound adjuvant 3 from Example 3 to a sterilized beaker. Under the action of a magnetic stirrer, slowly add the antigen protein PlpE to the beaker at a speed of 500 r / min and continue stirring for 5 min to obtain the water-adjuvanted immune vaccine 3 (rPlpE-0.5% kcal + 20.0 mg / mL iodine + 0.1% povidone-iodine). The final concentration of PlpE protein is 500.0 μg / mL.

[0047] Example 9 Preparation of Water-Adjuvanted Immunization Vaccine 4 Add the water-soluble compound adjuvant 4 from Example 4 to a sterilized beaker. Under the action of a magnetic stirrer, slowly add the antigen protein PlpE to the beaker at a speed of 500 r / min and continue stirring for 5 min to obtain the water-adjuvanted immune vaccine 4 (rPlpE-1% carboxylic acid + 20.0 mg / mL sodium + 0.1% povidone-iodine). The final concentration of PlpE protein is 500.0 μg / mL.

[0048] Example 10 Preparation of Water-Adjuvanted Immunization Vaccine 5 Add the water-soluble compound adjuvant 5 from Example 5 to a sterilized beaker. Under the action of a magnetic stirrer, slowly add the antigen protein PlpE to the beaker at a speed of 500 r / min and continue stirring for 5 min to obtain the water-adjuvanted immune vaccine 5 (rPlpE-0.5% kcal + 10.0 mg / mL eosin + 0.1% povidone-iodine). The final concentration of PlpE protein is 500.0 μg / mL.

[0049] Comparative Example 1 Preparation of adjuvant ISA206 vaccine The antigen protein PlpE was mixed with ISA206 adjuvant sterilized at 116℃ for 15 min in a 1:1 ratio and emulsified in a tissue homogenizer at a frequency of 60 Hz for 2 min to obtain the adjuvanted ISA206 vaccine (rPlpE-ISA206). The final concentration of PlpE protein was 500.0 μg / mL.

[0050] Comparative Example 2 Preparation of PBS vaccine The antigen protein PlpE was mixed with 0.01 M PBS solution at a ratio of 1:1 to obtain the PBS vaccine (rPlpE-PBS), with a final concentration of PlpE protein of 500.0 μg / mL.

[0051] Example 11 Performance screening of water-adjuvanted vaccines 1. Experimental animals: 6-8 week old SPF BALB / c mice were randomly divided into 7 groups, with 6 mice in each group, as follows: (1) Immunization group ①: Injected with the water adjuvant vaccine 1 of Example 6; (2) Immunization group ②: Injected with the water adjuvant vaccine 2 of Example 7; (3) Immunization group ③: Injected with the water adjuvant vaccine 3 of Example 8; (4) Immunization group ④: Injection of the aqueous adjuvant vaccine 4 of Example 9; (5) Positive control group: injected with the adjuvant ISA206 vaccine of comparison ratio 1; (6) Negative control group: injected with the PBS vaccine of comparison ratio 2; (7) Blank control group: injected with 0.01 M PBS solution.

[0052] 2. Experimental procedure: 200 μL of vaccine was injected subcutaneously into each group of mice, and the mice were immunized twice. Blood samples were collected from the mice on the 7th and 14th days after the first immunization to measure antibody levels. Blood samples were also collected on the 7th and 14th days after the second immunization to measure antibody levels.

[0053] The results are as follows Figure 1As shown, the immunization group induced higher antibody levels than the single antigen (negative control group). The water-adjuvanted vaccine 3 induced higher antibody levels than other adjuvanted vaccines 7 and 14 days after the second immunization. The antibody level induced by the fucoidan water-soluble complex adjuvant was significantly higher than that induced by the cimetidine water-soluble complex adjuvant 14 days after the second immunization. P <0.5), 7 days after the second immunization ( P <0.5) and 14 days after the second immunization ( P <0.0001), the water-adjuvanted vaccine 3 produced antibody levels significantly higher than those of the adjuvanted ISA206 vaccine.

[0054] 3. Measure the body weight of mice in each group before immunization and 7, 14, 21 and 28 days after the first immunization, observe the trend of body weight change, analyze whether the water-soluble compound adjuvant has an impact on the growth and development of mice, and necropsy the injection site and observe the local inflammatory response.

[0055] The results are as follows Figure 2 As shown, except for the water-adjuvanted vaccine group 1, whose body weight decreased slightly 7 days after the second immunization, the body weight of the other groups maintained a similar upward trend as the PBS group, indicating that the water-soluble compound adjuvant had no significant effect on the growth and development of mice.

[0056] Observation of the injection site in mice revealed ( Figure 3 (The local injection reaction diagram is compared with the antigen group alone to eliminate the influence of antigen factors.) All water adjuvant groups showed some mild inflammatory foci. Compared with the positive control group ISA206, the local reaction of the water adjuvant vaccine group 3 was the weakest, indicating that it was well absorbed and had fewer local purulent foci.

[0057] Example 12 Physical properties and long-term stability of water-soluble compound immunoadjuvants Water-soluble compound immune adjuvant 3 was selected as the final target adjuvant through Example 11. At the same time, in order to reduce the cost of adjuvant synthesis, the dosage of the immune enhancer fucoidan in the adjuvant was reduced to 10.0 mg / mL, i.e., water-soluble compound immune adjuvant 5 in Example 5. The physical properties and long-term storage stability of water-soluble compound immune adjuvant 5 (10.0 mg / mL CFP) and water-soluble compound immune adjuvant 3 (20.0 mg / mL CFP) were observed.

[0058] The results are as follows Figure 4 As shown, Figure 4 A shows that the water-soluble compound immune adjuvant 5 appears as a pale yellow, uniform, viscous, semi-transparent, fluid liquid. Figure 4 B shows that the water-soluble compound immune adjuvant 3 is a yellow, uniform, viscous, transparent, and fluid liquid. Both are dispersed in water and dissolve with slight stirring.

[0059] As shown in Table 1, water-soluble compound immunoadjuvant 5 and water-soluble compound immunoadjuvant 3 were stored at 4℃, room temperature and 37℃ for a long period of time. It was found that water-soluble compound immunoadjuvant 5 and water-soluble compound immunoadjuvant 3 were relatively stable within 6 months, and no layering, turbidity or flocculent precipitation occurred.

[0060] Table 1 Long-term stability Example 13 Immunization efficacy test of water-adjuvanted vaccines In Example 11, water-soluble compound immune adjuvant 3 was selected as the final target adjuvant. At the same time, in order to reduce the cost of adjuvant synthesis, the dosage of the immune enhancer fucoidan in the adjuvant was reduced, and the immune-enhancing effects of water-soluble compound immune adjuvant 3 and water-soluble compound immune adjuvant 5 on Pasteurella multocida were evaluated.

[0061] 1. Experimental animals: 6-8 week old SPF BALB / c mice were randomly divided into 5 groups, with 10 mice in each group, and were immunized as follows: (1) rPlpE-10.0 mg / mL CFP group: injected with the aqueous adjuvant vaccine of Example 10; (2) rPlpE-20.0 mg / mL CFP group: injected with the water adjuvant vaccine 3 of Example 8; (3) rPlpE-ISA206 group (positive control group): injected with the adjuvant ISA206 vaccine of comparison ratio 1; (4) rPlpE-PBS group (negative control group): injected with the PBS vaccine of comparison ratio 2; (5) PBS group (blank control group): 0.01 M PBS solution was injected.

[0062] 2. Each group of mice was subcutaneously injected with 200 μL of the corresponding vaccine, and immunized twice. Blood samples were collected from mice on days 7 and 14 after the first immunization to measure antibody levels. Blood samples were also collected on days 7 and 14 after the second immunization to measure antibody levels.

[0063] The results are as follows Figure 5As shown, the antibody levels in the rPlpE-20.0 mg / mL CFP group and the rPlpE-10.0 mg / mL CFP group were significantly higher than those in the negative control group. The antibody levels in the rPlpE-20.0 mg / mL CFP group were not significantly different from the positive control group 7 days, 14 days after the first immunization, and 7 days after the second immunization. The antibody levels in the rPlpE-10.0 mg / mL CFP group may have increased gradually after the second immunization due to the sustained-release effect of the main component (fucoidan can enhance the solubility of carbomer, so low concentrations of fucoidan water adjuvant may have stronger sustained-release properties), and the antibody levels 7 days after the second immunization were significantly higher than those in the positive control group. P <0.01).

[0064] 3. Antibody subtypes are a key factor in assessing immune levels. Among them, IgG1 antibody levels can reflect Th2 immunity, and IgG2a antibody levels can reflect Th1 immunity. The antibody subtype levels of each group of mice were measured.

[0065] The results are as follows Figure 7 As shown, there was no significant difference between the water-soluble compound immune adjuvant and the ISA206 adjuvant, indicating that similar antibody levels were induced. Compared with the single antigen group, the water-soluble compound immune adjuvant group induced higher levels of IgG1 and IgG2a, indicating that the water-soluble compound immune adjuvant induced both humoral immune response and a certain degree of cellular immune response.

[0066] 4. The spleen lymphocyte proliferation test is an important indicator for assessing cellular immunity. In step 2, 28 days after the first immunization, the spleen of the mouse was collected for the spleen cell proliferation test, and the supernatant secreted by the spleen lymphocytes was collected to determine the IFN-γ level.

[0067] The results are as follows Figure 6 As shown, the splenic lymphocyte proliferation level in the rPlpE-20.0 mg / mL CFP group was higher than that in the positive and negative control groups, but the proliferation level in the rPlpE-20.0 mg / mL CFP group was not significantly different from that in the positive control group. The splenic lymphocyte proliferation level in the rPlpE-10.0 mg / mL CFP group was significantly higher than that in the positive control group. P The results showed that the water-soluble compound immunoadjuvant induced a high level of splenic lymphocyte proliferation (<0.01) and a negative control group, further demonstrating that carbomer has strong sustained-release properties.

[0068] The results are as follows Figure 8As shown, both the rPlpE-20.0 mg / mL CFP group and the rPlpE-10.0 mg / mL CFP group induced high levels of IFN-γ, with the rPlpE-10.0 mg / mL CFP group inducing the highest interferon level. The IFN-γ levels induced by the rPlpE-20.0 mg / mL CFP group and the rPlpE-10.0 mg / mL CFP group were significantly higher than those in the positive control group. P <0.0001), indicating that the synthesized water-soluble complex adjuvant induced a high level of cellular immune response.

[0069] Example 14 Safety Study of Water-Soluble Compound Immunoadjuvants 1. Six- to eight-week-old SPF BALB / c mice were randomly divided into five groups of ten mice each, as described in Example 13. 28 days after immunization, serum samples were collected from each group to detect changes in biochemical indicators, and tissue sections of the heart, liver, spleen, lungs, and kidneys were collected for HE staining. The safety of the vaccine adjuvant was assessed by observing the local reaction at the injection site (the local reaction image was compared with the antigen-only group to eliminate the influence of antigen factors). At the same time, the weight changes of each group of mice were measured during the immunization cycle.

[0070] The results are as follows Figures 9-12 As shown, the trend of mouse body weight change in the formal experiment was consistent with that of the PBS group, indicating that the water-soluble compound adjuvant had no significant effect on the growth and development of mice during the immunization cycle. Furthermore, no obvious abnormalities were observed in the mice during the immunization cycle; all mice were in good health, with no cases of emaciation or death. Serum biochemical indicators showed no significant differences in AST, ALT, LDH, ALP, and UREA levels between the groups and the PBS group, and all were within the normal range. Histological sections showed no abnormalities in the vaccine adjuvant groups compared to the PBS group. Observation of local reactions at the subcutaneous injection site revealed that the side effects of the water-adjuvant vaccine 5 and water-adjuvant vaccine 3 were less than those of the adjuvant ISA206 vaccine. The immunostimulatory side effects of water-adjuvant vaccine 3 were the least compared to water-adjuvant vaccine 5 and adjuvant ISA206 vaccine.

[0071] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A water-soluble compound immune adjuvant, characterized in that: The water-soluble compound immune adjuvant includes carbomer, poloxamer, and an immune enhancer; In the water-soluble compound immunoadjuvant, the concentration of carbomer is 1.0~20.0 mg / mL, the concentration of poloxamer is 0.1~2.0 mg / mL, and the concentration of the immunostimulant is 1.0~40.0 mg / mL. The carbomer is any one of carbomer 974, carbomer 940, carbomer 971, carbomer 934, carbomer 980 and carbomer 941; The poloxamer is either poloxamer 188 or poloxamer 407; The immune enhancer is either cimetidine or fucoidan.

2. The water-soluble compound immunoadjuvant according to claim 1, characterized in that: In the water-soluble compound immunoadjuvant, the concentration of carbomer is 5.0~10.0 mg / mL, the concentration of poloxamer is 1.0 mg / mL, and the concentration of the immunostimulant is 10.0~20.0 mg / mL.

3. The water-soluble compound immunoadjuvant according to claim 2, characterized in that: In the water-soluble compound immunoadjuvant, the concentration of carbomer 974 is 5.0 mg / mL, the concentration of poloxamer 188 is 1.0 mg / mL, and the concentration of fucoidan is 20.0 mg / mL.

4. A method for preparing the water-soluble complex immune adjuvant according to any one of claims 1 to 3, characterized in that: Includes the following steps: (1) Dissolve carbomer in water, adjust the pH to 6.8-7.0, and sterilize to obtain a carbomer solution; (2) Dissolve the immune enhancer in a solvent to prepare an immune enhancer solution; (3) Dissolve poloxamer in water to obtain a poloxamer solution; (4) Mix carbomer solution, immune enhancer solution and poloxamer solution evenly to obtain water-soluble compound immune adjuvant.

5. The preparation method according to claim 4, characterized in that: In step (1), the concentration of the carbomer solution is 10.0~20.0 mg / mL; In step (2), the concentration of the immune enhancer solution is 12.5~50.0 mg / mL; In step (3), the concentration of the poloxamer solution is 10.0~20.0 mg / mL.

6. The preparation method according to claim 4, characterized in that: When the immune enhancer is cimetidine, the solvent is a 0.1-0.3 M hydrochloric acid solution, and the pH of the immune enhancer solution is adjusted to 6.8-7.0; When the immune enhancer is fucoidan, the solvent is water.

7. The use of the water-soluble composite adjuvant according to any one of claims 1 to 3 in the preparation of an aqueous adjuvant vaccine.

8. An immunizing aqueous adjuvant vaccine, characterized in that: The water-adjuvanted vaccine comprises the water-soluble compound adjuvant and antigen as described in any one of claims 1 to 3; The antigen is Pasteurella multocida PlpE protein; The nucleotide sequence of the protein PlpE is shown in SEQ ID NO: 1; In the aforementioned aqueous adjuvant vaccine, the concentration of the antigen is 400.0~600.0 μg / mL.

9. The aqueous adjuvant vaccine according to claim 8, characterized in that: In the aforementioned aqueous adjuvant vaccine, the concentration of antigen is 500.0 μg / mL.

10. A method for preparing an aqueous adjuvant vaccine according to any one of claims 8 to 9, characterized in that: The specific steps are as follows: at a speed of 400~600 r / min, the antigen is added to the water-soluble compound adjuvant and stirred for 5~8 minutes to obtain the water-adjuvanted immune vaccine.

Images