Oil-in-water nanoemulsion adjuvant, pasteurella nanoemulsion vaccine and preparation method and application thereof

Abstract

The application discloses an oil-in-water nanoemulsion adjuvant, a pasteurella nanoemulsion vaccine and a preparation method and application thereof. According to mass percentage, raw materials of the nanoemulsion adjuvant include 1.76-32.42% surfactants, 0.72-27.41% co-surfactants, 1.56-23.94% oil phases and the rest water phases. The oil-in-water nanoemulsion adjuvant of the application is clear and transparent, the particle size is between 1-100 nm, and the vaccine can be prepared by uniformly mixing the antigen, without complex process. After being injected into the animal body, the vaccine is easy to be absorbed. The synthesized nanoemulsion vaccine has good fluidity, high safety, can produce high level of humoral immunity, and has good application prospect.

Description

Technical Field

[0001] This invention relates to the field of bioimmunotechnology, specifically to an oil-in-water nanoemulsion adjuvant, a Pasteurella nanoemulsion vaccine, its preparation method, and its application. Background Technology

[0002] Conventional oil-emulsion adjuvants used in production often induce a series of adverse reactions and have side effects such as poor absorption by the body and high irritation to the body after immunization. Researching an adjuvant that is easily absorbed by the body and can enhance the body's immunity is currently needed in the market. Adjuvant systems have been shown to be able to resist antigen degradation and promote antigen delivery to antigen-presenting cells. Nanoemulsions are colloidal systems composed of oil, water and surfactants. Nanoemulsion droplets are small in size and can not only serve as antigen delivery carriers, but also enhance the solubility and stability of drugs.

[0003] As an antigen delivery system, nanoemulsion adjuvants often involve the antigen being adsorbed onto the surface of the nanoemulsion as an outer aqueous phase, with some antigen dispersed in the solvent. This adsorption mode makes it easier for antigen-presenting cells to absorb the antigen and can stimulate an immune response more quickly. The particle size of the emulsion is an important factor affecting the transport of vaccines in vivo and their phagocytosis by antigen-presenting cells. Nanoparticles smaller than 100 nm are more likely to act on immune lymphocytes through lymphatic vessels, thereby enhancing the immune effect.

[0004] Nanoemulsion adjuvants can be composed of surfactants, co-surfactants, an oil phase, and an aqueous phase. Surfactants reduce interfacial tension, allowing two immiscible liquids to form droplets. The lower the interfacial tension, the smaller the droplet size. Given the high toxicity of anionic and cationic surfactants to organisms, nonionic surfactants are chosen as components of nanoemulsions. Tweens and spans are widely used in nanoemulsion synthesis. Co-surfactants assist surfactants in reducing the tension at the oil-water interface, thereby forming stable nanodroplets. Alcohols are commonly used as co-surfactants, such as propylene glycol, polyethylene glycol 400, anhydrous ethanol, glycerol, n-butanol, and ethylene glycol. The oil phase, which generally has high drug solubility, is typically chosen. Commonly used oil phases include white oil, isopropyl myristate, isopropyl palmitate, oleic acid, fat-soluble vitamins, and vegetable oils.

[0005] With the extensive research on water-soluble immune enhancers, many formulations no longer use water as the aqueous phase of microemulsions. Instead, they select immune enhancers that can enhance immune responses as the aqueous phase for synthesizing nanoemulsions. Immune enhancers are generally classified into biological, chemical, and traditional Chinese medicine polysaccharide categories. Biological immune enhancers include cytokines, thymic hormones, bacterial and bacterial cell components, peptides and complexes, and nucleic acid preparations. However, biological immune enhancers are often very expensive and generally inconvenient for practical production. Chemical immune enhancers include levamisole, cimetidine, dioctadecyl dimethyl ammonium bromide, isoprotinin, and imiquimod. Polysaccharide immune enhancers include astragalus polysaccharides, wolfberry polysaccharides, β-glucan, fucoidan, lentinan, ganoderma lucidum polysaccharides, and chitosan. Using immune enhancers as the aqueous phase of nanoemulsions can enhance both the solubility of the immune enhancers and the immune-enhancing effect of the delivery system. Researching an adjuvant that can reduce animal stress and side effects while simultaneously stimulating cellular and humoral immunity is a current research direction.

[0006] Based on a comprehensive analysis of existing technologies, the following shortcomings still exist in the field of nanoemulsion adjuvants: (1) Existing conventional oil emulsion adjuvants mainly enhance humoral immune responses through sustained release and reservoir effects, but their effects on stimulating cellular immunity are limited, making it difficult to simultaneously meet the dual needs of humoral and cellular immunity; (2) Traditional mineral oil adjuvants have safety issues such as incomplete absorption at the injection site and local residues causing tissue damage; (3) The preparation process of some existing nanoemulsion adjuvants is complex and requires large equipment such as high-pressure homogenizers, which is not conducive to grassroots promotion and application; (4) Some nanoemulsion systems have poor stability during storage and are prone to phenomena such as layering and turbidity, which affect the shelf life and effectiveness of vaccines.

[0007] Therefore, developing a nanoemulsion adjuvant that is simple to prepare, safe, stable, and can simultaneously stimulate humoral and cellular immunity has significant theoretical and practical value. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an oil-in-water nanoemulsion adjuvant, a Pasteurella nanoemulsion vaccine, its preparation method, and its application. Compared with conventional oil-emulsion adjuvants, the oil-in-water nanoemulsion adjuvant of this invention adds an immunostimulant that can induce cellular immunity to the basis of conventional emulsions, aiming to simultaneously enhance the cellular and humoral immunity of the vaccine adjuvant. Furthermore, from a nanoscale perspective, nanoparticles are more easily absorbed by the body, resulting in fewer local side effects.

[0009] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides an oil-in-water nanoemulsion adjuvant, wherein, by mass percentage, the raw materials of the nanoemulsion adjuvant include 1.76~32.42% surfactant, 0.72~27.41% co-surfactant, 1.56~23.94% oil phase and the balance being an aqueous phase; the particle size of the nanoemulsion adjuvant is 1~100 nm. The surfactant is either Tween 60 or Tween 80; The co-surfactant is either polyethylene glycol 400 or glycerol; The oil phase is either vitamin E or squalene; The aqueous phase is either a cimetidine solution or a fucoidan solution.

[0010] Furthermore, the mass ratio of the surfactant to the co-surfactant is 1 to 3:1; The surfactant and co-surfactant form a surfactant-co-surfactant mixture, and the mass ratio of the surfactant-co-surfactant mixture to the oil phase is 2~9:1; The concentration of the cimetidine solution is 10.83~21.67 mg / mL, the pH of the cimetidine solution is 6.8~7.0, and the final concentration of cimetidine in the nanoemulsion adjuvant is 5.0~10.0 mg / mL. The concentration of the fucoidan solution is 21.67~43.33 mg / mL, and the final concentration of fucoidan in the nanoemulsion adjuvant is 10.0~20.0 mg / mL.

[0011] Furthermore, by mass percentage, the raw materials of the nanoemulsion adjuvant include 30.3% Tween 80, 15.15% polyethylene glycol 400, 9.09% vitamin E and 45.46% fucoidan solution; The concentration of the fucoidan solution was 43.33 mg / mL, and the final concentration of fucoidan in the nanoemulsion adjuvant was 20.0 mg / mL.

[0012] The present invention also provides a method for preparing the aforementioned nanoemulsion adjuvant, comprising the following steps: (1) Weigh out the surfactant, co-surfactant, oil phase and aqueous phase according to the above mass percentages; (2) Mix the surfactant and co-surfactant to obtain a surfactant-co-surfactant mixture; (3) Add the oil phase to the surface additive mixture, stir and emulsify to obtain an oil phase mixture; (4) Under stirring conditions, the aqueous phase is added dropwise to the oil phase mixture, and stirring is continued to obtain an oil-in-water nanoemulsion adjuvant.

[0013] Further, in step (1), when the aqueous phase is a cimetidine solution, the method for preparing the cimetidine solution is as follows: dissolve cimetidine in 0.1~0.3 M hydrochloric acid solution, adjust its pH to 6.8~7.0, and filter it using a 0.22 μm filter to obtain a cimetidine solution of 10.83~21.67 mg / mL; When the aqueous phase is a fucoidan solution, the fucoidan is prepared by dissolving fucoidan in water and filtering it through a 0.22 μm filter to obtain a fucoidan solution with a concentration of 21.67~43.33 mg / mL. In step (3), the stirring speed for emulsification is 400~600 r / min, and the stirring time for emulsification is 15~30 min; In step (4), the stirring speed is 400~600 r / min, and the stirring time is 30~45 min.

[0014] Furthermore, in step (1), the concentration of the hydrochloric acid solution is 0.15 M; In step (3), the stirring speed for emulsification is 500 r / min, and the stirring time for emulsification is 15 min. In step (4), the stirring speed is 500 r / min, and the stirring time is 30 min.

[0015] The present invention also provides the application of the nanoemulsion adjuvant described herein in delivering antigens to cells or in preparing nanoemulsion vaccines.

[0016] The present invention also provides a Pasteurella nanoemulsion vaccine, wherein the nanoemulsion vaccine comprises the aforementioned nanoemulsion adjuvant and Pasteurella antigen; wherein the antigen is protein PlpE; The concentration of the protein PlpE is 1.0~2.0 mg / mL, and the volume ratio of protein PlpE to nanoemulsion adjuvant is 1:1~3.

[0017] Furthermore, the concentration of the protein PlpE is 1.0 mg / mL, the volume ratio of protein PlpE to nanoemulsion adjuvant is 1:1, and the nucleotide sequence of the protein PlpE is shown in SEQ ID NO: 1.

[0018] The present invention also provides a method for preparing the nanoemulsion vaccine, wherein the preparation process is as follows: the nanoemulsion adjuvant and Pasteurella antigen are mixed according to the above volume ratio, and stirred at 400~600 r / min for 10~20 min to obtain the Pasteurella nanoemulsion vaccine.

[0019] The beneficial effects of this invention are: 1. The water-in-oil nanoemulsions of the present invention have a particle size mostly between 1 and 100 nm, which makes them easier to be absorbed and presented by antigen-presenting cells.

[0020] 2. The water-in-oil nanoemulsion synthesized in this invention is relatively stable at both 4°C and room temperature. After being placed at 4°C and room temperature for 3 months, no stratification, turbidity, or flocculent precipitation was observed, and its particle size and particle size dispersibility index (PDI) showed no significant changes.

[0021] 3. Pasteurella nanoemulsion vaccines formulated with oil-in-water nanoemulsions are easy to inject, have good fluidity, and are easier to inject and absorb than commercial mineral oil adjuvants. They also have good safety, and the vaccine is absorbed more completely after injection with no residue.

[0022] 4. The nanoemulsion adjuvant of the present invention can induce a higher level of humoral immune response than commercial adjuvants, and also has the ability to induce cellular immune response.

[0023] 5. The preparation process of this invention is simple and does not require complicated processes or large instruments. When preparing the vaccine, the oil phase and water phase are simply stirred and mixed evenly. Attached Figure Description

[0024] Figure 1 To fix the co-surfactant (polyethylene glycol 400) and Km value (surfactant to co-surfactant ratio of 2:1), the effect of surfactants Tween 60 and Tween 80 on the formation of nanoemulsion adjuvants is shown in the figure. In the figure, A is the pseudo-ternary phase diagram formed when Tween 60 is used as a surfactant, B is the pseudo-ternary phase diagram formed when Tween 80 is used as a surfactant, and the shaded area is the O / W nanoemulsion adjuvant region formed. Figure 2 Tyndall effect diagram of Tween 80 and co-surfactant with fixed Km value (2:1); Figure 3 The pseudo-ternary phase diagrams formed by Tween 80 and polyethylene glycol 400 at Km=1∶1, Km=2∶1 and Km=3∶1; In the figure, A is the pseudo-ternary phase diagram formed when Km=1∶1, B is the pseudo-ternary phase diagram formed when Km=2∶1, C is the pseudo-ternary phase diagram formed when Km=3∶1, and the shaded area is the O / W nanoemulsion adjuvant region. Figure 4 The physical properties of the synthesized cimetidine nanoemulsion adjuvant and fucoidan nanoemulsion adjuvant are shown in the diagram. Figure 5 This is a graph showing the antibody levels in mice in a preliminary experiment using cimetidine nanoemulsion adjuvant combined with fucoidan nanoemulsion adjuvant and Pasteurella PlpE antigen. Figure 6The figure shows the results of the preliminary experiment on the effects of cimetidine nanoemulsion vaccine and fucoidan nanoemulsion vaccine on the changes in mouse body weight. Figure 7 The figure shows the results of the preliminary experiment on the effects of cimetidine nanoemulsion vaccine and fucoidan nanoemulsion vaccine on absorption and residue at the injection site in mice. In the figure, 1 represents the PlpE + 10.0 mg / mL cimetidine nanoemulsion adjuvant group, 2 represents the PlpE + 20.0 mg / mL fucoidan nanoemulsion adjuvant group, 3 represents the PlpE + ISA206 group, and 4 represents the PlpE + PBS group; the yellow arrows indicate unabsorbed vaccine adjuvant or abscesses. Figure 8 Particle size distribution and microstructure of fucoidan nanoemulsion adjuvant; In the figure, A and C represent 10.0 mg / mL Fu-NE, and B and D represent 20.0 mg / mL Fu-NE. Figure 9 The particle size and PDI changes of fucoidan nanoemulsion adjuvants were observed over 3 months at 4°C and room temperature. In the figure, A and C are the changes in particle size and PDI at 4°C, and B and D are the changes in particle size and PDI at room temperature. Figure 10 This is a graph showing the changes in antibody levels in mice after the fucoidan nanoemulsion vaccine was tested in a formal trial. Figure 11 This is a graph showing the changes in antibody subtype levels of the fucoidan nanoemulsion vaccine in mice during a formal trial. Figure 12 This is a graph showing the effect of fucoidan nanoemulsion vaccine on the proliferation level of spleen lymphocytes in mice during a formal experiment. Figure 13 This is a graph showing the results of IFN-γ levels measured in the formal experiment; Figure 14 The figure shows the effect of the fucoidan nanoemulsion vaccine on the change in body weight in mice during a safety testing experiment. Figure 15 The graph shows the changes in serum biochemical parameters of mice after immunization with the nanoemulsion vaccine in a safety testing experiment. Figure 16 The figure shows the results of the effects of the nanoemulsion vaccine on the major organs of mice after immunization in a safety testing experiment. Detailed Implementation

[0025] 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.

[0026] Experimental materials description: Cimetidine (99%), Fucoidan (≥98%): Shanghai Aladdin Biochemical Technology Co., Ltd.; Vitamin E (97%): Shanghai Maclean Biochemical Technology Co., Ltd.; Twain 80: Anhui Hefei White Shark Biotechnology Co., Ltd.; Twain 60: Shanghai Aladdin Biochemical Technology Co., Ltd.; Span 85, Span 80: Shanghai Aladdin Biochemical Technology Co., Ltd.; Polyethylene glycol 400 (pharmaceutical grade): Shanghai Aladdin Biochemical Technology Co., Ltd.; Anhydrous ethanol, isopropanol, glycerol: Sinopharm Chemical Reagent Co., Ltd.; ISA206 adjuvant: Seppic, France; The nucleotide sequence of Pasteurella PlpE protein is shown in SEQ ID NO: 1.

[0027] Example 1 Screening of blank nanoemulsion adjuvant formulations I. Screening of Surfactants and Co-surfactants Vitamin E (α-tocopherol) was selected as the oil phase of the nanoemulsion. α-Tocopherol is a fat-soluble antioxidant, a type of vitamin E, and has an immune-enhancing effect. With the addition of α-tocopherol, the number of neutrophils, eosinophils, and monocytes induced by squalene adjuvant to drain into the lymph nodes significantly increased. The safe nonionic surfactants Tween 60 and Tween 80 were used as surfactants for screening nanoemulsion adjuvants. Co-surfactants were screened from polyethylene glycol 400, glycerol, anhydrous ethanol, and isopropanol.

[0028] 1. In this embodiment, the surfactant is Tween 60 or Tween 80, and the co-surfactant is polyethylene glycol 400, glycerol, anhydrous ethanol or isopropanol. The oil phase in this embodiment is vitamin E, and the aqueous phase is ultrapure water. The surfactant and co-surfactant are mixed in a mass ratio (Km value) of 2:1 to obtain a surfactant-co-surfactant mixture.

[0029] 2. Weigh the adjuvant mixture and oil phase according to the mass ratios of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9 respectively. Mix them thoroughly and stir. Add ultrapure water dropwise to the mixture using the water titration method. When a clear and transparent system with Tyndall effect is formed, it is preliminarily determined that a blank nanoemulsion adjuvant has been formed. The specific formulation is shown in Table 1.

[0030] Table 1 Formulations for screening surfactants and co-surfactants 3. Based on the area size formed by the pseudo-ternary phase diagram, select the optimal ratio of surfactant and co-surfactant, as well as the ratio of the surfactant-co-surfactant mixture to the oil phase. The pseudo-ternary phase diagram is divided into three points, with each vertex representing the aqueous phase, oil phase, and surfactant-co-surfactant mixture, respectively. The Km value is fixed at 2:1 (mass ratio of surfactant to co-surfactant). Record the mass of aqueous phase added when the solution in each formulation becomes clear, and calculate the mass ratio of each vertex to the total mixture to draw the pseudo-ternary phase diagram.

[0031] Experiments revealed that anhydrous ethanol and isopropanol, when used as co-surfactants, could not form a clear system. Glycerol could only form a clear blank nanoemulsion adjuvant when the ratio of the surfactant mixture to the oil phase was 9:1. Polyethylene glycol 400 could form a clear blank nanoemulsion adjuvant when the ratio of the surfactant mixture to the oil phase was 9:1, 8:2, 7:3, and 6:4, and exhibited the Tyndall effect when irradiated with laser. Figure 2 As shown.

[0032] When the co-surfactant is polyethylene glycol 400, the effect of the surfactant on the formation of nanoemulsion adjuvants is as follows: Figure 1 As shown, the optimal surfactant is Tween 80.

[0033] II. Km Filtering Based on the screened surfactant Tween 80 and co-surfactant polyethylene glycol 400, with vitamin E as the oil phase and ultrapure water as the aqueous phase, Km (mass ratio of surfactant to co-surfactant) was further screened to determine the optimal formulation ratio of the blank nanoemulsion adjuvant. The following formulation was set: Formula 1: Km = 1:1, surface additive mixture: oil phase = 9:1, Tween 80 + polyethylene glycol 400 (PEG-400) = 1.08 g, vitamin E = 0.12 g, aqueous phase (average of three replicates) = 2.0712 g.

[0034] Formula 2: Km = 1:1, surface additive mixture: oil phase = 8:2, Tween 80 + PEG-400 = 0.96 g, vitamin E = 0.24 g, and the resulting aqueous phase = 0.551 g.

[0035] Formula 3: Km = 1:1, surface additive mixture: oil phase = 7:3, Tween 80 + PEG-400 = 0.84 g, vitamin E = 0.36 g, and the resulting aqueous phase = 0.446 g.

[0036] Formula 4: Km = 1:1, surface additive mixture: oil phase = 6:4, Tween 80 + PEG-400 = 0.72 g, vitamin E = 0.48 g, aqueous phase = 19.228 g.

[0037] Formula 5: Km = 2:1, surface additive mixture: oil phase = 9:1, Tween 80 + PEG-400 = 1.08 g, vitamin E = 0.12 g, aqueous phase = 3.0726 g.

[0038] Formula 6: Km = 2:1, surface additive mixture: oil phase = 8:2, Tween 80 + PEG-400 = 0.96 g, vitamin E = 0.24 g, aqueous phase = 0.8073 g.

[0039] Formula 7: Km = 2:1, surface additive mixture: oil phase = 7:3, Tween 80 + PEG-400 = 0.84 g, vitamin E = 0.36 g, aqueous phase = 0.8884 g.

[0040] Formula 8: Km = 2:1, surface additive mixture: oil phase = 6:4, Tween 80 + PEG-400 = 0.72 g, vitamin E = 0.48 g, aqueous phase = 0.8048 g.

[0041] Formula 9: Km = 3:1, surface additive mixture: oil phase = 9:1, Tween 80 + PEG-400 = 1.08 g, vitamin E = 0.12 g, aqueous phase = 2.1026 g.

[0042] Formula 10: Km = 3:1, surface additive mixture: oil phase = 8:2, Tween 80 + PEG-400 = 0.96 g, vitamin E = 0.24 g, aqueous phase = 1.0211 g.

[0043] Formula 11: Km = 3:1, surface additive mixture: oil phase = 7:3, Tween 80 + PEG-400 = 0.84 g, Vitamin E = 0.36 g, aqueous phase = 1.1881 g.

[0044] Formula 12: Km = 3:1, surface additive mixture: oil phase = 6:4, Tween 80 + PEG-400 = 0.72 g, vitamin E = 0.48 g, aqueous phase = 1.1905 g.

[0045] Formula 13: Km = 3:1, surface additive mixture: oil phase = 5:5, Tween 80 + PEG-400 = 0.60 g, vitamin E = 0.60 g, aqueous phase = 19.6801 g.

[0046] Draw a pseudo-ternary phase diagram according to the above formula ratio, such as... Figure 3As shown, Km=1∶1 ( Figure 3 A) The area occupied by the phase diagram is 9.17%, Km=2∶1 ( Figure 3 B) The area occupied by the phase diagram is 9.88%, Km=3∶1 ( Figure 3 C) The area occupied by the phase diagram is 8.65%. The proportion with the largest phase diagram area is selected as the optimal formulation ratio, that is, Km=2∶1 is the optimal mass ratio of surfactant and co-surfactant.

[0047] III. Screening of the ratio of surface additive mixture to oil phase Using Tween 80 as the surfactant and polyethylene glycol 400 as the co-surfactant, with a Km ratio of 2:1, vitamin E as the oil phase and ultrapure water as the aqueous phase, the optimal mass ratio of the surfactant-co-surfactant mixture to the oil phase was screened from 2 to 9:1. The optimal ratio was determined by measuring the particle size and particle size dispersion index of the nanoemulsion adjuvant synthesized in each ratio, with priority given to nanoemulsion adjuvants with the highest oil phase content and the lowest particle size dispersion index. The following formulation was established: Formula 1: Km = 2:1, surface additive mixture: oil phase = 9:1, Tween 80 + PEG-400 = 1.08 g, vitamin E = 0.12 g, aqueous phase = 3.0726 g.

[0048] Formula 2: Km = 2:1, surface additive mixture: oil phase = 8:1, Tween 80 + PEG-400 = 1.07 g, vitamin E = 0.13 g, aqueous phase = 2.8930 g.

[0049] Formula 3: Km = 2:1, surface additive mixture: oil phase = 7:1, Tween 80 + PEG-400 = 1.05 g, vitamin E = 0.15 g, aqueous phase = 2.0518 g.

[0050] Formula 4: Km = 2:1, surface additive mixture: oil phase = 6:1, Tween 80 + PEG-400 = 1.03 g, vitamin E = 0.17 g, aqueous phase = 1.5448 g.

[0051] Formula 5: Km = 2:1, surface additive mixture: oil phase = 5:1, Tween 80 + PEG-400 = 1.00 g, vitamin E = 0.20 g, aqueous phase = 1.1847 g.

[0052] Formula 6: Km = 2:1, surface additive mixture: oil phase = 4:1, Tween 80 + PEG-400 = 0.96 g, vitamin E = 0.24 g, aqueous phase = 0.8073 g.

[0053] Formula 7: Km = 2:1, surface additive mixture: oil phase = 3:1, Tween 80 + PEG-400 = 0.90 g, vitamin E = 0.30 g, aqueous phase = 1.1884 g.

[0054] Formula 8: Km = 2:1, surface additive mixture: oil phase = 2:1, Tween 80 + PEG-400 = 0.80 g, vitamin E = 0.40 g, aqueous phase = 1.1376 g.

[0055] The results are shown in Table 2. When the mass ratio of the surface additive mixture to the oil phase is 5:1, the PDI is the lowest, the proportion of the oil phase is the highest, and the particle size is also relatively small. Therefore, 5:1 is the optimal mass ratio of the surface additive mixture to the oil phase.

[0056] Table 2. Particle size and PDI of the mixture and oil phase at different proportions. Example 2 Preparation of Cimetidine Nanoemulsion Adjuvant 1 1. Dissolve cimetidine powder in 0.15 M hydrochloric acid solution, adjust the pH to 6.8-7.0 with 1 M sodium hydroxide solution, filter through a 0.22 μm filter to obtain a cimetidine solution of 10.83 mg / mL.

[0057] 2. Weigh out 9.09% of the oil phase vitamin E, 30.30% of the surfactant Tween 80, 15.15% of the co-surfactant polyethylene glycol 400, and 45.46% of the aqueous phase according to the mass percentage. In this example, the aqueous phase is a 10.83 mg / mL cimetidine solution.

[0058] 3. Mix the surfactant Tween 80 and the co-surfactant polyethylene glycol 400 evenly to obtain a surfactant-co-surfactant mixture.

[0059] 4. Slowly add vitamin E to the additive mixture and emulsify thoroughly for 15 minutes at 500 r / min on a magnetic stirrer to obtain an oil phase mixture.

[0060] 5. The prepared cimetidine solution was slowly added dropwise to the oil phase mixture at a speed of 500 r / min on a magnetic stirrer. The solution became clear and transparent. Stirring was continued for 30 min. The final concentration of cimetidine in the nanoemulsion adjuvant was 5.0 mg / mL, and cimetidine nanoemulsion adjuvant 1 (5.0 mg / mL cimetidine nanoemulsion adjuvant, 5.0 mg / mL Ci-NE) was obtained.

[0061] Example 3 Preparation of Cimetidine Nanoemulsion Adjuvant 2 1. Dissolve cimetidine powder in 0.15 M hydrochloric acid solution, adjust the pH to 6.8-7.0 with 1 M sodium hydroxide solution, filter through a 0.22 μm filter to obtain a cimetidine solution of 21.67 mg / mL.

[0062] 2. Weigh out 9.09% of the oil phase vitamin E, 30.30% of the surfactant Tween 80, 15.15% of the co-surfactant polyethylene glycol 400, and 45.46% of the aqueous phase according to the mass percentage. In this example, the aqueous phase is a 21.67 mg / mL cimetidine solution.

[0063] 3. Mix the surfactant Tween 80 and the co-surfactant polyethylene glycol 400 evenly to obtain a surfactant-co-surfactant mixture.

[0064] 4. Slowly add vitamin E to the additive mixture and emulsify thoroughly for 15 minutes at 500 r / min on a magnetic stirrer to obtain an oil phase mixture.

[0065] 5. Using a magnetic stirrer, slowly add the prepared cimetidine solution dropwise to the oil phase mixture at a speed of 500 r / min. The solution becomes clear and transparent. Continue stirring for 30 min. The final concentration of cimetidine in the nanoemulsion adjuvant is 10.0 mg / mL, thus obtaining cimetidine nanoemulsion adjuvant 2 (10.0 mg / mL cimetidine nanoemulsion adjuvant, 10.0 mg / mL CTi-NE).

[0066] Example 4 Preparation of Fucoidan Nanoemulsion Adjuvant 1 1. Dissolve fucoidan powder in water and filter through a 0.22 μm filter to obtain a fucoidan solution of 21.67 mg / mL.

[0067] 2. Weigh out 9.09% of the oil phase vitamin E, 30.30% of the surfactant Tween 80, 15.15% of the co-surfactant polyethylene glycol 400, and 45.46% of the aqueous phase according to the mass percentage. In this example, the aqueous phase is a 21.67 mg / mL fucoidan solution.

[0068] 3. Mix the surfactant Tween 80 and the co-surfactant polyethylene glycol 400 evenly to obtain a surfactant-co-surfactant mixture.

[0069] 4. Slowly add vitamin E to the additive mixture and emulsify thoroughly for 15 minutes at 500 r / min on a magnetic stirrer to obtain an oil phase mixture.

[0070] 5. Using a magnetic stirrer, slowly add the prepared fucoidan solution dropwise to the oil phase mixture at a speed of 500 r / min. The solution becomes clear and transparent. Continue stirring for 30 min. The final concentration of fucoidan in the nanoemulsion adjuvant is 10.0 mg / mL, thus obtaining fucoidan nanoemulsion adjuvant 1 (10.0 mg / mL fucoidan nanoemulsion adjuvant, 10.0 mg / mL Fu-NE).

[0071] Example 5 Preparation of Fucoidan Nanoemulsion Adjuvant 2 1. Dissolve fucoidan powder in water and filter through a 0.22 μm filter to obtain a fucoidan solution with a concentration of 43.33 mg / mL.

[0072] 2. Weigh out 9.09% of the oil phase vitamin E, 30.30% of the surfactant Tween 80, 15.15% of the co-surfactant polyethylene glycol 400, and 45.46% of the aqueous phase according to the mass percentage. In this example, the aqueous phase is a fucoidan solution with a concentration of 43.33 mg / mL.

[0073] 3. Mix the surfactant Tween 80 and the co-surfactant polyethylene glycol 400 evenly to obtain a surfactant-co-surfactant mixture.

[0074] 4. Slowly add vitamin E to the additive mixture and emulsify thoroughly for 15 minutes at 500 r / min on a magnetic stirrer to obtain an oil phase mixture.

[0075] 5. Using a magnetic stirrer, slowly add the prepared fucoidan solution dropwise to the oil phase mixture at a speed of 500 r / min. The solution becomes clear and transparent. Continue stirring for 30 min. The final concentration of fucoidan in the nanoemulsion adjuvant is 20.0 mg / mL, thus obtaining fucoidan nanoemulsion adjuvant 2 (20.0 mg / mL fucoidan nanoemulsion adjuvant, 20.0 mg / mL Fu-NE).

[0076] The physical properties of cimetidine nanoemulsion adjuvant 1, cimetidine nanoemulsion adjuvant 2, fucoidan nanoemulsion adjuvant 1, and fucoidan nanoemulsion adjuvant 2 are as follows: Figure 4 As shown, the synthesized nanoemulsion adjuvants exhibited a transparent and uniform appearance. The 5.0 mg / mL and 10.0 mg / mL cimetidine nanoemulsion adjuvants presented a transparent and colorless system, the 10.0 mg / mL fucoidan nanoemulsion presented a pale yellow, clear and transparent system, and the 20.0 mg / mL fucoidan nanoemulsion presented a yellow, clear and transparent system.

[0077] The particle size and particle size dispersion index of cimetidine nanoemulsion adjuvant 1, cimetidine nanoemulsion adjuvant 2, fucoidan nanoemulsion adjuvant 1 and fucoidan nanoemulsion adjuvant 2 were detected, and the results are shown in Table 3. The particle sizes of the four nanoemulsion adjuvants prepared in this invention are all within the range of 1~100 nm.

[0078] Table 3. Particle size and particle size dispersion index of nanoemulsion adjuvants Example 6 Preparation of Cimetidine Nanoemulsion Vaccine 1 The 5.0 mg / mL cimetidine nanoemulsion adjuvant (prepared and used immediately) from Example 2 was mixed with 1.0 mg / mL antigen protein PlpE at a volume ratio of 1:1 and stirred for 10 min (500 r / min) to obtain cimetidine nanoemulsion vaccine 1.

[0079] Example 7 Preparation of Cimetidine Nanoemulsion Vaccine 2 The 10.0 mg / mL cimetidine nanoemulsion adjuvant (prepared and used immediately) from Example 3 was mixed with 1.0 mg / mL antigen protein PlpE at a volume ratio of 1:1 and stirred for 10 min (500 r / min) to obtain cimetidine nanoemulsion vaccine 2.

[0080] Example 8 Preparation of Fucoidan Nanoemulsion Vaccine 1 The 10.0 mg / mL fucoidan nanoemulsion adjuvant (prepared and used immediately) from Example 4 was mixed with the 1.0 mg / mL antigen protein PlpE at a volume ratio of 1:1 and stirred for 10 min (500 r / min) to obtain fucoidan nanoemulsion vaccine 1.

[0081] Example 9 Preparation of Fucoidan Nanoemulsion Vaccine 2 The 20.0 mg / mL fucoidan nanoemulsion adjuvant (prepared and used immediately) from Example 5 was mixed with the 1.0 mg / mL antigen protein PlpE at a volume ratio of 1:1 and stirred for 10 min (500 r / min) to obtain fucoidan nanoemulsion vaccine 2.

[0082] Comparative Example 1 Preparation of commercial nanoemulsion vaccines ISA206 was mixed with 1.0 mg / mL of antigen protein PlpE at a volume ratio of 1:1 and ground in a tissue homogenizer at a frequency of 60 Hz for 2 min with a 15 s pause in between to prepare a commercial nanoemulsion vaccine.

[0083] Comparative Example 2 Preparation of blank vaccine A blank vaccine was obtained by mixing 0.01 M PBS with 1.0 mg / mL antigen protein PlpE at a volume ratio of 1:1.

[0084] Example 10 Immunopotency test 1. BALB / c mice (30 mice): SPF grade, 6-8 weeks old, weighing 16-21 g, female, 6 mice per group. The experiment was divided into five groups: (1) PlpE + 10.0 mg / mL cimetidine nanoemulsion adjuvant group: cimetidine nanoemulsion vaccine 2; (2) PlpE + 20.0 mg / mL fucoidan nanoemulsion adjuvant group: fucoidan nanoemulsion vaccine 2; (3) PlpE+ISA206 group: commercial nanoemulsion vaccine; (4) PlpE+PBS group: blank vaccine; (5) PBS group: 0.01 M PBS.

[0085] 2. The immunization method was subcutaneous injection at multiple points on the back of mice, with an immunization dose of 200 μL. The first immunization was performed on day 0, and a second booster immunization was performed on day 14. Blood samples were collected from the orbital venous plexus of mice in each group before immunization, 7 days after the first immunization, 14 days after the first immunization, 7 days after the second immunization, and 14 days after the second immunization. Serum samples were collected to measure the IgG antibody levels in each group.

[0086] The results are as follows Figure 5 As shown, the antibody levels produced after the combined application of antigen and nanoemulsion adjuvant were higher than those in the blank vaccine group, and the antibody levels produced in the PlpE + 20.0 mg / mL fucoidan nanoemulsion adjuvant group (20.0 mg / mL Fu-NE) were higher 7 days after the first immunization. P <0.5), 14 days after the first vaccination ( P <0.001) and 7 days after the second immunization ( P The antibody levels in the 20.0 mg / mL Fu-NE group were significantly higher than those in the PlpE + 10.0 mg / mL cimetidine nanoemulsion adjuvant group (10.0 mg / mL Ci-NE), and the antibody levels in the 20.0 mg / mL Fu-NE group were slightly higher than those in the PlpE + ISA206 group.

[0087] Example 11 Evaluation of the effects of nanoemulsion vaccine on growth and development and local safety in mice during preliminary trials. In Example 10, the weight of mice was measured before immunization, 7 days after the first immunization, 14 days after the first immunization, 7 days after the second immunization, and 14 days after the second immunization. The trend of weight change was observed to determine whether the nanoemulsion adjuvant affected the growth and development of mice. The injection site of the mice was dissected 14 days after the second immunization to observe the local reaction at the injection site and to evaluate the local safety of the nanoemulsion vaccine.

[0088] The results are as follows Figure 6 As shown, all immunization groups and the PlpE+PBS group showed the same trend of weight gain. The mice in the nanoemulsion adjuvant groups (PlpE+10.0 mg / mL cimetidine nanoemulsion adjuvant group and PlpE+20.0 mg / mL fucoidan nanoemulsion adjuvant group) did not show a significant trend of weight loss, which proves that nanoemulsion adjuvants have no significant effect on the growth and development of mice.

[0089] The results are as follows Figure 7 As shown, observation of the injection sites in mice revealed that the PlpE + 10.0 mg / mL cimetidine nanoemulsion adjuvant group had fewer abscesses and inflammatory foci compared to the PlpE + ISA206 group. The PlpE + 20.0 mg / mL fucoidan nanoemulsion adjuvant group showed no obvious local inflammatory response, which was closest to the PlpE + PBS group. This indicates that 20.0 mg / mL Fu-NE has the best local safety in mice, and 20.0 mg / mL Fu-NE is more easily absorbed when injected subcutaneously.

[0090] In conclusion, 20.0 mg / mL Fu-NE offers the best efficacy and the best safety profile.

[0091] Example 12 Physicochemical characterization and stability evaluation of nanoemulsion adjuvants The optimal nanoemulsion adjuvant, 20.0 mg / mL Fu-NE, was screened from Examples 10 and 11 to investigate whether different aqueous phase concentrations of the same carrier would induce a comparable immune response.

[0092] 1. The particle size distribution of the nanoemulsion adjuvants at concentrations of 10.0 mg / mL Fu-NE and 20.0 mg / mL Fu-NE was determined, and their morphology and structure were reflected by transmission electron microscopy. The synthesized nanoemulsion adjuvants could be significantly diluted with water while maintaining their appearance, proving that they belong to the oil-in-water emulsion type. The nanoemulsion adjuvants were placed at 4°C, room temperature, and 37°C for 3 months to observe whether their physical properties changed, and to detect any changes in particle size and PDI.

[0093] The results are as follows Figure 8As shown, the particle size range of 10.0 mg / mL Fu-NE is mostly distributed between 8 and 50 nm, with an average particle size of 18.73 nm. The particle size range of 20.0 mg / mL Fu-NE is mostly distributed between 8 and 60 nm, with an average particle size of 19.38 nm. Transmission electron microscopy shows that the 10.0 mg / mL Fu-NE and 20.0 mg / mL Fu-NE nanoparticles exhibit a black, spherical morphology, with most falling within the particle size distribution range. A small portion may have formed larger particles due to demulsification.

[0094] 2. Centrifugal stability of nanoemulsion adjuvants: The prepared 10.0 mg / mL Fu-NE and 20.0 mg / mL Fu-NE were centrifuged at 12,000 × 10⁻⁶ rpm. g After centrifugation for 30 minutes, the solution remained clear and transparent, without any layering, turbidity, or flocculent precipitate, indicating good stability.

[0095] 3. The particle size and PDI of 10.0 mg / mL Fu-NE and 20.0 mg / mL Fu-NE were determined after being placed at 4°C or room temperature for 3 months.

[0096] The results are as follows Figure 9 As shown in Table 4, the particle size of 10.0 mg / mL Fu-NE and 20.0 mg / mL Fu-NE after being stored at room temperature for 3 months was not significantly different from that of the freshly prepared nanoemulsion adjuvant, and their PDI was also not significantly different, proving that the fucoidan nanoemulsion adjuvant can be stored at room temperature for 3 months. After being stored at 4°C for 3 months, the particle size of 10.0 mg / mL Fu-NE and 20.0 mg / mL Fu-NE increased slightly compared with the freshly prepared nanoemulsion adjuvant, but their PDI was still less than 0.3, and they still had good dispersibility. The particle size and PDI of 10.0 mg / mL Fu-NE and 20.0 mg / mL Fu-NE after being stored at 4°C for 6 months were relatively stable, further indicating that the synthesized fucoidan nanoemulsion adjuvant can be stored at 4°C for a long time.

[0097] Table 4. Stability of nanoemulsion adjuvants at different temperatures and times. Example 13 Evaluation of the immunogenicity of fucoidan nanoemulsion adjuvant in Pasteurella subunit vaccines In Examples 10 and 11, 20.0 mg / mL Fu-NE was selected as a nanoemulsion adjuvant for use in Pasteurella subunit vaccines to investigate and compare the immunization effects of fucoidan nanoemulsion vaccine 1 and fucoidan nanoemulsion vaccine 2.

[0098] I. ELISA detection of antibody levels IgG, IgG1, and IgG2a: 1. Animal grouping and immunization: 6-8 week old female BALB / c mice were divided into 6 groups, with 5 mice in each group, for immunization challenge. The animal experimental groups were as follows: (1) PlpE + 10.0 mg / mL Fu-NE group: Fucoidan nanoemulsion vaccine 1; (2) PlpE + 20.0 mg / mL Fu-NE group: Fucoidan nanoemulsion vaccine 2; (3) PlpE+ISA206 group: commercial nanoemulsion vaccine; (4) PlpE+PBS group: blank vaccine; (5) PBS group: 0.01 M PBS; (6) Blank control group: No immunization was performed.

[0099] 2. The immunization dosage and method were the same as in Example 10. Blood was collected from the orbital venous plexus of mice before immunization, 7 days after the first immunization, 14 days after the first immunization, 7 days after the second immunization, and 14 days after the second immunization. Serum from each group of mice was collected to measure IgG. Serum from mice 7 days and 14 days after the second immunization was collected to measure the antibody levels of IgG1 and IgG2a.

[0100] The results are as follows Figure 10 As shown, the antibody levels in the PlpE+20.0 mg / mL Fu-NE group were not significantly different from those in the positive control group PlpE+ISA206 group 7 days after the first and second immunizations. The antibody levels were slightly lower than those in the PlpE+ISA206 group 14 days after the first and second immunizations. As shown in the figure, the antibody levels in the adjuvant group were higher than those in the PlpE+PBS group, and the antibody levels in the PlpE+20.0 mg / mL Fu-NE group were higher than those in the PlpE+10.0 mg / mL Fu-NE group. This indicates that low-dose fucoidan aqueous phase cannot induce higher antibody levels.

[0101] The results of antibody subtypes are as follows Figure 11 As shown, there were no significant differences in IgG1 and IgG2a among the PlpE+10.0 mg / mL Fu-NE group, PlpE+20.0 mg / mL Fu-NE group, and PlpE+ISA206 group 7 days and 14 days after the second immunization. IgG1 can represent the Th2 immune response at the antibody level, while IgG2a can represent the Th1 immune response at the antibody level. The adjuvant group induced a certain cellular immune response compared to the PlpE+PBS group.

[0102] II. Measurement of splenic lymphocyte proliferation and cytokines: 1. Animal grouping and immunization: 6-8 week old female BALB / c mice were divided into 5 groups of 3 mice each for immunization. The immunization grouping and method were the same as in step one. 28 days after the first immunization, the spleens of mice in each group were collected, and the proliferation level of spleen lymphocytes was measured. The supernatant of spleen lymphocytes after PlpE stimulation was measured to assess the IFN-γ level, that is, to assess the level of cellular immunity.

[0103] The results are as follows Figure 12 and Figure 13 As shown, under PlpE stimulation, the splenic lymphocyte proliferation level induced by PlpE + 20.0 mg / mL Fu-NE was significantly higher than that in the PlpE + ISA206 group ( P <0.01) and PlpE+10.0 mg / mL Fu-NE group ( P <0.001), the IFN-γ assay results showed that the IFN-γ level induced by the PlpE+20.0 mg / mL Fu-NE group was higher than that PlpE+ISA206 group, further indicating that the synthesized fucoidan nanoemulsion adjuvant 2 has the ability to induce cellular immune response.

[0104] Example 14 Safety Study of Fucoidan Nanoemulsion Vaccine Animal grouping and immunization: 6-8 week old female BALB / c mice were divided into 5 groups of 3 mice each for immunization. The immunization grouping and method were the same as in Example 13. Serum and major organs (heart, liver, spleen, lung and kidney) of mice in each group were collected 28 days after the first immunization. The safety of the vaccine adjuvant was evaluated by measuring the change in mouse body weight during the immunization cycle, measuring biochemical indicators in serum after immunization, and observing HE-stained sections of major organs.

[0105] 1. The results are as follows Figure 14 As shown, the weight change trends of mice in each vaccine adjuvant group were consistent with those in the PBS group, indicating that the fucoidan nanoemulsion vaccine had no significant effect on the growth and development of mice during the immunization cycle. Furthermore, no significant abnormalities were observed in the mice during the immunization cycle; all mice were in good health, with no cases of emaciation or death.

[0106] 2. Results are as follows Figure 15 As shown, there were no significant differences in serum biochemical indicators between the vaccine adjuvant groups and the PBS group, and all were within the normal range. Among them, ALT represents liver function, AST represents cardiac function, LDH represents whether there is damage to major organs, ALP represents whether there is liver and gallbladder damage, and UREA represents kidney function.

[0107] 3. Major organs of the mouse, including the heart, liver, spleen, lungs, and kidneys, were collected. The results are as follows: Figure 16 As shown, there were no significant abnormalities in the major organs (heart, liver, spleen, lungs, and kidneys) of each vaccine adjuvant group compared to the PBS group; all organs were normal.

[0108] In summary, the nanoemulsion vaccine of the present invention has good safety and excellent immunization effect.

[0109] 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-in-oil nanoemulsion adjuvant, characterized in that: The nanoemulsion adjuvant comprises, by mass percentage, 1.76-32.42% surfactant, 0.72-27.41% co-surfactant, 1.56-23.94% oil phase and the balance being aqueous phase; the nanoemulsion adjuvant has a particle size of 1-100 nm. The surfactant is either Tween 60 or Tween 80; The co-surfactant is either polyethylene glycol 400 or glycerol; The oil phase is either vitamin E or squalene; The aqueous phase is either a cimetidine solution or a fucoidan solution.

2. The nanoemulsion adjuvant according to claim 1, characterized in that: The mass ratio of the surfactant to the co-surfactant is 1~3:1; The surfactant and co-surfactant form a surfactant-co-surfactant mixture, and the mass ratio of the surfactant-co-surfactant mixture to the oil phase is 2~9:1; The concentration of the cimetidine solution is 10.83~21.67 mg / mL, the pH of the cimetidine solution is 6.8~7.0, and the final concentration of cimetidine in the nanoemulsion adjuvant is 5.0~10.0 mg / mL. The concentration of the fucoidan solution is 21.67~43.33 mg / mL, and the final concentration of fucoidan in the nanoemulsion adjuvant is 10.0~20.0 mg / mL.

3. The nanoemulsion adjuvant according to claim 2, characterized in that: The nanoemulsion adjuvant comprises, by weight percentage, 30.3% Tween 80, 15.15% polyethylene glycol 400, 9.09% vitamin E and 45.46% fucoidan solution; The concentration of the fucoidan solution was 43.33 mg / mL, and the final concentration of fucoidan in the nanoemulsion adjuvant was 20.0 mg / mL.

4. A method for preparing the nanoemulsion adjuvant according to any one of claims 1 to 3, characterized in that: Includes the following steps: (1) Weigh out the surfactant, co-surfactant, oil phase and aqueous phase according to the above mass percentages; (2) Mix the surfactant and co-surfactant to obtain a surfactant-co-surfactant mixture; (3) Add the oil phase to the surface additive mixture, stir and emulsify to obtain an oil phase mixture; (4) Under stirring conditions, the aqueous phase is added dropwise to the oil phase mixture, and stirring is continued to obtain an oil-in-water nanoemulsion adjuvant.

5. The preparation method according to claim 4, characterized in that: In step (1), when the aqueous phase is a cimetidine solution, the method for preparing the cimetidine solution is as follows: dissolve cimetidine in 0.1~0.3 M hydrochloric acid solution, adjust its pH to 6.8~7.0, and filter it using a 0.22 μm filter to obtain a cimetidine solution of 10.83~21.67 mg / mL; When the aqueous phase is a fucoidan solution, the fucoidan is prepared by dissolving fucoidan in water and filtering it through a 0.22 μm filter to obtain a fucoidan solution with a concentration of 21.67~43.33 mg / mL. In step (3), the stirring speed for emulsification is 400~600 r / min, and the stirring time for emulsification is 15~30 min; In step (4), the stirring speed is 400~600 r / min, and the stirring time is 30~45 min.

6. The preparation method according to claim 5, characterized in that: In step (1), the concentration of the hydrochloric acid solution is 0.15 M; In step (3), the stirring speed for emulsification is 500 r / min, and the stirring time for emulsification is 15 min. In step (4), the stirring speed is 500 r / min, and the stirring time is 30 min.

7. The use of the nanoemulsion adjuvant according to any one of claims 1 to 3 in delivering antigens to cells or preparing nanoemulsion vaccines.

8. A Pasteurella nanoemulsion vaccine, characterized in that: The nanoemulsion vaccine comprises the nanoemulsion adjuvant as described in any one of claims 1 to 3 and Pasteurella antigen; the antigen is protein PlpE. The concentration of the protein PlpE is 1.0~2.0 mg / mL, and the volume ratio of protein PlpE to nanoemulsion adjuvant is 1:1~3.

9. The nanoemulsion vaccine according to claim 8, characterized in that: The concentration of the protein PlpE is 1.0 mg / mL, the volume ratio of protein PlpE to nanoemulsion adjuvant is 1:1, and the nucleotide sequence of the protein PlpE is shown in SEQ ID NO:

1.

10. A method for preparing a nanoemulsion vaccine according to any one of claims 8 to 9, characterized in that: The preparation process is as follows: the nanoemulsion adjuvant and Pasteurella antigen are mixed according to the above volume ratio, and stirred at 400~600 r / min for 10~20 min to obtain Pasteurella nanoemulsion vaccine.

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