Oil-in-water type veterinary adjuvant and preparation method thereof

Abstract

The invention belongs to the technical field of animal immunopotentiators, and particularly relates to an oil-in-water type veterinary adjuvant and a preparation method thereof. The oil-in-water type veterinary adjuvant comprises white oil, span 80, Tween 80, PEG400, astragalus polysaccharide and a phosphate buffer solution, the astragalus polysaccharide cooperates with a surfactant to activate a TLR4 / NF-kappa B pathway, Th1 / Th2 / Th17 three-way immune response is achieved, the serum IgG and mucous membrane sIgA antibody level is remarkably improved, and the problems that a traditional O / W adjuvant is high in layering rate (smaller than 5%), and the stability of the traditional O / W adjuvant is poor are solved through compound optimization of the HLB value (HLB = 11.5-12.0) of the HLB value of span 80 and Tween 80. And storage at 4 DEG C for 90 days) and poor freeze-thaw stability are solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal immune enhancer, and particularly relates to an oil-in-water type veterinary adjuvant and a preparation method thereof. BACKGROUND

[0002] An adjuvant is a kind of substance that can non-specifically enhance the intensity of the body's immune response to an antigen, and has core application value in the development of veterinary vaccines. Its core functions include: improving immunogenicity, promoting the activation of antigen-presenting cells (APC), enhancing antibody titer and immune persistence, reducing the amount of antigen, reducing the demand for unit dose of antigen through the effect of slow release or immune signal amplification, and directional regulation of immune response type.

[0003] Based on the demand of clinical application, the core technical standards of an ideal vaccine adjuvant should meet the following requirements at the same time: safety, local irritation reaction grading ≤ 1 (WHO standard); stability, no phase separation or titer decay after storage at 4 DEG C for ≥ 24 months; applicability, compatible with the existing vaccine cold chain transportation conditions (2-8 DEG C); and immunological efficacy, antibody titer of target animals is increased by ≥ 4 times.

[0004] Therefore, it is urgent to develop a new type of composite adjuvant to achieve a higher level of immune response through multi-mechanism synergistic effect under the premise of ensuring biological safety. SUMMARY

[0005] The present application provides an oil-in-water type veterinary adjuvant and a preparation method thereof, which provides a raw material source that is widely available, low in cost and has low toxicity and side effects compared with traditional adjuvants on the market. The adjuvant vaccine of the present application can reduce the oil amount of existing inactivated vaccines, accelerate the production speed of corresponding antibodies of immunized animals, enrich the selection diversity of immune enhancers, and reduce the cost of vaccine preparation. The oil-in-water type veterinary adjuvant specifically comprises the following components:

[0006] 20-30% of mineral oil, 10-20% of lipophilic surfactant, 20-40% of hydrophilic surfactant, 20-30% of stabilizer, and 0.3-15% of immune enhancer by mass.

[0007] Preferably, the HLB value of the oil-in-water type veterinary adjuvant is 9-15.

[0008] Preferably, the hydrophilic surfactant is Tween 80.

[0009] Preferably, the lipophilic surfactant is Span 80.

[0010] Preferably, the mineral oil is white oil.

[0011] Preferably, the stabilizer is PEG400.

[0012] Preferably, the immune enhancer is 1-50 mg / mL of an aqueous solution of astragalus polysaccharide. Most preferably, the aqueous solution of astragalus polysaccharide is prepared by dissolving astragalus polysaccharide in a phosphate buffer with a pH of 7.2-7.4 to make the concentration of astragalus polysaccharide 1-50 mg / mL.

[0013] Preferably, the phosphate buffer is prepared by weighing 8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 into 1000 mL of distilled water.

[0014] The preparation method of the oil-in-water veterinary adjuvant comprises the following steps:

[0015] Step one, add Span 80 and Tween 80 to white oil and heat to 55-65℃ to obtain an oil phase mixture.

[0016] Step two, mix astragalus polysaccharide with a phosphate buffer, then mix with PEG400 and heat to 40-50℃ to obtain an astragalus polysaccharide-PEG400 mixture.

[0017] Step three, mix the oil phase mixture and the astragalus polysaccharide-PEG400 mixture, stir at 18000-20000 rpm for 10-15 min, and stand still to obtain an oil-in-water veterinary adjuvant.

[0018] The beneficial effects of the present application are:

[0019] The oil-in-water veterinary adjuvant of the present application is stable and efficient, can significantly increase the production of immunoglobulin and promote the interaction between antigen presenting cells and T lymphocytes, increase the T lymphocyte transformation rate, and improve the immunity of animals. Compared with known vaccine adjuvants, it has the characteristics of safety and efficiency, simple preparation process, convenient use, easy product process quality control, and wide application prospect. The prepared vaccine has the characteristics of small viscosity, easy injection, good stability, safe and effective injection, no visible residue, etc. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only embodiments of the present application, and those skilled in the art can obtain other drawings according to the provided drawings without creative labor.

[0021] Figure 1 is the particle size determination diagram of the adjuvant vaccine of the present application.

[0022] Figure 2is the detection result chart of vaccine A, vaccine B PEDV IgG antibody titer.

[0023] Figure 3 is the detection result chart of vaccine A, vaccine B PEDV neutralizing antibody titer. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative efforts fall within the protection scope of the application.

[0025] The imported white oil used in the following examples and experiments is a product of Exxonmobil, France, and the model is marcol 52; the astragalus polysaccharide powder is provided by Deyang Hengyu Biotechnology Co., Ltd.; the porcine epidemic diarrhea virus inactivated liquid is provided by the College of Animal Medicine, China Agricultural University, and the antigen amount after inactivation is ≥10 ^6.0 TCID50.

[0026] The preparation method of the oil-in-water veterinary adjuvant in the following examples is as follows:

[0027] Step one, heat the white oil, Tween 80 and Span 80 to 60℃, then add them into the reaction container, and stir at 100 rpm for 30 min to obtain an oil phase mixture.

[0028] Step two, dissolve 1 part by weight of astragalus polysaccharide powder in 500 parts by weight of phosphate buffer (8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 dissolved in 1000 mL distilled water) to prepare an astragalus polysaccharide aqueous solution of 20 mg / mL, heat the astragalus polysaccharide aqueous solution and PEG400 to 45℃ and mix them to obtain an astragalus polysaccharide-PEG400 mixture;

[0029] Step three, stir the oil phase mixture and the astragalus polysaccharide-PEG400 mixture in a homogenizer at 20000 rpm for 15 min, and after standing for 30 min, wet heat sterilize them at 121℃ for 30 min to obtain the finished oil-in-water adjuvant.

[0030] Example 1

[0031] The raw material ratio of the oil-in-water veterinary adjuvant in this example is white oil 22%, Tween 80 34%, Span 80 15%, PEG400: 27%, astragalus polysaccharide aqueous solution 2%.

[0032] Example 2

[0033] The raw material ratio of the oil-in-water type veterinary adjuvant of this example is white oil 25%, Tween 80 30%, Span 80 13%, PEG400:26%, and astragalus polysaccharide aqueous solution 6%.

[0034] Example 3

[0035] The raw material ratio of the oil-in-water type veterinary adjuvant of this example is white oil 28%, Tween 80 31%, Span 80 11%, PEG400:24%, and astragalus polysaccharide aqueous solution 6%.

[0036] Test Example 1

[0037] The adjuvant prepared in Example 1 was subjected to particle size determination using a Mastersizer laser particle size instrument, and statistical analysis and plotting were performed, and the results are shown in Figure 1 .

[0038] Test Example 2

[0039] Twenty 6-8 week old SPF female Kunming mice were selected for testing, and were randomly divided into the following 4 groups: a blank control group, an Example 1 group, an Example 2 group, and an Example 3 group, with 5 mice in each group. The adjuvants of the Example 1 group, the Example 2 group, and the Example 3 group were injected intraperitoneally into each mouse at 0.2 mL, and the blank control group was injected intraperitoneally with an equal volume of physiological saline. After 14 days, the mice were sacrificed by cervical dislocation, and the thymus and spleen were removed, surface blood was absorbed, and the wet mass was weighed on an electronic balance, and the thymus and spleen indices were calculated, and the results are shown in Table 1.

[0040] Table 1: Immune organ indices of each group

[0041] Group n Thymus index (mg / g) Spleen index (mg / g) Blank control group 5 2.64±0.18 7.61±0.32 Example 1 group 5 3.85±0.41 11.81±0.44 Example 2 group 5 3.68±0.32 10.96±0.22 Example 3 group 5 3.71±0.29 9.91±0.54

[0042] Test Example 3

[0043] The spleen lymphocyte proliferation activity was determined as follows: the spleen was taken aseptically, and the spleen cells were washed with 3 mL of pre-cooled PBS buffer containing 1% double antibody until the spleen was white. The washing solution was collected in a 5 mL centrifuge tube, and centrifuged at 1500 r / min for 10 min. The supernatant was discarded, 2 mL of red blood cell lysis solution was added to each tube, and the cells were resuspended. After standing for 3 min, the cells were centrifuged at 1500 r / min for 10 min. The cells were washed twice with 1% double antibody-PBS. The cells were resuspended with 1% double antibody-containing 1640. The cell concentration was adjusted to 500,000 / mL. Six wells of a 96-well plate were selected and 200 μL of PBS was added as a blank control group, and 100 μL of cell suspension was added to the remaining wells. 100 μL of the drug of Example 1 group, Example 2 group, and Example 3 group was added to the experimental groups, and 100 μL of culture medium was added to the blank control group. Each group had 3 replicate wells. Incubation was performed at 37°C for 72 h. Four hours before the end of the culture, 10 μL / well of 5 mg / mL MTT solution was added to each well of the culture plate. Incubation was performed at 37°C for 4 h. The cells were centrifuged at 1500 r / min for 10 min, and the supernatant was discarded. 150 μL of DMSO was added to each well, and the cells were dissolved for 10 min. The absorbance (OD) value at 490 nm was determined within 30 min using an enzyme-labeled instrument. The results are shown in Table 2.

[0044] Table 2 Comparison of spleen lymphocyte proliferation activity of each group

[0045] Group OD 490 values Blank control group 0.61±0.18 Example 1 group 0.87±0.13 Example 2 group 0.94±0.15 Example 3 group 0.86±0.12

[0046] Test Example 4

[0047] Preparation of inactivated porcine epidemic diarrhea virus liquid, according to the relevant requirements of the "Veterinary Biological Quality Standards Compilation" (2006-2008), the inactivated porcine epidemic diarrhea virus liquid was prepared. The virus content of each milliliter of virus liquid before inactivation should be ≥10 6 TCID 50 .

[0048] Under sterile conditions, the oil-in-water veterinary adjuvant prepared in Example 1 above was heated to 30°C, and 30 g was placed into a 100 mL glass bottle, and 30 g of the prepared inactivated porcine epidemic diarrhea virus liquid was also placed into the glass bottle. Emulsification was performed using a rod homogenizer at 500 revolutions per minute for 15 minutes, and the vaccine A was obtained.

[0049] Under sterile conditions, the ISA206 adjuvant produced by the French SEPPIC company was heated to 30°C, and 30 g was placed into a 100 mL glass bottle, and 30 g of the prepared inactivated porcine epidemic diarrhea virus liquid was also placed into the glass bottle. Emulsification was performed using a rod homogenizer at 500 revolutions per minute for 15 minutes, and the vaccine B was obtained.

[0050] Select 60 6-8 weeks old SPF female Kunming mice for test, and randomly divide them into the following 4 groups: vaccine A group, vaccine B group, PEDV inactivated virus liquid group, and blank control group, 15 in each group. 5 mice in the vaccine A group and the vaccine B group are subcutaneously injected with 1 ml of adjuvant, 5 mice are subcutaneously injected with 2 ml of vaccine, and 5 mice are intraperitoneally injected with 1 ml of vaccine; 5 mice in the PEDV inactivated virus liquid group are subcutaneously injected with 1 ml of inactivated PEDV antigen liquid, 5 mice are subcutaneously injected with 2 ml of inactivated PEDV antigen liquid, and 5 mice are intraperitoneally injected with 1 ml of inactivated PEDV antigen liquid; 5 mice in the blank group are not treated. Observe whether the organs of the mice are abnormal after immunization, and observe abnormal reactions or death of the animals, and the results are shown in Table 3.

[0051] Table 3: Overdose safety test

[0052]

[0053]

[0054] Test Example 5

[0055] Select 20 healthy susceptible piglets of 40-50 days old (negative for antibody detection by using a commercial PEDV blocking ELISA detection kit) for test, and randomly divide them into the following 4 groups: vaccine A group, vaccine B group, PEDV inactivated virus liquid group, and blank control group, 5 in each group. The vaccines are injected into the muscles behind the ear roots of the piglets, 2 ml per piglet. Blood is collected and serum is separated 7 days, 14 days, 21 days, and 28 days after the first immunization, and the piglets are immunized again after blood collection and serum separation 28 days after the first immunization. Blood is collected and serum is separated 14 days and 21 days after the second immunization. The antibody titers of the piglets in each experimental group are determined by using a commercial blocking ELISA kit and a micro-neutralization test. The antibody results are shown in Table 4. Figure 2 、 Figure 3 .

[0056] The above description of disclosed embodiments enables those skilled in the art to carry out or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An oil-in-water veterinary adjuvant, characterized in that, The HLB value is 9-15, and the components include: 20-30% of mineral oil, 10-20% of oleophilic surfactant, 20-40% of hydrophilic surfactant, 20-30% of stabilizer, and 0.3-15% of immune enhancer by mass.

2. An oil-in-water veterinary adjuvant according to claim 1, characterised in that, The hydrophilic surfactant is Tween 80.

3. An oil-in-water veterinary adjuvant according to claim 1, characterised in that, The oleophilic surfactant is Span 80.

4. An oil-in-water veterinary adjuvant according to claim 1, characterised in that, The mineral oil is white oil.

5. An oil-in-water veterinary adjuvant according to claim 1, characterised in that, The stabilizer is PEG 400.

6. An oil-in-water veterinary adjuvant according to claim 1, characterised in that, The aqueous astragalus polysaccharide solution is 1-50 mg / mL.

7. An oil-in-water veterinary adjuvant according to claim 6, characterised in that, The preparation method of the aqueous astragalus polysaccharide solution is to dissolve astragalus polysaccharide in phosphate buffer with pH=7.2-7.4, so that the concentration of astragalus polysaccharide is 1-50 mg / mL.

8. The method for preparing the oil-in-water veterinary adjuvant according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: Step one, the oleophilic surfactant and the hydrophilic surfactant are added to the mineral oil and heated to obtain an oil phase mixture; Step two, the immune enhancer is first mixed with the phosphate buffer, then mixed with the stabilizer and heated to obtain an immune enhancer-stabilizer mixture; Step three, the oil phase mixture and the immune enhancer-stabilizer mixture are mixed and stirred, and then left to stand to obtain an oil-in-water type veterinary adjuvant.

9. The production method according to claim 8, characterized by, The heating temperature in step one is 55-65℃, and the heating temperature in step two is 40-50℃.

10. The preparation method according to claim 8, characterized in that In step three, the stirring speed is 18000-20000 rpm, and the stirring time is 10-15 min.

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