A compound adjuvant, its preparation method, and its application in vaccine preparation.

Abstract

This invention provides a composite adjuvant, its preparation method, and its application in vaccine preparation, belonging to the field of vaccine adjuvant technology. The composite adjuvant of this invention has low viscosity, only 12 mPa·s at 20°C, ensuring smooth injection; it exhibits high biocompatibility, with no deaths or organ abnormalities in acute toxicity experiments in mice, and no local or systemic adverse reactions or stress responses in chickens after injection; it also demonstrates excellent immunogenicity, achieving a 90% seroconversion rate for Mycoplasma cambogia and an 80% seroconversion rate for Mycoplasma synoviae 28 days after immunization with an inactivated Mycoplasma synoviae vaccine.

Description

Technical Field

[0001] This invention relates to the field of vaccine adjuvant technology, specifically to a compound adjuvant, its preparation method, and its application in vaccine preparation. Background Technology

[0002] Vaccines are preventive biological products that are immunogenic but do not cause disease in the body. The antigens in vaccines are mostly derived from inactivated or attenuated products of pathogenic microorganisms. When inactivated antigens are used to immunize animals alone, they are insufficient to produce immunogenicity, especially antigens with low immunogenicity and small molecular weight. Therefore, different types of adjuvants are needed to enhance the body's immune response to antigens.

[0003] Mycoplasma gallisepticum infection, also known as chronic respiratory disease in chickens, is a disease caused by Mycoplasma gallisepticum (MG). The main symptoms in infected chickens include coughing, runny nose, conjunctivitis, respiratory inflammation, decreased production performance, and increased feed conversion ratio in fattening chickens. This disease is prevalent in chicken farms in China and has become one of the most important infectious diseases threatening the poultry industry.

[0004] Mycoplasma synoviae infection, also known as infectious synovitis or infectious synovitis, is an acute and chronic infectious disease of chickens and turkeys, mainly affecting broilers and laying hens. It has a long course, and affected chickens show symptoms such as poor mental state, ruffled feathers, emaciation, lethargy, low mortality, swollen joints, and obvious inflammation of the synovial bursa and tendon sheath, which seriously affects the chicken's production capacity.

[0005] Immune adjuvants are substances that help vaccines enhance their immune efficacy by stimulating the body to initiate a protective response more quickly. Immunity is complex; the same antigen combined with different adjuvants can have synergistic or antagonistic effects, and different antigens require different adjuvants due to their different immune mechanisms.

[0006] Commonly used oil-emulsion adjuvant vaccines can be divided into three dosage forms: water-in-oil (W / O), oil-in-water (O / W), and water-in-oil-in-water (W / O / W). Oil-in-water (O / W) vaccines typically have low viscosity, are easily dispersed in the body after injection, and are less prone to residue. However, most W / O vaccines fail to achieve satisfactory immune responses. Water-in-oil-in-water (W / O / W) vaccines combine the advantages of both W / O and O / W vaccines. The conventional method involves first preparing the W / O vaccine, followed by a secondary emulsification process to disperse the antigen in the aqueous phase. This type of vaccine has low viscosity, is easy to inject, and provides high levels of immunity and long-lasting protection. However, its preparation technology is more challenging, and the vaccine stability is relatively poor. Water-in-oil (W / O) vaccines generally achieve high levels of immune response and long-lasting protection, but their high viscosity makes them difficult to inject, difficult to disperse and metabolize in the body, and can lead to significant adverse reactions in animals.

[0007] Therefore, there is a need for a vaccine for Mycoplasma gallisepticum and Mycoplasma synoviae, which maintains vaccine immunogenicity when combined with an inactivated vaccine to form a vaccine composition; remains stable during its shelf life; and has a significant effect in enhancing vaccine efficacy. Summary of the Invention

[0008] In view of this, the purpose of this invention is to provide an oil-in-water emulsion compound adjuvant that can induce a long-term immune response and has the advantages of low viscosity and low irritation compared with white oil Span-Tween adjuvant.

[0009] To achieve the above objectives, the present invention provides the following technical solution: A compound adjuvant comprising the following raw materials in parts by weight: 50-80 parts of injection oil, 1-18 parts of hydrophilic emulsifier, 2-20 parts of lipophilic emulsifier, 0.2-5 parts of amphoteric surfactant, 0.1-8 parts of immunomodulator, and 1-15 parts of viscosity modifier.

[0010] Preferably, the injectable oil is at least one of white oil, squalene, squalane, and medium-chain triglycerides.

[0011] More preferably, the injectable oil is a mixture of squalene and medium-chain triglycerides in a mass ratio of 1:1 to 1:3 to further reduce viscosity.

[0012] Preferably, the hydrophilic emulsifier is at least one selected from Tween-80, poloxamer F, Tween 20, and polyether.

[0013] Preferably, the lipophilic emulsifier is at least one selected from Span-80, Span-85, mannitol monooleate, and polyglycerol.

[0014] Preferably, the zwitterionic surfactant is lecithin or cocamidopropyl betaine.

[0015] Preferably, the immunomodulator is at least one of astragalus polysaccharide, ginsenoside, sodium alginate, and tocopherol.

[0016] Preferably, the viscosity modifier is glyceryl monostearate or polyglycerol ricinoleate.

[0017] Another object of the present invention is to provide a method for preparing a compound adjuvant, comprising the following steps: (1) Heat the oil for injection to 50-110℃, add lipophilic emulsifier and viscosity modifier, and stir at 200-400 rpm until completely dissolved to obtain the oil phase; (2) Add an amphoteric surfactant to the oil phase and stir at 300-500 rpm for 15-40 minutes at 50-70℃ to obtain a composite oil phase; (3) Add the immunomodulator to the oil phase; (4) The oil phase is homogenized 2-6 times at 40-70℃ and 50-120 MPa pressure to obtain an oil phase composite adjuvant with an average particle size of 200-500 nm.

[0018] A third objective of this invention is to provide the application of the aforementioned compound adjuvant in the preparation of mycoplasma vaccines, wherein the mycoplasma vaccines are Mycoplasma gallisepticum vaccine and Mycoplasma synoviae vaccine.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The composite adjuvant described in this invention has low viscosity, only 12 mPa·s at 20°C, ensuring smooth injection; it exhibits high biocompatibility, with no deaths or organ abnormalities observed in acute toxicity experiments in mice, and no local or systemic adverse reactions or stress responses in chickens after injection; it also demonstrates excellent immunogenicity, achieving a 90% seroconversion rate for Mycoplasma sarcoptica (MG) and an 80% seroconversion rate for Mycoplasma synoviae 28 days after immunization with the inactivated Mycoplasma sarcoptica vaccine. In summary, this invention achieves a balance between low viscosity, low stress, and highly efficient immune response, making it particularly suitable for the preparation of mycoplasma vaccines. Detailed Implementation

[0020] This invention provides a compound adjuvant, composed of the following raw materials in parts by weight: 50-80 parts of injection oil, 1-18 parts of hydrophilic emulsifier, 2-20 parts of lipophilic emulsifier, 0.2-5 parts of amphoteric surfactant, 0.1-8 parts of immunomodulator, and 1-15 parts of viscosity modifier.

[0021] Preferably, the injectable oil is at least one of white oil, squalene, squalane, and medium-chain triglycerides.

[0022] More preferably, the injectable oil is a mixture of squalene and medium-chain triglycerides in a mass ratio of 1:1 to 1:3 to further reduce viscosity.

[0023] Preferably, the hydrophilic emulsifier is at least one selected from Tween-80, poloxamer F127, Tween 20, and polyether.

[0024] Preferably, the lipophilic emulsifier is at least one selected from Span-80, Span-85, mannitol monooleate, and polyglycerol.

[0025] Preferably, the zwitterionic surfactant is lecithin or cocamidopropyl betaine.

[0026] Preferably, the immunomodulator is at least one of astragalus polysaccharide, ginsenoside, sodium alginate, and tocopherol.

[0027] Preferably, the viscosity modifier is glyceryl monostearate or polyglycerol ricinoleate.

[0028] The preparation method of the compound adjuvant includes the following steps: (1) Heat the oil for injection to 50-110℃, add lipophilic emulsifier and viscosity modifier, and stir at 200-400 rpm until completely dissolved to obtain the oil phase; (2) Add an amphoteric surfactant to the oil phase and stir at 300-500 rpm for 15-40 minutes at 50-70℃ to obtain a composite oil phase; (3) Add the immunomodulator to the oil phase; (4) The oil phase is homogenized 2-6 times at 40-70℃ and 50-120 MPa pressure to obtain an oil phase composite adjuvant with an average particle size of 200-500 nm.

[0029] Example 1 A compound adjuvant, comprising the following raw materials in parts by weight: 50 parts of injection oil, 10 parts of hydrophilic emulsifier Tween-80, 12 parts of lipophilic emulsifier mannitol monooleate, 0.5 parts of amphoteric surfactant lecithin, 0.5 parts of immunomodulator astragalus polysaccharide, and 10 parts of viscosity modifier glyceryl monostearate.

[0030] The injectable oil is a mixture of squalene and medium-chain triglycerides in a 1:2 mass ratio.

[0031] The preparation method of the compound adjuvant includes the following steps: (1) Heat the oil for injection to 60°C, add the lipophilic emulsifier and viscosity modifier, and stir at 300 rpm until completely dissolved to obtain the oil phase; (2) Add an amphoteric surfactant to the oil phase and stir at 500 rpm for 20 minutes at 60°C to obtain a composite oil phase; (3) Dissolve the immunomodulator and the hydrophilic emulsifier in water to obtain an aqueous phase; (4) The oil phase was homogenized five times at 70℃ and 100 MPa pressure to obtain an oil phase composite adjuvant with an average particle size of 200-500 nm.

[0032] Comparative Example 1 A composite adjuvant, with the same raw material composition and preparation method as in Example 1, except that the raw material in Comparative Example 1 does not contain zwitterionic surfactants.

[0033] Comparative Example 2 A composite adjuvant, with the same raw material composition and preparation method as in Example 1, except that sodium dodecyl sulfate is used in Comparative Example 2 to replace the zwitterionic surfactant in Example 1.

[0034] Comparative Example 3 A compound adjuvant, with the same raw material composition and preparation method as in Example 1, except that the amount of lecithin added in Comparative Example 3 is 0.1 parts.

[0035] Comparative Example 4 A composite adjuvant, with the same raw material composition and preparation method as in Example 1, except that the oil phase in Comparative Example 4 is squalene.

[0036] The performance of the compound adjuvant in Example 1 was tested, and the results are shown in Table 1. Table 1

[0037] The following experiments were conducted on the combined adjuvants of Example 1 and Comparative Examples 1-4: (1) Acute toxicity test (LD50) Experimental methods: 140 KM mice, half male and half female, weighing 18-22g, were randomly divided into 6 groups (5 experimental groups and 1 control group). The mice were fasted for 12 hours before the experiment. The 5 experimental groups were given the compound adjuvant of Example 1 and Comparative Examples 1-4 by gavage, with a gavage volume of 20ml / kg. The control group was given an equal volume of distilled water. The drugs were administered twice a day with an interval of 4 hours. The mice were observed for 14 days after administration, and the toxic reactions and number of deaths were recorded.

[0038] During the 14-day observation period, the mice showed normal food and water intake, weight gain, and no deaths. After 14 days, the animals were euthanized, and autopsies were performed. Macroscopic examination of vital organs such as the heart, liver, spleen, lungs, and kidneys revealed no abnormalities.

[0039] Experimental results showed that oral administration of the compound adjuvant to mice at a dose of 20 ml / kg (maximum daily dose of 40 ml / kg) indicated that the sample had extremely low acute toxicity and could be considered a non-toxic product.

[0040] (2) Clinical chicken safety experiment Seventy Hy-Line White chickens aged 35-45 days were randomly divided into 6 groups (5 experimental groups and 1 control group). Each chicken in the 5 experimental groups received an intramuscular injection of 1 ml of the vaccine to be tested (Mycoplasma gallisepticum antigen: adjuvant (V / V) = 1:2), and were observed for 14 consecutive days. No local or systemic adverse reactions were observed in either the control or experimental groups.

[0041] (3) Efficacy test of inactivated vaccines against Mycoplasma gallisepticum and Mycoplasma synoviae Several SPF chickens aged 28-42 days were randomly divided into 6 groups of 8 birds each. Each group received a subcutaneous injection of 0.3 mL of vaccine (containing one dose per bird). A control group was also included. 28 days after vaccination, serum was collected and antibodies against MG and MS were detected using an IDEXX ELISA kit, according to the kit's instructions. The results are shown in Table 2. Table 2

[0042] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A compound adjuvant, characterized in that, Including the following parts by weight of raw materials: 50-80 parts of injection oil, 1-18 parts of hydrophilic emulsifier, 2-20 parts of lipophilic emulsifier, 0.2-5 parts of amphoteric surfactant, 0.1-8 parts of immunomodulator, and 1-15 parts of viscosity modifier.

2. The compound adjuvant according to claim 1, characterized in that, The injectable oil is at least one of white oil, squalene, squalane, and medium-chain triglycerides.

3. The compound adjuvant according to claim 2, characterized in that, The injectable oil is a mixture of squalene and medium-chain triglycerides in a mass ratio of 1:1 to 1:3 to further reduce viscosity.

4. The compound adjuvant according to claim 1, characterized in that, The hydrophilic emulsifier is at least one of Tween-80, poloxamer F, Tween 20, and polyether.

5. The compound adjuvant according to claim 1, characterized in that, The lipophilic emulsifier is at least one of Span-80, Span-85, mannitol monooleate, and polyglycerol.

6. The compound adjuvant according to claim 1, characterized in that, The zwitterionic surfactant is lecithin or cocamidopropyl betaine.

7. The compound adjuvant according to claim 1, characterized in that, The immunomodulator is at least one of astragalus polysaccharide, ginsenoside, sodium alginate, and tocopherol.

8. The compound adjuvant according to claim 1, characterized in that, The viscosity modifier is glyceryl monostearate or polyglycerol ricinoleate.

9. The method for preparing the composite adjuvant according to any one of claims 1-8, characterized in that, Includes the following steps: (1) Heat the oil for injection to 50-110℃, add lipophilic emulsifier and viscosity modifier, and stir at 200-400 rpm until completely dissolved to obtain the oil phase; (2) Add an amphoteric surfactant to the oil phase and stir at 300-500 rpm for 15-40 minutes at 50-70℃ to obtain a composite oil phase; (3) Add the immunomodulator to the oil phase; (4) The oil phase is homogenized 2-6 times at 40-70℃ and 50-120 MPa pressure to obtain an oil phase composite adjuvant with an average particle size of 200-500 nm.

10. The use of the compound adjuvant according to any one of claims 1-8 in the preparation of mycoplasma vaccines.

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