Self-emulsifying vaccine adjuvant containing submicron oil-in-water two-phase emulsion oil phase and preparation method of self-emulsifying vaccine adjuvant

Abstract

The invention relates to the technical field of vaccine immunization, and discloses a self-emulsifying vaccine adjuvant containing a submicron oil-in-water two-phase emulsion oil phase and a preparation method of the self-emulsifying vaccine adjuvant. N-hexadecane; 5% of a lipophilic emulsifier: 20% of Span; 40, 27% of Span; 19% of Span 60; 7% of Span 65; 23% of Span 80; 4% of Span 85; 5% of a hydrophilic emulsifier: 15% of Tween 20; 40% and 15% of Tween; 47% of Tween 60; 23% of Tween 80; the immunopotentiator is VSP70, and the weight of the immunopotentiator is 3mg per part. The stable emulsion is generated, demulsification caused by interference of strong surfactants is avoided, and the final product is more stable due to correct dosage form selection and tiny and uniform particle size; the water phase and the oil phase can form a stable emulsified structure which is uniform and does not aggregate, so that the release time of the antigen is prolonged, and long-time immune protection is provided.

Description

Technical Field

[0001] The present invention belongs to the technical field of vaccine immunity, and in particular, relates to a self-emulsifying vaccine adjuvant containing a submicron water-in-oil-in-water two-phase emulsion oil phase and a preparation method thereof. Background Art

[0002] Vaccine immunization is one of the most effective measures to prevent and control major animal diseases. Modern vaccines have poor immunogenicity and need to be combined with adjuvants to enhance the immune efficacy of vaccines. As an important component of animal vaccines, adjuvants can enhance the immunogenicity of antigens, reduce the amount of antigens used, prolong the immune protection period, and play a key role in immune response. At present, the adjuvants used in most vaccines are oil emulsion adjuvants and composite adjuvants. Emulsions have certain advantages in effectiveness and economy as vaccine adjuvants. The mineral oil component can not only participate in sustained release delivery as an antigen carrier, but also can be used as an immunopotentiator to activate Th2 cells to a certain extent. However, emulsion adjuvants have poor targeting for cell uptake, cannot construct an effective cell uptake pathway, cannot construct a Th1 / Th2 immune balance, cannot induce other types of immunity while effectively inducing humoral immunity, and have poor immune efficacy. Composite adjuvants are composed of delivery carriers and targeted immunopotentiating substances. Since composite adjuvants can simultaneously increase the intensity of the body's immune response to antigens and build a higher degree of Th1 / Th2 balance, they have become a major trend in the development of vaccine adjuvants. It is worth noting that different types of immune responses have a certain degree of antagonism, among which the proportion of each component of the adjuvant and the rational design of the vaccine adjuvant structure are the key factors affecting the adjuvant effect. In addition, the particle size of the oil-emulsion adjuvant vaccine particles has a huge impact on the absorption and delivery of antigens and the effect of cellular immune response. Under the existing technology, the particle size of vaccine adjuvants is large, which is not conducive to cell endocytosis, and the duration of the immune enhancement effect is unstable, which greatly limits the vaccine-induced immune response.

[0003] Oil emulsion adjuvant is the most widely used adjuvant for veterinary vaccines. According to the dosage form, it can be divided into water-in-oil (W / O), oil-in-water (O / W), and water-in-oil-in-water (W / O / W). Water-in-oil (W / O) oil vaccine adjuvant is prepared by mixing antigen liquid, stabilizer and white oil under high shear force. Its immune mechanism is to store it at the inoculation site for a period of time, locally produce granulomas and inflammatory reactions, attract macrophages, lymphocytes, etc. to gather to recognize antigens and produce antibodies. At the same time, the slowly diffusing oil adjuvant prolongs the time for antigens to be transported to secondary lymphoid organs, constantly stimulating the body's immune system, thereby producing an immune efficacy with a long duration and high antibody levels. Although it can strongly enhance the level of antibody response, it is almost difficult to metabolize or be absorbed, may cause severe pain during vaccination, and cause a large number of sterile abscesses and granulomas at the injection site, which can easily have an adverse effect on the quality of animal carcasses. Oil-in-water (O / W) adjuvant vaccines have the advantages of small local reactions at the injection site and high stability, and are completely metabolizable and have good biocompatibility. However, the antigens contained in or exposed in the continuous phase of the oil-in-water adjuvant vaccine will quickly disperse at the injection site and be decomposed by the body fluid enzymes, making it difficult to provide long-term protective antibodies. Special immunostimulants and multiple booster immunizations are often required, and the preparation cost is often high, so it is not widely used. The water phase and oil phase inside the water-in-oil-in-water (W / O / W) adjuvant can form a stable emulsified structure, thereby prolonging the release time of the antigen and providing long-term immune protection. In addition, its structural characteristics make the antigen particles larger, improve the immunogenicity, and can more effectively activate the immune system to achieve better protection. However, under the prior art, the water-in-oil-in-water adjuvant has a more complicated emulsification process, stricter storage conditions, and unreasonable formulation ingredients and preparation processes. Therefore, it still has defects such as difficulty in injection, low immune efficacy, and poor stability compared with O / W or W / O adjuvants in the use link, which directly affects the production efficiency and quality of the vaccine.

[0004] The main problems existing in the existing technical solutions are: domestic animal adjuvants have low immune efficiency, poor safety, serious side effects, and are easily retained at the inoculation site, causing damage and inflammatory stress reactions;

[0005] Minor issues: 1. Traditional vaccines have poor thermal stability, are extremely dependent on the cold chain, and are prone to demulsification and failure; 2. There is a lack of new and highly efficient immune-enhancing substances, and the vaccine has poor immunogenicity, resulting in low antibody titers, and multiple vaccinations are required to achieve a protective effect; 3. The cost of preparing animal vaccines is high, and the production process needs to be upgraded urgently.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] In order to solve the above technical problems, the basic concept of the technical solution adopted by the present invention is:

[0008] A self-emulsifying vaccine adjuvant containing a submicron-sized water-in-oil-in-water biphasic emulsion oil phase, which is composed of the following components by weight percentage:

[0009] Mineral oil 90%;

[0010] n-Hexadecane;

[0011] Lipophilic emulsifier 5%: Span 20, 20%; Span 40, 27%; Span 60, 19%; Span 65, 7%; Span 80, 23%; Span 85, 4%;

[0012] Hydrophilic emulsifier 5%: Tween 20, 15%; Tween 40, 15%; Tween 60, 47%; Tween 80, 23%;

[0013] Immune enhancer: VSP70, 3 mg per serving.

[0014] As a preferred embodiment of the present invention, the oil phase of the submicron water-in-oil-in-water two-phase emulsion contains 0.5-50% mineral oil by mass, 0.1-10% two-component lipophilic emulsifier, and 0.05-25% cocktail combination hydrophilic non-ionic surfactant. After being combined with the aqueous vaccine antigen, it is naturally assembled into 1-5 internal water phases containing 0.00001-1% immunogens, and the water dispersed phase with a diameter of 5-30nm is wrapped in the oil dispersed phase; the oil dispersed phase is surrounded by the second water phase and the outer water continuous phase to form a three-phase emulsion of water-in-oil-in-water.

[0015] As a preferred embodiment of the present invention, the average droplet size of the submicron emulsion is in the range of about 0.1-0.5 μm, preferably in the range of 0.18-0.24 μm, with a peak value of 0.2 μm.

[0016] As a preferred embodiment of the present invention, in the oil phase of the water-in-oil-in-water two-phase emulsion, the content of oily particles with a size of 0.1-0.3 μm accounts for 80%-90%.

[0017] As a preferred embodiment of the present invention, the oil phase of the water-in-oil-in-water two-phase emulsion contains a plurality of surfactants of polyoxyethylene sorbitan monooleate and sorbitan monooleate to form a double cocktail emulsifier.

[0018] As a preferred embodiment of the present invention, the mineral oil is selected as an oily component that is liquid at room temperature of 15-25°C.

[0019] As a preferred embodiment of the present invention, it also includes adding surfactant saponin VSP70, which is composed of plant saponin VSP and nucleic acid VDN70 polysaccharide VPLA70.

[0020] As a preferred embodiment of the present invention, a method for preparing a self-emulsifying vaccine adjuvant containing a submicron water-in-oil-in-water two-phase emulsion oil phase is also disclosed, specifically, under low-speed stirring conditions, an inactivated antigen liquid is dropwise added to the water-in-oil-in-water type vaccine adjuvant, and after the dropwise addition is completed, low-speed stirring is performed for 30 minutes to obtain the vaccine.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention prepares a novel submicron water-in-oil-in-water two-phase emulsion oil phase, which is well absorbed at the injection site, overcomes the reactions of traditional adjuvants such as retention, damage and inflammatory stress at the vaccination site, does not affect the system health and body temperature of the vaccinated animal, and has good comfort, and can be used as a safe and effective adjuvant for livestock vaccines.

[0023] The present invention produces a stable emulsion that is not demulsified by interference from strong surfactants. The selection of the correct dosage form and the small and uniform particle size make the final product more stable. The water phase and the oil phase can form a stable emulsified structure that is uniform and does not aggregate, thereby extending the release time of the antigen and providing long-term immune protection.

[0024] The invention adds the immune enhancing substance VSP70, which can enhance the immunogenicity of the vaccine and improve its stability. The coordinated use of adjuvant and immune enhancing substance can increase the initial immune response rate and improve the animal antigen immune response, which has significant advantages in reducing the amount of antigen used and the number of immunizations.

[0025] The preferred technical solution provided by the present invention meets the needs of large-scale production, greatly reduces the preparation cost, has simple emulsification operation, and the emulsion is stable and has a strong ability to resist high temperature. It also has good antigen matching and can be used for almost all vaccines. It can be applied on an industrial scale, provide key technical support for the prevention and control of animal diseases, and ensure the healthy development of animal husbandry.

[0026] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In the attached picture:

[0028] Figure 1 Schematic diagram of the measurement of submicron water-in-oil-in-water two-phase emulsion foot-and-mouth disease vaccine by the Malvern Zetasizer particle size analyzer in the present invention;

[0029] Figure 2 Schematic diagram of the TSI particle size analyzer of the present invention for measuring submicron water-in-oil-in-water two-phase emulsion foot-and-mouth disease vaccine;

[0030] Figure 3The relationship between the HLB hydrophile-lipophile balance value and the function in the present invention is shown in FIG.

[0031] Figure 4 This is a comparison chart of the potency of the biphasic adjuvants Drakeol 6VR and n-hexadecane in the present invention; Figure 5 This is a diagram showing the results of foot-and-mouth disease antibody level detection in the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention.

[0033] Embodiment 1:

[0034] The present invention provides a submicron water-in-oil-in-water adjuvant for animal vaccines, wherein the oil phase of the two-phase emulsion contains about 0.5-50% of mineral oil, about 0.1-10% of a two-component lipophilic emulsifier, and about 0.05-25% of a cocktail combination of hydrophilic nonionic surfactants. After being combined with aqueous vaccine antigens, the oil phase is naturally assembled into an internal aqueous phase containing 1-5 0.00001-1% immunogens, and a water dispersion phase with a diameter of 5-30nm is wrapped in the oil dispersion phase. The oil dispersion phase is surrounded by a second aqueous phase (external aqueous continuous phase) to form a three-phase emulsion of water-in-oil-in-water. The average droplet size of the submicron emulsion ranges from about 0.1-0.5μm, preferably from 0.18-0.24μm, with a peak value of 0.2μm ( Figure 1 and Figure 2 ).

[0035] The detailed steps are as follows, and the combined emulsifier is modified according to the HLB technology to make it a series of emulsifier groups with slight differences. The term "W / O / W" means a water-in-oil-in-water emulsion, which is an emulsion in which small water droplets or water capsules (internal aqueous phase) are embedded in oily particles (oil phase) and dispersed in a continuous aqueous phase (external aqueous phase). The W / O / W emulsion is produced by a primary emulsion (wherein the aqueous phase is added to the oil phase), followed by at least one secondary emulsion (wherein the oil phase is dispersed in the aqueous phase). The specific W / O / W emulsion or composition of the present invention generally includes an internal aqueous phase (corresponding to a water capsule or droplet surrounded by a first surfactant film), an oil phase surrounded by a second surfactant film, and an external aqueous phase.

[0036] Typical W / O / W compositions are particulate compositions comprising water capsules embedded in oily particles, which are dispersed in an external aqueous phase. Preferably, the antigen or a portion thereof is contained in an internal aqueous phase embedded or encapsulated in the oily vesicle, while the additional adjuvant compound is in the external aqueous phase. In a preferred embodiment, the external aqueous phase comprises a portion of the at least one antigen and the additional adjuvant, preferably in the form of an antigen-adjuvant complex. According to a preferred embodiment, about 75% of the total amount of antigen in the composition is in the oily vesicle, while about 25% of the total amount of antigen in the composition is in the external aqueous phase, optionally compounded with an adjuvant compound.

[0037] W / O / W emulsions can have various size / distribution characteristics. However, in the preferred embodiment in the past, the size of the oily particles was 1-40 μm, more preferably 0.3-1 μm, and the size of the particles usually indicated its diameter. For a long time, due to the use of a single hydrophilic and lipophilic emulsifier, it has been impossible to accurately produce a small particle size (0.2 μm) highly stable emulsion that is easily absorbed and taken up by lymphocytes in the emulsification system. W / O / W multiple emulsions are systems in which W / O and O / W emulsions exist simultaneously. However, the main problem with its stability is the presence of two thermodynamically unstable interfaces, namely the W / O interface of the main emulsion and the O / W interface of the multiple emulsion. Therefore, two different groups of emulsifiers are needed to stabilize them: one emulsifier combination has a low HLB for the W / O interface, and the other group of emulsifiers has a high HLB for the O / W interface. When selecting a lipophilic surfactant and determining the rHLB of the main W / O emulsion in the multiple emulsion, the surfactant used for interface stabilization can be screened in the same manner as a simple W / O emulsion. However, determining the required HLB for W / O / W multiple emulsions, i.e. selecting the hydrophilic emulsifier necessary to stabilize the second interface and form the multiple emulsion, is more complicated. Both lipophilic and hydrophilic emulsifiers can be absorbed by the O / W interface. Therefore, if both hydrophilic and lipophilic emulsifiers are added, the HLB value of the O / W interface of the multiple emulsion is the sum of the HLB values ​​of all absorbed surfactants. However, the amount of lipophilic emulsifier absorbed to the O / W interface is not defined and depends on its total amount in the formulation and the phase volume ratio, among other things. The ideal HLB value of the hydrophilic emulsifier increases when the added lipophilic emulsifier concentration is higher or the amount of hydrophilic emulsifier is reduced ( Figure 3 ).

[0038] The present invention first utilizes the rHLB factor to screen suitable primary W / O emulsions. Secondly, in order to determine the rHLB that can incorporate the most suitable primary emulsion into the multiple emulsion, W / O / W emulsions are prepared with different hydrophilic emulsifier mixtures representing a range of HLB values, and the properties and long-term stability of the multiple W / O / W emulsions are studied. The stability of the formulation and the applicability of the emulsion rHLB value are determined based on parameters such as conductivity, multiple droplet size, emulsification volume and viscosity. The present invention shows that the 0.3-1 μm range can provide the best immune efficacy when administered in vivo. In the preferred W / O / W composition of the present invention, at least 80% of the oily particles have a size below 0.3 μm, more preferably at least 85%, and even more preferably at least 90% of the oily particles have a size between 0.1-0.3 μm. In addition, the present invention shows that a particularly strong immune response to the antigen can be generated by such size and distribution characteristics. Surfactants are usually selected, combined or used under conditions that provide an appropriate hydrophilic-lipophilic balance (HLB) for the formulation. The HLB of a surfactant or surfactant combination is a measure of its degree of hydrophilicity or lipophilicity and is determined by calculating the values ​​for different regions of the molecule. Most preferably, the surfactant combination used in the first emulsion has a low HLB value (usually between 4-7, preferably about 6.0), while the surfactant or surfactant combination used in the second emulsion has a high HLB value (usually between 7-15, preferably about 10.5).

[0039] The W / O / W emulsifier of the present invention comprises a plurality of surfactants of polyoxyethylene sorbitol monooleate and sorbitol monooleate to form a double cocktail emulsifier. The generated 200nm water-in-oil-in-water oil phase is self-emulsified after being mixed with an aqueous antigen, wherein the external water phase accounts for 30%, the internal water phase accounts for 70%, and the viscosity is less than 20mPa·s. The generated 400nm water-in-oil-in-water oil phase is self-emulsified after being mixed with an aqueous antigen, wherein the external water phase accounts for 17%, the internal water phase accounts for 83%, and the viscosity is less than 5mPa·s.

[0040] The lipophilic emulsifier components include:

[0041]

[0042]

[0043] The hydrophilic emulsifier component includes

[0044]

[0045]

[0046] In the present invention, the mineral oil is selected as an oily component that is liquid at room temperature (15-25°C), such as ester oily bases or non-ester oily bases commonly used in food, medicine, cosmetics, etc. Non-ester oily bases include light liquid mineral oil, squalene, squalane, polybutene, etc. Ester oily bases include various esters derived from medium-chain saturated fatty acids such as caprylic acid and capric acid or long-chain unsaturated fatty acids such as oleic acid and linoleic acid and alcohols; naturally occurring fatty acid esters, such as liquid vegetable oils such as peanut oil, olive oil, sunflower oil, safflower oil, jojoba oil, and liquid oils derived from animals such as orange snapper oil, these oily bases can be used alone or in combination. Depending on the purpose, oils such as ester derivatives of oleic acid and vegetable oils have various advantages, such as their relatively high stability to oxidation, high affinity for organ tissues, and can reduce local irritation and residual tendency. Therefore, it is preferred to use at least one component selected only from these metabolizable oils or mineral oils as the oil component. In addition, it is particularly preferred to use an ester type oil-based composition comprising an ester derived from a fatty acid containing not less than 85% by weight of cis-Δ9-octadecenoic acid and not less than 90% by weight of cis-Δ9-enoic acid, and an alcohol such as glycerol, diglycerol, propylene glycol, ethanol, decanol and oleyl alcohol containing not less than 85% by weight of cis-Δ9-octadecenoic alcohol and not less than 90% by weight of cis-Δ9-enol. Alternatively, a mixture of the aforementioned ester type oil and squalene can also be used in the present invention.

[0047] The invention selects white oil including Drakeol 6VR white mineral oil and n-hexadecane for comparison. By inoculating foot-and-mouth disease vaccine containing Drakeol 6VR and n-hexadecane, the antibody titer is compared, and n-hexadecane is preferred. Figure 4 ). The emulsion contains 80% Drakeol 6VR and n-hexadecane, 10% lipophilic composite emulsifier A or B, 10% hydrophilic composite emulsifier A or B, and 20μg / 2mL of foot-and-mouth disease antigen. Serum was collected 28 days after immunization for ELISA identification. The results showed that the antibody level of the foot-and-mouth disease vaccine inoculated with n-hexadecane water-in-oil-in-water two-phase emulsion was significantly higher than that of the Drakeol 6VR vaccine. At the same time, the combination of lipophilic composite emulsifier B and hydrophilic composite emulsifier B is the best choice for the two-phase adjuvant.

[0048] The adjuvant preparation and selection process was further optimized, and the water-in-oil-in-water vaccine adjuvant included the following components in weight percentage: 5% hydrophilic composite surfactant, 5% lipophilic composite surfactant, and 90% mineral oil.

[0049]

[0050] Embodiment 2:

[0051] The adjuvant invention described in Example 1 is further added with surfactant saponin VSP70, which is a compound of plant saponin VSP and nucleic acid VDN70 polysaccharide VPLA70. It has diversified action sites for activating immunity, and has a good balance between cellular and humoral immunity and the production of various antibodies. VSP70 is a low-amount, high-efficiency enhancer, which can be prepared into a solution and filtered when used, and emulsified with the antigen adjuvant for use.

[0052] The animal vaccine adjuvant of the present invention is composed of the following components by weight percentage:

[0053] Mineral oil 90%: n-hexadecane

[0054] Lipophilic emulsifier 5%: Span 20 (20%), Span 40 (27%), Span 60 (19%), Span 65 (7%), Span 80 (23%), Span 85 (4%)

[0055] Hydrophilic emulsifier 5%: Tween 20 (15%), Tween 40 (15%), Tween 60 (47%), Tween 80 (23%)

[0056] Immune booster: VS P70 (3 mg per serving)

[0057] The vaccine was prepared by mixing the invented adjuvant with foot-and-mouth disease antigen, and antibody immune evaluation was performed. Serum was collected on days 0, 14 and 28 after immunization for antibody level detection. The results showed that the prepared vaccine was stable and not demulsified by the strong surfactant saponin immunoenhancer. There were no abnormalities in the animals after vaccination, and no side effects such as fever were observed. Antibody level detection showed Figure 5 The present invention can significantly improve the antigen immune response of animals and produce high titer antibodies when using less antigen.

[0058] Embodiment 3:

[0059] The present invention provides the use of the above-mentioned water-in-oil-in-water vaccine adjuvant for preparing a vaccine. Preferably, the use comprises the following steps: under low-speed stirring conditions, dropwise adding an inactivated antigen liquid to the water-in-oil-in-water vaccine adjuvant, and stirring at a low speed for 30 minutes after the dropwise addition is completed, thereby obtaining the vaccine. The above steps are performed under a sterile environment, and the raw materials used for the preparation are also sterile raw materials.

[0060] The technical solution provided by the present invention preferably meets the needs of large-scale production and has an immune-enhancing effect. The addition method is designed according to the controlled release principle of water-in-oil-in-water microspheres, so that it can directly act on immune cells more effectively, achieving a longer-lasting effect and maximum bioavailability. Compared with traditional adjuvants, the water-in-oil-in-water vaccine adjuvant of the present invention is easier to inject, can induce short-term immunity and long-term immunity at the same time, improve immune efficacy and cellular immunity level, have a long duration of immunity, are not easy to cause irritation, and reduce the amount of oil used in existing inactivated vaccines.

[0061] The present invention selects raw materials that meet the needs of large-scale production, greatly reducing the preparation cost. At the same time, a method for preparing a vaccine using the same is matched. After repeated tests, the quantitative changes of the components and the changes in the sequence of the operating steps will affect the coating effect of the emulsion, the viscosity, stability, immune effect and absorption after injection of the finished seedling.

[0062] Large-scale emulsification can be emulsified by low-speed stirring (low cutting force of about 80-120rpm) for more than 30 minutes. Laboratory tests can be performed at about 300-500rpm for about 30 minutes. The simplest method is to mix the adjuvant and antigen directly by hand. Experiments have shown that hand-cranking and magnetic stirrers cannot guarantee the stability of the emulsion. Note: After the adjuvant and antigen are mixed, there is a "high viscosity transition period" of several tens of seconds. Continue to stir through this viscous transition period to form a normal emulsion. Avoid stirring too fast to produce bubbles during the emulsification process, as the self-emulsification process is easily destroyed by high shear force. The emulsion is best stored at 5-8°C, and freezing and high temperatures should be avoided. Repeated opening of the adjuvant in the same container may cause the unsaturated fatty acid component in the adjuvant to oxidize and become ineffective when exposed to air. The adjuvant can be sterilized by high-pressure steam sterilization at 121°C for 30-60 minutes, or by using a 0.2μm filter membrane for sterilization. If there is precipitation after sterilization at 121℃, it needs to be cooled to 30℃ and gently shaken to dissolve.

[0063] The water-in-oil-in-water vaccine adjuvant provided by the present invention has good safety, no local retention and side effects, and does not affect system health, body temperature and animal comfort. It overcomes the defects of poor needle permeability, high local residue, strong side effects, and affected carcass quality, weight gain, feed-to-meat ratio and reproductive capacity after slaughter of traditional white oil high-shear emulsions. The final product is more stable, and the stability depends on the correct dosage form and small and uniform particle size, which is uniform and does not aggregate. This characteristic also determines the antigen presentation effect and effectiveness of the emulsion. The emulsion is stable at 40°C for more than 30 days without abnormality. The self-emulsification process ensures a complete success rate, the emulsification operation is simple, the emulsion is stable and has a strong ability to resist high temperature, and has good antigen matching. The water-in-oil adjuvant can be used for all vaccines. It is more functional and can induce rapid and lasting protective immunity. The amount of antigen required for protective titer is lower, and cross protection between variants can be improved. The injection is simple, easy to vaccinate, and the viscosity can be ignored. Due to the precise balance of various emulsifiers, there will be no coagulation or clotting phenomenon caused by the original process defects in the early days of biphasic adjuvants.

[0064] Comparative Example 1:

[0065] This comparative example evaluates the safety and immunoenhancing effect of the adjuvant of the present invention by testing cattle, and evaluates the replacement of French SEPPIC MONTANIDE TM Possibility of ISA 206 VG adjuvant. The preparation method of the vaccine comprises the following steps

[0066] S1: Foot-and-mouth disease O type OS / 99 strain and Asia 1 type Jiangsu strain cytotoxic Asia / JSL / ZK / 06

[0067] Test strain O: OS / 99 suckling rat virus, Asia 1 Asia / JSL / GSZY / 06 suckling rat virus

[0068] S2: OS / 99 suspended inactivated antigen: K OS / 99 130503, pH 7.82, antigen provided by the workshop, for standby use. Asia-Ⅰ suspended inactivated antigen: KAsia-1 130502, pH 7.98, antigen provided by the workshop, for standby use.

[0069] S3: The OS / 99 and Asia / JSL / ZK / 06 cell venoms were purified, concentrated and inactivated according to the existing foot-and-mouth disease type O and Asia type 1 bivalent inactivated vaccine production procedures, and then prepared into 40,000 mL of bivalent antigen liquid, which was divided into two parts. One part was emulsified with SEPPIC 206 adjuvant to form a bivalent inactivated vaccine, and the other part was prepared with adjuvants according to Example 2 and emulsified into a bivalent inactivated vaccine according to the same antigen ratio. The above antigen adjuvants were mixed in a 1:1 ratio and diluted according to the conventional vaccine preparation ratio. The pH after dilution was 7.91, and the vaccine was prepared according to the scheme described in Example 3 to prepare 200 mL of vaccine.

[0070] 1. Comparison of physical and chemical properties

[0071]

[0072]

[0073]

[0074] The results showed that the moisture content of the present invention slightly exceeded the standard in various test indicators, while the rest met the quality standards. All the test indicators of SEPPIC met the quality standards. In the stability test, the test seedlings of the present invention had good stability when stored at 4°C, and slightly worse stability than the same batch of SEPPIC adjuvanted seedlings when stored at 37°. There was no significant difference in the centrifugation at 3000rpm for 15min on the second day and two months after the preparation of the two groups of vaccines. The sterility test results of the two groups of vaccines were both sterile, and there was no stratification or odor before and after the use of the adjuvant of the present invention.

[0075] 2. Safety inspection: Note: “↓” means no abnormality; “-” means no abnormality

[0076]

[0077] Psychological appetite and local reactions 24 hours after vaccination:

[0078]

[0079] The results showed that the body temperature of cattle increased after immunization with the vaccine, but there was no abnormality.

[0080] Table 1 Safety test on cattle (I)

[0081]

[0082] Table 2 Safety test on cattle (II)

[0083]

[0084] After the 2nd and 3rd doses were injected into the cattle, there was no abnormality in spirit and appetite, and no adverse reactions were observed at the injection site. After the 2nd and 3rd doses were injected into the cattle, there was no abnormality in body temperature increase, and after the 3rd doses were injected into the cattle, there was no abnormality in body temperature increase.

[0085] 3. Effectiveness test:

[0086] The vaccines prepared in the above two groups were used to immunize cattle with negative foot-and-mouth disease antibody test. The vaccine of the present invention was used to immunize 30 cattle, and SEPPIC ISA 206VG was used to immunize 10 cattle, of which 5 were immunized with 1 / 3 dose and 5 were immunized with 1 / 9 dose. The vaccine of the present invention and SEPPIC ISA 206VG were used for immunization, and the virus was challenged 21 days after immunization. After the virus was challenged, the serum was separated to measure the foot-and-mouth disease antibody. The vaccine of the present invention was used to immunize Asia-1 and OS / 99, and SEPPIC ISA 206VG was used to immunize Asia-1. The results are as follows:

[0087]

[0088] Results of antibody test before virus attack:

[0089]

[0090] According to the spirit of the notice of the Ministry of Agriculture and Rural Affairs on the issuance of the "2012 National Animal Disease Compulsory Immunization Plan", the antibody titer of the O-type foot-and-mouth disease inactivated vaccine antibody liquid phase blocking ELISA ≥ 26 is considered qualified, and the qualified rate of livestock immune antibodies ≥ 70% is considered qualified. The qualified rate and average antibody value of each immune group are shown in the following table:

[0091]

[0092]

[0093] The results show that the antibody level and qualified rate of the vaccine prepared by the present invention are higher than those of the SEPPIC ISA206VG vaccine immunized with the same dose.

[0094] Comparative Example 2:

[0095] This comparative example evaluates the safety and immunoenhancing effect of the adjuvant of the present invention by conducting a pig test, and evaluates the possibility of replacing the SEPPIC ISA 206VG adjuvant. The two vaccine adjuvants and the inactivated foot-and-mouth disease type O virus antigen MYA98 strain were used to prepare a foot-and-mouth disease type O inactivated vaccine in a mass ratio of 1:1.

[0096] The preparation method of the vaccine comprises the following steps

[0097] S1: Antigen is inactivated antigen of swine foot-and-mouth disease type O Mya98 strain, 6 times concentrated

[0098] S2: Adjuvant preparation, SEPPIC ISA 206VG adjuvanted vaccine was produced according to the workshop process at 800,000 ml, the adjuvanted vaccine of the present invention was prepared in the laboratory at 300 ml according to Example 2, and 100,000 ml was tested in the workshop according to Example 3, the aqueous phase was taken out from the aqueous phase tank in the workshop, and the aqueous phases of the two adjuvants were exactly the same.

[0099] S3: Adjuvant and antigen emulsification method

[0100] The adjuvant and antigen were placed in a constant temperature water bath and heated to 30°C. The aqueous phase antigen was slowly injected into the oil phase adjuvant at a weight ratio of 1:1 for mixing. The emulsion was stirred in a 30°C water bath with an electric stirrer (speed 500 rpm / min) for 40 min, then taken out and placed in 4°C ice water for cooling. The vaccine can be prepared with reference to Example 3, packaged in 50 ml / bottle, and sterilized and qualified in physical and chemical properties inspection for use.

[0101] 1. Comparison of physical and chemical properties:

[0102] The present invention SEPPIC ISA 206VG Viscosity (flow rate) 3-5 seconds 6-7 seconds Emulsified phase (water drops) Uniform diffusion, uniform mist Uniform diffusion, uniform mist Stability (centrifugal 4000 rpm) No stratification No stratification Stability at 37°C 3 days without delamination 17 days without stratification Uniformity (microscope inspection) Uniform Uniform

[0103] 2. Safety inspection

[0104] Two guinea pigs weighing 350-450g were used, and each was subcutaneously injected with 2.0mL of the vaccine; five mice weighing 18-22g were used, and each was subcutaneously injected with 0.5mL of the vaccine. The animals were observed for 10 consecutive days, and weight changes and adverse reactions were recorded. No deaths or obvious local and systemic adverse reactions caused by the vaccine should occur. The main observation was the absorption of the vaccine. The results showed that the guinea pigs absorbed the vaccine well at the injection site, and there were no obvious adverse reactions.

[0105] 3. Side effect test

[0106] In the efficacy test, the vaccination dose was 1 dose per group and the adverse reactions were measured at the same time. The body temperature was measured before immunization and 1 hour after injection. The measurement results were recorded, and the breathing, spirit, and feeding status were observed for 3 days. After the end of this experiment, the animals that did not get sick were injected with 10mL of vaccine and the adverse reactions were observed as above. The results showed that there were no obvious adverse reactions in all animals after single dose and 5 times large dose injection.

[0107] 4. Effectiveness test

[0108] The vaccines prepared in the above two groups were used to immunize piglets weighing about 40 kg with negative FMD antibody test results (cell neutralizing antibody titer not higher than 1:8, ELISA titer not higher than 1:8 or suckling mouse neutralizing antibody titer not higher than 1:4). The body temperature, spirit and appetite were normal before the test. Each group of vaccines immunized 15 pigs and divided them into 3 groups, with 5 pigs in each group. The vaccine was divided into three dose groups: 1 dose (2mL), 1 / 3 dose, and 1 / 9 dose. Five pigs were injected intramuscularly behind the ear roots in each dose group. 28 days after vaccination, together with 2 control pigs, each pig was injected intramuscularly with 1.0mL (containing 103.0ID50) of swine O type FMD virus behind the ear roots. After continuous observation for 10 days, the control pigs should have at least one hoof with blisters or ulcers. Immunized pigs were judged to be unprotected if they showed any symptoms of FMD. Isolate the sick pigs in time.

[0109]

[0110]

[0111]

[0112] The adjuvant of the present invention is slightly thicker than SEPPIC ISA 206VG adjuvant in physical properties, and there is no significant difference in other aspects, which may affect the absorption after immunization, but it may also be the fundamental factor for the high particle density and high potency. From the efficacy test, the adjuvant of the present invention has 13.97 PD50, which is significantly better than SEPPIC ISA 206VG adjuvant 5.96 PD50 (animals are randomly grouped).

Claims

1. A self-emulsifying vaccine adjuvant containing a submicron-sized water-in-oil-in-water biphasic emulsion oil phase, characterized in that: By weight percentage, the composition is as follows: Mineral oil 90%; n-Hexadecane; Lipophilic emulsifier 5%: Span 20, 20%; Span 40, 27%; Span 60, 19%; Span 65, 7%; Span 80, 23%; Span 85, 4%; Hydrophilic emulsifier 5%: Tween 20, 15%; Tween 40, 15%; Tween 60, 47%; Tween 80, 23%; Immune enhancer: VSP70, 3 mg per serving.

2. A self-emulsifying vaccine adjuvant containing a submicron-sized water-in-oil-in-water biphasic emulsion oil phase according to claim 1, characterized in that: The submicron water-in-oil-in-water two-phase emulsion oil phase contains 0.5-50% mineral oil by mass, 0.1-10% two-component lipophilic emulsifier, and 0.05-25% cocktail combination hydrophilic non-ionic surfactant. After being combined with aqueous vaccine antigens, it is naturally assembled into 1-5 internal water phases containing 0.00001-1% immunogens, and the water dispersed phase with a diameter of 5-30nm is wrapped in the oil dispersed phase; the oil dispersed phase is surrounded by the second water phase and the external water continuous phase to form a three-phase emulsion water-in-oil-in-water.

3. A self-emulsifying vaccine adjuvant containing a submicron-sized water-in-oil-in-water biphasic emulsion oil phase according to claim 3, characterized in that: The average droplet size of the submicron emulsion is in the range of about 0.1-0.5 μm, preferably in the range of 0.18-0.24 μm, with a peak value of 0.2 μm.

4. A self-emulsifying vaccine adjuvant containing a submicron-grade water-in-oil-in-water biphasic emulsion oil phase according to claim 2, characterized in that: In the oil phase of the water-in-oil-in-water two-phase emulsion, the content of oily particles with a size between 0.1 and 0.3 μm accounts for 80% to 90%.

5. A self-emulsifying vaccine adjuvant containing a submicron water-in-oil-in-water two-phase emulsion oil phase according to claim 2, characterized in that: The oil phase of the water-in-oil-in-water double-phase emulsion contains a plurality of surfactants of polyoxyethylene sorbitan monooleate and sorbitan monooleate to form a double cocktail emulsifier.

6. A self-emulsifying vaccine adjuvant containing a submicron-sized water-in-oil-in-water biphasic emulsion oil phase according to claim 1, characterized in that: The mineral oil is selected to be an oily component that is liquid at room temperature of 15-25°C.

7. A self-emulsifying vaccine adjuvant containing a submicron-sized water-in-oil-in-water biphasic emulsion oil phase according to claim 1, characterized in that: It also includes adding surfactant saponin VSP70, which is composed of plant saponin VSP and nucleic acid VDN70 polysaccharide VPLA70.

8. A method for preparing a self-emulsifying vaccine adjuvant containing a submicron-level water-in-oil-in-water two-phase emulsion oil phase, applied to the self-emulsifying vaccine adjuvant containing a submicron-level water-in-oil-in-water two-phase emulsion oil phase as claimed in claim 1, characterized in that: Under low-speed stirring conditions, the inactivated antigen liquid is added dropwise to the water-in-oil-in-water vaccine adjuvant, and after the addition is completed, the vaccine is obtained by stirring at a low speed for 30 minutes.

Images