KR20200057333A - A vaccine adjuvant composition for animals - Google Patents

Abstract

The present invention relates to a novel vaccine adjuvant composition. More specifically, the present invention relates to a vaccine adjuvant composition for animals with increased stability and immunogenicity. A vaccine adjuvant according to the present invention is a W/O emulsion vaccine adjuvant, which contains a non-ionic hydrophilic surfactant having a HLB value of 9 or higher, a non-ionic lipophilic surfactant, mineral oil and/or a stabilizer.

Description

Vaccine adjuvant composition for animals with increased stability and immunogenicity {A vaccine adjuvant composition for animals}

The present invention relates to novel vaccine adjuvant compositions. More specifically, the present invention relates to a vaccine adjuvant composition for animals with increased stability and immunogenicity.

When a vaccine for preventing or treating a disease is administered to an animal, the efficacy of the vaccine is important, but various in vivo environments that can reduce the efficacy of the vaccine should be considered. To this end, technologies have been developed to deliver vaccines using emulsions, liposomes, nanoparticles, etc., and recently, vaccine adjuvants for immunostimulation (adjuvants, adjuvants), and receptors ( Agonists using receptors have been developed (Seth A et al., Non-Carrier Nanoparticles Adjuvant Modular Protein Vaccine in a Particle-Dependent Manner.PloS one. 2015; 10). As an adjuvant or adjuvant, adjuvants can be prepared in a variety of ways and are widely used in vaccine production. In general, about 30 to 40% of the total weight of the vaccine composition is composed of an antigen, and about 60 to 70% is composed of a vaccine adjuvant, and the vaccine adjuvant should be able to be provided by an excellent reproducible manufacturing method. As such, the vaccine adjuvant does not constitute a specific antigen, but refers to an agent that increases immunogenicity and increases the half-life of the antigen in vivo, and stability and efficacy are the most important, and safety and economics are also important factors.

Various viruses are responsible for infectious diseases in animals. For example, a virus that causes infectious diseases in birds, such as Newcastle disease virus (ND), Avian influenza virus (H9N2), Infectious Bronchitis virus, or Scatter hypoviral virus ( Egg drop syndrome virus).

As an oil emulsion used as a vaccine adjuvant, a highly viscous Freud's adjuvant produced through a three-stage emulsion process step has been widely used, particularly in the manufacture of animal vaccines. Although the mechanism of action of the oil emulsion is not well known, it is believed to be similar to the mechanism of action of the aluminum salt, and it is known that a single inoculation causes the antigen to shed longer than the aluminum salt adjuvant and induce a higher immune response. It also appears to improve lymphocyte infiltration and antigenic expression of dendritic and macrophage cells. Subsequently, as a water in oil (W / O) emulsion mainly used in animal vaccine compositions, SEPPIC's MONTANIDE (trade name) ISA70 and ISA71 have been developed and are commercially available. The water-in-oil emulsion has a continuous emulsion state and induces a high level of continuous immune response, but has a high viscosity and is difficult to inject. To overcome these disadvantages, triple emulsions or water-in-oil emulsions have been developed. These emulsions have lower viscosity compared to water-in-oil emulsions, but have low stability and difficulty in production. It is also important to emulsify properly so that phase separation of the emulsion does not occur during storage. The recently developed oil-in-water emulsion type MF-59 has been reported to have superior antibody production capacity and to induce both humoral and cellular immune responses than aluminum salts, making it useful for vaccines such as HIV and HSV. It is considered safe for everyone.

In addition, refined mineral oils such as Darkerol 6 VR, Marcol 52, Marcol 82, Sontex 55, vestain A50B, and Whitetrex 307 are used for vaccine production, but recently, animal oils such as vegetable oils such as peanuts and sesame oil and shark liver oil Also developed as an emulsion adjuvant.

As a value indicating the hydrophilicity and lipophilicity of the surfactant, a HLB (Hydrophile Lipophile Balance) value is used. HLB value = 20 × defined as the molecular weight / molecular weight of the hydrophilic group, the HLB value is in the range of 0 to 20, and the larger the value, the stronger the hydrophilicity. In general, HLB values 1 to 3 are suitable as antifoaming agents, 3 to 7 are W / O emulsifiers, 7 to 9 are wetting agents, 8 to 18 are O / W type emulsifiers, 11 to 15 are cleaning agents, and 15 to 20 are solubilizing agents. It is said that.

The present inventors tried to develop a more stable and immunogenicity enhanced vaccine adjuvant. As a result, in preparing a W / O emulsion vaccine adjuvant, a nonionic hydrophilic surfactant having a HLB value of 9 or more, a nonionic lipophilic surfactant, a mineral oil, and a stabilizer are used together, and the aqueous phase ratio of the vaccine adjuvant is increased. Thus, the present invention was completed by preparing a vaccine adjuvant for animals having remarkably excellent properties.

Korean Patent Application No. 10-1998-0703215

Seth A, Ritchie FK, Wibowo N, Lua LHL, Middelberg APJ. Non-Carrier Nanoparticles Adjuvant Modular Protein Vaccine in a Particle-Dependent Manner. PloS one. 2015; 10.

As such, the present invention aims to provide an adjuvant vaccine for animals.

It is also an object of the present invention to provide a method for preparing an animal vaccine adjuvant.

It is also an object of the present invention to provide a vaccine composition comprising an animal vaccine adjuvant.

It is also an object of the present invention to provide a method for immunizing an animal with an animal vaccine composition.

In order to achieve the above object, the present invention is a mineral oil to surfactant is 50 ~ 90: 50 ~ 10, preferably 70 ~ 95: 30 ~ 5, more preferably 80: includes 20, the water phase ratio A water-in-oil (W / O) emulsion vaccine adjuvant that is 30 or more. In one embodiment related to this, the mineral oil to surfactant may be preferably 90:10. In another related embodiment, the vaccine adjuvant of the present invention is a mineral oil, a surfactant having a HLB value of 9 to 13 as a nonionic hydrophilic surfactant, a surfactant having a HLB value of more than 14 as a nonionic hydrophilic surfactant, a nonionic Lipophilic surfactants; And surfactants used as stabilizers. Preferably, the mineral oil is MARCOL 52® (Esso, France), MARCOL 82® (Esso, France), DRAKEOL 6VR® (Penreco), Castor Oil, Squalan, Polydecene, Sontax 55 , Vestain A50B and Whitertrex 307. Preferably, the surfactant having a HLB value of 9 to 13 as the nonionic hydrophilic surfactant is ethoxylated fatty acid monoester of sorbitan (especially 5 ethoxyl group) (e.g., Tween81 Ethoxylated sorbitan monooleate such as ®, ethoxylated fatty acid diesters of sorbitan, ethoxylated fatty acid ester of sorbitan triesters of sorbitan (especially 20 ethoxylated groups) (e.g., ethoxylated sorbitan trioleate, such as Tween85®, ethoxylated sorbitan trioleate, such as Tween65®, ethoxylated sorbitan tristearate, ethoxylated fatty alcohols (especially 5 to 10 ethoxyl groups) (e.g. Brij76®, Brij56®, Brij96®), ethoxylated fatty acids ( In particular, 5 to 10 ethoxyl groups) (e.g. Simulsol 2599®, Myrj45®), ethoxylated castor oil (especially 25 to 35 ethoxyl groups) (e.g. Arlatone650®, ArlatoneG ®) includes one or more selected from the group consisting of: Preferably, the surfactant having an HLB value of more than 14 as the nonionic hydrophilic surfactant is ethoxylated fatty acid monoesters of sorbitan. sorbitan) (especially 20 ethoxyl groups) (e.g., ethoxylated sorbitan monolaurate such as Tween20®, Tw ethoxylated sorbitan monopalmitate, such as een40®, ethoxylated sorbitan monostearate, such as Tween60®, ethoxylated sorbitan monostearate, such as Tween80® Ethoxylated sorbitan monooleate, ethoxylated fatty alcohols (especially 15 to 30 ethoxylated groups) (e.g. Brij78®, Brij98®, Brij721®), ethoxylated fatty acids acids) (especially 15-30 ethoxyl groups) (e.g. Myrj49®, Myrj51®, Myrj52®, Myrj53®), non-ionic block copolymers (e.g. LutrolF127®, LutrolF68 Polyoxyethylene / polyoxypropylenecopolymer (POE-POP)). Preferably, the nonionic lipophilic surfactant is sorbitan fatty acid esters of sorbitan (e.g., sorbitan monolaurate, such as Span20®, sorbitan monolaurate, such as Span40®). Sorbitan monopalmitate, sorbitan monostearate such as Span60®, sorbitan tristearate such as Span65®, sorbitan monooleate such as Span80®, sorbitan monooleate, Span85® Sorbitan trioleate, sorbitan monoisostearate, such as Arlacel987®, sorbitan isostearate, such as Crill6®), and fatty acid esters of mannide ( E.g. Montanide80®), mannide monooleate (e.g. ArlacelA®), mannide dioleate, mannide trioleate, mannide tetraoleate (mannide tetraoleate)), ethoxylated fatty acid esters of mannide (2,3 or 4-ethoxyl groups) (e.g. Montanide 888®, Montanide 103®, ethoxylated Ethoxylated mannide monooleate, ethoxylated mannide dioleate, ethoxylated mannide trioleate, ethoxylated mannide trioleate, ethoxylated mannide tetraoleate). Preferably, the surfactant used as the stabilizer is lecithin, stearic acid, stearyl alcohol, cholesterol, octanoic acid, cholesteryl linoleate (cholesteryl linoleate), cholesteryl oleate, palmitic acid, lauric acid, triton X-100, tricon X-102, octyl BD-glucopyranoside (octyl BD -glucopyranoside).

The present invention also provides a vaccine composition comprising the adjuvant and antigen protein of the present invention. In one related embodiment, the antigen protein may be avian influenza (AI) virus antigen, Newcastle disease (ND) virus antigen, chicken infectious bronchitis virus, or chicken spawning hypothesis (EDS) virus, but is not limited thereto. It does not work. In another embodiment related to this, the vaccine composition of the present invention may be 50 to 70: 30 to 50 (w / w) of the vaccine adjuvant to the antigen protein, but is not limited thereto.

The present invention also provides a method for immunizing an animal by administering the vaccine composition for animals of the present invention to an animal in need of administration of the vaccine.

The vaccine adjuvant of the present invention is excellent in immunogenicity, excellent in antigen amount and sustained release, and is adjustable. In addition, the vaccine adjuvant of the present invention has a viscosity of 21 cP and the viscosity of the vaccine emulsion is 20 to 40 cP. It was found to be stable for more than 6 weeks under severe conditions due to its excellent stability.

1 is a result of analyzing the release rate of the antigen protein from the vaccine composition prepared by varying the content of the vaccine adjuvant of the present invention.
2 is a result of performing an immunogenicity test for SPF chicken using a vaccine composition containing the vaccine adjuvant of the present invention.
3 is a result of performing an immunogenicity test for chickens of a clinical farmhouse using a vaccine composition containing the vaccine adjuvant of the present invention.
4 schematically shows an experiment of inoculating mice using a vaccine composition comprising the vaccine adjuvant of the present invention.
5 is a result of immunizing mice with a vaccine composition comprising the vaccine adjuvant of the present invention and analyzing the efficacy of the vaccine.
Figure 6 shows the results of a cellular immunoassay using a vaccine composition comprising the vaccine adjuvant of the present invention.

The present invention provides a vaccine adjuvant for providing a vaccine composition with increased stability and immunogenicity, comprising a mineral oil and a surfactant. In the present invention, mineral oil is MARCOL 52® (Esso, France), MARCOL 82® (Esso, France), DRAKEOL 6VR® (Penreco), Castor Oil, Squalan, Polydecene, Sontax 55, Vestain A50B Or selected from Whitertrex 307, the surfactant is a nonionic hydrophilic surfactant is a surfactant having a hydrophilic-lipophilic (HLB) value of 9 to 13, ethoxylatedfatty acid monoester of sorbitan of sorbitan (Especially 5 ethoxyl groups) (e.g., ethoxylated sorbitan monooleate, such as Tween81®, ethoxylated fatty acid diesters of sorbitan, sorbate) Ethoxylated fatty acid triesters of sorbitan (especially 20 ethoxyl groups) (e.g., ethoxylated sorbitan trioleate, Tween65®, such as Tween85®) Ethoxylated sorbitan tristearate, ethoxylated fatty alcohols (especially 5 to 10 ethoxyl groups) (e.g. Brij76®, Brij56®, Brij96® ), Ethoxylated fatty acids (especially 5 to 10 ethoxyl groups) (e.g. Simulsol2599®, Myrj45®), ethoxylated castor oil (especially 25 to 35) Ethoxyl group) (e.g., Arlatone650®, ArlatoneG®). Surfactants. In addition, the nonionic hydrophilic surfactant used in the present invention is a surfactant having a hydrophilic-lipophilic (HLB) value of more than 14, and ethoxylated fatty acid monoesters of sorbitan (especially 20). Ethoxyl groups) (e.g., ethoxylated sorbitan monolaurate, such as Tween20®, ethoxylated sorbitan monopalmitate, such as Tween40®, Tween60®, etc.) Ethoxylated sorbitan monostearate, ethoxylated sorbitan monooleate, such as Tween80®, ethoxylated fatty alcohols (especially 15 to 30 ethoxyl groups) (e.g. Brij78®, Brij98®, Brij721®), ethoxylated fatty acids (especially 15 to 30 ethoxyl groups) (e.g. Myrj49®, Myrj51® , Myrj52®, Myrj53®), non-ionic blockcopolymers (e.g., polyoxyethylene / polyoxypropylenecopolymer (POE-POP), e.g. LutrolF127®, LutrolF68 ®). In addition, nonionic lipophilic surfactants that can be used in the present invention include fatty acid esters of sorbitan (e.g., sorbitan monolaurate such as Span20®, Span40®). Sorbitan monopalmitate such as, sorbitan monostearate such as Span60®, sorbitan monostearate such as Span65®, sorbitan tristearate such as Span65®, sorbitan monooleate such as Span80® ( sorbitan monooleate, sorbitan trioleate such as Span85®, sorbitan monoisostearate such as Arlacel987®, sorbitan isostearate such as Crill6®, sorbitan isostearate Fatty acid esters of mannide (e.g. Montanide80®, mannide monooleate (e.g. ArlacelA®), mannide dioleate, mannide trioleate) , Mannide tetraoleate, ethoxylated fatty acid esters of mannide (2, 3 or 4-ethoxyl groups) (e.g. Montanide 888®, Montanide 103 ®, ethoxylated mannide monooleate, ethoxylated mannide dioleate, ethoxylated mannide trioleate, ethoxyle One or more surfactants selected from the group consisting of ethoxylated mannide tetraoleate can be used. Included as a stabilizer as other surfactants, Lecithin, Stearic acid, Stearyl alcohol, Cholesterol, Octanoic acid, Cholesteryl linoleate Linoleate, Cholesteryl Oleate, Palmitic acid, Lauric acid, Triton X-100, Triton X-102, and Octyl BD-glucopyranoside (OctylB-D- Glucopyranoside) may be one or more selected from the group consisting of.

Hereinafter, the present invention will be described based on Examples. The following examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these examples. It is needless to say that those skilled in the art to which the present technology pertains can be easily modified or substituted from the contents described in the detailed description including the drawings and claims of the present invention.

Manufacturing example:

Preparation Example 1: Preparation of vaccine adjuvant

As a mineral oil in the oil phase, white mineral oil (Penreco) or liquid paraffin (Liquid Paraffin) and surfactant (ethoxylated sorbitan trioleate, Croda) are matched with HLB (Hydrophilic Lipophilic Balance) and a high shear mixer ( High shear mixer) (PRIMIX HOMO MIXER Mark II) was mixed for several minutes. Next, sorbitan monoisostearate (Croda) and stabilizer octanoic acid are added as an amphiphilic surfactant in the water phase and oil phase, and then again for several minutes using a high shear mixer. Mixing to prepare a water-in-oil vaccine adjuvant. Vaccine adjuvant prepared according to this preparation example, mineral oil: HLB 9 to 13 nonionic hydrophilic surfactant; Nonionic hydrophilic surfactants with an HLB greater than 14; The ratio of the surfactant added as a nonionic lipophilic surfactant and a stabilizer was set to be 1 to 95: 1 to 10: 1 to 10: 0.1 to 2. In this embodiment, mineral oil was used MARCOL 52®, MARCOL 82®, DRAKEOL 6VR, Castor Oil, or squalene, and ethoxylated sorbitan monostearate, ethoxy as a nonionic hydrophilic surfactant. Ethoxylated sorbitan monooleate, ethoxylated fatty alcohol, or polyoxyethylene / polyoxypropylenecopolymer was used. Further, as the nonionic lipophilic surfactant, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, sorbitan trioleate , Or sorbitan monoisostearate was used, and lecithin, octanoic acid, palmitic acid, Triton X-100 or Triton X-102 were used as stabilizers. Became.

Preparation Example 2: Preparation of vaccine composition

A vaccine composition was prepared by mixing the antigen, which is a waterborne antigen, and the vaccine adjuvant, which is a cost. First, after adding the oil-based vaccine adjuvant prepared above to a beaker (500 mL) at 50 ~ 70 (w / w), while stirring at 1,000 ~ 3,000 rpm using a mixer, the waterborne avian influenza virus (Avian influenza virus H9N2 , National Veterinary Science and Quarantine Service) virus antigen, Newcastle disease La Sota (National Veterinary Science and Quarantine Service or rNDV-mF, Prof. Manseong Park, Hallym University) Virus antigen or Egg drop syndrome virus K11, National Veterinary Science and Quarantine Service virus Antigen was added slowly at a rate of 50-30 (w / w). After adding the antigen, the mixture was stirred and homogenized by stirring at a high shear rpm of 7,000 to 10,000 rpm for a few minutes to prepare a vaccine formulation in the form of a water-in-oil emulsion. As a control for comparison, an oil adjuvant Montanide ISA70 (manufactured by Seppic, France) was used and stirred with the antigen to prepare the same vaccine composition. Table 1 below (Preparation (% by weight) of a vaccine composition including an oil vaccine adjuvant) is a relative of these components in a vaccine composition prepared by mixing the vaccine adjuvant and commercial vaccine adjuvant of the present invention with avian influenza virus antigen or Newcastle disease virus antigen It shows the content.

ingredient Comparative Example 1 Comparative Example 2 Comparative Example 3 Preparation Example 1 Preparation Example 2 Preparation Example 3 Preparation Example 4 A
Vaccine Supplement Experimental group - - - 50 60 65 70 ISA70 50 60 70 - - - - B
antigen AI virus 10 10 10 10 10 10 10 ND virus 10 10 10 10 10 10 10 diluent ~ 100 ~ 100 ~ 100 ~ 100 ~ 100 ~ 100 ~ 100

* In the above, the experimental group means the vaccine adjuvant prepared in the preparation example according to the present invention, and ISA70 is a commercial vaccine adjuvant. AI stands for Avian Influenza, ND Newcastle Disease.

Preparation Example 3: Confirmation of emulsion formation

The vaccine adjuvant emulsion is a water-in-oil (W / O) type, distilled water is added to a transparent beaker, a few drops of the emulsion are dropped, and the emulsion is observed by visual observation. That is, when dropped in distilled water, it was confirmed that it is a W / O type by floating on the upper layer without mixing with distilled water. The homogeneity of the emulsion was observed using a microscope (Motic, Motic-AE-31, 400X), and as a result, the particle size was measured within 1 µm and was found to be uniformly distributed.

Preparation Example 4: Confirmation of the stability of the vaccine composition

The long-term storage stability of the vaccine composition of the present invention in emulsion form was evaluated. To this end, whether oil outflow, sedimentation, water outflow, or layer separation occurred was observed for 8 weeks under temperature cycling conditions circulating at 37 ℃, or 4 ℃ and 37 ℃, and the results were shown in Table 2 (Stability test of vaccine composition) Showed on.

Condition 4 ℃ 37 ℃ Temperature circulation Oil spill Water spill Layer separation Oil spill Water spill Layer separation Oil spill Water spill Layer separation Comparative Example 1 Instability Instability Instability Instability Instability Instability Instability Instability Instability Comparative Example 2 change
none change
none change
none Instability Instability Instability Instability Instability Instability Comparative Example 3 change
none change
none change
none change
none Instability Instability Instability Instability Instability Example 1 change
none change
none change
none change
none change
none change
none Instability Instability Instability Example 2 change
none change
none change
none change
none change
none change
none change
none change
none change
none Example 3 change
none change
none change
none change
none change
none change
none change
none change
none change
none Example 4 change
none change
none change
none change
none change
none change
none change
none change
none change
none

As a result, as shown in Table 2, in the case of the present composition containing a stabilizer, oil leakage and precipitation did not occur and were stable. Therefore, it can be confirmed that the composition according to the present invention has storage stability in long-term storage.

Example

Example 1: Safety test

For 6 to 8 week old SPF chickens (obtained from Namdeok SPF Co., Ltd.), one intramuscular inoculation of 2 minutes (1 ml) of the vaccine prepared in the above preparation example (the vaccine adjuvant of the present invention: the antigen is contained in a ratio of 7: 3) And observed for 14 days. The control group received the same amount of PBS. As a result, as shown in Table 3 below, no clinical signs such as death were observed in the group administered with the vaccine adjuvant of the present invention.

group Clinical signs (positive number / test number) Experimental group 0/5 Control 0/5

The SPF chickens aged 6 to 8 weeks were inoculated intramuscularly with the above vaccine 1 moisture (0.5 ml) and inoculated in the same manner after 2 weeks, and observed for 14 days. As a result, as shown in Table 4 below, no special clinical signs could be observed.

group Clinical signs (positive number / test number) Experimental group 0/5 Control 0/5

These results support that the vaccine adjuvant of the present invention has no safety problems.

Example 2: Vaccine composition antigen release rate and viscosity test

Confirmation of the rate at which antigen (algal influenza (AI) virus antigen, Newcastle disease (ND) virus antigen) or protein is released from the avian inactivated vaccine composition prepared with water-in-oil (W / O) emulsion according to the present invention To do this, an in vitro antigen release test was performed. A certain amount of the vaccine composition was put into a beaker containing a diluent, and the amount of the antigen released into the diluent was measured using a protein quantification kit by sampling over time while stirring at a constant rate. As shown in Fig. 1, the release rate of the antigen was different depending on the composition of the vaccine adjuvant (type and composition ratio of surfactant or stabilizer). The vaccine composition is a vaccine composition prepared according to the above preparation example, and the low, medium and high contents contained 10%, 4% and 1% of the vaccine adjuvant, respectively, based on the surfactant weight in the vaccine composition. These results indicate that the vaccine adjuvant of the present invention can effectively control the delay of antigen release according to its composition.

As a result of measuring the viscosity of the algae-inactivated vaccine composition prepared with a water-in-oil (W / O) emulsion containing a vaccine adjuvant of various compositions according to the present invention using a Brookfield viscometer according to the manufacturer's instructions, the vaccine composition was antigen-to-antigen. It was confirmed to have various viscosities of 22.2 to 67.9 cP depending on the content ratio of the vaccine adjuvant. In particular, when the ratio of the antigen to the vaccine adjuvant was 3: 7, it was found that the viscosity of the commercial adjuvant (ISA70) prepared at the same ratio was 38.2 cP, and the viscosity was low. These results suggest that the vaccine adjuvant of the present invention can be used for the preparation of a vaccine composition having a low viscosity, which can greatly reduce the incidence of injection and abnormal inoculation of the inoculation site.

Example 3: Immunogenicity test of vaccine composition

(3-1) Immunogenicity test in SPF chicken

A vaccine containing ISA70 as a vaccine adjuvant and a positive control according to the present invention was prepared according to the above preparation example, and then inoculated into the primary muscles of SPF chickens and bled 3 weeks after inoculation, resulting in a hemagglutination inhibition assay (HI) ) The titer was confirmed. PBS was inoculated as a negative control. The results are shown in FIG. 2. In Figure 2, WIO1 to WIO4 were prepared according to the above-described preparation example, so that each had the following composition. As a result, as shown in Figure 2, it was found that the vaccine containing the vaccine adjuvant according to the present invention has excellent immunogenicity. In particular, WIO3 appeared to be the best.

ingredient WIO1 WIO2 WIO3 WIO4 Mineral Oil (Drakeol 6VR) to 100 to 100 to 100 to 100 Nonionic hydrophilic surfactant (ethoxylated sorbitan trioleate) 10 8 8 6 Nonionic lipophilic surfactant (Sorbitan monoisostearate) One 1.6 4 6 Stabilizer (Octanoic acid) 2 2 2 2

(3-2) Immunogenicity test on clinical farm chickens

WIO3 judged to be the best in (3-1) was named CAvant WO-A1, and the immunogenicity of the vaccine containing the same was confirmed using chickens from a clinical farm. A vaccine composition comprising ISA70 (manufactured by Seppic, France) commercially available as a WO-A1 positive control of the present invention for 14 hens, 1,193 to 1,267 g, and 20 hens of domestic (Chungcheongbuk-do) farms according to the above preparation example Prepared and prepared. As the antigen, Newcastle virus antigen and avian influenza antigen were used. After pre-bleeding, the first inoculation, 2 weeks after the first inoculation, and the second inoculation, the results were compared with the inoculated control group (negative control group) for 14 weeks. The vaccine composition was inoculated with one or two minutes (0.5 ml). As a result, as shown in Fig. 3, the vaccine composition containing the vaccine adjuvant of the present invention showed excellent HI titers in both Newcastle disease and avian influenza virus.

(3-3) Immunogenicity test for mice

In order to evaluate the efficacy of the antigen protein and the vaccine adjuvant (WO-A1 or ISA70), mice (5 weeks old female BALB / c mice, Korea-Japan experimental animals) were inoculated with antigen proteins at primary and secondary intervals of 2 weeks, followed by secondary inoculation After 4 weeks, the body weight and survival rate of mice were compared for 1 LD ₅₀ infection of the influenza A virus H5N2 strain (A / bird / Korea / w81 / 2005) (Source: Prof. Young-Gi Choi, Lab., Chungbuk National University School of Medicine). For the experiment, (i) PBS control, (ii) antigenic protein, a group in which the vaccine adjuvant (WO-A1) according to the present invention was mixed, and (iii) three groups in which a commercial adjuvant (ISA70) was mixed in the antigenic protein. Was prepared. Each group consisted of 5 mice, and 5 groups were separately formed for ELISA measurement to measure antibody formation by the vaccine. Inoculation of the antigen protein was intramuscularly administered with purified M2HA2 protein (SEQ ID NO: 1, synthesized by Bioprogen) every two weeks. The dose of the antigen protein was set to 15 μg. Mixing of the antigen and the vaccine adjuvant was performed for 15 minutes using a 3-way stopcock and a syringe of the mixture prepared at the ratio of antigen 3 and adjuvant 7 (w / w). The total inoculation dose was 100 μl and the virus was infected 4 weeks after the last inoculation. Influenza virus H5N2 strain 1 LD ₅₀ dose of virus was used, the mice were anesthetized (Avertin, Sigma, 400 μl) and inoculated through the nasal cavity. After the virus infection, the mice were monitored and their body weight was measured. According to the policy of the Animal Ethics Experiment Committee, individuals who showed weight loss of more than 25% after virus infection were treated as euthanized and killed. As a result, as shown in Fig. 4, the survival rate of mice against influenza virus H5N2 strain 1 LD ₅₀ infection was significantly improved compared to the vaccine composition containing the control and commercial vaccine adjuvant, and weight loss compared to those of the vaccine composition. Appeared to be missing. These results support that the vaccine capacity of the antigenic protein is improved by the addition of the vaccine adjuvant of the present invention.

As a result of measuring the antibody titer in mice immediately before virus inoculation, a higher antibody was formed compared to the group inoculated with the antigen protein (M2HA2) alone in the group inoculated by mixing the vaccine adjuvant and commercial vaccine adjuvant of the present invention. Was confirmed.

Example  4: Mouse-related cellular immunity test

To determine whether T immune cells reacted by antigenic protein (M2HA2 protein) and participated in the immune function, splenocytes in the spleen of each group of mice (5 weeks old female BALB / c mice, 20 g body weight experimental animals) were isolated and antigens Protein-stimulated T-immune cell reaction experiments were performed. BD (trade name) ELISPOT set was used. The purified IFN-r antibody (imported by Sigma Aldrich) and purified IL-4 antibody (imported by Sigma Aldrich) were diluted 1: 200 in PBS and dispensed at 100 µl / well for coating. The next day, the spleen was isolated to obtain splenocytes, and the red blood cells were removed using ACK lysis buffer (Thermo Fisher Scientific), and then suspended in RPMI medium to spleen cells obtained from each individual at 100 µl / well. I was busy. The above-mentioned stimulants were equally diluted in RPMI medium (10% FBS) at a concentration of 3 μg / 100 μl and then dispensed at 100 μl / well. After storing in a 37%, 5% CO ₂ incubator for 24 hours, the detection antibody was reacted with streptabidin-HRP. Next, it was developed using AEC buffer (available from: Sigma Aldrich) to measure the number of spots caused by IFN-r and IL-4. Through the positive control mitogen (mitogen) stimulant (sigma-aldrich, importer, 1 μg / well) and the negative control (containing only the medium), it was confirmed that the experiment proceeded correctly. IFN-r and IL-4 were produced to confirm the formation of spots due to color development, which supports the contribution of antigenic proteins and adjuvants to the activated immune function of T immune cells.

Example  5: accelerated stability test

It was confirmed that the stability of the vaccine composition prepared in the W / O form was changed according to the ratio of the vaccine adjuvant (WO-A1) prepared according to the present invention and the antigen. First, a vaccine containing the vaccine adjuvant of the present invention and a commercially available ISA70 vaccine adjuvant and antigen at a ratio of 7: 3 and 6: 4, respectively, was prepared and prepared. In order to confirm long-term storage and stability for the prepared vaccine, 37 ° C, and 4 ° C, 37 ° C accelerated stability was observed for 6 weeks under test conditions. As a result, as shown in Table 6 below, it was confirmed that the vaccine containing the vaccine adjuvant of the present invention has superior stability compared to a vaccine containing a commercial vaccine adjuvant (ISA70) (positive control).

37 ℃ Temperature change (4 ℃ to 37 ℃) Vaccine adjuvant: antigen Oil spill Sedimentation Water spill Layer separation Oil spill Sedimentation Water spill Layer separation Positive control 7: 3 ○ ○ - - ○ ○ - ○ Positive control group 6: 4 ○ ○ ○ - ○ ○ ○ ○ Invention 7: 3 ○ - - - ○ - - - Invention 6: 4 ○ ○ - - ○ ○ - -

-: No change; ○: Change of properties, observation for 6 weeks

It was confirmed that the vaccine composition comprising the vaccine adjuvant and antigen according to the present invention showed increased viability and antibody titer as a result of administration to the animal. Therefore, the present invention may be widely used for enhancing immunogenicity in the preparation of a vaccine composition for animals.

<110> Choong Ang Vaccine Lab. <120> A vaccine adjuvant composition for animals <130> CVA18P02KR <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 609 <212> PRT <213> Artificial Sequence <220> <223> M2 + H5HA2 protein <400> 1 Thr Cys Ala Thr Thr Ala Thr Thr Ala Ala Cys Ala Gly Ala Ala Gly   1 5 10 15 Thr Thr Gly Ala Ala Ala Cys Ala Cys Cys Ala Ala Cys Ala Cys Gly              20 25 30 Thr Ala Ala Thr Gly Ala Ala Thr Gly Gly Gly Ala Ala Thr Gly Thr          35 40 45 Ala Ala Gly Thr Gly Thr Thr Cys Thr Gly Ala Thr Thr Cys Thr Thr      50 55 60 Cys Thr Gly Ala Ala Cys Cys Ala Gly Ala Thr Cys Gly Thr Thr Thr  65 70 75 80 Ala Thr Thr Thr Thr Thr Thr Ala Ala Ala Thr Gly Thr Ala Thr Thr                  85 90 95 Thr Ala Thr Cys Gly Thr Cys Gly Thr Cys Thr Thr Ala Ala Ala Thr             100 105 110 Ala Thr Gly Gly Thr Cys Thr Thr Ala Ala Ala Ala Gly Ala Gly Gly         115 120 125 Thr Cys Cys Ala Gly Cys Thr Ala Cys Ala Gly Cys Thr Gly Gly Thr     130 135 140 Gly Thr Thr Cys Cys Ala Gly Ala Ala Thr Cys Thr Ala Thr Gly Ala 145 150 155 160 Gly Ala Gly Ala Ala Gly Ala Ala Thr Ala Thr Ala Gly Ala Cys Ala                 165 170 175 Ala Gly Ala Ala Cys Ala Ala Cys Ala Ala Thr Cys Thr Gly Cys Thr             180 185 190 Gly Thr Cys Gly Ala Thr Gly Thr Cys Gly Ala Thr Gly Ala Thr Gly         195 200 205 Ala Thr Cys Ala Thr Thr Thr Thr Gly Thr Cys Ala Thr Thr Ala Thr     210 215 220 Thr Gly Ala Ala Cys Thr Thr Gly Ala Ala Gly Ala Ala Thr Thr Cys 225 230 235 240 Cys Ala Gly Gly Gly Ala Ala Thr Gly Gly Thr Ala Gly Ala Thr Gly                 245 250 255 Gly Thr Thr Gly Gly Thr Ala Thr Gly Gly Ala Thr Ala Cys Cys Ala             260 265 270 Cys Cys Ala Thr Ala Gly Cys Ala Ala Cys Gly Ala Gly Cys Ala Gly         275 280 285 Gly Gly Gly Ala Gly Thr Gly Gly Gly Thr Ala Cys Gly Cys Thr Gly     290 295 300 Cys Ala Gly Ala Cys Ala Ala Ala Gly Ala Ala Thr Cys Cys Ala Cys 305 310 315 320 Thr Cys Ala Ala Ala Ala Gly Gly Cys Ala Ala Thr Ala Gly Ala Thr                 325 330 335 Gly Gly Ala Gly Thr Cys Ala Cys Cys Ala Ala Thr Ala Ala Ala Gly             340 345 350 Thr Cys Ala Ala Cys Thr Cys Gly Ala Thr Cys Ala Thr Thr Gly Ala         355 360 365 Cys Ala Ala Ala Ala Thr Gly Ala Ala Cys Ala Cys Thr Cys Ala Ala     370 375 380 Thr Thr Thr Gly Ala Gly Gly Cys Cys Gly Thr Thr Gly Gly Ala Ala 385 390 395 400 Gly Gly Gly Ala Ala Thr Thr Thr Ala Ala Thr Ala Ala Cys Thr Thr                 405 410 415 Ala Gly Ala Ala Ala Gly Gly Ala Gly Ala Ala Thr Ala Gly Ala Gly             420 425 430 Ala Ala Thr Thr Thr Ala Ala Ala Cys Ala Ala Gly Ala Ala Gly Ala         435 440 445 Thr Gly Gly Ala Ala Gly Ala Cys Gly Gly Ala Thr Thr Thr Cys Thr     450 455 460 Ala Gly Ala Thr Gly Thr Cys Thr Gly Gly Ala Cys Thr Thr Ala Thr 465 470 475 480 Ala Ala Thr Gly Cys Thr Gly Ala Ala Cys Thr Thr Cys Thr Gly Gly                 485 490 495 Thr Thr Cys Thr Cys Ala Thr Gly Gly Ala Ala Ala Ala Thr Gly Ala             500 505 510 Gly Ala Gly Ala Ala Cys Thr Cys Thr Ala Gly Ala Cys Thr Thr Thr         515 520 525 Cys Ala Thr Gly Ala Cys Thr Cys Ala Ala Ala Thr Gly Thr Cys Ala     530 535 540 Ala Gly Ala Ala Cys Cys Thr Thr Thr Ahr Cys Gly Ala Cys Ala Ala 545 550 555 560 Gly Gly Thr Cys Cys Gly Ala Cys Thr Ala Cys Ala Gly Cys Thr Thr                 565 570 575 Ala Gly Gly Gly Ala Thr Ala Ala Thr Gly Cys Ala Ala Ala Gly Gly             580 585 590 Ala Gly Cys Thr Thr Gly Gly Thr Ala Ala Cys Gly Gly Thr Thr Gly         595 600 605 Thr    

Claims (15)

Mineral oil: Surfactant 70 ~ 90: 30 ~ 10 contained, the proportion of the aqueous phase is 30 or more, water-in-oil (W / O) emulsion vaccine adjuvant. The water-in-oil emulsion vaccine adjuvant according to claim 1, wherein the mineral oil to surfactant is 90:10. The method according to claim 1 or 2, wherein the mineral oil is MARCOL 52® (Esso, France), MARCOL 82® (Esso, France), DRAKEOL 6VR® (Penreco), Castor Oil, Squalan, Polydecene ( Polydecene), Sontax 55, Vestain A50B and Whitertrex 307, water-in-oil emulsion vaccine adjuvant. The ethoxylatedfatty acid monoester of sorbitan, ethoxylated sorbitan mono laurate according to claim 3, wherein the surfactant is a nonionic hydrophilic surfactant. ), Ethoxylated sorbitan mono stearate, ethoxylated sorbitan mono oleate, ethoxylated fatty acid triesters of sorbitan sorbitan), ethoxylated fatty alcohols, ethoxylated fatty acids, ethoxylated castor oil, ethoxylated sorbitan monopalmitate (ethoxylated sorbitan) monopalmitate) and non-ionic block copolymers (non-ionic block copolymers);
The surfactant is a nonionic lipophilic surfactant, fatty acid esters of sorbitan, fatty acid esters of mannide, and ethoxylated fatty acids of mannide. acid esters of mannide); or
The surfactant is a stabilizer, lecithin, stearic acid, steryl alcohol, cholesterol, octanoic acid, cholesteryl linoleate With cholesteryl oleate, palmitic acid, lauric acid, triton X-100, triton X-102, and octyl BD-Glucopyranoside One or more selected from the group consisting of; And
It comprises at least one surfactant selected from the nonionic hydrophilic surfactant, the nonionic lipophilic surfactant and the stabilizer,
Water-in-oil emulsion vaccine adjuvant. The surfactant according to claim 3, wherein the surfactant is fatty acid esters of sorbitan, fatty acid esters of mannide, and ethoxylated fatty acid esters of mannide. fatty acid esters of mannide), water-in-oil emulsion vaccine adjuvant. A vaccine composition comprising the vaccine adjuvant of claim 1 or 2 and an antigen protein. A vaccine composition comprising the vaccine adjuvant of claim 3 and an antigen protein. A vaccine composition comprising the vaccine adjuvant of claim 4 and an antigen protein. A vaccine composition comprising the vaccine adjuvant of claim 5 and an antigen protein. The vaccine composition of claim 6, wherein the antigenic protein is an avian influenza (AI) virus antigen, a Newcastle disease (ND) virus antigen or a chicken hen hypothyroidism (EDS) virus antigen. The vaccine composition according to claim 7, wherein the antigenic protein is an avian influenza virus antigen, a Newcastle disease virus antigen or a chicken spawning virus virus antigen. The vaccine composition according to claim 8, wherein the antigenic protein is an avian influenza virus antigen, a Newcastle disease virus antigen or a chicken spawning virus virus antigen. The vaccine composition according to claim 9, wherein the antigenic protein is an avian influenza virus antigen, a Newcastle disease virus antigen or a chicken spawning virus virus antigen. 7. The vaccine composition of claim 6, wherein the vaccine adjuvant to antigen protein is 7: 3 (w / w). A method of immunizing an animal, comprising administering the vaccine composition of claim 6 to an animal in need of immunization.

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