JPH10218788A - Production of immunopotentiator and immunopotentiation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable the simple production of an O/W type emulsion liquid immunopotentiator, excellent in immunopotentiating actions, immune sustaining actions and safety and useful for preparing an oil vaccine readily injectable in vivo by vigorously mixing a specific mixed oil with a physiological saline solution at a specified temperature. SOLUTION: (A) A mixed oil comprising anhydrous mannitol, an oleic acid ester and liquid paraffin is mixed with (B) a phosphoric acid buffer physiological saline solution so as to provide 40-45wt.% ingredient A and the resultant mixture is then heated at 45-50 deg.C temperature and vigorously mixed. The prepared immunopotentiator can subsequently be mixed with an antigenic solution (e.g. a virus, a bacterium, a live vaccine, an inactivated vaccine, a viral infected cell solubilized antigen or an antigenic protein derived therefrom) by hand shaking, etc., to afford a uniform oil adjuvant vaccine.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing an O / W emulsion (hereinafter referred to as "immune enhancer") and a method for enhancing immunity to animals.

[0002]

2. Description of the Related Art Conventionally, in order to enhance animal immunity, an aluminum gel adjuvant or an oil adjuvant is mixed with an antigen.
A method of introducing the obtained mixture into a living body to impart immunity to a target antigen has been adopted. The immunization method using aluminum gel adjuvant has been widely applied to inactivated vaccines. Although this method is excellent in safety, it has a problem that the immunopotentiating effect is weak. An oil adjuvant vaccine has been used as an immunization method using an oil adjuvant. According to this method, the effect of enhancing immunity to a living body is strong, but a special syringe is required due to its high viscosity, and further, inoculation reactions such as necrosis and induration of the inoculation site may be observed. In the production of an oil adjuvant vaccine, antigens, oils and emulsifiers, which are the main components of the vaccine, are produced by a special emulsifier. However, since the product is a vaccine, the oil, the emulsifier, and the like must be sterilized. For this purpose, it is necessary to sterilize oil and the like with a special sterilizer. Thus, the production of oil vaccines requires enormous capital investment.

[0003]

In order to solve the above problems, the present invention provides an O / W type by mixing anhydrous mannitol oleate, liquid paraffin, and phosphate buffered saline or purified water. A simple method for preparing an immunopotentiator, which is an emulsion of the present invention, was found. By mixing (hand-shaking) the immunopotentiator and the antigen produced by this production method, it has a strong immunity-enhancing effect on the living body, is excellent in immunity persistence and safety, and can be injected into the living body by injection or the like. An easy method for preparing an oil adjuvant vaccine and a method for enhancing immunity using the same were created. The method for preparing the immunopotentiator and the method for enhancing the immunity will be described in detail in the examples described below.

[0004]

【Example】

Example 1 Preparation of Immunity Enhancer A mixed oil consisting of anhydrous mannitol oleate and liquid paraffin and phosphate buffered saline (PBS)
Were mixed so that the mixed oil became 40 to 45 w / w%, and adjusted to 45 to 50 ° C. At this point, the PBS was in the lower layer and the oil was in the upper layer. As a result of vigorously shaking the mixture, a white, transparent and uniform emulsion was formed. The emulsion was allowed to stand at room temperature and was cooled to room temperature. The emulsified state was maintained even if the emulsion maintained at 4 ° C. or room temperature was mixed vigorously. In the emulsification method according to the present invention, a whitish transparent emulsion was obtained without foaming. In general, the size of the emulsified oil particles can be estimated from the color of the emulsion ("Surfactant Handbook", Kogaku Tosho Co., Ltd.). That is, when the emulsion is white, the diameter of the emulsified particles is 1-2.
Micron, and less than 0.1 micron when translucent. To date, none of the emulsions produced as oil adjuvants has a strong transparency. The immunopotentiator prepared by this method was a white, transparent, and uniform liquid, and its storage stability was stable for several years when kept at 4 ° C. or room temperature, and no change was observed. When an aseptic immunopotentiator is produced, a mixture of oil, an emulsifier and PBS is subjected to high-pressure steam sterilization, and then 45 to 50%.
After maintaining at ℃, the same O / W emulsion was obtained by the same method as described above.

Example 2 Preparation of Inactivated Oil Vaccine Pathogenic viruses such as Japanese encephalitis virus, swine parvovirus, Aujeszky's disease virus, swine reproductive and respiratory syndrome virus, swine epidemic diarrhea virus, and Actinobacillus brolonium pneumoniae Pathogenic bacteria such as bacteria (types 2 and 5) and Salmonella were cultured. The virus turns the culture
Alternatively, the bacteria destroyed with a homogenizer were inactivated with formalin, and the inactivated liquid and the immunopotentiator were mixed in equal amounts. The mixing method was simply performed by hand. The inactivated oil vaccine was a homogeneous emulsion and could be easily used as an injection. The production of an inactivated oil vaccine by this method was easy and instantaneous.

Example 3 Preparation of live oil vaccine Japanese encephalitis virus, swine parvovirus, Aujeszky's disease virus, swine reproductive and respiratory syndrome virus, swine epidemic diarrhea virus, bovine infectious rhinotracheitis virus, Akabane disease virus, etc. A culture solution of a virus strain (a commercially available vaccine or a virus solution obtained by culturing) obtained by attenuating the pathogenic virus was mixed with an equal amount of an immunopotentiator. The commercially available vaccine was prepared by dissolving the vaccine with the attached dissolution solution. The mixing method was simply performed by hand. The prepared live oil vaccine was a uniform emulsion and could be easily injected. The production of a live oil vaccine by this method was easy and instantaneous.

Example 4 Solubilized antigen of virus-infected cells
Preparation of Oil Vaccine When pathogenic viruses such as Aujeszky's disease virus, swine reproductive and respiratory syndrome virus, swine endemic diarrhea virus, and bovine infectious rhinotracheitis virus are inoculated into cells and the cytopathic effect has sufficiently occurred. Then, the cells adhering to the culture bottle were detached and suspended in the culture solution. The culture solution is 2,000 rpm for 10
Cells were pelleted by centrifugation for minutes. The cells were suspended in PBS containing 1% Nonidet P-40 or PBS containing 1% Triton X-100 so that the cells became 2%, and the cells were made into an emulsion using a homogenizer. Next, the mixture was stirred at 37 ° C. for 30 minutes using a magnetic stirrer. The cell emulsion was centrifuged at 5,000 rpm for 20 minutes to obtain a supernatant. This was used as a virus-infected cell solubilized antigen solution.
The virus-infected cell solubilized antigen solution and an equal amount of the immunopotentiator were mixed. The mixing method was simply performed by hand. The prepared virus-infected cell solubilized antigen oil vaccine was a homogeneous emulsion and could be easily injected. The preparation of a virus-infected cell solubilized antigen oil vaccine by this method was easy and instantaneous.

Example 5 Antibody of Inactivated Oil Vaccine
Production Example 2 results a variety of inactivated oil vaccine prepared were inoculated twice at 3 week intervals the animals, high antibody production than the conventional inactivated vaccine in all inactivated oil vaccine as shown in Table 1 Admitted.

[Table 1]

Example 6 Production of Antibodies from Live Oil Vaccine Using the various oil vaccines prepared in Example 4, animals were administered a live vaccine for the first vaccination, and three weeks later, the animals were vaccinated with the live oil vaccine. As shown in Table 2, all the live oil vaccines showed higher antibody production than the conventional live vaccines.

[Table 2]

Example 7 Solubilized antigen of virus-infected cells
Antibody Production of Oil Vaccine Solubilized Antigens of Various Virus-Infected Cells The oil vaccines prepared in Example 5 were inoculated twice into animals at three-week intervals. The vaccine showed high antibody production.

[Table 3] In the first inoculation, a live vaccine was administered, and three weeks later, a virus-infected cell solubilized antigen oil vaccine was inoculated. As shown in Table 4, when all the live vaccines were inoculated with the virus-infected cell solubilized antigen oil vaccine in combination, higher antibody production was observed than in the conventional live vaccination.

[Table 4]

As is apparent from the above description, the preparation of an immunopotentiator can be easily performed without requiring a special emulsifier. In addition, it has become possible to produce an oil vaccine by simply mixing an immunopotentiator and an antigen.
For this reason, a vaccine factory provides an innovative production method for oil vaccine production.

The immunopotentiating method using the present immunopotentiating agent can utilize many kinds of antigens, and has created a method capable of maximizing the ability of a vaccine expected to prevent infectious diseases in animals. It greatly contributes to the improvement of animal and livestock health and livestock productivity.

Claims (3)

[Claims] 1. A mixed oil consisting of anhydrous mannitol oleate and liquid paraffin and a phosphate buffered saline are mixed so that the mixed oil becomes 40 to 45 w / w%, and the mixture is heated to 45 to 50 ° C. A method for producing an immunopotentiator, which is an O / W emulsion, produced by heating and mixing vigorously. 2. An immunopotentiating method comprising mixing the emulsion obtained according to claim 1 and an antigen solution, and injecting the resulting mixture into a living body to enhance antibody production. 3. The method according to claim 2, wherein the antigen solution is a virus, a bacterium, a live vaccine derived therefrom, an inactivated vaccine, a virus-infected cell solubilized antigen or an antigenic protein.