KR860001564B1 - Preparation of hepatitis b virus vaccine - Google Patents

Abstract

Hepatitis B virus vaccine, which had HBsAg as a main component, was prepd. Thus, blood plasma contg. B-type hepatitis surface Ag was diluted with saline soln. to give Ag soln. This soln. was boiled at 102≰C for 2 min. and 40 sec. The resulting opalescent Ag soln. was centrifuged at 8000 g for 20 min. and the precipitate was resuspended with saline soln. to be 6 .g/ml. This suspension was equally mixed with 1.2 mg/ml aluminum phosphate gel to give 3.g/ml Ag and 0.6 mg/ml aluminum phosphate. This vaccine (1 .g, 0.25 .g, and 0.06 .g resp.) was injected into ICR mice and showed 17-30X higher activities compared to the pre-existing vaccine.

Description

Preparation method of hepatitis B vaccine with increased immunity

1 is an electron micrograph (magnification 195,000 times) of the purified antigen.

FIG. 2 is an electron micrograph (magnification of 195,000 times) of an antigen aggregated by heating for 2 minutes at 102 ° C.

FIG. 3 is an electron micrograph (magnification of 195,000 times) of the antigen which was heat-treated at 102 ° C. for 2 minutes and aggregated by centrifugation at 8000 g for 20 minutes.

4 is a graph of the effect of heat treatment and centrifugation on mouse immunity.

5 is a graph of the effect of heat treatment and centrifugation on the production of antibodies.

The present invention, in the production of hepatitis B vaccine mainly composed of HBsAg particles, through the step of removing the plasma components and hepatitis B infection components, the antigen purified to high purity is aggregated by heat treatment, 20- at 5,000-15,000 g The present invention relates to a method for preparing a hepatitis vaccine having a 17-30-fold increase in immunity by centrifugation for 60 minutes and reaggregation precipitation.

That is, an object of the present invention is to provide a hepatitis vaccine having high safety and low immunity at a low price.

For the virus causing hepatitis B, immunological and molecular biological studies have shown the characteristics and the structure of causing acute, chronic or cancer-causing diseases of the human liver, and hepatitis in the plasma of patients infected with hepatitis B virus. It is known that hepatitis B surface antigen particles, which are not infectious, are present together with sex dane particles.

In general, the hepatitis vaccine is prepared by purifying only hepatitis B surface antigen particles in the plasma of infected patients or by preparing a hepatitis B surface antigen using genetic engineering methods to inactivate the infectivity of the virus in a suitable manner and formulating it as an injection. It was.

As a method of inactivation, there is a method of treating with formalin for 4 days at 37 ° C as in US Pat. No. 4,164,565 or heating at 60-120 ° C as in Japanese Patent Publication 55-23,253. There are many attempts to reduce immunity and heat treatment, such as Krugman's report in 1971 (see J. Am. Med. Ass. 217). (Table 1).

TABLE 1

Effect of Heat Treatment on Antigen Value

* Specific antigen = antigen / antigen protein

However, the present invention focuses on the increase in specific immunogenicity when many antigens aggregate in polyvalent form, so that the mono-spherical particles of the antigen when heat treated for inactivation of infectious agents that may remain. Were found to be fused to each other in a filamentous form to increase immunity. Purified pure hepatitis B surface antigen and heat treatment resulted in a large number of single spherical particles of the antigen (FIG. 1). After reaggregation and precipitation into a straight, branched or cyclic filament type (Fig. 3), the precipitate is resuspended in physiological saline and adsorbed onto an aluminum phosphate gel (prepared by the method of Rijks Instituut voor de Volkgezondheid). Injected into ICR Swiss mice and subjected to antibody-generated immunoassay, heat treatment reduced the dose required for 50% seroconversion of mice by 7.4 times (Table 2). A 6-fold increase in body weight was also obtained (Table 3), and the abundance of aggregated antigens was further increased by centrifugation. As a result, hepatitis vaccines with an increased immunity of 17-30 times as shown in Tables 2 and 3 were prepared. .

In addition, the stock solution of the hepatitis vaccine was confirmed by the chimpanzee test.

TABLE 2

Antigen amount required for transformation of 50% of mice

TABLE 3

Antigen amount required for antibody to produce 1.5 Log10 mIU / ml

In addition, the present invention is to defibrin the serum or plasma of a healthy person containing hepatitis B surface antigen, and then polyethylene glycol is added to remove infectious dane particles and plasma protein components and bio-gel (Bio-gel HT, USA) Bio-Rad) to increase the purity and concentration, and then pure antigen obtained by isotropic gradient ultracentrifugation with potassium bromide heated at 100-104 ℃ for 2-6 minutes and centrifuged pale milky-white aggregated antigen. Supernatants were collected separately and resuspended antigen was resuspended to prepare a hepatitis vaccine stock solution.

Hereinafter, the present invention will be described in detail with reference to Examples.

The antibody titer used in the present invention was tested by radioimmunoassay (RIA) using an AUSAB test kit (manufactured by Evote, USA), and the WHO International HBIG Standard contained 100 IU (International units) per ml. Antigen titer was tested by radioimmunoassay using the AUSRIA test kit (manufactured by Ebbott, USA), and quantitatively compared to the standard provided by the US FDA.

)와 바이오래드(BIORAD)단백 정량법으로 정량하였다.In addition, the amount of antigenic protein has an absorbance value of OD ₂₈₀ (

) And Biorad (protein) quantitation method.

Example 1

20 ml of the purified and concentrated antigen in the plasma of healthy humans containing hepatitis B surface antigen was diluted with physiological saline and the protein content was absorbed by OD ₂₈₀ .

의 항원액 100㎖을 조제하였다. 이 조제액을 전자 현미경으로 관찰한 결과 직경 18-24nm의 단일 구형 입자로 구성되어 있으며 단백 파편은 존재하지 않았다. 이 조제액을 간염백신원액으로 하여 제균 여과하고 연속 가열 냉각 장치를 사용하여 102℃에서 2분간 연속적으로 가열처리하였다. 이때 102℃에 달하는 예비시간 40초를 감안하여 2분 40초간 가열처리하였다.

100 mL of the antigen solution was prepared. The preparation was observed under an electron microscope and consisted of single spherical particles with a diameter of 18-24 nm, and no protein fragments were present. This preparation solution was used as a hepatitis vaccine stock solution for disinfection, followed by continuous heat treatment at 102 DEG C for 2 minutes using a continuous heating and cooling apparatus. At this time, the heat treatment was performed for 2 minutes and 40 seconds in consideration of the preliminary time 40 seconds reaching 102 ℃.

As a result of the heat treatment, 30 ml of pale milky white antigen solution showing protein aggregation was centrifuged at 8,000 g for 20 minutes to collect the supernatant separately, and the re-aggregated precipitate was added to 30 ml with physiological saline isotonic solution and resuspended.

Dilute this suspension with physiological saline to 6 μg / mL, add 1.2 mg / mL aluminum phosphate gel as an immunosuppressive agent, and prepare the antigen to be 3 μg / mL and aluminum phosphate gel 0.6 mg / mL. A small hepatitis vaccine formulation was made.

The vaccine thus prepared was intraperitoneally injected into mice (ICR) by 0.5 cc to measure antibody titers.

At this time, the amount of the antigen injected was 1 μg, 0.25 μg, 0.06 μg. As a result, the vaccine produced 17-30-fold increase in antibody production immunity.

Claims (1)

In the preparation of hepatitis vaccine with high purity purified hepatitis B surface antigen, the method of producing hepatitis vaccine, characterized in that the immunity is increased by agglutination by heat treatment and centrifugation at 5,000-15,000 g for 20-60 minutes for reaggregation and precipitation. .

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