KR20010054305A - Method for producing VZV-HAV combined vaccine - Google Patents

Abstract

PURPOSE: Provided is a process for producing Varicella-Zoster Virus(VZV)-Hepatitis A Virus(HAV) combined vaccine by cultivating, refining, and inactivating each of the viruses and then adsorbing each of the viruses on aluminium gel. CONSTITUTION: The process comprises the steps of: cultivating each of the VZV and the HAV in media containing 30-80mM disaccharide; refining and inactivating each of the viruses, wherein the refined HAV is treated by using formaldehyde as an inactivating agent; diluting each of the viruses by using phosphoric buffer solution(0.01M, pH 7.8) containing white sugar and lactose and then mixing the VZV contained solution and the HAV contained solution, wherein the last Varicella-Zoster antigen is 100EIA unit and the last Hepatitis A antigen is 50EIA unit; adding 500microgram/ml of the aluminium gel and stirring at 4deg.C for 2 hours.

Description

Method for producing chickenpox-hepatitis combined vaccine {Method for producing VZV-HAV combined vaccine}

There is currently no development of mixed vaccines for the above-mentioned vaccines, and Varicella-Zoster Virus (hereinafter referred to as VZV) belongs to the alphaherpes family of humans, Chickenpox and Shingles. ) Is a virus that causes. VZV is very contagious and, when infected, causes rashes and fever.

VZV is a primary viremia that is transmitted through the respiratory system and travels through the limbs and through the blood. It is transported to the lungs or liver through the fluid, followed by a full multiplication, and then the second viral viremia is transported through the blood to the mucous membranes or skin of the nose or mouth.

This secondary viremia soon disappears by cellular or humoral immune mechanisms.

After the completion of secondary viral hyperemia, VZV incubates in satellite cells that wrap around nerve cells in the brain.

On the other hand, in elderly people or patients with markedly decreased cellular immune function, reactivation of VZV takes place and the skin moves and spreads in the reverse order of the latent process, resulting in the development of zoster (Lawrence 2 ^nd , (1990)). , 2011-2054).

This reactivated VZV causes complications such as pneumonia, encephalitis and cerebellar ataxia in patients with hydrocephalus.

In order to prevent such diseases caused by chickenpox virus, the prevention of primary infection is the most effective and the development of a vaccine for this is required.

VZV belongs to the Herpes virus group, which consists of 125,000 base pairs and reveals the entire genome. 1) The virus is characterized by: 1) an inverted terminal base with an 80 ± 3x10 ⁶ Dalton linear genome inside; With double DNA molecules, 2) short replication cycles, and 3) frequent latent infection of the perceptual nervous system. The cell nucleus is approximately 100 nm in diameter and has 162 hexagonal capsomers with 5: 3: 2-axis symmetrical holes. It is composed of icosahedron together.

Complete VZV particles range from 180-200 nm in diameter and at least 5 of the 30 different polypeptides observed are glycoproteins.

Oka, the chickenpox attenuated vaccine strain Oka developed by Takahashi in 1974, has been vaccinated in Japan and Korea for about 20 years and has been shown to be immune to shingles.

Accordingly, in 1975, Oka strain, an attenuated virus, was developed at the University of Osaka, Japan using animal cell culture technology, and it is developed as an attenuated live vaccine and used as a varicella virus vaccine.

However, chickenpox vaccines using these live viruses have problems in efficacy, safety and stability.

In clinical trials involving 437 leukaemia children, 36 patients received chickenpox after vaccination (28%). (The New England Journal of Madicine (1989) vol. 320, No. 14,892-897 ).

Similar results were found in clinical trials in Belgium (Postgraduate Madical Journal (1985) 61, Suppl. 4,97-102).

Therefore, the present invention also has the technique of solving the problem of this part.

Hepatitis A Virus (hereinafter referred to as HAV) belongs to the piconavirdae family and is the virus responsible for hepatitis A. HAV is asymptomatic under 6 years of age and the majority of symptoms are mild and rarely recognized as hepatitis.However, after 6 years of age, more than 70% of patients have typical hepatitis symptoms. And lethality increase gradually.

HAV is most commonly an oral infection and refers to a viremia that passes through the liver and into the blood. Hepatitis A RNA virus is a virus with a small number of specific mutations and is known to have about 10 nucleotide variations in 10 years.

Hepatitis A is a disease in which fatality increases with age, and the economic loss is severe. Therefore, national measures are urgently needed.

HAV has two sites in the structure where neutralizing antibodies bind, and these sites bind to antibodies under specific three-dimensional arrangement. It is this characteristic that makes it difficult to manufacture a gene vaccine for hepatitis A. However, this specific part is well preserved, which makes it possible to produce an effective vaccine using any strain.HVRIX: SmithKline Beecham, VQTA: Merck & Company Inc. has already been proven to be safe and effective for tens of thousands of people in Europe and Southeast Asia. After human proliferation from embryonic cells

This vaccine is inactivated and the manufacturing process is not simple and the selling price is high.

Therefore, the present invention also has the technique of solving the problem of this part.

It is an object of the present invention to provide a method for producing a combination vaccine which is more effective and safer than what was previously present in each vaccine.

In the present invention, human embryonic lung fibroblast (hereinafter referred to as MRC-5) is used as a host cell for chickenpox and hepatitis A virus, and Oca strain (ATCC VR-795) is used as chickenpox virus strain and HM175 (ATCC: VR-1402) strain is used as hepatitis A virus strain.

In order to achieve the above object, the step of culturing chickenpox and hepatitis A virus, inactivating the virus, purely separating and purifying the virus, preparing two viral antigens, and finally preparing the antigen-immunopotentiator, aluminum It provides a method for producing chickenpox and hepatitis A mixed vaccine comprising the step of adsorbing to the gel.

Example 1 Culture of Host Cells and Varicella Virus

First, MRC-5 cells are cultured in a culture flask (T175: Falcon) using DME medium containing 5% fetal calf serum and antibiotics.

After incubation at 37 ° C for 5 hours in a 5% Co ₂ incubator to form a cell monolayer, inoculated with an appropriate amount of virus-infected cells, 1% fetal bovine serum and mixed disaccharides (white sugar: 30 mM, lactose: 20 mM) Add one DME medium and incubate in Co ₂ incubator at 37 ° C. Incubate for 2-5 days and harvest the infected cells with trypsin-EDTA solution after 50-80% of the cell lesion effect. Remove the liquid.

Example 2 Culture of Host Cells and Hepatitis A Virus

First, MRC-5 cells are cultured in a culture flask (T175: Falcon) using DME medium containing 5% fetal calf serum and antibiotics.

After culturing at 37 ° C in a 5% CO ₂ incubator for a certain period of time to form a cell monolayer, inoculating an appropriate amount of virus-infected cells, 1% fetal calf serum and mixed disaccharides (white sugar: 30 mM, lactose: 20 mM) Add one DME medium and incubate in a 37 ° C. CO ₂ incubator. Incubate for 25 days to harvest infected cells with trypsin-EDTA solution. The harvested cells are centrifuged to remove supernatant.

Example 3: Pure Purification and Inactivation of Chicken Pox Virus

In the past, freeze-thaw crushing and ultrasonic emulsification were used for the elution of the virus, but for more efficient mass processing, Triton X-100 was treated with 0.1 ~ 0.5% concentration at 4 ℃ for 10 minutes and centrifuged. The precipitate was discarded and only the upper layer was recovered.

The recovered supernatant was concentrated and dialyzed at 100,000MWCOL to remove residual Triton X-100. The restriction enzyme was also treated to remove residual nucleic acid.

The restriction enzyme treatment solution completely removed residual nucleic acids and enzymes using an ion exchange resin method.

Some purified virus suspension is treated with polyethylene glycol (PEG) to a final 2% to 10% (w / v) to precipitate the virus and discard the supernatant.

The precipitate is added to the buffer solution (0.01 M, pH 7.8 PBS) and resuspended after crushing the virus using a cell disruption system in an ice bath.

Resuspension was purified pure virus through 20% sucrose gradient ultrafast centrifugation.

The virus part was extracted and concentrated again to 100,000MWCOL using phosphate buffer solution (0.01M, pH 7.8 PBS), and sucrose was removed by dialysis.

Here, the final formalization of formalin as an inactivating agent to be 0.02% (v / v), and then disinfection filtration.

After formalin treatment, inactivation is usually completed at 4 ° C. for about 10 days, but it was treated for at least 50 days to ensure inactivation.

When inactivation is complete, to remove formalin, concentrated and dialysis was performed at 100,000 MWCOL using a phosphate buffer solution (0.01 M, pH 7.8 PBS) to complete the preparation of the concentrate.

Example 4 Pure Purification and Inactivation of Hepatitis A Virus

In the past, freeze-thaw crushing and ultrasonic emulsification were used for the elution of the virus, but for more efficient mass processing, NP40 (Nonidet P40) was treated at 1% concentration for 10 minutes at 4 ℃ and 10 minutes at 1000rpm. The precipitate was discarded by centrifugation and only the upper layer was recovered.

The formaldehyde (formaaldehyde) concentration in the recovered upper layer was 0.04% and left for 50 days in refrigeration.

After inactivation is completed, high-speed centrifugation is performed for 5 hours with a 20% sucrose gradient at 25,000 rpm, and only the virus part is separated. Acetone at -20 ° C was added at a ratio of 10: 1, shaken and left in an ice box for 10 minutes. Centrifugation at 2,000 rpm for 10 minutes was carried out and dried under a negative pressure. A crude drug was prepared by dissolving it in a phosphate buffer solution. .

Example 5 Measurement and Mixing Preparation of Chicken Pox Antigen and Hepatitis A Antigen Using Immunofluorescence Antibody (EIA)

First, the antigen values of inactivated chickenpox antigen and hepatitis A antigen were measured and diluted with phosphate buffer solution (0.01M, pH 7.8 PBS) to which stabilizer such as sugar and lactose was added so that the final EIA antigen value of chickenpox antigen was 200EIA unit. Solution A was prepared.

In addition, solution B was prepared by diluting with a phosphate buffer solution (0.01M, pH 7.8 PBS) to which the final EIA antigen value of hepatitis A antigen was 100EIA unit, and added stabilizers such as sucrose and lactose.

The same amount of solution A and B were mixed to prepare a final chickenpox antigen of 100EIA unit and hepatitis A antigen of 50EIA unit.

Example 6 Adsorption of Immunostimulator Aluminum Gel

500 ug / ml aluminum gel was added and stirred at 4 ° C. for 2 hours, and then samples were taken from the supernatant to confirm that each virus antigen was adsorbed onto the gel.

Effect Test Example 1: Animal experiments were performed as follows to determine whether the mixed vaccine prepared in Examples 1, 2, and 3 was reliable in vivo.

First, weaned mice were assigned to 10 rats in each treatment group to perform intracranial and subcutaneous injections.

Each treatment group was injected with a pseudo vaccine consisting of a mixed vaccine prepared in the above example and a physiological saline containing no virus as a control.

However, the mixed vaccine was injected twice at 10 day intervals.

At 12 months after the last injection, an autopsy was conducted to confirm the abnormality of all organs.

Table 1

Vaccine Type Autopsy result after 12 months Mixed vaccine clear Pseudo vaccine clear Unvaccinated clear

Effect Test Example 2 Animal experiments were performed as follows to determine whether the combined vaccine prepared in Examples 1, 2, and 3 exhibited high titers in each vaccine even in vivo.

First, to confirm the effects of chickenpox vaccine, 10 weeks old guinea pigs were allocated to each treatment group, and subcutaneous or intramuscular injection was performed.

Each treatment group was injected with a mixed vaccine prepared in Example and a pseudo vaccine consisting of a Viken chicken pox vaccine and a physiological saline containing no virus.

However, the mixed vaccine was immunized twice at 10 day intervals.

Four weeks after the last injection, blood was collected from guinea pigs and ELISA was performed to determine the amount of antibodies against chickenpox virus in the blood.

The antibody production rate of each vaccine was measured using the EIA varicella kit commercially available from Bio-Whittaker.

The results show that the combined vaccine production rate is higher.

[Table 2]

Vaccine Type Absorbance (490nm) Mixed vaccine 1.86 Viken attenuated live vaccine 1.425 Pseudo vaccine 0.21

To confirm the effects of the mixed vaccine on hepatitis A vaccine, 10 female mature mice (weight 25g ~ 30g) were grouped into 1 group and intraperitoneally injected.

Each treatment group was intraperitoneally injected with a pseudo vaccine consisting of a mixed vaccine prepared in the above Example and a HAVRIX (SKB) vaccine and a physiological saline containing no virus as a control.

Four weeks after the last injection, blood was collected from the mice, and a competitive RIA method was performed to determine the amount of antibody against hepatitis A virus in the blood.

The antibody production rate of each vaccine was measured using the AB-HAVK kit sold by SORIN biomedica.

The results show that the combined vaccine production rate is higher.

Table 3

Vaccine Type Net counts per min Mixed vaccine 343 Havrix 365 Voice control 10533

In the present invention, the chickenpox vaccine and hepatitis A vaccine, which existed as the respective vaccines, are prepared under optimal conditions by culturing, purifying, and inactivating antigens of each virus, and then adsorbed onto aluminum gel to prepare one vaccine. The development of a method that can be used to produce a combination vaccine that can prevent both diseases.

Claims (4)

Development method of mixed vaccine incubated with chickenpox and hepatitis A virus, inactivated, mixed, formulated and adsorbed on aluminum gel The method according to claim 1, wherein the disaccharide is cultured using a concentration of 30 to 80 mM in the culture of chickenpox and hepatitis A virus. The method of claim 1, wherein the pure alcoholic hepatitis A virus is inactivated by treating the final formaldehyde concentration of 0.02% to 0.08 (v / v) as an inactivating agent. The inactivated chickenpox virus of claim 1 was diluted with a phosphate buffer solution (0.01M, pH 7.8 PBS) added with a stabilizer such as sucrose and lactose so that the final chickenpox antigen was 100EIA unit and the hepatitis A antigen was 50EIA unit. After adding the aluminum gel (alhydrogel) to 500ug / ml and stirred at 4 ^o C for 2 hours to prepare a mixed vaccine adsorbed on aluminum gel