KR100242879B1 - Method for stabilizing live varicella zoster virus using human diploid cells - Google Patents

Abstract

The present invention relates to a method for stabilizing varicella virus using normal cells, and to stabilize by crushing together with infected cells by adding normal cells uninfected with the virus during cell disruption to separate extracellular varicella virus from chickenpox virus infected cells. This can effectively separate large quantities of live pox virus while at the same time significantly increasing the activity of the extracellular virus to be isolated.

Description

Stabilization of varicella virus using normal cells

The present invention relates to a method for stabilizing varicella virus using normal cells, and more specifically, to isolate extracellular varicella virus from chickenpox virus infected cells, by adding normal cells not infected with virus during ultrasonic cell disruption, The present invention relates to a method for effectively separating large quantities of live chickenpox virus while maintaining the activity of the virus by stabilizing the chickenpox virus by crushing the cells together.

Chicken pox, commonly called `` water mama, '' is a herpes virus that weakens children and immunity by Varicella Zoster Virus ("VZV"), a DNA virus with a size of 200 nm. It is a highly infectious viral disease that affects elderly and elderly patients. About 14 days after the VZV infection, a fever develops very similar to the symptoms of a cold, and a rash in the form of spots develops in the whole body, which eventually develops into an acne form and forms a 'sac'.

VZV mainly begins to proliferate in the respiratory system, which is the site of primary infection, flows into the blood, migrates to the lymph glands, and spreads, resulting in primary viral symptoms. Viremia is a virus that contains a virus in the blood, usually characterized by sickness (病 感), fever, stomach pain (통 部 痛) and limbs (四 脂 痛). When the cellular immune response is weakened or deficient, VZV moves to the next stage, the reticulo endothelial system, where full multiplication occurs in the skin and extends to the lungs and liver, resulting in secondary beremia. In general, in normal people, this secondary vilemia phenomenon disappears within three days by cellular or humoral immune defense mechanisms. In particular, cellular immune responses are known to play a more important role in the inhibition of chicken pox virus. However, even after the second virememia disappears, two weeks after infection, the virus penetrates into the ganglion cells of the brain and escapes into the ganglion cells of the brain and lurks in satellite cells surrounding the nerve cells. The proliferation of the virus is suppressed for a long time by the immune response, thus entering the incubation period without damaging the host cell. This reactivation of the latent virus cannot be prevented by the humoral immune response, but only by the cellular immune response. That is, several cytokines are induced by cellular immune mechanisms to bind to virus-infected neurons, thereby inhibiting virus proliferation. On the other hand, in older people or patients whose cellular immunity is significantly lowered to the extent that virus proliferation is not suppressed, even when a specific antibody against VZV exists, VZV is reactivated and thus multiplies in ganglion cells, thus incubating the process as described above. It moves back to the skin and spreads by the reverse order of herpes zoster. [H. John et al., Virol. 5 , 241 (1991); and DG Lawrence et al., Virology , 2 , 2011-2054 (1990)].

VZV reactivated in ganglion cells causes more severe symptoms in patients with hydrocephalus, such as primary hydrocephalus pneumonia. Primary nervous system abnormalities such as tremors, hypotonia (paralysis), and acute cerebellar degeneration appear primarily, but in some cases, are more severe, such as cerebral hemorrhage and peripheral blood cell infiltration, leading to neuritis or encephalomyelitis, leading to death. It can also be a fatal disease. In the United States, about 4 million chickenpox and 1.2 million shingles occur every year, and more than 100 die each year from hydrocephalus, especially when leukemia patients need to take immunosuppressive drugs. It is reported to be fatal. [G. Fleisher et al. Am. J. Dis. Child. 135 , 896 (1981); RP Stephen et al., Pediatrics , 68 , 14 (1981); and PA Patel et al., Pediatrics , 94 , 223 (1979).

Currently, the drugs that can suppress the VZV such as vidarabine (Adenosine arabinoside), Acyclovir (Acyclovir), etc. have been developed, but because of their strong toxicity causes severe side effects on the kidneys and brain central nervous system, treatment using them Very limited (TH Weller et al., New England J. Med., 309 , 1434 (1983)). Therefore, development of a preventive chickenpox vaccine is essential, and many studies have been made to prevent hydrocephalus. However, these conventional studies are based on classical live attenuated vaccines, not on recombinant vaccines using advanced genetic engineering techniques. The live chickenpox vaccine was first developed in 1975 by the Takahashi Group of the University of Osaka, Japan [M. Takahashi et al., Biken J., 18 , 25 (1975)] is producing Oka strain from Biken. In the United States, the chickenpox vaccine (KMcC strain) developed by Merck has been used for immunosuppressive patients in a limited way.In early 1994, the patent was transferred from Japan's Baiken Company and received FAD approval (Oka / Merck strain). Its safety and effectiveness have been proven to contribute to the prevention of chickenpox disease.

In addition, some recent reports show that shingles, which are generally known to be more common in older people, are caused by the reactivation of the virus by attenuating cellular immune responses, thus re-stimulating immunity with vaccination of live chickenpox vaccines. Can prevent the development of herpes zoster (SA Plotkin et al., Science , 265 , 1383 (1994)). The chickenpox vaccine has a cellular immune response within 5 days of inoculation and neutralizing antibodies are produced within 7 days. When the chickenpox virus is naturally infected, the virus multiplies at the site of the first infection and spreads throughout the body.Therefore, it is reported that the vaccine can be used to treat chickenpox by only vaccination within 5 days after infection. [M. Takahashi et al., Postpraduate Med. J., 61, 37 (1985); and Y. Asano et al., Biken J., 25 , 43 (1982)], the chicken pox vaccine market is expected to grow in the future.

On the other hand, VZV has been studied in earnest since 1986 (AJ Davison et al., J. Gen. Virol., 67 , 1759 (1986)), whose DNA sequence was completely revealed by AJ Davison . VZV has six major glycoproteins (gpI-gpV, IE-62) made by the three genes gA, gB and gC on the surface of the viral membrane, in particular gpI and gpIV contain specific neutralizing antibodies against VZV. Because of its potential as a recombinant vaccine [A. Vafai et al., Vaccine , 11, 937 (1993)], a number of studies are underway.The method of proliferation of VZV using a cell culture system is for example human amnion (amnion). Cells, human thyroid cells, human fetal lung cells, human cervix hela cells, and monkey kidney cells have been reported to be possible [EV Meurisse et al. ., J. Microbiol., 2, 317 (1969)], which includes vaccines during development Each company will be the trend of the current which developed a unique cell applications in vaccine development because the diploid cells are suitable.

Such VZV has cell-dependent properties [MC Cook et al., J. Virol., 2, 1458 (1968)]. Stabilized extracellular virus that is well-activated because of its minimal viral activity outside the cell. The establishment of a method to obtain a large amount of and the development of a high cell culture technology capable of proliferating a large amount of VZV using a cell culture system is the most important factor in the development of chickenpox vaccine.

In particular, since the production of stabilized extracellular varicella virus is essential for vaccine development, many studies have been actively conducted to stabilize the chickenpox virus. As a method for obtaining extracellular varicella virus, for example, ultrasonic disruption [C. Grose et al., Infection and Immunity, 19, 199 (1978); NJ Schmidt et al., Infection and Immunity, 14, 709 (1976); and C. Grose et al., J. Gen. Virol., 43, 15 (1979)]. Although glass bead crushing and cell homogeneous crushing have been reported, ultrasonic crushing is known to be the most effective. Stabilizers for stabilizing varicella virus include 5% to 7.5% sucrose upon disruption of infected cells [MR Hilleman et al., US Pat. No. 4,000,256 (1975); K. Takahashi et al., US Pat. No. 3,985,615 (1975); And Nathan Zygraich et al., US Pat. No. 3,915,794 (1973) or 5% Sorbitol [D'Hondt Erik et al., European Patent No. 0,252,059 A2 (1987); C. Grose et al., J. Gen. Virol., 43, 15 (1979).

On the other hand, as mentioned above, chickenpox virus VZV, unlike other viruses, has a vertical-dependent property and exists in a stabilized state in the cell, but the virus activity is very small outside the cell. It can be estimated that the components play a very important role in the stabilization of VZV.

Therefore, the present inventors while studying the stabilizing effect of VZV by normal cells under the assumption that there are components that play an important role in the stabilization of VZV in the cell, normal cells that are not infected with chickenpox virus at all when the infected cell disrupted When crushing was added together, it was confirmed that the activity of the isolated extracellular pox virus was effectively maintained, and it was found that normal cells can effectively stabilize the virus to complete the present invention.

Accordingly, it is an object of the present invention to provide a method for isolating large quantities of live chickenpox virus while maintaining virus activity from virus infected cells.

In order to achieve the above object, in the present invention, in the method of separating extracellular pox virus from chickenpox virus-infected cells by centrifuging the lysate after crushing the virus-infected cells, virus-infected cells are not infected when the virus is crushed. It provides a method for stabilizing varicella virus, characterized in that the normal cells are added at the same time and crushed together with the infected cells.

In the method of stabilizing varicella virus according to the present invention, the amount of normal cells added upon disrupting infected cells is preferably 10 to 60% based on the total number of cells to be disrupted.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

In the present invention, the present invention is a self-developed human embryonic lung fibroblast (hereinafter referred to as 'LBHEL') by the applicant's primary culture from the lung tissue of the human fetus from the host cell against the virus; November 9, 1994 Korea Deposited in Accession No. KCTC 0127BP to the Genetic Bank of Genetic Engineering Research Institute affiliated to the Institute of Science and Technology, and Varicella Oka strain (ACTC VR-795, Lot No. 6w) is used as the viral cell line.

According to the present invention, when the extracellular varicella virus is isolated from the cells infected with the chickenpox virus, the process of stabilizing the extracellular chickenpox virus using normal cells when the infected cells are disrupted is as follows.

First, the LBHEL cells were cultured in a culture flask (T 80, TAB) using DMEM (GIBCO BRL 12800-082) medium containing 5% FBS (GIBCO BRL 16000-028) and ABAM (antibiotic-antimycotic, GIBCO BRL 15240-013). Nunc 178905, 80 cm 2) or multilayer embryo plates (CF-10, Nunc 164327, 6000 cm 2). After maintaining the culture temperature of 37 ℃ and CO ₂ concentration of 5% incubator (Nuaire TM) for a predetermined time to form a cell monolayer, and then phosphate buffer (D-PBS, GIBCO BRL 450-1600 EA) 3 Wash twice and inoculate an appropriate amount of virus infected cells. After culturing for about 48 hours in the culture conditions, when the cytopathic effect reached 50 to 75%, the resultant was washed three times with the phosphate buffer solution and treated with 0.02% EDTA (GIBCO BORL 890-12661M) solution. The infected cells are harvested after standing in the incubator for 10 minutes.

The harvested infected cells were then subjected to SPGA (pH 7.2, 7.5% (w / v) sucrose, 0.0038 M potassium phosphate (1 base), 0.0072 M potassium phosphate (2 base), 0.0049 M sodium glutamate as cell disruption solution. , 0.2% EDTA, and 1% (w / v) human serum albumin) in 1 ml per T 80 flasks to make a cell suspension, then normal cells without virus infection 10 to 60 based on the total cell number. Add in weight percent and disrupt until circular cells are barely visible using an ultrasonic cell crusher (Branson sonifier 450). Thereafter, the cell lysate is centrifuged at a speed of 1000 rpm for 10 minutes at a temperature of 4 ° C to discard the precipitate and recover only the virus suspension.

Next, LBHEL cultured using DMEM medium in the same procedure as above in a Petri dish 6.0 cm in diameter to determine the plaque forming cell (PFC) and extracellular viral activity (plaque forrming unit (PFU)). The cell monolayer is washed three times with PBS and then inoculated with an appropriate amount of serially diluted infected cells and virus suspension. After culturing for 1 to 2 hours under the same culture conditions as above, add 3% FBS-containing DMEM medium, and incubate for 7 to 9 days in the incubator, and count the plaques with 100 magnification microscope (Nicon, TMS) to infect the cells. Calculate the viral activity of the virus suspension. At this time, to reduce the error of the experiment is repeated three times for each sample.

As a result of the above experiments, when extracellular varicella virus was scavenged by adding an appropriate amount of normal cells at the time of ultrasonic cell disruption, which is a step for obtaining extracellular varicella virus from the infected cell, extracellular pox virus activity is effectively maintained to provide excellent virus stabilization effect. Appeared to be.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but these examples are only for illustrating the present invention in detail and the scope of the present invention is not limited thereto.

Example 1

Culture and Harvest of Host Cells

As host cells in T 80 flasks (N _UNC 178905, 80 cm 2) or multilayer culture plates (CF-10, Nunc 164327, 6000 cm 3) using DMEM (GIBCO BRL 12800-082) medium containing 5% FBS and ABAM Human fetal lung fibroblasts (LBHEL, Accession No. KCTC 0127BP) were incubated at 37 ° C. for 3 to 5 days in the presence of 5% CO ₂ . Once the cells formed a monolayer, they were washed three times with PBS, treated with 0.25% trypsin solution (GIBCO BRL 15090-012) and harvested cells after about 2-3 minutes. The harvested cells were centrifuged at 1000 rpm for 3 minutes, and the precipitated cells were stored at -180 ° C in 10% glycerol solution containing 20% FBS and used when necessary.

Example 2

Cultivation of inoculated virus (seed virus)

Varicella Oka strain (ATCC VR-795, Lot No. 6w) as varicella virus was inoculated into the LBHEL cell monolayer previously prepared by the method of Example 1, incubated for 2 to 3 days, and then inoculated three times with PBS. The cells were washed, then treated with 0.02% EDTA, left in the incubator and the infected cells harvested after 5-10 minutes. Virus passage was performed three times in the same manner to propagate the virus, and then stored at -180 ° C in a 10% glycerol solution containing 20% FBS as in Example 1 and used as the inoculation virus if necessary.

Example 3

Virus Culture and Cell Shredding

Step 1. Virus Inoculation and Harvesting of Infected Cells

The LBHEL cell monolayer prepared in the same manner as in Example 1 was washed three times with PBS, and then 2 ml of the inoculum of the virus-infected cells prepared in Example 2 was dispensed so that the viral activity of the inoculum was 100,000 to 200,000 PFC per T 80 flask. Inoculated as much as possible. After 1 to 2 hours of inoculation, DMEM medium containing 3% FBS was added and incubated for 48 hours in an incubator, washed three times with PBS again and treated with 0.02% EDTA when showing a 50-75% cell lesion effect. . The infected cells were harvested by standing for 5 to 10 minutes and then centrifuged at 1000 rpm for 3 minutes.

Step 2. Ultrasonic Shredding

Ultrasonic disruption was performed to separate the extracellular virus from the infected cells obtained in step 1 above. First, the infected cells were disrupted with SPGA (pH 7.2, 7.5% (w / v) sucrose, 0.0038 M potassium phosphate (1 base), 0.0072 M potassium phosphate (2 base), 0.0049 M sodium glutamate, 0.2% EDTA. , And 1% (w / v) human serum albumin) to a concentration of 5-10 × 10 ₆ cells / ml to form a cell suspension, followed by microtips (for T 80 flasks) or horns (multilayer plates). In case of), the cells were disrupted with an ultrasonic cell crusher (Branson sonifier 450) until almost no circular cells were seen. At this time, the shredding solution was kept in an ice container and kept cold.

In this example, the amount of infected cells to be disrupted is 20, 40, 60, 80 and 100% based on the total cell numbers, respectively. Ultrasonic disruption was performed at 80, 60, 40, 20 and 0% of normal cells. Then, each obtained cell lysate was centrifuged at 1000 rpm for 10 minutes at 4 ° C, and only the supernatant was taken to examine virus activity.

Example 4

Viral activity (infectious) investigation

LBHEL cell monolayers were formed in a Petri dish with a diameter of 6.0 cm in the same manner as in Example 1, and then washed three times with PBS, and each virus suspension prepared in Step 2 of Example 3 was serially diluted five times to activate infected cells. Was measured at 0.3 ml / dish and extracellular virus activity was measured at an amount of 0.1 ml / dish. After 30 minutes to 1 hour of incubation, 3% FBS-containing DMEM medium was added, and after 7-9 days, plaques were counted under a microscope at 100 magnification, indicating the formation of plaque forming cells (PFCs) and extracellular viral activity (plaque forming units). , PFU) was measured and the results are shown in Tables 1 and 2 below. At this time, the infecting cell activity of the T 80 flask was 750,000 PFC / T 80, the multilayer culture plate was 6,450,000 PFC / T 80, the multilayer culture plate was 6,450,000 PFC / CF-10.

TABLE 1

TABLE 2

* ND: not detected

From the results of Tables 1 and 2 above, when virus infected cells were disrupted and normal cells were added and disrupted together, compared to the case where only infected cells were broken, virus activity was significantly increased as a whole, thereby effectively stabilizing VZV by normal cells. It can be seen that. In addition, as shown in the results of Table 2, it can be seen that the method of the present invention has an equivalent effect even in mass production.

As described above, when the chicken pox virus infected cells are crushed, normal cells that are not infected with the chickenpox virus are added and crushed together with the infected cells to stabilize the chickenpox virus, thereby significantly increasing the activity of the isolated extracellular virus. At the same time, a large amount of live chickenpox virus can be effectively isolated.

Claims (2)

In the method of separating extracellular varicella virus from chickenpox virus-infected cells by crushing virus-infected cells and then centrifuging the lysate, the virus-infected cells are crushed together with the infected cells by adding normal cells not infected with virus. Stabilization method of chickenpox virus, characterized in that. The method of claim 1, The amount of normal cells added during the cell disruption is characterized in that 10 to 60% based on the total cell number.