KR100349776B1 - Process for getting high titer of varicella-zoster virus using tartaric acid - Google Patents

Abstract

PURPOSE: A method for increasing the activity of Varicella zoster virus(VZV) causing chicken pox using tartaric acid is provided, thereby preparing stable extracellular Varicella zoster virus for the development of chicken pox vaccine. CONSTITUTION: A method for increasing the activity of Varicella zoster virus(VZV) causing chicken pox treating Varicella zoster virus infected cells or Varicella zoster virus is characterized by using tartaric acid as a lysosome acidic phosphatase inhibitor, wherein a tartaric acid solution is added in the amount of 1 micromole into the Varicella zoster virus infected cells or Varicella zoster virus 24 hours before the inoculation or 1 to 2 hours after the inoculation.

Description

PROTESS FOR GETTING HIGH TITER OF VARICELLA-ZOSTER VIRUS USING TARTARIC ACID}

The present invention relates to a method for enhancing the activity of chickenpox virus using tartaric acid, and more particularly, a method for enhancing the activity of chickenpox virus by adding a small amount of tartaric acid to a cell monolayer before or after inoculation of an infected cell or chickenpox virus. It is about.

Chicken pox, commonly referred to as 'water mama', is a herpes virus that causes children and immunity to be caused by Varicella Zoster Virus ("VZV"). It is a highly infectious and seasonal viral disease that affects weakened older or patients.

About 14 days after VZV infection, fever develops very similar to the symptoms of a cold, and a rash in the form of spots develops in the form of acne, which eventually develops into a form of acne, which causes fatal scars.

VZV mainly begins to proliferate in the respiratory system, the site of primary infection, flows into the blood, moves to the lymph gland, and spreads, resulting in primary viremia. 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). At this stage, if the cellular immune response is weakened or deficient, it moves to the next stage, the reticulo endothelial system, whereby full-scale multiplication occurs in the skin and extends to the lungs and liver, resulting in secondary beremia. Generally, in normal people, secondary vilemia phenomena disappear 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. In other words, various cytokines are induced by cellular immune mechanisms to bind virus-infected neurons to inhibit 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. The reverse order of migrating back to the skin leads to herpes zoster [H. John et al., Virol. 5,241 (1991); and D.G. 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); R.P. Stephen et al., Pediatrics, 68, 14 (1981); and P.A. Patel et al., Pediatrics, 94, 223 (1979).

Currently, therapeutic agents that can suppress such VZV have been developed, such as vidarabine (Adenosine arabinoside) and acyclovir (Acyclovir), but because of their strong toxicity causes severe side effects on the kidneys and brain central nervous system Is very limited [TH Weller et al, New England J. Med., 309, 1434 (1983). Therefore, development of 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 fresh chickenpox vaccine was first developed in 1975 by the Takahashi Group of Osaka University, Japan [M. Takahashi et al., Biken. J., 18, 25 (1975)] Biken is producing Oka strain. In the United States, Merck's chickenpox vaccine (KMcC strain) has been used in limited immunosuppressive patients.In early 1994, the company obtained a patent from the Japanese company Biken and received FDA approval (Oka / Merck strain). Its safety and effectiveness have been proven to contribute to the prevention of chickenpox disease. In Japan and Korea, more than 2 million have already been vaccinated. Currently, more than 600,000 newborns are known to receive chickenpox vaccine in Korea.

In addition, several recent interesting reports suggest that shingles, which are commonly known to be common in older people, are due to reactivation of the virus by attenuating cellular immune responses, thus restimulating immunity with vaccination of live chickenpox vaccine. This prevents the development of shingles in older people [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.

VZV, on the other hand, was completely uncovered in 1986 by A.J. Davison [A.J. Davison et al., J. Gen. Virol., 67, 1759 (1986)]. VZV has six major glycoproteins (gpI-gpV, IE-62) produced by the three genes gA, gB and gC on the surface of the viral membrane, in particular gpI and gpIV make specific neutralizing antibodies against VZV Because there is possibility as recombinant vaccine [A. Vafai et al., Vaccine, 11, 937 (1993)].

Methods of proliferation of VZV using cell culture systems include, for example, human amnion cells, human thyroid cells, human fetal lung cells, human cervix, hela cells, and monkey kidneys. It has been reported to be possible in many cells, such as Meurisse et al., J. Microbiol., 2, 317 (1969)], since human diploid cells are suitable for vaccine development, current companies are developing unique cell lines and applying them to vaccine development.

VZV, on the other hand, has cell-dependent properties [M.C. Cook et al., J. Virol., 2, 1458 (1968)] Establishment of methods for obtaining large quantities of stabilized extracellular viruses with proper activity, and cell culture because of their minimal extracellular activity. The development of advanced cell culture technology that can multiply VZV using the 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); N.J. Schmidt et al., Infection and Immunity, 14, 709 (1976); and C. Grose et al., J. Gen. Virol., 43, 15 (1979)], glass bead crushing, cell homogeneous crushing, etc. have been reported, of which ultrasonic crushing is known to be the most effective. Sucrose and sorbitol have been mainly used as stabilizers to stabilize the chickenpox virus, for example 5 to 7.5% sucrose [M.R. Hilleman et al., US Pat. No. 4,000,256 (1975); K. Takahashi et al., US Pat. No. 3,985,615 (1978); 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)] has been added as a stabilizer.

As mentioned above, all the results so far are controlled by controlling various factors that may be present in cell crushing, which is a step for obtaining extracellular virus from virus infected cells, such as cell crushing method and stabilizer type. Activity enhancement was aimed at.

Chickenpox virus, unlike other herpes viruses of the same family, has a complete form of the total virus particles present in the cell, so that intact viruses are present in small amounts [M. L. Cook et al., J. Virol. 2, 1458 (1968). In other words, the chickenpox virus present in the cell is incomplete in that the large amount does not already have a complete virus form, the envelope and the capsid of the virus are damaged, or the central high density nucleus is missing. In particular, the outer membrane is very unstable and is damaged as it is secreted out of the cell, thus the infectivity of the extracellular virus is almost disappeared. This fact explains the cell-dependency of chickenpox virus.

In the case of other herpes viruses, the infection rate of all extracellular viruses is known to be about 1/50 [D. H. Watson et al., Virol., 19, 250 (1968)], and the case of chickenpox virus is reported to be 1 / 1,000,000 [D. G. Lawrence et al., Virology 2nd, 2011-2054 (1990)]. In other words, in the case of chickenpox virus, only one in a million viruses are secreted out of the cell in a fully infectious form.

There is no experimental evidence available to explain why chickenpox virus loses its infectivity as it is secreted out of the cell. Chickenpox virus binds with acidic phosphatase, a representative enzyme of lysosomes, forms a capsule in the Golgi apparatus, migrates to the cytoplasm, gathers near the cell membrane, and is secreted out of the cell. Existence has been experimentally demonstrated [A. Gershon et al., J. Gen. Virol., 18, 21 (1973). This suggests the possibility that the chickenpox virus can be degraded by lysosomal enzymes at the stage of secretion outside the cell. The other herpes virus, which has almost the same mechanism as chickenpox virus, is thought to remain infectious outside the cell because of its resistance to lysosomal enzymes.

On the other hand, children with chickenpox have cold symptoms, and when aspirin (acetylsalicylic acid) is taken, the symptoms become more severe. Aspirin inhibits the release of lysosomal enzymes from lysosomes [A. J. Dewar et al., Exp. Eye Res., 20, 63 (1975)], as a result, the chickenpox virus is not degraded by lysosomal enzymes and can be further activated. Indeed, in vitro cell experiments have been shown to increase the activity of chicken pox virus when aspirin is added after viral inoculation [M. A. Walz-Cicconi et al., Proc. Natl. Acad., 81, 5223 (1984)].

Lysosomal acidic phosphatase is found in several tissues of various species, and is classified into tartaric acid-inhibiting phosphatase and tartaric acid-resistant phosphatase depending on its sensitivity to the inhibitor tartaric acid [F. R. Susi et al., Am. J. Physiol., 211, 959 (1986)]. For human fibroblasts, 51% of acidic phosphatase in whole lysosomes and 80% of acidic phosphatase in lysosomes are reported to be tartaric acid-inhibiting phosphatase [B. M. Kelley et al., Mol. Cellular Biochem., 87, 171 (1989)].

Based on the above-mentioned facts, the present inventors believe that chickenpox virus is degraded if it can inhibit the activity of lysosomal acidic phosphatase in the stage of virus secretion out of the cell, that is, the virus is exposed to phosphatase and degraded. As a result of making the assumption that it can increase the viral activity by preventing the virus, the present invention was completed by confirming that tartaric acid, an inhibitor of lysosomal acid phosphatase, can increase the activity of chickenpox virus. .

It is an object of the present invention to provide a method of increasing the activity of chickenpox virus infected cells or chickenpox virus.

In order to achieve the above object, the present invention provides a method for enhancing the activity of chickenpox virus, characterized in that tartaric acid treatment is performed on chickenpox virus-infected cells or chickenpox virus.

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

In the present invention, MRC-5 (ATC CCL171), which is a human embryonic lung fibroblast, is used as a host cell for a virus, and a varicella Oka strain (ACTC VR-795, Lot No. 6w) is used as a viral cell line. Use

First, the MRC-5 was cultured in a culture flask (T 80) using DMEM (GIBCO BRL 12800-082) medium containing 10% FBS (GIBCO BRL 16000-028) and ABAM (antibiotic-antimycotic, GIBCO BRL 15240-013). , Nunc 178905, 80 cm ² ). Incubation temperature is 37 ℃, while maintaining a CO ₂ concentration of 5% incubator for a period of time in the incubator (Nuaire TM) to form a cell monolayer, phosphate buffer (D-PBS, GIBCO BRL 450-1600 EA) Wash three times 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%, washed three times again with the phosphate buffer solution and treated with 0.02% EDTA (GIBCO BRL 890-12661M) solution. Incubate the infected cells after standing for 10 minutes in the incubator.

Harvested infected cells were then subjected to cell disruption solution 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 1 ml per T 80 flask was added to% EDTA and 1% (w / v) human serum albumin) to make a cell suspension, and then disrupted until an almost circular cell was not seen using an ultrasonic longitudinal crusher (Branson sonifier 450). do. Then, the infected cell lysate is centrifuged for 10 minutes at a rate of 1000 rpm at a temperature of 4 ℃ to discard the precipitate and recover only the virus suspension.

Next, MRC cultured using DMEM medium in the same procedure in a petri dish 6.0 cm in diameter to determine the plaque forming cell (PFC) and extracellular viral activity (plaque forming unit (PFU)). The -5 cell monolayer is washed three times with PBS and then inoculated with an appropriate amount of serially diluted infected cells and virus suspension. At this time, in order to see the effect of tartaric acid, tartaric acid is added at least 24 hours before or 1 to 2 hours after inoculation. DMEM medium containing 3% FBS is added and cultured for 7 to 9 days in an incubator, and the plaques are counted by 100 magnification microscope (Nicon, TMS) to calculate viral activity of infected cells and virus suspensions. At this time, to reduce the error of the experiment is repeated three times per sample.

As a result, when tartaric acid was added before or after virus inoculation, the activity of infected cells (Plaque Forming Cell, PFC) was increased by 1.3 to 2.4 times, and the activity of extracellular virus (Plaque Forming Unit, PFU) was 1.2 to 1.7. It was found to increase by a factor of two.

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 Culturing and Harvesting Host Cells

MRC-5 (ATCCCCL171), a human fetal lung fibroblast cell, was used as a host cell and T 80 flask (Nunc 178905, 80 cm ² ) using DMEM (GIBCO BRL 12800-082) medium containing 10% FBS and ABAM. Incubated at 37 ° C. in the presence of 5% CO ₂ for 3 to 5 days. Once the cells had formed a monolayer, they were washed three times with PBS and treated with 0.25% trypsin solution (GIBCO BRL 15090-012) to harvest the cells after about 2-3 minutes. The harvested cells were centrifuged at 1000 rpm for 3 minutes and the precipitated cells were subcultured for the next experiment, or stored at −180 ° C. for a long time in a 10% glycerol solution containing 20% FBS, a cell storage solution, and used if necessary.

Example 2 Cultivation of Seed Virus

Varicella Oka strain (ATCC VR-795, Lot No. 6w) was used as chickenpox virus. Inoculated with the virus-infected cells on MRC-5 prepared in advance in the cell monolayer by the method of Example 1 and incubated for 2 to 3 days, washed three times with PBS, treated with 0.02% EDTA and left in the incubator 5 to Infected cells were harvested after 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 Crushing

Step 1. Virus Inoculation and Harvesting of Infected Cells

The cell monolayer prepared in the same manner as in Example 1 was washed three times with PBS, and then the virus infected cells prepared in Example 2 were inoculated so that the viral activity of the inoculum was 20,000 to 50,000 PFC / T 80 (2 ml). 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, crushed solution 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 add the infected cells to a concentration of 4-6 x 10 ⁶ cells / ml to make a cell suspension, and then using a microtip using an ultrasonic cell crusher (Branson sonifier 450). The cells were disrupted until the circular cells were almost invisible. At this time, the shredding solution was kept in an ice container and kept cold.

Infected cell lysate was centrifuged at 1000 rpm for 10 minutes at 4 ° C, and only the supernatant was taken for virus activity. At this time, the infected cell activity was 500,000 PFC / T 80 flasks, and the extracellular viral activity was 8,500 PFU / T 80 flasks.

Example 4 Investigating Virus Activity (Infectious)

After forming a cell monolayer of MRC-5 in a Petri dish 6.0 cm in diameter in the same manner as in Example 1, washed three times with PBS, 5 ⁿ of the infected cells and virus suspension prepared in steps 1 and 2 of Example 3 Serial dilutions (to 50 PFC, to PFU / dish). This was inoculated in an amount of 0.3 ml / dish when measuring the activity of infected cells and 0.1 ml / dish when measuring the activity of extracellular virus. After incubation for 30 minutes to 1 hour, DMEM medium containing 5% FBS was added.

At this time, in order to see the effect of tartaric acid, dissolve tartaric acid (Aldrich # 25138-0) in 1 mM in DMEM medium containing 5% FBS, and continuously dilute the diluted solution with 10 ^{n of} 0.6 ml per dish 24 hours before inoculation. Or 1 to 2 hours after inoculation.

After 7 to 9 days, plaques were counted under a microscope at 100 magnification to measure infecting cell activity (PFC) and extracellular virus activity (PFU). The results are shown in Tables 1 and 2 below.

As shown in the results of Tables 1 and 2, the activity of the infected cells (PFC) increased 1.3 to 1.6 times at a concentration of 10 μM or more at the pretreatment of tartaric acid, 2.1 to 2.4 times at a concentration of 1 μM or more at the post-treatment, Extracellular viral activity (PFU) was found to increase by 1.2 to 1.7 times in post-treatment at concentrations of 1 μM or more of tartaric acid. It was also shown that the activity of chickenpox virus showed a concentration-dependent tendency by tartaric acid.

As described above, according to the present invention, the method of treating varicella virus-infected cells or viruses with tartaric acid before or after inoculating the cells significantly increases the activity of chickenpox virus-infected cells and the activity of extracellular viruses. It can be seen that.

Claims (3)

A method of enhancing the activity of chickenpox virus, characterized by performing tartaric acid treatment on chickenpox virus-infected cells or chickenpox virus. The method of claim 1, The tartaric acid treatment is characterized in that to add a tartaric acid solution of more than 1 μM concentration. The method according to claim 1 or 2, The tartaric acid treatment is performed 24 hours or more before the inoculation or 1 to 2 hours after the inoculation.