KR100859345B1 - Method of producing smallpox vaccine using cell culture and the smallpox vaccine produced by the method - Google Patents

Abstract

The present invention comprises inoculating and culturing vaccinia virus NYCBOH strain in a human-derived cell line; And recovering the attenuated vaccinia virus from the culture, a method for producing an attenuated vaccinia virus, and the prevention of a head wound comprising attenuated vaccinia virus and a stabilizer produced by the method or The present invention relates to a therapeutic immunogenic composition.

Vaccine vaccine, vaccinia virus, berryola virus, cell culture

Description

Method of producing smallpox vaccine using cell culture and the smallpox vaccine produced by the method}

Figure 1 shows the results of performing the crmB genome PCR of the NYCBOH virus, Lister / Elstree virus, WR strain (left) and the analysis results (right) after treatment of the crmB genome PCR product with NlaIII restriction enzyme .

2 is a diagram showing the results of measuring virus production after infecting a vaccine for head vaccine with M.O.I (multiplicity of infection) at various concentrations.

3 is a diagram showing the result of measuring the virus production according to the culture time after infecting the virus for the head vaccine with MRC-5 cell line 0.01 M.O.I.

4 shows the results of measuring the degree of virus titer retention of six candidate stabilizers.

5 is a schematic diagram of a vaccine production process for the vaccine.

The present invention relates to a method for preparing a vaccine against the vaccine from New York City Board of Health Strain vaccinia virus (NYCBOH) using cell culture, and to a vaccine produced by the method.

Acute infectious disease caused by a virus is a papule rash after 7 to 17 days of incubation period (average 12 days), followed by blister pustules, and more than 30% mortality. Leave The bacillus pathogen, variola virus, belongs to the Orthopoxvirus genus, along with monkeypox, cowpox, and vaccinia virus. Orthopox virus viruses are characterized by inducing strong cross-immunity with each other, so vaccines made from vaccinia virus, which is less pathogenic than that of the berryola virus, have been used in the global program for eradicating cramps. The WHO proves its effectiveness by declaring its complete disappearance (WHO. Declaration of global eradication of smallpox. Wkly Epideminol Rec 1980: 55: 148).

Veriola virus, a pathogenic bacterium, is 1) stable in aerosol conditions, 2) easy to mass-produce, 3) capable of infecting in trace amounts, 4) highly contagious among humans, and 5) long (7 to 17 days) incubation period. 6) It has characteristics that are easy to be developed as biological weapons, such as high mortality, and there has always been a concern about the risk of spillage.

Uncertainty about the eradication of poisons and the dangers of biological terrorist weapons have led to stockpiling of vaccines in countries for emergency use. In the US, some generations of cows are inoculated with cows from the first generation of vaccines. Some developed countries, including Japan, also develop their own vaccines or purchase first-generation vaccines. It is known that measures are taken.

The first generation of vaccines used in the WP eradication program was vaccinia virus produced from the skin of animals such as cattle, rabbits and sheep. This method using live animal skin was effective in vaccine production, but there were concerns about quality, including microbial contamination. In addition, due to the limited supply and expiration of the current validity of the first-generation vaccine, the development of a second-generation vaccine for vaccines using MRC-5 (human lung diploid fibroblast), Vero cells, etc. It is an urgent situation.

Accordingly, the present inventors cultured and cultivated the New York City Board of Health (NYCBOH), the same virus strain as the original virus strain of the first generation vaccine DRYVAX ^R , in an MRC-5 cell line, in order to develop a high purity and safe vaccine against the vaccine produced by cell culture. The technology to purify was developed. Vaccines produced through the manufacturing process developed by the present inventors have been found to be safe and effective as a result of preclinical studies, and are currently undergoing clinical trials based on preclinical results.

It is an object of the present invention to provide a method for producing a vaccine against the NYCBOH virus strain using cell culture.

It is still another object of the present invention to provide a vaccine for vaccines produced from NYCBOH virus strains using cell culture.

In order to achieve the above object, the present invention comprises the steps of inoculating and culturing vaccinia virus NYCBOH strain in human-derived cell line; And recovering the attenuated vaccinia virus from the culture.

The method for producing attenuated vaccinia virus of the present invention comprises inoculating and culturing vaccinia virus NYCBOH strain in a human derived cell line.

In the present invention, the human-derived cell line may be an MRC-5 cell line which is a cell line of diploid fibroblasts of human fetal lung tissue.

In the present invention, the human-derived cell line may be cultured for 2 days to 3 days at 37 ° C., 5% CO ₂ incubator with DMEM medium containing 10% serum, and then cultured to 100% confluency.

In the present invention, the vaccinia virus NYCBOH strain may be one of ATCC VR-118 or CJ-MVB-SPX. The CJ-MVB-SPX is a NYCBOH master virus bank prepared by passage four times the NYCBOH virus line (ATCC VR-118) obtained from ATCC in MRC-5 cell line.

The New York City Board of Health strain (NYCBOH) virus is a vaccinia virus belonging to the poxvirus family, also known as the Lederle-Chorioallantoic strain. It is known to be derived from humans and grows well in CE, RabK, HaK, KB, WI-38, LLC-MK2, and vero cells. According to the virus characteristics provided by the ATCC, NYCBOH has a passage history of alternating culture in cattle, rabbits, humans and CE.

The vaccinia virus infecting MRC-5 cells of the invention can be derived from the CJ-MVB-SPX master virus bank.

In the present invention, MRC-5 cells can be infected by inoculating vaccinia virus at 0.001 to 0.1 M.O.I (Multiplicity of Infection). Preferably, the cultured MRC-5 cells can be infected with vaccinia virus of 0.001 to 0.01 M.O.I, more preferably 0.01 M.O.I vaccinia virus.

The method for producing attenuated vaccinia virus of the present invention may comprise culturing MRC-5 cells infected with vaccinia virus derived from NYCBOH virus strain in serum-free medium.

In the present invention, the serum-free medium may be DMEM.

In the present invention, the step of culturing the MRC-5 cells infected with vaccinia virus in a serum-free medium may be performed for 40 hours to 50 hours. Preferably, the culture of MRC-5 cells may be performed for 46 hours to 48 hours.

The method for producing the attenuated vaccinia virus of the present invention is inoculated with 0.01 MOI of an attenuated NYCBOH strain derived from CJ-MVB-SPX to a human-derived MRC-5 cell line cultured to 100% confluence and infected with the above. The infected cells may be incubated for 48 hours in serum-free medium.

The method for producing attenuated vaccinia virus of the present invention may comprise recovering the attenuated vaccinia virus from a culture obtained by culturing MRC-5 cells infected with vaccinia virus.

In the present invention, recovering the attenuated vaccinia virus produced may be performed by releasing the virus through freezing and thawing from the infected cells recovered in the culture. Freezing and thawing to release such viruses can be repeated three to five times. Preferably the freezing and thawing step is performed three times, followed by sonication after repeated freezing and thawing steps.

The invention also provides an attenuated vaccinia virus produced by the method.

The attenuated vaccinia virus produced by the method of the invention may be a virus of CJ-50300 (KCCM 10797).

The attenuated vaccinia virus of the present invention has a substantially equivalent or improved immunogenicity and stability to NYCBOH or existing Nancy Vaxinia Berna (Beran Biotech Ltd., Beran) derived from NYCBOH.

The present invention also provides an immunogenic composition for the prevention or treatment of head pain, comprising the attenuated vaccinia virus produced by the method and a stabilizer.

Since the vaccine produced by cell culture is an attenuated live virus vaccine, it should be harmless to the living body and can be preserved for a long time. According to the WHO guidelines, the virus is less than 10 times of the starting titer for 1 month when stored at 37 ℃ after freeze-drying. It must ensure that the basic conditions are maintained to ensure that the pH and moisture content remain stable. Therefore, the vaccine can be suspended and lyophilized and stored in a stabilizer capable of stably maintaining the vaccine.

In the present invention, the immunogenic composition for preventing or treating apox may include a stabilizer having the following composition:

NaCl 6.0-7.0 g

KCl 0.1-0.2 g

Na ₂ HPO ₄ 12H ₂ O 2.0-3.0 g

KH ₂ PO ₄ 0.1-0.2 g

Sucrose 40-60 g

Sodium L-Glutamate 0.1-2.0 g

1.0-3.0 g of gelatin

20-30 g of hydrolyzed gelatin

EDTA trisodium salt 0.01-0.2 g

Amount to be 1,000 ml of distilled water.

Preferably, the stabilizer in the present invention is 6.4g NaCl; KCl 0.16 g; 2.3 g Na ₂ HPO ₄ .12H ₂ O; 0.16 g KH ₂ PO ₄ ; 50 g sucrose; 1.0 g sodium L-glutamate; 2.0 g of gelatin; 25 g of hydrolyzed gelatin; 0.1 g of EDTA trisodium salt of EDTA; The composition may have an amount of 1,000 ml of distilled water.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these embodiments are intended to illustrate the present invention by way of example, but the scope of the present invention is not limited to these embodiments.

Example

Example  One. Viralism  Confirm

Dryvax ^R is a vaccinia vaccine approved by the U.S. Food and Drug Administration (FDA) derived from the New York City Board of Health (NYCBOH) virus strain, which was used by the bovine calf lymph method until 1982. (Wyeth-Lederle, ATCC Accession No. VR-325). Dryvax ^r Consists of live, attenuated vaccinia virus and does not exist as a cell culture product. The inventors were the most attenuated virus line among the vaccinia virus used in the WP eradication campaign, and it was able to induce cell culture and strong cross immunity, and select the available NYCBOH virus line as the WP vaccine production virus line.

The present inventors signed a contract for the use of viral strains from the American Type Culture Collection (ATCC) to use NYCBOH for vaccine development.

The NYCBOH virus line obtained from ATCC was passaged four times in MRC-5 cell line to prepare a master virus bank, which was named CJ-MVB-SPX.

A rapid and accurate method for detecting and distinguishing the virus of the orthopoxvirus family to which the virus belongs, to identify the CJ-MVB-SPX virus at the genetic level, and the WHO collaborating center (Atlanta, Ga.) PCR-RFLP analysis of the crmB gene was adopted and used (Vladimir N. Journal of Clinical Microbiology, 2001; 39: 94-100). DNA isolation was performed using EzWay Genomic DNA Mini Kit V (Virus) from KOMA Biotech Inc., and was directly isolated from virus stock. PCR was performed using Biioner's PCR-premix kit with 20 μL of DNA isolated from the final 40 μL in 250 μL of viral stock as a template. The composition of the reaction mixture was 20 μL of DNA solution, 28 μL of PCR-premix suspended in distilled water, and 2 μL of 1 μL of the primers of SEQ ID NO: 1 and SEQ ID NO: 2 of 10 pmol / μL, respectively, and the total volume was 50 μL. It was. The reaction mixture was subjected to a program procedure in which denaturation (94 ° C., 1 minute), binding (55 ° C., 1 minute), and extension (72 ° C., 1 minute) were repeated a total of 30 times to obtain an amplified PCR product. The amplified gene was extracted with DNA using QIAGEN's QIAquick PCR purification kit, and the extracted DNA was separated on a 1% agarose gel to confirm its size.

Virus species of the orthopoxvirus family have different genome-size crmB gene PCR products. Among these, vaccinia virus has a size of 1210 bp. The PCR fragment size of the crmB gene having cross-specificity was 1210 bp in all of the CJ-MVB-SPX, Lister / Elstree, and WR viruses, as shown in the photograph of FIG. However, when the PCR product of the crmB gene was treated with restriction enzyme NlaIII, NYCBOH virus had DNA fragments of the size shown in Table 1, similar to Lister / Elstree virus, showing DNA characteristics distinct from WR strain. By using the NlaIII-RFLP pattern analysis method for the crmB gene, the genome DNA of CJ-MVB-SPX showed restriction fragment length polymorphism (RFLP) similar to Lister virus among vaccinia virus. there was.

Table 1. PCR-RFLP Analysis of the crmB Gene

Virus strain Size markers NYCBOH WR Lister DNA fragment size (bp) 500 400 300 200 100 423 256 243 96 77 38 35 4 1 406 267 220 96 77 38 35 23 4 1 423 267 243 97 77 38 35 4 1

Example  2. Establishment of Vaccine Manufacturing Process: Process Step Conditional Test

MRC-5 cells were selected and used as a cell line for the production of vaccine. The most optimal vaccine production conditions through studies confirming the optimal culture and purification conditions of the virus were 2 to 3 days in a 37 ° C., 5% CO ₂ incubator with DMEM medium containing 10% serum, as indicated in Table 2. Serum-free DMEM medium after infecting MRC-5 cells cultured to 100% confluency with 0.01 MOI (Multiplicity of Infection) derived from CJ-MVP-SPX prepared in Example 1 (serum free media) by changing the medium was confirmed that the conditions for 48 hours in 37 ℃, 5% CO ₂ incubator. Viruses cultured in optimal condition were released via four sonications, 20 seconds each time after three freeze / thaw cycles. Virus micrographs confirmed that the virus in the vaccine stock removed cell debris by centrifugation showed a typical brick shape. The viral titer of the obtained vaccine stock satisfies the WHO specification> 1 × 10 ⁸ PFU / ml.

As described above, the virus derived from CJ-MVB-SPX, a master virus bank prepared by subcultured with NYCBOH, was infected with MRC-5 cell line to establish optimal culture conditions and purification conditions for producing a vaccine by cell culture. . Based on this, preclinical samples were produced on a scale of 1/6 of the actual scale, and a safety test and an efficacy test were performed in mice as follows, and manufacturing was started in a KGMP facility according to the results. In addition, changchang vaccine prepared in KGMP facility was used for the safety and efficacy test in monkeys.

   Table 2. Optimal Cell Culture Conditions for Poison Vaccine Production

Item Condition / Method Cell line MRC -5 Inoculation potency 0.01 M.O.I Incubation time 48 hours Production medium Serum free DMEM  ( Serum free ) Virus release method 3 freeze / thaw cycles

2.1 Optimal M.O.I. decision

Remove the medium from T185 flasks (7 × 10 ⁶ cells / T185 flasks) in which MRC-5 cells were cultured to 100% confluence and place 0.001, 0.01, 0.1, 1, and 10 MOI in serum-free Dulbeccos Modification of Eagles Medium (DMEM) medium. Inoculated with virus diluted. Incubation time after inoculation was 2 hours, and the flask was shaken every 30 minutes after inoculation to allow the virus solution to come into contact with the cells. After the viral infection, remove the virus solution into the inhaler (aspirator) behind, 37 ℃, CO ₂ Incubated for 48 hours in the incubator. Infected cells were cultured and removed using a scraper after removing the culture medium. The recovered infected cells were transferred to a centrifuge bottle, centrifuged at 3,500 rpm for 10 minutes at 4 ° C. to remove the supernatant, and then crushed three times by freezing / thawing. Cell lysates were indirectly sonicated (Sonication) and then centrifuged at 3,000 rpm for 15 minutes at 4 ℃, the supernatant was removed. The plaque vaccine stock thus obtained was subjected to a plaque test to determine the titer. For plaque testing, first, Vero cells were seeded in 6-well plates at a concentration of 0.8 × 10 ⁶ cells / well, followed by 37 ° C., CO ₂ The incubator was incubated for about 24-30 hours until 100% confluency. Remove the medium from each well and wash once with serum-free DMEM, then add 200 μL of virus dilution (10 ^{-2-10 -7} ) per well and press 37 ° C, CO ₂ Infected for 2 hours in the incubator. Every two minutes, the cells were gently shaken every 30 minutes to spread the virus fluid evenly. After the infection was completed, the virus infection solution was removed, and 3 mL of overlay medium containing 1% volume of 2% LMT (Low melting temperature) agarose solution and 2X Medium 199 was added to each well. If the gel is hardened by standing at room temperature for 20 ~ 30 minutes, 37 ℃, CO ₂ Transfer to the incubator and incubated for 3 days. A fixed solution (5% formalin solution in saline) was added to each well and left at room temperature for 3 hours, and then the agarose gel was separated and washed with water and stained solution (5% formalin, 1% crystal violet, 20% saline solution). Ethanol) was added to the bottom of the well soaked for 5 minutes and washed with water. After the remaining solution was removed well and dried, the number of plaques was counted.

2 is a diagram showing the titer of virus infection concentration. As shown in Figure 2, it showed high productivity upon infection with a concentration of 0.01 M.O.I.

2.2 Determining optimal virus incubation time

Medium was removed from T185 flasks (7 × 10 ⁶ cells / T185 flasks) in which MRC-5 cells were cultured to 100% confluence and inoculated with virus diluted with 0.001, 0.01, 0.1, 1 and 10 MOI in serum free DMEM medium. . Incubation time after inoculation was 2 hours, and the flask was shaken every 30 minutes after inoculation to allow the virus solution to come into contact with the cells. After the viral infection, remove the virus solution into the inhaler (aspirator) behind, 37 ℃, CO ₂ Incubators were incubated for 0, 24, 30, 48 and 72 hours, respectively. Infected cells were cultured and removed using a scraper after removing the culture medium. The recovered infected cells were transferred to a centrifuge bottle, centrifuged at 3,500 rpm for 10 minutes at 4 ° C. to remove the supernatant, and then crushed three times by freezing / thawing. The cell lysate was indirectly sonicated and then centrifuged at 3,000 rpm for 15 minutes at 4 ° C. to remove the supernatant. As in 2.1 above, the titer of the stock vaccine vaccine obtained thereby was determined via a plaque test.

3 is a diagram showing the titer of each culture time after viral infection. As shown in FIG. 3, after infection at a concentration of 0.01 M.O.I, high productivity was observed at 48 hours of incubation.

2.3 Determine optimal virus release conditions

The cells harvested to release the virus were repeated three times in a -70 ° C deep freezer for three hours and three times in a 25 ° C water bath for 15 to 20 minutes. Ultrasonicated. The release of the virus by sonication after the repeated freeze / thaw process showed a low likelihood of bacterial contamination in the vaccine production and good yield. Therefore, this was determined as the optimal virus release condition.

Example  3. Selection of the best stabilizer

Since the vaccine produced by cell culture is attenuated live virus vaccine, it should be harmless to the living body and can be preserved for a long time. According to the WHO guidelines, it guarantees the loss of 1 x log10 ¹⁰ or less for 1 month when stored at 37 ℃ after lyophilization according to WHO guidelines. It must meet the basic conditions for keeping pH and moisture content stable.

Comparative experiments were carried out by preparing six stabilizers known to be used as stabilizers for live virus vaccines through patents and related documents. The six stabilizer compositions are shown in Table 3.

Fourteen T185 flasks in which MRC-5 cells were cultured to 100% confluence (1.78 × 10 ⁷ / flask) were inoculated with dilutions of NYCBOH vaccinia virus, CJ-MVB-SPX to 0.01 MOI, followed by incubation for 2 hours. After removing the inoculated virus solution, 40ml of DMEM containing 1% NEAA (non essential amino acid) was added and the virus was propagated in 5% CO ₂ , 37 ° C incubator for 48 hours. After removing the supernatant, the cells were washed twice with PBS (pH 7.2) containing 0.2% gelatin, and the buffers used were collected in tubes kept on ice. To isolate virus infected cells, PBS containing 0.2% gelatin (pH7.2) was added 3 ml per T185 flask, shaken every 30 minutes, and reacted in a 5% CO ₂ , 37 ° C. incubator for 3 hours. After the reaction, the solution containing the isolated infected cells and the buffer solution used for washing are collected and dispensed into 6 tubes. The supernatant is removed by centrifugation at 2,000 rpm for 10 minutes under cooling (4 ° C). Recovered. The recovered infected cells were suspended in 3.2 ml of 6 prepared post-security tablet solutions. Three freeze / thaw cycles were performed to release the amplified virus from the cells, followed by ultrasonic treatment. Centrifuged at 1,000 rpm for 10 minutes in a cold state (4 ° C) to obtain a stock vaccine vaccine. 0.4 ml per vial was dispensed from the obtained vaccine for vaccine for dialysis and lyophilized. The lyophilized vaccine virus was stored at 37 ° C., and the vials were taken out at weekly intervals and dissolved in 0.55 ml of PBS to confirm virus titer through plaque assay using Vero cells.

As shown in Table 4, stabilizers 1 and 6 showed high stability. In case of stabilizer No. 6, the freeze-dried vaccine virus was distorted when stored at 37 ° C. for one week due to high water absorption due to excessive content of sorbitol, and finally stabilizer 1 was selected as an optimal stabilizer.

Table 3. Composition of candidate stabilizers

stabilizator Furtherance One NaCl 6.4 g KCl 0.16 g Na ₂ HPO ₄ .12H ₂ O 2.3 g KH ₂ PO ₄ 0.16 g Sucrose 50 g Sodium L-glutamate 1.0 g Gelatin 2 g Hydrolyzed gelatin 25 g EDTA Trisodium salt 0.1 g 1,000 ml . 2 _{NaCl 6.4g KCl 0.16g Na 2 HPO 4} .12H 2 O 2.3g KH 2 PO 4 0.16g lactose 50g D- sorbitol 20g L- cysteine 2g of sodium L- glutamate, 1.0g gelatin 7.5g hydrolyzed gelatin 25g EDTA trisodium salt Distilled water is added to make 0.1 g 1,000 ml. 3 Nail 12.8g KCl 0.32g Na ₂ HPO ₄ .12H ₂ O 4.6g KH ₂ PO ₄ 0.32g Lactose 100g D-sorbitol 40g L-Cysteine 4g Sodium L-Glutamate 2.0g Gelatin 15g Hydrolyzed Gelatin 50g EDTA Trisodium 0.2 Distilled water is added to make 1,000 ml. 4 Lactose 8% Sorbitol 2% Mannitol 2% EDTA 0.01% Human Albumin Solution 0.2% L-Arginine 0.5% L-Cysteine-HCl 0.01% Kanamycin 0.0035% KH₂PO₄ 0.007% Na₂HPO₄0.004% MgSO₄.2H₂O 0.006% NaCl 0.5% KCl 0.02% CaCl₂.2H₂O 0.005% 5 3 times the concentration of the post-security tablet No. 4  6 5% Sorbitol, 5% Peptone

Table 4. Determination of Optimum Stabilizers

 stabilizator  Before freeze drying Titer log (pfu / ml) Days of storing at 37 ^o C after freeze drying 0 7 14 21 28 One 8.0 7.8 7.4 7.0 7.0 7.0 2 7.8 7.6 7.2 6.7 6.7 6.5 3 8.0 7.6 7.1 6.6 6.7 6.7 4 8.6 8.4 8.2 7.6 7.9 7.0 5 8.6 8.5 7.6 <6 <6 <6 6 8.2 8.1 7.5 7.6 7.9 7.7

To check whether the selected stabilizer is stable in terms of humidity and pH, the free virus-free stabilizer was lyophilized. The lyophilized stabilizer was stored at 4 ° C. (long-term stability), 25 ° C. (acceleration condition), and 37 ° C. (severe condition), and pH and humidity were measured for 4 weeks. As a result, as shown in Table 5 and Table 6, the selected stabilizer was confirmed that the pH or the humidity is kept stable even under accelerated and harsh conditions.

Table 5. Moisture stability test results of stabilizers

Storage condition (temperature) Week 0 1 week 2 weeks 3 weeks 4 Weeks 4 ℃ 0.8 1.16 0.94 1.16 1.51 25 ℃ 0.8 2.04 1.99 2.4 2.16 37 ℃ 0.8 1.34 1.36 1.62 1.72

Table 6. pH stability test results of stabilizers

Storage condition (temperature) Week 0 1 week 2 weeks 3 weeks 4 Weeks 4 ℃ 7.24 7.09 7.15 7.15 7.19 25 ℃ 7.24 7.12 7.14 7.18 7.15 37 ℃ 7.24 7.09 7.09 7.19 7.14

Example  4. Final manufacturing process

Through experiments to confirm the optimal culture conditions of cells and viruses, the optimal vaccine production condition is that the cells were cultured to 100% confluence and infected with the virus for 0.01 MOI and replaced with serum-free medium for 48 hours. Confirmed. Vaccine virus cultured under optimal conditions was released from cells by sonication after three freeze / thaw cycles to prepare attenuated vaccinia virus CJ-50300.

Figure 5 is a schematic process diagram of the vaccine production by the vaccine by the cell culture established by the inventors. After establishing the optimum culture and purification conditions for CJ-50300, preclinical samples were produced at 1 / 6th the actual production scale, and safety and efficacy tests were performed in mice. Started. In addition, the vaccine was prepared in the KGMP facility for safety and efficacy tests in monkeys.

Example  5. pock  Validity test of the vaccine

In order to confirm the immune response for evaluating the effectiveness of the vaccine prepared in Example 4, IgG ELISA using a mouse as a bovine animal, a neutralizing antibody test, and production of alpha-IFN using lymphocytes of the mouse spleen Enzyme-linked immunospot assay (ELISPOT) and fluorescence activated cell sorting assay (FACS) were performed for cell analysis and infection defense tests using the lethal intranasal vaccinia (strain WR) challenge model. Was performed. As a large animal, the cyanomolgus monkey was used to measure the antibody titer and neutralizing antibody titer. Detailed evaluation items and results are summarized in Table 7 below. Lancy Vaxinia Berna, used as a positive control in the following tests, belongs to the orthopox virus family, but is different from NYCBOH, and is the first generation of vaccines that were officially licensed in the UK and used in the vaccine eradication program.

Table 7. Validation Summary Table

animal Test Items Way result mouse ELISA Negative control (1: 1 mixture of solutions and stabilizers used for virus dilution) and positive control (Lancy Vaxinia Berna) were administered to mice transdermally to confirm vaccinia specific antibody formation (ELISA) Negative control not detected Specific antibody identified in specimen and positive control Neutralizing antibody test For the measurement of B-cell mediated immune responses after transcutaneous administration of Lancy Vaxinia to mice, expressed as 50% neutralizable serum dilution multiples CJ-50300 shows an inferior B-cell mediated immune response compared to the positive control group (2 ¹¹ ~ 2 ¹² vs 2 ⁹ ~ 2 ¹⁰ ) γ-IFN inducibility For the purpose of measuring T-cell mediated immune response after mouse transdermal administration of Lancy Vaxinia FACS ratio of γ-IFN-expressing splenocytes CJ-50300 showed inferior T-cell mediated immune response compared to positive control (4.73% vs 3.96%) Major Cutaneous Reaction Observed after transcutaneous administration of Lancy Vaxinia mice Excellent major skin reactions observed on day 8 in both groups Infection Defense Test Negative control, Lancy Vaxinia mouse transdermal administration, post-immunization 50xLD _{50 ,} 100xLD ₅₀ Infecting WR virus to observe infection defenses All negative controls died; both CJ-50300 and positive controls survived; Infection Defense monkey ELISA Percutaneous administration of Lancy Vaxinia monkeys confirmed formation of vaccinia-specific antibodies after immunization (ELISA) Specific antibody identified in positive control group and test substance administration group Neutralizing antibody test Percutaneous administration of Lancy Vaxinia monkeys for measurement of post-immune B-cell mediated immune responses, expressed as 50% neutralizable serum dilution multiples The neutralizing antibody was identified in the non-inferior positive control group and the test substance administration group compared to the positive control group. The titers were similar.

Example  6. pock  Safety test of the vaccine

In order to confirm the safety of the vaccine prepared by the above manufacturing process was measured whether single toxicity, repeat toxicity, neurotoxicity in mice, single toxicity test in monkeys, immunotoxicity and genotoxicity in guinea pig. Table 8 summarizes the details and the results.

Table 8. Summary Table of Biological Safety of Poison Vaccine Producing Virus Lines

Test Items Way result Single dose toxicity (mouse) Negative control mouse transdermal administration 5.0 x 10 ⁵ PFU / head 21 days death, weight change, general symptoms observed, autopsy findings No toxicity was found No general symptoms noted in both control and test groups, no deaths, no weight changes Single dose toxicity (monkey) Negative control monkey transdermal administration 2.5 x 10 ⁵ PFU / 2.5ul / head 6 weeks general symptoms, urine test, blood test, ECG test, autopsy No toxicity was found. No significant symptoms, including death and weight changes, were observed in both the control and test groups. Repeat dose toxicity (mouse) Negative control, mouse repeated transdermal administration, repeated 4 times, 4 weeks 0, 2.5 x 10 ⁵ PFU, 5.0 x 10 ⁵ PFU / head General symptoms, urine test, blood test, autopsy No change other than reversible change in spleen weight related to pharmacological action. Non-toxic amount is estimated to be 5.0 x 10 ⁵ PFU / head for both male and female. Immunotoxicity (Gunier Pig) Percutaneous and intravenous administration to negative control, positive control (OVA) guinea pigs 0, 2.5 x 10 ⁵ PFU, 5.0 x 10 ⁵ PFU / head 5.0 x 10 ⁵ PFU / head + FCS, OVA + FCS anaphylaxis shock response test No immunotoxicity Anaphylactic shock response result Negative control, moderate abnormality observed in test substance group, which is a natural physiological phenomenon after 10 minutes Positive control group 5/5 cases died within 10 minutes Neurotoxicity (mouse) Evaluation of local replication in the brain Single control in the brain of the negative control group, WR strain, and Lancy Vaxina mice 10 ² PFU / 20ul 28-day survival rate, virus titer measurement in the brain All 15 cases of non-inferior WR strain group died compared to Lancy Vaxina. Both CJ-50300 and Lancy Vaxina survive 8/15. There was no significant difference in neurotoxicity between the two groups when comparing results such as virus titer measurements. Neuroinvasiveness Assessment Negative Control, WR strain, and Lancy vaxina Mice Intranasal Administration 2.5 x 10 ⁵ PFU / 20ul 14-day survival rate, virus titer in brain, lung and blood No non-inferior test group death animals compared to Lancy Vaxina.Blood, lung and blood results show that BBB permeability is highest in the positive control group, and there is no significant difference between the test and comparison groups. Genotoxicity Inoculate SOS chromotest, negative control (1: 1 mixture of solution and stabilizer used for virus dilution), positive control (4-nitroquinoline 1-oxide) E. coli , PQ37 in Escherichia coli at the following concentrations: Incubator 2 x 10 ⁶ PFU / Analysis, 1 x 10 ⁶ PFU / Analysis, 5 x 10 ⁶ PFU / Analysis No genotoxicity. IF value did not increase by more than 1.5 at all concentrations, it was negative. Peripheral blood micronucleus test for mice, positive control group (MMC intraperitoneal administration) mouse transdermal administration, 5 x 10 ⁵ PFU / head microscopy to increase the number of micronuclei in red blood cells No induction of micronucleus formation Positive control increased the number of micronuclei (3.6 → 47.8), test group showed no micronucleus number (3.6 → 3.6)

According to the vaccine production method of the present invention, it is possible to produce a safe and high quality vaccine by cell culture. The head immunogenic composition comprising the head vaccine and stabilizer produced by the method of the present invention can be stably maintained even after prolonged storage without weakening and denaturing immunogenicity.

<110> CJ Corporation <120> Method of producing smallpox vaccine using cell culture and the          smallpox vaccine produced by the method <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for crmB <400> 1 acatgcatgc caggac 16 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for crmB <400> 2 accattacaa acattatcc 19  

Claims (7)

delete delete delete delete The diploid fibroblast MRC-5 cell line in human lungs was inoculated with the attenuated vaccinia virus New York City Board of Health (NYCBOH) strain from the Vaccinia Virus Master Virus Bank at 0.001 to 0.1 Multiplicity of Infection (MOI). And culturing in serum-free medium; And Attenuated vaccinia virus produced according to the method for producing attenuated vaccinia virus comprising the step of freezing and thawing cells recovered from the culture, followed by sonication to release and recover the attenuated vaccinia virus. KCCM 10797. delete An attenuated vaccinia virus and stabilizer according to claim 5, wherein the stabilizer has the following composition: NaCl 6.0-7.0 g KCl 0.1-0.2 g Na ₂ HPO ₄ 12H ₂ O 2.0-3.0 g KH ₂ PO ₄ 0.1-0.2 g Sucrose 40-60 g Sodium L-Glutamate 0.1-2.0 g 1.0-3.0 g of gelatin 20-30 g of hydrolyzed gelatin EDTA trisodium salt 0.01-0.2 g Amount to be 1,000 ml of distilled water.

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