KR19990038070A - Attenuated Japanese encephalitis virus strain and method for producing the vaccine containing the same - Google Patents

Abstract

The present invention relates to a new Japanese encephalitis strain capable of mass production and having an easier immunization schedule, and a method for producing a vaccine using the same, wherein Japanese encephalitis virus strain SA ₁₄ isolated from a Japanese encephalitis mosquito (Culex pipens) And a new Japanese encephalitis host SA ₁₄ -14-2 (strain accession number: KCTC 0316BP) is obtained by cultivating and attenuating the Japanese encephalitis virus, and the Japanese encephalitis vaccine is produced by applying it to the vaccine, .

Description

Attenuated Japanese encephalitis virus strain and method for producing the vaccine containing the same

FIG. 1 is a diagram showing RNA Sequence of attenuated Japanese encephalitis virus strain SA ₁₄ -14-2 according to the present invention.

The present invention relates to an attenuated Japanese encephalitis virus strain and a method for producing the vaccine containing the same.

Japanese encephalitis originates from mosquitoes and is a summer disease of the Arbovirus family, which has an important meaning in the national health in Asia. The lesion is most strongly present in the cerebral nucleus and changes in the cerebral cortex and cerebellum Symptoms of meningitis also appear strongly. It is known that infection rate is more than 60% and about 25 ~ 40% of survivors have permanent neurological or psychiatric diseases. Since Japanese encephalitis occurred in Japan and Korea in the summer of 1924, Japan, Korea, as well as recently China, India, Indonesia, Thailand, Malaysia and all of the Asian outbreaks of the disease has so far killed a lot of young people under the age of 10. In recent research efforts, the Japanese encephalitis virus from Culex pipens, a radio-mediator, has been isolated and inoculated with prophylactic vaccine to reduce the incidence of actual Japanese encephalitis.

The inventor of the present invention has conducted intensive studies to overcome the above-mentioned viral diseases. As a result, the present inventors have developed a preventive vaccine for preventing or limiting infection from these pathogens and filed the patent as a patent.

In order to prevent Japanese encephalitis, an inactivated vaccine obtained by inoculating and culturing a Nakayama strain in a mouse brain has been produced and spread internationally. This vaccine is inoculated three times in the first immunization and every two years in order to maintain the immune system, as well as the high price of production, so many countries were unable to carry out large-scale immunization and patient management was difficult, The simplest and easiest way to mass-produce the vaccine is to make cheap vaccines.

It is an object of the present invention to provide an economical and efficient method for mass production of a novel encephalitis strain and a vaccine using the same, which can be mass-produced and have an easier immune schedule.

The present inventor has obtained SA ₁₄ -14-2 through submerged culture and attenuation using the Japanese encephalitis virus strain SA ₁₄ isolated from the Japanese encephalitis mosquito (Culex pipens) larva. Respectively.

SA ₁₄ was inoculated in a number of 11 pigs in a mouse, and 100 cells in PHK cells were attenuated to produce HKC ₁₀₀ and purified three times in CE (Primary Chick Embryo) cells to select Clone 12-1-7. Again, the clone 12-1-7-17-4 was obtained from the CE cell twice, and the suspension was intraperitoneally injected into the mouse, and the virus suspension of the mouse spleen was harvested by one pass, Clone 2 was selected by cell plaque purification once, followed by three rounds of plasmid purification on CE cells to obtain Clone 9, which was subcutaneously injected into mice to pass one strain, and then the virus suspension obtained from mouse skin was transferred to CE cells After clone 9-7 was obtained, it was orally infected with hamster, and the virus suspension of the hamster spleen was transplanted for 6 times. Then, the virus suspension of the obtained hamster spleen was purified by two rounds of plasmid purification on PHK cells to obtain Clone 5-3, plaque purified and passaged 5 times in a row, after conducting a cloned twice by getting clone 14-2 this microbial JE SA ₁₄ -14-2 live virus strains termed, and international deposit agency, Korea Institute of Science and technology 12/12/1997 Here was deposited with Accession No. KCTC 0316 BP.

SA ₁₄ -14-2 virus that raw state SA ₁₄ with attenuated viruses of the invention with the following differences:

First, the nucleotide sequence of the nucleic acid (see FIG. 1) shows a difference of 57 nucleotides, so that there are 24 amino acid differences (see Table 1).

Table 1: Comparison of representative amino acid positions

Note: a- Location in the encrypted state

b-the amino acid in the translated sequence of the nucleotide in the untranslated sequence

SA (A) - Raw virus stock

SA (V) -SA ₁₄ -14-2 weeks

On the other hand, it can be called the SA ₁₄ -14-2 of a mutant derived from the native viral SA main precursor SA ₁₄ -5-3 The virus has several parameters were compared with experimental viral SA ₁₄ 14-2 CAUTION sustainability is immune Compared to SA ₁₄ -14-2, the dielectric stability was evaluated similarly. In monkey neuronpower experiments, the venom was more potent than SA ₁₄ -14-2. The Japanese encephalitis virus vaccine of the present invention is prepared by lyophilizing the attenuated strain SA ₁₄ -14-2 as a main ingredient with a stabilizer and the like, dissolving in water for injection at the time of injection, and subcutaneously injecting 0.5 to 1 ml thereof.

Hereinafter, the method for obtaining the virus strain of the present invention is described as an example, and its preparation examples are described. Toxicity experiment examples and clinical trial examples concerning stability are described below.

[Example 1]

How to obtain mutants from parental strains

A: Recipe summary

B: Detailed recipe

SA ₁₄ Virus strain was isolated from mosquitoes in Xi'an district in 1953, then it was comparatively tested with 14 domestic and foreign virus strains. As a result, its antigenicity was relatively broad, immunity was good, (neutropism) proved relatively weak.

This strain has good proliferation in the hamster kidney epithelial cells and has a remarkable action to cause cell lesion, so that it is convenient for culturing the cells in a serial manner.

First, a brain suspension for the 11th generation in the mouse brain is prepared, and this is used as a primitive virus (SA14A). Next, monolayer kidney tissue epithelial cells are prepared according to the Dulbecco's Pancreatin Digestion method using hamster kidney tissue. That is, a comprehensive medium containing 10-20% of bovine serum 199 as growth promoting particles is used as a cell growth solution, and cells are densely grown and then decantation is performed to remove the growth liquid. Thereafter, 0.2 ml of the virus suspension obtained above is added, and 0.8 ml of a medium containing 2-5% of rabbit serum 199 (growth promoting factor) is added to obtain a retentate. After incubation for 3-4 days in a CO ₂ incubator at 35-37 ° C, the resulting virus strain was repeatedly attenuated by the same procedure up to 100 times to obtain HKC ₁₀₀ .

It has the suspension of mutants for SA14A HKC ₁₀₀ based on the viral culture fetal cells to go through the selected clones (clone purification). In other words, three clones with weak neurovirulence or neurotropism are isolated through a test for mouse poisoning and a test for hamster infection (TCID ₅₀ experiment) (No. 9, No. 10, No. 12) The method is as follows.

First, 9-10 day old fetal cells are used for digestion with 0.25% pancreatin, and then a medium containing 10% bovine serum 199 as a growth promoting agent is added (dilution due to the medium is about 4 million cells per ml ), Take 12-15 ml of cell suspension, divide into 80 mm diameter petri dishes and dry. Remove oxygen from the dryer (combustion method) and incubate at 36-37 ° C for 3-4 days. When the cells are dense, remove the culture medium, inoculate 2 ml of diluted virus solution (HKC ₁₀₀ ) per plate, and incubate at 37 ° C for 1-2 hours. After completion of virus adsorption, remove the culture medium and add agar overlay.

Clone 10 showed viral particles with increased neurotoxicity during refinement process, while No. 9 had little change in neurotoxicity but had a large size of plaque. Thus, we chose Clone 12 and continued to refine, resulting in Clone 12-1-7. Clone 12-1-7 was cultured in the same manner as described above. Clones were isolated and two of the clones were selected and purified to obtain Clone 12-1-17-4.

The clone 12-17-4 was intraperitoneally injected into the mouse, and the virus suspension was harvested from the mouse spleen. Then, the above-described fetal cells were cultured, and Clone 2 was obtained through a single round of plasmid purification.

Clone 2-1-9 was obtained after three rounds of plasmid purification in fetal cells, and the clone was re-injected into the mouse for one pass, and then the virus suspension was obtained from mouse skin. Clone 9-7 was obtained from this harvest solution through the plagioclase once in the fetal cells by the above method.

Clone 9-7 did not meet the expected immunogenicity. So, we clone 5-3 with this clone, and the process is as follows.

Clone 9-7 was orally infected to hamsters, and after 6 passages, the virus suspension was harvested from the spleen of the experimental animals, and the harvest solution was cultured in PHK cells by the above-mentioned method. 3 is obtained.

The vaccine was prepared with the clone 5-3, and the immunity to human was investigated. As a result, the vaccine showed more than 85% seroconversion rate in the Ponto area, but only 61% in the non-climate area. The low immunity seen in non-climatic regions without antibody to Flavivirus prior to inoculation has shown that the SA14-5-3 virus is overexpressed and therefore does not reproduce a certain response in humans. Moreover, the process of practical application, the virus content of 5.0 logTCID ₅₀ / 20ml or less Once there was a drawback that can not get a good immune response.

Therefore, in order to obtain better immunity, Clone 14-2 was obtained by the following procedure.

First, clonal selection is carried out with a 5-year-old Korean virus suspension in a group cultured in infant mouse subcutaneous tissues for 7 days. In other words, mice were immunized to select a clone having a relatively good immunity.

Again, one was attenuated with PHK cells by the above plaque selection method to obtain Clone 14-2.

Clone 14-2 thus obtained excelled in human immunity while satisfying the criteria for safe attenuation (90% or more seroconversion rate when administered to the primary vaccine)

Clone 14-2 obtained was further subcultured six times in PHK cells by the above method to obtain master seed virus (PHK6)

10-12 days old healthy Syrian hamsters were dissected and the kidneys were taken out, trypsinized, washed several times, changed into maintenance solution, and cultured at 37 ° C to obtain the original hamster kidney monolayer cells (hereinafter referred to as PHK cells). The master seed virus strain in the lyophilized state is diluted, and the prepared PHK cell monolayer is infected and cultured in an EMEM medium containing 0.25% human serum albumin. The virus-infected culture is cultured at 35 ° C for 78-96 hours, harvested, collected and filtered. The stock solution is mixed, diluted, and added with 0.1% gelatin and 5% sucrose as a stabilizer. The obtained liquid vaccine is lyophilized and filled into vials. This vial was deposited with the Korean Gene Bank. (International Depositary Agency: Korea Institute of Science and Technology, Deposition Date: Feb. 12, 1997, KCTC 0316 BP)

[Example 2]

A. Manufacturing Process Summary

1. Preparation of Seed virus

B. Manufacturing process of formulation

(Note: Korean Pharmacopoeia Aseptic Test (3) is the same as Myco Plasma Test)

Virus note

SA14-14-2 A virus stock that is considered to be a state or equivalent is used, and a seed lot is set for this use case.

The following tests were carried out on the seed lot.

1. Aseptic test

According to the pharmacopoeial aseptic test methods (1), (2) and (3), it was appropriate.

2. Virus content test

The virus content was measured according to APPENDIX 1. When measuring the number of PFU in the sample, the value is within the range of 7.0-7.2 log PFU / ml.

3. Identification test

According to APPENDIX 2, when the specimens were mixed with non-kinetic anti-Japanese encephalitis virus (P3) immunized serum and grown using appropriate cell culture, the proliferation was neutralized by serum.

4. Inoculation test

4-1. Mouse Inoculation Test (Neurovirluence test)

0.03 ml each of 10 mice weighing 12-14 g were inoculated into each brain and survived for 14 days. It was not a problem to die within 3 days after the inoculation, and if there was a mouse that developed after 3 days, it was killed and the brain power was taken and the poisoning power was measured. The intracerebral brain power of the mice did not exceed 3.0 log LD ₅₀ per 0.03 ml of sample. ^10-1-fold dilution of a suspension of brain disease mice were injected subcutaneously at the same time, each of the 10 per 0.1ml mouse. No signs of infection by Japanese encephalitis virus were observed when they were observed for 14 days.

4-2. Neurotropism test

0.1 ml was injected subcutaneously into each of 10 mice weighing 10-20 g, and at the same time, the blood brain barrier was pierced and observed for survival for 14 days.

4-3. Atavism test

Three to five-day-old infant mice are injected with 0.2 ml each in the brain, and three infant mice are killed, and brain fluids are taken to measure the poisoning power.

Neuroprotective ability of mice weighing 12-14 g after brain-inoculation did not exceed 3.0 log LD ₅₀ per 0.03 ml of each sample. At the same time, brain suspension of each mouse in each of 10 mice was diluted 10 ^-1 times and injected subcutaneously in 0.1 ml each. No signs of infection by the Japanese encephalitis virus were observed at 14 days of observation.

5. Adultery virus testing

The sample was previously neutralized by mixing with non-kinetic anti-Japanese encephalitis virus immunized serum and tested as a sample. The antisera used should be obtained from animals other than humans, monkeys, and hamsters.

5-1. Animal Inoculation Test

5-1-1. Mature mouse vaccination test

Over 10 mice weighing 12-14 g were intravenously injected with 0.5 ml of sample per mouse and 0.03 ml into the brain and observed for 21 days. In the meantime, none of the animals showed infection by exogenous pathogens, and more than 80% of the animals survived.

5-1-2. Infant Mouse Inoculation Test

0.1 ml of sample per mouse was injected into the abdominal cavity and 0.01 ml was injected into the brain for more than 10 mouse infants within 24 hours after birth and observed for 14 days. In the meantime, none of the animals should show infection by exogenous pathogens, and more than 80% of the animals survived.

5-1-3. Guinea pig intraperitoneal inoculation test

In 5 or more guinea pigs weighing about 400 g, 5 ml of sample was injected into the abdominal cavity and 0.11 ml was injected into the brain, respectively, and observed for 21 days. In the meantime, none of the animals should show infection with M. tuberculosis and more than 80% of the animals survived.

5-1-4. Egg Inoculation Test

At least 10 eggs of about 11 days of incubation were injected into the urine membrane 0.5 ml per sample and observed for 3 days. Eggs of about 6 days of incubation were injected into the yolk sac in an amount of 0.5 ml per sample and observed for 9 days. In all of these tests, none of the eggs showed any change due to the presence of adventitious virus.

5-2. Cell culture inoculation test

5-2-1. Human cell culture test

10 ml of the sample was inoculated into human embryonic stem cells and observed for 14 days. The retentate (culture solution) was taken from each vessel of the cell culture for which the observation period was completed, frozen and thawed, and another human was inoculated to the culture of the embryoid cells for another 14 days. On the last day of observation, guinea pig red blood cells were added to confirm that hemocyte adsorption did not occur, and no cytotoxicity was observed by the exogenous virus during the entire observation period. In addition, over 20% of the cell cultures were not observable due to non-specific or incidental reasons.

5-2-2. Hamster kidney cell culture test

10 ml of the sample was inoculated into the hamster kidney primary culture and tested according to 5-2-1.

5-2-3. Monkey kidney cell culture test

10 ml of sample was inoculated into African green monkey kidney cell culture and tested according to 5-2-1.

6. Detection of retrovirus (Murine rumemia virus) by extracorporeal dilatation induction

It was suitable when tested according to APPENDIX 3.

7. Anti-retroviral replication enzyme test by double template

Appended when tested according to APPENDIX 4.

8. Monkey nerve poisoning test

The Japanese encephalitis virus has no antibodies. 0.5 ml of each specimen per animal is injected into each hemispheric sphincter and 0.25 ml is injected into the cerebellum in each of 10 animals or more for 21 days. During this period all animals should not exhibit paralysis or other neurological disturbances, and more than 80% of the animals must survive. In addition, animals that have undergone the observation period should not be admitted to the central nervous system with an inoculation virus or an abnormal lesion caused by an exogenous microorganism in the sample.

Viruses should not be used for more than three generations from seedlot.

Working Seed Virus shall be in accordance with the requirements set out above and may be stored at -20 ° C for 2 years in freeze-dried form and stored for -60 to 6 months in liquid phase.

Animal and kidney

Use a kidney of a Syrian hamster or a hamster of equivalent sensitivity. Animals should be healthy and tuberculin response negative. In addition, only autologous kidneys should be used when autopsy is not possible. If kidney anomalies or frostbite are found, discard them.

Culture solution

For cell culture, use EMEM medium (pH 7.2-7.4) suitable for cell culture. The cell culture medium may contain a cell growth factor (FBS), 0.002w.v% or less of phenol sulfophthaline, and a minimum amount of antibiotics required. Penicillin or β-lactam antibiotics should not be added. When the differentiation serum or its fraction is used as a cell growth factor, it should be operated in the manufacturing process so as to be 0.0001w / v% or less in the final stock solution. The culture medium of the virus is EMEM medium suitable for virus culture. Add the appropriate stabilizer and minimum amount of antibiotic to the viral culture. However, the heterologous serum or its fractions and penicillin or β-lactam antibiotics should not be added.

Virus suspension

For viral cultivation, a large-scale hamster kidney cell culture suitable for the virus strain is used. The cells are incubated at 35 ° C for 3-4 days. Cells are harvested when specific lesions (CPE) appear and then cooled to 2-5 ° C.

An amount corresponding to 5-10% of the cell culture per individual was used as a control cell culture, and the following test was performed:

1. Culture observation

Control cell cultures without virus inoculation were observed under the same condition as virus culture for 7 days and 14 days, showing no cytotoxicity due to adventitious virus. During the observation period, more than 20% of the control cell cultures were not observable due to nonspecific or accidental reasons.

2. Hemocyte adsorption virus negative test

It was confirmed that the guinea pig red blood cells were added to the control cell cultures after the observation period was completed, so that the hemocyte adsorption did not occur. If red blood cells are stored, they should be stored at 2-8 ° C within 7 days.

3. Non-hemocyte adhesion virus negative test

The retentate (culture medium) was taken from each container of the control cell cultures for which observation period was completed and mixed when necessary. When this was used as a sample, it was tested according to the extrinsic virus negative test (see above).

4. Detection of retrovirus (Murine vulgaris) by in vitro expansion

It was suitable when tested according to APPENDIX 3.

5. Anti-retroviral replication test by double template

Appended when tested according to APPENDIX 4.

Collect the harvested virus suspension and use it as virus suspension.

The following tests were performed on the virus suspension of each individual.

1. Aseptic test

(1), (2), (3) and (4) of the Aspergillus aseptic test.

2. Identification test

According to APPENDIX 2, when the specimens were mixed with non-kinetic anti-Japanese encephalitis virus (P3) immunized serum and grown using appropriate cell culture, the proliferation was neutralized by serum.

3. Virus content test

The virus content was measured according to APPENDIX 1. When measuring the number of PFU in the sample, the value is within the range of 7.0-7.2 log PFU / ml.

Collecting the virus suspension by object is called virus suspension before filtration. At this time, it is possible to add 0.002 w / v% or less of phenol sulfone phthalazine as a coloring agent, human serum albumin as a stabilizer and a necessary minimum amount of antibiotic.

The virus suspension before filtration was subjected to the following tests.

1. Aseptic test

(1), (2), (3) and (4) of the Pharmacopoeia Test.

2. Adulteration virus negative test

The sample was previously neutralized by mixing with non-kinetic anti-Japanese encephalitis virus immunized serum and tested as a sample. The antisera used should be obtained from animals other than humans, monkeys, and hamsters.

2-1. Animal Inoculation Test

2-1-1. Mature mouse vaccination test

Over 10 mice weighing 15-20 g were intraperitoneally injected with 0.5 ml of a sample per mouse, and 0.03 ml was injected into the brain and observed for 21 days. In the meantime, none of the animals showed infection by exogenous pathogens and more than 80% of the animals survived.

2-1-2. Infant Mouse Inoculation Test

0.1 ml of sample per mouse was injected into the abdominal cavity and 0.01 ml was injected into the brain for more than 10 mouse infants within 24 hours after birth and observed for 14 days. In the meantime, none of the animals showed infection by exogenous pathogens and more than 80% of the animals survived.

2-1-3. Guinea pig intraperitoneal inoculation test

In 5 or more guinea pigs weighing about 400 g, 5 ml of sample was injected into the abdominal cavity and 0.11 ml was injected into the brain, respectively, and observed for 21 days. In the meantime, none of the animals showed infection with Mycobacterium tuberculosis and more than 80% of the animals survived.

2-1-4. Egg Inoculation Test

At least 10 eggs of about 11 days of incubation were injected into the urine membrane 0.5 ml per sample and observed for 3 days. Eggs of about 6 days of incubation were injected into the yolk sac in an amount of 0.5 ml per sample and observed for 9 days. In all of these tests, none of the eggs showed any change due to the presence of adventitious virus.

2-2. Cell culture inoculation test

2-2-1. Human cell culture test

10 ml of the sample was inoculated into human embryonic stem cells and observed for 14 days. The retentate (culture solution) was taken from each vessel of the cell culture for which the observation period was completed, frozen and thawed, and another human was inoculated to the culture of the embryoid cells for another 14 days. On the last day of observation, guinea pig red blood cells were added to confirm that hemocyte adsorption did not occur, and no cytotoxicity was observed by the exogenous virus during the entire observation period. In addition, over 20% of the cell cultures were not observable due to nonspecific or incidental reasons.

2-2-2. Hamster kidney cell culture test

10 ml of the sample was inoculated into the hamster kidney primary culture and tested according to 2-2-1.

2-2-3. Monkey kidney cell culture test

10 ml of the sample was inoculated into african green monkey kidney cell culture and tested according to 2-2-1.

percolation

The virus suspension before filtration is centrifuged, the cells are removed by filtration, and the cells are combined to make the undiluted solution. The stock solution shall be tested.

1. Aseptic test

(1), (2), (3) and (4), and shall be appropriate.

2. Dyeing test

It shall be in accordance with the Yaku Dyeing Test Method and shall be appropriate.

Final stock solution and freeze-drying

Mix the stock solution and dilute if necessary to make the final stock solution. At this time, 1% gelatin and 5% sucrose are added as a stabilizer. This final stock solution is aseptically dispensed and lyophilized. The final stock solution was subjected to the following test before drying:

1. Aseptic test

(1), (2), (3) and (4) of the Pharmacopoeia Test.

2. Inoculation test

2-1. Mouse Inoculation Test (Neurovirluence test)

0.03 ml each of 10 mice weighing 12-14 g were inoculated into each brain and survived for 14 days. It was not a problem to die within 3 days after the inoculation, and if there was a mouse that developed after 3 days, it was killed and the brain power was taken and the poisoning power was measured. The intracerebral brain power of the mice did not exceed 3.0 log LD ₅₀ per 0.03 ml of sample. ^10-1-fold dilution of a suspension of brain disease mice were injected subcutaneously at the same time, each of the 10 per 0.1ml mouse. No signs of infection by Japanese encephalitis virus were observed when they were observed for 14 days.

2-2. Neurotropism test

0.1 ml was injected subcutaneously into each of 10 mice weighing 10-20 g, and at the same time, the blood brain barrier was pierced and observed for survival for 14 days.

2-3. Atavism test

Three to five-day-old infants were injected with 0.2 ml each in the brains of 10 mice to kill three infant mice. Brain suspension was taken and the poisoning power was measured. Neuroprotective ability of mice weighing 12-14 g after brain-inoculation did not exceed 3.0 log LD ₅₀ per 0.03 ml of each sample. At the same time, brain suspension of each mouse in each of 10 mice was diluted 10 ^-1 times and injected subcutaneously in 0.1 ml each. No signs of infection by the Japanese encephalitis virus were observed at 14 days of observation.

3. Residual Serum Protein Content Test

When analyzed by RPHA method according to APPENDIX 5 using ax antiserum against bovine serum proteins, residual bovine serum protein content was less than 50 ng per ml of sample.

black

1. Humidity test

It shall be not more than 3% according to the method of measuring the humidity of plating.

2. pH measurement test

According to the pharmacopoeial pH measurement, the pH should be 7.2-8.0.

When the number of plaques in human inoculation volume is measured using appropriate cell culture after stepwise dilution of the sample according to APPENDIX 1, the value should be within the range of 5.4-6.2 log PFU.

4. Stability test

According to APPENDIX 6, the lyophilized vaccine is left at 37 ± 1 ° C and tested 7 days later according to the '3-Activity Test'. At this time, the decrease in virus content should not exceed 1.0 log PFU compared to 7 days before.

5. Aseptic testing

It shall be in accordance with the Pharmacopoeia aseptic test methods (1) and (2) and shall be appropriate.

6. Abnormal Toxicity Testing

(2) of the Pharmacodynamic Abnormal Toxicity Test, and shall comply with this.

7. Retroviral (Murine rumemia virus) malignancy test by induction of ex vivo deaf lesion.

Appropriate for testing according to APPENDIX 3.

8. Anti-retroviral replication enzyme test by double template

Appropriate when testing according to APPENDIX 4.

9.. Identification test

According to APPENDIX 2, when the specimens were mixed with non-kinetic anti-Japanese encephalitis virus (P3) immunized serum and grown using appropriate cell culture, the proliferation was neutralized by serum.

<APPENDIX 1>

Virus content test

1) Overview

The virus content test should measure the potency by plaque formation in BHK ₂₁ cells.

The result should be expressed in units of log PFU of human inoculation dose.

2) Materials & Devices

* Cell culture medium

Eagle's MEM (10 ^-1 ) 10 ml

5% Lactalbumin Hydrosylate 5ml

3% L-Glutamine 1ml

7.5% NaHCO ₃ 2ml

Penicillin (10 ⁵ U / ml) 0.1 ml

Sterilized purified water 100ml

5% FBS 10ml

* Cell culture bottles

* 6-well tissue culture plate

* Pipette

* CO ₂ Incubation

* BHK ₂₁ cells

* 0.25% Trypsin-Earle's Solution

1% trypsin 25%

Earle's Solution 73%

7.5% NaHCO ₃ 2%

* Virus diluent

20% human serum albumin 2.5ml

5% Latalbumin Hydroxylate 2ml

7.5 ml of ₃ % NaHCO3

Penicillin (10 ⁵ U / ml) 0.1 ml

Sterilized purified water 100ml

* Overlay badge

1.5% Carboxymethyl Cellulose 100ml

Eagle's MEM (10 ^-1 ) m 13.5 ml

3% -L-Glutamine 1.5ml

7.5% NaHCO ₃ 4.5ml

Penicillin (10 ⁵ U / ml) 0.2 ml

15% FBS 5%

Sterilized distillation flask

Total 150ml

* 1% Crystal Violet Solution

5% Crystal Violet 100ml

0.85% NaCl 375 ml

Formaldehyde 25ml

3) Test method

<Cell Culture>

The cell culture medium should be placed in a cell culture container with 0.25% Trypsin-Earle's Solution. After digesting at ambient temperature for 1 to 3 minutes, discard Trypsin-Earle's Solution.

BHK ₂₁ cells should be mixed by shaking the container after inoculation. 50 ml of cell culture medium should be placed in the distributed cells and mixed evenly.

<Inoculation>

The analytical sample should be redissolved and diluted in EMEM.

Each 0.2 ml of vaccine diluent should be inoculated into 2-4 wells using a 6 or 24-well plate as a test sample.

Add 1.0 ml of the BHK ₂₁ cell suspension to the wells. The culture plate is incubated for 90 minutes under the condition of 5% CO ₂ at a temperature suitable for culture of Japanese encephalitis virus.

The medium is then removed from each well and replaced with 4 ml of medium containing the appropriate Overlay (1% carboxymethyl cellulose), and the culture plate is incubated for 5 days.

<Fixed, Dyeing, Counting>

The cells are fixed and stained to visualize the plaque. The commonly used reagent is a formaldehyde solution of 1.0% Crystal Violet.

First, the medium is removed from each well and the cells are carefully washed.

2 ml of 1% Crystal Violet formaldehyde solution is added and the plate is allowed to stand at room temperature for 15-20 minutes.

Then, the staining is removed, the cells are carefully washed with water, and the culture plate is dried. Count the number of plagues and calculate PFU per human inoculum volume by the following formula.

[log PFU / 0.5 ml = log A + log (No. of Plague x 0.5 / 0.2)]

<APPENDIX 2>

Identification test

1) Principle

Japanese encephalitis virus appears as a method of neutralizing BHK ₂₁ cell culture using a specific antibody against Japanese encephalitis virus.

In other words, when the vaccine virus is mixed with an antibody specific to Japanese encephalitis virus, it is possible to neutralize the cytotoxicity caused by the virus seen in the control group.

2) Method

The test sample is mixed with the same amount of guinea pig-derived serum specific for the Japanese encephalitis virus (P3) strain and incubated at 37 ° C for 90 minutes.

Place BHK ₂₁ cells in a 6-well cell culture plate and inoculate 0.3 ml each of the neutralized test sample.

The non-neutralized test sample is set as a control group and subjected to a plague test at the same time and then compared.

3) Results and Interpretation

There should be no plug when observed 5-7 days after inoculation.

<APPENDIX 3>

Retrovirus by in vitro expansion

(Murine leukemia virus) negative test

(Extended In Vitro Focus Induction Assay for Murine leukemia Virus)

1) Purpose

Detection of the presence of xentropic or amphotropic retroviruses derived from murine or other species present in the specimen. Mink MiCL1 S + / L- cells were expanded in MV1LU cells (Mink lung cells) through S + / L- Is observed.

2) Control setting

① positive control group

xenotropic retrovirus

amphotropic retrovirus

② Negative control group

RPMI 1640 medium

③ Test cell

Mink Lung MiCL1 S + / L-cells

Mink Lung (MV1LU) cells

3) Examination Process

① Test method

The presence of S + / L- lesion formation is determined by direct and extended assays.

② Test period

21st

③ Direct test method

Test substances and positive and negative controls were inoculated on a culture plate containing Mink S + / L-cells prepared by adding polybrene for 24 hours and adsorbed for 60 minutes. After the adsorption is completed, add the polybrene-containing growth medium and change the medium as necessary. After 8 days, count the lesions generated by microscopic observation.

④ Extended test method

The test material and the positive and negative controls are inoculated into a 25 cm 2 tissue culture flask containing Mink lung cells pre-treated with polybrene, and the viruses and negative control cells infected for a minimum period of 10-14 days, which is necessary for virus multiplication, are proliferated. Cells are incubated at least three times during this period. After the final pass, the culture is harvested and inoculated into Mink CiCL1 S + / L- cells and tested for the lesion-forming virus by direct assay.

4) Evaluation of the test

The presence of S + / L-lesion forming virus formed in positive and negative controls is determined by comparison with the case of the test material.

<APPENDIX 4>

Negative test for retroviral replication enzyme by double template

(Testing for Reverse Transcriptase by Dual Template)

1) Purpose of examination

The presence of murine and human retroviruses is determined via retroviral replication enzymes.

2) Control setting

① positive control group

A suspension of cell cultures known to be positive for retrovirus or retroviral replication enzyme (M-MLV)

- Manganese Divalent Ion Constituent Retrovirus or equivalent virus

- magnesium divalent ionic retrovirus or equivalent virus

-DNA polymerase

- Substances which are spiked with murine retrovirus or suquile monkey retrovirus in test substance

② Negative control group

Cell culture supernatant known to be negative for retroviral replication enzyme

3) Examination Process

① Test method

The presence of an exogenous or endogenous retrovirus is tested indirectly through retroviral replication enzymes.

Since the DNA polymerase is not specific to either DNA or RNA template, the density of a specific cell culture is high, and a large amount of intracellular DNA generated by self-decomposition may replicate the polymerase and be incorporated into the cell culture medium. Since the polymerase derived from the DNA thus incorporated exhibits fluorescence activity, the activity of the DNA polymerase originating from the RNA can be obscured, so that the analysis of the results is made clear when the double-stranded template is used instead of a single template.

② Test period

one week

③ Negative test for retrovirus replication enzyme

The test material, positive, negative control, and spiked materials are centrifuged (12,000 rpm, 2 hours, 4 ° C).

The precipitate is resuspended in 4 ° C buffer and divided into 96-well culture plates in appropriate amounts. And the material containing the template precursor is incubated in a 96-well culture plate for 1 hour at 37 占 2 占 폚.

After incubation, the cells are separated from the material contained in each well, transferred to mini-fold 1 containing DEAE 81 paper, and dried under vacuum. When the culture is injected into the filter, the fluorescence activity after washing, drying and cutting is measured by Ambis System or Beckmann Sxintillation Counter.

4) Test result

① Validation of test

The validity of the test is determined according to the results of the control group.

② Positive judgment

The centrifuged sample should be at least 4 times the negative control.

Results in RNA templates in duplicate templates should be at least 1000 fold.

The result ratio for the DAN template of the RNA template should be at least 0.6.

<APPENDIX 5>

Residual Bleeding Test

(RPHA test: Reverse Passive Hamagglutination Test)

1) Principle

During production of the vaccine, animal serum can be added as a component of the cell growth promoter in the cell growth medium. This must be removed before the final stock solution stage to rule out the possibility of chilling.

The minimum vanity content does not exceed 50 ng of bovine serum albumin per ml of final stock solution.

The rabbit-derived anti-serum antibody is adsorbed to the 'aldehyde red blood cells' using tannic acid. Aldehyde red blood cells can specifically aggregate bovine serum.

2) Materials & Devices

* Diagnostic hemocyte cell device

: Tannin blood cells, glutaraldehyde blood cells, sensitized blood cells.

* V-type micro-agglutinating plate

* Pipette

* pipette

* Vibrator

* Watering device

* Positive control of test vaccine

* Bovine serum positive control

* Standard bovine serum

* Negative control of test vaccine

3) Course

① Preparation of Sensitized Blood Cell

&Lt; aldehyde &lt; / RTI &gt; Double-aldehyde &gt;

Positive blood cells should be removed, the fibers removed, washed five times with physiological saline (NS), and centrifuged (1,500 rpm, 15 min).

After centrifugation (1,000 rpm, 10 min), the cells were washed three times with sterile purified water to obtain 10 ml of 10% glutaraldehyde solution, % Cell suspension (PBS (pH 7.2)).

Each 100 ml of 10% hemocyte suspension is mixed with 1.0 ml of 35% formaldehyde (formalin), and the plate is washed at 37 ° C for 1 hour in a water bath, then twice with NS, and twice with PBS (pH 7.2).

Thrombocytes (10: ⁴ ) are added to 10% hemocyte suspension, mixed evenly and stored at 4 ℃.

<Tannin Reaction of Blood Cells>

10% double-aldehyde blood cells are washed with PBS (pH 7.2), centrifuged (1, 500 rpm, 2 min) and then suspended in 5% cell suspension.

Tannic acid (1:20, 000) equivalent is mixed with the cell suspension, left to stand in a water bath at 37 ° C for 12 minutes, and then washed three times with NS solution to obtain 2% tannin blood cells (PBS, pH 6.4).

<Reduction of hemocyte cells>

Dilute the digested, purified rabbit anti-bovine serum antibody with appropriate PBS (pH 6.4). The antibody was mixed with 2% tannin blood cells at a ratio of 1: 1. The mixture was incubated in a water bath at 37 ° C for 45 minutes, washed twice with PBS (pH 7.2), and then washed with NS solution containing rabbit serum to obtain 1% .

(2) Determination of the degree of cell hematopoiesis; Agglutination potency of standard bovine serum

Standard bovine serum should be diluted stepwise in micro-agar plates with the same amount of sensitized erythrocyte and incubated for 1 hour at 37 ° C.

Coagulation completion is determined by "++".

("++") standardized bovine serum diluted with sensitized blood cells should be taken as the sensitivity (aggregation potency) of the blood cells.

③ Negative control test of test vaccine

0.25 μl of the test vaccine is mixed with 0.25 μl of 1% tannin blood cells or glutaraldehyde hemocytes and cultured at 37 ° C for 1 hour.

If the coagulation reaction is negative, perform the following operation.

④ Operation

The test vaccine is diluted 2-fold and added to each well of the V-type microaggregation plate.

Add 1% sensitized cells (25 μl) each to the same plate and mix evenly with the test sample diluent using a vibrator.

Incubate at 37 ° C for 1 hour.

After coagulation is completed ("++"), the bovine serum protein content is calculated using the following equation.

① Calculation of bovine serum protein content

&Lt; Bovine serum content (ng / ml) >

= Susceptibility of sensitized blood cells × A

(A: maximum dilution degree in which the aggregation result shows "++")

&Lt; Bovine serum protein content (ng / ml) >

= Bovine serum content x 0.05

<APPENDIX 6>

Stability test

1) Principles and methods

Three vials are taken from the lyophilized vaccine lot and left at 36-38 ° C for 7 days before measuring the titer.

Measure the titers of vials not exposed to heat simultaneously.

2) Results and judgment

The results should be expressed in PFU per human dose.

[log PEU / 0.5 ml = -logA + log (No. of Plague x 0.5 / 0.2)]

The "arithmetic mean viral titer" of this vial should be greater than the minimum required value of the inoculated amount of human inoculum and the arithmetic mean viral load of the vaccine should not be increased by more than 1.0 log during the incubation period.

C. 1. Amount of raw material in vial

Main ingredient: Inoculated Japanese encephalitis virus 5.4 LogPFU or higher

(Virus name: SA ₁₄ -14-2)

Stabilizer: Gelatin 5.0 mg

Sucrose 25 mg

Human serum albumin 2.5 mg

0.03 mg of sulfuric acid kanamycin

Antimicrobial Gentamycin 12u

Colorant: 0.003 mg of phenol sulfonophthalene

Attachment: Injection 0.7ml

2. Efficacy · Effect

Prevention of Japanese encephalitis

<Acute Toxicity Experimental Example>

1) Test animals: 120 RAT (SD, female, 60 male): 5 to 6 weeks

MOUSE 120 (ICR, ♀, ♂ 60 each): 4 weeks

2) Test method

3) Duration of administration: once a day

4) Observation period

(1) Oral administration: 6 hours after administration and 14 days from the next day of administration

(2) Intraperitoneal injection: 6 hours after administration and 7 days from the next day of administration

5) Test result

6) Conclusion

LD ₅₀ values in PO and I. P were higher than 8.40 log PEU / kg (50 ml / kg) in mood mode and LD ₅₀ values in PO and I. P in rats were 8.40 log PEU / kg (50 ml / kg). Mortality and body weight were not observed due to the administration of the test substance and were not considered to be a specific response by the test substance itself.

&Lt; Antigenicity Experimental Example &

Passive Cutaneous Anaphylaxis (PCA) response using mouse-rat system

1) Test animals: Mouse- BALB / C (sensitization)

Rat-SD (for PCA reaction)

2) Test method

a. Sensitization of mouse

OVA: Ovalbumin,

Alum: Aluminum

b. The rat's PCA response

c. Observation and inspection items

① General symptoms and observation of dead animals

② Weight change

③ Determination of PCA response: Observation of blue half-hour after 30 minutes of PCA reaction

- Judged as positive if the average value of long diameter and short diameter of the emerging blue bar is 5 mm or more

3) Results

a. GENERAL SYMPTOMS AND DEATH ANIMALS: No abnormal signs and deaths were observed in the experimental group during the test period.

b. Weight change: No significant change

c. Determination of PCA response: In the positive control group (OVA + alum), antibodies were detected from 10 to 640 times dilution, but no antibody was detected in all other groups.

<Experimental Study on the Efficiency of Japanese Encephalitis Virus Vaccine>

1. Summary

The effect of one injection was 80% (95% CI 44 ~ 93%) and the effect of two injections was 97.5% (86 ~ 99.6%). Several times the test results were found to be efficient. We have found that administering two doses of the Japanese encephalitis virus vaccine, one year after a single injection, can prevent clinically significant diseases.

2. Patients and Methods

1) Research model

We conducted case-control studies in rural areas surrounding the Sichuan province of Sichuan in China.

2) Check the case

Two city hospitals and 24 rural hospitals were recruited for rural residents around Chengdu, China, to confirm the suspected cases of Japanese encephalitis. The estimated cases were all children under the age of 15. Most of them have abnormalities in the central nervous system, and they show symptoms such as headache, epilepsy, coma, delirium, and gait, which are accompanied by a high temperature of 37.5 ℃. For serologic confirmation, we included only patients with at least one serum or CSF sample (unconfirmed or suspected) as part of the clinical treatment.

3) Serological confirmation

The neutralization antibody against Japanese encephalitis was measured by the plaque reduction experiment. If the neutralizing antibody level of the serum is above 80, the case that meets the clinical criteria is classified as domestic encephalitis, and if two serum samples exhibit a 4-fold change in neutralizing antibody changes and neutralizing antibody If politics is more than 10, the case is classified as authentic Japanese encephalitis.

4) Control group

Because each village experienced only one case of encephalitis during the study period, the control group consisted of all children of a given age who were at risk of developing encephalitis at the time of the occurrence of the case. This strategy is called "DENSITY SAMPLING".

5) Statistical analysis

Vaccination efficacy as a randomized trial was calculated as 1-RR (RR is the incidence of the group in which the vaccine was isolated from the incidence of non-vaccinated group). If DENSTTY SAMPLING is used in this case and the control group, the exposure odd ratio (OR) and RR value are considered to be a measure of error. We used conditionl logistic regression to calculate the OR and 95% CIs compared to once, twice, and three live vaccines for vaccination and unvaccinated for each case. We used conditional logistic regression of multiple variants to control and calculate the effect of conditional logistic regression. In the first analysis, we calculated two effects on uninoculation and used EGRET.

3. Results

158 cases of potential Japanese encephalitis after clinical inoculation with at least one biological sample were clinically isolated. 59 potential cases (57%) were excluded because serologic evidence for recent infections by Japanese encephalitis virus was not an unreliable estimate. 39 (25%) potential cases were also serologically estimated and excluded from the vaccine effect analysis. Only 60 potential cases (38%) are serologically positive.

Table 2. Serological effects experiments

NE = Not calculated due to insufficient data.

52% of chronic cases were women and the average age of all cases was 4.7 years. In four chronic cases, no treatment was useless. 1, 299 control groups corresponding to the remaining 56 chronic cases were prepared. The mean age of the control group was also 4.7 years old and 57% of the control group was female

The table shows the degree of previous vaccination between case and control. 68% of the cases never received the vaccine compared to 47% of the control. The efficacy of a single vaccination was 80% (95% CI 44 to 93%) and the twice vaccination effect was 97.5% (86 to 99.6%). In the past, inoculation with inactivated vaccine was 71% (21 to 90%) in one live vaccine and 97.6% in two.

In the past, the efficacy of once inoculated vaccines was 61% (-14 to 86%), except for the case of inoculation of small doses of inactivated vaccines. The effect of the second vaccination was 97.5% (86 to 99.6%). One inoculation effect in the village with a vaccination record score of 5 (the first vaccine record) was 82% (12 to 96%) and the second vaccination effect was 94% (16 to 99%). The results of the third inoculation were not calculated because there were few cases in which three inoculations were made.

Claims (3)

The attenuated Japanese encephalitis virus strain SA ₁₄ -14-2 (KCTC 0316 BP) Japanese encephalitis vaccine containing attenuated Japanese encephalitis virus strain SA ₁₄ -14-2 (KCTC 0316 BP). In vitro in vitro focus induction assay for murine leukemia virus (KCTC 0316 BP) and the Japanese encephalitis vaccine containing the enriched Japanese encephalitis virus strain SA ₁₄ -14-2 (KCTC 0316 BP) ) And the use of dual template for testing for reverse transcriptase by dual template.