WO2012088763A1

WO2012088763A1 - Hepatitis a virus strain sh and method for adapting same to diploid cells

Info

Publication number: WO2012088763A1
Application number: PCT/CN2011/002181
Authority: WO
Inventors: 张现臣; 魏文进; 黄秋香; 刘雨; 钟汉斌; 王春雨; 孟红彦
Original assignee: 深圳康泰生物制品股份有限公司; 北京民海生物科技有限公司
Priority date: 2010-12-28
Filing date: 2011-12-26
Publication date: 2012-07-05
Also published as: CN102174477A; CN102174477B

Abstract

Disclosed are a novel hepatitis A vaccine virus strain SH, a method for isolating the same, and a method for adapting the same to MRC-5 cells. The virus was isolated from the feces of a patient with an acute infection of hepatitis A and then transmitted to human diploid MRC-5 cells for adapted culture. In the adaptation process, the culture period for the early generation subculture was 35 days, and the culture period after the eighth passage was shortened to 24 days. With eight further passages, the antigen titer reached 1:512-1:1024, and the virus infectious titer reached 7.0-8.0lgCCID₅₀/ml. Immunogenicity and cross protection assays showed that the hepatitis A inactivated vaccine produced from the virus strain has a good protective effect of immunogenicity. The virus strain is an ideal strain for the large-scale industrial production of hepatitis A inactivated vaccine.

Description

Hepatitis A virus strain SH and its diploid cell adaptation method

The present invention relates to the field of microorganisms, and in particular to a hepatitis A virus strain SH and a diploid cell adaptation method thereof. Background technique

Hepatitis A, referred to as hepatitis A, is an acute infectious disease caused by hepatitis A virus that seriously endangers human health. Hepatitis A mainly spreads through the "fecal-mouth" route, or between individuals, or from water or food contaminated with hepatitis A virus. After infection of hepatitis A in older children and adults, more than 70% are clinically infected, with a mortality rate of 0.3% to 0.6%; the mortality rate of patients over 50 years old is 1.8%; the risk of acute liver failure after chronic hepatitis patients infected with hepatitis A Raise. With the improvement of living conditions, the number of adults infected with hepatitis A has increased, and the proportion of clinical hepatitis A has increased, making hepatitis A a serious public health problem.

China is a high-risk area for hepatitis A. At least 240,000 people suffer from hepatitis A every year, causing huge economic losses and social harm. Therefore, the development and development of effective vaccines and drugs for the prevention and treatment of hepatitis A has become an urgent problem to be solved. .

At present, the inactivated vaccine for hepatitis A produced in China is of different quality due to the different strains, cell matrices and production processes used by various manufacturers. China has a large population, and the hepatitis A vaccine is far from meeting the needs of the domestic market. Summary of the invention

It is an object of the present invention to provide a novel Hepatitis A virus SH strain.

Another object of the present invention is to provide a method for adapting a Hepatitis A virus SH strain on diploid cells.

A further object of the present invention is to provide an application of the Hepatitis A virus SH strain and its passaged virus in the preparation of a vaccine or medicament for preventing and treating hepatitis A.

In order to achieve the object of the present invention, a novel hepatitis A virus SH strain of the present invention, It has been deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee. It is located at the Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing, China. The deposit number is CGMCC NO. 4501. The deposit period is December 21, 2010.

The method for isolating the virus strain comprises the following steps:

(a) Taking 30-50 g of feces from patients with acute hepatitis A infection, adding 40 and 5 volumes of PBS with a diameter of 3 mm and shaking for 10 minutes;

(b) Centrifuge at 3000 rpm for 20 minutes to collect the supernatant;

(c) treating the precipitate twice as described above, collecting and combining the supernatant three times;

(d) Add PEG6000 lOg/lOOml and NaC1 2.3g/100ml to the supernatant, and set it to 4'C for several hours after all dissolution;

(e) Centrifuge at 8000 rpm for 30 minutes, discard the supernatant;

(f) The precipitate was resuspended in 50 ml PBS, shaken for 20 minutes by adding an equal volume of chloroform; centrifuged at 3000 rpm for 30 minutes, the upper liquid phase was collected, and PEG6000 10g/100ml and NaCl 2.3g/100ml were added, dissolved and placed at 4 ° C overnight. ;

(g) The supernatant was centrifuged at 8000 rpm for 30 minutes the next day, and the precipitate was added with 10 ml of PBS to prepare a virus suspension. Dialysis for 15-18 hours, change dialysate PBS 3 times, then add ΙΟχΜΕΜ;

(h) Filter sterilization, add 1% P.S.

Wherein, the MEM is 0.03% Glu, 0.08% NaHCO ₃ , 0.01% penicillin and 0.01% streptomycin; PS is penicillin and streptomycin, wherein the molar ratio of penicillin to streptomycin is 1:1.

The present invention also provides a method for adapting a Hepatitis A virus SH strain on diploid cells, comprising the following steps:

1) MRC-5 cells were cultured in a conventional manner, and the cells were grown into a single layer and then inoculated with a suspension of Hepatitis A virus SH, a culture solution of 1 ml of virus solution in a 40 cm ² flask, adsorbed at 37 ° C for 2 hours, and a maintenance solution, 35 C. Culture, change the virus maintenance solution once every 7 days, culture for 35 days, wash the cell surface 3 times with PBS, digest the cells with trypsin, add the MEM suspension culture cells to the culture area of 0.01 ml/cm ² to obtain the virus cell suspension. , ultrasonically disrupting cells, preparing a virus suspension; 2) Serially transfer 15-22 generations to MRC-5 cells by the method of step 1).

Wherein, the maintenance solution is MEM plus 2% calf serum.

The virus adapted to the above cell culture can be used as a vaccine production candidate after being tested.

The novel hepatitis A virus SH strain obtained by the invention has a virus antigen titer of 1:512-1:1024, and a virus infection titer of 7.0-8.01 gCCID ₅ . /ml, the immunogenicity and cross-protection test, the hepatitis A inactivated vaccine produced by the strain has good immunogenic protective effect, and the strain has a short proliferation cycle and reproductive efficiency on MRC-5 cells. High characteristics, suitable for use as a large-scale industrialized production of hepatitis A inactivated vaccine, is an ideal strain for the production of inactivated hepatitis A vaccine. detailed description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1 Method for isolating hepatitis A virus strain SH

(a) A total of four clinical hepatitis A patients (patients admitted to the Hepatitis Department of the Sixth People's Hospital of Shenyang, China) were collected for 30-50 g of acute feces, and 40 and 5 volumes of PBS with a diameter of 3 mm were added and oscillated. 10 minutes;

(b) Centrifuge at 3000 rpm for 20 minutes, collect the supernatant;

(d) Add PEG6000 lOg/lOOml and NaC12.3g/100ml to the supernatant, and set it to 4 °C for several hours after all dissolution;

(e) Centrifuge at 8000 rpm for 30 minutes, discard the supernatant;

(f) The precipitate was resuspended in 50 ml of PBS, shaken for 20 minutes by adding an equal volume of chloroform; centrifuged at 3000 rpm for 30 minutes, the upper liquid phase was collected, and PEG 6000 lOg/lOOml and NaCl 2.3 g/100 ml were added, dissolved and placed at 4 ° C overnight;

(h) Filtration sterilization, adding 1% PS. Wherein, the MEM is 0.03% Glu, 0.08% NaHCO ₃ , 0.01% penicillin and 0.01% streptomycin. (MEM was purchased from Beijing Qingda Tianyi Biotechnology Co., Ltd., production batch number: 080302). RS. is penicillin and streptomycin, wherein the molar ratio of penicillin to streptomycin is 1:1.

Example 2 Adaptation of Hepatitis A Virus Strain SH on MRC-5 Cells

The cryotube containing MRC-5 cells was quickly placed in a 40 ° C water bath and then thawed, and then added to a MEM cell culture medium pre-warmed to 30-37 ° C, and placed in a 37 ° C culture, through 3-4 After growing into a single layer, inoculate the suspension of the Hepatitis A virus strain SH strain isolated in Example 1, 40 ml ² culture flask 1 ml virus suspension, adsorb at 37 ° C for 2 hours, add 20 ml of maintenance solution (MEM plus 2% calf Serum), cultured at 35 °C, change the virus maintenance solution once every 7 days, culture for 35 days, wash the cell surface with PBS 3 times, then digest the cells with 0.125w/v%J enzyme, press 0.01ml/cm ² The culture area was added to the MEM suspension-precipitated cells, that is, the virus cell suspension, and the cells were ultrasonically disrupted to prepare a virus suspension.

According to the above method, the Hepatitis A virus strain SH strain isolated in Example 1 was continuously uploaded on the MRC-5 cells for 15 generations, and finally a MHC-5 cell-adapted hepatitis A virus was obtained.

The hepatitis A virus strain SH of the present invention is characterized by:

Virus particle diameter: 27-32 nm;

Acid resistance: Under the condition of pH 3.0, the effect of 2-8'C for 8 hours, the infection titer did not change significantly;

Alkali resistance: No significant change in infection titer after 8 hours at 2-8 °C in ether; Neutralization test: Neutralized by HAV-specific antibody;

Gene sequence analysis: The gene sequence homology with the wild-type hepatitis A virus strain HM-175 is as high as 95.92%, and belongs to the I B gene subtype.

Antigen titer: 1:512-1:1024;

Infectious titer: 7.0-8.01g CCID ₅₀ /ml;

Peak period of value added: 21-24 days; Optimum culture temperature: 35'C-36 °C;

Value-added parts: intracellular cytoplasm;

Has the characteristics of hepatitis A virus.

The antigen titer and virus infection titer of different generation SH strains of the present invention are shown in Table 1.

Table 1 Different antigenic titers of SH strains and virus infection titer Subatomic antigen titer (EIA) Virus infection titer (logCCID ₅₀ /ml)

1 - 1.0

2 1:16 1.5

3 1:32 2.0

4 1:64 2.0

5 1:64 2.5

6 1:128 3.0

7 1:128 3.33

8 1:256 3.5

9 1:256 4.0

10 1:256 5.5

11 1:512 6.33

12 1:512 7.0

13 1:512 7.5

14 1:1024 8.0

15 1:1024 8.0

The results of the 15th generation proliferating peak antigen titer and virus infection titer of the SH strain of the present invention are shown in Table 2.

Table 2 SH strain 15 generation proliferative peak period test results days antigen titer (EIA) virus infection titer (logCCID ₅₀ /ml)

8 1:128 5.33

12 1:128 5.5

16 1:256 7.0

20 1:512 8.0

24 1:1024 8.2

28 1:1024 8.0 The test results of the SH strain immunized mice of the present invention are shown in Table 3. Mouse immunogenicity test results of SH strain

(L-A-1 strain is a control strain, and is derived from the Chinese Center for Disease Control and Prevention, Inc. Viral Disease Prevention and Control Institute.) The cross-protection effect of the SH strain of the present invention against other strains is shown in Table 4.

Table 4 Cross-protection of SH strains against other strains Name of strain Neutralizing antibody titer

SH-3 1024

SH-4 512

Dragon A strain 1024

JN-2 512

JN-12 512

(The above strains are all from the Center for Viral Disease Control and Prevention of the Chinese Center for Disease Control and Prevention.) Example 3 Application of Hepatitis A virus strain SH in the preparation of vaccines and drugs for the prevention and treatment of hepatitis A

Human embryonic lung diploid cells (MRC-5) cultured in a single-layer cell culture plant were digested with 0.125 w/v °/(^ protease-EDTA, and MEM cell culture medium was added to make the cell concentration per 亳The cells contain 100-1.5 million cells, and the cell fluid is mixed with Hepatitis A at 0.05-0.1 MOI for 30-90 minutes at a temperature of 20-3 CTC. The mixed adsorbed cell-virus mixture is mixed with 10% calf serum. The MEM cell culture solution was diluted 10-fold and then inoculated into a 2-40-layer cell factory to proliferate hepatitis A virus at 0.5 MOI, and allowed to stand at 35 ° C ± 0.5 ° C for 21-24 days. At the peak of virus proliferation, 0.125 w / _{V was used.} % of trypsin was routinely digested, and 20 μl of 0.01 M PBS pH 7.2 was added to collect the cell virus mixture per square centimeter area. The sonicated cell virus mixture was sonicated and sonicated to an output of 1500 W, each time in the ice for 5 minutes. Broken cells, 5 times in total. After centrifugation at 2000 rpm for 10 minutes, the supernatant virus was taken. Liquid, virus solution was added to chloroform extraction, mixed with chloroform and virus solution 1:2, shaken for 20 minutes, centrifuged at 4500 rpm for 20 minutes, and the upper aqueous phase was aspirated; protein phase was pumped again. The extraction buffer (0.01M PBS, pH 7.4) was extracted 4 times, and most of the virus was recovered by extracting 4 times, and the upper aqueous phase was combined. The extract was concentrated by ultrafiltration on an ultrafiltration membrane with a molecular weight cut off of 50-100 KD for 10-40 times, and then purified by Phenyl Sepharose 6FF hydrophobic gel chromatography column, using 0.9 mol/L PB (pH 6.8) as a loading buffer. O.Olmol/L PBS (pH 6.8) was used as a gradient eluent to collect the virus elution peak. The purified hepatitis A virus solution was sterilized by 0.2 μηι filter membrane and added to the final concentration under aseptic conditions. Inactivated for 8 (^g/ml of formaldehyde 37'C for 12 days. After inactivation of the hepatitis A virus solution, it was diluted to 640 EU/ml according to the antigenic titer level of the virus, and adsorbed by aluminum hydroxide adjuvant. In semi-finished products, semi-finished products are packaged in sterile vials to make a finished vaccine.

The finished product was subjected to a mouse in vivo efficacy test, and the terminal dilution was calculated to observe the immunogenicity. The km mice were purchased from the Institute of Pathogenic Microorganisms of the Chinese Academy of Military Medical Sciences, half male and half female, weighing 16-18 g, randomly divided into groups. The test was divided into 9 groups, 10 in each group, 4 groups in the test seedlings: the vaccine prepared in Example 3 was injected separately; the reference seedling group: the foreign hepatitis A inactivated vaccine (purchased from the Beijing Military Region Center for Disease Control and Prevention, Aibasu) : Product Lot Number: 3001424.02 ). The test vaccine and the reference vaccine were diluted 4 times with vaccine diluent, and the total dilution was 4 times, 1:4, 1:16, 1:64. Each mouse was intraperitoneally injected with 1.0 ml, and after 4 weeks of immunization, blood was collected from the eye, and the serum was separated by centrifugation and stored at -20 ° C until use. The other 10 were used as blank control groups.检测 Anti-HAV antibodies were detected by the ELIS A competition inhibition method, and the endpoint dilution was calculated according to the Reed-Muench method. The results are shown in Table 5.

The efficacy test result of the hepatitis A inactivated vaccine mouse of the present invention

ED ₅₀

Sample antigen content dilution factor antibody positive / number of inoculation

(end point dilution)

0 10/10

1/4 6/10

Control vaccine 24 (IU)

1/16 3/10 6.93

1/64 0/10

640 ( EU ) 0 10/10 Example 3 vaccine 1/4 7/10 7.94

1/16 3/10

1/64 0/10

Experimental example

The mice were intraperitoneally injected with the vaccine of Example 3, the vaccination amount was 640 EU/ml/only, and 1 ml of physiological saline was intraperitoneally injected as a control, and the hepatitis A virus SH virus was diluted with physiological saline to 1×10 ⁵ CCID ⁵ O/ml. 10 mice in the immunoprotective group (vaccination) and the control group (injected with normal saline), the body temperature of each mouse was measured every day after inoculation of the virus solution, and the clinical symptoms of each mouse were observed. The number of deaths was observed. 28 days. After the hepatitis A virus SH strain was challenged, there was no significant change in body temperature and clinical symptoms in the vaccine immunoprotective group. All the 28 days were observed to be healthy. In the control group, 3 mice recovered from body temperature 39.3'C~40.5 °C on the 4th day after challenge, and recovered after 8 days. The body temperature of 2 mice rose to 38.4 °C~39.8 °C, and the appetite decreased, and the recovery was maintained for 6 days. 5 mice after the 3rd to 5th day after the attack, the body temperature began to increase 40.5 ° C ~ 41. 8 ° C, lasted 4 to 10 days, and showed weight loss, cough, jaundice symptoms, in the 10th to 15th day death.

Although the present invention has been described in detail with reference to the preferred embodiments of the present invention, it will be apparent to those skilled in the art. Therefore, such modifications or improvements made without departing from the spirit of the invention are intended to be within the scope of the invention.

Industrial applicability

The invention provides a novel hepatitis A vaccine virus strain SH, an isolation method thereof and an MRC-5 cell adaptation method. The virus was isolated from the stool of patients with acute hepatitis A infection and then transferred to human diploid MRC-5 cells for adaptation. During the adaptation process, the early culture period was 35 days, and the culture period was shortened to 24 days after the passage to 8 generations. Continue to culture for 8 generations, the antigen titer can reach 1:512-1:1024, and the virus infection titer can reach 7.0-8.01gCCID ₅₀ /ml. The immunogenicity and cross protection test showed that the hepatitis A inactivated vaccine produced by the strain has good immunogenic protective effect and is suitable for use as a large-scale industrialized production of hepatitis A inactivated vaccine virus. The ideal strain for inactivated hepatitis A vaccine.

Claims

Claim

1. Hepatitis A virus SH strain, the accession number is CGMCC NO. 4501.

2. The method of adapting a hepatitis A virus SH strain according to claim 1 to diploid cells, comprising the steps of:

1) MRC-5 cells were cultured by conventional methods. After growing into a monolayer, the cells were inoculated with a suspension of Hepatitis A virus strain SH, and a lcm virus solution of 40 cm ² culture flask was adsorbed at 37 ° C for 2 hours, and a maintenance solution was added, 35 °. C culture, change the virus maintenance solution once every 7 days, culture for 35 days, wash the cell surface 3 times with PBS, digest the cells with trypsin, add the MEM suspension culture cells to the culture area of 0.01 ml/cm ² to obtain the virus cell suspension. Liquid, sonicated the cells, and prepared into a virus suspension;

2) 15-22 generations of continuous delivery in MRC-5 cells by the method of step 1); wherein, the MEM is 0.03% Glu, 0.08% NaHCO ₃ , 0.01% penicillin and 0.01% streptomycin; Add 2% calf serum to MEM.

The use of the virus strain according to claim 1 or the passaged virus according to claim 2 for the preparation of a vaccine or a medicament for preventing or treating hepatitis A.