The immune effect of inactivated hepatitis A vaccine has been internationally recognized. Sencibaoxiong of Japan infected marmosets with hepatitis A virus and formed ideal animal model of hepatitis A. Korzaya used Macaca to form animal model of hepatitis A. Mao Jiangsen used red face monkey as sensitive animal of hepatitis A virus. Lian Youhui used Macaca mulatta and Common Marmoset to carry through comparing study of animal model of hepatitis A and proved that common marmoset was more obvious than Macaca mulatta in pathologic reaction of liver, hepatitis A antibody and change of liver function. So we selected Common Marmoset and Macaca mulatta as laboratory animals to carry through study on inactivated hepatitis A vaccine. Here is the results report.

Materials and methods

1. Inactivated hepatitis A vaccine

Culture the thirtieth generation of the strain TZ84 of hepatitis A in human embryo lung diploid cell 2BS strain. Then treat them with PEG and molecular sieve, purify them with ultrafiltrate and inactivate them with 1:4000 of foermaldehyde. The content of hepatitis A antigen is 1000 unit/ml through quantitative determine. At last produce inactivated hepatitis A vaccine absorbed by aluminium hydroxide that contains 1.0mg/ml and the separate loading is 1.0 ml/bottle. Storage it in 4℃.

2. Hepatitis A virus wild strain

2.1 Strain TZ84: culture three generations in human embryo lung diploid cell 2BS strain continuously, harvest by freeze and thaw, gather hepatitis A virus by 20,000 r/min ultra acceleration and quantify the antigen to be 1500 unit/ml.

2.2 Luxing strain: Medical biology institute of Chinese academy of medical sciences produced it by gathering feces of hepatitis A patients in acute stage. It has been proved to contain hepatitis A virus through biology. It can be used as invasive wild strain of hepatitis A virus.

3. Laboratory animal

3.1 Common Marmoset: filial generation in Tianjin medical university laboratory animal center that was imported from Australian. They were one to three years old and weighed 250 to 400 grams. Determine anti-HAV to be negative and liver function and biopsy of liver tissue to be normal before vaccination.

3.2 Macaca Mulatta: supplied by Medical biology institute of Chinese academy of medical sciences laboratory animal center. They were one to two years old and weighed 1.5 to 2 kilograms. Their anti-HAV were negative and serum ALT and biopsy of liver tissue were normal before vaccination.

4. Vaccinate methods and doses

Vaccinate 1.0ml of the vaccine intramuscular to every monkey. Twenty-eight days after vaccination, challenge them with wild virus strain through intravenous injection and 1.0 ml every monkey.

5. Sample collection

5.1 Serum: take blood before and after vaccination and once a week after challenge with empty stomach. Take blood once every two weeks after eight weeks. Place it in room temperature over night and separate the serum in the next day.

5.2 Biopsy of liver tissue: carry through liver aspiration biopsy once a month before and after vaccination. Execute the monkeys after four months. Take out their liver, heart, lung, stomach, duodenum, kidney, mesenterium and lymph node to carry through liver tissue pathologic examination.

5.3 Feces collection: collect feces once a day from the second day after challenge and collect continuously for one month. Then collect once a week for one month. Storage them in –20℃ for test.

6. Determination methods

6.1 Quantitative determination of hepatitis A antigen: use EIA quantitative determination kit of hepatitis A antigen produced by Tangshan Yian bioengineering Co Ltd to determine it. Construct a standard curve basing on reference standard of hepatitis A antigen. Use parallel method to calculate the antigen content of samples and show it with U/ml.

6.2 Anti-HAV IgM: use EIA determination kit of anti-HAV IgM produced by Tangshan Yian bioengineering Co Ltd to determine it.

6.3 Anti-HAV: use EIA determination kit of anti-HAV kit produced by Tangshan Yian bioengineering Co Ltd to determine it.

6.4 ALT: adopt Encore Ⅱ auto-biochemical analyzer

6.5 ICD: adopt biochemical analysis method

6.6 Determination of hepatitis A virus in feces of monkey: prepare the feces of monkey into 20% suspending liquid with PBS. Freeze and thaw in -70℃ three times and centrifugate it thirty minutes with speed of 10,000r/min. Collect supernatant fluid and dilute it to one tenth. Culture it in 2BS cell for two generations. Harvest by freeze and thaw and determine the hepatitis A antigen with EIA methods.

6.7 Pathology of liver tissue: refer to graduating methods of liver disease. Krawezymski, 1981.

7. Experiment grouping

7.1 Immune group: two Common Marmosets and four Macaca Mulattas were vaccinated a bottle of inactivated hepatitis A vaccine.

7.2 Booster immune group: Common Marmosets and Macaca Mulattas were vaccinated a bottle of inactivated hepatitis A vaccine. Four weeks later, one of the common marmosets and one of the Macaca mulattas were booster vaccinated once.

7.3 Prevention study group: Common marmosets and Macaca mulattas were vaccinated a bottle of inactivated hepatitis A vaccine. Four weeks later, one of the common marmosets and two of the Macaca mulattas were challenged with Luxing strain (20% feces suspending liquid, 1.0ml/monkey).

7.4 Control group: Luxing strain challenged two common marmosets and two Macaca mulattas directly.

7.5 Virus infection group: the third generation strain TZ84 infected two common marmosets and three Macaca mulattas.

Results

1. Immunoreaction of common marmosets

After two common marmosets were vaccinated inactivated hepatitis A vaccine, it appeared seroconversion of anti-HAV in the second week and the antibody titer reached 625 to 2500 mIU/ml in the fourth week. It appeared seroconversion of anti-HAV IgM two to three weeks after vaccination and the highest titer is 1:1000. Anti-HAV IgM lasted for five to ten weeks and disappeared. There was no abnormal increase of ALT. There was no pathologic change in liver biopsy.

Four weeks later, one common marmoset was vaccinated a bottle of inactivated hepatitis A vaccine again. Challenge one common marmoset with hepatitis A virus wild Luxing strain. Both common marmosets appeared peak value reaction of HAV. After challenge or booster vaccination, anti-HAV reached 10000mIU/ml in the second week and last for four weeks. Then it decreased to 2500mIU/ml. There was no abnormal increasing of ALT. There was no pathologic change in liver biopsy after challenge or booster vaccination. See Fig 1 and Fig 2 . There was no hepatitis A virus in feces of common marmoset after challenge.

Fig 1. Antibody response of common marmosets to inactivated hepatitis A vaccine

Fig 2. Section of liver tissue of immunized marmostes after being chall enged with hepatitis A wild virus

2. Immunoreaction of Macaca mulatta

After four Macaca mulattas were vaccinated inactivated hepatitis A vaccine, it appeared seroconversion of anti-HAV in the second week and the antibody titer reached 625 to 2500 mIU/ml in the fourth week. It appeared seroconversion of anti-HAV IgM two to three weeks after vaccination and the highest titer is 1:1000. Anti-HAV IgM lasted for one to three weeks and disappeared. There was no abnormal increase of ALT. There was no pathologic change in liver biopsy.

Four weeks later, one Macaca mulatta was vaccinated a bottle inactivated hepatitis A vaccine again. It appeared peak value reaction of HAV. After vaccination, anti-HAV reached 40000mIU/ml in the second week and last for four weeks. Then it decreased to 10000mIU/ml. There was no abnormal increasing of ALT. There was no pathologic change in liver biopsy.

Four weeks later, challenge two Macaca mulattas with hepatitis A virus wild Luxing strain. The result was that anti-HAV increased four times. After challenge, anti-HAV titer reached 10000mIU/ml in the second week. It began to decrease two weeks later and maintain 2500mIU/ml. See Fig 3.  There was no abnormal increasing of ALT in Macaca mulattas after challenge. There was no pathologic change in liver biopsy. There was no hepatitis A virus in feces.

Fig 3. Antibody response of macaca Mulatta to inactivated hepatitis A vaccine

3. Immunoreaction of common marmosets and Macaca mulattas after wild virus strain infection

After two Common Marmosets were inoculated feces suspending liquid of hepatitis A virus Luxing strain, one of them appeared seroconversion of anti-HAV in the third week and the antibody titer reached 1:16384 in the twelfth week. It appeared seroconversion of anti-HAV IgM in the second week after inoculation and the titer was 1:20000. It lasted to the tenth week and decreased a little to 1:10000 in twelve week. There was no abnormal increasing of ALT. It appeared pathologic change in liver biopsy from the sixth week. It appeared hepatitis A virus in feces from the seventh day to the twenty-sixth day through determination of feces suspending liquid. Another common marmoset appeared seroconversion of anti-HAV in the fourth week and the antibody titer was 1:64 in the twelfth week. It appeared pathologic change in liver biopsy from the tenth week. It appeared hepatitis A virus in feces from the sixth day to twenty-two day through determination of feces suspending liquid.

After Macaca mulattas were inoculated feces suspending liquid of hepatitis A virus Luxing strain, it appeared seroconversion of anti-HAV in the second week and maintained in low level. The antibody titer reached 156 to 625mIU/ml in the eighth week. It never appeared seroconversion of anti-HAV IgM. There was no abnormal reaction of ALT. There was no pathologic change in liver biopsy. It appeared hepatitis A virus in feces from the ninth day to fifteenth day. See Table 1 and Fig 4.

 Table 1 Reactions of common marmosets and macaca mulatta infected with strain Luxing of hepatitis A virus.

Fig 4. Section of liver tissue of immunized marmosts after being challenged with strain Luxing of hepatitis A virus

4. Immunoreaction of common marmosets infected by the third generation strain TZ84

After two common marmosets were inoculated with the third generation strain TZ84, it appeared seroconversion of anti-HAV two weeks later and the antibody titer reached 2500mIU/ml in the fourth week. The peak value was 10000mIU/ml and lasted for two weeks. Then it maintained in the level of 2500mIU/ml. It appeared seroconversion of anti-HAV IgM in the second week after inoculation and the titer is 1:1000. The peak value in the fourth week was 1:5000 and it disappeared in the fifth to sixth week. ALT of one common marmoset increased a little in the fourth to sixth week and then trended to be normal. There was no pathologic change in liver biopsy. It appeared hepatitis A virus in feces from the third day to twentieth day.

5. Immunoreaction of Macaca mulatta infected by the third generation strain TZ84

After three Macaca mulatta were inoculated with the third generation strain TZ84, it appeared seroconversion of anti-HAV in the second week and the antibody titer reached 625 to 2500mIU/ml in the fourth week. The peak value was 2500 to 10000mIU/ml and lasted for six to eight weeks. Then it began to decrease and maintained in the level of 2500 to 10000mIU/ml. It appeared seroconversion of anti-HAV IgM in the second week after inoculation and the titer is 1:1000. The peak value in the fourth week was 1:5000 to 1:25000 and it disappeared in the tenth week. ALT of one common marmoset increased a little in the first to second week and then trended to be normal. There was no pathologic change in liver biopsy. It appeared hepatitis A virus in feces from the ninth day to fifteenth day.

Discussion

Hepatitis A virus strain TZ84 cultured to the third generation in 2BS cell. There was no pathologic change of liver in common marmosets and Macaca mulatta. There were only temporal change of ALT and ICD. It showed that the pathogenicity of the third generation of hepatitis A virus strain TZ84 had decreased a little and it was more safe to be used to produce inactivated vaccine.

There was obvious pathologic change of liver in Common Marmoset challenged by hepatitis A virus Luxing strain. Hepatitis A antibody titers of two Common Marmosets were 1:64 and 1:16348 respectively. Both ALT and ICD increased and there was pathologic change of liver. It proved that hepatitis A virus Luxing strain had pathogenicity and it could be used as hepatitis A wild virus strain to challenge.

        There are fewer study on common marmoset in China. This kind of monkey weighs light and its feeding condition is very strict. It is expensive too. The reference ranges of its ALT and ICD have not been confirmed exactly. The source of Macaca Mulatta is easier and its feeding condition is simple. It is cheap too. Its immunoreaction is similar to human. The results of study in monkey showed: the hepatitis A antibody level of Common Marmoset is equal to the Macaca mulatta basically. But its pathologic reaction of liver is sensitive than Macaca mulatta. So when carry through study on immunogenicity of inactivated hepatitis A vaccine, it is favorable to select both Common Marmoset and Macaca Mulatta to make up each other.

References

1. Wiedemann G. Ambroach F. Andre et. al. Persenes of  vaccine induced antibody to hepatitis A virus. Vaccine, 1992. 10 ( suppl): 129-

2.Van Desnme P. Thockn S, Gnurun M, et al, Inactivated hepatitis A vaccine: Reackgenicity, immunogenicity and long-term antibody presisitence. J Med Virol 1994,44:446-

3.Tilaey AJ, planner Sj, Rurrow S, et al, Effect of hepatitis A vaccinution schedules on immuse responses, Vaccine, 1992 10( suppl): 121-

4.Mao JS, Xie RY, Hunng HY, et al. Studies in monkey of atternzated hepatitis A vatinants Sci, Sin (B), 1998.31:338-