Application Baculovirus Surface Display Technology To Produce Human Bird Flu Vaccine

The outbreaks of H5N1 subtype avian influenza in Hong Kong in 1997 and in Asia since 2004 have broken the traditional acknowledgements to avian influenza viruses, because it is always known that avian influenza virus could not crossed the species barrier to infect people. As the events of human infected by H5 subtype of influenza virus never happened, the human lacks the H5 subtype of influenza virus antibodies. The consequence is very serious once the infection happens.It was worrying that the mutation of H5N1 virus eventually got the ability of infecting mammals and is close to the standards of a pandemic virus: cause people to disease, spread from person to person. But H5N1 has not caused a pandemic, because they can not be spread from person to person. From 2004 to so far, the world had reported 378 cases, 238 cases of death, and mortality 63.0%. We have more concern on whether H5N1 avian influenza virus has the ability of causing human influenza pandemic once again. If the H5N1 avian influenza virus can spread from person to person in an effective and sustained way, it would be a disaster for human. Safe and effective vaccine is a good strategy to reduce mortality, morbidity, and the economic loss.Baculovirus surface display system was developed as a new eukaryotic display system in recent years, through inserting into the baculovirus glycoprotein gp64 with heterologous protein, the two coexpressed or integration with the specific location of the anchor, fuse the heterologous protein to the surface of recombinant baculovirus. Application of the expression of specific proteins by recombinant baculovirus can select the heterologous protein. It can be used to display complex eukaryotic proteins requiring post translational processing including glycosylation and efficient folding for functional activity and build peptide library, antibody library and so on.1. Construction of recombinant baculovirus to express HA protein and the establishment of the vaccine production processThe recombinant baculovirus Bmgp64HA were constructed which can express H5N1 human avian influenza virus though baculovirus surface display system. The study use pupa of silkworm as a bioreactor to produce recombinant baculovirus, which to be used avian influenza vaccine for human, and establish the process to produce avian influenza vaccine.The Synthetic fusion gene which contains baculovirus signal peptide gp64, transmembrane region and A/Zhejiang/16/06(H5N1) strain of the HA gene was constructed to be recombinant transfer vector, the recombinant transfer vector and the Linear wild-type baculovirus BacPAK6 were cotransfected into silkworm BmN cells. After several rounds of screening, the recombinant baculovirus Bmgp64HA which can express HA protein were got. Pupas were inoculated by the combatant baculovirus, 5-6days later, the pupas were harvest. We could got combatant baculovirus after multi-step centrifugal, ultracentrifugation, the zone centrifugal, ultrafiltration. The production is per 100g pupa can produce 8-16mg recombinant baculovirus.2. Validity assessment of the recombinant avian influenza vaccine before Clinical trialsThere are eight BALB/C rats in every group. The mice were divided into the highest, median and lowest three groups by the dose of avian influenza vaccine. They were immune avian influenza vaccine separately 150μg / kg body weight, 30μg / kg body weight, 6μg / kg body weight which total volume were 1 ml. They were immune 3 times, each time interval two weeks. We infected the rats when antibody titers reached a peak and observe the immune protective effect of the vaccine. Recombinant vaccine has a better security to the rats. Results of separation of drugs, RT-PCR showed that the recombinant vaccine might against a virus attack.In simulative clinical way, rhesus monkeys were divided into the highest, median and lowest three groups by the dose of recombinant vaccine (480μg/kg body weight, 160μg/kg body weight, 53μg/kg body weight). There are four monkeys each group. When the antibody titers reached 1:100, monkeys were infected by A/tiger/harbin/ (01)/2002(H5Nl) stain. After immunity and infection, the blood biochemistry and blood routine examination of each group of animal were all in the normal ranges. The animals did not appear unusual clinical manifestations. It revealed that the recombinant vaccine has good security. The second day after infection, in highest, median and lowest three groups, there were 2, 1, 1 monkeys respectively whose neutralizing antibody reach 1:4. The fifth day in the highest group and lowest group, there were 1 monkey both whose neutralizing antibody reach 1:4. The 7th day after infection, in highest, median and lowest three groups, there were 4, 2, 1 monkeys respectively whose neutralizing antibody reach 1:8. The 14th day after infection, in highest, median and lowest three groups, there were 2, 1, 1 monkeys respectively whose neutralizing antibody reach 1:16. The second day after virus inoculation, in median group, there were a monkey whose throat swab was positive, the other two groups did not secrete virus. All of the model groups were positive, after 5th, 7th and 14th day, their throat swab did not secrete virus. The 7th day after virus inoculation, in the lowest group and the model groups, there were 1 animal respectively whose lung tissue secreted virus. The 14th day ,the results of lung tissue was negative. The results showed that: the animals in the highest, median groups attack the virus effectively.3. Validity assessment of the recombinant avian influenza vaccine before Clinical trials(1) The impact of avian influenza vaccine on nervous and mental systemMice general conduct of activities, self-hypnosis and under the threshold dose of pentobarbital sodium coordination test and mice rotating coordinating function test showed that: Subcutaneous injection of avian influenza vaccine (16, 8, 4 mg/kg), solvent(0.5 mL/kg) had no significant effect on mice nervous and mental system.(2) The impact of avian influenza vaccine on cardiovascular system, respiratory system and body temperatureThe tests of monkey cardiovascular and respiratory system indicate that: Subcutaneous injection of avian influenza vaccine (3.2,1.6,0.8 mg/kg),solvent(0.5 mL/kg) had no significant effect on indexes of cardiovascular system and respiratory system, their blood pressure, respiratory rate, volume of breathing and indexes of ECG were similar to pre-administration (P>0.05); Subcutaneous injection had no significant effect on body temperature, the mean body temperature in during the same survey time was similar to pre-administration (P>0.05), also similar to control groups. In the condition of the test dose, avian influenza vaccine with the batch number 20061207 has no significant effect on mice nervous and mental system, cardiovascular system, respiratory system and body temperature.(3) The acute toxicity tests of avian influenza vaccine on rats and micesAfter single time subcutaneous injection of 94mg/kg and 141mg/kg avian influenza vaccine into clean and health SD rats and ICR rats, the major toxic side effect was there was a granular gelosis with the diameter of 0.1-0.3cm on the injection site. The maximum tolerated dose for subcutaneous injection to rats and mices is respectively: rats >94mg/kg (equal to 18800 times clinical dosage for human, equal to 250 times effective dose for rats); mice >141mg/kg (equal to 28200 times clinical dosage for human, equal to 263 times effective dose for rats).(4) Long Term Toxicity Test on avian influenza vaccine in monkeyCynomolgus Monkeys were successive administrated respectively for 8 times (Every time Interval 9 days) with subcutaneous injection of avian influenza vaccine dose of 3.2,1.6,0.8 mg/(kg·time), solvent control and 0.9% NaCl Injection 1 ml/ (kg·time) . Among them, the monkeys in three dosage groups and solvent control group had some degree of abnormal reaction. The sub lethal dose of avian influenza vaccine was no lower than 3.2mg/kg. The major toxic side effect with condition of the dose equal to 18800 times clinical dosage for human was only incrustation of injecting region and erythema and so on.(5) Long Term Toxicity Test on avian influenza vaccine in ratsSD rats were successive administrated for 8 times with subcutaneous injection of avian influenza vaccine which batch number were 20061002,20061201. The major toxic side effect was nodes like Granuloma were formed in injection site, which would tend to decreasing after drug withdrawal, Individual haematological indexes (Grn↑, MCV↓) and organ weight and coefficients(liver coefficient, weight and coefficient of spleen↑) would be effected. The toxic target organs of avian influenza vaccine are spleen, but with effect of the reversibility. The toxic dose had no obvious display. The dosage of toxic reaction was 1.88mg/ (kg.time).The safe dosage was no lower than 0.375mg/(kg.time).(6) The ASA test data of avian influenza vaccine on guinea pigHealth white guinea pigs were successive administrated with subcutaneous injection of avian influenza vaccine with the dosage of 1700μg,340μg/(kg.time), BSA normal saline with the dosage of 60mg(2ml)/(kg.time)and solvent with the dosage of 2ml/(kg.time), once every other day for a total of 3 times to sensitize. The 12th day after last sensitization, the pigs were excitated by the single injection of correspondent substance which equal to 4 times of sensitization via coccygeal vein. The results showed that Guinea pigs showed positive reaction to influenza vaccine, and the reactions were relative to the doses.