应用家蚕杆状病毒表面展示技术生产人用禽流感疫苗的研究
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摘要
1997年香港H5N1以及2004年以来亚洲H5N1禽流感事件的暴发打破了人们对禽流感病毒的传统的认识模式,因为多年来人们一直认为禽流感病毒不能突破种间屏障直接感染人。由于从来没有H5亚型流感病毒感染人类的事件发生,所以人体内缺乏H5亚型流感病毒的抗体,一旦感染后果非常严重。
令人担忧的是H5N1病毒不断变异最终获得感染哺乳动物的能力,这一病毒正接近大流行病毒的标准:使人发病,能在人与人间传播。但H5N1目前还没有造成大流行,因为还不能在人与人传播。2004年至今,全球累计报告378例,死亡238例,病死率63.0%。我们对H5N1禽流感病毒是否具有引起人类流感再一次大流行的能力倍加关注。如果H5N1禽流感病毒能在人间有效传播并持续,对人类将是一场灾难。安全有效的疫苗是降低死亡率、发病率、经济损失的好策略。
杆状病毒表面展示系统是近几年发展起来的一种新的真核展示系统,通过在杆状病毒囊膜糖蛋白gp64插入外源蛋白、二者融合表达或与特异性的锚定部位结合,将外源蛋白展示在重组杆状病毒的表面,通过筛选表达特异蛋白的重组杆状病毒粒子得到外源蛋白,可用来展示需糖基化、二硫键异构化等翻译后修饰才表现功能活性的复杂真核蛋白及构建多肽文库、抗体库等。
1.表达HA蛋白的重组杆状病毒的构建及疫苗生产工艺的建立
本研究运用杆状病毒表面展示技术构建表达H5N1型人禽流感病毒HA蛋白的重组杆状病毒Bmgp64HA,以家蚕蛹作为生物反应器生产重组杆状病毒,并以此重组杆状病毒作为人用禽流感疫苗,同时建立人用禽流感疫苗的生产工艺。
人工合成含有杆状病毒gp64信号肽、跨膜区与A/Zhejiang/16/06(H5N1)毒株的HA基因的融合基因,构建重组转移载体pBacPAK-gp64-HA,与线性化的野生型杆状病毒BacPAK6共转染家蚕BmN细胞,经过多轮筛选,获得表达HA蛋白的重组杆状病毒Bmgp64HA。重组杆状病毒接种家蚕蛹,建立以家蚕蛹为生物反应器生产重组疫苗的生产工艺。将重组杆状病毒接种蚕蛹后5-6天收获蚕蛹,经过多步离心、超速离心、区带离心、超滤等方法收获重组杆状病毒。每100g蚕蛹生产重组杆状病毒8-16mg。
2.重组禽流感疫苗临床前的有效性评价
每组14只的BALB/C小鼠按高中低剂量组分别以150μg/kg体重,30μg/kg体重,6μg/kg体重免疫总体积1ml的禽流感疫苗,免疫3次,每次间隔2周,抗体效价达到最高峰时攻毒,观察疫苗的免疫保护效果。重组疫苗对小鼠具有较好的安全性。病毒分离、RT-PCR结果显示重组疫苗可能有抵御病毒攻击的作用。
模拟临床途径将重组疫苗分高、中、低剂量(480μg/kg体重、160μg/kg体重、53μg/kg体重)进行免疫恒河猴,每组4只,当抗体效价达到1∶100时用A/tiger/harbin/(01)/2002(H5N1)毒株进行攻毒。免疫及攻毒后,各组试验动物的血生化、血常规指标均在正常范围内。免疫后动物未出现异常的临床表现,提示疫苗具有较好的安全性。攻毒后第2天高、中、低剂量组分别有2、1、1只动物中和抗体达到1∶4,攻毒后第5天高、低剂量组各有1只动物中和抗体达到1∶4,攻毒后第7天高、中、低剂量组分别有4、2、1只动物中和抗体达到1∶8,攻毒后第14天高、中、低剂量组分别有2、1、1只动物中和抗体达到1∶16。病毒接种后第2天,中剂量组各有1只动物的咽拭子为阳性,高、低剂量组未分离出病毒,模型对照组均为阳性,第5、7、14天的咽拭子均未分离出病毒。病毒接种后第7天,低剂量组和模型对照组各有1只动物的肺组织中分离出病毒。14天时肺组织的病毒分离结果均为阴性。试验结果表明疫苗组的高、中剂量组有抵御病毒攻击的作用。
3.重组禽流感疫苗临床前的安全性评价
(1)禽流感疫苗对精神神经系统的影响
小鼠一般行为、自主活动和阈下催眠剂量戊巴比妥钠协同试验和小鼠旋转协调机能试验结果显示:小鼠经皮下注射给予禽流感疫苗16、8、4mg/kg和溶媒5ml/kg剂量,对小鼠精神神经系统无明显影响。
(2)禽流感疫苗对心血管、呼吸系统及体温的影响
猴心血管及呼吸系统试验表明:猴分别皮下注射给予禽流感疫苗3.2、1.6、0.8mg/kg和溶媒1ml/kg剂量,对猴心血管系统和呼吸系统的各项指标均无明显影响,其血压、呼吸频率、呼吸幅度和心电各指标均与给药前相似(P＞0.05);对猴体温也无明显影响,其各测量时间段的平均体温与给药前相似(P＞0.05),也与相应时间对照组相似(P＞0.05)。
在本试验剂量条件下,批号为20061207的禽流感疫苗对小鼠精神神经系统无明显影响。对猴心血管系统、呼吸系统和体温也无明显影响。
(3)禽流感疫苗大鼠、小鼠急性毒性试验
健康清洁级SD大鼠和ICR小鼠分别单次皮下注射(sc)给予禽流感疫苗94mg/kg和141mg/kg剂量,主要的毒副反应为注射部位出现直径为0.1～0.3cm颗粒状硬块。对大鼠、小鼠皮下注射的最大耐受剂量分别为:大鼠＞94mg/kg(相当于人临床拟用剂量的18800倍、大鼠有效剂量的250倍);小鼠＞141mg/kg(相当于人临床拟用剂量的28200倍、小鼠等效剂量的263倍)。
(4)禽流感疫苗猴长期毒性试验
食蟹猴连续8次(每10天给药1次)分别皮下注射给予禽流感疫苗3.2、1.6、0.8 mg/(kg·次)、溶媒对照和0.9%NaCl注射液1 ml/(kg·次)剂量,其中禽流感疫苗三个剂量组和溶媒对照组对注射部位有一定的异常作用。安全无毒剂量大于3.2 mg/kg。禽流感疫苗在高达人临床拟用剂量640倍的剂量下对猴的主要毒性反应仅表现为注射部位结痂、红斑等损伤。
(5)禽流感疫苗大鼠长期毒性试验
SD大鼠连续八次皮下注射给予批号为20061002、20061201的禽流感疫苗,对大鼠的主要毒副反应为注射部位出现肉芽肿样结节但停药有逐渐缩小的趋势,个别血液学指标(Grn↑,MCV↓)和脏器的重量及系数(肝脏系数、脾脏重量及系数↑)的升降影响。毒性靶器官为脾脏,但呈可逆性影响。中毒剂量未明显显示。毒副反应剂量为1.88 mg/(kg.次),无毒(影响)剂量大于0.375mg/(kg.次)。
(6)禽流感疫苗豚鼠全身主动过敏试验资料
健康白色豚鼠分别皮下注射禽流感疫苗1700μg、340μg/(kg.次),牛血清白蛋白生理盐水溶液60mg(2ml)/(kg.次)和溶媒2ml/(kg.次)剂量,隔日一次,连续3次进行致敏。末次致敏后第12天由静脉单次注射4倍致敏剂量的以上相应受试物进行激发。结果显示:禽流感疫苗对豚鼠过敏反应为极强阳性,呈剂量反应关系。
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 process
The 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 trials
There 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 system
Mice 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 temperature
The 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 mices
After 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 monkey
Cynomolgus 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 rats
SD 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 pig
Health 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.
令人担忧的是H5N1病毒不断变异最终获得感染哺乳动物的能力,这一病毒正接近大流行病毒的标准:使人发病,能在人与人间传播。但H5N1目前还没有造成大流行,因为还不能在人与人传播。2004年至今,全球累计报告378例,死亡238例,病死率63.0%。我们对H5N1禽流感病毒是否具有引起人类流感再一次大流行的能力倍加关注。如果H5N1禽流感病毒能在人间有效传播并持续,对人类将是一场灾难。安全有效的疫苗是降低死亡率、发病率、经济损失的好策略。
杆状病毒表面展示系统是近几年发展起来的一种新的真核展示系统,通过在杆状病毒囊膜糖蛋白gp64插入外源蛋白、二者融合表达或与特异性的锚定部位结合,将外源蛋白展示在重组杆状病毒的表面,通过筛选表达特异蛋白的重组杆状病毒粒子得到外源蛋白,可用来展示需糖基化、二硫键异构化等翻译后修饰才表现功能活性的复杂真核蛋白及构建多肽文库、抗体库等。
1.表达HA蛋白的重组杆状病毒的构建及疫苗生产工艺的建立
本研究运用杆状病毒表面展示技术构建表达H5N1型人禽流感病毒HA蛋白的重组杆状病毒Bmgp64HA,以家蚕蛹作为生物反应器生产重组杆状病毒,并以此重组杆状病毒作为人用禽流感疫苗,同时建立人用禽流感疫苗的生产工艺。
人工合成含有杆状病毒gp64信号肽、跨膜区与A/Zhejiang/16/06(H5N1)毒株的HA基因的融合基因,构建重组转移载体pBacPAK-gp64-HA,与线性化的野生型杆状病毒BacPAK6共转染家蚕BmN细胞,经过多轮筛选,获得表达HA蛋白的重组杆状病毒Bmgp64HA。重组杆状病毒接种家蚕蛹,建立以家蚕蛹为生物反应器生产重组疫苗的生产工艺。将重组杆状病毒接种蚕蛹后5-6天收获蚕蛹,经过多步离心、超速离心、区带离心、超滤等方法收获重组杆状病毒。每100g蚕蛹生产重组杆状病毒8-16mg。
2.重组禽流感疫苗临床前的有效性评价
每组14只的BALB/C小鼠按高中低剂量组分别以150μg/kg体重,30μg/kg体重,6μg/kg体重免疫总体积1ml的禽流感疫苗,免疫3次,每次间隔2周,抗体效价达到最高峰时攻毒,观察疫苗的免疫保护效果。重组疫苗对小鼠具有较好的安全性。病毒分离、RT-PCR结果显示重组疫苗可能有抵御病毒攻击的作用。
模拟临床途径将重组疫苗分高、中、低剂量(480μg/kg体重、160μg/kg体重、53μg/kg体重)进行免疫恒河猴,每组4只,当抗体效价达到1∶100时用A/tiger/harbin/(01)/2002(H5N1)毒株进行攻毒。免疫及攻毒后,各组试验动物的血生化、血常规指标均在正常范围内。免疫后动物未出现异常的临床表现,提示疫苗具有较好的安全性。攻毒后第2天高、中、低剂量组分别有2、1、1只动物中和抗体达到1∶4,攻毒后第5天高、低剂量组各有1只动物中和抗体达到1∶4,攻毒后第7天高、中、低剂量组分别有4、2、1只动物中和抗体达到1∶8,攻毒后第14天高、中、低剂量组分别有2、1、1只动物中和抗体达到1∶16。病毒接种后第2天,中剂量组各有1只动物的咽拭子为阳性,高、低剂量组未分离出病毒,模型对照组均为阳性,第5、7、14天的咽拭子均未分离出病毒。病毒接种后第7天,低剂量组和模型对照组各有1只动物的肺组织中分离出病毒。14天时肺组织的病毒分离结果均为阴性。试验结果表明疫苗组的高、中剂量组有抵御病毒攻击的作用。
3.重组禽流感疫苗临床前的安全性评价
(1)禽流感疫苗对精神神经系统的影响
小鼠一般行为、自主活动和阈下催眠剂量戊巴比妥钠协同试验和小鼠旋转协调机能试验结果显示:小鼠经皮下注射给予禽流感疫苗16、8、4mg/kg和溶媒5ml/kg剂量,对小鼠精神神经系统无明显影响。
(2)禽流感疫苗对心血管、呼吸系统及体温的影响
猴心血管及呼吸系统试验表明:猴分别皮下注射给予禽流感疫苗3.2、1.6、0.8mg/kg和溶媒1ml/kg剂量,对猴心血管系统和呼吸系统的各项指标均无明显影响,其血压、呼吸频率、呼吸幅度和心电各指标均与给药前相似(P＞0.05);对猴体温也无明显影响,其各测量时间段的平均体温与给药前相似(P＞0.05),也与相应时间对照组相似(P＞0.05)。
在本试验剂量条件下,批号为20061207的禽流感疫苗对小鼠精神神经系统无明显影响。对猴心血管系统、呼吸系统和体温也无明显影响。
(3)禽流感疫苗大鼠、小鼠急性毒性试验
健康清洁级SD大鼠和ICR小鼠分别单次皮下注射(sc)给予禽流感疫苗94mg/kg和141mg/kg剂量,主要的毒副反应为注射部位出现直径为0.1～0.3cm颗粒状硬块。对大鼠、小鼠皮下注射的最大耐受剂量分别为:大鼠＞94mg/kg(相当于人临床拟用剂量的18800倍、大鼠有效剂量的250倍);小鼠＞141mg/kg(相当于人临床拟用剂量的28200倍、小鼠等效剂量的263倍)。
(4)禽流感疫苗猴长期毒性试验
食蟹猴连续8次(每10天给药1次)分别皮下注射给予禽流感疫苗3.2、1.6、0.8 mg/(kg·次)、溶媒对照和0.9%NaCl注射液1 ml/(kg·次)剂量,其中禽流感疫苗三个剂量组和溶媒对照组对注射部位有一定的异常作用。安全无毒剂量大于3.2 mg/kg。禽流感疫苗在高达人临床拟用剂量640倍的剂量下对猴的主要毒性反应仅表现为注射部位结痂、红斑等损伤。
(5)禽流感疫苗大鼠长期毒性试验
SD大鼠连续八次皮下注射给予批号为20061002、20061201的禽流感疫苗,对大鼠的主要毒副反应为注射部位出现肉芽肿样结节但停药有逐渐缩小的趋势,个别血液学指标(Grn↑,MCV↓)和脏器的重量及系数(肝脏系数、脾脏重量及系数↑)的升降影响。毒性靶器官为脾脏,但呈可逆性影响。中毒剂量未明显显示。毒副反应剂量为1.88 mg/(kg.次),无毒(影响)剂量大于0.375mg/(kg.次)。
(6)禽流感疫苗豚鼠全身主动过敏试验资料
健康白色豚鼠分别皮下注射禽流感疫苗1700μg、340μg/(kg.次),牛血清白蛋白生理盐水溶液60mg(2ml)/(kg.次)和溶媒2ml/(kg.次)剂量,隔日一次,连续3次进行致敏。末次致敏后第12天由静脉单次注射4倍致敏剂量的以上相应受试物进行激发。结果显示:禽流感疫苗对豚鼠过敏反应为极强阳性,呈剂量反应关系。
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 process
The 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 trials
There 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 system
Mice 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 temperature
The 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 mices
After 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 monkey
Cynomolgus 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 rats
SD 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 pig
Health 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.
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