乙型肝炎核酸疫苗的安全性研究
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摘要
核酸疫苗是90年代兴起的新型疫苗,已在多种疾病的防治中显示出巨大的潜能。然而核酸疫苗是一个新生事物,目前还存在许多尚未解决的问题,其中安全性的问题最为重要,直接关系到核酸疫苗的应用。全面评价乙肝DNA疫苗的安全性,我们进行了以下的毒理研究:1.急性毒性试验
本研究分别考察了乙肝DNA疫苗肌肉注射或皮下注射免疫小鼠或豚鼠的急性毒性,结果表明乙肝DNA疫苗在小鼠或豚鼠上未见任何明显急性毒性反应。半数致死量是推荐临床剂量的几百倍。
2.重复剂量的长期毒性试验
分别用三个剂量组的DNA疫苗(4.5m乙肝g/kg、1.5mg/kg、0.5mg/kg)肌注免疫小鼠56天(每1周给药2次,之后每周1次),从实验开始至停药后30天内进行毒性观察。结果表明小鼠体重、食量及粪便与对照组之间未见明显改变。血液学及临床生化指标均属正常范围内波动,未发现与受试药相关的毒性反应。尸检、器官重量检查未发现有与受试药物有关的毒性改变。观察了在上述给药条件下小鼠的多项疫苗免疫指标,发现乙肝DNA疫苗未发现有产生免疫耐受的迹象;不能导致自身免疫;不会诱导抗核抗体;不会引起慢性炎症及非特异性的免疫激活等免疫病理状况。
以PCR法检测质粒DNA的染色体整合情况,结果表明1μg基因组DNA中整合质粒低于10拷贝(即PCR的最大灵敏度);说明质粒DNA整合到宿主细胞基因组上并使细胞发生恶性转化的可能性微乎其微。
3.致突变及生殖毒性实验
致突变试验的结果表明乙肝DNA疫苗在-S9和+S9系统对CHL细胞染色体畸变均小于5%。乙肝DNA疫苗对染色体无畸变作用。乙肝DNA疫苗不引起母体毒性和胚胎毒性的无毒剂量(NOAEL)大于5.4 mg/kg。
Nucleic acid vaccine is a new generation of vaccine beginning in the 90's, which has shows powerful potency in the prevention and therapy of many diseases. Because nucleic acid vaccine is a new kind of vaccine, there has been several issues unsolved. And among these issues, the security of nuclei acid is most important since it is related with the application of nucleic acid. We have done the research of toxicology as following in order to give the overall assessment of the security of HBV DNA vaccine.1. acute-term toxicity testThe acute toxicity of immune mice or guinea pig by intramuscular injection or hypodermic injection was evaluated according acute-term toxicity test. There was any obviously acute toxic reaction upon the HBV DNA vaccine immune mice or guinea pig. And half lethal dose was several hundred folds than the clinical dose.2. long-term toxicity test of repeated doseThe immune mice were divided into three groups according the intramuscular injection dose of HBV DNA vaccine (4.5mg/kg、 1.5mg/kg、 0.5mg/kg) in 56 days (the dosage is twice per week, then once per week). The toxicity was observed from the beginning of the test to post-discontinuation within 30 days. The result was demonstrated that there was no obvious alteration in body weigh, appetite and faeces of mice compared with control group. Clinical biochemical indicator and hematology indicator was fluctuated within normal limits. The toxic reaction related to HBV DNA vaccine didn't detect. Also the toxic revised didn't detect related to HBV DNA vaccine according autopsy and the test of organ weight. There wasn't the phenomena of immune tolerance in the immune mice, didn't detect antinuclear antibody, didn't induce the phenomena of immunopathogenesis such as chronic inflammation, nonspecific immune activation and so on. The chromosomal integration with plasmid was tested by PCR, and the result was confirmed that 20 copy plasmid confused into 1 μg genome DNA confuse could be detected, that is to say the probability that DNA integrated into the cytogene of host cell and let the host cell malignant transformation
本研究分别考察了乙肝DNA疫苗肌肉注射或皮下注射免疫小鼠或豚鼠的急性毒性,结果表明乙肝DNA疫苗在小鼠或豚鼠上未见任何明显急性毒性反应。半数致死量是推荐临床剂量的几百倍。
2.重复剂量的长期毒性试验
分别用三个剂量组的DNA疫苗(4.5m乙肝g/kg、1.5mg/kg、0.5mg/kg)肌注免疫小鼠56天(每1周给药2次,之后每周1次),从实验开始至停药后30天内进行毒性观察。结果表明小鼠体重、食量及粪便与对照组之间未见明显改变。血液学及临床生化指标均属正常范围内波动,未发现与受试药相关的毒性反应。尸检、器官重量检查未发现有与受试药物有关的毒性改变。观察了在上述给药条件下小鼠的多项疫苗免疫指标,发现乙肝DNA疫苗未发现有产生免疫耐受的迹象;不能导致自身免疫;不会诱导抗核抗体;不会引起慢性炎症及非特异性的免疫激活等免疫病理状况。
以PCR法检测质粒DNA的染色体整合情况,结果表明1μg基因组DNA中整合质粒低于10拷贝(即PCR的最大灵敏度);说明质粒DNA整合到宿主细胞基因组上并使细胞发生恶性转化的可能性微乎其微。
3.致突变及生殖毒性实验
致突变试验的结果表明乙肝DNA疫苗在-S9和+S9系统对CHL细胞染色体畸变均小于5%。乙肝DNA疫苗对染色体无畸变作用。乙肝DNA疫苗不引起母体毒性和胚胎毒性的无毒剂量(NOAEL)大于5.4 mg/kg。
Nucleic acid vaccine is a new generation of vaccine beginning in the 90's, which has shows powerful potency in the prevention and therapy of many diseases. Because nucleic acid vaccine is a new kind of vaccine, there has been several issues unsolved. And among these issues, the security of nuclei acid is most important since it is related with the application of nucleic acid. We have done the research of toxicology as following in order to give the overall assessment of the security of HBV DNA vaccine.1. acute-term toxicity testThe acute toxicity of immune mice or guinea pig by intramuscular injection or hypodermic injection was evaluated according acute-term toxicity test. There was any obviously acute toxic reaction upon the HBV DNA vaccine immune mice or guinea pig. And half lethal dose was several hundred folds than the clinical dose.2. long-term toxicity test of repeated doseThe immune mice were divided into three groups according the intramuscular injection dose of HBV DNA vaccine (4.5mg/kg、 1.5mg/kg、 0.5mg/kg) in 56 days (the dosage is twice per week, then once per week). The toxicity was observed from the beginning of the test to post-discontinuation within 30 days. The result was demonstrated that there was no obvious alteration in body weigh, appetite and faeces of mice compared with control group. Clinical biochemical indicator and hematology indicator was fluctuated within normal limits. The toxic reaction related to HBV DNA vaccine didn't detect. Also the toxic revised didn't detect related to HBV DNA vaccine according autopsy and the test of organ weight. There wasn't the phenomena of immune tolerance in the immune mice, didn't detect antinuclear antibody, didn't induce the phenomena of immunopathogenesis such as chronic inflammation, nonspecific immune activation and so on. The chromosomal integration with plasmid was tested by PCR, and the result was confirmed that 20 copy plasmid confused into 1 μg genome DNA confuse could be detected, that is to say the probability that DNA integrated into the cytogene of host cell and let the host cell malignant transformation
引文
1.孙树汉 核酸疫苗 2001,第二军医大学出版社。
2. Martin T, Parker SE. Plasmid DNA malaria vaccine: the potential for genomic interaction after intramuscular injection. Human Gene Therapy, 1999,10:759.
3.新药(西药)临床前研究指导原则汇编(药学药理学毒理学) 中华人民共和国卫生部药政局1993,P199-205
4.袁伯俊 王治乔主编 新药临床前安全性评价与实践 军事医学科学出版社
5.1997,p43-62
6.秦伯益 主编 新药评价概论(第二版)人民卫生出版社 1998,p387-392
2. Martin T, Parker SE. Plasmid DNA malaria vaccine: the potential for genomic interaction after intramuscular injection. Human Gene Therapy, 1999,10:759.
3.新药(西药)临床前研究指导原则汇编(药学药理学毒理学) 中华人民共和国卫生部药政局1993,P199-205
4.袁伯俊 王治乔主编 新药临床前安全性评价与实践 军事医学科学出版社
5.1997,p43-62
6.秦伯益 主编 新药评价概论(第二版)人民卫生出版社 1998,p387-392