腺病毒载体疫苗在恒河猴体内的长期毒性试验

Repeat administration toxicity study of an adenovirus vector vaccine Ad5-LMP2 in rhesus monkey

目的探讨以5型腺病毒为载体的人用疫苗Ad5-LMP2在恒河猴体内的药效学作用和安全性。方法恒河猴经im给予4.5×1011、1.5×1011和0.5×1011v.p·(kg·次)-1的Ad5-LMP2和等体积的磷酸盐缓冲液(对照组),每5d给药1次,共给药6次。在停药次日和停药15d时进行尿常规、血液、眼科、心电图、脏器重量和系数、大体观和病理组织学检查;给药前后定期采取猴血清并从剖检猴的脾脏分离淋巴细胞,用ELISA、病毒中和试验和ELISPOT检测腺病毒载体和目的基因蛋白产物LMP2诱导的体液和细胞免疫应答;通过PCR扩增各器官总DNA中的lmp2基因来检测Ad5-LMP2在体内的生物分布。结果在整个试验期间试验猴均未出现任何异常反应,也无动物死亡;给药组动物在整个给药期间的摄食、饮水和体重增长及上述各项常规毒理学检测中均未出现明显异常。受试物在试验猴体内诱发了较高水平的抗腺病毒中和抗体,但目的蛋白LMP2诱导的特异性细胞免疫应答未受抗腺病毒中和抗体的明显抑制;在猴的心、肝、脾、肺、肾、脑、睾丸(或卵巢)及给药部位均可检测到Ad5-LMP2并维持到停药后15d。结论在保证受试物药理学作用的前提下,恒河猴连续1mon肌肉注射相当人临床拟用剂量37.5倍的Ad5-LMP2,未见明显不良反应,中毒靶器官和中毒剂量未明显显示,其安全剂量大于4.5×1011v.p·(kg·次)-1。

Aim To explore the pharmacological effect and safety of an adenovirus vector vaccine Ad5-LMP2 in rhesus monkey. Methods Sixteen rhesus monkeys were divided randomly into 4 groups and injected intramuscularly with Ad5-LMP2 (4.5×1011, 1.5×1011, 0.5×1011 vp·kg-1) and PBS buffer in equal volume, respectively. The vaccine was administered every 5-day for 6 times. The examinations for urine, blood, cardiogram and ophthalmic test were carried out periodically and animal organs were examined with regard to weight, facies and histology at the day after the last administration and the end of recovery period. Serum samples and lymphocytes derived from spleen were collected periodically and titrated by ELISA, neutralizing test and ELISPOT, respectively, for humoral and cellular immune responses. Total DNA extracted from different organs was amplified with LMP2-specific primers for biological distribution of the vaccine. Results There was no abnormal reaction or animal death appeared in the whole test duration. No toxicological reaction was found in the above tests and although the viral vector induced high neutralizing antibody against the vaccine, a LMP2-specific cellular immune response was dramatically activated. It′s suggested by PCR that Ad5-LMP2 can distribute in heart, liver, spleen, lung, kidney, brain and testis/ovary till 15 d after the last administration. Conclusion The vaccine Ad5-LMP2 appeared no toxicological effect in rhesus monkey while its pharmacological effect reserved, and the safe dose is over 4.5×1011 vp·kg-1 for each time.