Study on Neurovirulence and Safety of Recombinant Vaccinia Virus in Monkeys

为评价重组痘苗病毒（RVV）作为强毒活疫苗的安全性，对表达B肝炎病毒表面抗原、乙型脑炎病毒E蛋白和HIV-1 gag/pol蛋白的RVV进行了小鼠、豚鼠和家兔毒力试验和体外培养实验室减毒标志物试验。RVV是由强毒WR株、Lister（埃尔斯特里）疫苗株（LO）和牛痘病毒的低神经毒力LC 16 m0株（m0）构建的。实验动物和原代培养的兔肾细胞接种来自WR，LO和mO的RVV显示，神经毒力和皮肤病变，并在体外减毒标志物，分别增加，在这个顺序。RVV保留了与亲本野生型列车几乎相同或较低程度的毒力。毒力最低的RVV-mO的病毒抗原仅定位于皮内接种动物皮肤的注射部位。根据实验动物的毒力试验结果，使用食蟹猴进行了RVV-mO的安全性试验。皮下或静脉接种的猴子既没有表现出临床症状，如腿麻痹，也没有体重减轻。从大脑中未回收到病毒。在7天的观察期内，脑内接种RVV-mO在猴中未诱导临床体征。猴脑标本的组织学检查显示脑膜中弥漫性单核细胞浸润和脑实质中轻度细胞袖套。免疫荧光和原位杂交结果表明，RVV-mO株病毒的神经毒力较低，可作为一种安全的重组活疫苗。

In order to evaluate the safety of recombinant vaccinia virus(RVV) as a potent live vaccine, RVVs expressing hepatitis B virus surface antigen, Japanese encephalitis virus E protein or HIV-1 gag/pol proteins were tested for the virulence in mice, guinea pigs and rabbits, and for laboratory attenuation markers in in vitro cultures. RVVs were constructed from the virulent WR strain, the Lister(Elstree) vaccine strain(LO) and the low neurovirulent LC16mO strain (mO) of vaccinia virus. Experimental animals and primary cultures of rabbit kidney cells inoculated with RVVs derived from WR, LO and mO showed the decreasing neurovirulence and skin lesions, and the increasing in vitro attenuation markers, respectively, in this order. RVVs reserved almost the same or low degree of virulence as parental wild-type trains. Virus antigens of the least virulent RVV-mO was localized only at the injection sites of the skin in intradermally inoculated animals. Even when inoculated intravenously or intraperitoneally, RVV-mO was not detected in the brain.Based on results of the virulence tests in experimental animals, safety tests of RVV-mO were done using cynomolgus monkeys. The monkeys inoculated subcutaneously or intravenously showed neither clinical signs such as paralysis of the legs nor weight loss. No virus was recovered from the brain. Intra-cerebral inoculation of RVV-mO induced no clinical signs in monkeys during the observation period of 7 days. Histopathological examination of the monkey brain specimen showed diffuse infiltration of mononuclear cells in the meninges and mild cellular cuffing in the parenchyma. Small amounts of the RVV-mO antigens and viral genome DNA were localized at the lesions by immunoflucence and in situ hybridization analyses.These results indicate low neurovirulence of RVV constructed from the mO strain of vaccinia virus, suggesting safety as a recombinant live vaccine for humans.