Study on Neurovirulence and Safety of Recombinant Vaccinia Virus in Monkeys

为评价重组痘苗病毒(RVV)作为一种有效的活疫苗的安全性，对表达乙肝病毒表面抗原、乙型脑炎病毒E蛋白和HIV-1 Gag/Poll蛋白的重组痘苗病毒在小鼠、豚鼠和兔体内进行了毒力测定，并在体外培养条件下进行了实验室减毒指标的测定。用痘苗病毒WR强毒株、Lister(Elstree)疫苗株(LO)和神经毒力低的LC16mO株(Mo)构建RVV。实验动物和原代培养的兔肾细胞接种WR、LO和Mo来源的RVV后，其神经毒性和皮肤损伤程度依次降低，体外衰减指标依次升高。RVV与亲本野生型毒株的毒力几乎相同或更低。RVV-Mo毒力最弱的病毒抗原仅定位于皮内接种动物的皮肤注射部位。根据实验动物的毒力试验结果，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没有出现腿部瘫痪或体重减轻等临床症状。没有从大脑中发现病毒。猴脑内接种RVV-Mo后，在7天的观察期内未出现临床症状。猴脑标本的组织病理学检查显示，单个核细胞在脑膜中弥漫性渗透，实质中有轻微的细胞袖带。通过免疫荧光和原位杂交分析，发现少量RVV-Mo抗原和病毒基因组DNA定位于皮损处。这些结果表明，由痘苗病毒Mo株构建的RVV具有较低的神经毒力，是一种对人类安全的重组活疫苗。

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.