Study on Neurovirulence and Safety of Recombinant Vaccinia Virus in Monkeys

为了评价重组痘苗病毒（RVV）作为强效活疫苗的安全性，在小鼠、豚鼠和家兔身上检测了表达乙型肝炎病毒表面抗原、乙型脑炎病毒E蛋白或HIV-1 gag/pol蛋白的重组痘苗病毒的毒力，并在体外培养中检测了实验室衰减标志物。用牛痘病毒强毒WR株、利斯特（Elstree）疫苗株（LO）和低神经毒力LC16mO株（mO）构建RVVs。实验动物和兔肾细胞原代培养物接种WR、LO和mO衍生的rvv后，其神经毒力和皮肤损伤依次降低，体外衰减标志物依次增加。RVVs与亲本野生型毒株具有几乎相同或较低的毒力。在皮内接种动物中，毒性最低的RVV-mO病毒抗原仅定位于皮肤注射部位。即使静脉或腹腔接种，RVV-mO在大脑中也未被检测到。在实验动物毒力试验的基础上，利用食蟹猴进行了RVV-mO的安全性试验。注射皮下或静脉注射的猴子没有表现出腿部瘫痪或体重减轻等临床症状。脑部没有发现病毒。在7天的观察期内，猴脑内接种RVV-mO未引起任何临床症状。猴脑标本的组织病理学检查显示脑膜内单个核细胞弥漫性浸润，实质有轻度细胞割伤。通过免疫影响和原位杂交分析，将少量RVV-mO抗原和病毒基因组DNA定位在病变处。这些结果表明，由牛痘病毒mO株构建的RVV具有较低的神经毒力，提示作为人用重组活疫苗是安全的。

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.