Infectious cDNA technology and RNA virus vaccines

总结： 以前，我们与B Puri合作，从1型登革热病毒（DEN 1 WP）和减毒活DEN 1疫苗候选物（DEN 1 PDK 20）中制备了全长cDNA克隆，后者已适应在狗肾细胞中生长。用从这些克隆体外转录的RNA转染细胞产生登革热感染。克隆衍生的病毒在生长曲线上表现得与相应的亲本DEN 1相似，并且回收的DEN 1 PDK 20具有与其亲本相同的小空斑表型。我们在WP和PDK 20感染性克隆之间制备了一系列重组体，并表征了所得嵌合病毒的噬斑大小。 大多数嵌合体有一个中等大小的斑块，这表明，多个突变参与了这种表型的确定。 与NIH的S Whitehead和B Murphy合作，我们将3'非编码区的30 nt缺失引入DEN 1 WP克隆中，他们已经表明该缺失对于DEN 4感染性克隆是减毒的。 缺失突变病毒比其亲本生长更慢，并且产生更小的噬斑尺寸。 将在猴中检测该病毒的免疫原性和减毒作用。 与B Puri的另一项合作涉及DEN 4 PDK 20候选疫苗。到目前为止，我们已经对PDK 20病毒及其亲本进行了测序，并制备了该疫苗的感染性全长cDNA克隆。 由于B Puri已经在专利局找到了一份工作，因此不太可能在这方面做进一步的工作。 与E Kelly合作，我们对DEN 2 PDK 50候选疫苗及其强毒亲本进行了测序和感染性克隆。 克隆衍生的病毒正在与它们的亲本在组织培养细胞中的生长动力学进行比较。克隆的DEN 2 PDK 50由K.埃克尔斯和他的合作者，这种病毒正在猴子身上进行减毒和免疫原性测试。 最后，我们最近完成了陆军候选日本脑炎病毒（JEV）减毒活疫苗的感染性克隆，这是中国JEV活疫苗株SA 14 14-2的PDK细胞衍生物的Vero细胞适应版本。 克隆衍生的病毒在细胞培养物中以与其亲本相同的动力学生长。 这种病毒正在小鼠中进行免疫原性试验。

Summary: Previously, in collaboration with B Puri we made full-length cDNA clones from a virulent dengue type 1 virus (DEN1 WP) and from a live-attenuated DEN1 vaccine candidate, which had been adapted to grow in dog kidney cells (DEN1 PDK20). Transfection of cells with RNA transcribed in vitro from these clones produces a dengue infection. Clone-derived viruses behave like the corresponding parent DEN1 in growth curves, and the recovered DEN1 PDK20 has the same small plaque phenotype as its parent. We made a series of recombinants between the WP and PDK20 infectious clones, and characterized the plaque size of the resulting chimeric viruses. Most chimeras had an intermediate plaque size, suggesting that multiple mutations are involved in the determination of this phenotype. In collaboration with S Whitehead and B Murphy at the NIH we introduced a 30 nt deletion in the 3' non-coding region, which they had shown was attenuating for a DEN4 infectious clone, into the DEN1 WP clone. The deletion mutant virus grows more slowly than its parent, and makes a smaller plaque size. This virus is to be tested for immunogenicity and attenuation in monkeys. Another collaboration with B Puri involved a DEN4 PDK20 vaccine candidate. So far, we have sequenced the PDK20 virus and its parent, and we made an infectious full-length cDNA clone of the vaccine. Further work on this is unlikely, as B Puri has taken a job at the patent office. In collaboration with E Kelly, we have sequenced and made infectious clone of a DEN2 PDK50 vaccine candidate and its virulent parent. Clone-derived viruses are being compared to their parents for growth kinetics in tissue culture cells. The cloned DEN2 PDK50 was manufactured under GMP by K. Eckels and his collaborators, and this virus is being tested in monkeys for attenuation and immunogenicity. Finally, we recently completed an infectious clone of the Army's candidate live attenuated Japanese encephalitis virus (JEV) vaccine, a vero cell adapted version of a PDK cells derivative of the Chinese JEV live vaccine strain SA14 14-2. Clone-derived virus grows with the same kinetics as its parent in cell cultures. This virus is being tested in mice for immunogenicity.