Infectious cDNA technology and RNA virus vaccines

摘要：之前，我们与B Puri合作，从一种强毒的1型登革热病毒(DEN1 WP)和一种已适应在狗肾细胞(DEN1 PDK20)中生长的减毒DEN1候选疫苗中进行了全长cDNA克隆。用这些克隆的体外转录的RNA转染细胞会产生登革热感染。克隆衍生病毒在生长曲线上的表现与相应的亲本DEN1相似，恢复的DEN1 PDK20具有与其亲本相同的小斑块表型。我们在WP和PDK20感染性克隆之间构建了一系列重组体，并对所得到的嵌合病毒的空斑大小进行了鉴定。大多数嵌合体有一个中等大小的斑块，这表明这种表型的确定涉及多个突变。与国立卫生研究院的S·怀特黑德和B·墨菲合作，我们在DEN1WP克隆中引入了3‘非编码区30个核苷酸的缺失，他们已经证明这对DEN4感染性克隆是弱化的。缺失突变病毒的生长速度比其亲本慢，并且形成了更小的斑块尺寸。这种病毒将在猴子身上进行免疫原性和减毒试验。与B Puri的另一项合作涉及一种DEN4 PDK20候选疫苗。到目前为止，我们已经对PDK20病毒及其亲本进行了测序，并获得了具有感染性的疫苗全长cDNA克隆。这方面的进一步工作不太可能进行，因为B Puri已经在专利局找到了一份工作。在与E Kelly的合作下，我们已经对DEN2PDK50候选疫苗及其强毒亲本进行了测序和感染性克隆。克隆衍生病毒在组织培养细胞中的生长动力学正在与它们的亲本进行比较。克隆化的DEN2PDK50是由K·埃克尔斯和他的合作者在GMP下制造的，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乙脑减毒活疫苗(JEV)的感染性克隆，这是中国乙脑病毒活疫苗株SA14 14-2的PDK细胞衍生物的Vero细胞适应版本。克隆衍生病毒在细胞培养中以与其亲本相同的动力学生长。这种病毒正在小鼠身上进行免疫原性测试。

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.