Applications of infectious cDNA technology to RNA virus

以前，我们（BF和BP）从1型登革热病毒（DEN 1 WP）和减毒活DEN 1候选疫苗（DEN 1 PDK 20）中制备了全长“感染性”cDNA克隆，该候选疫苗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。在生长曲线上，被回收的病毒表现为相应的亲本DEN 1，并且回收的DEN 1 PDK 20具有与其亲本相同的小空斑表型。通过在WP和PDK 20感染性克隆之间制造嵌合体，工作已经开始绘制导致这种小斑块表型的突变。此外，在人体临床试验中，PDK 20病毒的反应原性略高，而PDK 26病毒则过度减毒。 我们计划将进一步的减毒突变引入PDK 20克隆，以恢复正确的减毒疫苗株，首先我们将使用在PDK 26中发现的突变子集，这些突变在PDK 20中没有。 目前正在与B Puri合作的另一个项目是制造DEN 4型PDK适应性疫苗候选物的感染性克隆。到目前为止，我们已经成功地制作了全长cDNA克隆;目前正在研究转录本的感染性。 与E Kelly合作，我们已经制作了另一种感染性克隆，即PDK适应的DEN 2疫苗候选物。 将克隆的序列与亲本候选疫苗的群体平均序列进行比较，并且正在修复几个差异。 接下来的议程是在组织培养细胞和小动物中将这种病毒与其亲本进行比较。克隆的最终计划是根据GMP生产候选疫苗（由K. Eckels），并在人体试验中将转录衍生病毒与其未克隆的亲本进行比较。 最后，去年我们（BF和CZ）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。 正在细胞生长曲线和小鼠免疫原性研究中研究从该克隆中回收的病毒表型。

Previously, we (BF and BP) made full length "infectious" 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. Recovered viruses behave like the corresponding parent DEN1 in growth curves, and the recovered DEN1 PDK20 has the same small plaque phenotype as its parent. Work has begun to map the mutations responsible for this small plaque phenotype, by making chimeras between the WP and PDK20 infectious clones. Also, in human clinical trials the PDK20 virus is slightly too reactogenic, yet a PDK26 virus is overattenuated. We plan to introduce further attenuating mutations into the PDK20 clone in an effort to recover a correctly attenuated vaccine strain, and at first we will use subsets of the mutations found in PDK26 which are not in PDK20. Another project currently underway in collaboration with B Puri is to make an infectious clone of a DEN type 4 PDK-adapted vaccine candidate. So far, we have succeeded in making a full-length cDNA clone; the infectivity of transcripts is currently being investigated. In collaboration with E Kelly, we have made another infectious clone, of a PDK adapted DEN2 vaccine candidate. The sequence of the clone was compared to the population average sequence of the parent vaccine candidate, and several differences are being repaired. Next on the agenda is to compare this virus to its parent in tissue culture cells and in small animals. The ultimate plan for this clone is to manufacture a candidate vaccine under GMP (to be done at WRAIR by K. Eckels) and to compare the transcript- derived virus to its uncloned parent in human trials. Finally, last year we (BF and CZ) 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. The phenotype of virus recovered from this clone is being investigated in growth curves in cells and in mouse immunogenicity studies.