Applications of infectious cDNA technology to RNA virus

此前，我们(BF和BP)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候选疫苗中获得了全长的“感染性”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。恢复的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。通过在WP和PDK20感染性克隆之间制作嵌合体，已经开始绘制导致这种小斑块表型的突变图谱。此外，在人体临床试验中，PDK20病毒略有过度反应性，而PDK26病毒则过度减弱。我们计划在PDK20克隆中引入进一步的减毒突变，以努力恢复正确的减毒疫苗毒株，首先我们将使用在PDK26中发现的突变的子集，这些突变不在PDK20中。目前正在与B Puri合作的另一个项目是制作一种具有感染性的4型PDK适应疫苗的克隆。到目前为止，我们已经成功地克隆了全长的cdna；转录本的感染性目前正在调查中。在与E Kelly的合作中，我们已经制作了另一个具有感染性的克隆，即PDK适应的DEN2候选疫苗。克隆的序列与亲本候选疫苗的群体平均序列进行了比较，几个差异正在修复中。议程上的下一步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。这一克隆的最终计划是根据GMP制造候选疫苗(由K.Eockels在WRAIR完成)，并在人体试验中将转录本衍生病毒与其未克隆的亲本进行比较。最后，我们(BF和CZ)去年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JEV活疫苗株SA14-14-2的PDK细胞衍生株的Vero细胞适应版本。从这个克隆中回收的病毒的表型正在细胞生长曲线和小鼠免疫原性研究中进行研究。

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.