Applications of infectious cDNA technology to RNA virus

此前，我们（BF和BP）从一种强毒登革热1型病毒（DEN1 WP）和一种已适应在狗肾细胞中生长的DEN1减毒候选疫苗（DEN1 PDK20）中获得了全长的“传染性”cDNA克隆。用体外从这些克隆中转录的RNA转染细胞会产生登革热感染。恢复的病毒在生长曲线上表现得与对应的亲本DEN1相似，并且恢复的DEN1 PDK20具有与其亲本相同的小斑块表型。通过在WP和PDK20感染性克隆之间制造嵌合体，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，PDK20病毒的反应性略强，而PDK26病毒的反应性过弱。我们计划在PDK20克隆中引入更多的减毒突变，以恢复正确的减毒疫苗株，首先我们将使用PDK26中发现的PDK20中没有的突变子集。目前正在与B Puri合作的另一个项目是制作DEN 4型pdk适应候选疫苗的传染性克隆。到目前为止，我们已经成功地制作了一个全长cDNA克隆；目前正在研究转录本的传染性。在与E Kelly的合作下，我们制作了另一种传染性克隆，一种PDK改编的DEN2候选疫苗。克隆序列与亲本候选疫苗的群体平均序列进行了比较，并修复了一些差异。下一个议程是在组织培养细胞和小动物体内将这种病毒与其亲本进行比较。该克隆的最终计划是在GMP下制造一种候选疫苗（将在WRAIR由K. Eckels完成），并在人体试验中将转录本衍生病毒与其未克隆的母体进行比较。最后，去年我们（BF和CZ）完成了陆军候选减毒日本脑炎病毒（JEV）活疫苗的传染性克隆，这是中国JEV活疫苗菌株SA14-14-2的PDK细胞衍生物的vero细胞改编版本。从该克隆中恢复的病毒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.