A Synthetic Human Cytomegalovirus Vaccine Platform

描述（由申请方提供）：本项目的目标是基于基因组序列信息合成一种人巨细胞病毒（HCMV）毒株，该毒株已证明能够在血清阳性个体中建立持续感染。所得合成产物将形成减毒HCMV疫苗开发的基础。创新的合成生物学方法将克服当前技术的缺点，即需要大量的HCMV体外传代，这通常会导致选择不太可能具有感染性的组织培养适应性病毒变体。作为原理证明，我们将重新创建先前在临床试验中使用的临床分离株Toledo的野生型版本。实验数据表明，Toledo的早期传代包含显示组织培养适应和非适应基因型的基因组的混合物。我们将确定该混合物中存在的基因型的基因组序列，并使用寡核苷酸合成、聚合酶链反应、体外和体内组装的组合，以逐步的方式重新创建与人类中使用的原始野生型序列最相似的HCMV基因组。将在体外重构所得病毒以及扩散缺陷型变体，并使用人源化NOD/SCID/IL2Rg-nul小鼠在体外和体内将其生长特征与实验室适应性毒株和其他临床分离株的生长特征进行比较。如果成功，该项目将a）极大地促进直接从序列信息产生CMV的分子克隆，而无需预先组织培养，B）导致迄今为止合成的最大病毒基因组，c）彻底改变疱疹病毒基因组以及其他大DNA病毒基因组的遗传操作。

DESCRIPTION (provided by applicant): The goal of this project is to synthesize, based on genomic sequence information, a human cytomegalovirus (HCMV) strain with demonstrated ability to establish persistent infection in sero-positive individuals. The resulting synthetic product will form the basis for the development of attenuated HCMV vaccines. Innovative synthetic biology methods will overcome a shortcoming of current technologies that require extensive in vitro passaging of HCMV which often results in selecting tissue-culture adapted viral variants that are unlikely to be infectious. As proof-of-principle we will recreate the wild-ype version of the clinical isolate Toledo which has been used previously in clinical trials. Preliminay data suggest that early passages of Toledo contain a mixture of genomes displaying both tissue-culture adapted and non-adapted genotypes. We will determine the genomic sequence of the genotypes present in this mixture and use a combination of oligonucleotide synthesis, polymerase chain reaction, in vitro and in vivo assembly to recreate in a step-wise fashion a HCMV genome that will most closely resemble the original, wild-type sequence used in humans. The resulting virus, as well as spread-deficient variants, will be reconstituted in vitro and its growth characteristics will be compared to that of laboratory-adapted strains and other clinical isolates in vitro and in vivo using humanized NOD/SCID/IL2Rg-nul mice. If successful, this project would a) greatly facilitate the generation of molecular clones of CMV directly from sequence information without prior tissue culture, b) result in the largest viral genome synthesized to date, c) revolutionize the genetic manipulation of herpesviral genomes as well as that of other large DNA viruses.