A Synthetic Human Cytomegalovirus Vaccine Platform

描述（由申请人提供）：该项目的目标是根据基因组序列信息合成一种人类巨细胞病毒（HCMV）毒株，该毒株具有在血清阳性个体中建立持续感染的能力。由此产生的合成产品将成为开发HCMV减毒疫苗的基础。创新的合成生物学方法将克服当前技术的一个缺点，即需要大量的HCMV体外传代，这往往导致选择适合组织培养的病毒变体，而这些变体不太可能具有传染性。作为原理证明，我们将重新创建野生型临床分离物托莱多之前已用于临床试验。初步数据表明，托莱多的早期传代包含了显示组织培养适应和非适应基因型的基因组混合物。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，以逐步方式重建HCMV基因组，该基因组将最接近于人类中使用的原始野生型序列。由此产生的病毒，以及传播缺陷变异，将在体外重组，并将其生长特性与实验室适应株和其他临床分离株在体外和体内使用人源化NOD/SCID/IL2Rg-nul小鼠进行比较。如果成功，该项目将a)极大地促进CMV分子克隆的产生，而无需事先组织培养，b)导致迄今为止最大的病毒基因组合成，c)彻底改变疱疹病毒基因组以及其他大型DNA病毒的遗传操作。

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.