A Synthetic Human Cytomegalovirus Vaccine Platform

描述(由申请人提供)：该项目的目标是基于基因组序列信息合成一种具有在血清阳性个体中建立持续感染能力的人巨细胞病毒(HCMV)株。由此合成的产品将成为开发减毒HCMV疫苗的基础。创新的合成生物学方法将克服目前技术的一个缺点，即需要在体外广泛传代人巨细胞病毒，这往往导致选择组织培养适应的病毒变种，这些变种不太可能具有传染性。作为原则证明，我们将重新创建临床分离株托莱多的野生型版本，该版本以前曾用于临床试验。Preliminay数据表明，托莱多的早期传代含有混合的基因组，显示出组织培养适应和非适应的基因类型。我们将确定这种混合物中存在的基因类型的基因组序列，并使用寡核苷酸合成、聚合酶链式反应、体外和体内组装的组合，以循序渐进的方式重建与人类使用的原始野生型序列最相似的HCMV基因组。由此产生的病毒以及传播缺陷的变种将在体外重组，并将使用人源化NOD/SCID/IL2RG-NUL小鼠在体外和体内将其生长特性与实验室适应的毒株和其他临床分离株进行比较。如果成功，该项目将极大地促进直接从序列信息中产生CMV的分子克隆，而无需事先进行组织培养，b)产生迄今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.