A Synthetic Human Cytomegalovirus Vaccine Platform

合成人巨细胞病毒疫苗平台

• 批准号: 8687582
• 负责人: Sanjay Vashee
• 金额: $ 27.31万
• 依托单位: TOMEGAVAX, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8687582
• 关键词: Attenuated; Attenuated Vaccines; Bacterial Artificial Chromosomes; Biology; Blood Circulation; Bone Marrow Transplantation; Cell Line; Cells; Characteristics; Chromosomes, Human, Pair 4; Clinical; Clinical Trials; Cloning; Complement; Complication; Congenital Abnormality; Cytomegalovirus; Cytomegalovirus Vaccines; DNA; DNA Sequence; DNA Viruses; DNA biosynthesis; Data; Development; Disease; Fibroblasts; Frequencies; Generations; Genes; Genome; Genome Stability; Genomics; Genotype; Goals; Growth; Herpesviridae; Human; IL2RA gene; Immune response; Immune system; Immunocompromised Host; Immunology; In Vitro; Individual; Infection; Institutes; Laboratories; Lesion; Life; Methods; Minor; Modification; Molecular; Molecular Cloning; Mus; Oligonucleotides; Organ; Outcome; Phase; Polymerase Chain Reaction; Recombinants; Sampling; Solid; Symptoms; Technology; Time; Transfection; Transplant Recipients; Tropism; Vaccines; Variant; Viral; Viral Genome; Virus; base; cell growth; cell type; congenital infection; deep sequencing; design; gene synthesis; genetic manipulation; genome sequencing; immunogenicity; immunosuppressed; in vivo; innovation; mouse model; public health relevance; reconstitution; synthetic biology; synthetic construct; tissue culture; vaccine development; vector vaccine; volunteer

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.

描述（由申请人提供）：该项目的目的是基于基因组序列信息合成人类巨细胞病毒（HCMV）菌株，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构成衰减HCMV疫苗开发的基础。创新的合成生物学方法将克服需要广泛体外HCMV传播的当前技术的缺点，这通常会导致选择组织培养适应的病毒变体，而病毒变体不太可能具有感染性。作为原理证明，我们将重新创建以前在临床试验中使用的临床分离式托莱多的野生版本。预序数据表明，托莱多的早期段落包含一种基因组的混合物，这些基因组既适合组织培养和非适应性基因型。我们将确定这种混合物中存在的基因型的基因组序列，并结合寡核苷酸合成，聚合酶链反应，体外和体内组装以逐步的hCMV基因组重现，该基因组将与原始的，野生型序列相似，以hcmv基因组重现。所得的病毒以及扩散缺陷的变体将在体外重构，其生长特征将与实验室适应的菌株和其他临床分离株进行比较，并使用人源化/SCID/SCID/IL2RG-NUL小鼠在体外和体内进行比较。如果成功的话，该项目将a）极大地促进没有先前组织培养的序列信息的CMV的分子克隆的产生，b）导致迄今为止合成的最大病毒基因组，c）彻底改变了疱疹病毒基因组的遗传操纵以及其他大型DNA病毒的遗传操纵。

项目成果

Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination.

• DOI: 10.1128/mspheredirect.00331-17
• 发表时间: 2017-09
• 期刊: mSphere
• 影响因子: 4.8
• 作者: Vashee S;Stockwell TB;Alperovich N;Denisova EA;Gibson DG;Cady KC;Miller K;Kannan K;Malouli D;Crawford LB;Voorhies AA;Bruening E;Caposio P;Früh K
• 通讯作者: Früh K