Project 3: Dr. Li

项目3：通过甲基转移酶突变来减毒RSV PI：李建荣，DVM，博士 摘要： 人类呼吸道合胞病毒(RSV)是引起儿童呼吸道疾病的主要病原体。 全世界。尽管做出了重大努力，但目前还没有疫苗可以对抗这种病毒。带电衰减 疫苗是RSV最有希望的候选疫苗。然而，要找出 一种在减毒和免疫原性之间达到最佳平衡的减毒RSV株。这个 该项目的目标是通过靶向病毒mRNA帽甲基转移酶来合理地减弱RSV。 (MTase)，并开发MTase缺陷病毒作为RSV的新型减毒活疫苗候选。这个 RSV mRNA帽子结构在鸟嘌呤-N-7(G-N-7)和核糖2‘-O位上由单个甲基化 保守区VI(CR-VI)位于L的大聚合酶蛋白中。人体必需的氨基酸残基 将鉴定G-N-7和/或2‘-O MTase的活性，并将其引入到感染性cDNA中 呼吸道合胞病毒的克隆。G-N-7和/或2‘-O MTase活性缺陷的重组RSV将被恢复，并 它们在细胞培养中的衰减将被表征。这些重组病毒将被测试其 在原代、分化良好的人呼吸道上皮(HAE)培养中的传染性和传播能力。下一首, 它们的复制、致病机制、免疫原性和诱导保护的能力将在棉花上进行检验。 大鼠是目前最好的呼吸道合胞病毒小动物模型。通过添加产生干扰素的NS1突变(项目 1)和修复G突变(项目2)到最有希望的L到RSV的MTase突变，我们预计 我们将产生一组理想的RSV疫苗株，它们在Vero细胞中生长良好，并在体外减毒 在体内，遗传稳定，高度免疫原性。这些新的、经过改进的活RSV候选疫苗 然后可以用于未来在非人类灵长类动物身上的试验。

Project 3: Attenuating RSV with Mutations in Its Methyltransferase PI: Jianrong Li, DVM, PhD Abstract: Human respiratory syncytial virus (RSV) is the leading causative agent of pediatric respiratory tract disease worldwide. Despite major efforts, there is no vaccine currently available to combat this virus. Live attenuated vaccines are the most promising vaccine candidates for RSV. However, it has been a challenge to identify an attenuated RSV strain that has an optimal balance between attenuation and immunogenicity. The objectives of this project are to rationally attenuate RSV by targeting the viral mRNA cap methyltransferase (MTase) and to develop MTase-defective viruses as novel live attenuated vaccine candidates for RSV. The RSV mRNA cap structure is methylated at the guanine-N-7 (G-N-7) and ribose 2’-O positions by a single conserved region VI (CR-VI) located in the large (L) polymerase protein. Amino acid residues essential for G-N-7 and/or 2’-O MTase activities will be characterized and will be introduced into an infectious cDNA clone of RSV. Recombinant RSVs defective in G-N-7 and/or 2’-O MTase activities will be recovered, and their attenuation in cell culture will be characterized. These recombinant viruses will be tested for their infectivity and ability to spread in primary, well differentiated, human airway epithelial (HAE) cultures. Next, their replication, pathogenesis, immunogenicity, and capacity to induce protection will be examined in cotton rats, the best available small animal model for RSV. By adding interferon-producing NS1 mutations (Project 1) and ‘repaired G’ mutations (Project 2) to the most promising MTase mutations in L to RSV, we expect that we will generate a panel of ideal RSV vaccine strains that grow well in Vero cells, are attenuated in vitro and in vivo, genetically stable, and highly immunogenic. These new, improved live RSV vaccine candidates can then be used for future trials in nonhuman primates.