Role of nonstructural protein in limited genetic diversity of yellow fever 17D vaccine virus

非结构蛋白在黄热病17D疫苗病毒有限遗传多样性中的作用

• 批准号: 10372589
• 负责人: Alan D.T. Barrett
• 金额: $ 20万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-22 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10372589
• 关键词: Acute; Amino Acid Substitution; Amino Acids; Antiviral Agents; Attenuated; Attenuated Live Virus Vaccine; Attenuated Vaccines; Base Sequence; Capsid; Category B pathogen; ChimeriVax; Complex; Dengvaxia; Development; Disease; Family member; Fatality rate; Fever; Flaviviridae; Flavivirus; Future; Gene Mutation; Genes; Genetic; Genetic Determinism; Genetic Variation; Genome; Goals; Hemorrhage; Human; Human poliovirus; Investigation; Maps; Modeling; Molecular; Mus; Mutation; National Institute of Allergy and Infectious Disease; Nonstructural Protein; Nucleotides; Paper; Pathogenicity; Phenotype; Population; Predisposition; Quality Control; RNA; RNA Viruses; RNA-Directed RNA Polymerase; Resistance; Ribavirin; Role; Safety; Structural Genes; Syndrome; Technology; Tissues; Vaccines; Vertebral column; Viral; Viral Genes; Virulence; Virus; Virus Replication; West Nile virus; Yellow Fever; Yellow Fever Virus Infection; Yellow fever virus; attenuation; base; improved; liver injury; mosquito-borne; mutant; next generation sequencing; novel vaccines; prototype; rational design; single molecule; vaccine development; viral RNA

ABSTRACT Many successful live attenuated vaccines (LAVs) were derived empirically and little is known about their mechanisms of attenuation. The best characterized of these LAVs is poliovirus where attenuation has been mapped to the 5’NCR and capsid genes. Increased understanding the mechanism of attenuation of licensed LAVs will help in the rational development of future LAVs. The disease yellow fever is controlled by the use of a live attenuated vaccine, strain 17D, derived from wild-type (WT) strain Asibi, and differ by 20 amino acids; 9 in the structural genes and 11 in the nonstructural (NS) genes. Our overall goal is to understand the mechanism of attenuation of 17D vaccine, which is poorly understood. Next Generation Sequencing (NGS) technology has great applications to vaccine development and quality control and safety of LAVs. We have compared WT Asibi and 17D vaccine by NGS and found that Asibi is a typical RNA virus with a quasispecies population while, surprisingly, 17D vaccine has very little evidence of quasispecies, and we believe that this may contribute to the attenuated phenotype of the vaccine virus. We have shown that the restricted quasispecies in 17D vaccine virus is due to mutation(s) in the NS proteins of the replication complex as a whole, rather than the RNA dependent RNA polymerase (RdRp) alone. In addition, ribavirin is an antiviral drug that introduces mutations into RNA genomes during replication due to the lack of fidelity of the viral RdRp. We have shown that WT Asibi virus is sensitive to ribavirin while 17D vaccine virus is relatively resistant suggesting that a high fidelity replication complex potentially contributes to the attenuated phenotype of 17D vaccine. We believe that investigation of the mechanism of limited quasispecies in 17D vaccine virus may have important applications to understanding the molecular basis of attenuation of 17D vaccine, other LAVs, and development of future flavivirus LAVs. In this application we will identify the viral genes that contribute to limited quasispecies of 17D vaccine. We hypothesize that the restricted quasispecies in 17D vaccine virus is due to multiple mutations in the NS proteins of the replication complex as a whole, rather than the RdRp alone, encodes an attenuated phenotype, and multiple mutations contribute to the very low rate of reversion to virulence. The objective of this proposal is to perform targeted studies to identify which YFV NS genes contribute to the restricted quasispecies of 17D vaccine to propose a hypothesis to investigate the role of NS genes in the mechanism of attenuation of 17D vaccine. This will be achieved via three specific aims: Aim 1 will investigate the quasispecies population and ribavirin sensitivity of Asibi/17D mutants to identify residues in NS genes that contribute to restricted quasispecies population and ribavirin resistance of 17D vaccine; Aim 2 will generate Asibi mutants that are resistant to ribavirin, identify the nucleotide changes responsible for resistance, and characterize the quasispecies population of the mutants; and Aim 3 will investigate the phenotype of Asibi/17D mutants in AG129 mice to determine if ribavirin resistance and/or lack of quasispecies correlates with an attenuated phenotype.

摘要 许多成功的减毒活疫苗（LAV）是经验性地获得的，并且关于它们的特性知之甚少。 衰减机制。这些LAV的最佳特征是脊髓灰质炎病毒，其中减毒已被证实。 定位于5 'NCR和衣壳基因。增加了对许可证衰减机制的了解 LAV将有助于未来LAV的合理发展。黄热病是通过使用 活减毒疫苗，菌株17 D，来源于野生型（WT）菌株Asibi，并且相差20个氨基酸; 结构基因11个，非结构基因11个。我们的总体目标是了解 17 D疫苗的减毒，这是知之甚少。下一代测序（NGS）技术 在LAV的疫苗开发、质量控制和安全性方面有很大的应用。我们比较了WT Asibi 和17 D疫苗，发现Asibi是一个典型的RNA病毒，具有准种群体， 令人惊讶的是，17 D疫苗几乎没有准种的证据，我们相信这可能有助于 疫苗病毒的减毒表型。我们已经表明，17 D疫苗病毒中的限制性准种 是由于复制复合物的NS蛋白作为一个整体的突变，而不是RNA依赖的 单独使用RNA聚合酶（RdRp）。此外，利巴韦林是一种抗病毒药物，它会将突变引入RNA 由于病毒RdRp缺乏保真度，在复制过程中基因组被破坏。我们已经证明WT Asibi病毒是 对利巴韦林敏感，而17 D疫苗病毒相对耐药，表明高保真复制 复合物可能有助于17 D疫苗的减毒表型。我们认为， 17 D疫苗病毒中有限准种的机制可能对理解 17 D疫苗、其他LAV减毒的分子基础，以及未来黄病毒LAV的开发。在这 应用中，我们将鉴定有助于17 D疫苗的有限准种的病毒基因。我们假设 17 D疫苗病毒中的限制性准种是由于17 D疫苗病毒的NS蛋白中的多个突变所致。 复制复合物作为一个整体，而不是单独的RdRp，编码减毒表型，和多个 突变导致毒力返强率非常低。本提案的目的是执行 有针对性的研究，以确定哪些YFV NS基因有助于17 D疫苗的限制性准种， 提出一个假设来研究NS基因在17 D疫苗减毒机制中的作用。这 将通过三个具体目标实现：目标1将调查准种群体和利巴韦林敏感性 Asibi/17 D突变体的突变，以确定NS基因中有助于限制准种种群的残基， Aim 2将产生对利巴韦林具有抗性的Asibi突变体， 负责抗性的核苷酸变化，并表征突变体的准种群体;以及 目的3研究Asibi/17 D突变体在AG 129小鼠中的表型，以确定是否存在利巴韦林耐药 和/或缺乏准种与减毒表型相关。