Development of reassortment-deficient influenza vaccine candidates: Rewiring packaging signals

项目摘要 流感是世界范围内发病和死亡的主要原因，季节性流行病估计造成 每年有29万到65万人死亡。由于抗原性，每年的疫苗效力可低至10%。 流行的流感病毒株和疫苗株之间的不匹配，强调需要一个普遍的 流感病毒疫苗。我们的实验室已经开发了一种新的疫苗接种策略，利用嵌合血凝素 (HA)将宿主免疫应答从抗原性可变的HA头部结构域转移到HA头部结构域的构建体。 保守的HA茎。用表达这些的减毒活流感病毒疫苗（LAIV）接种 在动物模型中构建了针对不同流感病毒株的广泛保护。然而，关注 关于LAIV与野生型循环病毒的重配潜力，阻碍了广泛的考虑， 大规模接种含有新型HA的LAIV，并将这些疫苗的临床试验限制在 控制装置我们实验室以前的工作提供了证据，包装信号位于3'， 病毒基因组RNA片段的5 ′末端可用于控制流感病毒重配。通过 交换两个片段的包装信号，我们防止了这些片段的自由重配。基于 根据这些数据，我已经生成了更多的病毒，它们具有额外的重新布线的包装信号，以定义它们的 在假设它们将是重配缺陷的情况下，使用共感染研究的重配潜力。 为了更好地理解选择性包装的机械基础，我将进行转座子 对HA vRNA片段进行诱变，并绘制关键RNA-RNA中涉及的特异性序列 包装相互作用。本项目的最终目标是建立一个新的平台， LAIV不能自由重组和理解核苷酸相互作用， 流感基因组包装和重组。这将允许我们创建LAIV候选项， 对人群而言是新的，用于普遍或大流行前疫苗接种目的。

Project Summary Influenza is a major cause of morbidity and mortality worldwide, with seasonal epidemics causing an estimated 290,000 to 650,000 deaths every year. Annual vaccine efficacy can be as low as 10% due to antigenic mismatch between circulating influenza strains and vaccine strains, underscoring the need for a universal influenza virus vaccine. Our lab has developed a novel vaccination strategy utilizing chimeric hemagglutinin (HA) constructs to shift the host immune response from the antigenically variable HA head domain to the conserved HA stalk. Vaccination with live attenuated influenza virus vaccines (LAIVs) expressing these constructs elicits broad protection against different influenza virus strains in animal models. However, concern regarding the reassortment potential of LAIVs with wild-type circulating viruses impedes consideration of wide- scale vaccination with LAIVs containing novel HAs and confines clinical trials with these vaccines to containment units. Previous work from our lab has provided evidence that packaging signals located at the 3' and 5' termini of the viral genomic RNA segments can be utilized to control influenza virus reassortment. By swapping the packaging signals of two segments, we prevented free reassortment of those segments. Based on these data, I have generated more viruses with additional rewired packaging signals to define their reassortment potential using coinfection studies under the hypothesis that they will be reassortment-deficient. To better understand the mechanistic underpinnings of selective packaging, I will perform transposon mutagenesis on the HA vRNA segment and map the specific sequences implicated in key RNA-RNA packaging interactions. The ultimate goal of this project is to establish a novel platform for the development of LAIVs that are unable to freely reassort and gain an understanding of the nucleotide interactions at play in influenza genome packaging and reassortment. This would allow us to create LAIV candidates that contain HAs novel to the human population for either universal or prepandemic vaccination purposes.