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

项目摘要 流感是世界范围内发病率和死亡率的主要原因，据估计，季节性流行病造成 每年有29万到65万人死亡。由于抗原性，疫苗的年度效力可低至10% 流行的流感毒株和疫苗毒株之间的不匹配，突显出需要一种普遍的 流感病毒疫苗。我们的实验室利用嵌合血凝素开发了一种新的疫苗接种策略 (HA)构建将宿主免疫反应从抗原性可变的HA头部结构域转移到 保存好的腐植酸茎。用表达这些基因的减毒流感病毒活疫苗(LAIV)接种 构建物在动物模型中对不同的流感病毒株具有广泛的保护作用。然而，令人担忧的是 关于LAIV与野生型传播病毒的重新组合的潜力阻碍了对广泛的 用含有新型HAS的LAIV进行大规模接种，并将这些疫苗的临床试验限制在 控制单元。我们实验室以前的工作提供了证据，证明包装信号位于3‘ 病毒基因组RNA片段的5‘端可用于控制流感病毒的重排。通过 交换了两个片段的包装信号，我们防止了这些片段的自由重新分类。基座 在这些数据上，我已经生成了更多带有额外重新连接的包装信号的病毒，以定义它们的 利用混合感染进行的重组潜力研究假设它们将是重组缺陷的。 为了更好地理解选择性包装的机制基础，我将执行转座子 对HA vRNA片段进行突变，并定位关键RNA-RNA所含的特定序列 包装相互作用。该项目的最终目标是建立一个新的开发平台 LAIV不能自由地重新排序和了解在其中起作用的核苷酸相互作用 流感基因组的包装和重新排序。这将允许我们创建包含以下内容的LAIV候选 无论是出于普遍接种还是流行前接种的目的，都对人类人群产生了新的影响。

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.