Development of Universal Influenza Virus Vaccines Using Nucleoside-Modified Messenger RNA

摘要 流感病毒是人类最重要的传染病之一。流感死亡率估计为 全球每年约有650,000人感染，此外，偶尔发生的全球大流行可感染多达20- 占世界人口的40%。获得许可的流感病毒疫苗需要每年重新配制，并 由于免疫球蛋白寿命差和相关病毒缺乏中和作用而导致的重新管理。我们已经开发出一种 使用编码抗原的核苷修饰的mRNA的流感病毒疫苗新途径 包裹在脂质纳米粒(LNPs)中。这种疫苗形式完全由生理成分组成 这是简单和成本效益高的生产，应该不会引起不良事件后给药。 此外，信使核糖核酸疫苗的生产是独立于序列的，不需要卵子、细胞培养或 蛋白质提纯。我们证明了编码全长的单价mRNA-LNP疫苗 血凝素(HA)诱导针对免疫抑制的HA茎区域的有效免疫反应，1 在通用流感病毒疫苗的目标中，这与预防同源病毒有关， 小鼠体内的异源和异亚型流感病毒。此外，我们还表明，单个 核苷修饰的mRNA-LNP疫苗免疫诱导持久保护性HA茎特异性 抗体反应。最后，我们证明了核苷修饰的mRNALNP免疫诱导了高表达。 对生发中心的产生至关重要的抗原特异性T滤泡辅助细胞(TFH)水平， 免疫球蛋白类开关和长时记忆，这是发展中的关键因素 有效的流感疫苗。在这笔赠款中，我们的目标是开发和评估新一代，具有广泛的保护性 使用编码血凝素的核苷修饰的mRNA-LNPs在小鼠和雪貂中接种流感病毒疫苗， 神经氨酸酶、核蛋白和M2的胞外区。此外，我们的目标是研究其机制。 MRNA-LNP流感病毒疫苗的作用。我们将有3个具体目标：1)设计和制作 流感病毒保守区的信使核糖核酸免疫原。2)免疫原性和保护效果 小鼠和雪貂核苷修饰的mRNALNP流感病毒疫苗。3)调查 核苷修饰的mRNALNP流感疫苗的作用机制。这项提议将发展安全， 易于制造和管理并可预防的新一代流感病毒疫苗 抗原性相距遥远的流感病毒。产生的数据将能够将这种疫苗方法转移到 临床试验开发。

ABSTRACT Influenza virus is one of the most important human infectious diseases. The influenza mortality is estimated to be approximately 650,000 per year worldwide, in addition, occasional global pandemics can infect up to 20- 40% of the world's population. Licensed influenza virus vaccines require annual reformulation and readministration due to poor IgG longevity and lack of neutralization of related viruses. We have developed a new vaccine approach against influenza virus that uses antigen-encoding nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs). This vaccine format composed entirely of physiologic components that is simple and cost effective to produce and should induce no adverse events after administration. Additionally, mRNA vaccine production is sequence-independent and does not require eggs, cell culture or protein purification. We demonstrated that monovalent mRNA-LNP vaccines encoding full-length hemagglutinin (HA) induced potent immune responses against the immunosubdominant HA stalk region, one of the targets of universal influenza virus vaccines, which was associated with protection against homologous, heterologous and heterosubtypic influenza viruses in mice. Furthermore, we showed that a single immunization with nucleoside-modified mRNA-LNP vaccines induced durable protective HA stalk-specific antibody responses. Finally, we demonstrated that nucleoside-modified mRNA-LNP immunization induced high levels of antigen-specific T follicular helper (Tfh) cells that are critical for the generation of germinal centers, immunoglobulin class switch, and long-term memory, which are vital elements in the development of an effective influenza vaccine. In this grant, we aim to develop and evaluate new generation, broadly protective influenza virus vaccines in mice and ferrets using nucleoside-modified mRNA-LNPs encoding hemagglutinin, neuraminidase, nucleoprotein and the ectodomain of M2. Additionally, we aim to investigate the mechanisms of action of mRNA-LNP influenza virus vaccines. We will have 3 specific aims: 1) Design and production of mRNA immunogens for conserved regions of influenza virus. 2) Immunogenicity and protective efficacy of nucleoside-modified mRNA-LNP influenza virus vaccines in mice and ferrets. 3) Investigation of the mechanisms of action of nucleoside-modified mRNA-LNP influenza vaccines. This proposal will develop safe, new generation influenza virus vaccines that are easy to manufacture and administer and can protect against antigenically distant influenza viruses. The data generated will be capable of moving this vaccine approach to clinical trial development.