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，此外，偶尔的全球大流行病可感染多达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， 由于IgG寿命短和缺乏相关病毒的中和作用而再次给药。我们已经开发出一种 使用编码抗原的核苷修饰的mRNA的抗流感病毒的新疫苗方法 包封在脂质纳米颗粒（LNP）中。这种疫苗形式完全由生理成分组成 其制备简单且成本有效，并且在给药后不会引起不良事件。 此外，mRNA疫苗的生产是不依赖于序列的，并且不需要卵、细胞培养或细胞培养。 蛋白质纯化我们证明了编码全长LNP的单价mRNA-LNP疫苗， 血凝素（HA）诱导针对免疫亚显性HA茎区的有效免疫应答， 通用流感病毒疫苗的靶点，与针对同源， 异源和异亚型流感病毒。此外，我们还发现， 核苷修饰的mRNA-LNP疫苗免疫诱导持久的保护性HA茎特异性 抗体反应。最后，我们证明了核苷修饰的mRNA-LNP免疫诱导了高水平的免疫应答。 抗原特异性T滤泡辅助（Tfh）细胞的水平，所述细胞对于生成生发中心至关重要， 免疫球蛋白类别转换和长期记忆，这是发展中的重要因素， 有效的流感疫苗。在这项赠款中，我们的目标是开发和评估新一代，广泛保护， 使用编码血凝素的核苷修饰的mRNA-LNP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的胞外域。此外，我们的目标是调查的机制， mRNA-LNP流感病毒疫苗的作用。我们将有3个具体目标：1）设计和生产 流感病毒保守区的mRNA免疫原。2)免疫原性和保护效力 核苷修饰的mRNA-LNP流感病毒疫苗在小鼠和雪貂中的应用。3)调查 核苷修饰的mRNA-LNP流感疫苗的作用机制。这项建议将发展安全， 新一代流感病毒疫苗易于生产和使用， 抗原性远的流感病毒。所产生的数据将能够将这种疫苗方法 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.