Development of an RSV Vaccine by Molecular Manipulation of the Viral Matrix Prot

人呼吸道合胞病毒（HRSV）是小儿毛细支气管炎的单一最大病毒病因， 肺炎据估计，全世界每年有超过100，000名儿童死亡， HRSV疫苗是优先事项。在当前的方法中，实况衰减是有吸引力的，因为，不像 灭活疫苗，它有望诱导广泛和平衡的免疫反应。然而，活HRSV 迄今为止，疫苗还不能完全预防对婴儿和儿童的潜在危险副作用。 本研究的长期目标是克服减毒活HRSV的安全性挑战 疫苗和贡献HRSV生命周期的基础知识，以设计替代抗HRSV 接近。为了实现这些目标，本提案集中于病毒基质的分子操作 (M)蛋白质，以提高减毒活疫苗的安全性。M蛋白是复制和发挥重要作用的蛋白质。 在病毒体组装过程中起着重要作用，其中许多过程与生产高度相关， 病毒颗粒的组成、释放和可能的稳定性。更好地理解M函数 为疫苗的发展提供了巨大的潜力。一种新的系统是基于一种传染性的 缺乏M基因的病毒（M-null），其允许快速筛选和操纵M功能。通过提供 将表达M突变体的质粒转染到用M-无效病毒感染的细胞中，初步筛选鉴定出M 具有调节活病毒的感染性子代产生水平的潜力的突变。这项建议 利用基于M-null的系统来同样地识别和操作与汇编相关的M函数，并测试 通过以下具体目的（略），在体内的翻译潜力：1）确定M功能 和突变，以及调节病毒组装、组成和释放的潜在机制。（二） 根据M测定对传播缺陷活病毒的免疫应答质量 突变，在体外和体内。3)测试有希望的M突变病毒在攻击后保护小鼠的能力 野生型HRSV。这些目标将共同提高我们对HRSV复制的基本理解， 测试M蛋白操纵的潜力，以有助于产生安全的减毒活疫苗。

Human Respiratory Syncytial Virus (HRSV) is the single largest viral cause of pediatric bronchiolitis and pneumonia. With an estimated mortality of >100,000 children per year worldwide, development of an anti- HRSV vaccine is a priority. Among the current approaches, live-attenuation is attractive because, unlike inactivated vaccines, it promises to induce a broad and balanced immune response. However, live HRSV vaccines have so far been unable to fully prevent potentially dangerous side-effects in infants and children. The long-term objectives of this research are to overcome the safety challenges of live-attenuated HRSV vaccines and to contribute fundamental knowledge of the HRSV life cycle to design alternative anti-HRSV approaches. To meet these objectives, this proposal focusses on molecular manipulation of the viral matrix (M) protein to enhance safety of live-attenuated vaccines. The M protein is essential for replication and plays a prominent role in virion-assembly processes, many of which are highly relevant for the production, composition, release, and perhaps stability of virus particles. A better understanding of M functions therefore offers significant potential for vaccine advancements. A novel system was developed based on an infectious virus lacking the M gene (M-null) which allows rapid screening and manipulation of M functions. By providing plasmids expressing M mutants to cells infected with the M-null virus, a preliminary screen identified M mutations with potential to regulate the level of infectious progeny production of a live virus. This proposal utilizes the M-null based system to likewise identify and manipulate assembly-relevant M functions and test the translational potential in vivo, through the following Specific Aims (abbreviated): 1) Identify M functions and mutations, and the underlying mechanisms, that regulate virus assembly, composition, and release. 2) Determine the quality ofthe immune response to live viruses with transmission-deficiencies based on M mutations, in vitro and in vivo. 3) Test promising M mutant viruses for ability to protect mice after challenge with wildtype HRSV. Together these aims will raise our fundamental understanding of HRSV replication and test the potential of M protein manipulation to contribute to the generation of a safe live-attenuated vaccine.