REPLICATION,VIRULENCE & IMMUNOGENICITY IN RECOMBINANT RESPIRATORY SYNCYTIAL VIRU

复制、毒力

• 批准号: 6431557
• 负责人: PETER LEON COLLINS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6431557
• 关键词: Pneumovirus vaccine; attenuated microorganism; complementary DNA; gene mutation; laboratory mouse; live vaccine; microorganism culture; nucleocapsid; recombinant virus; respiratory syncytial virus; site directed mutagenesis; tissue /cell culture; transfection; vaccine development; virulence; virus antigen; virus cytopathogenic effect; virus genetics; virus protein; virus replication

Human respiratory syncytial virus (RSV) is the most important viral agent of pediatric respiratory tract disease worldwide and is responsible for a huge burden of morbidity and significant mortality. A licensed vaccine or effective antiviral therapy is unavailable. Obstacles to vaccine development include the poor growth of the virus in cell culture, the semi-permissive nature of infection in most animal models, the difficulty of achieving an appropriate balance between immunogenicity and attenuation, and the inefficiency of the immune response in the very young infant. We developed a method for producing infectious recombinant RSV by the intracellular coexpression of cDNAs encoding a complete RSV replicative intermediate RNA (antigenome) and the N, P, L and M2-1 proteins, which together constitute a nucleocapsid that is fully competent for RNA synthesis. This provides an important tool for basic molecular and pathogenesis studies as well as a method for fine-tuning the level of attenuation of candidate vaccine viruses. We showed that RSV encodes ten mRNAs and eleven unique proteins (the M2 mRNA contains two overlapping ORFs encoding the M2-1 and M2-2 proteins). Five RSV genes, namely NS1, NS2, SH, M2-2 and G, could be "knocked out" (deleted) singly and in some cases in combination without ablating the ability of the virus to grow in cell culture. However, in most cases growth was less efficient. Deletion of the NS1 and NS2 genes singly or in combination was found to be highly attenuating in mice and chimpanzees. These deletions are candidates for inclusion in a live-attenuated vaccine. The M2-2 deletion virus exhibited a shift favoring transcription over replication. This implicates this protein as a regulatory factor in RNA synthesis. This mutation has the novel property of reducing growth while increasing, rather than decreasing, gene expression, and thus might make a vaccine that is "better than nature". The G knockout virus demonstrated cell-specific differences in growth efficiency, implying that it is using an alternative receptor whose distribution is cell-specific. Thus, these findings have important implications for virus assembly and receptor usage. Fully-viable chimeric viruses were constructed between human RSVs representing the two antigenic subgroups, making it possible to use a single attenuated backbone to express the major antigenic determinants of each of the subgroups. Fully-viable chimeras also were constructed between human RSV and bovine RSV, a virus that has a host range restriction that renders it highly attenuated in primates and thus represents a new method of attenuating an RSV vaccine. As another approach to making an RSV vaccine "better than nature", recombinant RSV was engineered to express various cytokines and chemokines in order to enhance immunogenicity and, in some cases, attenuate the virus.

人类呼吸道合胞病毒(RSV)是全球范围内引起儿童呼吸道疾病的最重要的病原体，造成了巨大的发病率负担和显著的死亡率。目前还没有获得许可的疫苗或有效的抗病毒疗法。疫苗开发的障碍包括病毒在细胞培养中生长不良，在大多数动物模型中感染的半允许性，难以在免疫原性和衰减性之间实现适当的平衡，以及幼儿免疫反应的低效。我们发展了一种通过编码完整的RSV复制中间RNA(抗基因组)的cDNA与N、P、L和M2-1蛋白在细胞内共表达来产生具有感染性的重组RSV的方法，这些蛋白共同构成了完全能够合成RNA的核衣壳。这为基础分子和发病机制研究提供了重要工具，也为微调候选疫苗病毒的减毒水平提供了一种方法。我们发现RSV编码10个mRNAs和11个独特的蛋白(M2mRNA包含两个重叠的ORF，编码M2-1和M2-2蛋白)。5个RSV基因，即NS1、NS2、SH、M2-2和G，可以单独“敲除”(删除)，在某些情况下可以组合在一起，而不会削弱病毒在细胞培养中的生长能力。然而，在大多数情况下，增长效率较低。在小鼠和黑猩猩中，NS1和NS2基因的单独或联合缺失被发现是高度减弱的。这些缺失是纳入减毒活疫苗的候选。M2-2缺失病毒表现出倾向于转录而不是复制的转变。这意味着该蛋白质是RNA合成中的一个调节因子。这种突变具有一种新奇的特性，即在增加而不是减少基因表达的同时抑制生长，从而可能制造出一种“比自然更好”的疫苗。G基因敲除病毒在生长效率上表现出细胞特异性差异，这意味着它使用的是一种替代受体，其分布是细胞特异性的。因此，这些发现对病毒组装和受体的使用具有重要的意义。在代表两个抗原亚群的人RSV之间构建了完全活性的嵌合病毒，使得使用单个减毒的骨架来表达每个亚群的主要抗原决定簇成为可能。人RSV和牛RSV之间也构建了完全可行的嵌合体，这种病毒有宿主范围限制，使其在灵长类动物中高度减毒，从而代表了一种减毒RSV疫苗的新方法。作为制造比自然更好的RSV疫苗的另一种方法，重组RSV被改造成表达各种细胞因子和趋化因子，以增强免疫原性，在某些情况下，还能减弱病毒。