REPLICATION,VIRULENCE & IMMUNOGENICITY IN RECOMBINANT RESPIRATORY SYNCYTIAL VIRU

人呼吸道合胞病毒（RSV）是世界范围内儿科呼吸道疾病的最重要的病毒因子，并造成巨大的发病率和死亡率。没有获得许可的疫苗或有效的抗病毒治疗。疫苗开发的障碍包括病毒在细胞培养物中生长不良，大多数动物模型中感染的半容许性质，难以在免疫原性和减毒之间达到适当的平衡，以及非常年幼的婴儿中免疫应答的效率低下。我们开发了一种通过胞内共表达编码完整RSV复制中间体RNA（反基因组）和N、P、L和M2-1蛋白的cDNA来产生感染性重组RSV的方法，所述N、P、L和M2-1蛋白一起构成完全胜任RNA合成的核衣壳。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方法。我们发现RSV编码10种mRNA和11种独特的蛋白质（M2 mRNA包含编码M2-1和M2-2蛋白质的两个重叠ORF）。五种RSV基因，即NS 1、NS 2、SH、M2-2和G，可以单独地和在某些情况下组合地被“敲除”（缺失），而不消除病毒在细胞培养物中生长的能力。然而，在大多数情况下，增长的效率较低。在小鼠和黑猩猩中，发现单独或组合缺失NS 1和NS 2基因是高度减毒的。这些缺失是包含在减毒活疫苗中的候选者。M2-2缺失病毒表现出有利于转录而非复制的转变。这暗示该蛋白质是RNA合成中的调节因子。这种突变具有减少生长的新特性，同时增加而不是减少基因表达，因此可能会制造出“比自然更好”的疫苗。G基因敲除病毒表现出细胞特异性生长效率的差异，这意味着它正在使用一种替代受体，其分布是细胞特异性的。因此，这些发现对病毒组装和受体使用具有重要意义。在代表两个抗原亚组的人RSV之间构建完全存活的嵌合病毒，使得可以使用单个减毒骨架来表达每个亚组的主要抗原决定簇。还在人RSV和牛RSV之间构建了完全存活的嵌合体，牛RSV是一种具有宿主范围限制的病毒，使其在灵长类动物中高度减毒，因此代表了减毒RSV疫苗的新方法。作为制备“优于天然”的RSV疫苗的另一种方法，重组RSV被工程化以表达各种细胞因子和趋化因子，以增强免疫原性，并且在一些情况下，使病毒减毒。

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.