REPLICATION,VIRULENCE & IMMUNOGENICITY IN RECOMBINANT RESPIRATORY SYNCYTIAL V

复制、毒力

基本信息

批准号：
6098927
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
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

Human respiratory syncytial virus (RSV) is an important agent of pediatric respiratory tract disease worldwide and is responsible for a huge burden of morbidity and significant mortality. There is no licensed vaccine. Obstacles to vaccine development include the poor growth of the virus in cell culture, the semi- permissive nature of the infection in convenient experimental animals, and the difficulty of achieving an appropriate balance between immunogenicity (which depends on reasonable levels of virus replication) and attenuation (which depends on reduced levels of virus replication). We recently developed a method for producing 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. RSV encodes ten mRNAs encoding eleven proteins (the M2 mRNA contains two overlapping ORFs encoding two separate proteins, M2- 1 and M2-2). We investigated whether individual RSV genes could be ?knocked out? without ablating the ability of the virus to grow in cell culture. To date, four RSV genes can be individually knocked out without loss of infectivity, namely NS1, NS2, SH and G. This was done in two cases (NS1 and NS2) by introducing stop codons into the translational open reading frame (ORF), and in all four cases also was done by completely deleting the gene, such that the genome is shorter and encodes one fewer mRNA. The NS1 knockout virus grows somewhat less well than the parent in cell culture, and its analysis is in progress. The NS2 knockout virus grows somewhat more slowly than does the wild type parent in a single step growth curve and forms pinpoint plaques. The version of the NS2 knockout virus in which stop codons had been introduced exhibits reversion to an NS2+ phenotype by reversion of the stop codons to sense. This was not observed with the gene deletion, illustrating its stability against reversion. The SH knockout virus forms plaques that were larger than wild type, and it grows as well or slightly better than wild type in cell culture in a single step growth curve. In mice, the SH knockout grew as well as wild type in the lower respiratory tract but was significantly restricted in the upper respiratory tract. It was equivalent to wild type with regard to the ability to induce RSV-specific serum antibodies and protection against challenge virus replication. In chimpanzees, the only experimental animal which approaches humans with regard to permissiveness for RSV infection and disease, the SH knockout virus was only slightly attenuated. Interestingly, however, it was associated with significantly reduced disease, i.e. rhinorrhea, which would be an ideal feature for a live-attenuated vaccine virus. The G knockout virus grows less well than its wild type parent in cell culture. Nonetheless, it does form plaques and can be propagated. The fact that its attachment activity is not essential for in vitro growth implies that one of the other surface glycoproteins can serve an auxiliary attachment function. We showed that RSV can accept the insertion of sequence encoding an additional, foreign mRNA. Chimeric genes were constructed in which the ORF encoding chloramphenicol acetyl transferase (CAT), green fluorescent protein (GFP) or luciferase (LUC) was engineered to be flanked by the RSV gene-start (GS) and gene-end (GE) transcription signals. Each transcription cassette was inserted into the leader-NS1, SH-G or F-M2 junctions in the antigenomic cDNA. Infectious recombinant viruses which each contained one foreign insert were recovered. Each foreign gene was expressed as an additional mRNA, and each protein was expressed to a level comparable to that of the RSV proteins. The presence of the short CAT or GFP genes (~750 bp) resulted in a small decrease in plaque size and a 20-fold reduction in virus yield. In contrast, the presence of the longer (~1750 bp) LUC gene reduced virus growth to the point that the virus could be propagated but not significantly amplified. This shows that the insertion of an additional gene can be a method of attenuation that appears to be length-dependent. We hypothesize that this restriction occurs at the level of packaging, which is currently being investigated. Insert stability was investigated for the CAT gene and found to be remarkably high. Deletion of foreign sequence was not observed, and the accumulation of point mutations was not rapid, such that after seven passages each gene had on average a single nucleotide substitution which, in the 25 plaques examined, did not affect protein expression or enzymatic activity. The gene deletions and insertions described above each resulted in a change in genome length and in the number of expressed mRNAs. We are examining transcription by these viruses to determine what effects, in any, these changes have on sequential transcription. We also are examining the effects of introducing longer-than-natural intergenic regions as possible attenuating mutations and to shed light on polymerase activities at the gene junctions. We previously showed that one of the attenuating mutations of the biologically-derived cptsRSV vaccine virus is a single nucleotide change (A9 to G, negative-sense) in the GS signal of the M2 gene. This mutation has now been inserted into the GS signal of the NS1, or NS2, or both genes, and evaluation of the resulting mutants is in progress. We previously showed that the naturally-occurring GE signals for these two genes are only 60% as efficient in directing transcriptional termination as are the signals for the other eight RSV genes. This is associated with an increased frequency of synthesis of the following readthrough mRNAs: NS1-NS2, NS2-N, NS1-NS2-N. Since internal ORFs of eukaryotic mRNAs are not efficiently translated, this would have the effect of reducing the amount of mRNA capable of synthesizing the NS2 and N proteins.

人呼吸道合胞病毒（RSV）是全球小儿呼吸道疾病的重要因素负责巨大的发病率和重大负担死亡。没有持牌疫苗。疫苗的障碍发展包括病毒在细胞培养中的不良生长，方便感染的半允许性质实验动物，以及实现适当的难度免疫原性之间的平衡（这取决于合理病毒复制水平）和衰减（取决于病毒复制水平降低）。我们最近开发了一个通过细胞内共表达产生RSV的方法编码完整的RSV复制中间RNA的cDNA （抗原组）和N，P，L和M2-1蛋白，它们一起构成一个完全合成RNA合成的核包膜。这为基本分子和发病机理研究以及一种微调的方法候选疫苗病毒的衰减。 RSV编码十个mRNA 编码11种蛋白（M2 mRNA包含两个重叠 ORF编码两个单独的蛋白质M2-1和M2-2）。我们调查了是否可以敲定单个RSV基因？不消除病毒在细胞培养中生长的能力。到日期，可以单独淘汰四个RSV基因而不会损失感染性，即NS1，NS2，SH和G。案例（NS1和NS2）通过将停止密码子引入翻译开放式阅读框（ORF），在所有四种情况下也是通过完全删除基因来完成的，以使基因组是较短和编码的mRNA少。 NS1敲除病毒在细胞培养中的生长要比父母少一些，它分析正在进行中。 NS2敲除病毒生长有些比单一步骤生长的野生型父母要慢曲线并形成精确斑块。 NS2的版本淘汰病毒在其中引入了停止密码子通过将终止密码子归还为NS2+表型感觉。基因缺失没有观察到这一点，说明了它的稳定性反向回归。 SH淘汰病毒形成斑块比野生型大，它也会增长或略微在单步生长曲线中，在细胞培养中比野生型更好。在小鼠，sh淘汰赛正在生长，较低的野生型呼吸道，但在上部受到显着限制呼吸道。相当于野生类型能够诱导RSV特异性血清抗体和保护反对挑战病毒复制。在黑猩猩，唯一实验动物，接近人类 RSV感染和疾病的允许性，SH淘汰赛病毒仅略有减弱。有趣的是，这是与疾病显着降低有关，即鼻炎，这是对于活触及的疫苗病毒，将是理想的特征。 G 基因敲除病毒的生长不如细胞中的野生型父母好文化。尽管如此，它确实会形成斑块，并且可以传播。它的依恋活动对于体外不是必不可少的事实生长意味着另一个表面糖蛋白可以使用辅助附件功能。我们表明RSV可以接受编码额外的外源mRNA的序列的插入。构建了ORF编码的嵌合基因氯霉素乙酰基转移酶（CAT），绿色荧光蛋白质（GFP）或荧光素酶（LUC）被设计为侧翼 RSV基因启动（GS）和基因端（GE）转录信号。每个转录盒插入了Leader-NS1，SH-G 或抗原组cDNA中的F-M2连接。感染力每个包含一个外插入物的重组病毒是恢复。每个外国基因被表示为附加 mRNA，每种蛋白质的水平与 RSV蛋白。短猫或GFP的存在基因（〜750 bp）导致斑块尺寸较小，A 病毒产量降低20倍。相反，存在更长（〜1750 bp）Luc基因降低了病毒的生长至该病毒可以被传播，但不能显着扩增。这表明插入附加基因可以是衰减似乎是长度依赖的。我们假设这种限制发生在包装级别，即目前正在调查。研究了插入稳定性的猫基因，发现非常高。删除外国未观察到序列，并且点的积累突变不是快速的，因此每个基因七个段落后平均有一个核苷酸取代检查的斑块不影响蛋白质表达或酶促活动。上面描述的基因缺失和插入导致基因组长度的变化和数量表示mRNA。我们正在检查这些病毒的转录为了确定这些变化对顺序有什么影响转录。我们还在研究引入的效果尽可能减弱的基因间区域长期衰减突变并阐明基因的聚合酶活性连接。我们以前表明其中一个衰减生物衍生的CPTSRSV疫苗病毒的突变是一种 GS信号中的单核苷酸变化（A9至G，负态） M2基因。现在将该突变插入了GS NS1或NS2或两个基因的信号，以及评估产生的突变体正在进行中。我们以前证明了这两个基因的自然出现的GE信号仅为60％有效地指导转录终止以及信号其他八个RSV基因。这与增加有关以下读取mRNA的合成频率： NS1-NS2，NS2-N，NS1-NS2-N。由于真核生物的内部ORF mRNA没有有效地翻译，这将具有减少能够合成NS2和的mRNA量 N蛋白质。