Metapneumovirus Biology and Vaccine Development

偏肺病毒生物学和疫苗开发

• 批准号: 7592141
• 负责人: PETER LEON COLLINS
• 金额: $ 69.59万
• 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/7592141
• 关键词: Antigens; Attenuated; Attenuated Live Virus Vaccine; Base Sequence; Biological; Biology; Cells; Cercopithecus pygerythrus; Characteristics; Childhood; Chimera organism; Chimeric Proteins; Clinical Trials; Cloning; Collaborations; Computer Systems Development; Condition; Consensus; Consensus Sequence; Cultured Cells; Dendritic Cells; Development; Epithelial Cells; Evaluation; Event; Fibrinogen; Frail Elderly; Frequencies; Gene Expression; Genes; Genetic; Genome; Genome engineering; Glycoproteins; Goals; Growth; Hamsters; Heterogeneity; Hot Spot; Human; Human Metapneumovirus; Human Volunteers; Human respiratory syncytial virus; Immune response; Immunocompromised Host; In Vitro; Individual; Industry; Length; Life; M2 protein; Messenger RNA; Metapneumovirus; Methods; Molecular; Molecular Biology; Monitor; Mutation; Nature; Northern Blotting; Nucleoproteins; Nucleotides; Numbers; Oligonucleotides; Open Reading Frames; Pathogenesis; Phase I Clinical Trials; Phosphoproteins; Point Mutation; Polymerase; Population; Positioning Attribute; Preparation; Primates; Proteins; RNA; RNA chemical synthesis; Range; Reading Frames; Reagent; Recombinant DNA; Recombinants; Relative (related person); Replicon; Reporting; Research Design; Respiratory Tract Diseases; Role; Safety; Sequence Alignment; Sequence Analysis; Signal Transduction; Site; Subgroup; System; Tail; Translating; Turkey rhinotracheitis virus; Vaccines; Viral; Viral Genes; Viral Proteins; Virus; Virus Diseases; attenuation; base; genetic manipulation; immunogenicity; improved; in vivo; macrophage; mutant; positional cloning; pre-clinical; preparation 31; recombinant virus; research clinical testing; response; size; vaccine development; viral RNA; virus host interaction

Human metapneumovirus (HMPV) was first reported in 2001 and has quickly come to be recognized as a significant agent of respiratory tract disease worldwide, especially in the pediatric population, in immunocompromised individuals, and in the frail elderly. We are using recombinant DNA methods to characterize viral molecular biology and pathogenesis and to develop attenuated derivatives of HMPV for use as a live intranasal pediatric vaccine. HMPV is an enveloped virus with a genome that is a single negative-sense strand of RNA of approximately 13.3 kb. We previously developed the first complete HMPV consensus sequences representng the two genetic subgroups, A and B. Molecular studies by ourselves and others showed that HMPV encodes eight mRNAs that are translated into nine proteins (the M2 mRNA contains two separate overlapping open reading frames encoding two distinct proteins, M2-1 and M2-2). By analogy to human respiratory syncytial virus, its better-known relative, the HMPV proteins are: N, nucleoprotein; P, phosphoprotein; M, matrix protein; F, fusion protein; M2-1, RNA synthesis factor; M2-2, RNA synthesis factor; SH, small hydrophobic protein; G, attachment glycoprotein; and L, viral polymerase. We developed a reverse genetic system for HMPV whereby complete infectious virus can be generated in cell culture entirely from cloned cDNAs. This provides a method for engineering the genome in pursuit of basic studies and for designing vaccines. We found that four viral genes could be deleted individually and in various combinations with little or no effect on viral replication in vitro, namely: G, SH, M2-1 and M2-2. Evaluation of the attenuation and immunogenicity of these viruses in hamsters and African green monkeys indicated that the del-G virus (with or without the additional deletion of the SH gene) and del-M2-2 virus are promising candidates to be live attenuated vaccines against HMPV. Additional vaccine candidates were generated by replacing the N or P open reading frame of HMPV with its counterpart from the closely related avian metapneumovirus (AMPV) subgroup C. AMPV is attenuated in primates due to a natural host range restriction, and it was hoped that the chimeric viruses would have a host range restriction due to the AMPV N or P gene. Evaluation in hamsters and African green monkeys showed that this indeed was the case, and the HMPV-AMPV-P virus in particular is a promising vaccine candidate. Preparations of the del-M2-2, and HMPVAMPV-P viruses have been produced under conditions appropriate for products for human use and will be amplified to make clinical trial material for phase I studies of safety, attenuation and immunogenicity in human volunteers. The P chimera is an attractive candidate because it combines improved growth in vitro that is characteristic of AMPV with attenuation in vivo. The del-M2 virus is attractive because M2-2 appears to be involved in regulating viral RNA synthesis, and its deletion up-regulates gene expression resulting in increased antigen synthesis. Analysis of intracellular viral mRNAs made in response to virus lacking the M2-1 protein showed that they contained polyA tails that were approximately 25 nucleotides in length compared to the 150-250-nucleotide length that is characteristic of eukaryotic mRNAs. This was confirmed with a mini-replicon system. Thus, HMPV M2-1 appears to have a role in the synthesis or stability of the polyA tail. This is being further investigated. Northern blot analysis of intracellular HMPV RNA from infected cells showed that the various viral genes differ considerably with regard their representation in monocistronc mRNA versus readthough mRNAs. Studies with a mini-replicon system showed that differences in the efficiency of termination appeared to be due completely to differences in the gene-end (GE) signal (and in particular does not involve the various intergenic and gene-start signals). The GE signals of the M2 and SH genes are particularly inefficient, resulting in very low levels of expression of the respective downstream SH and G genes. Alignment of the sequences of the various GE signals indicated that they usually differ by only one or two nucleotides, indicating that these differences presumably are the basis for the functional differences. We evaluated the biological significance of these inefficient signals by making recombinant viruses in which an efficient signal (in the M gene) and an inefficient signal (in SH) were swapped in various combinations. Any change from the natural arrangement was associated with a decrease in plaque size in vitro, although there was essentially no effect on virus yield. Any change from the natural arrangement also was associated with decreased replication in hamsters. The greatest effect on replication in hamsters was observed with any arrangement in which the GE signal of the M gene was inefficient. This effect presumably was due to reduced expression of the next-downstream F gene. These studies provided the first demonstration that differences in the efficiency of the GE signals appear to confer an optimal ratio of expression of the various viral genes, such that changes result in suboptimal replication in vivo. During the preparation of a number of recombinant HMPVs, consensus nucleotide sequencing of the recovered RNA genomes provided evidence of frequent sequence heterogeneity at a number of genome positions. This was suggestive of sizable subpopulations containing mutations. Most of the mutations occurred in the SH gene. For example, partial consensus sequencing of 40 independent preparations of recombinant HMPV (wild type and various derivatives) showed that 31 of these preparations contained a total of 41 instances of small insertions in the SH gene and a total of five small insertions elsewhere. In each of these 31 preparations, there was at least one insert in SH that changed the reading frame and would yield a truncated protein. Nearly all of these insertions involved adding one or more A residues to various tracks of four or more A residues, with the most frequent site being a tract of seven A residues. There also were two instances of nucleotide deletion and numerous instances of nucleotide substitution point mutations, mostly in the SH gene. Analysis of molecularly cloned cDNAs derived from these preparations confirmed that the additional peaks on the sequencing electropherograms indeed represented subpopulations of molecules containing the various changes. The biologically derived virus on which the recombinant system is based also contained sizeable mutant subpopulations, whose presence was confirmed by biological cloning and nucleotide sequencing. The occurrence of mutant subpopulations was greatly reduced by substitution of the SH gene with a synthetic version in which these oligonucleotide tracts were eliminated by silent nucleotide changes. The fact that SH is dispensable for replication in vitro likely in one factor in this high frequency of recovered mutations. However, this is not sufficient explanation, since G also is dispensable but accumulated far fewer mutations. In addition, most of the observed mutations occurred in the SH open reading frame and were such that they ablated expression of SH by frame-shifts. Thus, there appeared to be a preferential accumulation of mutations silencing SH, suggesting that this conferred a (modest) growth advantage in vitro (but not in vivo, since silencing of SH does not seem to occur in nature). In any event, these results indicate the need to carefully monitor vaccine virus by sequence analysis and to proactively remove mutational hot spots, which usually involve A or U tracts in sequence that is non-essential for replication in vitro.

人类元病毒（HMPV）于2001年首次报道，并迅速被公认为是全球呼吸道疾病的重要药物，尤其是在儿科人群中，在免疫功能低下的个体中，以及在脆弱的老人中。我们正在使用重组DNA方法来表征病毒分子生物学和发病机理，并开发HMPV的衰减衍生物，以用作生命的鼻内小儿小儿疫苗。 HMPV是一种带有基因组的包膜病毒，它是一条大约13.3 kb的RNA的单个负义链。我们以前开发了第一个完整的HMPV共识序列，代表了我们自己的两个遗传亚组，A和B的分子研究，而其他人则表明，HMPV编码八个mRNA，这些mRNA被翻译成九个蛋白质（M2 mRNA包含两个独立的重叠的开放式读取框架，编码两个不同的蛋白质，M2-1和M2和M2）。通过类似于人类呼吸道合胞病毒，其知名亲戚的HMPV蛋白是：N，核蛋白； P，磷蛋白； M，基质蛋白； F，融合蛋白； M2-1，RNA合成因子； M2-2，RNA合成因子； SH，小疏水蛋白； G，附着糖蛋白；和L，病毒聚合酶。 我们开发了一种用于HMPV的反向遗传系统，从而完全通过克隆的cDNA在细胞培养中产生完全感染性病毒。这提供了一种在追求基础研究和设计疫苗的方法来设计基因组的方法。我们发现，可以单独删除四个病毒基因，并以各种组合删除，对体外病毒复制的影响很小或没有影响：G，SH，M2-1和M2-2。对仓鼠和非洲绿猴中这些病毒的衰减和免疫原性的评估表明，DEL-G病毒（有或没有其他SH基因的额外缺失）和DEL-M2-2病毒是有希望的候选者，可以将其疫苗减弱。 通过替换与密切相关的禽类元中病毒（AMPV）子组的对应物替换N或P开放式阅读框来产生其他候选疫苗C. AMPV在自然宿主范围内会在灵长类动物中衰减，并且希望该嵌合病毒能够造成宿主范围限制Ampv n或P genee genee genee。对仓鼠和非洲绿猴的评估表明确实是这种情况，而HMPV-AMPV-P病毒尤其是有希望的疫苗候选者。 DEL-M2-2和HMPVAMPV-P病毒的制剂已在适合人类使用的条件下产生，并将被放大，以制造人类志愿者的安全性，衰减和免疫原性的I期研究临床试验材料。 P嵌合体是一个有吸引力的候选者，因为它结合了体外的改善生长，这是AMPV的特征和体内衰减。 DEL-M2病毒具有吸引力，因为M2-2似乎参与了调节病毒RNA合成，其缺失上调基因表达，从而导致抗原合成增加。 对缺乏M2-1蛋白的病毒作出的细胞内病毒mRNA的分析表明，与150-250-核苷酸长度相比，它们的长度约为25个核苷酸，而真核mRNA的特征。这是通过迷你修复系统确认的。因此，HMPV M2-1似乎在Polya尾巴的合成或稳定性中起作用。这正在进一步研究。 来自感染细胞的细胞内HMPV RNA的北印迹分析表明，各种病毒基因在单科传播mRNA中的表示与读取mRNA时的表示相差很大。对小修复系统的研究表明，终止效率的差异似乎完全是由于基因末端（GE）信号的差异（尤其不涉及各种基因间和基因启动信号）。 M2和SHES的GE信号效率特别低，导致相应下游SH和G基因的表达水平非常低。各种GE信号的序列的比对表明它们通常仅通过一个或两个核苷酸差异，表明这些差异可能是功能差异的基础。 我们通过制作重组病毒来评估这些无效信号的生物学意义，其中有效信号（在M基因中）和效率低下的信号（在SH中）被以各种组合交换。自然排列的任何变化都与体外斑块大小的减少有关，尽管基本上对病毒产量没有影响。自然布置的任何变化也与仓鼠复制减少有关。观察到仓鼠复制的最大影响是通过任何排列，其中M基因的GE信号效率低下。这种效应大概是由于隔壁f基因的表达降低所致。这些研究提供了第一次证明，GE信号效率的差异似乎赋予了各种病毒基因表达的最佳比例，因此变化会导致体内次优复制。 在制备许多重组HMPV时，回收的RNA基因组的共有核苷酸测序为许多基因组位置提供了频繁序列异质性的证据。这暗示着含有突变的大量亚群。大多数突变发生在SH基因中。例如，重组HMPV（野生型和各种衍生物）的40个独立制剂的部分共识测序表明，其中31种制剂包含SH基因中的41个小插入实例，在其他地方共有5个小插入。在这31种制剂中的每一个中，SH中至少有一个插入物可以改变阅读框并产生截短的蛋白质。几乎所有这些插入都涉及在四个或多个A残基的各种轨道中添加一个或多个A残基，其中最常见的位点是七个A残基的区域。还有两个核苷酸缺失的实例和许多核苷酸取代点突变的实例，主要是在SH基因中。对这些制剂的分子克隆cDNA的分析证实，测序电图上的附加峰确实代表了包含各种变化的分子的亚群。重组系统所基于的生物学衍生的病毒还包含较大的突变体亚群，其存在通过生物克隆和核苷酸测序证实。 通过替代SH基因的合成版本，突变亚群的发生大大降低了，其中这些寡核苷酸束被静音核苷酸变化消除。 SH可以在体外复制可分配的事实，在这种恢复突变的高频中可能是一个因素。但是，这不是足够的解释，因为g也可分配，但积累的突变却少得多。此外，大多数观察到的突变发生在SH开放式阅读框架中，因此它们通过框架移动烧杯表达。因此，似乎突变沉默的SH似乎优先积累，这表明这赋予了体外（但不是体内）的（适度）生长优势（因为SH的沉默似乎在自然界中并不是发生的）。无论如何，这些结果表明需要通过序列分析仔细监测疫苗病毒，并主动去除突变热点，这通常涉及序列的A或U段，这是不必要在体外复制的。