Metapneumovirus Biology and Vaccine Development

偏肺病毒生物学和疫苗开发

基本信息

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
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

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

项目摘要

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.

人类偏肺病毒 (HMPV) 于 2001 年首次报道，很快就被认为是全世界呼吸道疾病的重要病原体，特别是在儿科人群、免疫功能低下的个体和体弱的老年人中。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制，并开发 HMPV 减毒衍生物，用作鼻内儿科活疫苗。 HMPV 是一种有包膜病毒，其基因组是大约 13.3 kb 的单条负义链 RNA。我们之前开发了第一个完整的 HMPV 共有序列，代表两个遗传亚组 A 和 B。我们自己和其他人的分子研究表明，HMPV 编码 8 个 mRNA，这些 mRNA 被翻译成 9 个蛋白质（M2 mRNA 包含两个独立的重叠开放阅读框，编码两个不同的蛋白质，M2-1 和 M2-2)。与人类呼吸道合胞病毒（其更为人所知的近亲）类比，HMPV 蛋白为： 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； L，病毒聚合酶。我们开发了一种 HMPV 反向遗传系统，可以在细胞培养物中完全从克隆的 cDNA 中产生完整的感染性病毒。这提供了一种基因组工程方法以进行基础研究和设计疫苗。我们发现四种病毒基因可以单独删除或以各种组合删除，对病毒体外复制影响很小或没有影响，即：G、SH、M2-1 和 M2-2。对这些病毒在仓鼠和非洲绿猴中的减毒和免疫原性的评估表明，del-G 病毒（有或没有额外删除 SH 基因）和 del-M2-2 病毒是有前途的候选减毒活疫苗HMPV。通过将 HMPV 的 N 或 P 开放阅读框替换为密切相关的禽类偏肺病毒 (AMPV) C 亚型的对应物，生成了其他候选疫苗。由于自然宿主范围限制，AMPV 在灵长类动物中减弱，希望由于 AMPV N 或 P 基因，嵌合病毒将具有宿主范围限制。对仓鼠和非洲绿猴的评估表明情况确实如此，HMPV-AMPV-P 病毒尤其是一种有前途的候选疫苗。 del-M2-2和HMPVAMPV-P病毒的制剂已在适合人类使用的产品的条件下生产，并将被放大以制作临床试验材料，用于人类志愿者的安全性、减毒和免疫原性的I期研究。 P嵌合体是一个有吸引力的候选者，因为它结合了AMPV特征的体外生长改善和体内衰减。 del-M2病毒很有吸引力，因为M2-2似乎参与调节病毒RNA合成，并且其缺失会上调基因表达，从而导致抗原合成增加。针对缺乏 M2-1 蛋白的病毒而进行的细胞内病毒 mRNA 分析表明，它们含有长度约为 25 个核苷酸的多聚腺苷酸尾，而真核 mRNA 的特征长度为 150-250 个核苷酸。这已通过微型复制子系统得到证实。因此，HMPV M2-1 似乎在多聚腺苷酸尾部的合成或稳定性中发挥作用。此事正在进一步调查中。对受感染细胞的细胞内 HMPV RNA 进行 Northern 印迹分析表明，各种病毒基因在单顺反子 mRNA 与读通 mRNA 中的表达存在显着差异。微型复制子系统的研究表明，终止效率的差异似乎完全是由于基因末端（GE）信号的差异（特别是不涉及各种基因间和基因起始信号）。 M2 和 SH 基因的 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信号的序列比对表明它们通常仅存在一两个核苷酸的差异，这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低效信号的生物学意义，其中有效信号（在 M 基因中）和低效信号（在 SH 中）以各种组合进行交换。自然排列的任何变化都与体外噬菌斑大小的减小有关，尽管对病毒产量基本上没有影响。自然排列的任何变化也与仓鼠复制的减少有关。在任何 M 基因的 GE 信号效率低下的排列中，都观察到对仓鼠复制的最大影响。这种效应可能是由于下一个下游 F 基因的表达减少所致。这些研究首次证明，GE 信号效率的差异似乎赋予了各种病毒基因的最佳表达比例，从而导致体内复制不理想。在许多重组HMPV的制备过程中，对回收的RNA基因组进行共有核苷酸测序，提供了许多基因组位置上频繁出现的序列异质性的证据。这表明存在相当大的亚群含有突变。大多数突变发生在SH基因中。例如，对 40 个独立的重组 HMPV 制剂（野生型和各种衍生物）进行部分一致测序表明，其中 31 个制剂在 SH 基因中包含总共 41 个小插入实例，在其他地方总共包含 5 个小插入实例。在这 31 种制剂中，SH 中至少有一个插入片段改变了阅读框并产生截短的蛋白质。几乎所有这些插入都涉及将一个或多个A残基添加到四个或多个A残基的各种轨道上，最常见的位点是七个A残基的区域。还有两个核苷酸缺失实例和许多核苷酸取代点突变实例，主要发生在 SH 基因中。对源自这些制剂的分子克隆 cDNA 的分析证实，测序电泳图上的额外峰确实代表了包含各种变化的分子亚群。重组系统所基于的生物衍生病毒还包含相当大的突变亚群，其存在已通过生物克隆和核苷酸测序得到证实。通过用合成版本替换 SH 基因，突变亚群的出现大大减少，其中这些寡核苷酸片段通过沉默核苷酸变化而被消除。事实上，SH 对于体外复制来说是可有可无的，这可能是这种高频率恢复突变的一个因素。然而，这还不足以解释，因为 G 也是可有可无的，但积累的突变要少得多。此外，大多数观察到的突变发生在 SH 开放阅读框内，并且通过移码消除了 SH 的表达。因此，沉默SH的突变似乎优先积累，表明这在体外赋予了（适度的）生长优势（但在体内则不然，因为自然界中似乎不会发生SH沉默）。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，并主动消除突变热点，这些突变热点通常涉及序列中的 A 或 U 束，这对体外复制来说不是必需的。