Metapneumovirus Biology and Vaccine Development

偏肺病毒生物学和疫苗开发

• 批准号: 6985263
• 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/6985263
• 关键词: Cercopithecidae; Paramyxoviridae; Paramyxoviridae disease; attenuated microorganism; biotechnology; gene expression; genetic regulation; hamsters; host organism interaction; live vaccine; neutralizing antibody; pediatrics; protein structure function; recombinant virus; respiratory infections; tissue /cell culture; vaccine development; vaccine evaluation; viral vaccines; virus genetics; virus infection mechanism; virus protein; virus replication; western blottings

Human metapneumovirus (HMPV) was first identified in the Netherlands in 2001 and soon after was isolated in patients with respiratory tract disease throughout the world, particularly in the pediatric population. HMPV replicates inefficiently in cell culture, posing a challenge to research. The contribution of HMPV to human disease remains to be defined but is approximately similar to that of human parainfluenza virus type 3, and thus there is a need for an HMPV vaccine, especially for the pediatric population. An HMPV vaccine likely would be a live-attenuated strain that would be given intranasally, likely in combination with live-attenuated vaccines that are being developed against human respiratory syncytial virus (HRSV) and the human parainfluenza viruses (HPIVs). HMPV is an enveloped virus with a genome that is a single negative-sense strand of RNA, and is classified in the paramyxovirus family together with HRSV and the HPIVs. We recently described the first complete sequence of the HMPV genome, and prepared complete consensus sequences for viruses (CAN97-83 and CAN97-75) representing the two genetic subgroups of HMPV (A and B, respectively). The HMPV genome sequenced to date range in length from 13,280-13,335 nt. The genome contains 8 genes that are in the order 3?-N-P-M-F-M2-SH-G-L-5? and have open reading frames corresponding to 9 major proteins. By analogy to HRSV, which has been studied in much greater detail, 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 of unknown function; G, attachment glycoprotein; and L, viral polymerase. The HMPV proteins have only been deduced from the nucleotide sequence and had not been characterized directly with regard to their biochemistry or function. Compared to HRSV, HMPV lacks the non-structural NS1 and NS2 genes and has the F, M2, SH and G genes in the order F-M2-SH-G compared to SH-F-G-M2 for HRSV. The two HMPV subgroups share 81% nucleotide identity and 88% aggregate amino acid identity, similar to the respective values of 81% and 88% for the two HRSV subgroups. We developed a reverse genetic system for the CAN97-83 isolate, whereby changes can be introduced into the genome of infectious virus by recombinant DNA techniques. We designed a version of HMPV, rHMPV-GFP, in which the enhanced green fluorescent protein (GFP) was expressed from a transcription cassette placed 58 nt from the 3' end of the genome. The ability to monitor GFP expression in living cells greatly facilitated the initial recovery and characterization of this slow-growing virus. In addition, the ability to express a foreign gene from an engineered transcription cassette confirmed the identification of the HMPV transcription signals, and the ability to recover virus containing a foreign insert in this position indicated that the viral promoter is contained within the 3'-terminal 57 nt of the genome. The rHMPV-GFP virus was used to develop a more rapid and reliable assay for HMPV-neutralizing antibodies We also recovered a version of HMPV without the added GFP gene. This virus replicated in vitro as efficiently as biologically-derived HMPV (showing that we had made a correct virus), whereas the kinetics and final yield of rHMPV-GFP were reduced several-fold (showing that the addition of an extra gene was slightly inhibitory). Another version of HMPV, rHMPV+G1F23, was recovered that contained a second copy of the G gene and two extra copies of F in the promoter proximal position in the order G1-F2-F3. Thus, this recombinant genome would encode 11 mRNAs rather than eight and would be 17.3 kb in length, 30% longer than that of the natural virus. This rHMPV+G1F23 virus replicated in vitro with an efficiency that was only modestly reduced compared to rHMPV and was essentially the same as rHMPV-GFP. Thus, it should be feasible to construct an HMPV vaccine virus containing extra copies of the G and F putative protective antigen genes in order to increase gene dose or to provide representation of additional antigenic lineages or subgroups of HMPV. The ability to produce infectious HMPV from full-length cDNA provides a method to investigate the functions of individual HMPV proteins and to develop attenuating mutations for the purposes of constructing a live vaccine. As a first step, we engineered a HMPV to delete the SH or G genes individually or in combination. The del-SH, del-G, and del-SH/G deletion mutants were readily recovered and were found to replicate efficiently during multicycle growth in cell culture. Indeed, the del-G virus grew marginally better, and the del-SH virus unambigously better, than their wild-type parent, whereas the double deletion mutant replicated marginally less efficiently. Thus, the SH and G proteins are not essential for efficient growth in cell culture. The SH, G and F proteins were identified for the first time by immunoprecipitation using peptide-specific sera. This showed that the SH protein accumulates in a variety of forms that range in apparent electrophoretic mobility from 23-220 kDa, with the differences appearing to be due to glycosylation. The G protein also appeared to be heavily glycosylated. Apart from the absence of the deleted protein(s), the virions produced by the gene-deletion mutants were very similar by protein yield and gel electrophoresis protein profile to wild-type HMPV. This showed that neither SH nor G is essential for the efficient production of virus particles. However, subtle differences in yield and in sucrose sedimentation were noted that will be further investigated. When administered intranasally to hamsters, the del-SH virus replicated at least as efficiently as wild-type rHMPV. This indicates that SH is completely dispensable in vivo and that its deletion does not confer a significant attenuating effect, at least in this rodent model. The del-G and del-SH/G mutants also replicated in both the upper and lower respiratory tract, showing that HMPV containing F as the sole viral surface protein is competent for replication in vivo. However, both viruses were found to be strongly attenuated for replication in both the upper and lower respiratory tract (at least 600-fold and 40-fold reduction, respectively, of mean titer on day 3 post infection compared to wild-type rHMPV). The immunogenicity of the del-SH virus was comparable to that wild-type rHMPV, consistent with its high level of replication. Although the del-G and del-SH/G viruses were strongly attenuated, they also induced high titers of HMPV-neutralizing serum antibodies and conferred complete protection against replication of wild-type HMPV challenge virus in the lungs. Thus, the del-G and del-SH/G viruses represent promising vaccine candidates that will be prepared for clinical evaluation. Additional mutants were made involving the M2 gene, which encodes an mRNA with two overlapping ORFs that have the potential to encode two separate proteins M2-1 and M2-2. Expression of M2-1 was confirmed for the first time by immunoprecipitation with antiserum raised against HMPV, whereas expression of the M2-2 protein from recombinant HMPV was visualized by adding an epitope tag added to its carboxy-terminus. Recombinant HMPV were generated in which expression of M2-1 and M2-2 was silenced individually or together. This showed that neither protein is required for HMPV replication. These deletion viruses are being evaluated to characterize the effects of these deletions in vitro and in vivo and to determine the potential of these viruses as candidate vaccines.

人类元瘤病毒（HMPV）于2001年首次在荷兰发现，不久之后，在世界各地的呼吸道疾病患者中，特别是在儿科人群中分离出来。 HMPV在细胞培养中效率低下，对研究构成挑战。 HMPV对人类疾病的贡献仍有待定义，但与3型人类Parainfluenza病毒的贡献大致相似，因此需要HMPV疫苗，尤其是针对儿科人群。 HMPV疫苗可能是一种活体菌株，可以在鼻内给予，可能与针对人呼吸道合胞病毒（HRSV）和人类副parainfluenza病毒（HPIV）相结合的实时销售疫苗（HPIV）。 HMPV是一种带有基因组的包膜病毒，它是单个RNA的一个负义链，并将其分类在Paramyxovirus家族中，以及HRSV和HPIVS。我们最近描述了HMPV基因组的第一个完整序列，并为病毒（CAN97-83和CAN97-75）制备了完整的共有序列，该序列代表了HMPV的两个遗传亚组（A和B分别）。 HMPV基因组测序的日期长度为13,280-13,335 nt。基因组包含8个基因，该基因是3？-n-p-m-f-m2-sh-g-l-5？并具有与9个主要蛋白质相对应的开放式阅读框架。与HRSV相比，已经对HRSV进行了更详细的研究，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，功能未知的小疏水蛋白； G，附着糖蛋白；和L，病毒聚合酶。 HMPV蛋白仅从核苷酸序列推导出来，并且没有直接表征其生物化学或功能。与HRSV相比，HMPV缺乏非结构性NS1和NS2基因，并且与HRSV相比，F-M2-SH-G的速度为F，M2，SH和G基因。这两个HMPV亚组具有81％的核苷酸同一性和88％的聚集氨基酸身份，类似于两个HRSV亚组的相应值81％和88％。 我们为CAN97-83分离株开发了一个反向遗传系统，可以通过重组DNA技术将变化引入传染病的基因组中。我们设计了HMPV RHMPV-GFP的版本，其中增强的绿色荧光蛋白（GFP）是从基因组3'末端放置在58 nt的转录盒中表达的。监测活细胞中GFP表达的能力极大地促进了这种缓慢生长的病毒的初始恢复和表征。此外，从工程转录盒中表达外国基因的能力证实了HMPV转录信号的鉴定，并且在该位置恢复含有外插入的病毒的病毒的能力表明，病毒启动子包含基因组的3'-末端57 nt中。 RHMPV-GFP病毒用于开发更快，更可靠的HMPV中和抗体测定法 我们还恢复了没有添加GFP基因的HMPV版本。该病毒在体外复制，就像生物学上衍生的HMPV一样有效（表明我们已经形成了正确的病毒），而RHMPV-GFP的动力学和最终产率降低了几倍（表明添加了额外的基因略有抑制）。恢复了另一个版本的HMPV RHMPV+G1F23，该版本包含G基因的第二份副本，并在启动子中以G1-F2-F3的顺序近端位置中的F副本F副本。因此，该重组基因组将编码11个mRNA，而不是8个mRNA，长度为17.3 kb，比天然病毒长30％。这种RHMPV+G1F23病毒在体外复制，其效率仅与RHMPV相比仅适度降低，并且与RHMPV-GFP基本相同。因此，应构建包含G和F推定的保护性抗原基因的额外副本的HMPV疫苗病毒，以增加基因剂量或提供其他抗原性谱系或HMPV亚组的表示。 从全长cDNA产生感染性HMPV的能力提供了一种方法来研究单个HMPV蛋白的功能并开发衰减突变，以构建活疫苗。作为第一步，我们设计了HMPV，以单独或组合删除SH或G基因。 DEL-SH，DEL-G和DEL-SH/G缺失突变体很容易恢复，并被发现在细胞培养中多环生长期间有效地复制。的确，DEL-G病毒的增长略略有，并且与野生型母亲相比，DEL-SH病毒越来越好，而双层删除突变体复制的效率较小。因此，SH和G蛋白对于细胞培养的有效生长并不是必需的。首次使用肽特异性血清进行免疫沉淀，首次鉴定出SH，G和F蛋白。这表明SH蛋白以各种形式积累，这些形式在23-220 kDa的表观电泳迁移率中范围，差异似乎是由于糖基化引起的。 G蛋白似乎也被大量糖基化。除了缺乏已删除的蛋白质（S）外，基因 - 脱落突变产生的病毒体非常相似，蛋白质产量和凝胶电泳蛋白谱与野生型HMPV非常相似。这表明SH和G对病毒颗粒的有效产生都不是必不可少的。然而，注意到将进一步研究的产量和蔗糖沉积中的细微差异。 当对仓鼠内经麻时，DEL-SH病毒至少与野生型RHMPV一样有效。这表明SH在体内完全可分配，并且至少在此啮齿动物模型中，其缺失不会赋予显着的衰减效果。 DEL-G和DEL-SH/G突变体也在上呼吸道和下呼吸道中复制，表明含有F作为唯一病毒表面蛋白的HMPV具有在体内复制的能力。然而，发现两种病毒在上呼吸道和下呼吸道中都被强烈减弱以复制（与野生型RHMPV相比，感染后第3天，平均滴度至少降低了600倍和40倍。 DEL-SH病毒的免疫原性与野生型RHMPV相当，与其高水平的复制水平一致。尽管DEL-G和DEL-SH/G病毒严重减弱，但它们还诱导了HMPV中和血清抗体的高滴度，并赋予了完全保护肺中野生型HMPV挑战病毒的完全保护。因此，DEL-G和DEL-SH/G病毒代表了有希望的疫苗候选物，这些疫苗将为临床评估做好准备。 涉及M2基因的其他突变体，该突变体编码具有两个重叠的ORF的mRNA，它们具有编码两个单独的蛋白质M2-1和M2-2的潜力。首次通过对HMPV提出的抗血清的免疫沉淀来证实M2-1的表达，而通过在其羧基末端添加一个表位标签来可视化M2-2蛋白从重组HMPV中的表达。生成重组HMPV，其中M2-1和M2-2的表达单独或一起沉默。这表明HMPV复制不需要蛋白质。正在评估这些缺失病毒，以表征这些缺失在体外和体内的影响，并确定这些病毒作为候选疫苗的潜力。