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.

人类偏肺病毒 (HMPV) 于 2001 年在荷兰首次被发现，不久后在世界各地的呼吸道疾病患者中分离出来，特别是在儿科人群中。 HMPV 在细胞培养中复制效率低下，给研究带来了挑战。 HMPV 对人类疾病的贡献仍有待确定，但与人副流感病毒 3 型大致相似，因此需要一种 HMPV 疫苗，特别是针对儿科人群。 HMPV 疫苗可能是一种鼻内注射的减毒活毒株，可能与正在开发的针对人类呼吸道合胞病毒 (HRSV) 和人类副流感病毒 (HPIV) 的减毒活疫苗结合使用。 HMPV 是一种有包膜病毒，其基因组为单条负义 RNA 链，与 HRSV 和 HPIV 一起属于副粘病毒科。我们最近描述了 HMPV 基因组的第一个完整序列，并准备了代表 HMPV 两个遗传亚群（分别为 A 和 B）的病毒（CAN97-83 和 CAN97-75）的完整共有序列。迄今为止，HMPV 基因组测序长度范围为 13,280-13,335 nt。基因组包含8个基因，其顺序为3？-N-P-M-F-M2-SH-G-L-5？并具有对应9种主要蛋白质的开放阅读框。与已进行更详细研究的 HRSV 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，功能未知的小疏水蛋白； G，附着糖蛋白； L，病毒聚合酶。 HMPV 蛋白仅从核苷酸序列中推导，尚未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，并且具有F、M2、SH和G基因，与HRSV的SH-F-G-M2相比，顺序为F-M2-SH-G。两个 HMPV 亚群具有 81% 的核苷酸同一性和 88% 的总氨基酸同一性，与两个 HRSV 亚群各自的 81% 和 88% 的值相似。 我们为 CAN97-83 分离株开发了一个反向遗传系统，通过重组 DNA 技术可以将变化引入传染性病毒的基因组中。我们设计了 HMPV 的一个版本，rHMPV-GFP，其中增强型绿色荧光蛋白 (GFP) 从位于距离基因组 3' 端 58 nt 处的转录盒表达。监测活细胞中 GFP 表达的能力极大地促进了这种生长缓慢的病毒的初步恢复和表征。此外，从工程转录盒表达外源基因的能力证实了HMPV转录信号的识别，并且回收在该位置含有外源插入片段的病毒的能力表明病毒启动子包含在基因组的3'端57nt内。 rHMPV-GFP 病毒用于开发更快速、更可靠的 HMPV 中和抗体检测方法 我们还恢复了未添加 GFP 基因的 HMPV 版本。这种病毒在体外的复制效率与生物来源的 HMPV 一样有效（表明我们制造了正确的病毒），而 rHMPV-GFP 的动力学和最终产量降低了数倍（表明添加额外基因具有轻微的抑制作用）。 HMPV 的另一个版本，rHMPV+G1F23，被发现含有第二个 G 基因拷贝和两个额外的 F 拷贝，位于启动子近端位置，顺序为 G1-F2-F3。因此，这种重组基因组将编码 11 个 mRNA，而不是 8 个，长度为 17.3 kb，比天然病毒长 30%。这种 rHMPV+G1F23 病毒在体外复制的效率与 rHMPV 相比仅略有降低，并且与 rHMPV-GFP 基本相同。因此，构建含有G和F推定保护性抗原基因的额外拷贝的HMPV疫苗病毒应该是可行的，以增加基因剂量或提供HMPV的额外抗原谱系或亚群的代表。 从全长 cDNA 产生感染性 HMPV 的能力提供了一种研究单个 HMPV 蛋白功能并开发减毒突变以构建活疫苗的方法。第一步，我们设计了 HMPV 来单独或组合删除 SH 或 G 基因。 del-SH、del-G 和 del-SH/G 缺失突变体很容易恢复，并且被发现在细胞培养物的多周期生长过程中有效复制。事实上，与野生型亲本相比，del-G 病毒和 del-SH 病毒的生长速度稍好，而双缺失突变体的复制效率稍低。因此，SH 和 G 蛋白对于细胞培养物的有效生长并不是必需的。首次使用肽特异性血清通过免疫沉淀鉴定了 SH、G 和 F 蛋白。这表明 SH 蛋白以多种形式积累，表观电泳迁移率范围为 23-220 kDa，差异似乎是由于糖基化造成的。 G 蛋白似乎也被严重糖基化。除了不存在缺失的蛋白质外，基因缺失突变体产生的病毒体在蛋白质产量和凝胶电泳蛋白质谱上与野生型 HMPV 非常相似。这表明SH和G对于病毒颗粒的有效生产都不是必需的。然而，我们注意到产量和蔗糖沉降方面存在细微差异，这将进一步研究。 当对仓鼠进行鼻内给药时，del-SH 病毒的复制效率至少与野生型 rHMPV 相同。这表明SH在体内是完全可有可无的，并且它的缺失不会带来显着的减毒作用，至少在该啮齿动物模型中是这样。 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能够在体内复制。然而，发现这两种病毒在上呼吸道和下呼吸道中的复制均被强烈减弱（与野生型 rHMPV 相比，感染后第 3 天的平均滴度分别降低了至少 600 倍和 40 倍）。 del-SH 病毒的免疫原性与野生型 rHMPV 相当，与其高水平的复制一致。尽管del-G和del-SH/G病毒被强烈减毒，但它们也诱导高滴度的HMPV中和血清抗体，并提供针对野生型HMPV攻击病毒在肺部复制的完全保护。因此，del-G 和 del-SH/G 病毒代表了有前途的候选疫苗，将为临床评估做好准备。 还制作了涉及 M2 基因的其他突变体，该基因编码具有两个重叠 ORF 的 mRNA，这两个 ORF 有可能编码两种独立的蛋白质 M2-1 和 M2-2。 M2-1 的表达首次通过使用 HMPV 抗血清进行免疫沉淀得到证实，而重组 HMPV 的 M2-2 蛋白的表达则通过在其羧基末端添加表位标签来可视化。产生了重组HMPV，其中M2-1和M2-2的表达被单独或一起沉默。这表明这两种蛋白质都不是 HMPV 复制所必需的。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。