Metapneumovirus Biology and Vaccine Development

偏肺病毒生物学和疫苗开发

• 批准号: 7192840
• 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/7192840
• 关键词: Cercopithecidae; Paramyxoviridae; Paramyxoviridae disease; Pneumovirus; attenuated microorganism; biotechnology; gene expression; genetic regulation; hamsters; host organism interaction; live vaccine; neutralizing antibody; pediatrics; protein structure function; recombinant virus; respiratory infections; site directed mutagenesis; 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 reported 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 it appears to account for approximately 5 to 15% of pediatric hospitalizations due to respiratory tract disease. We are using reverse genetic methods to develop attenuated strains of HMPV for use as a live intranasal pediatric vaccine, one that would be given in combination with live vaccines currently being developed by LID/NIAID 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. It 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 genomes 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 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 complete infectious virus can be generated entirely from cloned cDNAs transfected into cultured cells. This provides a method for introducing predetermined changes into infectious HMPV for the purpose of basic molecular genetic studies and for designing vaccines. In one study, 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. This helped define the viral cis-acting signals necessary to transcribe a gene into mRNA. This virus is being used to monitor viral infection directly in living cells. In addition, we used the HMPV-GFP virus to develop a more rapid and reliable assay for detecting HMPV-neutralizing antibodies. In another study, HMPV was engineered to delete the SH and G genes in their entirely individually and in combination. The del-SH, del-G, and del-SH/G deletion mutants were readily recovered and were found to replicate in vitro with an efficiency that was approximately equivalent to that of wild type virus. This showed that 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. 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 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. It is feasible to consider an HMPV vaccine virus lacking one or both of these surface proteins because other ongoing work indicates that F is the major neutralization and protective antigen whereas, somewhat surprisingly, SH and G do not appear to be significant neutralization and protective antigens. 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 both ORFs was confirmed. Recombinant HMPVs were generated in which expression of M2-1 and M2-2 was silenced individually or together (del-M2-1, del-M2-2, and del-M2[1+2]). Each mutant virus directed efficient multi-cycle growth in Vero cells, showing that neither protein is required for HMPV replication. The del-M2-2 virus exhibited a 3- to 9-fold increase in the accumulation of mRNA normalized to the genome template, suggesting that M2-2 has a role in regulating RNA synthesis. Replication and immunogenicity were tested in the hamster model. Animals infected intranasally with del-M2-1 or del-M2(1+2) did not have recoverable virus in the lungs or nasal turbinates on days 3 or 5 post-infection and did not develop HMPV-neutralizing serum antibodies or resistance to HMPV challenge. Thus, M2-1 appears to be essential for significant virus replication in vivo. In animals infected with del-M2-2, virus was recovered from only 1 of 12 animals, and only in the nasal turbinates on a single day. However, these animals developed a high titer of HMPV-neutralizing serum antibodies and were highly protected against challenge with wild-type HMPV. The del-SH, del-G and del-M2-2 viruses were analyzed further in African green monkeys, an experimental animal that is anatomically and phylogenetically more closely related to humans. Del-SH replicated to a level comparable to that of the parental virus, whereas del-G was slightly attenuated in the upper respiratory tract and more than 1000-fold attenuated in the lower respiratory tract. The del-M2-2 virus was 160-fold attenuated in the upper respiratory tract, and 4000-fold attenuated in the lower respiratory tract. This confirmed that SH, G and M2-2 are nonessential accessory proteins. Induction of neutralizing antibodies by each mutant virus was efficient and comparable to wild-type HMPV. Upon challenge with wild-type HMPV, each of the three deletion mutants conferred essentially complete protection of the lower respiratory tract and complete protection (del-SH) or greater than 1000-fold reduction of challenge virus replication (del-G, del-M2-2) in the upper respiratory tract. Thus, at least two promising HMPV vaccine candidates with attenuation based on independent mutations involving the G or M2 genes are available and will be developed for phase 1 clinical testing in humans.

2001年在荷兰首次在荷兰报道了人元病毒（HMPV），不久之后，全世界的呼吸道疾病患者，尤其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，但由于呼吸道疾病，大约占小儿住院的5％至15％。我们正在使用反向遗传学方法来开发HMPV菌株的衰减菌株，以用作实时鼻内小儿疫苗，该疫苗将与目前由针对人呼吸道疾病综合病毒（HRSV）和HumanParlafluenza Viruse（Hpivs（Hpivs）相结合的实时疫苗（HPIVS）与目前开发的活疫苗相结合。 HMPV是一种包膜病毒，其基因组是RNA的单个负义链。它与HRSV和HPIV一起在Paramyxovirus家族中分类。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基因组对日期的测序范围为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。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，从而完全由转染到培养细胞的克隆的CDNA中产生完全传染病。这提供了一种将预定的变化引入传染性HMPV的方法，以进行基本的分子遗传研究和设计疫苗。 在一项研究中，我们设计了HMPV的RHMPV-GFP版本，其中增强的绿色荧光蛋白（GFP）是从基因组3'末端放置58 nt的转录盒中表达的。这有助于定义将基因转录到mRNA所需的病毒顺式作用信号。该病毒用于直接监测活细胞中的病毒感染。此外，我们使用HMPV-GFP病毒开发了一种更快，更可靠的测定法，以检测HMPV中和抗体。 在另一项研究中，HMPV经过精心设计，以完全单独和组合删除SH和G基因。 DEL-SH，DEL-G和DEL-SH/G缺失突变体很容易恢复，并被发现在体外复制，其效率大致相当于野生型病毒。这表明SH和G蛋白对于细胞培养的有效生长不是必不可少的。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。 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。可以考虑缺乏一种或两种表面蛋白的HMPV疫苗病毒，因为其他正在进行的工作表明F是主要中和和保护性抗原，而有些令人惊讶的是，SH和G似乎没有明显的中和化为中和抗原。 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。确认了两个ORF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（DEL-M2-1，DEL-M2-2和DEL-M2 [1+2]）。每种突变病毒都在Vero细胞中定向有效的多周期生长，表明HMPV复制不需要蛋白质。 DEL-M2-2病毒在标准化为基因组模板的mRNA的积累中表现出3至9倍，这表明M2-2在调节RNA合成中起作用。在仓鼠模型中测试了复制和免疫原性。感染后第3或第5天，用DEL-M2-1或DEL-M2内经室感染的动物在肺部或鼻涡轮上没有可回收的病毒，并且不会产生HMPV中和中和中和的血清抗体或对HMPV挑战的耐药性。因此，M2-1似乎对于体内的重要病毒复制至关重要。在感染DEL-M2-2的动物中，仅从12只动物中的1种中回收了病毒，并且仅在一天中才能在鼻涡轮上回收。然而，这些动物产生了HMPV中和血清抗体的高滴度，并受到了野生型HMPV的挑战的高度保护。 在非洲绿猴中进一步分析了DEL-SH，DEL-G和DEL-M2-2病毒，这是一种实验性动物，在解剖学和系统发育上与人类更加紧密相关。 DEL-SH复制到与亲本病毒相当的水平，而DEL-G在上呼吸道中略有减弱，下呼吸道中的1000倍以上。 DEL-M2-2病毒在上呼吸道中衰减160倍，下呼吸道减弱4000倍。这证实了SH，G和M2-2是非必需的辅助蛋白。每种突变病毒诱导中和抗体的诱导效率且与野生型HMPV相当。在挑战野生型HMPV时，三个缺失突变体中的每个突变体基本上赋予了下呼吸道和完全保护（DEL-SH），或在上呼吸道中降低了挑战病毒复制（DEL-G，DEL-M2-2）的1000倍。因此，至少有两个有希望的HMPV疫苗候选者，基于涉及G或M2基因的独立突变的衰减，可用，并将开发用于人类的1期临床测试。