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.

人类偏肺病毒 (HMPV) 于 2001 年在荷兰首次报道，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 对人类疾病的影响仍有待确定，但似乎约占因呼吸道疾病住院的儿童的 5% 至 15%。我们正在使用反向遗传方法开发 HMPV 减毒株，用作鼻内儿科活疫苗，该疫苗将与 LID/NIAID 目前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) 的活疫苗联合使用。 HMPV 是一种有包膜病毒，其基因组是单条负义 RNA 链。它与 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。迄今为止已测序的 HMPV 基因组长度范围为 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。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 分离株开发了反向遗传系统，通过转染培养细胞的克隆 cDNA 可以完全产生完整的感染性病毒。这提供了一种将预定变化引入传染性 HMPV 的方法，用于基本分子遗传学研究和设计疫苗。 在一项研究中，我们设计了 HMPV 的一个版本，即 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。这有助于定义将基因转录为 mRNA 所需的病毒顺式作用信号。这种病毒被用来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速、更可靠的检测方法来检测 HMPV 中和抗体。 在另一项研究中，HMPV 被设计为完全单独或组合删除 SH 和 G 基因。 del-SH、del-G 和 del-SH/G 缺失突变体很容易回收，并且发现其在体外复制的效率大约与野生型病毒相当。这表明 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。 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。考虑缺乏这些表面蛋白中的一种或两种的 HMPV 疫苗病毒是可行的，因为其他正在进行的工作表明 F 是主要的中和和保护性抗原，而令人惊讶的是，SH 和 G 似乎并不是重要的中和和保护性抗原。 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。两个 ORF 的表达均得到证实。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]）。每种突变病毒都能在 Vero 细胞中进行有效的多周期生长，表明这两种蛋白质都不是 HMPV 复制所必需的。 del-M2-2病毒表现出标准化至基因组模板的mRNA积累增加3至9倍，表明M2-2在调节RNA合成中发挥作用。在仓鼠模型中测试了复制和免疫原性。鼻内感染 del-M2-1 或 del-M2(1+2) 的动物在感染后第 3 天或第 5 天肺部或鼻甲中没有可恢复的病毒，并且没有产生 HMPV 中和血清抗体或对 HMPV 攻击的抵抗力。因此，M2-1 似乎对于体内病毒的显着复制至关重要。在感染 del-M2-2 的动物中，12 只动物中只有 1 只在一天内仅在鼻甲中发现了病毒。然而，这些动物产生了高滴度的 HMPV 中和血清抗体，并且受到高度保护，免受野生型 HMPV 的攻击。 del-SH、del-G 和 del-M2-2 病毒在非洲绿猴身上进行了进一步分析，非洲绿猴是一种在解剖学和系统发育上与人类关系更密切的实验动物。 Del-SH 的复制水平与亲代病毒相当，而 del-G 在上呼吸道中轻微减弱，在下呼吸道中减弱超过 1000 倍。 del-M2-2病毒在上呼吸道减毒160倍，在下呼吸道减毒4000倍。这证实了 SH、G 和 M2-2 是非必需辅助蛋白。每种突变病毒诱导中和抗体的效率与野生型 HMPV 相当。在用野生型HMPV攻击后，三种缺失突变体中的每一种都赋予下呼吸道基本上完全的保护和上呼吸道的攻击病毒复制（del-G、del-M2-2）的完全保护（del-SH）或减少1000倍以上。因此，至少有两种基于 G 或 M2 基因独立突变而具有减毒作用的有前景的 HMPV 候选疫苗已经上市，并将被开发用于人体 1 期临床测试。