Laboratory Studies of Human Respiratory Syncytial Virus and Other Pneumoviruses

人类呼吸道合胞病毒和其他肺病毒的实验室研究

• 批准号: 8745290
• 负责人: PETER LEON COLLINS
• 金额: $ 191.68万
• 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/8745290
• 关键词: Acetylglucosamine; Affect; Age; Age-Months; Amino Acids; Animals; Antigens; Antiviral Agents; Antiviral Therapy; Arsenites; Attenuated; Attenuated Live Virus Vaccine; Basic Science; Binding; Biology; Cell physiology; Cercopithecus pygerythrus; Child; Childhood; Clinical Trials; Cloning; Code; Codon Nucleotides; Complementary DNA; Cytoplasm; Cytoplasmic Granules; Development; Elderly; Electrons; Elongation Factor; Enzymes; Evaluation; Exhibits; FDA approved; Family; Gene Deletion; Genes; Genetic; Genetic Code; Genetic Transcription; Genetic Variation; Genome; Glycoproteins; Goals; Growth; Hand; Human Metapneumovirus; Human poliovirus; Human respiratory syncytial virus; Immunity; Immunocompromised Host; In Vitro; Inclusion Bodies; Individual; Inflammatory; Inflammatory Response; Laboratory Study; Left; Life; Link; Lower respiratory tract structure; MAP Kinase Gene; MAPK14 gene; Messenger RNA; Methods; Missense Mutation; Mitogen-Activated Protein Kinases; Molecular; Molecular Biology; Molecular Genetics; Morbidity - disease rate; Mus; Mutation; Nature; Nonstructural Protein; Nucleocapsid; Nucleoproteins; Nucleotides; Open Reading Frames; Oxidative Stress; PTPN11 gene; Paramyxovirus; Phenotype; Phosphoproteins; Phosphorylation; Physiological; Play; Pneumovirus; Polioviruses; Polymerase; Polymerase Gene; Population; Protein Kinase; Proteins; RNA; RNA Viruses; RNA chemical synthesis; Reagent; Recombinant DNA; Recombinants; Refractory; Reporting; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus Vaccines; Respiratory Tract Diseases; Respiratory syncytial virus; Respiratory syncytial virus RSV proteins; Ribosomes; Role; Serial Passage; Signal Pathway; Signal Transduction; Site; Stress; Stress Tests; Structure; System; TIS11 protein; Temperature; Time; Transcription Elongation; Transfer RNA; Transferase; Translations; Vaccine Design; Vaccines; Viral; Viral Genome; Viral Inclusion Bodies; Viral Proteins; Virus; Virus Diseases; Work; Zinc; attenuation; base; biological adaptation to stress; clinical lot; design; genome sequencing; glycoprotein G; human MAPK14 protein; improved; influenzavirus; mRNA Stability; mitogen-activated protein kinase p38; mortality; multiple myeloma M Protein; mutant; novel vaccines; pathogen; phase 1 study; positional cloning; preclinical study; research and development; research clinical testing; response; vaccine candidate; vaccine development; viral RNA

RSV and HMPV are cytoplasmic enveloped RNA viruses of the paramyxovirus family. Their genomes are single strands of negative-sense RNA of 15.2 kb (RSV) and 13.3 kb (HMPV) that encode 10 mRNAs and 11 unique proteins (RSV) or 8 mRNAs and 9 unique proteins (HMPV). Each virus encodes a nucleoprotein N, phosphoprotein P, matrix protein M, small hydrophobic protein SH, major glycoprotein G, fusion glycoprotein F, M2-1 and M2-2 polymerase factors, and polymerase L. In addition, RSV encodes two nonstructural proteins NS1 and NS2. 
We have been working to develop attenuating mutations in RSV that have improved genetic and phenotypic stability, for the purpose of producing live-attenuated vaccine strains with increased stability. In one approach, we worked to create attenuating mutations by deleting one or several codons at a given locus in coding sequence in the viral genome, using known missense mutations as a guide. Most of the attempted codon-deletions proved to be lethal to the virus, but we found that deletion of codon 1313 in the L polymerase gene (del1313) resulted in a virus that replicated with wt-like efficiency at the permissive temperature of 32C but was restricted at 37C. In addition, it was restricted 50-fold and 150-fold in the upper and lower respiratory tract, respectively, of mice. We combined this del1313 mutation with the previously described attenuating NS2 gene deletion (delNS2) to produce the recombinant live-attenuated RSV vaccine candidate delNS2/del1313. During in vitro stress tests involving serial passage at incrementally increasing temperatures to evaluate genetic stability, a second-site compensatory mutation was detected in close proximity of del1313, namely I1314T. This site was genetically and phenotypically stabilized by an I1314L substitution. Combination of I1314L with delNS2/del1313 yielded genetic stability at physiological temperature. This stabilized vaccine candidate was moderately temperature-sensitive and had a level of restriction in experimental animals comparable to that of MEDI-559 and RSV cps2, two promising RSV vaccine candidate that presently are in or entering clinical trials. The level of attenuation and the genetic stability of delNS2/del1313 indicate that it is a promising candidate suitable for evaluation in pediatric phase I studies. This virus has been manufactured into a clinical lot and has been approved by the FDA for studies beginning this year in seropositive children 6-59 months of age. 
 We evaluated the strategy of codon-pair deoptimization (CPD) as a means of developing genetically and phenotypically stable attenuated RSV strains. It is well known that there is a bias in codon-pair usage in nature. Specifically, any given pair of amino acids has the possibility to be encoded by a variety of different combinations of synonymous codons due to the degeneracy of the genetic code, but the observed usage of codon-pairs typically is biased to favor a subset of the possible combinations. One factor in this bias is thought to be translational efficiency and accuracy, because certain combinations of tRNA pairs are favored at the A and P sites in the ribosome due to tRNA geometry and other factors. CPD involves the deliberate introduction of under-represented synonymous codon-pairs into numerous sites in protein-coding sequence to achieve sub-optimal expression. These substitutions only involve the ORFS, and thus non-protein-coding genome regions are not affected. Also, CPD involves only synonymous codon substitutions, and thus amino acid coding is unaffected and the antigens remain unchanged. In addition, CPD applied to one or several genes typically involves hundreds or thousands of nucleotide changes, and thus should be highly refractory to de-attenuation. Recently, CPD was applied to poliovirus and influenza virus and was shown to result in attenuated strains. We designed the following set of four CPD RSV genomes in which the indicated ORFs were recoded: (i) Min A; NS1, NS2, N, P, M, and SH (i.e., the left-hand third of the genome); (ii) Min B; G and F (located in the middle of the genome); (iii) Min L; L (located at the right-hand end of the genome); and (iv) Min FLC; all ORFs except M2-1 and M2-2. The recoded genome regions were synthesized commercially and the four CPD viruses were constructed and recovered by reverse genetics. All of the CPD viruses were temperature-sensitive (level of sensitivity: Min FLC>Min L>Min B>Min A) for replication in vitro. This was unexpected given the lack of change in amino acid coding, which was confirmed by re-sequencing. All of the CPD mutants grew less efficiently in vitro than wild type (wt) RSV, even at the permissive temperature of 32C (growth efficiency: wt>Min L>Min A>Min FLC>Min B). Thus, CDP of G and F ORFs provided the greatest effect. The CPD viruses exhibited a range of restriction in mice and African Green Monkeys (AGM) and induced immunity against wt RSV. This study identified new vaccine candidates for RSV and showed that CPD of a nonsegmented negative-strand RNA virus can rapidly generate vaccine candidates with a range of attenuation phenotypes. RSV infection results in the formation of viral inclusion bodies (IBs) that appear as large, prominent, electron-dense structures in the cytoplasm. IBs are thought to be sites of nucleocapsid accumulation and viral RNA synthesis. We found that, during RSV infection, the IBs also were the sites of major sequestration of two proteins involved in cellular signaling pathways. These are phosphorylated p38 mitogen-activated protein kinase (MAPK) (p38-P), a key regulator of cellular inflammatory and stress responses, and O-linked N-acetylglucosamine (OGN) transferase (OGT), an enzyme that catalyzes the post-translational addition of OGN to protein targets to regulate cellular processes including signal transduction, transcription, translation, proteasomal degradation, and the stress response. The virus-induced sequestration of p38-P in viral IBs resulted in a substantial reduction in the accumulation of a downstream signaling substrate, MAPK-activated protein kinase 2 (MK2). Sequestration of OGT in IBs was associated with suppression of stress granule (SG) formation. Thus, while the RSV IBs are thought to play an essential role in viral replication, the present results show that they also play a role in suppressing the cellular response to viral infection. The sequestration of p38-P and OGT in IBs appeared to be reversible: severe oxidative stress resulting from a brief arsenite treatment transformed large IBs into a scattering of smaller bodies, suggestive of partial disassembly, and this was associated with MK2 phosphorylation and OGN-addition. Unexpectedly, the RSV M2-1 protein was found to localize in SGs that formed during oxidative stress. This protein was previously shown to be a viral transcription elongation factor, and the present findings provide the first evidence of possible involvement in SG activities during RSV infection. This involvement is intriguing because the M2-1 protein has some structural similarity - including the presence of a CCCH zinc-binding motif - with the cellular protein tristetraprolin. Tristetraprolin is a SG-associated protein that helps regulate the stability of certain cellular mRNAs including a number encoding pro-inflammatory and antiviral proteins. This suggests a possible role for the viral M2-1 mRNA in mRNA stability.

RSV 和 HMPV 是副粘病毒科的细胞质有包膜 RNA 病毒。它们的基因组是 15.2 kb (RSV) 和 13.3 kb (HMPV) 的单链负义 RNA，编码 10 个 mRNA 和 11 个独特蛋白 (RSV) 或 8 个 mRNA 和 9 个独特蛋白 (HMPV)。每种病毒均编码核蛋白 N、磷蛋白 P、基质蛋白 M、小疏水蛋白 SH、主要糖蛋白 G、融合糖蛋白 F、M2-1 和 M2-2 聚合酶因子以及聚合酶 L。此外，RSV 还编码两种非结构蛋白 NS1 和 NS2。 
我们一直致力于开发 RSV 减毒突变，以提高遗传和表型稳定性，以生产稳定性更高的减毒活疫苗株。在一种方法中，我们使用已知的错义突变作为指导，通过删除病毒基因组编码序列中给定基因座的一个或多个密码子来创建减毒突变。大多数尝试的密码子删除被证明对病毒是致命的，但我们发现 L 聚合酶基因 (del1313) 中密码子 1313 的删除导致病毒在 32°C 的允许温度下以类似 wt 的效率复制，但在 37°C 时受到限制。此外，它在小鼠的上呼吸道和下呼吸道中分别被限制了50倍和150倍。我们将此 del1313 突变与先前描述的减毒 NS2 基因缺失 (delNS2) 结合起来，产生重组减毒活 RSV 候选疫苗 delNS2/del1313。在涉及在逐渐升高的温度下连续传代以评估遗传稳定性的体外压力测试中，在 del1313 附近检测到第二位点补偿突变，即 I1314T。该位点通过 I1314L 取代在遗传和表型上保持稳定。 I1314L 与 delNS2/del1313 的组合在生理温度下产生了遗传稳定性。这种稳定的候选疫苗对温度具有中等敏感性，并且对实验动物的限制水平与 MEDI-559 和 RSV cps2 相当，这两种候选疫苗目前正处于或进入临床试验。 delNS2/del1313 的减毒水平和遗传稳定性表明它是适合儿科 I 期研究评估的有前途的候选药物。该病毒已投入临床批量生产，并已获得 FDA 批准，从今年开始在 6-59 个月大的血清反应呈阳性的儿童中进行研究。 
 我们评估了密码子对去优化 (CPD) 策略作为开发遗传和表型稳定的减毒 RSV 菌株的一种手段。众所周知，自然界中密码子对的使用存在偏差。具体而言，由于遗传密码的简并性，任何给定的氨基酸对都有可能由同义密码子的多种不同组合编码，但观察到的密码子对的使用通常偏向于可能的组合的子集。造成这种偏差的一个因素被认为是翻译效率和准确性，因为由于 tRNA 几何形状和其他因素，核糖体中的 A 和 P 位点有利于 tRNA 对的某些组合。 CPD 涉及故意将代表性不足的同义密码子对引入蛋白质编码序列的许多位点，以实现次优表达。这些替换仅涉及 ORFS，因此非蛋白质编码基因组区域不受影响。此外，CPD仅涉及同义密码子替换，因此氨基酸编码不受影响并且抗原保持不变。此外，应用于一个或多个基因的 CPD 通常涉及数百或数千个核苷酸变化，因此应该高度难以去衰减。最近，CPD 被应用于脊髓灰质炎病毒和流感病毒，并被证明可以产生减毒株。我们设计了以下四个 CPD RSV 基因组，其中指定的 ORF 被重新编码：（i）Min A； NS1、NS2、N、P、M 和 SH（即基因组的左侧三分之一）； (ii) 最小 B； G和F（位于基因组中部）； (iii) 最小 L； L（位于基因组的右端）； (iv) 最小 FLC；除 M2-1 和 M2-2 之外的所有 ORF。重新编码的基因组区域是商业合成的，四种 CPD 病毒是通过反向遗传学构建和恢复的。所有 CPD 病毒对于体外复制都是温度敏感的（敏感度水平：Min FLC>Min L>Min B>Min A）。这是出乎意料的，因为氨基酸编码没有变化，这通过重新测序得到了证实。即使在 32°C 的允许温度下，所有 CPD 突变体在体外的生长效率也低于野生型 (wt) RSV（生长效率：wt>Min L>Min A>Min FLC>Min B）。因此，G和F ORF的CDP提供了最大的效果。 CPD 病毒对小鼠和非洲绿猴 (AGM) 表现出一系列限制，并诱导针对 wt RSV 的免疫力。这项研究确定了 RSV 的新候选疫苗，并表明非节段负链 RNA 病毒的 CPD 可以快速生成具有一系列减毒表型的候选疫苗。 RSV 感染导致病毒包涵体 (IB) 的形成，这些包涵体在细胞质中表现为大的、突出的电子致密结构。 IB被认为是核衣壳积累和病毒RNA合成的场所。我们发现，在 RSV 感染期间，IB 也是参与细胞信号传导途径的两种蛋白质的主要隔离位点。它们是磷酸化 p38 丝裂原激活蛋白激酶 (MAPK) (p38-P)（细胞炎症和应激反应的关键调节因子）和 O-连接 N-乙酰氨基葡萄糖 (OGN) 转移酶 (OGT)，这是一种催化 OGN 翻译后添加到蛋白质靶标上以调节细胞过程（包括信号转导）的酶。 转录、翻译、蛋白酶体降解和应激反应。病毒诱导的病毒 IB 中 p38-P 的隔离导致下游信号底物 MAPK 激活蛋白激酶 2 (MK2) 的积累大幅减少。 IB 中 OGT 的隔离与应激颗粒 (SG) 形成的抑制有关。因此，虽然RSV IB被认为在病毒复制中发挥重要作用，但目前的结果表明它们还在抑制细胞对病毒感染的反应中发挥作用。 IB 中 p38-P 和 OGT 的隔离似乎是可逆的：短暂亚砷酸盐处理导致的严重氧化应激将大 IB 转化为分散的较小物体，表明部分分解，这与 MK2 磷酸化和 OGN 添加有关。出乎意料的是，RSV M2-1 蛋白被发现定位于氧化应激期间形成的 SG 中。该蛋白先前被证明是一种病毒转录延伸因子，目前的发现提供了第一个证据，表明 RSV 感染期间可能参与 SG 活动。这种参与很有趣，因为 M2-1 蛋白与细胞蛋白 tristetraprolin 有一些结构相似性，包括 CCCH 锌结合基序的存在。 Tristetraprolin 是一种 SG 相关蛋白，有助于调节某些细胞 mRNA 的稳定性，包括许多编码促炎和抗病毒蛋白。这表明病毒 M2-1 mRNA 在 mRNA 稳定性中可能发挥作用。