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: 8946258
• 负责人: PETER LEON COLLINS
• 金额: $ 107.29万
• 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/8946258
• 关键词: Affect; Age; Amino Acids; Antiviral Therapy; Attenuated; Attenuated Live Virus Vaccine; Autologous; Basic Science; CD209 gene; CD4 Positive T Lymphocytes; Cell Maturation; Cercopithecus pygerythrus; Childhood; Cloning; Code; Codon Nucleotides; Complementary DNA; Confocal Microscopy; Dendritic Cells; Development; Elderly; Endocytosis; Exhibits; Family; Gene Deletion; Genes; Genetic; Genetic Code; Genetic Variation; Genome; Geometry; Glycoproteins; Goals; Growth; Hand; Human; Human Metapneumovirus; Human poliovirus; Human respiratory syncytial virus; Immune; Immune response; Immunity; Immunocompromised Host; In Vitro; Individual; Laboratory Study; Left; Life; Mediating; Messenger RNA; Methods; Minor; Molecular; Molecular Biology; Molecular Genetics; Morbidity - disease rate; Mus; Nature; Nonstructural Protein; Nucleoproteins; Nucleotides; Open Reading Frames; PTPN11 gene; Paramyxovirus; Pathway interactions; Phenotype; Phosphoproteins; Pneumovirus; Polioviruses; Polymerase; Population; Proteins; RNA; RNA Viruses; Reagent; Recombinant DNA; Refractory; Reporting; Respiratory Syncytial Virus Vaccines; Respiratory Tract Diseases; Respiratory syncytial virus; Respiratory syncytial virus RSV proteins; Ribosomes; Role; Site; System; T Virus; T-Cell Proliferation; T-Lymphocyte; Temperature; Time; Transfer RNA; Translations; Vaccine Design; Vaccines; Viral; Viral Proteins; Virus; Virus Replication; attenuation; base; design; genome sequencing; glycoprotein G; human disease; immunogenicity; immunological synapse; improved; influenzavirus; interest; memory CD4 T lymphocyte; monocyte; mortality; multiple myeloma M Protein; mutant; novel vaccines; pathogen; polypeptide; positional cloning; preclinical study; protein folding; research and development; research clinical testing; uptake; vaccine candidate; vaccine development

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) or 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, polymerase factors M2-1 and M2-2, and the polymerase protein L. In addition, RSV encodes two nonstructural proteins NS1 and NS2. 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 in a polypeptide chain 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. 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. We speculate that CDP may slow down the rate of translation sufficiently to create protein-folding problems that are exacerbated by increased temperature. 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, CPD 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. We used gene-deletion HMPV strains to evaluate the role of the attachment G and small hydrophobic SH glycoproteins on HMPV uptake by primary human monocyte-derived dendritic cells (MDDC) in vitro, and on subsequent MDDC maturation and activation of autologous T cells. Deletion of G and SH (delSHG) conferred increased infectivity but had little effect on MDDC maturation. However, MDDC stimulated with ΔSHG induced increased proliferation of autologous Th1-polarized CD4+ T cells. This effect was independent of virus replication. Increased T cell proliferation was strictly dependent on contact between virus-stimulated MDDC and CD4+ T cells. Confocal microscopy revealed that deletion of SH and G was associated with an increased number of immunological synapses between memory CD4+ T cells and virus-stimulated MDDC. Uptake of HMPV by MDDC was found to be primarily by macropinocytosis. Uptake of wild-type (WT) virus was reduced compared to ∆SHG, indicative of inhibition by the SH and G glycoproteins. In addition, DC-SIGN-mediated endocytosis provided a minor alternative pathway that depended on SH and/or G and thus operated only for WT. Altogether our results show that SH and G glycoproteins reduce the ability of HMPV to be internalized by MDDC, resulting in a reduced ability of the HMPV-stimulated MDDC to activate CD4+ T cells. This study describes a previously unknown mechanism of virus immune evasion. This is of interest because reinfection by HMPV is common throughout life without need for significant antigenic change, suggesting that protective immune response to HMPV is incomplete and short-lived. The present study provides a mechanism that might contribute to suppressing host immunity.

RSV和HMPV是副粘病毒科的胞质包膜RNA病毒。它们的基因组是15.2kb（RSV）或13.3kb（HMPV）的单链负义RNA，编码10种mRNA和11种独特蛋白（RSV）或8种mRNA和9种独特蛋白（HMPV）。每种病毒编码核蛋白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、聚合酶因子M2-1和M2-2以及聚合酶蛋白L。另外，RSV编码两种非结构蛋白NS 1和NS 2。 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毒株的一种手段。众所周知，在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，多肽链中任何给定的氨基酸对都有可能由同义密码子的各种不同组合编码，但观察到的密码子对的使用通常偏向于有利于可能组合的子集。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的几何形状和其他因素，tRNA对的某些组合在核糖体中的A和P位点是有利的。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中的许多位点以实现次优表达。这些取代仅涉及ORFS，因此非蛋白质编码基因组区域不受影响。此外，CPD仅涉及同义密码子取代，因此氨基酸编码不受影响。此外，应用于一个或几个基因的CPD通常涉及数百或数千个核苷酸变化，因此对于去减毒应该是高度难治的。最近，CPD被应用于脊髓灰质炎病毒和流感病毒，并显示产生减毒毒株。 我们设计了以下一组四个CPD RSV基因组，其中所示的ORF被重新编码：（i）Min A; NS 1、NS 2、N、P、M和SH（即，基因组的左手三分之一）;（ii）Min B; G和F（位于基因组的中间）;（iii）Min L; L（位于基因组的右手端）;和（iv）Min FLC;除M2-1和M2-2之外的所有ORF。商业合成重新编码的基因组区域，并通过反向遗传学构建和回收四种CPD病毒。所有CPD病毒在体外复制时都是温度敏感的（敏感性水平：Min FLC>Min L>Min B>Min A）。我们推测，CDP可能会减慢翻译的速度，足以产生蛋白质折叠的问题，加剧了温度升高。所有CPD突变体的体外生长效率低于野生型（wt）RSV，甚至在32 ℃的允许温度下也是如此（生长效率：wt>Min L>Min A>Min FLC>Min B）。因此，G和F ORF的CPD提供了最大的效果。CPD病毒在小鼠和非洲绿色猴（AGM）中表现出一系列限制，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，并表明非分段负链RNA病毒的CPD可以快速产生具有一系列减毒表型的候选疫苗。 我们使用基因缺失的HMPV毒株来评估附着G和小疏水性SH糖蛋白对体外原代人单核细胞来源的树突状细胞（MDDC）摄取HMPV的作用，以及对随后的MDDC成熟和自体T细胞活化的作用。G和SH的缺失（delSHG）赋予增加的感染性，但对MDDC的成熟几乎没有影响。然而，用SHG刺激的MDDC诱导自体Th 1极化的CD 4 + T细胞增殖增加。这种效应与病毒复制无关。增加的T细胞增殖严格依赖于病毒刺激的MDDC和CD 4 + T细胞之间的接触。共聚焦显微镜显示，SH和G的缺失与记忆性CD 4 + T细胞和病毒刺激的MDDC之间的免疫突触数量增加有关。MDDC对HMPV的摄取主要通过巨胞饮作用。与SHG相比，野生型（WT）病毒的摄取减少，表明SH和G糖蛋白的抑制。此外，DC-SIGN介导的内吞作用提供了一个依赖于SH和/或G的次要替代途径，因此仅对WT起作用。总之，我们的结果表明，SH和G糖蛋白降低HMPV被MDDC内化的能力，导致HMPV刺激的MDDC活化CD 4 + T细胞的能力降低。这项研究描述了一种以前未知的病毒免疫逃避机制。这是令人感兴趣的，因为HMPV的再感染在整个生命中是常见的，而不需要显著的抗原变化，这表明对HMPV的保护性免疫应答是不完全的和短暂的。本研究提供了一种可能有助于抑制宿主免疫的机制。