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是帕托马病毒家族的细胞质包膜RNA病毒。它们的基因组是15.2 kb（RSV）的负义RNA或13.3 kb（HMPV）的单链，它们编码10个mRNA和11个独特的蛋白质（RSV）或8个mRNA和9个独特蛋白质（HMPV）。每种病毒均编码核蛋白N，磷酸蛋白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和聚合酶蛋白L.其他。 我们评估了密码子除外（CPD）的策略，以此作为开发遗传和表型稳定的衰减RSV菌株的一种手段。众所周知，自然界的密码子使用情况存在偏见。具体而言，多肽链中的任何给定的氨基酸都有可能由遗传密码退化而被各种不同的同义密码子组合编码，但是通常观察到的密码子对使用通常会偏向于可能组合的子集。这种偏见中的一个因素被认为是转化效率和准确性，因为由于tRNA几何形状和其他因素，核糖体的A和P位点的某些组合在核糖体的A和P位点受到青睐。 CPD涉及故意将代表性不足的同义密码子对以蛋白质编码序列的众多位点引入，以实现亚最佳表达。这些替代仅涉及ORF，因此非蛋白质编码基因组区域不受影响。同样，CPD仅涉及同义密码子取代，因此氨基酸编码不受影响。另外，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，因此应高度难以耐受性去衰减。最近，将CPD应用于脊髓灰质炎病毒和流感病毒，并被证明会导致菌株减弱。 我们设计了以下四个CPD RSV基因组，其中重新编码了指示的ORF：（i）min a； NS1，NS2，N，P，M和SH（即基因组的左手三分之一）； （ii）最小b; G和F（位于基因组中间）； （iii）最小L; L（位于基因组的右端）； （iv）min flc;除M2-1和M2-2以外的所有ORF。重新编码的基因组区域是商业合成的，并通过反向遗传学构建并回收了四个CPD病毒。所有CPD病毒均对温度敏感（灵敏度：min flc> min l> min b> min b> min a）用于体外复制。我们推测CDP可能会降低翻译速率，从而产生蛋白质折叠问题，而温度升高会加剧。即使在32c的允许温度下，所有CPD突变体在体外的效率均低于野生型（WT）RSV（生长效率：wt> min l> min a> min a> min a> min a> 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猴（AGM）中表现出一系列限制，并诱导了针对WT RSV的免疫力。这项研究确定了针对RSV的新疫苗候选物，并表明非细分负链RNA病毒的CPD可以迅速产生具有一系列衰减表型的候选疫苗。 我们使用基因脱落HMPV菌株来评估原代人单核细胞衍生的树突状细胞（MDDC）在体外以及随后的MDDC成熟和自体T细胞激活中，评估了原代人单核细胞衍生的树突状细胞（MDDC）对HMPV摄取的作用。 G和SH（DELSHG）的缺失赋予了增加的感染力，但对MDDC的成熟几乎没有影响。然而，用ΔSHG刺激的MDDC诱导自体Th1极化CD4+ T细胞的增殖增加。这种作用与病毒复制无关。 T细胞增殖的增加严格取决于病毒刺激的MDDC和CD4+ T细胞之间的接触。共聚焦显微镜表明，SH和G的缺失与记忆CD4+ T细胞和病毒刺激的MDDC之间的免疫突触数量增加有关。发现MDDC对HMPV的摄取主要是通过大型细胞增多症。与∆SHG相比，野生型（WT）病毒的摄取减少，表明SH和G糖蛋白抑制。此外，DC鉴定介导的内吞作用提供了次要的替代途径，该途径取决于SH和/或G，因此仅用于WT。总之，我们的结果表明，SH和G糖蛋白降低了HMPV被MDDC内化的能力，从而降低了HMPV刺激的MDDC激活CD4+ T细胞的能力。这项研究描述了先前未知的病毒免疫逃避机制。这很有趣，因为HMPV的重新感染在整个生命中都是常见的，而无需重大的抗原变化，这表明对HMPV的保护性免疫反应是不完整且短暂的。本研究提供了一种可能有助于抑制宿主免疫力的机制。