Maturational Intermediates of Trimeric HIV-1 Envelope as Unique Immunogens

三聚体 HIV-1 包膜的成熟中间体作为独特的免疫原

• 批准号: 8790245
• 负责人: MARK AYER MUESING
• 金额: $ 28.48万
• 依托单位: AARON DIAMOND AIDS RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-06 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8790245
• 关键词: Adoption; Affinity; Amino Acids; Antibodies; Antigens; Binding; Biochemical; Biological; Biological Assay; Biological Preservation; Cell membrane; Cells; Collection; Complex; Coupled; DNA Sequence Rearrangement; Endoglycosidases; Endoplasmic Reticulum; Engineering; Epitopes; Event; Funding; Generations; Genetic; Glycoproteins; Golgi Apparatus; Growth; HIV Envelope Protein gp120; HIV Infections; HIV-1; Human; Immunofluorescence Immunologic; Immunoprecipitation; Infection; Interruption; Laboratories; Life; Location; Mannose; Maps; Masks; Mass Spectrum Analysis; Measures; Mediating; Membrane Fusion; Methodology; Methods; Modeling; Molecular; Molecular Chaperones; Molecular Sieve Chromatography; N-terminal; New Zealand; Oryctolagus cuniculus; Pathway interactions; Pattern; Peptides; Polysaccharides; Post-Translational Protein Processing; Process; Production; Property; Protein Region; Proteins; Proteolytic Processing; Proteome; Proteomics; Recombinant Proteins; Recombinants; Series; Solvents; Source; Specific qualifier value; Structure; Suspension substance; Suspensions; System; Techniques; Technology; Testing; Time; Translations; Viral; Viral Envelope Proteins; Viral Proteins; Virion; Virus; base; cryogenics; env Gene Products; glycosylation; gp160; immunogenic; immunogenicity; magnetic beads; neutralizing antibody; programs; prototype; public health relevance; receptor; recombinant virus; response; spatiotemporal; transmission process; vaccine development

DESCRIPTION: We have previously developed methodology to engineer the incorporation of a potent, foreign affinity tag (3xFLAG) into the HIV-1 proteome. As these engineered viruses are generated through a process based on viability, the tagged and essential viral protein must remain functional, likely to undergo the same interactions encountered by the wild type virus. Furthermore, because the foreign tag is stably accommodated over several cycles of viral growth, high-level expression and purification of a specific tagged viral protein can be obtained through biological amplification of the recombinant virus. In conjunction with cryogenic preservation of transient viral-host interactions and differential isotopic mass spectrometry techniques that can differentiate specific from nonspecific protein interactions, unequivocal identification of host complexes that specifically interact with the viral machinery during progressive infection has been made possible. In this proposal, we focus on another powerful attribute of our epitope-tagged viruses-the potential for selective and quantitative purification of maturational intermediates of a given vira protein directly from infected cells. Our focus here is the viral envelope protein (Env), arguably the most structurally and biochemically diversified protein of the virus and as the only solvent exposed protein of the virion, the most relevant target for vaccine development. Remarkably and unpredictably, our prototype Env 3xFLAG-tagged virus, Env-3xF, has provided us with the ability to preferentially recover and purify immature Env derivative(s) from a defined step-soon after its translation in the endoplasmic reticulum-at a point when the protein is neither proteolytically processed nor fully glycosylated but exists as a high-mannose, trimerized derivative of gp160. Here, we propose a model whereby neutralization-specific epitopes may be exposed at steps along the Env maturation pathway, when extensive glycosylation is limited and chaperone-mediated folding of structural intermediates of the glycoprotein is in progress. In addition to our prototype Env-3xF virus, we propose to construct and select an additional set of viruses derived from our original TCLA proviral clone as well as a transmitted viral founder clone (CH077) targeting different functional domains of gp120 or gp41 for 3xFLAG tag incorporation. Selective immunopurification of native maturational intermediates of Env may enrich for those rudimentary molecules in which rare, neutralization-sensitive epitopes, masked in the fully mature protein, are exposed and in a proper context to elicit a relevant immunological response. Such a panel of replication competent viruses may offer a wide spectrum of unique biochemical and/or immunological properties. Thus, the modified viruses will be expanded by biological amplification, the Env protein immunopurified and eluted under native conditions and then evaluated for the generation of broadly neutralizing antibodies in rabbits.

描述：我们以前已经开发了方法来设计将有效的外国亲和力标签（3xflag）纳入HIV-1蛋白质组。由于这些工程病毒是通过基于生存力的过程产生的，因此标记的和必需的病毒蛋白必须保持功能性，可能会经历野生型病毒遇到的相同相互作用。此外，由于外国标签在病毒生长的几个循环中稳定稳定，因此可以通过重组病毒的生物学扩增获得高级表达和特定标记的病毒蛋白的纯化。结合瞬态病毒宿主相互作用的低温保存和差异同位素质谱技术，这些技术可以将特异性与非特异性蛋白质相互作用区分开来，使宿主复合物的明确鉴定在促进感染过程中特异性相互作用的宿主鉴定已成为可能。 在此提案中，我们关注了表位标记的病毒的另一个强大属性 - 直接从受感染细胞中直接从受感染细胞中的给定VIRA蛋白的成熟中间体进行选择性和定量纯化的潜力。我们这里的重点是病毒包膜蛋白（ENV），可以说是该病毒的结构和生化多样化的蛋白质，也是Virion的唯一溶剂暴露蛋白，这是疫苗发育的最相关靶标。值得注意的，不可预测的是，我们的原型ENV 3XFLAG标记的病毒Env-3XF为我们提供了能够从定义的继承人（S）从蛋白质中的蛋白质中的蛋白质既不是在蛋白质上均不完全加工的，但是在蛋白质上均不完全凝聚的，但可以优先恢复和净化未成熟的ENV衍生物（S） GP160的导数。 在这里，我们提出了一个模型，当广泛的糖基化受到限制并且伴侣介导的糖蛋白结构中间体的折叠正在进行时，可以在沿ENV成熟途径的步骤中暴露于中和特异性表位的模型。除了我们 原型Env-3XF病毒，我们建议构建并选择一组来自我们原始的TCLA病毒病毒克隆的病毒，以及针对3xFlag Tag Incoriantion的GP120或GP41不同功能域的传播病毒创建者克隆（CH077）。 ENV的天然成熟中间体的选择性免疫性可能会富集那些基本分子，在这种基本分子中，在完全成熟的蛋白质中掩盖的罕见，中和敏感的表位是暴露的，并且在适当的环境中引起相关的免疫学反应。这样的复制胜任病毒可能会提供各种独特的生化和/或免疫学特性。因此，修饰的病毒将通过生物学扩增扩展，在天然条件下进行ENV蛋白免疫促进和洗脱，然后评估兔子中广泛中和抗体的生成。