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.

产品说明：我们以前已经开发了一种方法，将一种有效的外源亲和标签（3xFLAG）掺入HIV-1蛋白质组。由于这些工程病毒是通过基于活力的过程产生的，因此标记的和必需的病毒蛋白必须保持功能，可能经历野生型病毒遇到的相同相互作用。此外，由于外源标签在病毒生长的几个循环中被稳定地容纳，因此可以通过重组病毒的生物扩增获得特异性标记的病毒蛋白的高水平表达和纯化。结合低温保存的瞬时病毒-宿主相互作用和差分同位素质谱技术，可以区分特异性和非特异性蛋白质相互作用，明确识别的主机复合物，特别是在进行性感染过程中与病毒机制相互作用已成为可能。 在这个建议中，我们专注于我们的表位标记的病毒的另一个强大的属性-选择性和定量纯化的潜力，直接从受感染的细胞给定的病毒蛋白的成熟中间体。我们的重点是病毒包膜蛋白（Env），可以说是病毒结构和生物化学上最多样化的蛋白质，也是病毒粒子唯一暴露于溶剂的蛋白质，是疫苗开发最相关的靶点。值得注意的是，不可预测的是，我们的原型Env 3xFLAG标记的病毒，Env-3xF，为我们提供了优先回收和纯化未成熟的Env衍生物的能力，从一个定义的步骤后不久，在内质网翻译的蛋白质既不是蛋白水解加工，也不是完全糖基化，但作为gp 160的高甘露糖，三聚衍生物存在的点。 在这里，我们提出了一个模型，其中中和特异性表位可能会暴露在步骤沿着的Env成熟途径，当广泛的糖基化是有限的，分子伴侣介导的折叠的糖蛋白的结构中间体正在进行中。除了我们 原型Env-3xF病毒，我们建议构建和选择衍生自我们的原始TCLA前病毒克隆以及靶向gp 120或gp 41的不同功能结构域用于3xFLAG标签掺入的传播病毒创始人克隆（CH 077）的另外一组病毒。Env天然成熟中间体的选择性免疫纯化可以富集那些基本分子，其中在完全成熟的蛋白质中掩蔽的罕见的中和敏感表位被暴露并且在适当的环境中引发相关的免疫应答。这样一组有复制能力的病毒可以提供广泛的独特的生物化学和/或免疫学特性。因此，将通过生物扩增扩增修饰的病毒，在天然条件下免疫纯化和洗脱Env蛋白，然后评价在兔中产生的广泛中和抗体。