Maturational Intermediates of Trimeric HIV-1 Envelope as Unique Immunogens

描述：我们之前已经开发了一种方法来设计将一个有效的外来亲和标签(3xFLAG)结合到HIV-1蛋白质组中。由于这些工程病毒是通过基于生存能力的过程产生的，标记的和必要的病毒蛋白必须保持功能，很可能经历与野生型病毒相同的相互作用。此外，由于外源标签在病毒生长的几个周期中被稳定地容纳，因此通过重组病毒的生物扩增可以获得特定标签病毒蛋白的高水平表达和纯化。结合对瞬时病毒-宿主相互作用的低温保存和可以区分特定和非特定蛋白质相互作用的差示同位素质谱学技术，可以明确识别在进行性感染过程中与病毒机制特异相互作用的宿主复合体。 在这项建议中，我们关注于我们的表位标记病毒的另一个强大属性--直接从感染细胞中选择性和定量地纯化给定病毒蛋白的成熟中间产物的可能性。这里我们的重点是病毒包膜蛋白(Env)，它可以说是病毒结构和生物化学最多样化的蛋白，也是病毒粒子的唯一溶剂暴露蛋白，是疫苗开发的最相关靶点。值得注意和不可预测的是，我们的原型Env 3xFLAG标记病毒Env-3XF为我们提供了优先从定义的步骤中恢复和纯化未成熟的Env衍生物(S)的能力-在其翻译到内质网后不久-在蛋白质既没有经过蛋白质水解性加工也没有完全糖基化的时候，而是以gp160的高甘露糖三聚衍生物的形式存在。 在这里，我们提出了一个模型，在这个模型中，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折叠正在进行时，中和特异性表位可能会在包膜成熟途径的各个步骤中暴露出来。除了我们的 对于原型Env-3XF病毒，我们建议从我们最初的TCLA前病毒克隆和针对gp120或gp41不同功能结构域的传播性病毒方正克隆(CH077)中额外构建和选择一组病毒，用于3xFLAG标签的掺入。对Env天然成熟中间体的选择性免疫纯化可以丰富那些在完全成熟的蛋白质中暴露的稀有的中和敏感表位的原始分子，并在适当的背景下引发相关的免疫学反应。这样一组具有复制能力的病毒可以提供广泛的独特的生化和/或免疫学特性。因此，修饰后的病毒将通过生物扩增、免疫纯化和自然条件下洗脱的Env蛋白进行扩增，然后用于在兔体内产生广谱中和抗体。

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.