Stabilized gp41 Structural Motif for HIV-1 Vaccine

描述（由申请人提供）：能够引发广泛中和抗体HIV-1的免疫原的开发仍然是当今生物医学研究面临的最困难的挑战之一。尽管如此，已经从HIV-1感染的个体中分离出少量人单克隆抗体（mab），可以中和来自不同遗传亚型的HIV-1原代分离株。在这些罕见的单克隆抗体中，两个（2F5和4E10）识别gp41外域的膜近端区域的表位。然而，迄今为止，所有通过接种多肽或简单线性表位来诱导类似的抗gp41抗体的尝试都失败了。我们的免疫原设计方法是由我们最近鉴定和结构确定的一种新的双链线圈结构域（C55）指导的，该结构域包括gp41的膜近端表位区域。然而，我们已经确定C55二聚体结构太不稳定，不适合用于免疫原性研究。本研究计划的总体目标是详细了解C55线圈结构域的结构和热力学性质，并利用这些知识设计和生产稳定的免疫原，以激发广泛中和的HIV-1抗体。我们的中心假设是，在这种新的结构状态下，2F5和4E10识别的高度保守序列的出现，为发现和开发新的免疫原来诱导广泛反应性中和抗体开辟了一条有希望的途径。本研究的具体目的是：(1)鉴定和开发包含2F5和4E10表位的C55 coil -coil结构域的稳定版本。我们将识别并将极性侧链取代纳入二聚体的疏水核心，以稳定线圈状结构。我们还将筛选半胱氨酸取代，以在两个相互作用的螺旋之间引入二硫键。我们的重点是产生稳定的分子，既保留二聚体构象，又保留膜近端区域的临界表面暴露残留物。(2)评价稳定的C55螺旋变异体在小动物体内引起的免疫应答。我们将在兔子和豚鼠身上进行免疫原性研究，以确定稳定的二聚体结构是否可以引发中和抗体。这些研究将涉及使用可溶性蛋白质对动物进行免疫接种。我们还将评估稳定的盘绕线圈分子捕获到纳米级珠上作为颗粒免疫原的免疫原性。

DESCRIPTION (provided by applicant): The development of immunogens capable of eliciting broadly neutralizing antibodies to HIV-1 remains one of the most difficult challenges confronting biomedical research today. Nonetheless, a small number of human monoclonal antibodies (mAbs) have been isolated from HIV-1-infected individuals that can neutralize primary isolates of HIV-1 from different genetic subtypes. Of these rare mAbs, two (2F5 and 4E10) recognize epitopes on the membrane-proximal region of the gp41 ectodomain. However, all attempts to elicit comparable anti-gp41 antibodies by vaccination with peptides or simple linear epitopes have thus far failed. Our approach to immunogen design is guided by our recent identification and structural determination of a novel two-stranded coiled-coil domain (C55) that includes the membrane-proximal epitope region of gp41. However, we have determined that the C55 dimer structure is too unstable to be suitable for immunogenicity studies. The overall goal of this research plan is to gain a detailed understanding of the structural and thermodynamic properties of the C55 coiled-coil domain, and to use this knowledge to design and produce stable immunogens for the elicitation of broadly neutralizing HIV-1 antibodies. Our central hypothesis is that presentation of the highly conserved sequences recognized by 2F5 and 4E10 in this new structural state opens a promising avenue for the discovery and development of novel immunogens to induce broadly reactive neutralizing antibodies. Specific aims of this research are: (1) To identify and develop stabilized versions of the C55 coiled-coil domain encompassing the 2F5 and 4E10 epitopes. We will identify and incorporate apolar side chain substitutions into the buried hydrophobic core of the dimer to stabilize the coiled-coil structure. We will also screen cysteine substitutions to introduce a disulfide bond between the two interacting helices. Our emphasis is to generate stable molecules that preserve both the dimer conformation and the critical surface-exposed residues of the membrane-proximal region. (2) To evaluate the immunological responses elicited by stabilized C55 coiled-coil variants in small animals. We will conduct immunogenicity studies in rabbits and guinea-pigs to determine whether the stabilized dimer structure can elicit neutralizing antibodies. These studies will involve immunization of the animals using soluble proteins. We will also evaluate the immunogenicity of the stable coiled-coil molecules captured onto nanometer-sized beads as particulate immunogens.