Stabilized gp41 Structural Motif for HIV-1 Vaccine

描述(申请人提供)：开发能够诱导针对HIV-1的广泛中和抗体的免疫原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感染者身上分离出少量的人类单抗，这些抗体可以中和来自不同基因亚型的HIV-1初级分离株。在这些罕见的单抗中，有两个(2F5和4E10)识别gp41胞外结构域膜近端的表位。然而，所有尝试通过接种多肽或简单的线性表位来诱导类似的抗gp41抗体的尝试到目前为止都失败了。我们的免疫原设计方法是以我们最近鉴定和结构确定的一个新的双链螺旋卷曲结构域(C55)为指导的，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，不适合免疫原性研究。这项研究计划的总体目标是详细了解C55螺旋线圈结构域的结构和热力学性质，并利用这一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识别的高度保守的序列的出现，为发现和开发新的免疫原来诱导广泛反应的中和抗体开辟了一条有希望的途径。这项研究的具体目的是：(1)确定和开发包含2F5和4E10表位的C55螺旋线圈结构域的稳定版本。我们将识别并将非极性侧链取代引入到二聚体的埋藏的疏水核心中，以稳定卷曲螺旋结构。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，既保持二聚体的构象，又保持膜近端区域表面暴露的关键残基。(2)评价稳定的C55螺旋线圈变异体在小动物体内的免疫应答。我们将在兔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诱导中和抗体。这些研究将涉及使用可溶性蛋白质对动物进行免疫。我们还将评估作为颗粒免疫原捕获到纳米微珠上的稳定盘绕分子的免疫原性。

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.