Engineered Envelope Glycoprotein Trimers for HIV-1 Vaccine Immunogens

描述（由申请人提供）：开发有效的HIV-1疫苗的一个主要障碍是难以引起广泛中和的抗体反应。包膜糖蛋白（Env）复合物是HIV-1中和抗体的唯一靶标，以不同的构象状态存在于细胞或病毒粒子表面。不同的Env寡聚形式被认为反映了gp41外域两个七重复（HR）区域的多形性相互作用。大量的免疫原设计工作已经指向了可溶性重组Env （gp140）蛋白的创建，该蛋白形成了功能尖峰中发现的成熟三聚体结构。这个建议直接来自于我们最近对gp41 C端HR1区域的两种状态的晶体结构的测定：一种是三聚体卷曲线圈，另一种是四聚体卷曲线圈。我们的初步结果支持这种结构二态性可以影响低聚Env复合物的稳定性和特异性的假设。我们建议利用这些见解来设计和生产稳定形式的裂解HIV-1 gp140三聚体，作为诱导广泛中和抗体的新型候选免疫原。提出的研究的具体目的是：(1)为免疫原性研究创建稳定的、裂解的HIV-1 gp140三聚体的均匀制剂。我们将使用突变分析来剖析gp41 HR1序列基序中两个新发现的低聚物卷曲线圈相互作用的决定因素。我们将在摩尔的SOS方法的基础上，通过选择和测试上述确定的稳定gp41亚基间相互作用的HR1取代，构建gp140的三聚体稳定形式。我们还将对gp140三聚体进行修改，以减少多形性和/或灵活性。(2)评价稳定的、裂解的HIV-1 gp140三聚体在小动物体内的免疫原性，并利用所得数据改进免疫原设计。我们将在家兔和豚鼠中进行免疫原性研究，以确定gp41-gp41相互作用的稳定性是否会影响HIV-1 gp140三聚体引发中和抗体的能力。我们还将评估三聚体gp140复合物作为颗粒免疫原捕获到纳米级珠上的免疫原性。

DESCRIPTION (provided by applicant): One major obstacle to the development of an effective HIV-1 vaccine is the difficulty in eliciting a broadly neutralizing antibody response. The envelope glycoprotein (Env) complex, the only HIV-1 target for neutralizing antibodies, exists in distinct conformational states on the cell or virion surface. Different oligomeric forms of Env are thought to reflect pleomorphic interactions of two heptad-repeat (HR) regions in the ectodomain of gp41. Intensive immunogen design efforts have been directed toward the creation of a soluble recombinant Env (gp140) protein that forms the mature trimeric structure found in functional spikes. This proposal grows directly out of our recent determination of the crystal structures of the two states of the C- terminal HR1 region of gp41: one, a trimeric coiled coil and the other, a tetrameric coiled coil. Our preliminary results favor the hypothesis that this structural dimorphism can inflence the stability and specificity of the oligomeric Env complex. We propose to use these insights to engineer and produce stabilized forms of the cleaved HIV-1 gp140 trimer as novel candidate immunogens for the induction of broadly neutralizing antibodies. The Specific Aims of the proposed research are: (1) To create homogeneous preparations of stabilized, cleaved HIV-1 gp140 trimers for immunogenicity studies. We will use mutational analysis to dissect the determinants of each of the two newly identified oligomeric coiled-coil interactions in the gp41 HR1 sequence motif. We will build on Moore's SOS method for making a trimeric, stabilized form of gp140 by selecting and testing the HR1 substitutions identified above that stabilize inter-subunit interactions in gp41. We will also make modifications of gp140 trimers in order to reduce pleomorphism and/or flexibility. (2) To evaluate the immunogenicity of stable, cleaved HIV-1 gp140 trimers in small animals and use the resulting data to improve immunogen design. We will conduct immunogenicity studies in rabbits and guinea-pigs to determine whether the stabilization of the gp41-gp41 interactions affect the ability of HIV-1 gp140 trimers to elicit neutralizing antibodies. We will also evaluate the immunogenicity of trimeric gp140 complexes captured onto nanometer-sized beads as particulate immunogens. PUBLIC HEALTH RELEVANCE: The research and public health communities concur that a preventive vaccine is the obvious long-term solution to bring the global HIV-1 epidemic under control (1-4). Unfortunately, this goal has proven elusive and no such vaccine is available. Overcoming this important biomedical problem will require new and imaginative design strategies to bring us closer to the goal of a successful AIDS vaccine.