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的中和抗体反应。包膜糖蛋白(Env)复合体是HIV-1病毒中和抗体的唯一靶标，在细胞或病毒粒子表面以不同的构象存在。不同形式的Env被认为反映了gp41胞外区两个七重复序列(HR)区域的多态相互作用。密集的免疫原设计工作一直致力于创造一种可溶的重组Env(Gp140)蛋白，这种蛋白形成在功能棘突中发现的成熟的三聚体结构。这一提议直接源于我们最近对gp41 C-末端HR1区两种态的晶体结构的测定：一种是三聚螺旋线圈，另一种是四聚螺旋线圈。我们的初步结果支持这样的假设，即这种结构二型性可以影响寡聚Env复合体的稳定性和特异性。我们建议利用这些洞察力来设计和生产稳定形式的裂解HIV-1gp140三聚体，作为诱导广谱中和抗体的新候选免疫原。拟议研究的具体目标是：(1)创造稳定的、裂解的HIV-1 gp140三聚体的均一制剂，用于免疫原性研究。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寡聚螺旋线圈相互作用的决定因素。我们将在Moore的SOS方法的基础上，通过选择和测试上面确定的稳定gp41亚基间相互作用的HR1取代来制造三聚体稳定形式的gp140。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)评估稳定的、裂解的HIV-1gp140三聚体在小动物中的免疫原性，并利用所得数据改进免疫原设计。我们将在兔和豚鼠身上进行免疫原性研究，以确定gp41-gp41相互作用的稳定性是否会影响HIV-1 gp140三聚体诱导中和抗体的能力。我们还将评估作为颗粒免疫原捕获到纳米微珠上的三聚体gp140复合体的免疫原性。

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.