Engineered Envelope Glycoprotein Trimers for HIV-1 Vaccine Immunogens

描述（由申请人提供）：开发有效HIV-1疫苗的一个主要障碍是难以引发广泛中和抗体应答。包膜糖蛋白（Env）复合物是中和抗体的唯一HIV-1靶标，以不同的构象状态存在于细胞或病毒体表面。Env的不同寡聚体形式被认为反映了gp 41胞外域中两个七重重复（HR）区域的多形性相互作用。密集的免疫原设计工作已经指向创建可溶性重组Env（gp 140）蛋白，其形成在功能性尖峰中发现的成熟三聚体结构。这一建议直接源于我们最近对gp 41的C-末端HR 1区域的两种状态的晶体结构的测定：一种是三聚体卷曲螺旋，另一种是四聚体卷曲螺旋。我们的初步结果支持这样的假设，即这种结构二态性可以影响寡聚Env复合物的稳定性和特异性。我们建议使用这些见解，工程师和生产稳定形式的切割HIV-1 gp 140三聚体作为新的候选免疫原诱导广泛中和抗体。本研究的具体目的是：（1）制备稳定的、裂解的HIV-1 gp 140三聚体的均质制剂，用于免疫原性研究。我们将使用突变分析来剖析gp 41 HR 1序列基序中两个新发现的寡聚卷曲螺旋相互作用的决定因素。我们将建立在摩尔的SOS方法，通过选择和测试上述确定的稳定gp 41亚基间相互作用的HR 1取代，制备三聚体的稳定形式的gp 140。我们还将对gp 140三聚体进行修饰，以减少多态性和/或灵活性。(2)在小动物中评价稳定、裂解的HIV-1 gp 140三聚体的免疫原性，并使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gp 41-gp 41相互作用的稳定性是否影响HIV-1 gp 140三聚体引发中和抗体的能力。我们还将评估三聚体gp 140复合物作为颗粒免疫原捕获到纳米大小的珠子上的免疫原性。 公共卫生相关性：研究和公共卫生界一致认为，预防性疫苗是控制全球HIV-1流行病的明显长期解决方案（1-4）。不幸的是，这一目标已被证明是难以捉摸的，没有这样的疫苗可用。克服这一重要的生物医学问题将需要新的和富有想象力的设计策略，使我们更接近成功的艾滋病疫苗的目标。

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.