Engineering Stable, Soluble, and Trimeric HIV gp140 by Paired Cys Scanning

描述(申请人提供)：当呈现合适的抗原膜(Env)结构时，人类免疫系统可以产生HIV无法逃避的保护性抗体。迄今为止已发现的许多最有效的中和HIV抗体优先结合Env的三聚体结构。因此，将Env的天然三聚体结构概括为 存在于病毒和细胞表面是开发能够产生广泛保护性体液免疫的免疫原的主要目标。开发一种可溶的、三聚体的Env结构，它概括了Env的天然融合前结构，已成为HIV疫苗发展的主要目标。然而，gp120和gp41亚单位复合体固有的不稳定性已经成为设计一种能够向免疫系统呈递有效抗原结构的可溶性Env三聚体(Gp140)的主要障碍。该项目的目标是设计一种具有二硫键的gp140变体，以稳定三聚体，产生构象完整的三聚体gp140。为了实现这个项目的目标，我们将设计和测试多达10,000个gp140变体，这些变体已经被系统地突变，以包含成对的二硫键，旨在共价稳定蛋白质。每个突变体都将在人类细胞中单独表达，以维持天然的翻译后加工，并将测试每个突变体是否保持其融合前的构象、抗原完整性和三聚体结构。 公共卫生相关性：该项目将通过确定改进的gp140免疫原作为候选疫苗，为人类健康作出贡献。作为一种改进的gp140免疫原，它作为一个纯的、共价结合的三聚体是稳定的，它代表了天然的gp160结构，因为它存在于病毒粒子和细胞上，存在于其受体前结合，融合前构象可能激发出针对HIV的有效和广泛的中和抗体反应。

DESCRIPTION (provided by applicant): When presented with a suitable antigenic Envelope (Env) structure, the human immune system can produce protective antibodies that HIV is unable to evade. Many of the most potently neutralizing HIV antibodies identified to date preferentially bind the trimeric structure of Env. Thus, recapitulating the native trimeric structure of Env as it exists on the virus and cell surface is a primary objective in developing an immunogen capable of generating broadly protective humoral immunity. The development of a soluble, trimeric Env structure that recapitulates the native, pre-fusion structure of Env has become a major goal for HIV vaccine advancement. However, the inherent instability of the gp120 and gp41subunit complex has posed a major obstacle in designing a soluble Env trimer (gp140) capable of presenting an effective antigenic structure to the immune system. The goal of this project is to engineer a gp140 variant with disulfide bonds that stabilize the trimer to yield conformationally intact, trimeric gp140. To accomplish the Aims of this project, we will engineer and test up to 10,000 gp140 variants that have been systematically mutated to contain paired disulfide bonds designed to covalently stabilize the protein. Each mutant will be individually expressed in human cells to maintain native post-translational processing, and each mutant will be tested for retention of its pre-fusion conformation, antigenic integrity, and trimeric structure. PUBLIC HEALTH RELEVANCE: This project will contribute to human health by identifying improved gp140 immunogens as vaccine candidates. An improved gp140 immunogen that is stable as a pure, covalently associated trimer and that represents the native gp160 structure as it exists on virions and cells in its pre-receptor binding, pre-fusion conformation is likely to elcit a potent and broadly neutralizing antibody response against HIV.