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

描述（由申请人提供）：当存在合适的抗原包膜（Env）结构时，人体免疫系统可以产生HIV无法逃避的保护性抗体。迄今为止鉴定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，重现Env的天然三聚体结构，因为它 存在于病毒和细胞表面上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Env结构，其重现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亚基复合物的固有不稳定性已经在设计能够向免疫系统呈递有效抗原结构的可溶性Env三聚体（gp140）方面造成了主要障碍。该项目的目标是设计一个具有二硫键的gp140变体，以稳定三聚体，从而产生构象完整的三聚体gp140。为了实现该项目的目标，我们将设计和测试多达10，000个gp140变体，这些变体已被系统地突变为含有旨在共价稳定蛋白质的成对二硫键。每种突变体将在人细胞中单独表达，以维持天然翻译后加工，并将检测每种突变体的融合前构象、抗原完整性和三聚体结构的保留。

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.