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

通过配对半胱氨酸扫描工程设计稳定、可溶和三聚体 HIV gp140

• 批准号: 8507144
• 负责人: Benjamin Jacob Doranz
• 金额: $ 24.63万
• 依托单位: INTEGRAL MOLECULAR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-06 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8507144
• 关键词: Abbreviations; Animal Testing; Antibody Formation; Antigens; Binding; Biochemical; Cell surface; Cells; Characteristics; Complex; Dengue; Detergents; Development; Diagnostic; Elements; Engineering; Ensure; Epitope Mapping; Epitopes; Extracellular Domain; Goals; HIV; HIV Antibodies; HIV Envelope Protein gp120; HIV vaccine; HIV-1; Health; Human; Humoral Immunities; Immune response; Immune system; Libraries; Mediating; Membrane; Molecular Conformation; Monoclonal Antibodies; Mutagenesis; Mutate; Mutation; Phase; Process; Property; Protein Engineering; Proteins; Scanning; Shotguns; Sodium Chloride; Structure; Surface; Technology; Temperature; Testing; Time; TimeLine; Variant; Viral; Virion; Virus; antibody-dependent cell cytotoxicity; design; disulfide bond; gp160; immunogenic; improved; mutant; neutralizing antibody; new technology; receptor binding; stability testing; vaccine candidate

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.

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。