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: 8404028
• 负责人: Benjamin Jacob Doranz
• 金额: $ 30万
• 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 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8404028
• 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. 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.

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。 公共卫生相关性：该项目将通过鉴定改进的gp140免疫原作为候选疫苗，为人类健康做出贡献。一种改进的gp140免疫原，其作为纯的、共价结合的三聚体是稳定的，并且代表天然gp160结构，因为它以其受体结合前、融合前构象存在于病毒体和细胞上，可能引发针对HIV的有效和广泛中和的抗体应答。