Computational design of novel antigens targeting mature and germline b12

针对成熟和种系 b12 的新型抗原的计算设计

• 批准号: 8463113
• 负责人: WILLIAM R. SCHIEF
• 金额: $ 63.97万
• 依托单位: SEATTLE BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8463113
• 关键词: Address; Affect; Affinity; Antibodies; Antibody Binding Sites; Antigen Targeting; Antigens; B-Lymphocytes; Binding; Binding Sites; Complex; Dissociation; Engineering; Epitopes; Feedback; Grant; HIV; HIV Envelope Protein gp120; HIV vaccine; Human; Immunization; In Vitro; Infusion procedures; Kinetics; Libraries; Macaca; Mannose; Membrane; Mus; Mutagenesis; Names; Oryctolagus cuniculus; Polysaccharides; Proteins; Receptors, Antigen, B-Cell; Scaffolding Protein; Serum; Specificity; Structure; Testing; Transplantation; V3 Loop; Variant; Yeasts; antigen binding; base; crosslink; design; directed evolution; env Gene Products; human monoclonal antibodies; in vivo; neutralizing antibody; novel; particle; scaffold; screening; simian human immunodeficiency virus; vaccine development

Here we focus on immunogen design to elicit b12-like antibodies against the conserved cd4 binding site (cd4bs) of HIV Envelope. Many different engineered variants of HIV Envelope have failed to elicit such antibodies, but here we develop two novel types of b12 antigen that have not previously been tested - non-HIV protein scaffolds onto which the b12 epitope has been transplanted, and minimized, stabilized variants of core gp120. Further, we employ a combination of computational protein design and yeast display directed evolution that has not been employed for b12 antigen design previously. We will test the following hypotheses: (i) to induce b12-like antibodies rather than non- or narrowly-neutralizing antibodies against the cd4bs, it will be necessary to design antigens that bind b12 but not other cd4bs antibodies (ii) to optimally stimulate b12 B-cells and elicit b12-like antibodies will require antigens that stabilize the structure of the b12 epitope and optimize the affinity and kinetics of the antigen-b12 interaction (iii) to stimulate naive B cells to develop into those producing the mature broadly-neutralizing form of b12, antigens that bind both germline b12 and mature b12 will be required; (iv) to maximally stimulate b12 B cells by crosslinking B cell receptors, it will be necessary to multimerize b12-antigens on particles in an oriented fashion with the epitope facing outward. Our (Project 1) design efforts will be informed by structural and biophysical analysis of b12 antigens and their interactions with b12 and with mouse MAbs elicited by designed antigens (Project 2), by analysis of b12 antigen stimulation of B cells in vitro and in vivo (Project 3), and by binding specificity and neutralization analysis of rabbit sera elicited by selected b12 antigens (Core B).

在这里，我们重点关注免疫原设计，以引发针对 HIV 包膜保守 cd4 结合位点 (cd4bs) 的 b12 样抗体。 HIV 包膜的许多不同的工程变体未能引发此类抗体，但在这里，我们开发了两种以前未测试过的新型 b12 抗原 - 已移植 b12 表位的非 HIV 蛋白支架，以及最小化的稳定变体核心GP120。此外，我们采用了计算蛋白质设计和酵母展示定向进化的组合，这在之前的 b12 抗原设计中尚未采用。我们将测试以下假设：（i）为了诱导 b12 样抗体而不是针对 cd4bs 的非中和或窄中和抗体，有必要设计结合 b12 但不结合其他 cd4bs 抗体的抗原（ii）以最佳地刺激b12 B 细胞并引发 b12 样抗体将需要稳定 b12 表位结构并优化抗原 b12 的亲和力和动力学的抗原相互作用 (iii) 刺激初始 B 细胞发育成产生成熟广泛中和形式的 b12 的细胞，b12 是结合两种种系的抗原 需要b12和成熟的b12； (iv) 为了通过交联 B 细胞受体最大限度地刺激 b12 B 细胞，有必要以表位朝外的定向方式在颗粒上多聚化 b12 抗原。我们（项目 1）的设计工作将通过对 b12 抗原及其与 b12 和设计抗原引发的小鼠单克隆抗体的相互作用进行结构和生物物理分析（项目 2），通过分析 b12 抗原对 B 细胞的体外和体内刺激进行分析（项目 3），并通过对选定的 b12 抗原引发的兔血清进行结合特异性和中和分析（核心 B）。