Novel evolutionary platforms and computational strategies for the engineering of quaternary epitope-focused HIV immunogens

 描述（由申请人提供）：本赠款申请响应计划公告编号PA-11-110（露丝L。Kirschstein National Research Service Awards for Individual Predoctoral MD/PhD and Other Dual Doctoral Degree Fellows），提出开发新的进化平台，并应用计算建模策略设计可溶性HIV gp 140三聚体和表位支架模拟物，提高关键功能保守的四元广泛中和抗体（bNAb）表位的稳定性和可及性。这些表位集中的抗原预期比天然病毒三聚体更强地引发广泛中和反应，天然病毒三聚体通过提供对功能关键表位的有限接近来逃避免疫识别。表位聚焦抗原可用作候选疫苗、用于分离新bNAb的诱饵和表征血清抗体应答的表位探针。为了工程化表位聚焦的三聚体，将开发用于酵母展示三聚体gp 140包膜的平台，并用于产生随机和合理突变的三聚体文库。这些文库将被进化以增强与四级表位特异性bNAb的结合，以分离表位聚焦的候选三聚体。为了设计以表位为中心的模拟物，合作者开发的计算蛋白质剥离策略将被应用于生成四级表位的重布线表位支架。我们将生产并测试所设计的表位支架的表达、稳定性、聚集倾向以及与构象单克隆bNAb的结合。然后将表现最好的候选物进行小鼠免疫原性研究，以确定引发的抗体应答是否靶向预期的表位并识别天然折叠的三聚体。到拟议的资助期结束时，我们预计已经产生了一个广泛适用的新平台，用于多聚体蛋白的定向进化，一种改进的计算蛋白质剥离方法，用于产生由我们的结果所告知的重连接表位-支架模拟物，以及多个以四元表位为重点的候选疫苗。这些进展将为全球社会服务，为开发艾滋病毒疫苗提供战略和候选疫苗，并改善保护性广泛中和抗体反应的激发。

 DESCRIPTION (provided by applicant): This grant application responds to Program Announcement Number PA-11-110 (Ruth L. Kirschstein National Research Service Awards for Individual Predoctoral MD/PhD and Other Dual Doctoral Degree Fellows) by proposing to develop novel evolutionary platforms and apply computational modeling strategies to engineer soluble HIV gp140 trimers and epitope-scaffold mimetics with improved stability and accessibility of key functionally conserved quaternary broadly neutralizing antibody (bNAb) epitopes. These epitope-focused antigens are expected to elicit broadly neutralizing responses more strongly than native viral trimers, which evade immune recognition by providing limited access to functionally critical epitopes. Epitope-focused antigens may be used as vaccine candidates, baits for isolation of new bNAbs, and epitope probes to characterize serum antibody responses. To engineer epitope-focused trimers, a platform for yeast display of trimeric gp140 envelope will be developed and used to generate both randomly and rationally mutated libraries of trimers. These libraries will be evolved for enhanced binding to quaternary epitope-specific bNAbs to isolate epitope-focused candidate trimers. To engineer epitope-focused mimetics, a computational protein peeling strategy developed by collaborators will be applied to generate rewired epitope-scaffolds of quaternary epitopes. We will produce and test the designed epitope-scaffolds for expression, stability, aggregation propensity, and binding to conformational monoclonal bNAbs. The top-performing candidates will then be carried into immunogenicity studies in mice to determine whether elicited antibody responses target the intended epitopes and recognize natively folded trimers. By the end of the proposed funding period, we expect to have generated a widely applicable novel platform for directed evolution of multimeric proteins, an improved computational protein peeling method for generation of rewired epitope-scaffold mimetics informed by our results, and multiple quaternary epitope-focused vaccine candidates. These advances will serve the global community by offering both strategies towards and candidates for the development of HIV vaccines with improved elicitation of protective broadly neutralizing antibody responses.