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

 描述(由申请人提供)：这项拨款申请回应了PA-11-110号计划(Ruth L.Kirschstein国家研究服务奖授予个人博士前医学博士/博士学位和其他双博士学位研究员)，建议开发新的进化平台并应用计算建模策略来设计可溶的HIV gp140三聚体和表位-支架模拟，提高关键功能保守的四元广义中和抗体(BNAb)表位的稳定性和可获得性。这些集中于表位的抗原有望比天然病毒三聚体更强地诱导广泛的中和反应，天然病毒三聚体通过提供对功能关键表位的有限访问来逃避免疫识别。针对表位的抗原可用作疫苗候选、分离新的bNAbs的诱饵，以及用于表征血清抗体反应的表位探针。为了设计以表位为中心的三聚体，将开发一个酵母展示三聚体gp140外膜的平台，并用于产生随机和合理突变的三聚体文库。这些文库将进化为增强与第四系表位特异性bNAbs的结合，以分离出专注于表位的候选三聚体。为了设计专注于表位的模拟，将应用合作者开发的计算蛋白质剥离策略来生成重新连接的四元表位的表位支架。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抗的结合。然后，表现最好的候选者将被带到小鼠的免疫原性研究中，以确定引发的抗体反应是否针对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，我们预计将产生一个广泛适用的多聚体蛋白质定向进化的新平台，一个根据我们的结果用于产生重新连接的表位-支架模拟物的改进的计算蛋白质剥离方法，以及多个以表位为重点的第四系候选疫苗。这些进展将为全球社会服务，为艾滋病毒疫苗的开发提供战略和候选方案，并改进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.