Structure-guided vaccine design of HCV E1E2 to induce broadly neutralizing antibodies (bNAbs)

摘要-项目1 一种有效的预防慢性感染的丙型肝炎疫苗需要在短期内诱导强大的T和B细胞反应 暴露在易感宿主后，这款U19的总体目标是。项目1的重点是以结构为导向的方法来开发一种免疫原，以增加诱导的中和效力/亲和力 针对保守表位，特别是针对这些表位的广谱中和抗体(BNAbs) 与中和协同作用有关。它将是一种优化的突变体(MT)E1E2异源二聚体，可以保持 多个bNAb表位的天然构象结构，以最大限度地减少病毒逃逸。不断增长的 关于人类单抗(HMAbs)可能产生的E2晶体结构的信息 等人已经分离出并鉴定为bNAbs，为分子研究提供了坚实的基础 理性设计中的方法。一个关键的和补充的因素是高分辨率的可用性， E2和E1E2中bNAb和非中和表位的功能图谱。我们已经开发了一个大型的 关于E2免疫原域的数据库，该数据库是通过与一大批 E2bNAbs。我们还开发了一大批针对E1E2的bNAbs，这将有助于对 项目4中的E1E2异二聚体。此外，项目1建立在与Native的实质性成果的基础上 重组野生型E1E2异源二聚体作为丙型肝炎疫苗的研究我们已经开发出了必要的 生产和纯化过程，检测表明，我们的wt E1E2表达了许多bNAbs 免疫小鼠、豚鼠、黑猩猩和人类产生的抗血清中含有 BNAbs。然而，对某些2型、3型和7型丙型肝炎病毒分离株的保护范围存在差距。 因此，一个基准是设计一种mtE1E2，它能诱导出更能中和这些基因分离株的抗血清。 同时保持对其他基因型分离株的高水平中和，通过wt E1E2实现。我们 建议通过以下具体目标来实现这一目标：目标1.以结构为导向的建模以重新设计 E1E2结构以优化E1E2bNAb表位的呈现；目的2.表达、纯化和 修饰/突变的E1E2异源二聚体的特性；和目的3.免疫学特性 候选E1E2疫苗以选择优化的E1E2。最后，我们的目标是将优化后的E1E2与强大的 增强中和抗体效价的佐剂(项目3)。目标4是免疫学上的 NHP接受针对bNAbs和T细胞反应优化的丙型肝炎疫苗的特征

ABSTRACT - PROJECT 1 An effective HCV vaccine to prevent chronic infection will need to induce robust T and B cell responses shortly after exposure in the susceptible host, the overall goal of this U19. Project 1 focuses on a structure-guided approach to develop an immunogen that increases the neutralization potency/affinity of elicited broadly neutralizing antibodies (bNAbs) to conserved epitopes and particularly to those epitopes associated with neutralization synergy. It will be an optimized mutant (mt) E1E2 heterodimer that maintains native conformational structures for multiple bNAb epitopes to minimize viral escape. The increasing information on E2 crystal structures made possible by human monoclonal antibodies (HMAbs) that we and others have isolated and characterized as bNAbs, provides a strong foundation for the molecular approaches in rational design. A critical and complementary element is the availability of a high-resolution, functional map of both bNAb and non-neutralizing epitopes in E2 and E1E2. We have developed a large database on immunogenic domains on E2 that is derived from epitope mapping studies with a large panel of E2 bNAbs. We have also developed a large panel of bNAbs to E1E2 that will contribute to structural studies on E1E2 heterodimer in Project 4. In addition, Project 1 is built on substantial achievements with native recombinant wild-type (wt) E1E2 heterodimer as a vaccine against HCV. We have developed the necessary production and purification processes and tested to show that our wt E1E2 expresses many bNAbs epitopes and that antisera generated from vaccinated mice, guinea pigs, chimpanzees and humans contains bNAbs. However, there are gaps in the breadth of protection against some genotypes 2, 3 and 7 HCV isolates. Thus, a benchmark is to design a mt E1E2 that elicits antisera more able to neutralize these genotype isolates while maintaining a high level of neutralization towards the other genotype isolates achieved with wt E1E2. We propose to accomplish this goal by the following Specific Aims: Aim 1. Structure-guided modeling to redesign E1E2 structures to optimize presentation of E1E2 bNAb epitopes; Aim 2. Expression, purification, and characterization of modified/mutant E1E2 heterodimers; and Aim 3. Immunological characterization of candidate E1E2 vaccines to select optimized E1E2. Finally, we aim to combine optimized E1E2 with powerful adjuvants to enhance neutralizing antibody titers (Project 3). Aim 4 is then the immunological characterization of NHP receiving an HCV vaccine optimized for bNAbs and T cell responses