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

摘要-项目1 有效预防慢性感染的HCV疫苗需要在短期内诱导强有力的T和B细胞应答 在易感宿主暴露后，本U19的总体目标。项目1的重点是一种结构导向的方法，以开发一种免疫原，增加中和效力/亲和力的诱导 针对保守表位的广泛中和抗体（bNAb）， 与中和协同作用有关。它将是一种优化的突变体（mt）E1 E2异二聚体，它维持 多个bNAb表位的天然构象结构以使病毒逃逸最小化。日益 关于E2晶体结构的信息， 和其他人已经分离并鉴定为bNAb，为分子生物学提供了强有力的基础。 合理设计的方法。一个关键的补充因素是提供高分辨率， E2和E1 E2中bNAb和非中和表位的功能图。我们开发了一个大型 E2上免疫原性结构域的数据库，该数据库来源于表位作图研究， E2 bNAb。我们还开发了一个针对E1 E2的bNAb的大型面板，这将有助于对E1 E2的结构研究。 项目4中的E1 E2异二聚体。此外，项目1是建立在与当地的实质性成就， 重组野生型（wt）E1 E2异二聚体作为针对HCV的疫苗。我们开发了必要的 我们的野生型E1 E2表达许多bNAb 免疫小鼠、豚鼠、黑猩猩和人产生的抗血清含有 bNAb。然而，在针对一些基因型2、3和7 HCV分离株的保护广度方面存在差距。 因此，一个基准是设计一种mt E1 E2，其产生的抗血清更能中和这些基因型分离株 同时保持用wt E1 E2实现的对其它基因型分离株的高水平中和。我们 我建议通过以下具体目标来实现这一目标：目标1。结构导向的重新设计建模 E1 E2结构以优化E1 E2 bNAb表位的呈递;目的2.表达、纯化和 修饰的/突变的E1 E2异二聚体的表征;和目的3.免疫学特性 候选E1 E2疫苗以选择优化的E1 E2。最后，我们的目标是将联合收割机优化的E1 E2与强大的 增强中和抗体滴度的佐剂（项目3）。目的4是免疫学 接受针对bNAb和T细胞应答优化的HCV疫苗的NHP的表征

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