Structure-based design of an epitope-focused Hepatitis C virus vaccine

据估计，全世界有1.7亿人感染丙型肝炎病毒（HCV），慢性HCV感染是肝硬化和肝癌的主要原因。针对HCV的直接作用的抗病毒药物达到了令人印象深刻的治愈率;然而，它们的高成本和耐药病毒的出现加强了开发安全有效的HCV疫苗的迫切需要。最近的证据已经强调了中和抗体（nAb）在保护免于持续HCV感染中的作用。与表位肽或HCV糖蛋白E2复合的nAb片段的结构研究为合理的疫苗设计策略提供了基础。大多数nAb靶向E2内的受体结合位点（CD 81），但病毒已开发出许多机制来逃避中和抗体应答，从而阻止E2成为有效的免疫原，例如广泛的聚糖屏蔽，免疫显性“诱饵”表位和明显的构象灵活性。用于免疫原优化的新型计算设计方法为基于结构的疫苗设计铺平了道路。这些新的方法允许特定的表位移植到支架蛋白集中的抗体反应的重要中和epitopes.The项目的目标是利用这些新的计算方法来设计抗原表位为重点的免疫原，提高HCV中和抗体的诱导。我们将从HCV糖蛋白E1和E2（分别为一个和四个表位）移植中和表位，包括线性和构象表位作为两个不同的战略机会。对于这些表位中的每一个，表位聚焦的免疫原将被计算设计和实验测试。最有希望的候选人将进行生物药理学，生物化学和结构表征;我们随后将选择具有所需生物化学特征的免疫原，并测试其在小鼠中诱导特异性抗体应答的能力。我们将确定免疫后血清结合E2的能力，更重要的是，评估中和和交叉中和活性。本研究将通过分离抗原特异性记忆B细胞，在单克隆水平上研究诱导抗体应答的机制，从结构和功能两个方面对诱导抗体进行表征，为基于结构的HCV疫苗设计提供新的途径。此外，由于HCV糖蛋白内中和表位的结构数据很少，新的免疫原与靶向所展示表位的充分表征的抗体片段的共结晶将大大增加我们对体液免疫系统和HCV糖蛋白之间相互作用的理解。该项目开发的策略也可能有助于为缺乏有效疫苗的其他传染病（如艾滋病毒、流感或疟疾）设计优化的免疫原。

An estimated 170 million people worldwide are infected with the hepatitis C virus (HCV), and chronic HCV infection is a major cause of liver cirrhosis and liver cancer. The direct-acting antivirals against HCV reach impressive cure rates; however, their high-costs and the emergence of drug-resistant viruses reinforce the urgent need to develop a safe and efficient HCV vaccine. Recent evidence has highlighted the role of neutralizing antibodies (nAbs) in protection from persistent HCV infection. Structural studies of nAb fragments in complex with epitope peptides or HCV glycoprotein E2 have provided the foundation to enable rational vaccine design strategies. The majority of nAbs target the receptor-binding site (CD81) within E2, but the virus has developed a number of mechanisms to evade the neutralizing antibody response that preclude E2 from being an efficient immunogen, such as an extensive glycan shield, immunodominant "decoy" epitopes and a pronounced conformational flexibility. Novel computational design methodologies used for immunogen optimization have paved the way for structure-based vaccine design. These novel approaches allow for the transplantation of specific epitopes to scaffold proteins to focus the antibody response on important neutralization epitopes.The goal of this project is to leverage these novel computational methods to design epitope-focused immunogens that enhance the elicitation of HCV neutralizing antibodies. We will transplant neutralization epitopes from the HCV glycoproteins E1 and E2 (one and four epitopes, respectively), including linear and conformational epitopes as two distinct strategic opportunities. For each of these epitopes, epitope-focused immunogens will be computationally designed and experimentally tested. The most promising candidates will be biophysically, biochemically and structurally characterized; we will subsequently select immunogens with the desired biochemical profile and test their ability to induce a specific antibody response in mice. We will determine the capacity of post-immunization sera to bind E2 and more importantly, assess neutralizing and cross-neutralizing activity. We will investigate the induced antibody responses at the monoclonal level by isolating antigen-specific memory B cells, to characterize the elicited antibodies both structurally and functionally.The proposed project will provide a novel approach for structure-based design of an efficient HCV vaccine. In addition, as structural data on neutralization epitopes within HCV glycoproteins are scarce, co-crystallization of novel immunogens with well-characterized antibody fragments targeting the displayed epitopes will greatly increase our understanding of the interactions between the humoral immune system and the HCV glycoproteins. The strategies developed with this project may also be useful to design optimized immunogens for other infectious diseases that lack efficacious vaccines, such as HIV, Influenza, or malaria.