Development of germline-targeting HCV E2 immunogens to drive neutralizing antibody evolution

丙型肝炎病毒（HCV）的慢性感染与包括肝细胞癌在内的严重肝脏疾病相关，已成为肝移植的主要原因之一。最近获得许可的抗病毒联合治疗达到治愈率大于95%。然而，治疗是昂贵的，并不能防止病毒再次感染。此外，许多患者没有得到诊断，也没有意识到自己的感染，病毒传播率仍然很高。因此，开发预防性HCV疫苗是一个未满足的医疗需求。越来越多的证据支持中和抗体在自然感染期间清除HCV的重要性。值得注意的是，20-30%的暴露个体自然清除感染，中和抗体可以通过免疫接种引发，这增加了开发预防性HCV疫苗的希望。然而，血清中诱导的抗体应答在广度和效力方面通常是适度的，尽管已经鉴定出具有高效力的广泛中和的单克隆人和鼠抗体（bnAb）。此类bnAb在需要克隆选择和亲和力成熟以优化抗体-抗原相互作用的微调的长过程中通过抗原结合区的体细胞超突变从所谓的推断生殖系B细胞受体（iGL BCR）发展而来。靶向HCV糖蛋白的许多bnAb的序列分析揭示，亲和力成熟需要很少的体细胞突变，这表明使用所谓的生殖系靶向免疫原，即，靶向特异性iGL BCR的工程化免疫原可以增强bnAb的诱导。因此，本项目的目标是提供概念验证，即使用此类工程化的生殖系靶向免疫原可增强相应bnAb的开发，并可能增加血清的中和能力。我们将重组HCV糖蛋白开发成生殖系靶向免疫原，其以更高的亲和力与iGL BCR结合，因此具有更高的刺激相应B细胞的能力。为此，我们将并行使用1）基于结构的蛋白质工程策略和2）基于文库的表面展示策略。我们将在最近开发的产生人抗体的转基因人源化小鼠模型（埃尔朗格mighty mouse antibody platform; EMMA）中研究所得免疫原的免疫原性。这些小鼠将使我们能够通过单细胞B细胞测序来跟踪抗体成熟，从而评估单个免疫原对bnAbs发展的影响。我们的研究结果将有助于更好地了解HCV和人类免疫系统之间的相互作用，以及通过靶向刺激某些B细胞群体对后者的可操作性。因此，它们将为开发新的预防和治疗疫苗以对抗HCV感染铺平道路。

Chronic infection by the hepatitis C virus (HCV) is associated with severe liver disease including hepatocellular carcinoma and it has become one of the leading causes for liver transplantation. Recently licensed antiviral combination therapies reach cure rates greater than 95%. However, treatment is costly and does not protect from viral re-infection. Moreover, many patients are not diagnosed and unaware of their infection, and virus transmission remains high. Therefore, the development of a prophylactic HCV vaccine is an unmet medical need. Accumulating evidence underpins the importance of neutralizing antibodies for the clearance of HCV during natural infection. Notably, 20-30% of exposed individuals naturally clear the infection and neutralizing antibodies can be triggered by immunization, raising hopes that a prophylactic HCV vaccine can be developed. However, the induced antibody responses in serum are usually modest with regard to breadth and potency, although broadly neutralizing monoclonal human and murine antibodies (bnAbs) with high potency have been identified. Such bnAbs develop from the so-called inferred germline B cell receptor (iGL BCR) via somatic hypermutation of the antigen-binding regions in a long process that requires clonal selection and affinity maturation for an optimized fine-tuning of the antibody-antigen interaction. Sequence analysis of numerous bnAbs targeting HCV glycoproteins revealed that few somatic mutations are required for affinity maturation, indicating that the usage of so-called germline-targeting immunogens, i.e., engineered immunogens targeting a specific iGL BCR, could potentiate the induction of bnAbs. The goal of this project is therefore to provide the proof-of-concept that usage of such engineered germline-targeting immunogens results in an enhanced development of the respective bnAb and potentially an increased neutralization capacity of serum. We will develop recombinant HCV glycoproteins into germline-targeting immunogens that bind to iGL BCR with higher affinity and therefore have a higher capacity to stimulate the corresponding B cells. For this purpose we will use in parallel 1) a structure-based protein engineering strategy and 2) a library-based surface display strategy. We will investigate the immunogenicity of the resulting immunogens in a recently developed transgenic humanized mouse model that produces human antibodies (Erlanger mighty mouse antibody platform; EMMA). These mice will allow us to follow antibody maturation by single cell B cell sequencing and therefore assess the impact of individual immunogens on the development of bnAbs.Our results will contribute to a better understanding of the interplay between HCV and the human immune system as well as the manipulability of the latter by targeted stimulation of certain B cell populations. They will therefore pave the way for the development of a novel prophylactic and therapeutic vaccine to combat HCV infection.