Precision Glycoengineering of an HCV Envelope-Based Nanoparticle Vaccine

摘要 全球有7100多万人感染丙型肝炎病毒，新增1-200万人 每年都会发生感染。这一重大的健康问题需要开发有效的疫苗。 由于丙型肝炎病毒迅速积累变异，疫苗必须诱导产生广泛的中和抗体。 以可复制的方式。病毒包膜E1E2糖蛋白是天然的中和靶点 抗体。然而，生产E1E2等被膜蛋白的两个主要挑战如下。第一, 随着候选蛋白质的发展，获得稳定的、可重复的、同质的产品将是至关重要的 具有产生有效和广谱中和抗体反应的高潜力的糖体。这 确保临床前和临床试验中各批次效力相当的最佳强效疫苗；以及 使其在制造过程中更容易保持葡聚糖谱。然而，宿主细胞之间的差异，甚至 克隆，可导致糖基化的显著变化。第二，在识别出提供 想要广泛中和抗体反应，可能很难获得有效的表达宿主 以经济的方式以功能性形式生产疫苗亚单位蛋白。在这里，我们正在应对这些挑战 通过在一组具有良好特性的新的天然类分泌型E1E2(SE1E2)复合体中 糖工程哺乳动物细胞株获得更均一的糖基化 结构。在生产了所有糖形式的临床前材料后，将确定最有效的糖形式。 通过严格的生物分析，结合纳米颗粒，并接受免疫学评估 糖工程化CHO细胞系野生型CHO表达的sE1E2免疫动物的多克隆血清 HEK 293和HuH7为对照细胞系。因此，这项拟议的研究将确定最佳的糖型 并帮助建立生产有效的泛丙型肝炎疫苗的平台细胞系。

ABSTRACT Globally, more than 71 million people are infected with Hepatitis C virus (HCV), with 1-2 million new infections occurring each year. This major health concern necessitates the development of an effective vaccine. Since HCV rapidly accumulates mutations, vaccines must elicit the production of broadly neutralizing antibodies (bnAbs) in a reproducible fashion. The viral envelope E1E2 glycoprotein is a natural target of neutralizing antibodies. However, two major challenges in production of envelope proteins such as E1E2 are as follows. First, as the candidate protein advances, it will be critical to obtain a product with stable, reproducible, homogenous glycoforms that show high potential for yielding a potent and broadly neutralizing antibody response. This ensures an optimally potent vaccine with comparable efficacy across batches in preclinical and clinical trials, and makes it easier to retain the glycan profile through manufacturing. However, variation across host cells, even clones, can lead to substantial variation in glycosylation. Second, upon identifying a glycoform that provides the desired broadly neutralizing antibody response, it can be difficult to obtain an effective expression host that can economically produce the vaccine subunit proteins in a functional form. Here we are addressing these challenges by producing a well-characterized novel native-like secreted E1E2 (sE1E2) complex in a panel of glycoengineered mammalian cell lines to obtain more homogeneous glycosylation with predictable and defined structures. After production of preclinical material of all glycoforms, the most effective glycoforms will be identified by rigorous bioanalytical analysis, coupled to nanoparticles, and subjected to immunological assessment of polyclonal sera from animals immunized with sE1E2 produced in glycoengineered CHO cell lines, wild-type CHO cell lines, and HEK 293 and Huh7 control cell lines. Thus, this proposed research will identify optimal glyforms and help establish a platform cell line for manufacturing an effective pan-HCV vaccine.