Institute of Immunity and Transplantation, Division of Infection and Immunity, The Royal Free Hospital, University College London, London, UK.
Department of Medical Microbiology, Amsterdam Infection & Immunity Institute, Amsterdam UMC, Amsterdam, University of Amsterdam, Amsterdam, The Netherlands.
J Gen Virol. 2021 Jan;102(1). doi: 10.1099/jgv.0.001512.
Great strides have been made in understanding and treating hepatitis C virus (HCV) thanks to the development of various experimental systems including cell-culture-proficient HCV, the HCV pseudoparticle system and soluble envelope glycoproteins. The HCV pseudoparticle (HCVpp) system is a platform used extensively in studies of cell entry, screening of novel entry inhibitors, assessing the phenotypes of clinically observed E1 and E2 glycoproteins and, most pertinently, in characterizing neutralizing antibody breadth induced upon vaccination and natural infection in patients. Nonetheless, some patient-derived clones produce pseudoparticles that are either non-infectious or exhibit infectivity too low for meaningful phenotyping. The mechanisms governing whether any particular clone produces infectious pseudoparticles are poorly understood. Here we show that endogenous expression of CD81, an HCV receptor and a cognate-binding partner of E2, in producer HEK 293T cells is detrimental to the infectivity of recovered HCVpp for most strains. Many HCVpp clones exhibited increased infectivity or had their infectivity rescued when they were produced in 293T cells CRISPR/Cas9 engineered to ablate CD81 expression (293T). Clones made in 293T cells were antigenically very similar to their matched counterparts made parental cells and appear to honour the accepted HCV entry pathway. Deletion of CD81 did not appreciably increase the recovered titres of soluble E2 (sE2). However, we did, unexpectedly, find that monomeric sE2 made in 293T cells and Freestyle 293-F (293-F) cells exhibit important differences. We found that 293-F-produced sE2 harbours mostly complex-type glycans whilst 293T-produced sE2 displays a heterogeneous mixture of both complex-type glycans and high-mannose or hybrid-type glycans. Moreover, sE2 produced in 293T cells is antigenically superior; exhibiting increased binding to conformational antibodies and the large extracellular loop of CD81. In summary, this work describes an optimal cell line for the production of HCVpp and reveals that sE2 made in 293T and 293-F cells are not antigenic equals. Our findings have implications for functional studies of E1E2 and the production of candidate immunogens.
由于各种实验系统的发展，包括能够在细胞中培养的丙型肝炎病毒（HCV）、HCV 假病毒系统和可溶性包膜糖蛋白，人们在理解和治疗丙型肝炎病毒方面取得了重大进展。HCV 假病毒（HCVpp）系统是广泛用于研究细胞进入、筛选新型进入抑制剂、评估临床观察到的 E1 和 E2 糖蛋白表型的平台，最相关的是，该系统用于描述接种疫苗和自然感染后诱导的中和抗体广度。尽管如此，一些患者来源的克隆产生的假病毒要么无感染性，要么感染性太低，无法进行有意义的表型分析。目前尚不清楚哪些特定的克隆会产生有感染性的假病毒。在这里，我们发现，在产生 HCVpp 的 HEK 293T 细胞中，内源性表达 HCV 的受体 CD81及其与 E2 的同源结合伴侣，对大多数毒株的恢复 HCVpp 的感染性有害。许多 HCVpp 克隆在 293T 细胞中表达时，其感染性增加或恢复感染性（293T 细胞中经 CRISPR/Cas9 工程敲除 CD81 表达）。在 293T 细胞中产生的克隆与在亲本细胞中产生的匹配克隆在抗原上非常相似，并且似乎遵循公认的 HCV 进入途径。CD81 的缺失并没有显著增加回收可溶性 E2（sE2）的滴度。然而，出乎意料的是，我们发现，在 293T 细胞和 Freestyle 293-F（293-F）细胞中产生的单体 sE2 存在重要差异。我们发现，293-F 产生的 sE2 主要含有复杂型糖，而 293T 产生的 sE2 显示出复杂型糖和高甘露糖或杂合型糖的混合。此外，在 293T 细胞中产生的 sE2 具有更好的抗原性；表现出与构象抗体和 CD81 大细胞外环的结合增加。总之，这项工作描述了一种用于生产 HCVpp 的最佳细胞系，并揭示了在 293T 和 293-F 细胞中产生的 sE2 不是抗原等价物。我们的发现对 E1E2 的功能研究和候选免疫原的生产具有重要意义。
