Optimized cell systems for the investigation of hepatitis C virus E1E2 glycoproteins.
Optimized cell systems for the investigation of hepatitis C virus E1E2 glycoproteins.
复制标题
DOI：
10.1099/jgv.0.001512
复制
发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Grove J
中科院分区：
文献类型：
--
作者：
Kalemera MD;Capella-Pujol J;Chumbe A;Underwood A;Bull RA;Schinkel J;Sliepen K;Grove J
由于各种实验系统的发展，包括细胞培养熟练的HCV， HCV假颗粒系统和可溶性包膜糖蛋白，在理解和治疗丙型肝炎病毒（HCV）方面取得了巨大的进步。HCV伪颗粒（HCVpp）系统是一个广泛用于细胞进入研究、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，最重要的是，用于表征疫苗接种和患者自然感染诱导的中和抗体广度。尽管如此，一些患者衍生的克隆产生假颗粒，这些假颗粒要么没有传染性，要么表现出传染性太低，无法进行有意义的表型分析。控制任何特定克隆是否产生传染性假粒子的机制尚不清楚。本研究表明，在HEK 293T细胞中，CD81（一种HCV受体和E2的同源结合伙伴）的内源性表达对大多数菌株回收的HCVpp的感染性有害。许多HCVpp克隆在293T细胞中产生时，表现出增加的传染性或恢复的传染性，这些细胞经过CRISPR/Cas9工程改造，可以去除CD81的表达（293TCD81KO）。293TCD81KO细胞克隆在抗原性上与亲本细胞非常相似，似乎遵循公认的HCV进入途径。CD81的缺失没有明显增加可溶性E2 （sE2）的恢复滴度。然而，我们出乎意料地发现，在293T细胞和Freestyle 293-F （293-F）细胞中产生的单体sE2表现出重要的差异。我们发现293- f产生的sE2主要含有络合型聚糖，而293t产生的sE2显示出络合型聚糖和高甘露糖或杂合型聚糖的异质混合物。此外，293T细胞中产生的sE2具有抗原性优势；表现出与构象抗体的结合增加和CD81的大细胞外环。总之，本研究描述了一种生产HCVpp的最佳细胞系，并揭示了293T和293-F细胞中产生的sE2在抗原性上并不相等。我们的发现对E1E2的功能研究和候选免疫原的产生具有启示意义。
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 (293TCD81KO). Clones made in 293TCD81KO 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.
登录
查看更多内容
DOI：
10.1007/978-1-4939-8976-8_30
发表时间：
2019-01-01
期刊：
HEPATITIS C VIRUS PROTOCOLS
影响因子：
--
作者：
Bailey, Justin R.;Urbanowicz, Richard A.;Foung, Steven K. H.
通讯作者：
Foung, Steven K. H.
影响因子：
64.5
作者：
de Taeye SW;Ozorowski G;Torrents de la Peña A;Guttman M;Julien JP;van den Kerkhof TL;Burger JA;Pritchard LK;Pugach P;Yasmeen A;Crampton J;Hu J;Bontjer I;Torres JL;Arendt H;DeStefano J;Koff WC;Schuitemaker H;Eggink D;Berkhout B;Dean H;LaBranche C;Crotty S;Crispin M;Montefiori DC;Klasse PJ;Lee KK;Moore JP;Wilson IA;Ward AB;Sanders RW
通讯作者：
Sanders RW
影响因子：
5.4
作者：
Andreadis, S;Lavery, T;Morgan, JR
通讯作者：
Morgan, JR
影响因子：
5.4
作者：
Goffard, A;Callens, N;Dubuisson, J
通讯作者：
Dubuisson, J
影响因子：
4.3
作者：
Kalemera, Mphatso;Mincheva, Dilyana;Illingworth, Christopher J. R.
通讯作者：
Illingworth, Christopher J. R.