Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, California, United States of America.
Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, California, United States of America.
PLoS One. 2020 May 29;15(5):e0233866. doi: 10.1371/journal.pone.0233866. eCollection 2020.
Several candidate HIV subunit vaccines based on recombinant envelope (Env) glycoproteins have been advanced into human clinical trials. To facilitate biopharmaceutical production, it is necessary to produce these in CHO (Chinese Hamster Ovary) cells, the cellular substrate used for the manufacturing of most recombinant protein therapeutics. However, previous studies have shown that when recombinant Env proteins from clade B viruses, the major subtype represented in North America, Europe, and other parts of the world, are expressed in CHO cells, they are proteolyzed and lack important glycan-dependent epitopes present on virions. Previously, we identified C1s, a serine protease in the complement pathway, as the endogenous CHO protease responsible for the cleavage of clade B laboratory isolates of -recombinant gp120s (rgp120s) expressed in stable CHO-S cell lines. In this paper, we describe the development of two novel CHOK1 cell lines with the C1s gene inactivated by gene editing, that are suitable for the production of any protein susceptible to C1s proteolysis. One cell line, C1s-/- CHOK1 2.E7, contains a deletion in the C1s gene. The other cell line, C1s-/- MGAT1- CHOK1 1.A1, contains a deletion in both the C1s gene and the MGAT1 gene, which limits glycosylation to mannose-5 or earlier intermediates in the N-linked glycosylation pathway. In addition, we compare the substrate specificity of C1s with thrombin on the cleavage of both rgp120 and human Factor VIII, two recombinant proteins known to undergo unintended proteolysis (clipping) when expressed in CHO cells. Finally, we demonstrate the utility and practicality of the C1s-/- MGAT1- CHOK1 1.A1 cell line for the expression of clinical isolates of clade B Envs from rare individuals that possess broadly neutralizing antibodies and are able to control virus replication without anti-retroviral drugs (elite neutralizer/controller phenotypes). The Envs represent unique HIV vaccine immunogens suitable for further immunogenicity and efficacy studies.
几种基于重组包膜（Env）糖蛋白的候选 HIV 亚单位疫苗已被推进到人体临床试验中。为了促进生物制药生产，有必要在 CHO（中国仓鼠卵巢）细胞中生产这些糖蛋白，CHO 细胞是用于制造大多数重组蛋白治疗药物的细胞基质。然而，先前的研究表明，当来自 B 型病毒的重组 Env 蛋白在 CHO 细胞中表达时，它们会被蛋白水解，并且缺乏存在于病毒粒子上的重要糖依赖性表位。以前，我们鉴定出补体途径中的 C1s 作为内源性 CHO 蛋白酶，负责切割在稳定的 CHO-S 细胞系中表达的 B 型实验室分离株-rgp120s（rgp120s）的裂解。在本文中，我们描述了两种新型 CHO-K1 细胞系的开发，这些细胞系通过基因编辑使 C1s 基因失活，适合生产任何易受 C1s 蛋白水解作用影响的蛋白质。一种细胞系 C1s-/-CHOK1 2.E7 含有 C1s 基因缺失。另一种细胞系 C1s-/-MGAT1-CHOK1 1.A1 含有 C1s 基因和 MGAT1 基因的缺失，这限制了糖基化至甘露糖-5 或 N-连接糖基化途径中的早期中间体。此外，我们比较了 C1s 和凝血酶对 rgp120 和人 Factor VIII 的切割的底物特异性，这两种重组蛋白在 CHO 细胞中表达时已知会发生意外的蛋白水解（剪接）。最后，我们证明了 C1s-/-MGAT1-CHOK1 1.A1 细胞系在表达具有广泛中和抗体的稀有个体的 B 型 clade Envs 临床分离株中的实用性和实用性，这些个体能够在没有抗逆转录病毒药物的情况下控制病毒复制（精英中和/控制器表型）。Env 代表独特的 HIV 疫苗免疫原，适合进一步进行免疫原性和疗效研究。
