Gene editing in CHO cells to prevent proteolysis and enhance glycosylation: Production of HIV envelope proteins as vaccine immunogens

• Type of Material: Article
• Authors: Sophia W. Li, University of California Santa Cruz; Meredith Wright, University of California Santa Cruz; John F. Healey, Emory University; Jennie M. Hutchinson, University of California Santa Cruz; Sara O'Rourke, University of California Santa Cruz; Kathryn Mesa, University of California Santa Cruz; John Lollar, Emory University; Phillip W. Berman, University of California Santa Cruz
• Language: English
• Date: 2020-05-29
• Publisher: Public Library of Science
• Publication Version: Final Published Version
• Copyright Statement: © 2020 Li et al.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1371/journal.pone.0233866
• Title of Journal or Parent Work: PLoS One
• Volume: 15
• Issue: 5
• Start Page: e0233866
• End Page: e0233866
• Grant/Funding Information: Funding was provided by the National Institute of Drug Abuse through Grant R01DA036335 to Phillip W. Berman. Funding was also provided by the National Institute of Allergy and Infectious Diseases through Grant R01AI113893 to Phillip W. Berman.
• Keywords: Science & Technology; Broadly neutralizing antibodies; Coagulation factor VIII; Recombinant; Structural bias; Multidisciplinary Sciences; Glycoproteins; Complement; Substrate Specificity; GP120; Science & Technology - Other Topics; Protease; Potent
• Research Categories: Biology, Cell; Biology, Genetics; Chemistry, Biochemistry; Engineering, Biomedical

Abstract

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 Nlinked 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.