Engineering of CHO Cells for the Production of Recombinant Glycoprotein Vaccines with Xylosylated N-glycans

Abstract

Xylose is a general component of O-glycans in mammals. Core-xylosylation of N-glycans is only found in plants and helminth. Consequently, xylosylated N-glycans cause immunological response in humans. We have used the F-protein of the human respiratory syncytial virus (RSV), one of the main causes of respiratory tract infection in infants and elderly, as a model protein for vaccination. The RSV-F protein was expressed in CHO-DG44 cells, which were further modified by co-expression of β1,2-xylosyltransferase from Nicotiana tabacum. Xylosylation of RSV-F N-glycans was shown by monosaccharide analysis and MALDI-TOF mass spectrometry. In immunogenic studies with a human artificial lymph node model, the engineered RSV-F protein revealed improved vaccination efficacy.

Analysis of the N-Glycoforms and Immunoactivity of Chinese Yam (Dioscorea opposita Thunb.) Glycoprotein 30CYGP

“…However, other studies have reported that soybean glycoprotein contained a high-mannose-type N -glycan proportion (76%), while the complex type N -glycans were lower (24%) . Additionally, the storage glycoprotein N -glycan MMXF3 derived from Ginkgo seeds has been shown to continuously increase during the entire period of seed development; however, high-mannose-type N -glycan content was most abundant in the mature seed. ,, Furthermore, research using glycoengineered cells showed that the RSV-F glycoprotein containing xylosylated N -glycans displayed stronger immunogenicity than non-xylosylated N -glycans . We used the NaOH-PMP method developed by our lab to release the O -glycan of the yam glycoprotein 30CYGP .…”

“…21,32,33 Furthermore, research using glycoengineered cells showed that the RSV-F glycoprotein containing xylosylated N-glycans displayed stronger immunogenicity than non-xylosylated N-glycans. 34 We used the NaOH-PMP method developed by our lab to release the O-glycan of the yam glycoprotein 30CYGP. 35 As a result, Oglycan was not detected via mass spectrometry but contained a special form of N-glycan modified only by xylose and fucose that may be related to the observed biological activity.…”

In Vitro Evaluation of Glycoengineered RSV-F in the Human Artificial Lymph Node Reactor

“…Cells for expression of soluble RSV-F were obtained according to Sandig et al [ 5 ]. In brief, a cDNA was constructed containing the N-terminal extracellular part of RSV-F, an N-terminal Mellitin signal sequence, and a C-terminal Factor Xa cleavage site fused with a 6xHis-tag.…”

“…However, glycans with and without fucose are both common in humans and, therefore, a direct adjuvant role is highly unlikely. This is in contrast to monosaccharides artificial to humans, e.g., N-glycan-bound xylose, which may work as adjuvants [ 4 , 5 , 6 ].…”

Cell Line Techniques and Gene Editing Tools for Antibody Production: A Review

“…The benefits of cell line engineering are however not limited to antibody production only, but have also been explored in production of other biopharmaceutical products such as other recombinant proteins, vaccines, fusion proteins, growth factors etc. ( Xiao et al, 2014 ; Genzel, 2015 ; Johari et al, 2015 ; Sandig et al, 2017 ). Cell engineering approaches have also been used to obtain a desired attribute or quality in the target product produced by a cell, reduce those host cell proteins which could act as impurity and adversely impact the final product during downstream processing, or identify of an optimal site in host cell genome in order to target a transgene to that location ( Gadgil, 2017 ).…”