Production of SARS-CoV-2 spike protein receptor binding domain variants in Nicotiana tabacum Bright Yellow 2 suspension cells

Details

Supervisors

Chaumont, François ; Peeters, Marie

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a virus from the Coronaviridae family, responsible for the Coronavirus disease 2019 (COVID-19), and that leads to several variants. The virus is composed of four structural proteins, namely the spike (S), membrane, envelope and nucleocapsid proteins. The S protein allows the virus to enter into host cells by binding to the human angiotensin-converting enzyme 2 (hACE2) receptor. It is a highly glycosylated trimer, with 66 N-linked glycosylation sites. These glycans play an important role in different properties of the protein, notably, its conformational changes. The domain of the S protein that interacts with the hACE2 receptor is the receptor binding domain (RBD), which presents different mutations between different SARS-CoV-2 variants. This work focused on the production of the RBD from three variants: the delta variant, the omicron variant, and a third variant, named the SARS-CoV variant, which consists in the reintroduction of the N370 glycosylation site. This N-glycosylation site was present in SARS-CoV RBD, but not in the SARS-CoV-2 RBD. To produce the different RBD proteins, two molecular farming hosts were used, namely Nicotiana benthamiana leaves and Nicotiana tabacum Bright Yellow 2 (BY-2) suspension cells. BY-2 suspension cells offer several advantages over traditional production platforms, including low cultivation and purification costs, low risk contamination and the ability to produce complex proteins or proteins requiring complex post-translational modifications. The three variants were transiently produced in N. benthamiana leaves, and it was observed that the delta RBD was better produced than the other variants. Subsequently, the delta RBD was produced in wild-type (wt) and several engineered (RDR-KO 12.6 and RDR-KO 22 lines) and glycoengineered BY-2 cell lines (XylT/FucT-KO 29 and GnTI/FucT-KO 10 Cre 16 lines). These two glycoengineered cell lines are expected to give rise to proteins bearing N-glycans devoid of β1,2-xylose and α1,3-fucose residues, and high-mannose N-glycans, respectively. The glycoengineered lines gave a higher delta RBD yield as compared to the wt line. Finally, a first analysis of the N-glycans present on the delta RBD produced in the different glycoengineered cell lines confirmed the expected N-glycosylation profile of the RBD.