Cell-Free Glycoengineering of the Recombinant SARS-CoV-2 Spike Glycoprotein

Abstract

The baculovirus-insect cell expression system is readily utilized to produce viral glycoproteins for research as well as for subunit vaccines and vaccine candidates, for instance against SARS-CoV-2 infections. However, the glycoforms of recombinant proteins derived from this expression system are inherently different from mammalian cell-derived glycoforms with mainly complex-type N-glycans attached, and the impact of these differences in protein glycosylation on the immunogenicity is severely under investigated. This applies also to the SARS-CoV-2 spike glycoprotein, which is the antigen target of all licensed vaccines and vaccine candidates including virus like particles and subunit vaccines that are variants of the spike protein. Here, we expressed the transmembrane-deleted human β-1,2 N-acetlyglucosamintransferases I and II (MGAT1ΔTM and MGAT2ΔTM) and the β-1,4-galactosyltransferase (GalTΔTM) in E. coli to in-vitro remodel the N-glycans of a recombinant SARS-CoV-2 spike glycoprotein derived from insect cells. In a cell-free sequential one-pot reaction, fucosylated and afucosylated paucimannose-type N-glycans were converted to complex-type galactosylated N-glycans. In the future, this in-vitro glycoengineering approach can be used to efficiently generate a wide range of N-glycans on antigens considered as vaccine candidates for animal trials and preclinical testing to better characterize the impact of N-glycosylation on immunity and to improve the efficacy of protein subunit vaccines.

Cell-free N-glycosylation of peptides using synthetic lipid-linked hybrid and complex N-glycans

“…It is unclear whether a reaction equilibrium was obtained or whether the reaction was still ongoing. However, the result is in line with previous experiments by our group ( Ruhnau et al, 2021 ). Moreover, small amounts of intermediates such as LL-Man2 (7% TPH) (140 MTU”) and LL-Man5 (25% TPH) (248 MTU’’) that originate from the LL-Man3 stock solution were identified.…”

“…Except for ALG2, transmembrane deleted variants were overexpressed. Information regarding the in vivo and in vitro activity of the enzymes has been described previously ( Stanley et al, 2015 ; Ruhnau et al, 2021 ).…”

Human adenovirus type 7 subunit vaccine induces dendritic cell maturation through the TLR4/NF-κB pathway is highly immunogenic

“…successfully expressed Escherichia coli β –galactosidase by using insect cells, which marked the birth of baculovirus-insect cell expression system( Pennock et al., 1984 ). In recent years, the expression system has also been used in many virulent infectious disease vaccines, such as Covid-19 vaccine and influenza virus vaccine ( Ruhnau et al., 2021 ; Yang et al., 2022 ; Pavot et al., 2023 ). The expression system has also been widely used in the field of animal vaccines.…”

Evaluation of Variant-Specific Peptides for Detection of SARS-CoV-2 Variants of Concern

“…As proteomics lacks an amplification strategy similar to PCR, efficient recovery of viral proteins is critical. Sample preparation methods reported include precipitation, , and solid-phase extraction. , Both selective, e.g., antibody based extraction of N-protein, and nonselective, e.g., hydrophilic interaction chromatography (HILIC) extraction targeting total protein have been reported. − …”