In Planta Production of the Receptor-Binding Domain From SARS-CoV-2 With Human Blood Group A Glycan Structures

Abstract

Glycosylation of viral envelope proteins is important for infectivity and immune evasion. The SARS-CoV-2 spike protein is heavily glycosylated and host-derived glycan modifications contribute to the formation of specific immunogenic epitopes, enhance the virus-cell interaction or affect virus transmission. On recombinant viral antigens used as subunit vaccines or for serological assays, distinct glycan structures may enhance the immunogenicity and are recognized by naturally occurring antibodies in human sera. Here, we performed an in vivo glycoengineering approach to produce recombinant variants of the SARS-CoV-2 receptor-binding domain (RBD) with blood group antigens in Nicotiana benthamiana plants. SARS-CoV-2 RBD and human glycosyltransferases for the blood group ABH antigen formation were transiently co-expressed in N. benthamiana leaves. Recombinant RBD was purified and the formation of complex N-glycans carrying blood group A antigens was shown by immunoblotting and MS analysis. Binding to the cellular ACE2 receptor and the conformation-dependent CR3022 antibody showed that the RBD glycosylation variants carrying blood group antigens were functional. Analysis of sera from RBD-positive and RBD-negative individuals revealed further that non-infected RBD-negative blood group O individuals have antibodies that strongly bind to RBD modified with blood group A antigen structures. The binding of IgGs derived from sera of non-infected RBD-negative blood group O individuals to blood group A antigens on SARS-CoV-2 RBD suggests that these antibodies could provide some degree of protection from virus infection.

Investigation of the N-Glycosylation of the SARS-CoV-2 S Protein Contained in VLPs Produced in Nicotiana benthamiana

“…With regard to the expression of the SARS-CoV-2 S related proteins expressed in plant systems, the functional receptor-binding domain (RBD) has been successfully produced in tobacco [9][10][11][12], as well as in a plant glycosylation mutant impaired in core xylosyl-and fucosyltranferases [12] and in plants expressing glycosyltransferases responsible for the biosynthesis of human blood group A epitopes [13]. N-glycosylation of RBD in plants was demonstrated to be important for its proper folding.…”

Stable production of recombinant SARS-CoV-2 receptor-binding domain in mammalian cells with co-expression of a fluorescent reporter and its validation as antigenic target for COVID-19 serology testing

“…One advantage of HEK 293T cells as expression system is related to the achievement of proper post-translational modifications, a feature which could have a potential influence in diverse applications when proteins are produced in non-human cell lines [ 20 ]. The use of other eukaryotic expression systems as yeast or plant cells, even if advantageous in costs and up-scaling, often requires additional elements or modifications in order to achieve adequate physicochemical characteristics and biological responses [ 17 , 24 , 25 , 26 ]. Our system allows stable expression of a minimally modified RBD, saving time and resources needed for the generation of the transfection grade DNA used by the transient expression approaches.…”

“…SARS-CoV-2 RBD can be also used in serological assays, such as enzyme-linked immunosorbent assays (ELISA) [ 15 ] or surrogate neutralizing antibodies assays [ 16 ] that are of critical importance to help define previous exposure to SARS-CoV-2 or monitoring vaccine responses in populations. Strategies for recombinant production of RBD have been assessed using different expression systems such as E. coli [ 17 ], baculovirus-insect cells [15 , 16 , 18 ] mammalian cells [15 , 16 , 19 , 20 , 21 , 22 , 23] yeast cells [ 19 , 24 , 25 ] and plants [ 21 , 26 , 27 , 28 ]. Mammalian cells have been shown to have higher yields of RBD expression than insect cells [15 , 29] .…”

Production of the SARS-CoV-2 Spike protein and its Receptor Binding Domain in plant cell suspension cultures

“…Receptor Binding Domain (RBD) Ceballo et al, 2022Diego-Martin et al, 2020Khorattanakulchai et al, 2022König-Beihammer et al, 2022Makatsa et al, 2021Maharjan et al, 2021Mamedov et al, 2021Mardanova et al, 2021Rattanapisit et al, 2020Rattanapisit et al, 2021Royal et al, 2021Schwestka et al, 2021Siriwattananon et al, 2021 Spike protein Rattanapisit et al, 2020Shanmugaraj et al, 2020 stability (Wrapp et al, 2020). The transmembrane and cytoplasmic domains of the S protein were replaced by a thrombin cleavage site and a T4 foldon sequence for trimerization (Amanat et al, 2020).…”