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. 2023 Oct 6:14:1275228.
doi: 10.3389/fpls.2023.1275228. eCollection 2023.

Impact of mutations on the plant-based production of recombinant SARS-CoV-2 RBDs

Valentina Ruocco  1 , Ulrike Vavra  1 , Julia König-Beihammer  1 , Omayra C Bolaños Martínez  1 , Somanath Kallolimath  1 , Daniel Maresch  2 , Clemens Grünwald-Gruber  2 , Richard Strasser  1
Affiliations

Impact of mutations on the plant-based production of recombinant SARS-CoV-2 RBDs

Valentina Ruocco et al. Front Plant Sci. .

Abstract

Subunit vaccines based on recombinant viral antigens are valuable interventions to fight existing and evolving viruses and can be produced at large-scale in plant-based expression systems. The recombinant viral antigens are often derived from glycosylated envelope proteins of the virus and glycosylation plays an important role for the immunogenicity by shielding protein epitopes. The receptor-binding domain (RBD) of the SARS-CoV-2 spike is a principal target for vaccine development and has been produced in plants, but the yields of recombinant RBD variants were low and the role of the N-glycosylation in RBD from different SARS-CoV-2 variants of concern is less studied. Here, we investigated the expression and glycosylation of six different RBD variants transiently expressed in leaves of Nicotiana benthamiana. All of the purified RBD variants were functional in terms of receptor binding and displayed almost full N-glycan occupancy at both glycosylation sites with predominately complex N-glycans. Despite the high structural sequence conservation of the RBD variants, we detected a variation in yield which can be attributed to lower expression and differences in unintentional proteolytic processing of the C-terminal polyhistidine tag used for purification. Glycoengineering towards a human-type complex N-glycan profile with core α1,6-fucose, showed that the reactivity of the neutralizing antibody S309 differs depending on the N-glycan profile and the RBD variant.

Keywords: Nicotiana benthamiana; antigen; glycoprotein; glycosylation; spike protein; vaccine; virus.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
Comparison of expressed RBD-215 variants. (A) Schematic illustration of the expressed SARS-CoV-2 RBD-215 protein. SP, signal peptide; “Y”, N-glycosylation sites, numbering according to the full-length SARS-CoV-2 spike sequence (UniProt: P0DTC2); 6xHis, polyhistidine tag composed of six histidine residues. (B) Sequence alignment of the amino acid region 319-533 from different SARS-CoV-2 variants was done using T-Coffee (https://www.ebi.ac.uk/Tools/msa/tcoffee/) and displayed using ESPript3.0 (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). N-glycosylation sites are marked by blue triangles. (C) Phylogenetic tree of RBD-215 sequences constructed using Phylogeny.fr (https://www.phylogeny.fr/). (D) Comparison of RBD-215 models produced by AlphaFold CoLab, showcasing their distinct and common structures and secondary features. The models were superimposed using the MatchMaker tool from UCSF Chimera (https://www.cgl.ucsf.edu/chimera/), which allows a direct comparison, highlighting conserved regions and structural variations (see also Table S1 ).
Figure 2
Figure 2
SDS-PAGE of purified RBD-215 variants. IMAC-purified RBD-215 variants were subjected to SDS-PAGE under non-reducing and reducing conditions and stained with Coomassie Brilliant Blue (CBB).
Figure 3
Figure 3
RBD-215 variants display differences in expression and yield after purification. (A) Immunoblot analysis (anti-histidine and anti-RBD antibodies) and SDS-PAGE with CBB-staining of isolated apoplastic fluid and purified RBD-215 variants. (B) Immunoblot of total soluble protein (TSP). Leaves from infiltrated N. benthamiana ΔXT/FT plants were harvested 4 days after infiltration and TSP was analysed with anti-histidine antibody. (C, D) Immunoblot of Endo H or PNGase F digested TSP. TSP was obtained by infiltration of N. benthamiana ΔXT/FT with Agrobacteria carrying the expression vectors for the indicated RBD-215 proteins. (E) Immunoblot and SDS-PAGE with CBB staining of the apoplastic fluid. The apoplastic fluid or the purified proteins were incubated for 2 h at 4°C followed by SDS-PAGE and immunoblotting or IMAC-purification (re-purified) and SDS-PAGE with CBB staining.
Figure 4
Figure 4
MS spectra of the RBD-215 glycopeptide containing N-glycosylation site N331 of the SARS-CoV-2 spike protein. The assigned N-glycan structures were labelled according to the ProGlycAn nomenclature (http://www.proglycan.com/). A cartoon illustration (filled green circle, mannose; filled blue square, GlcNAc; for details see http://www.functionalglycomics.org/) highlights the main N-glycan structures detected for each peptide.
Figure 5
Figure 5
ACE2-Fc receptor binding of RBD-215 variants. SPR sensorgrams are shown and the KD values (mean ± SD, n = 3) are given (red line fitted curves).
Figure 6
Figure 6
Thermal stability of RBD-215 variants. DSF profiles of the RBD-215 variants show changes in protein intrinsic fluorescence. For each RBD-215 protein, three overlaid curves (technical replicates) are shown and the Tm is given for every protein (mean ± SD, n = 3).
Figure 7
Figure 7
Glycoengineering results in RBD-215 variants with oligomannosidic or fucosylated complex N-glycans whose Tm and KD values are not affected by changes in N-glycan structures. (A) MS spectra of RBD-215 + kif, Omicron BA.1 + kif, RBD-215 + FUT8, Omicron BA.1 + FUT8. A cartoon illustration (filled green circle, mannose; filled blue square, GlcNAc; red triangle, fucose) highlights the main N-glycan structures detected for each peptide (B) SPR analysis of ACE2-Fc binding to RBD-215 variants and (C) DSF curves and values (mean ± SD, n = 3) of RBD-215 + kif, Omicron BA.1 + kif, RBD-215 + FUT8, Omicron BA.1 + FUT8.
Figure 8
Figure 8
ELISA showing differences in binding of S309 antibody to glycoengineered Omicron BA.1. Binding to antibody P5C3 was used for comparison. (A) ELISA binding curves. (B) EC50 values (mean values, n = 3).
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