The impact of N-glycans on the immune response of plant-produced SARS-CoV-2 RBD-Fc proteins

Plant-based manufacturing has the advantage of post-translational modifications. While plant-specific N-glycans have been associated with allergic reactions, their effect on the specific immune response upon vaccination is not yet understood. In this study, we produced an RBD-Fc subunit vaccine in both wildtype (WT) and glycoengineered (∆XF) Nicotiana benthamiana plants. The N-glycan analysis: RBD-Fc carrying the ER retention peptide mainly displayed high mannose. When produced in WT RBD-Fc displayed complex-type (GnGnXF) N-glycans. In contrast, ∆XF plants produced RBD-Fc with humanized complex N-glycans that lack potentially immunogenic xylose and core fucose residues (GnGn). The three recombinant RBD-Fc glycovariants were tested. Immunization with any of the RBD-Fc proteins resulted in a similar titer of anti-RBD antibodies in mice. Likewise, antisera from subunit RBD-Fc vaccines also demonstrated comparable neutralization against SARS-CoV-2. Thus, we conclude that N-glycan modifications of the RBD-Fc protein have no impact on their capacity to activate immune responses and induce neutralizing antibody production.