Impact of SARS-CoV-2 RBD Mutations on the Production of a Recombinant RBD Fusion Protein in Mammalian Cells

Abstract

SARS-CoV-2 receptor-binding domain (RBD) is a major target for the development of diagnostics, vaccines and therapeutics directed against COVID-19. Important efforts have been dedicated to the rapid and efficient production of recombinant RBD proteins for clinical and diagnostic applications. One of the main challenges is the ongoing emergence of SARS-CoV-2 variants that carry mutations within the RBD, resulting in the constant need to design and optimise the production of new recombinant protein variants. We describe here the impact of naturally occurring RBD mutations on the secretion of a recombinant Fc-tagged RBD protein expressed in HEK 293 cells. We show that mutation E484K of the B.1.351 variant interferes with the proper disulphide bond formation and folding of the recombinant protein, resulting in its retention into the endoplasmic reticulum (ER) and reduced protein secretion. Accumulation of the recombinant B.1.351 RBD-Fc fusion protein in the ER correlated with the upregulation of endogenous ER chaperones, suggestive of the unfolded protein response (UPR). Overexpression of the chaperone and protein disulphide isomerase PDIA2 further impaired protein secretion by altering disulphide bond formation and increasing ER retention. This work contributes to a better understanding of the challenges faced in producing mutant RBD proteins and can assist in the design of optimisation protocols.

Immunogenicity of novel DNA vaccines encoding receptor‐binding domain (RBD) dimer‐Fc fusing antigens derived from different SARS‐CoV‐2 variants of concern

“…Thus, the immunogenicity of 2omRBD‐Fc protein is also suboptimal. It is reported that the E484K mutation in RBD interferes with the proper disulphide‐bond formation and folding of the B.1.351 RBD‐Fc fusion protein, resulting in the reduction of its thermostability 54 . We speculate that similar structural alterations also occurred in 2omRBD‐Fc as the Omicron strains contain E484A mutation, therefore contributed to its low immunogenicity.…”

“…It is reported that the E484K mutation in RBD interferes with the proper disulphide-bond formation and folding of the B.1.351 RBD-Fc fusion protein, resulting in the reduction of its thermostability. 54 We speculate that similar structural alterations also occurred in 2omRBD-Fc as the Omicron strains contain E484A mutation, therefore contributed to its low immunogenicity. Thus, further optimization of omicron-derived antigens, or utilizing other VOC antigens with high immunogenicity, may enhance the immune responses induced by sequential immunization.…”

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

“…Preclinical studies have shown that RBD-Fc-based vaccines are effective [ [5] , [6] , [7] ], and clinical research are currently underway. In addition, different platforms were used to generate RBD-based recombinant proteins, including yeast [ 8 ], mammalian cells [ 9 , 10 ], and plants [ 11 , 12 ]. Plant expression systems offer several advantages for recombinant protein biosynthesis.…”