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

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.

Comparative study of chikungunya Virus-Like Particles and Pseudotyped-Particles used for serological detection of specific immunoglobulin M.

The incidence of chikungunya virus (CHIKV) infection has increased dramatically in recent decades. Effective diagnostic methods must be available to optimize patient management. IgM-capture Enzyme-Linked Immunosorbent Assay (MAC-ELISA) is routinely used for the detection of specific CHIKV IgM. This method requires inactivated CHIKV viral lysate (VL). The use of viral bioparticles such as Virus-Like Particles (VLPs) and Pseudotyped-Particles (PPs) could represent an alternative to VL. Bioparticles performances were established by MAC-ELISA; physico-chemical characterizations were performed by field-flow fractionation (HF5) and confirmed by electron microscopy. Non-purified PPs give a detection signal higher than for VL. Results suggested that the signal difference observed in MAC-ELISA was probably due to the intrinsic antigenic properties of particles. The use of CHIKV bioparticles such as VLPs and PPs represents an attractive alternative to VL. Compared to VL and VLPs, non-purified PPs have proven to be more powerful antigens for specific IgM capture.