Deglycosylated RBD produced in Pichia pastoris as a COVID-19 diagnosis tool and a vaccine candidate

TOMMY IDROVO-HIDALGO; MARÍA F. PIGNATARO; LUIS M. BREDESTON; FERNANDA ELIAS; MARÍA G. HERRERA; MARÍA F. PAVAN; SABRINA FOSCALDI; MAYRA SUIRESZCZ; NATALIA FERNÁNDEZ; DIANA WETZLER; CARLOS PAVÁN; PATRICIO CRAIG; ERNESTO ROMAN; LUCAS RUBERTO; DIEGO G. NOSEDA; ALEJANDRO NADRA; SANTOS, JAVIER; D'ALESSIO CECILIA

Congreso; Congress of the Society for Glycobiology; 2023

Society for Glycobiology

During the COVID-19 pandemic various tools including protein-based vaccines were developed with varying degrees of effectiveness. The yeast Pichia pastoris is known to be a cost-effective and scalable eukaryotic system for producing recombinant proteins. It offers the advantages of an efficient secretion system and assistance in protein folding through the eukaryotic cell´s secretory pathway. In a prior study, we expressed the Receptor Binding Domain (RBD) of the SARS-CoV-2 Spike protein in both P. pastoris and human cells. Although there were differences in size and glycosylation patterns, the structural and conformational characteristics of the proteins were remarkably similar. Here we addressed potential issues related to yeast-added high mannose glycan moieties, which could trigger non-specific immune responses, by deglycosylating yRBD under native conditions. This resulted in a highly pure, uniform, and well-folded monomeric protein with circular dichroism spectra matching those of glycosylated RBD and RBD produced in human cells. The deglycosylated RBD was obtained in a single-step process, yielding high quantities, and it effectively differentiated between sera from SARS-CoV-2-negative and -positive patients. Furthermore, when used as an immunogen, the deglycosylated variant elicited a humoral immune response stronger than the glycosylated RBD, produced antibodies with greater neutralizing capacity, and induced a better cellular response. This approach can be employed to cost- effectively produce numerous antigens that require glycosylation for proper folding and expressionbut not for recognition purposes.