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

IDROVO-HIDALGO, TOMMY; PIGNATARO, MARÍA F; BREDESTON, LUIS M; ELIAS, FERNANDA; HERRERA, MARÍA G; PAVAN, MARÍA F; FOSCALDI, SABRINA; SUIRESZCZ, MAYRA; FERNÁNDEZ, NATALIA B; WETZLER, DIANA E; PAVÁN, CARLOS H; CRAIG, PATRICIO O; ROMAN, ERNESTO A; RUBERTO, LUCAS A M; NOSEDA, DIEGO G; IBAÑEZ, LORENA I; CZIBENER, CECILIA; UGALDE, JUAN E; NADRA, ALEJANDRO D; SANTOS, JAVIER; D?ALESSIO, CECILIA

Glycobiology

Oxford University Press,

Año: 2023

DuringtheCOVID-19outbreak,numerous tolos including protein-basedvaccines have been developed.The methylotrophic yeast Pichiapastoris (synonymous to Komagataella phaffii) is aneukaryotic cost-effective and scalable system for recombinant proteinproduction, with the advantages of an efficient secretion system and theprotein folding assistance of the secretory pathway of eukaryotic cells. In aprevious work, we compared the expression of SARS-CoV-2 Spike Receptor BindingDomain in P. pastoris with that in human cells. Althoughthe size and glycosylation pattern was different between them, their proteinstructural and conformational features were indistinguishable. Nevertheless,since high mannose glycan extensions in proteins expressed by yeast may be thecause of a nonspecific immune recognition, we deglycosylated RBD in nativeconditions. This resulted in a highly pure, homogenous, properly folded andmonomeric stable protein.This was confirmed by circular dichroism andtryptophan fluorescence spectra and by SEC-HPLC, which were similar to those ofRBD proteins produced in yeast or human cells. Deglycosylated RBD was obtainedat high yields in a single step, and it was efficient in distinguishing betweenSARS-CoV-2-negative and positive sera from patients. Moreover, when thedeglycosylated variant was used as an immunogen, it elicited a humoral immuneresponse ten times greater than the glycosylated form, producing antibodieswith enhanced neutralizing power and eliciting a more robust cellular response.The proposed approach may be used to produce at a low cost, many antigens thatrequire glycosylation to fold and express, but do not require glycans forrecognition purposes.