Horizontal and Collaborative against the clock work in pandemic: Production, characterization and distribution of a low-cost SARS-CoV-2 antigen by the Argentinian AntiCovid Consortium

ARGENTINIAN ANTICOVID CONSORTIUM; ERNESTO A. ROMAN

Ciudad Autonoma de Buenos Aires

Congreso; Sociedad Argentina de Biofisica; 2020

Sociedad Argentina de Biofisica

In December 19th 2019, an epidemiological alert came from China to the whole world: A new virus causing Severe Acute Respiratory Syndrome may be evolving. On January 10th the world got to know the genome of the SARS CoV2. Shortly after the installed quarantine period in Argentina, a group of researchers from different institutions and with diverse relevant expertise began a collaborative work to contribute in the development of local accessible high-quality molecular tools against SARS-CoV-2. Our work was mainly aimed at producing the Receptor Binding Domain (RBD) of the SARS-CoV-2 Spike protein at low cost. In this work, we produced RBD from SARS-CoV-2 Spike protein in both the well-established HEK-293T mammalian cells system and also in the yeast Pichia pastoris. P. pastoris is a cost-effective and easily scalable system for recombinant protein production. Importantly, we found that that (i) RBD produced in both systems was properly folded as judged by CD spectroscopy; (ii) mass spectrometry analysis and glycosidase digestion suggested that both forms are differentially glycosylated; (iii) both variants are conformationally stable and their thermal stabilities depend on the ionic strength in the same way, and (iv) antibodies generated in mice immunized with RBD from yeast recognize the protein produced in mammalian cells and vice versa. Simultaneously, the production of RBD in P. pastoris was scaled-up in a stirred-tank bioreactor, with yields above 100 mg/L of recombinant protein (∼90% of purity), thus allowing many collaborations with different projects regarding: the development of an anti SARS-CoV-2 antibody detection kit, hens immunizations and IgY production, vaccine investigations, and monoclonal antibody developments. Also, some insights in the design of an improved antigen with better immunogenic characteristics, compared with those of wild type monomeric RBD, obtained through the covalent coupling of RBD to other proteins will be presented.