Cloning, Expression and Biophysical Characterization of a Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain COVID-19 Vaccine Candidate

Chen, W.-H.; Wei, J.; Kundu, R. T.; Adhikari, R.; Liu, Z.; Lee, J.; Versteeg, L.; Poveda, C.; Keegan, B.; Villar, M. J.; de Araujo Leao, A. C.; Altieri Rivera, J.; Gillespie, P. M.; Pollet, J.; Strych, U.; Zhan, B.; Hotez, P. J.; Bottazzi, M. E.

doi:10.1101/2020.11.09.373449

bioRxiv · 10.1101/2020.11.09.373449

Cloning, Expression and Biophysical Characterization of a Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain COVID-19 Vaccine Candidate

Chen, W.-H.·Wei, J.·Kundu, R. T.·Adhikari, R.·Liu, Z.·Lee, J.·Versteeg, L.·Poveda, C.·Keegan, B.·Villar, M. J.·de Araujo Leao, A. C.·Altieri Rivera, J.·Gillespie, P. M.·Pollet, J.·Strych, U.·Zhan, B.·Hotez, P. J.·Bottazzi, M. E.

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Abstract

BackgroundCoronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has now spread worldwide to infect over 110 million people, with approximately 2.5 million reported deaths. A safe and effective vaccine remains urgently needed. MethodWe constructed three variants of the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (residues 331-549) in yeast as follows: (1) a "wild type" RBD (RBD219-WT), (2) a deglycosylated form (RBD219-N1) by deleting the first N-glycosylation site, and (3) a combined deglycosylated and cysteine-mutagenized form (C538A-mutated variant (RBD219-N1C1)). We compared the expression yields, biophysical characteristics, and functionality of the proteins produced from these constructs. Results and conclusionsThese three recombinant RBDs showed similar secondary and tertiary structure thermal stability and had the same affinity to their receptor, angiotensin-converting enzyme 2 (ACE-2), suggesting that the selected deletion or mutations did not cause any significant structural changes or alteration of function. However, RBD219-N1C1 had a higher fermentation yield, was easier to purify, was not hyperglycosylated, and had a lower tendency to form oligomers, and thus was selected for further vaccine development and evaluation. General significanceBy genetic modification, we were able to design a better-controlled and more stable vaccine candidate, which is an essential and important criterion for any process and manufacturing of biologics or drugs for human use.

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Chen, W.-H., Wei, J., Kundu, R. T., Adhikari, R., Liu, Z., Lee, J., Versteeg, L., Poveda, C., Keegan, B., Villar, M. J., de Araujo Leao, A. C., Altieri Rivera, J., Gillespie, P. M., Pollet, J., Strych, U., Zhan, B., Hotez, P. J., Bottazzi, M. E.. 2020-11-09. Cloning, Expression and Biophysical Characterization of a Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain COVID-19 Vaccine Candidate. https://doi.org/10.1101/2020.11.09.373449

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