Genetic modification to design a stable yeast-expressed recombinant SARS-CoV-2 receptor binding domain as a COVID-19 vaccine candidate.
Biophysical characterization
Biotechnology
Coronavirus
P. pastoris
Journal
Biochimica et biophysica acta. General subjects
ISSN: 1872-8006
Titre abrégé: Biochim Biophys Acta Gen Subj
Pays: Netherlands
ID NLM: 101731726
Informations de publication
Date de publication:
06 2021
06 2021
Historique:
received:
07
01
2021
revised:
08
03
2021
accepted:
11
03
2021
pubmed:
19
3
2021
medline:
29
4
2021
entrez:
18
3
2021
Statut:
ppublish
Résumé
Coronavirus 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. We 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. These 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. By 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.
Sections du résumé
BACKGROUND
Coronavirus 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.
METHOD
We 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 CONCLUSIONS
These 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 SIGNIFICANCE
By 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.
Identifiants
pubmed: 33731300
pii: S0304-4165(21)00051-9
doi: 10.1016/j.bbagen.2021.129893
pmc: PMC7955913
mid: NIHMS1683434
pii:
doi:
Substances chimiques
COVID-19 Vaccines
0
Recombinant Proteins
0
Spike Glycoprotein, Coronavirus
0
spike protein, SARS-CoV-2
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
129893Subventions
Organisme : NIAID NIH HHS
ID : R56 AI140872
Pays : United States
Informations de copyright
Copyright © 2021 Elsevier B.V. All rights reserved.
Références
J Pharm Sci. 2020 May;109(5):1673-1680
pubmed: 32070701
Hum Vaccin Immunother. 2021 Aug 3;17(8):2356-2366
pubmed: 33847226
Nature. 2020 Jul;583(7815):290-295
pubmed: 32422645
Vaccine. 2018 Mar 27;36(14):1853-1862
pubmed: 29496347
J Immunol. 2005 Dec 1;175(11):7496-503
pubmed: 16301657
Nature. 2020 Oct;586(7830):572-577
pubmed: 32726802
Cell. 2020 Aug 6;182(3):722-733.e11
pubmed: 32645327
Hong Kong Med J. 2012 Feb;18 Suppl 2:31-6
pubmed: 22311359
PLoS Negl Trop Dis. 2020 Apr 10;14(4):e0008271
pubmed: 32275667
Nature. 2020 Mar;579(7798):270-273
pubmed: 32015507
Science. 2020 Mar 13;367(6483):1260-1263
pubmed: 32075877
Biophys J. 2010 Oct 20;99(8):2657-65
pubmed: 20959107
Sci Transl Med. 2020 Nov 4;12(568):
pubmed: 33077678
Elife. 2020 Oct 28;9:
pubmed: 33112236
Viral Immunol. 2010 Apr;23(2):211-9
pubmed: 20374001
Anal Biochem. 1987 Dec;167(2):228-34
pubmed: 3442318
Curr Trop Med Rep. 2020;7(2):61-64
pubmed: 32219057
Nature. 2020 Aug;584(7821):450-456
pubmed: 32698192
J Biol Chem. 2004 Jan 30;279(5):3197-201
pubmed: 14670965
Cell Host Microbe. 2021 Mar 10;29(3):463-476.e6
pubmed: 33592168
Trends Immunol. 2020 May;41(5):355-359
pubmed: 32249063
J Virol. 2020 Mar 17;94(7):
pubmed: 31996437
Hum Vaccin Immunother. 2014;10(3):648-58
pubmed: 24355931
Expert Rev Vaccines. 2012 Dec;11(12):1405-13
pubmed: 23252385
Vaccine. 2020 Nov 3;38(47):7533-7541
pubmed: 33039209
Lancet Infect Dis. 2020 May;20(5):533-534
pubmed: 32087114
Proc Natl Acad Sci U S A. 2007 Jul 17;104(29):12123-8
pubmed: 17620608
Cell. 2021 Apr 29;184(9):2348-2361.e6
pubmed: 33730597
Cell Mol Immunol. 2020 Jun;17(6):613-620
pubmed: 32203189
Vaccine. 2007 Apr 12;25(15):2832-8
pubmed: 17092615
N Engl J Med. 2020 Dec 10;383(24):2320-2332
pubmed: 32877576
Hum Vaccin Immunother. 2014;10(7):1914-25
pubmed: 25424799
Biochem Biophys Res Commun. 2009 Jul 10;384(4):486-90
pubmed: 19422787
Virology. 2009 Oct 10;393(1):144-50
pubmed: 19683779
Adv Drug Deliv Rev. 2021 Mar;170:71-82
pubmed: 33421475
Hum Vaccin Immunother. 2020 Jun 2;16(6):1239-1242
pubmed: 32298218
PLoS One. 2012;7(4):e35421
pubmed: 22536382
ACS Infect Dis. 2016 May 13;2(5):361-76
pubmed: 27627203
Nature. 2020 Dec;588(7839):682-687
pubmed: 33045718
Trends Biotechnol. 2017 Apr;35(4):334-346
pubmed: 28277249
Science. 2020 Jul 17;369(6501):330-333
pubmed: 32366695
Biochim Biophys Acta. 1980 Jul 24;624(1):13-20
pubmed: 7407231
Appl Environ Microbiol. 2004 May;70(5):2639-46
pubmed: 15128513
Cell. 2020 Apr 16;181(2):271-280.e8
pubmed: 32142651
J Pharm Sci. 2017 Aug;106(8):1961-1970
pubmed: 28456726