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Development and characterization of a multimeric recombinant protein using the spike protein receptor binding domain as an antigen to induce SARS-CoV-2 neutralization.
- Source :
-
Immunity, inflammation and disease [Immun Inflamm Dis] 2024 Jul; Vol. 12 (7), pp. e1353. - Publication Year :
- 2024
-
Abstract
- Background: SARS-CoV2 virus, responsible for the COVID-19 pandemic, has four structural proteins and 16 nonstructural proteins. S-protein is one of the structural proteins exposed on the virus surface and is the main target for producing neutralizing antibodies and vaccines. The S-protein forms a trimer that can bind the angiotensin-converting enzyme 2 (ACE2) through its receptor binding domain (RBD) for cell entry.<br />Aims: The goal of this study was to express in HEK293 cells a new RBD recombinant protein in a constitutive and stable manner in order to use it as an alternative immunogen and diagnostic tool for COVID-19.<br />Materials & Methods: The protein was designed to contain an immunoglobulin signal sequence, an explanded C-terminal section of the RBD, a region responsible for the bacteriophage T4 trimerization inducer, and six histidines in the pCDNA-3.1 plasmid. Following transformation, the cells were selected with geneticin-G418 and purified from serum-fre culture supernatants using Ni2+-agarand size exclusion chromatography. The protein was structurally identified by cross-linking and circular dichroism experiments, and utilized to immunize mice in conjuction with AS03 or alum adjuvants. The mice sera were examined for antibody recognition, receptor-binding inhibition, and virus neutralization, while spleens were evaluated for γ-interferon production in the presence of RBD.<br />Results: The protein released in the culture supernatant of cells, and exhibited a molecular mass of 135 kDa with a secondary structure like the monomeric and trimeric RBD. After purification, it formed a multimeric structure comprising trimers and hexamers, which were able to bind the ACE2 receptor. It generated high antibody titers in mice when combined with AS03 adjuvant (up to 1:50,000). The sera were capable of inhibiting binding of biotin-labeled ACE2 to the virus S1 subunit and could neutralize the entry of the Wuhan virus strain into cells at dilutions up to 1:2000. It produced specific IFN-γ producing cells in immunized mouse splenocytes.<br />Discussion: Our data describe a new RBD containing protein, forming trimers and hexamers, which are able to induce a protective humoral and cellular response against SARS-CoV2.<br />Conclusion: These results add a new arsenal to combat COVID-19, as an alternative immunogen or antigen for diagnosis.<br /> (© 2024 The Author(s). Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)
- Subjects :
- Animals
Humans
Mice
HEK293 Cells
COVID-19 Vaccines immunology
Mice, Inbred BALB C
Female
Protein Multimerization
Protein Domains immunology
Protein Binding
Spike Glycoprotein, Coronavirus immunology
Spike Glycoprotein, Coronavirus genetics
Spike Glycoprotein, Coronavirus chemistry
Antibodies, Neutralizing immunology
SARS-CoV-2 immunology
COVID-19 immunology
COVID-19 prevention & control
Recombinant Proteins immunology
Recombinant Proteins genetics
Recombinant Proteins chemistry
Angiotensin-Converting Enzyme 2 metabolism
Angiotensin-Converting Enzyme 2 immunology
Antibodies, Viral immunology
Subjects
Details
- Language :
- English
- ISSN :
- 2050-4527
- Volume :
- 12
- Issue :
- 7
- Database :
- MEDLINE
- Journal :
- Immunity, inflammation and disease
- Publication Type :
- Academic Journal
- Accession number :
- 39056544
- Full Text :
- https://doi.org/10.1002/iid3.1353