Veronica A. de Lima, João P. S. Nunes, Daniela S. Rosa, Rodrigo Ferreira, Maria L. V. Oliva, Robert Andreata-Santos, Marcia Duarte-Barbosa, Luiz M. R. Janini, Juliana T. Maricato, Milena A. Akamatsu, Paulo L. Ho, Sergio Schenkman
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The S-protein forms a trimer that can bind the angiotensin-converting enzyme 2 (ACE2) through its receptor binding domain (RBD) for cell entry.</p>\n </section>\n \n <section>\n \n <h3> Aims</h3>\n \n <p>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.</p>\n </section>\n \n <section>\n \n <h3> Materials & Methods</h3>\n \n <p>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.</p>\n </section>\n \n <section>\n \n <h3> Results</h3>\n \n <p>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.</p>\n </section>\n \n <section>\n \n <h3> Discussion</h3>\n \n <p>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.</p>\n </section>\n \n <section>\n \n <h3> Conclusion</h3>\n \n <p>These results add a new arsenal to combat COVID-19, as an alternative immunogen or antigen for diagnosis.</p>\n </section>\n </div>","PeriodicalId":13289,"journal":{"name":"Immunity, Inflammation and Disease","volume":null,"pages":null},"PeriodicalIF":3.1000,"publicationDate":"2024-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11273545/pdf/","citationCount":"0","resultStr":"{\"title\":\"Development and characterization of a multimeric recombinant protein using the spike protein receptor binding domain as an antigen to induce SARS-CoV-2 neutralization\",\"authors\":\"Veronica A. de Lima, João P. 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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
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.
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.
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.
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.
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.
Conclusion
These results add a new arsenal to combat COVID-19, as an alternative immunogen or antigen for diagnosis.
期刊介绍:
Immunity, Inflammation and Disease is a peer-reviewed, open access, interdisciplinary journal providing rapid publication of research across the broad field of immunology. Immunity, Inflammation and Disease gives rapid consideration to papers in all areas of clinical and basic research. The journal is indexed in Medline and the Science Citation Index Expanded (part of Web of Science), among others. It welcomes original work that enhances the understanding of immunology in areas including:
• cellular and molecular immunology
• clinical immunology
• allergy
• immunochemistry
• immunogenetics
• immune signalling
• immune development
• imaging
• mathematical modelling
• autoimmunity
• transplantation immunology
• cancer immunology