RESUMO
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.
Assuntos
Enzima de Conversão de Angiotensina 2 , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , Proteínas Recombinantes , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , Humanos , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/química , Camundongos , Anticorpos Neutralizantes/imunologia , SARS-CoV-2/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Células HEK293 , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/imunologia , Camundongos Endogâmicos BALB C , Feminino , Multimerização Proteica , Domínios Proteicos/imunologia , Ligação ProteicaRESUMO
Background: SARS‐CoV2 virus, responsible for the COVID‐19 pandemic, hasfour structural proteins and 16 nonstructural proteins. S‐protein is one of thestructural proteins exposed on the virus surface and is the main target forproducing neutralizing antibodies and vaccines. The S‐protein forms a trimerthat can bind the angiotensin‐converting enzyme 2 (ACE2) through itsreceptor binding domain (RBD) for cell entry.Aims: The goal of this study was to express in HEK293 cells a new RBDrecombinant protein in a constitutive and stable manner in order to use it asan alternative immunogen and diagnostic tool for COVID‐19.Materials & Methods: The protein was designed to contain an immuno-globulin signal sequence, an explanded C‐terminal section of the RBD, aregion responsible for the bacteriophage T4 trimerization inducer, and sixhistidines in the pCDNA‐3.1 plasmid. Following transformation, the cells wereselected with geneticin‐G418 and purified from serum‐fre culture super-natants using Ni2+‐agarand size exclusion chromatography. The protein wasstructurally 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‐bindinginhibition, and virus neutralization, while spleens were evaluated forγ‐interferon production in the presence of RBD. Results: The protein released in the culture supernatant of cells, andexhibited a molecular mass of 135 kDa with a secondary structure like themonomeric and trimeric RBD. After purification, it formed a multimericstructure comprising trimers and hexamers, which were able to bind the ACE2receptor. It generated high antibody titers in mice when combined with AS03adjuvant (up to 1:50,000). The sera were capable of inhibiting binding ofbiotin‐labeled ACE2 to the virus S1 subunit and could neutralize the entry ofthe Wuhan virus strain into cells at dilutions up to 1:2000. It produced specificIFN‐γ producing cells in immunized mouse splenocytes.Discussion: Our data describe a new RBD containing protein, formingtrimers and hexamers, which are able to induce a protective humoral andcellular response against SARS‐CoV2.Conclusion: These results add a new arsenal to combat COVID‐19, as analternative immunogen or antigen for diagnosis.