Development and characterization of a multimeric recombinant protein using the spike protein receptor binding domain as an antigen to induce SARS-CoV-2 neutralization

BackgroundSARS-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.AimsThe 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 & MethodsThe 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 gamma-interferon production in the presence of RBD.ResultsThe 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-gamma producing cells in immunized mouse splenocytes.DiscussionOur 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.ConclusionThese results add a new arsenal to combat COVID-19, as an alternative immunogen or antigen for diagnosis. We generated a new trimeric recombinant continuously expressed in HEK-293 cells corresponding to the receptor binding domain and the C-terminus of S1 protein SARS-Cov2. It was able to generate neutralizing antibodies and suitable for diagnostic procedures of Covid-19. image