Development and characterization of a multimeric recombinant protein based on the spike protein receptor binding domain of SARS-CoV-2 that can neutralize virus infection (preprint)

ABSTRACT

Background: The SARS-CoV-2 virus, responsible for the COVID-19 pandemic, has four structural proteins and sixteen non-structural proteins. The S-protein is one of the structural proteins exposed on the surface of the virus 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. Methods: We stably expressed in a constitutive manner in HEK293 cells a new recombinant protein containing a signal sequence of immunoglobulin to produce an extended C-terminal portion of the RBD followed by a region responsible for the trimerization inducer of the bacteriophage T4, and a sequence of 6 histidines. The protein was produced and released in the culture supernatant of cells and was purified by Ni-agarose column and exclusion chromatography. It was then characterized by SDS-polyacrylamide gel and used as antigen to generate protective antibodies to inhibit ACE2 receptor interaction and virus entry into Vero cells. Results: The purified protein displayed a molecular mass of 135 kDa and with a secondary structure like the monomeric RBD. Electrophoresis analysis in SDS-polyacrylamide gel with and without reducing agents, and in the presence of crosslinkers indicated that it forms a multimeric structure composed of trimers and hexamers. The purified protein was able to bind the ACE2 receptor and generated high antibody titers in mice (110000), capable of inhibiting the binding of biotin labeled ACE2 to the virus S1 subunit, and to neutralize the entry of the SARS-CoV-2 Wuhan strain into cells. Conclusion: Our results characterize a new multimeric protein based on S1 subunit to combat COVID-19, as a possible immunogen or antigen for diagnosis.