Designed SARS-CoV-2 receptor binding domain variants form stable monomers

Area of Science:

• Biochemistry
• Immunology
• Virology

Background:

• The SARS-CoV-2 spike protein's receptor binding domain (RBD) is a key target for neutralizing antibodies.
• Recombinant RBD expression is vital for COVID-19 immunosurveillance and vaccine development.
• Mammalian cell expression systems are particularly effective for producing functional RBD.

Purpose of the Study:

• To optimize the SARS-CoV-2 RBD produced in HEK293-6E cells for stability and homogeneity.
• To investigate the role of O-glycosylation and cysteine residue 538 in RBD structure and function.
• To enhance RBD variants for improved serological assays and vaccine design.

Main Methods:

• Engineered modifications to the RBD sequence, focusing on O-glycosylation site T323 and cysteine residue C538.
• Utilized HEK293-6E cells for recombinant protein expression.
• Employed bead-based and enzyme-linked immunosorbent assays (ELISA) to evaluate antibody detection.

Main Results:

• An intact O-glycosylation site at T323 is essential for maintaining RBD as a stable monomer.
• Deletion or substitution of C538 significantly reduced RBD aggregation, increasing monomer yield.
• Optimized RBD variants demonstrated high performance in detecting low levels of SARS-CoV-2 antibodies in convalescent sera.

Conclusions:

• Optimized RBD variants facilitate the development of sensitive serological tests for SARS-CoV-2.
• These improved RBD preparations can inform the design of next-generation RBD-based COVID-19 vaccines.
• The study provides insights into stabilizing RBD for diagnostic and therapeutic applications.