Design and characterization of stabilized mutants of the SARS-CoV-2 Receptor Binding Domain

Abstract

The global pandemic caused by SARS-CoV-2 has resulted in millions of deaths worldwide, highlighting the urgent need for effective vaccines. The trimeric spike glycoprotein, particularly the receptor-binding domain (RBD), is a key target for neutralizing antibodies. Glycosylation of viral proteins, including the RBD, plays critical roles in viral pathobiology, immune evasion, and vaccine development. Our study investigates the role of N-linked glycans on RBD stability and immunogenicity, comparing proteins expressed in Pichia and mammalian cells. Through second-site saturation-suppressor mutagenesis (SSSM), stabilizing mutations in the RBD are identified, enhancing thermal stability, and facilitating mass production. Vaccination studies demonstrate that stabilized RBD proteins induce neutralizing antibodies against variants, with mammalian-expressed versions offering broader protection. Additionally, glycan removal impacts virus infectivity and antibody neutralization, while different glycan types have influenced immunogen localization and immune responses. Engineering Pichia strain to mimic mammalian-type complex N-linked glycans holds promise for enhancing subunit vaccine efficacy. Moreover, trimeric RBD formulations with various adjuvants elicit robust B and T-cell responses in comparison to monomeric RBD. These findings underscore the importance of glycosylation in vaccine design and highlight a promising RBD vaccine candidate with enhanced stability, immunogenicity, and choice of adjuvants offering insights for future vaccine development against emerging viral threats.