Glycosylated SARS-CoV-2 RBD Antigens Expressed in Glycoengineered Yeast Induce Strong Immune Responses Through High Antigen–Alum Adsorption

Glycosylation plays a pivotal role in regulating the functions and immunogenicity of antigens. Targeting the receptor-binding domain (RBD) of the spike protein (S protein) of SARS-CoV-2, we examined the impact of different glycoforms on RBD antigen immunogenicity and the underlying mechanisms. IgG-specific antibody titers and pseudovirus neutralization were compared in mice immunized with RBD antigens bearing different glycoforms, which were prepared using glycoengineering-capable <i>Pichia pastoris</i> and mammalian cell expression systems with distinct glycosylation pathways. The glycosylation impacted the surface charges of the RBD antigen, and influenced its adsorption onto alum. This may further lead to variations in the antigen's immunogenicity. The high-mannose variant of the RBD antigen (H-MAN/RBD) expressed in wild-type <i>Pichia pastoris</i> induced significantly higher IgG-specific antibody titers and pseudovirus neutralization activity compared with the complex RBD variant (Complex/RBD) expressed in mammalian cells (293F) or glycoengineering-capable <i>Pichia pastoris</i>. The rate of H-MAN/RBD adsorption onto aluminum hydroxide (alum) adjuvant was significantly higher than that of Complex/RBD. It was assumed that H-MAN/RBD might carry more negative charges because of its phosphomannose-modified surfaces, leading to a higher rate of adsorption onto the positively charged alum and enhancing the immune response. To assess the impact of phosphomannose modification on antigen immunogenicity, a yeast strain was engineered to prepare a low-mannose RBD antigen (L-MAN/RBD); additionally, a yeast strain was constructed to generate a low-phosphomannose-modified RBD antigen (L-MAN-P/RBD). In conclusion, phosphomannose modification substantially enhanced the immunogenicity of RBD by altering the surface charges of the RBD antigen and facilitating its adsorption onto alum. These findings offer novel insights and strategies for vaccine design and immunotherapeutic approaches.