Semisynthesis of homogeneous spike RBD glycoforms from SARS-CoV-2 for profiling the correlations between glycan composition and function.

Vaccines have been the primary remedy in the global fight against coronavirus disease 2019 (COVID-19). The receptor-binding domain (RBD) of the spike protein, a critical viral immunogen, is affected by the heterogeneity of its glycan structures and relatively low immunogenicity. Here, we describe a scalable synthetic platform that enables the precise synthesis of homogeneously glycosylated RBD, facilitating the elucidation of carbohydrate structure-function relationships. Five homogeneously glycosylated RBDs bearing biantennary glycans were prepared, three of which were conjugated to T-helper epitope (T pep ) from tetanus toxoid to improve their weak immune response. Relative to natural HEK293-derived RBD, synthetic RBDs with biantennary N -glycan elicited a higher level of neutralising antibodies against SARS-CoV-2 in mice. Furthermore, RBDs containing T pep elicited significant immune responses in transgenic mice expressing human angiotensin-converting enzyme 2. Our collective data suggest that trimming the N -glycans and T pep conjugation of RBD could potentially serve as an effective strategy for developing subunit vaccines providing efficient protection.