NSRRC Activity Report 2022

046 NSRRC ACTIVITY REPORT 2022 glycan shield on its surface acting as a mask, the research team adopted an enzymatic digestion method to trim down the N-glycan coat of the spike glycoprotein from the fully glycosylated form (S FG ) to the mono-GlcNAc (S MG ) glycoform ( Fig. 1(a) ). The processed S MG protein was subsequently tested as a vaccine antigen, and its immune response elicitation in different animal models was analyzed, including BALB/c mice as a common immune response assessment model, Syrian hamsters as a mild SARS-CoV-2 infection model, and hACE2 transgenic mice as a severe SARS-CoV-2 infection model ( Fig. 1(b) ). In the BALB/c mice, compared with the S FG vaccine, the S MG vaccine induced a more favorable immune response against SARS- CoV-2 wildtype (WT) and VOCs, with increased anti-spike IgG titer ( Fig. 1(c) ) and SARS-CoV-2 pseudovirus neutralization titer ( Fig. 1(d) ). During WT SARS-CoV-2 infection, both the S FG and S MG vaccines effectively decreased weight loss and viral load in the hamster lungs compared with the control (phosphate-buffered saline, PBS) group, indicating that both vaccines can provide sufficient protection against WT SARS-CoV-2 infection. However, during challenges with SARS-CoV-2 VOCs, the S MG -vaccinated hACE2 transgenic mice exhibited lower weight loss and a higher survival rate than the S FG -vaccinated mice. For example, in the alpha variant real virus challenge, all of the S MG -vaccinated mice survived, whereas the S FG -vaccinated mice experienced severe weight loss, and only 60% of them survived. These results provide proof of concept that the S MG vaccine has great potential for fighting against SARS-CoV-2 variants. Fig. 2 : (a) Overview of the single B-cell screening platform. Single spike protein-specific B cells from the spleens of immunized mice were subjected to fluorescence-activated cell sorting (FACS). After sequencing, cDNAs from the variable regions of the IgH or IgL genes were subcloned into expression vectors. The chimeric monoclonal antibody was produced by Expi293, and its binding to spike-expressing 293T cells was measured using FACS. The isolated m31A7 is one of the broad-spectrum antibody candidates. (b) Cryo-EM map fitted with the m31A7–Fab/S protein complex structure and an enlarged view of the receptor-binding domain (RBD)-m31A7 interface. Heavy chain, dark green; light chain, light green; RBD, red; NTD, orange; remainder of S1, light gray; S2, dark gray; and N-glycans, blue. The star marks the vicinity between the m31A7 light chain and the N165-glycan. (c) View of the m31A7–Fab-bound RBD crystal structure. (d) Footprint comparison of COV253 (pink) and m31A7 (green) on RBD (gray) showing similarity, with residues of VOCs labeled and drawn as red spheres. [Reproduced from Ref. 1] To explore how S MG works as a broad-spectrum weapon against variants, the research group analyzed the B-cell repertoire after S MG immunization through a spike-specific B-cell sorting assay ( Fig. 2(a) ). Several monoclonal antibodies with broader-spectrum effectiveness were identified from a uniquely abundant locus, namely IGHV1-18 ; one of the most effective antibodies, m31A7, was found to neutralize WT SARS-CoV-2 and its VOCs with astonishing subpicomolar potency. Subsequently, the research team led by Ma reported the cryo-electron microscopy (cryo-EM) and crystal structures of the m31A7-Fab-bound spike protein, and the diffraction data were collected at TLS 15A1 of the NSRRC ( Figs. 2(b) and 2(c) ). The structural information revealed that m31A7 targeted the spike receptor-binding domain (RBD) only when it was in the “up” position ( Figs. 2(b) and 2(c) ). Footprint mapping indicated that m31A7 approached the RBD from a unique angle and that the contact area bypassed the important mutated residues of most VOCs, such as E484 and K417, contributing to escaping (a) (b) (c) (d)