Mosaic RBD nanoparticle elicits immunodominant antibody responses across sarbecoviruses

Universal vaccines cross-protecting against sarbecoviruses including SARS-CoV-2 are in great need under continuous emergence of SARS-CoV-2 variants and potential novel coronavirus. Nanoparicle vaccines displaying mosaic receptor-binding domains (RBDs) or spike (S) proteins from SARS-CoV-2 and other sarbecoviruses were used for preparedness to emergent zoonotic outbreak. Here, we describe a self-assembling nanoparticle using lumazine synthase (LuS) as the scaffold to display RBDs from different sarbecoviruses. The mosaic LuS-RBD vaccines induced cross-reactive binding and neutralizing antibody responses to sarbecoviruses. Single B cell sequencing revealed that mosaic LuS-RBD elicited B-cell receptor (BCR) repertoire using an immunodominant germline gene pair of IGHV14-3: IGKV14-111 in mice. Most of the tested IGHV14-3: IGKV14-111 monoclonal antibodies (mAbs) are broadly cross-reactive to the clade 1a, 1b and 3 sarbecoviruses. By antibody binning and cryo-electron microscopy, we determined a reprensentative IGHV14-3: IGKV14-111 mAb, M2-7, bound to an conserved epitope on RBD largely overlapping with a pan-sarbecovirus mAb S2H97, which suggested that mosaic nanoparticles expended B cells recognizing the common epitopes shared by different clades of sarbecoviruses. These results provide immunological insights into the cross-reactive responses elicited by mosaic nanoparticle against emerging sarbecoviruses. To identify the potential mechanisms contributing to the broad-spectrum neutralization induced by the mosaic nanoparticle vaccines, we analyzed the antigen-specific antibody repertoire using single-cell BCR profiling. BALB/c mice were immunized with two doses of LuS-RBD or LuS-mRBD-B. At fourteen days post-vaccination, using RBD proteins conjugated with fluorochrome as the probe, antigen-reactive germinal center B cells (BGC) from lymph nodes were isolated and sorted by flow cytometry (GL-7+B220hiCD38loIgD−CD93−CD138−). To obtain the paired variable (V)-regions, chromium immune profiling solution (10X Genomics) was applied to these BGC cells for single-cell BCR sequencing (scBCR-seq). Sequences covering the full-length V(D)J segments for both heavy and light chains were analyzed.