Two cross-neutralizing antibodies isolated from a COVID-19 convalescent via single B cell sorting.

The ongoing emergence of Omicron subvariants, which exhibit significant resistance to existing therapeutic antibodies, underscores the urgent need to develop new reagents capable of broadly and effectively neutralizing current subvariants. Antigen-specific memory B cells were sorted by flow cytometry. The heavy and light variable region genes of monoclonal antibodies (mAbs) were amplified and cloned into expression vectors. Following protein expression, the binding and neutralizing activities of the mAbs were assessed using ELISA, biolayer interferometry (BLI), and neutralization assays. Additionally, the neutralizing mechanism of mAb C12 was analyzed through protein modeling. We identified two receptor-binding domain (RBD)-targeting cross-neutralizing antibodies (cross-NAbs), B5 and C12. C12 demonstrated potent neutralization against SARS-CoV-2 wild type (WT), BA.1, BA.2, BA.4/5, and BF.7, with IC values below 0.0300 µg/mL, and against XBB and EG.5, with IC values of 0.2340 µg/mL and 0.2762 µg/mL, respectively. B5 exhibited strong neutralizing activity against WT, BA.1, and BA.2, with IC values below 0.0900 µg/mL, and moderate neutralizing ability against BA.4/5, XBB, and EG.5. BLI experiments revealed that both B5 and C12 bind strongly to the RBD of WT and BA.1, with K values below 1.0 × 10 M. Structural modeling of the C12-RBD complex suggested that the full contact of the heavy chain complementarity-determining region 3 (HCDR3) loop with the RBD, together with region between the light chain complementarity-determining region 3 (LCDR3) and the RBD, may contribute to the cross-neutralizing activity of C12. These findings highlight the therapeutic potential of B5 and C12 against evolving Omicron subvariants and provide insights for antibody engineering and vaccine design.