Breakthrough infection elicits synergistic broadly neutralizing antibodies targeting non-dominant epitopes on RBD and NTD in an individual of inactivated SARS-CoV-2 vaccine

Background/Objectives Identification and characterization of broadly neutralizing monoclonal antibodies from individuals exposed to SARS-CoV-2, either by infection or vaccination, can inform the development of next-generation vaccines and antibody therapeutics with pan-SARS-CoV-2 protection.

Through single B cell sorting and RT-PCR, monoclonal antibodies (mAbs) were isolated from a donor who experienced BA.5 or BF.7 breakthrough infection after three doses of inactivated vaccines. Their binding and neutralizing capacities were respectively measured with ELISA and pseudovirus-based neutralization assay. Their epitopes were mapped by competition ELISA and site- directed mutation.

Among a total of 67 spike-specific mAbs cloned from the donor, four mAbs (KXD643, KXD652, KXD681, and KXD686) can neutralize all tested SARS-CoV-2 variants from wild-type to KP.3. Moreover, KXD643, KXD652, and KXD681 belong to a clonotype encoded by IGHV5-51 and IGKV1-13, and recognize the cryptic and conserved RBD-8 epitope on the receptor-binding domain (RBD). In contrast, KXD686 is encoded by IGHV1-69 and IGKV3-20, and targets a conserved epitope (NTD Site iv) outside the antigenic supersite (NTD Site i) of the N-terminal domain (NTD). Notably, antibody cocktails containing these two groups of mAbs can neutralize SARS-CoV-2 more potently due to synergistic effects. In addition, bispecific antibodies derived from KXD643 and KXD686 demonstrate further improved neutralizing potency compared to antibody cocktails.

These four mAbs can be developed as candidates of pan-SARS-CoV-2 antibody therapeutics through further antibody engineering. On the other hand, vaccines designed to simultaneously elicit neutralizing antibodies towards RBD-8 and NTD Site iv have the potential to provide pan-SARS-CoV-2 protection. Competing Interest Statement The authors have declared no competing interest.