Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes (1)
The SARS-CoV-2 spike ectodomain (S-ECD) folds into a multidomain architecture and includes the receptor-binding domain (RBD), which is essential for viral infectivity, and the structurally adjacent NTD, which plays an uncertain role. Humoral immunity to the spike (S) surface glycoprotein can correlate with protection, and it is the primary antigenic target for most vaccines and monoclonal antibodies (mAbs).
IgG lineages detected by Ig-Seq in the S-ECD fraction but absent from the RBD fraction were deemed to be reactive with spike epitopes outside the RBD. In subject P3, we detected six IgG lineages that bound to S-ECD. Bulk serology ELISAs recapitulated the Ig-Seq result and demonstrated similarly high levels of non-RBD-binding IgG. On average, 84% of the anti-S-ECD plasma IgG repertoire bound to epitopes outside the RBD. CM30 potently neutralized authentic SARS-CoV-2 in vitro (IC50 = 0.83 μg/ml), CM32 was slightly less potent (2.1 μg/ml), whereas CM29 and CM31 showed minimal neutralization activity. The neutralizing mAb CM30, derived from the top-ranking NTD-targeting IgG lineage (21% abundance), was the sole plasma antibody that conferred complete protection to MA10 viral challenge. Interestingly, administration of a cocktail comprising the top non-RBD plasma mAbs CM29–CM31.
Subject P2, with ~10-fold higher neutralizing titer compared to subject P3 (fig. S1 and table S1), displayed a more polyclonal IgG response. MAbs CM25 and CM17, representative of two NTD-targeting lineages each comprising ~2.5% of the response at day 56 (Ig-Seq Lin.6 and Lin.9), were both encoded by unmutated or near-germline IGHV1-24. We found an additional NTD-targeting unmutated IGHV1-24 plasma mAb (CM58) in subject P4. CM17, CM25 and CM58 bound S-ECD with similar single-digit nM affinity and all three potently neutralized SARS-CoV-2 virus, with IC50 values of 0.01–0.81 μg/ml comparable to S309 anti-RBD control.
IGHV1-24 plasma antibodies was detected only in S-ECD fractions (mean 3.7%), but not among anti-RBD IgGs. A “checkerboard” binding-competition experiment indicated the presence of at least two epitope clusters on the NTD, including one targeted by all of the tested IGHV1-24 mAbs (4A8, CM25, CM17, CM58, and 1-68) and the IGHV3-11 mAb CM30.
A cryo-EM structure of CM25 Fabs bound to trimeric S-ECD was determined. SARS-CoV-2 variants of concern contain mutations in the NTD N3 and N5 loops, including Y144/Y145Δ and K147E (UK lineage B.1.1.7), W152C (California B.1.429), and 242-244Δ or R246I (South Africa B.1.351). Alanine substitutions at several of these positions ablated binding or reduced affinity more than fivefold by public IGHV1-24 antibodies as exemplified by 4A8, CM17, and CM25, a result consistent with the CM25-NTD and 4A8-NTD structures. Additionally, we confirmed that an engineered N3-N5 double-mutant and native B.1.351 both evade neutralization by mAbs CM25 and 4A8. Thus, mutations in SARS-CoV-2 variants confer escape from public neutralizing anti-NTD antibodies.
Numerous other NTD mutations—which overlap with the structural epitope recognized by the public IGHV1-24 antibody class—have been described in additional circulating variants, in laboratory escape mutants, and in immunocompromised patients.
1. W. N. Voss, Y. J. Hou, N. V. Johnson, G. Delidakis, J. E. Kim, K. Javanmardi, A. P. Horton, F. Bartzoka, C. J. Paresi, Y. Tanno, C.-W. Chou, S. A. Abbasi, W. Pickens, K. George, D. R. Boutz, D. M. Towers, J. R. McDaniel, D. Billick, J. Goike, L. Rowe, D. Batra, J. Pohl, J. Lee, S. Gangappa, S. Sambhara, M. Gadush, N. Wang, M. D. Person, B. L. Iverson, J. D. Gollihar, J. Dye, A. Herbert, I. J. Finkelstein, R. S. Baric, J. S. McLellan, G. Georgiou, J. J. Lavinder, G. C. Ippolito, Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes. Science (2021), doi:10.1126/science.abg5268.