Dissociation kinetics and avidity gate SARS-CoV-2 neutralization by HR2 stem helix antibodies

Abstract

SARS-CoV-2 evolution has reduced the efficacy of clinical monoclonal antibodies, underscoring the need for therapeutics targeting conserved viral regions. The Spike (S) heptad repeat 2 (HR2) stem helix is highly conserved across SARS-CoV-2 variants and related betacoronaviruses. Although neutralizing antibodies to this region have been identified, the evolution of humoral responses to HR2 and the determinants of effective neutralization at this site remain poorly understood. We previously identified human neutralizing antibodies to a conserved peptide within this region (HR2 coldspot). Here, longitudinal analysis over 30 months shows that HR2-specific antibodies persist and undergo somatic hypermutation, yet antibodies isolated at later time points did not surpass the breadth or potency of hr2.016, which emerged shortly after primary infection. Crystal structures of four HR2 stem-helix antibodies revealed convergent recognition across distinct antibody lineages. However, comparison of hr2.016 with its non-neutralizing clonal relative hr2.086 showed that adopting this shared binding mode is not sufficient for effective neutralization. Characterization of this antibody pair through surface plasmon resonance and molecular dynamics simulations revealed that robust neutralization requires slow intrinsic dissociation reinforced by avidity. Together, these findings show that continued evolution of HR2-specific responses does not necessarily enhance antibody breadth or potency and that, despite convergent epitope recognition, effective neutralization requires slow intrinsic dissociation reinforced by avidity, highlighting kinetic stability as a key criterion for antibody discovery and vaccine design against viral epitopes. Significance statement SARS-CoV-2 spike HR2 stem helix is highly conserved and targeted by broadly neutralizing antibodies, but how HR2 responses evolve and what converts its recognition into neutralization remain unclear. We show that HR2 antibodies remain rare after repeated antigen exposure and can accumulate somatic mutations without gaining breadth or potency. Although distinct lineages converge on the same epitope and binding mode, structural similarity does not ensure neutralization. Instead, antiviral activity requires prolonged HR2 engagement, generated by slow intrinsic dissociation and amplified by IgG avidity. These findings establish antibody residence time as a critical determinant of neutralization at the conserved HR2 fusion epitope, providing a kinetic principle to guide antibody discovery and vaccine design.