Deciphering the Mechanistic Continuum of Broadly Neutralizing Class 4 Antibodies Targeting Conserved Cryptic Epitopes of the SARS-CoV-2 Spike Protein : Operating at the Intersection of Binding, Allostery and Immune Escape

Understanding atomistic basis of multi-layer mechanisms employed by broadly reactive neutralizing antibodies of SARS-CoV-2 spike protein without directly blocking receptor engagement remains an important challenge in coronavirus immunology. Class 4 antibodies represent an intriguing case: they target a deeply conserved, cryptic epitope on the receptor-binding domain yet exhibit variable neutralization potency across subgroups F1 (CR3022, EY6A, COVA1-16), F2 (DH1047), and F3 (S2X259). In this study, we employed a multi-modal computational approach combining atomistic and coarse-grained simulations, mutational scanning of binding interfaces, binding free energy calculations, and allosteric modeling using dynamic network analysis to map the allosteric landscapes and binding hotspots of these antibodies. Through this approach, our data revealed that distal binding can influence ACE2 engagement and immune escape traits through the confluence of direct interfacial interactions and allosteric effects. We found that group F1 antibodies can operate via classic allostery by modulating flexibility in the receptor binding domain loop regions and indirectly interfering with ACE2 binding using long-range effects. Group F2 antibody DH1047 represents an intermediate mechanism, engaging residues T376, R408, V503, and Y508 hotspots which are both critical for ACE2 binding and under immune pressure. Mutational scanning and rigorous binding free energy calculations highlight the synergy between hydrophobic and electrostatic interactions, while dynamic network modeling reveals a shift toward localized communication pathways connecting the cryptic site to the ACE2 interface. Our results demonstrate how group F3 antibody S2X259 achieves efficient synergistic mechanism through confluence of direct competition with ACE2 and localized allosteric effects leading to stabilization of the spike protein at the cost of increased escape vulnerability. Dynamic network analysis identifies a shared “allosteric ring” embedded in the core of the receptor binding domain and serving a conserved structural scaffold mediating long-range signal propagation with antibody-specific extensions propagating toward the ACE2 interface. The findings of this study support a modular model of antibody-induced allostery where neutralization strategies evolve via refinement of peripheral network connections, rather than complete redesign of the epitope itself. Taken together, this study establishes a robust computational framework for atomistic understanding of mechanisms underlying neutralization activity and immune escape for class 4 antibodies which harness conformational dynamics, binding energetics, and allosteric modulation to influence viral entry. The findings highlight the modular evolution of neutralization strategies, where progressive refinement of peripheral interactions enhances potency but increases susceptibility to immune pressure.