Aug 2026· Proceedings of the National Academy of Sciences of the United States of America· Vol 123 32, pp.
e2535385123
· 0 citations· 57 references
Medicine
TL;DR
Mechanistic studies reveal that NTD-5 and NTD-9 antibodies neutralize by inducing S1 shedding, thereby extending this mechanism to selected NTD-directed antibodies and providing a structural and mechanistic framework for rational vaccine and antibody design resilient to antigenic drift.
Abstract
The N-terminal domain (NTD) of the SARS-CoV-2 spike (S) is a critical antibody target, yet its epitope organization, neutralization mechanisms, and immune evasion strategies remain incompletely resolved. Here, we classify NTD antibodies into nine spatially distinct classes (designated as NTD-1 to NTD-9), including a cryptic epitope defined here (NTD-8). Mechanistic studies reveal that NTD-5 and NTD-9 antibodies neutralize by inducing S1 shedding, thereby extending this mechanism to selected NTD-directed antibodies. Format profiling shows that while most NTD antibodies require bivalency, selected antibodies from NTD-3, NTD-5, and NTD-9 retain neutralizing activity in Fab form. Profiling 41 antibodies across prototype, Delta, and 17 Omicron subvariants defines an epitope-resolved escape landscape and enables dissection of three convergent evasion strategies: contact residue disruption, glycan shielding, and conformational remodeling. Notably, the KP.3.1.1 subvariant uses a dual escape mechanism in which ∆S31 introduces N30 glycosylation and substantially remodels the S27-R34 region, undermining recognition by both NTD-5 and NTD-9 antibodies. These findings provide a structural and mechanistic framework for rational vaccine and antibody design resilient to antigenic drift.
Neutralizing antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) exhibit distinct patterns of potency, breadth, and resilience, yet the molecular mechanisms underlying these differences remain incompletely understood. Here, we employ an integrated computational framework combining structural analysis, conformational dynamics, mutational scanning, binding energetics, and allosteric network modeling to systematically dissect Class 3 and Class 4 antibodies. Analysis of single-antibody complexes (COV2-3835, COV2-3891, COV2-3906) and synergistic dual-antibody pairs reveals a fundamental mechanistic dichotomy: Class 3 antibodies exert localized mechanical constraint strictly confined to the binding interface, whereas Class 4 antibodies propagate long-range allosteric destabilization from an evolutionarily conserved hydrophobic core through the β-sheet core to the RBM loop. Mutational scanning and energetic analyses demonstrate that Class 4 epitopes are anchored by an immutable hydrophobic core exquisitely sensitive to mutation yet conserved across viruses, conferring ultra-broad binding and limited escape potential, while Class 3 epitopes exhibit a plastic periphery with variable sensitivity that creates multiple escape pathways. Predictions show excellent agreement with deep mutational scanning data, validating our approach. Allosteric network analysis identifies β-sheet core residues as essential communication hubs, where convergence of high centrality and extreme perturbation sensitivity establishes critical hotspots resistant to mutation. This multi-pronged framework provides a generalizable paradigm for understanding antibody neutralization mechanisms, predicting immune escape, and guiding rational design of next-generation therapeutics that balance potency, breadth, and resilience.
Mohammed Alshahrani, Will Gatlin, Max Ludwick et al.· Physical Chemistry, Chemical...· 0 citations
Two human-derived monoclonal antibodies are characterized that recognize conserved epitopes on the SARS-CoV-2 RBD and retain activity across antigenically distinct variants, and conserved, mutationally constrained epitopes may serve as targets for vaccines designed to elicit antibody responses resilient to ongoing SARS-CoV-2 evolution and future sarbecovirus emergence.
M. Abernathy, William B. Foreman, Jasmyn A. Lopez et al.· bioRxiv· 0 citations
The N-terminal domain (NTD) of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike harbors a neutralizing antibody supersite and is a recurrent site of antigenic change. However, how sequence variability across pre- and post-vaccination immune landscapes reshapes antibody recognition remains incompletely characterized.
Clinical-genomic surveillance was combined with structural modeling and molecular dynamics to identify antigenically relevant NTD changes and probe their structural impact on the neutralizing antibody interface. SARS-CoV-2 profiles were compared across two periods reflecting contrasting immune contexts: a pre-vaccination period dominated by B.1.1.33 and a post-vaccination period dominated by the Omicron sublineage BQ.1.1. A local cohort (n=126) supported descriptive characterization. An expanded cohort integrating local and GISAID genomes with harmonized clinical metadata (n=534; 401 pre-vaccination, 133 post-vaccination) was used to prioritize candidate loci by their association with clinical severity.
In adjusted models, age remained independently associated with severity in both periods, whereas sex did not. In the pre-vaccination cohort, Spike S in the NTD was associated with greater severity, without phylogenetic segregation by outcome. This association is exploratory and was used to prioritize an antigenically located NTD change for structural analysis. Q23K was regionally concentrated in Espírito Santo (57.4%) relative to other Brazilian states (2.7%) and international genomes (1.4%). In the post-vaccination cohort, an overlapping deletion event (ORF9b -29/N -33) was associated with non-severe disease, but should be interpreted cautiously given metadata heterogeneity, cohort structure, and the nature of the locus. However, in this period clinical outcome and sequencing source were partially confounded. A source-stratified sensitivity analysis restricted to GISAID-only samples showed the association persisted, but could not rule out a source- or pipeline-dependent indel-calling artifact, so the finding is reported as exploratory. Although Q23K itself did not persist, adjacent NTD changes (T19I/A27S/D24 -26) became recurrent post-vaccination, indicating remodeling of the same antigenic neighborhood rather than persistence of a single mutation. Structural modeling and molecular dynamics of the NTD-antibody 1 -87 complex supported reorganization of the modeled antibody-antigen interface, with redistribution of supersite contacts rather than loss of structural compatibility.
These findings link NTD variability across immune contexts to structural reorganization of the modeled antibody-antigen interface and support integrating genomic surveillance with structural analysis to prioritize antigenically relevant changes for functional testing.
J. S. Ardisson, Brena Ramos Athaydes, Mariane Vedovatti Monfardini Sagrillo et al.· Frontiers in Cellular and In...· 0 citations
Comparing wild-type XG83 and modified MuXG83 shows how allosteric tuning affects antibody-antigen compatibility in developing variations like Omicron, and indicates that non-epitope (potentially allosteric) changes to CDRH3 are also important while investigating potential development and neutralization before advancement.
Muhammad Waqas Nasir, Qi-Yun Liang, Jun He et al.· Microbiology spectrum· 0 citations
Background The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the COVID-19 pandemic, which resulted in millions of deaths globally and had profound social, economic, and political consequences. Although effective vaccines and antiviral therapies have substantially reduced the global burden of COVID-19, the continued emergence of viral variants highlights the need for next-generation effective vaccine strategies capable of providing broader and more durable immune response. Methods In this work, we provide an immunoinformatic approach for multi-epitope vaccine (MEV) design and prediction. Based on the spike (S) and nucleocapsid (N) proteins of SARS-CoV-2, immunoinformatic methods were used to identify the epitopes for B cells, cytotoxic T lymphocytes (CTL), and helper T lymphocytes (HTL). The B cell, CTL, and HTL epitopes were conjugated with flexible linkers GSG, GSGG, and a Gb-1 peptide conjugated to the C-terminal of the MEV ccandidate. Results The final MEV candidate exhibited favorable predicted characteristics, with a molecular weight of approximately 55.47 kDa and a length of 498 amino acid residues. Computational analyses indicated that the designed construct was antigenic, non-toxic, non-allergenic, and possessed suitable physicochemical properties and predicted solubility, supporting its potential as a vaccine candidate for further investigation. Molecular docking analysis demonstrated favorable interactions between the MEV construct and selected Toll-like receptors (TLRs), while molecular dynamics (MD) simulations suggested the stability of the vaccine-receptor complexes throughout the simulation period. Furthermore, C-ImmSim-based immune simulation predicted the induction of both humoral and cellular immune responses following the proposed immunization schedule. Collectively, these findings highlight the potential of the designed MEV construct as a computationally optimized vaccine candidate and provide a framework for future experimental evaluation. Conclusion This study presents a computationally designed MEV candidate against SARS-CoV-2 by integrating immunoinformatics approaches, structural modeling, molecular docking, molecular dynamics simulations, and immune response prediction. The findings suggest that the proposed MEV construct may possess favorable immunogenic and structural properties; however, experimental validation through in vitro and in vivo studies remains essential to confirm its safety, immunogenicity, and protective efficacy. The proposed approach provides a valuable strategy for accelerating rational vaccine design and may serve as a foundation for future development of experimentally validated vaccine candidates.
Analysis suggests mAbs generically defined as class 1/4 mAbs may be separated into two classes, like Class 1/4 mAbs, and class 4/1 mAbs that make extensive interactions with the class-4 epitope and limited contacts with the class-1 knob498-596.
M. Piepenbrink, Yao Ma, Simran Panjwani et al.· iScience· 0 citations
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