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.
Abstract
Broadly reactive antibodies elicited by SARS-CoV-2 infection or vaccination can reveal conserved viral vulnerabilities and inform vaccines with broad coronavirus coverage. Here, we characterize two human-derived monoclonal antibodies, B2014 and C5078, that recognize conserved epitopes on the SARS-CoV-2 RBD and retain activity across antigenically distinct variants. Notably, C5078 also recognizes diverse sarbecoviruses and remains active against currently circulating variants, including XFG and NB.1.8.1. Cryo-EM structures reveal that B2014 recognizes an epitope adjacent to the class 3 antibody site, whereas C5078 targets the highly conserved, cryptic site V epitope. Structural analysis defines how C5078 uses affinity-matured interactions to engage conserved RBD residues, providing a molecular basis for its exceptional breadth. Deep mutational scanning across multiple SARS-CoV-2 variant backgrounds further defines potential pathways of antibody escape, explaining the loss of B2014 activity against antigenically evolved variants while revealing a high barrier to escape from C5078. Together, these findings define distinct structural solutions for broad RBD recognition and highlight conserved, mutationally constrained epitopes that may serve as targets for vaccines designed to elicit antibody responses resilient to ongoing SARS-CoV-2 evolution and future sarbecovirus emergence.
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.
Jianjie Zhou, Wenyu Li, Xiaoyu Wang et al.· Proceedings of the National...· 0 citations
Broadly neutralizing antibodies (bnAbs) targeting conserved regions of the betacoronavirus spike are important for pan-betacoronavirus protection and pandemic preparedness. Here, we report the isolation of a human monoclonal antibody, CC65.1, from a SARS-CoV-2 convalescent donor that targets the conserved S2 stem helix region. CC65.1 neutralizes various sarbecoviruses, including SARS-CoV-2, and binds to the MERS-CoV spike but lacks MERS-CoV-neutralizing activity due to insufficient binding affinity. We utilized directed evolution to enhance the binding affinity of CC65.1 for the MERS-CoV S2 stem helix, yielding engineered antibody variants with newly acquired MERS-CoV-neutralizing activity. High-resolution structural analysis reveals key paratope mutations that enhance binding and stabilize epitope engagement. Our findings demonstrate the potential of in vitro affinity maturation to expand the neutralization breadth of stem-helix-targeting antibodies across divergent betacoronaviruses. This work supports the development of engineered bnAbs for broadly protective betacoronavirus countermeasures and provides a strategy for achieving cross-lineage neutralization.
Panpan Zhou, M. Yuan, Yuexiu Zhang et al.· PLoS Pathogens· 0 citations
A combined experimental and computational characterization of SARS-CoV-2 variant-specific neutralisation across infection, vaccination and hybrid immunity-driven cohorts is provided, offering a hypothesis-generating framework to contextualize observed differences in antibody responses and epitope recognition across variants.
Jyoti Sawant, Ajit Patil, Madhuri Thakar et al.· Frontiers in Immunology· 0 citations
ABSTRACT Rapid antigenic drift of the SARS‐CoV‐2 receptor‐binding domain (RBD) underlies immune escape and continues to challenge the durability of antibody‐mediated protection. Among the major classes of RBD‐directed antibodies, germline‐encoded IGHV3‐53 responses are highly potent against early SARS‐CoV‐2 variants but are generally compromised by Omicron‐associated mutations. Here, we identify an intrinsically cross‐reactive IGHV3‐53 germline antibody that recognizes multiple pre‐Omicron variants, including SARS‐CoV‐2 wild‐type, Alpha, and Delta. Notably, we demonstrate that targeted somatic evolution can further expand this breadth to overcome the immune escape of different Omicron variants. Guided by integrated structural and sequence analyses, we introduce four somatic mutations (G26E, T28I, S53P, and Y58F) into the germline antibody, resulting in markedly enhanced binding and neutralization of Omicron BA.1, and BA.4/5. High‐resolution crystal structures reveal that these mutations re‐establish interactions disrupted by substitutions in the Omicron RBD and improve binding at a remodeled epitope interface. Collectively, our findings define the structural basis by which specific mutations enhance cross‐variant recognition of SARS‐CoV‐2. This work highlights the underappreciated breadth encoded within the naïve B‐cell repertoire and provides a conceptual framework for engineering and eliciting antibody responses resilient to future antigenic drift.
Huibin Lv, Ziqi Feng, Q. Teo et al.· Advancement of science· 0 citations
The bridging domain (BD) region on hepatitis C virus (HCV) E1E2 glycoprotein complex is a conserved, E1-dependent conformational site of vulnerability formed by E2 residues 646-704. Given its importance for rational vaccine design, here, we characterized the genetic, structural, functional, and epitope features of 25 BD-directed nAbs isolated from HCV-infected individuals or immunized macaques. These antibodies derive from diverse B cell lineages but frequently display recurrent CDRH3 motifs and recognize overlapping epitopes spanning antigenic regions AR4 and AR5, with variable engagement of the E2 back layer (BL) region. A crystal structure of the macaque nAb RM3-26 bound to E2 core domain reveals a BL-directed binding mode analogous to the human nAb hcab40. Importantly, BD-directed nAbs achieve broad neutralization with minimal somatic hypermutation at contact residues and act additively with neutralizing face-directed antibodies. Together, these findings underscore BD as a promising target for rational HCV vaccine design.
Fang Chen, Yen Thi Kim Nguyen, Yi-Zong Lee et al.· Cell Reports· 0 citations
ABSTRACT SARS-CoV-2 antigenic evolution continues to erode the activity of first-generation monoclonal antibodies, underscoring the value of antibodies that recognize conserved features within the spike receptor-binding domain (RBD). As a model for breadth-oriented engineering, we assessed the RBD-directed antibody XG83, which has a CDRH3-dominated paratope. Utilizing the XG83-Wuhan RBD crystal structure as a reference, we integrated MOE alanine scanning and residue scanning with CDRH3 (A116-Y132) to identify chemically reasonable replacements and test interaction to an Omicron BA.1 RBD. The parental CDRH3-centric pose in comparative docking had a better score (−280.18) than the mutant MuXG83 (−254.2), along with 100-ns molecular dynamics showed that MuXG83 was less stable (with higher RMSD/RMSF with less favorable interaction energy). The ELISA results against Omicron BA.1 RBD demonstrated that XG83 had 40% stronger binding than MuXG83; convergence happened only at the highest concentration, concordant with SPR studies and the fact that mutation increased dissociation. According to this binding gap, BA.1 pseudovirus neutralization demonstrated that XG83 was much more powerful than MuXG83, showing that the E118L/F130H CDR-H3 alterations decreased functional activity against Omicron BA.1. These results imply that allosteric influences on interface stability and conformational dynamics by non-epitope CDR-H3 residues can affect antibody performance. Functional testing was confined to Omicron BA.1; therefore, larger variant-panel studies are needed to ascertain if such mutations affect antibody breadth. Our findings highlight a structure-guided approach for optimizing paratopes and indicate that non-epitope (potentially allosteric) changes to CDRH3 are also important while investigating potential development and neutralization before advancement. IMPORTANCE This study shows how modest allosteric characteristics in CDR-H3 control the delicate balance between neutralizing potency and breadth, making a timely and significant addition to SARS-CoV-2 antibody engineering. Using crystallography, alanine scanning, residue scanning, docking, molecular dynamics, and experimental ELISA and neutralization assays, this study offers a structure-guided framework for rational paratope optimization. The discovery of CDR-H3 residues that regulate long-range stability rather than just direct epitope contacts reveals an unappreciated aspect of antibody design and explains why some alterations improve anticipated interactions but degrade functional performance. Importantly, the comparison of wild-type XG83 and modified MuXG83 shows how allosteric tuning affects antibody-antigen compatibility in developing variations like Omicron. These findings illuminate conserved RBD recognition and offer strategies for building next-generation therapeutic antibodies that are more resistant to viral evolution. This study shows how modest allosteric characteristics in CDR-H3 control the delicate balance between neutralizing potency and breadth, making a timely and significant addition to SARS-CoV-2 antibody engineering. Using crystallography, alanine scanning, residue scanning, docking, molecular dynamics, and experimental ELISA and neutralization assays, this study offers a structure-guided framework for rational paratope optimization. The discovery of CDR-H3 residues that regulate long-range stability rather than just direct epitope contacts reveals an unappreciated aspect of antibody design and explains why some alterations improve anticipated interactions but degrade functional performance. Importantly, the comparison of wild-type XG83 and modified MuXG83 shows how allosteric tuning affects antibody-antigen compatibility in developing variations like Omicron. These findings illuminate conserved RBD recognition and offer strategies for building next-generation therapeutic antibodies that are more resistant to viral evolution.
Muhammad Waqas Nasir, Qiyun Liang, Jun He et al.· Microbiology spectrum· 0 citations
