Structural characterization of antibodies binding the conserved central helix and membrane-proximal lower stalk of coronavirus spike glycoprotein 2248624
Monoclonal antibodies from COVID-19 convalescent donors are identified that target two conserved epitopes in the S2 domain of the coronavirus Spike glycoprotein: the central helix and the lower stalk, advancing knowledge of two conserved and structurally vulnerable sites within the coronavirus S2 subunit.
Abstract
The ongoing threat of zoonotic coronavirus spillover into humans, exemplified by severe acute respiratory syndrome virus 2 (SARS-CoV-2), underscores the urgent need for pan-coronavirus therapeutics that can be deployed to mitigate future pandemics.
Here, we identified monoclonal antibodies from COVID-19 convalescent donors that target two conserved epitopes in the S2 domain of the coronavirus Spike glycoprotein: the central helix (CH) and a membrane-proximal epitope in the heptad repeat 2 (HR2), which we designate the lower stalk (LS).
CH-directed antibodies exhibited broad cross-reactivity across betacoronaviruses, whereas LS-directed antibodies demonstrated reactivity primarily within sarbecoviruses. Using cryogenic electron microscopy (cryo-EM), we determined sub-4 Å structures of three cross-reactive CH antibodies–ch.005, ch.007, and ch.010–bound to the prefusion-stabilized SARS-CoV-2 S2 protein, revealing distinct binding poses and contact residues relative to previously described CH antibodies. In parallel, X-ray crystallography studies yielded a sub-2 Å structure of the ls.019 Fab in complex with the LS peptide, providing, to our knowledge, the first structural visualization of a human monoclonal antibody engaging this epitope.
Together, these findings advance our knowledge of two conserved and structurally vulnerable sites within the coronavirus S2 subunit—the central helix and the lower stalk—that can guide the development of broad-spectrum antibody therapeutics and vaccines against current and emerging coronaviruses.
Howard Hughes Medical Institute
Vaccines and Immunotherapy (VAC)
Conserved epitopes within the coronavirus spike S2 domain elicit broadly reactive antibodies, yet many characterized responses show limited neutralizing and variable protective activity, leaving their contribution to antiviral immunity unclear. Building on our previous mapping of evolutionarily conserved spike “coldspots”, we isolated human monoclonal antibodies targeting four conserved epitopes in the spike S2 domain: the internal fusion peptide (iFP), the central helix (CH), the connector domain (CD), and a membrane-proximal epitope in the heptad repeat 2 that we term the lower stalk (LS). A crystal structure of an LS-directed antibody defined a previously unresolved mode of antibody recognition of this membrane-proximal epitope, while cryogenic electron microscopy (cryo-EM) structures revealed that genetically diverse CH-specific antibodies use distinct binding modes to converge on conserved features of the prefusion S2 apex. Despite minimal neutralizing activity, CH- and LS-directed antibodies exhibited distinct antiviral functions. LS-directed antibodies mediated Fcγ receptor-dependent effector activity in vitro, whereas the broadly reactive CH-directed antibody ch.007 lacked detectable antibody-dependent cellular cytotoxicity (ADCC) or cellular phagocytosis (ADCP) activity yet protected mice from lethal SARS-CoV-2 MA10 challenge, with protection abrogated by Fcγ receptor-silencing mutations. Together, these findings expand the genetic, structural and functional landscape of human antibody responses to conserved coronavirus S2 epitopes and demonstrate that CH-directed antibodies can contribute to protective immunity through Fc-dependent mechanisms not predicted by in vitro neutralization or conventional in vitro Fc effector assays.
Adonis A. Rubio, Virginia Crivelli, Václav Hönig et al.· bioRxiv· 0 citations
We previously generated mouse monoclonal antibody N179 against the SARS-CoV-2 nucleocapsid (N) protein and developed a colloidal gold-based immunochromatographic test strip with a 2 ng/mL detection limit and 98% concordance with RT-qPCR. However, the precise epitope recognized by N179 remained undefined. Using GST-fused N protein truncation fragments, Western blotting, and ELISA, we mapped the linear antibody-reactive region to the C-terminal tail of the N protein and identified 390QTVTLL395 as the smallest reactive region under the truncation-mapping conditions used. Multiple sequence alignment of 11 representative SARS-CoV-2 N protein sequences showed complete conservation of this motif across the sequences analyzed. EMBOSS WATER analysis further demonstrated a perfect 6/6 match only in SARS-CoV-2; no identical sequence was found in six other human coronaviruses, four influenza viruses, or five bat coronaviruses. Computational structural analyses predicted that this region may be surface accessible, and all six residues exceeded the default BepiPred 3.0 threshold. These findings identify 390QTVTLL395 as the smallest reactive region defined by truncation mapping and show that this sequence is conserved among the representative SARS-CoV-2 lineages analyzed. Together with the sequence comparison and structural predictions, these results provide sequence-level information that may help explain the previously observed recognition profile of N179. This motif may serve as a reference for the selection and evaluation of diagnostic antibodies targeting conserved regions of the SARS-CoV-2 N protein.
Zhoujun Su, Junhao Guo, Ning Li et al.· Viruses· 0 citations
The results show the value of nanobody technology for identifying novel neutralising epitopes in the S2 region of beta-coronaviruses with potential for the development of new selective anti-viral agents.
John D. Clarke, Luke M. Jones, I. Buckle et al.· Scientific Reports· 0 citations
The isolation of a human monoclonal antibody from a SARS-CoV-2 convalescent donor that targets the conserved S2 stem helix region is reported, demonstrating the potential of in vitro affinity maturation to expand the neutralization breadth of stem-helix-targeting antibodies across divergent betacoronaviruses.
Panpan Zhou, M. Yuan, Yue-Xiu Zhang et al.· PLoS Pathogens· 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
ABSTRACT Human parainfluenza viruses (PIVs) are a leading cause of respiratory illness, particularly in vulnerable populations, where infection can lead to severe disease. Despite their clinical impact, there are currently no licensed vaccines or effective antiviral treatments available. PIVs have two large surface proteins, the fusion and hemagglutinin-neuraminidase (HN) proteins, both of which are targets of neutralizing antibodies. In this study, we identified and characterized two human monoclonal antibodies (mAbs), 5217-2 and 5217-9, which bind recombinant PIV3 HN protein, recognize PIV3-infected cells, and neutralize in vitro. We determined the antigenic sites of the PIV3 HN-specific mAbs via biolayer interferometry and found mAb 5217-9 targets a previously defined neutralizing antigenic site, while mAb 5217-2 binds a distinct antigenic site, expanding the known antigenic landscape. To further understand the newly defined epitope, we determined a cryo-electron microscopy (cryo-EM) structure of mAb 5217-2 in complex with PIV3 HN, revealing an epitope adjacent to the PIV3 HN protein receptor binding site. We also determined the structure of the previously discovered anti-PIV3 HN mAb PIV3HN-09, previously shown to be partially protective in vivo. In a hamster challenge model of PIV3, mAb 5217-2 significantly reduced lung viral titers, demonstrating its protective capacity. Furthermore, as the mAb PIV3HN-05 was previously shown to cross-neutralize PIV1, we evaluated its protective efficacy in a PIV1 challenge model, demonstrating reduced lung viral titers. Overall, these findings provide new insights into PIV3 HN epitopes to support structure-based vaccine design and demonstrate protective mAbs for both PIV3 and PIV1. IMPORTANCE Human parainfluenza viruses (PIVs) are a major cause of respiratory illness, yet there are currently no licensed vaccines or targeted antiviral therapies. A better understanding of how antibodies recognize viral surface proteins is critical for developing effective interventions. In this study, we identified human monoclonal antibodies that neutralize PIV3 and mapped the specific regions of the viral hemagglutinin-neuraminidase (HN) protein they recognize. Structural analysis revealed a previously uncharacterized antibody-binding site near the HN active site and expanded the known antigenic landscape of this protein. We also demonstrate protective activity of these antibodies in animal models against PIV3 and PIV1 infection. These findings provide new targets and structural insights that can guide the development of vaccines and antibody-based therapies against PIVs. Human parainfluenza viruses (PIVs) are a major cause of respiratory illness, yet there are currently no licensed vaccines or targeted antiviral therapies. A better understanding of how antibodies recognize viral surface proteins is critical for developing effective interventions. In this study, we identified human monoclonal antibodies that neutralize PIV3 and mapped the specific regions of the viral hemagglutinin-neuraminidase (HN) protein they recognize. Structural analysis revealed a previously uncharacterized antibody-binding site near the HN active site and expanded the known antigenic landscape of this protein. We also demonstrate protective activity of these antibodies in animal models against PIV3 and PIV1 infection. These findings provide new targets and structural insights that can guide the development of vaccines and antibody-based therapies against PIVs.
Katelyn D. McCaffrey, B. G. Esfahani, Mohamed A. Elbehairy et al.· Journal of Virology· 0 citations
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