Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Jan 20;26(1):105783.
doi: 10.1016/j.isci.2022.105783. Epub 2022 Dec 9.

Molecular basis for antiviral activity of two pediatric neutralizing antibodies targeting SARS-CoV-2 Spike RBD

Yaozong Chen  1 , Jérémie Prévost  2   3 , Irfan Ullah  4 , Hugo Romero  5 , Veronique Lisi  5 , William D Tolbert  1 , Jonathan R Grover  6 , Shilei Ding  2 , Shang Yu Gong  2   7 , Guillaume Beaudoin-Bussières  2   3 , Romain Gasser  2   3 , Mehdi Benlarbi  2 , Dani Vézina  2 , Sai Priya Anand  2   7 , Debashree Chatterjee  2 , Guillaume Goyette  2 , Michael W Grunst  6 , Ziwei Yang  6 , Yuxia Bo  8 , Fei Zhou  9 , Kathie Béland  5 , Xiaoyun Bai  10 , Allison R Zeher  9   10 , Rick K Huang  9   10 , Dung N Nguyen  1 , Rebekah Sherburn  1 , Di Wu  11 , Grzegorz Piszczek  11 , Bastien Paré  12 , Doreen Matthies  9 , Di Xia  10 , Jonathan Richard  2   3 , Priti Kumar  4 , Walther Mothes  6 , Marceline Côté  8 , Pradeep D Uchil  6 , Vincent-Philippe Lavallée  5   13   14 , Martin A Smith  5   12 , Marzena Pazgier  1 , Elie Haddad  3   14 , Andrés Finzi  2   3   7
Affiliations

Molecular basis for antiviral activity of two pediatric neutralizing antibodies targeting SARS-CoV-2 Spike RBD

Yaozong Chen et al. iScience. .

Abstract

Neutralizing antibodies (NAbs) hold great promise for clinical interventions against SARS-CoV-2 variants of concern (VOCs). Understanding NAb epitope-dependent antiviral mechanisms is crucial for developing vaccines and therapeutics against VOCs. Here we characterized two potent NAbs, EH3 and EH8, isolated from an unvaccinated pediatric patient with exceptional plasma neutralization activity. EH3 and EH8 cross-neutralize the early VOCs and mediate strong Fc-dependent effector activity in vitro. Structural analyses of EH3 and EH8 in complex with the receptor-binding domain (RBD) revealed the molecular determinants of the epitope-driven protection and VOC evasion. While EH3 represents the prevalent IGHV3-53 NAb whose epitope substantially overlaps with the ACE2 binding site, EH8 recognizes a narrow epitope exposed in both RBD-up and RBD-down conformations. When tested in vivo, a single-dose prophylactic administration of EH3 fully protected stringent K18-hACE2 mice from lethal challenge with Delta VOC. Our study demonstrates that protective NAbs responses converge in pediatric and adult SARS-CoV-2 patients.

Keywords: Immunology; Virology.

PubMed Disclaimer

Conflict of interest statement

A.F. has filed a provisional patent application on the following monoclonal antibodies: CV3-1 and CV3-25. A.F., J.P., V.L. M.A.S., V.-P.L., and E.H. have filed a provisional patent application on the following monoclonal antibodies: EH3 and EH8.

Figures

None
Graphical abstract
Figure 1
Figure 1
Isolation of RBD-specific mAbs from a pediatric patient (A) Pseudoviruses encoding the luciferase gene (Luc+) and bearing SARS-CoV-2 full-length S (Wuhan-Hu-1 strain) were used to infect 293T-hACE2 cells in presence of increasing dilutions of indicated COVID-19+ plasma samples at 37°C for 1 h prior infection of 293T-hACE2 cells. Fitted curves and half maximal inhibitory dilution (ID50) values were determined using a normalized nonlinear regression. (B) Indirect ELISA was performed using recombinant SARS-CoV-2 RBD protein and incubation with COVID-19-positive plasma samples from two adult donors (S002, S006 [CV3]) and the pediatric patient of interest (Patient 12). Anti-RBD antibody binding was detected using horseradish peroxidase (HRP)-conjugated anti-human IgG. Relative light unit (RLU) values obtained with BSA (negative control) were subtracted and further normalized to the signal obtained with the anti-RBD mAb CR3022 present in each plate. Seropositivity thresholds were calculated using ten pre-pandemic COVID-19-negative plasma samples. (C) Cryopreserved PBMCs obtained from Patient 12 were stained for the expression of cell-surface markers (CD3, CD14, CD19, IgD, IgG) and probed with fluorescently labeled SARS-CoV-2 RBD proteins. RBD-specific B cells (CD3− CD14− CD19+ IgD- IgG+ RBD-AF488+ RBD-AF647+) were individually sorted into a 96-well plate, followed by targeted B cell receptor sequencing. (D) Pseudoviruses Luc+ bearing SARS-CoV-2 full-length S (Wuhan-Hu-1 strain) were used to infect 293T-hACE2 cells in presence of increasing concentrations of indicated mAbs isolated from Patient 12 at 37°C for 1 h prior infection of 293T-hACE2 cells. Fitted curves and half maximal inhibitory antibody concentration (IC50) values were determined using a normalized nonlinear regression. Error bars indicate means ± SEM.
Figure 2
Figure 2
Characterization of potent pediatric RBD-specific neutralizing mAbs (A and B) Cell-surface staining of 293T cells expressing full-length S from indicated variants using EH3 (A) and EH8 (B) mAbs. The graphs show the median fluorescence intensities (MFI). Dashed lines indicate the reference value obtained with S D614G. Statistical significance was tested using mixed-effects ANOVA with a Dunnett post-test (∗∗∗∗p < 0.0001). (C and D) Pseudoviruses encoding the luciferase gene (Luc+) and bearing SARS-CoV-2 full-length S from indicated variants were used to infect 293T-hACE2 cells in presence of increasing concentrations of EH3 (C) or EH8 (D) at 37°C for 1 h prior infection of 293T-ACE2 cells. (E) Cell-to-cell fusion was measured between 293T effector cells expressing HIV-1 Tat and SARS-CoV-2 S D614G, which were incubated in presence of increasing concentrations of CV3-1, EH3, or EH8 at 37°C for 1 h prior coculture with TZM-bl-hACE2 target cells. (C–E) Fitted curves and IC50 values were determined using a normalized nonlinear regression. (F) Cell-surface staining of CEM.NKr-Spike (Wuhan-Hu-1 strain) using increasing concentrations of CV3-1, EH3, or EH8 mAbs. (G) Parental CEM.NKr cells were mixed at a 1:1 ratio with CEM.NKr-Spike cells and were used as target cells. Cryopreserved PBMCs from an uninfected donor were used as effector cells in a fluorescence-activated cell sorting (FACS)-based ADCC assay. The graphs shown represent the percentages of ADCC obtained in the presence of increasing concentrations of CV3-1, EH3, or EH8 mAbs. These results were obtained in at least 3 independent experiments. Error bars indicate means ± SEM.
Figure 3
Figure 3
Molecular basis of SARS-CoV-2 S recognition by EH3 and EH8 antibodies (A) Crystal structures of EH3 Fab-RBD and EH8 Fab-RBD complexes. Structures were superimposed based upon the RBD domain and are shown as ribbons. The molecular surfaces are displayed over the variable regions of both Fabs (constant domains were omitted for simplicity). The Cα atoms of the RBD that contribute to receptor ACE2 binding residues are highlighted as red spheres. (B) Epitope footprints of EH3 (yellow line) and EH8 (blue line) contoured over the RBD surface (green). ACE2 binding sites are colored in red, and the major RBD residues known to contribute to Fab-RBD interactions are labeled. (C) Diagram of the buried surface area (BSA) of RBD residues contributing to EH3-RBD (yellow), EH8-RBD (blue), and ACE2-RBD (red) interfaces. The BSA of individual RBD residues was calculated using PISA and is given as the sum contributed by the heavy and light chains of the antibody or as the average contributed by ACE2 in two high-resolution structures (PDB IDs 6VW1 and 6M0J21). Residue mutations found in VOCs are denoted above with those occurring in the Omicron variant underlined. (D and E) The interaction network at the (D) EH3-RBD and (E) EH8-RBD interface. The Fab-RBD structures are shown in the center with close-up views showing contacts mediated by the antibody’s CDRs to the RBD (green). Hydrogen bonds and salt bridges with bond lengths <3.5 Å are depicted as yellow dashed lines, and the VOCs mutations are marked with red asterisks. IGHV3-53-conserved residues in D are highlighted by red boxes.
Figure 4
Figure 4
Epitope-driven S conformational preference by RBM-binding EH3 and Ridge-directed EH8 (A and B) EH8, but not EH3, recognized both RBD-up and RBD-down S. Structural superimposition of (A) EH3 (left panel) and (B) EH8 (right panel) onto SARS-CoV-2 Spike in fully closed conformation (6VXX, left) and “one-RBD-up” conformation (6VSB, right). Epitopes of EH3 and EH8 are colored in yellow and blue, respectively. Structural alignments show potential clashes for the heavy chain of EH3 with the adjacent RBD when bound to the closed Spike, whereas the ridge-binding EH8 can bind to both the RBD-up and RBD-down Spike with no steric clashes. (C) The total buried surface area (BSA) at the Fab-RBD interface contributed by heavy/light chains of two nAbs and ACE2 (average of two ACE2-RBD crystal structures 6M0J and 6VW120). (D and E) EH3 in context of other IGHV3-53 antibodies. (D) Structures of EH3-RBD and the IGHV3-53 antibodies available in PDB in complexes with SARS-Cov-2 antigen were superimposed based upon the RBD. The constant regions of the Fabs were omitted for clarity. (E) The table (right panel) shows a list of IGHV3-53 antibodies used for the structural alignment. EH3 is shown (blue) superimposed on the 26 structurally available IGHV3-53 derived antibodies (semi-transparent gray ribbons) that bind with similar angle. Two antibodies, COVA2-39 and C144, which bind using different angles of approach, are colored pink and orange, respectively. (F) Heavy chain CDRs sequence alignments of EH3 with 16 selected RBD-specific IGHV3-53 antibodies. The identical amino acids as compared with EH3 are shaded in colors. Residues involved in salt bridges or H-bonds to the RBD are marked above the sequence with (+) for the side chain, (−) for the main chain, and (±) for both. (G) Comparison of four structurally resolved RBD-directed IGHV1-18 antibodies, including EH8, Beta-44 (7PS6), BG7-15 (7M6G), CV07-250 (6XKQ), and S309 (6WPT), showing distinct RBD epitopes and angles of approach. The constant regions of the Fabs were omitted for clarity.
Figure 5
Figure 5
Epitope mapping of RBD-specific mAbs by site-directed mutagenesis (A and B) Cell-surface staining of 293T cells expressing selected full-length SARS-CoV-2 S harboring RBM mutations using EH3 (A) and EH8 (B). The graphs shown represent the median fluorescence intensities (MFI) corrected for cell-surface S expression of the corresponding mutant using the CV3-25 mAb and further normalized to the MFI obtained with S D614G (WT). Dashed lines indicate the reference value obtained with S D614G (WT). Error bars indicate means ± SEM. These results were obtained in at least three independent experiments. Statistical significance was tested using one-way ANOVA with a Dunnett post-test. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001). (C) Structural representation of SARS-CoV-2 RBD depicted as a surface model (PDB: 6VW1) using UCSF ChimeraX. Amino acid substitutions able to significantly decrease the binding of indicated ligands are colored in red (if decrease is more than 50% compared with WT) or in yellow (if decrease is less than 50% compared with WT).
Figure 6
Figure 6
Prophylactic administration of EH3 effectively protected K18-hACE2 mice from lethal challenge of SARS-CoV-2 Delta variant (A) Schematic presenting the experimental design for testing in vivo efficacy of the EH3 antibody delivered at 12.5 mg/kg body weight intraperitoneally (i.p.) in a prophylactic regimen 12 h before challenging K18-hACE2 mice with 1 × 105 PFU SARS-CoV-2 Delta-nLuc VOC. Human IgG1-treated (n = 6) mice were used as control. (B) Representative BLI images of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions. (C and D) Temporal quantification of nLuc signal as flux (photons/sec) computed non-invasively. (E) Temporal changes in mouse body weight with initial body weight set to 100% for an experiment shown in A. (F) Kaplan-Meier survival curves of mice (n = 4–6 per group) statistically compared by log rank (Mantel-Cox) test as in A. (G and H) Ex vivo imaging of indicated organs and quantification of nLuc signal as flux (photons/sec) after necropsy as per A. (I) Fold changes in nucleocapsid mRNA expression in brain, lung, and nasal cavity tissues. Data were normalized to Gapdh mRNA in the same sample and in non-infected mice after necropsy. (J) Viral loads (nLuc activity/mg) from indicated tissues using Vero E6 cells as targets. Virus loads in indicated tissues were determined when they succumbed to infection and at 10 days post-infection (dpi) for surviving mice. (K and L) Fold-change of cytokine mRNA expression in brain and lung tissues. Data were normalized to Gapdh mRNA in the same sample and in non-infected mice after necropsy. Cytokines in indicated tissues were determined when they succumbed to infection and at 10 dpi for surviving mice. Grouped data in (C–E) were analyzed by two-way ANOVA followed by Tukey’s multiple comparison tests. The data in (H–L) were analyzed by t test followed by non-parametric Mann-Whitney U tests (∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001). Mean values ±SD are depicted.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Waterfield T., Watson C., Moore R., Ferris K., Tonry C., Watt A., McGinn C., Foster S., Evans J., Lyttle M.D., et al. Seroprevalence of SARS-CoV-2 antibodies in children: a prospective multicentre cohort study. Arch. Dis. Child. 2021;106:680–686. doi: 10.1136/archdischild-2020-320558. - DOI - PubMed
    1. Weisberg S.P., Connors T.J., Zhu Y., Baldwin M.R., Lin W.H., Wontakal S., Szabo P.A., Wells S.B., Dogra P., Gray J., et al. Distinct antibody responses to SARS-CoV-2 in children and adults across the COVID-19 clinical spectrum. Nat. Immunol. 2021;22:25–31. doi: 10.1038/s41590-020-00826-9. - DOI - PMC - PubMed
    1. Yuan M., Liu H., Wu N.C., Lee C.C.D., Zhu X., Zhao F., Huang D., Yu W., Hua Y., Tien H., et al. Structural basis of a shared antibody response to SARS-CoV-2. Science. 2020;369:1119–1123. doi: 10.1126/science.abd2321. - DOI - PMC - PubMed
    1. Zhang Q., Ju B., Ge J., Chan J.F.W., Cheng L., Wang R., Huang W., Fang M., Chen P., Zhou B., et al. Potent and protective IGHV3-53/3-66 public antibodies and their shared escape mutant on the spike of SARS-CoV-2. Nat. Commun. 2021;12:4210. doi: 10.1038/s41467-021-24514-w. - DOI - PMC - PubMed
    1. Parameswaran P., Liu Y., Roskin K.M., Jackson K.K.L., Dixit V.P., Lee J.Y., Artiles K.L., Zompi S., Vargas M.J., Simen B.B., et al. Convergent antibody signatures in human dengue. Cell Host Microbe. 2013;13:691–700. doi: 10.1016/j.chom.2013.05.008. - DOI - PMC - PubMed

LinkOut - more resources