Nanobody repertoire generated against the spike protein of ancestral SARS-CoV-2 remains efficacious against the rapidly evolving virus
- Laboratory of Cellular and Structural Biology, The Rockefeller University, United States
- Center for Global Infectious Disease Research, Seattle Children's Research Institute, United States
- Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, California Institute for Quantitative Biosciences, Byers Hall, University of California, San Francisco, United States
- Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, United States
- Department of Pediatrics, University of Washington, United States
- Department of Biochemistry, University of Washington, United States
Figures
Figure 1
Nanobody repertoires generated against wild-type SARS-CoV-2 remain efficacious.
Nanobodies targeting the S1-RBD, S1 non-RBD, and S2 regions of spike effectively neutralize lentivirus pseudotyped with delta and omicron BA.1 SARS-CoV-2 spikes (PSV) from infecting angiotensin-converting enzyme 2 (ACE2)-expressing HEK293T cells. (A) The half-maximal inhibitory concentration (IC50) is reported for the indicated nanobodies against wild-type (Mast et al., 2021), delta, and omicron BA.1 PSV. These values are summarized in Figure 2—source data 1. Nanobodies are grouped by epitope and arranged within each epitope by neutralization efficacy against the wild-type PSV. n ≥ 4 (B) The structural differences in the receptor-binding domain (RBD) of the delta (PDB ID: 7SBO) and omicron BA.1 (PDB ID: 7T9K) variants are depicted. Nanobody epitopes are heat-mapped ranging from pale white (epitopes with weak neutralization against SARS-CoV-2) to dark red (indicating strong neutralization). Boxed in gray are mutations specific to each variant mapped in blue on the aforementioned structures. The nanobodies that contributed to epitope mapping are in bold in panel A. The color bar scale for each epitope is the neutralizing strength of each nanobody epitope, calculated as the normalized −log10 ratio of nanobody binding (IC50) to variant versus wild-type SARS-CoV-2 Spike S1. For groups with multiple nanobodies, the average −log10 (IC50) is first calculated for the nanobodies within that group, then normalized to a neutralization score within the 0–100 range using the min and max average −log10 (IC50) for that group. A higher score indicates more potent neutralization of the variant relative to the wild-type. All structural representations were created on ChimeraX (Pettersen et al., 2021).
Figure 2
Nanobody epitope groups and mAb epitope classes mapped on receptor-binding domain (RBD).
(A) Nanobody epitope groups overlapping with the three mAb epitope classes (classes 1, 2, and 3). Nanobody groups are highlighted in gold, while mAb class footprints are outlined in black. mAb epitopes are taken from Cox et al. (B) A single RBD subunit with the angiotensin-converting enzyme 2 (ACE2) footprint/RBM mapped in green. All epitopes are represented on the structure of wild-type RBD (PDB ID: 6M0J). All structure representations were generated using ChimeraX (Pettersen et al.).
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Figure 2—source data 1
Nanobody binding and neutralization characterization; related to Figures 2 and 3.
- https://cdn.elifesciences.org/articles/89423/elife-89423-fig2-data1-v1.xlsx
Figure 3
Affinities of the nanobody repertoire against SARS-CoV-2 variants.
(A) Each nanobody is plotted against their affinity (KD) for SARS-CoV-2 Spike S1 from wild-type, delta and omicron BA.1, BA.4, XBB, and BQ.1.1 strains. Kinetic values are summarized in . Nanobodies are characterized into their respective epitope groups as described previously (Mast et al., 2021). (B) Displayed are the structures of the receptor-binding domain (RBD) of spike delta (PDB ID: 7SBO), omicron BA.1 (PDB ID: 7T9K), omicron BA.4 RBD modeled using AlphaFold (Jumper et al., 2021), omicron XBB (PDB ID: 8IOU), and omicron BQ.1.1 (PDB ID: 8FXC). The structures feature heat-mapped epitopes of binding, ranging from pale white (weak binding to SARS-CoV-2) to dark red (strong binding to SARS-CoV-2). In the gray box, mutations specific to each variant are highlighted in blue. The nanobodies that contributed to epitope mapping are in bold in panel A. The color bar scale indicates each epitope’s binding affinity strength, represented as the normalized −log10 ratio of nanobody binding (KD) of variant versus wild-type SARS-CoV-2 Spike S1. For groups with multiple nanobodies, the average −log10 (KD) for the nanobodies within that group was calculated, then normalized to an affinity score ranging from 0 to 100 using the min and max average −log10 (KD) for that group. Higher −log10 ratios indicate stronger binding of the nanobody to the variant versus wild-type. S1-RBD-16 bound omicron BA.1 and BA.4/5 in ELISA. S1-RBD-11 was not tested against omicron BA.4. S1-65 was not tested against BA.1. Only S1-1, S1-RBD-22, S1-RBD-9, S1-4, S1-35, S1-39, S1-RBD-6, S1-RBD-5, S1-23, S1-RBD-40, S1-RBD-46, and S1-46 were tested against omicron XBB and BQ.1.1. All structure representations were generated using ChimeraX (Pettersen et al., 2021).
Figure 4
Potent neutralization by broadly active nanobodies.
Nanobodies targeting the S1-RBD of spike and raised against the original wild-type sequence remain highly efficacious in neutralizing an evolved variant of SARS-CoV-2, omicron variant BA.5. The derived neutralization curves are plotted from the results of a plaque-forming reduction neutralization test with the indicated nanobodies. Serial dilutions of each nanobody were incubated with ~200 SARS-CoV-2 virions for 60 min and then overlaid on a monolayer of TMPRSS2-expressing Vero E6 cells. After 72 hr, cells were fixed and stained with crystal violet stain (1% wt/vol in 20% ethanol) allowing for the enumeration of viral plaques. The percent plaque inhibition for each nanobody dilution, summarized in Figure 4—source data 1, was used to fit the neutralization curves depicted in the figure. The colored shaded areas denote 90% confidence intervals for each fitted curve. n ≥ 3.
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Figure 4—source data 1
Neutralization data from the plaque reduction neutralization test (PRNT) assay with omicron BA.5 virus.
- https://cdn.elifesciences.org/articles/89423/elife-89423-fig4-data1-v1.csv
Figure 5 with 1 supplement
Refining epitopes of the nanobody repertoire.
All epitopes are mapped on the structure of wild-type receptor-binding domain (RBD) (PDB ID: 6M0J). (A) The original six RBD nanobody epitope groups capable of binding and/or neutralizing one or more variants are highlighted in dark gray. Further refinement of four groups: I, III, IV, and V led to the identification of six additional epitope groups – resulting in a total of 12 epitope groups able to bind one or more variants of concern. (B) Summary of variant specific and broadly reactive nanobodies. (C) Nanobody groups predicted to bind/neutralize the circulating omicron variants EG.5 and HV.1. All structure representations were created on ChimeraX (Pettersen et al., 2021).
Figure 5—figure supplement 1
Mapping of variant-specific amino acid changes on different SARS-CoV-2 variants.
(A) Nanobody epitopes predicted to retain binding/neutralization to current circulating SARS-CoV-2 variants of concern (VoC) mapped on wild-type Spike receptor-binding domain (RBD) (PDB ID: 6M0J). (B) The RBD of each variant of concern is colored gray and shown in four different orientations. Amino acid differences of each variant are colored blue. The mutations were mapped on the RBDs of spike omicron BA.4 RBD (modeled), omicron XBB.1, EG.5, and HV.1 (all three mapped on PDB ID: 8IOU) and omicron BQ.1.1 (PDB ID: 8FXC). All structure representations were created on ChimeraX (Pettersen et al., 2021).
Figure 6
Persistence of synergistic neutralization with nanobody cocktails against SARS-CoV-2 variants of concern.
(A) S1-1 synergizes with S1-23 in neutralizing SARS-CoV-2 PSV. The upper left panel shows two representations of spike with the accessible S1-1 (dark goldenrod) and S1-23 (sky blue) epitopes (PDB ID: 6VYB). The measured affinities for S1-1 and S1-23for the receptor-binding domain (RBD) of wild-type, delta, and omicron BA.1 are displayed. Both S1-1 and S1-23 neutralize wild-type (i), whereas only S1-1 neutralizes delta at the concentrations shown (ii). In spite of a lack of neutralization at these concentrations, S1-23, synergizes with S1-1 and enhances its neutralization of delta SARS-CoV-2 PSV (ii). As neither S1-1 nor S1-23 are able to bind to the RBD of omicron BA.1, neither nanobody neutralizes omicron BA.1 SARS-CoV-2 PSV (iii). (B) S1-36 synergizes with S1-RBD-22 in neutralizing SARS-CoV-2 PSV. As in A, the upper left panel shows two representations of spike with the accessible S1-36 (cornflower blue) and S1-RBD-22 (sandy brown) epitopes. The measured affinities for S1-36 and S1-RBD-22 are displayed. Both S1-36 and S1-RBD-22 neutralize wild-type (i), whereas only S1-RBD-22 effectively neutralizes delta and omicron BA.1 SARS-CoV-2 PSV at the concentrations shown (ii and iii, respectively). However, S1-36 synergizes with S1-RBD-22 and enhances its neutralization of the three depicted SARS-CoV-2 pseudoviruses (i, ii, and iii). (C) An example of no interactions (synergistic or antagonistic) between S1-1 and LaM2 (Fridy et al., 2014), a non-specific nanobody that does not bind the RBD of delta. (D) An example of antagonism, where higher concentrations of S1-23 interferes with the ability of S1-RBD-16 to neutralize delta SARS-CoV-2 PSV. These nanobodies have adjacent epitopes on the RBD of spike and were previously shown to interfere with each other’s binding to their respective epitope (Mast et al., 2021). n = 4. Source data in .
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Figure 6—source data 1
Neutralization data from synergy experiment.
- https://cdn.elifesciences.org/articles/89423/elife-89423-fig6-data1-v1.xlsx
Figure 7
Nanobodies effective against circulation variants of concern.
The nanobody epitopes (red) that retain effectiveness against wild-type (PDB ID: 7KNB), delta (PDB ID: 7V7O), omicron BA.1, omicron BA.4 (the nanobody epitopes were mapped on to PDB ID: 7XO5 for both BA.1 and BA.4 due to the lack of a suitable BA.4 structure),omicron XBB.1 and BQ.1.1 (the nanobody epitopes were mapped on to PDB ID: 8IOU for both XBB.1 and BQ.1.1 due to the lack of a suitable BQ.1.1 structure). The spike trimer (silver) where glycans are represented as light blue sticks for each variant is displayed in three views: top = side view of spike trimer; middle = birds eye view looking down on the S1 domain and; bottom = birds eye view (same as middle view), with the S1 domain rendered transparent to enable visualization of the S2 domain. All structure representations were created on ChimeraX (Pettersen et al., 2021).
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Nanobody repertoire generated against the spike protein of ancestral SARS-CoV-2 remains efficacious against the rapidly evolving virus
eLife 12:RP89423.
https://doi.org/10.7554/eLife.89423.3
