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. 2020 Aug 7;369(6504):731-736.
doi: 10.1126/science.abc7424. Epub 2020 Jun 15.

Broad neutralization of SARS-related viruses by human monoclonal antibodies

Anna Z Wec  1 , Daniel Wrapp  2 , Andrew S Herbert  3 , Daniel P Maurer  1 , Denise Haslwanter  4 , Mrunal Sakharkar  1 , Rohit K Jangra  4 , M Eugenia Dieterle  4 , Asparouh Lilov  1 , Deli Huang  5 , Longping V Tse  6 , Nicole V Johnson  2 , Ching-Lin Hsieh  2 , Nianshuang Wang  2 , Juergen H Nett  1 , Elizabeth Champney  1 , Irina Burnina  1 , Michael Brown  1 , Shu Lin  1 , Melanie Sinclair  1 , Carl Johnson  1 , Sarat Pudi  1 , Robert Bortz 3rd  4 , Ariel S Wirchnianski  4 , Ethan Laudermilch  4 , Catalina Florez  4 , J Maximilian Fels  4 , Cecilia M O'Brien  3 , Barney S Graham  7 , David Nemazee  5 , Dennis R Burton  5   8   9   10 , Ralph S Baric  6   11 , James E Voss  5 , Kartik Chandran  4 , John M Dye  3 , Jason S McLellan  2 , Laura M Walker  12
Affiliations

Broad neutralization of SARS-related viruses by human monoclonal antibodies

Anna Z Wec et al. Science. .

Abstract

Broadly protective vaccines against known and preemergent human coronaviruses (HCoVs) are urgently needed. To gain a deeper understanding of cross-neutralizing antibody responses, we mined the memory B cell repertoire of a convalescent severe acute respiratory syndrome (SARS) donor and identified 200 SARS coronavirus 2 (SARS-CoV-2) binding antibodies that _target multiple conserved sites on the spike (S) protein. A large proportion of the non-neutralizing antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of preexisting memory B cells elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a _target for the rational design of pan-sarbecovirus vaccines.

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Figures

Fig. 1
Fig. 1. Isolation of SARS-CoV-2 S-specific antibodies.
(A) Frequency of SARS-CoV-2 S-reactive B cells in donor 84 and a SARS-CoV–naïve donor. Fluorescence-activated cell sorting plots are gated on CD19+CD20+IgD−IgM− B cells. swIg, switched immunoglobulin. (B) Binding of 315 isolated antibodies to SARS-CoV-2 S, as determined by BLI. The dashed line indicates the threshold for designating binders (0.1 nm). (C) Clonal lineage analysis. Each lineage is represented as a segment proportional to the lineage size. The total number of antibodies is shown in the center of the pie. Clonal lineages were defined based on the following criteria: identical VH and VL germline genes, identical CDR H3 (third complementarity-determining region of the heavy chain) length, and CDR H3 amino acid identity ≥80%. (D) Somatic mutation load, expressed as the number of nucleotide substitutions in VH, in unique antibodies and members of expanded clonal lineages. Red bars indicate medians. Statistical comparisons were made using the Mann-Whitney test (****P < 0.0001). (E) Proportion of SARS-CoV-2 S binders derived from IgG+ and IgA+ B cells, as determined by index sorting.
Fig. 2
Fig. 2. Binding properties of SARS-CoV-2 S-specific antibodies.
(A) Apparent binding affinities (KDApps) of SARS-CoV-2 S-specific IgGs for prefusion-stabilized SARS-CoV and SARS-CoV-2 S proteins, as determined by BLI. Low-affinity clones for which binding curves could not be fit are designated as “poor fit” (p.f.) on the plot. n.b., nonbinder. (B) IgG KDApps for SARS-CoV-2, SARS-CoV, 229E, HKU1, NL63, and OC43 S proteins. Germline gene usage, clonality, and SHM are presented in the three leftmost columns. SHM load is represented as the number of nucleotide substitutions in VH. (C) Load of somatic mutations in broadly cross-reactive and SARS-CoV– and SARS-CoV-2–specific antibodies. Red bars indicate medians. (D) Degree of clonal expansion in broadly cross-reactive and SARS-CoV– and SARS-CoV-2–specific antibodies. Each lineage is represented as a segment proportional to the lineage size. The total number of antibodies is shown in the center of the pie. (E) Proportion of broadly cross-reactive and SARS-CoV– and SARS-CoV-2–specific antibodies derived from IgG+ and IgA+ B cells, as determined by index sorting. (F) Load of somatic mutations in SARS-CoV-2 S-reactive antibodies isolated from three naïve donors and donor 84. Antibodies from healthy donors were combined for this analysis. (G) Binding activity of antibodies isolated from SARS-CoV-2 S-reactive B cells in donor 84 and three naïve donors to SARS-CoV and SARS-CoV-2 S proteins, as determined by BLI. Statistical comparisons were made using the Mann-Whitney test (**P < 0.01; ***P < 0.001; ****P < 0.0001).
Fig. 3
Fig. 3. Epitope mapping and neutralization screening.
(A) Proportion of SARS-CoV-2 S-specific antibodies _targeting each of the indicated antigenic sites. (B) Heat map showing the competitive binding profiles of the RBD-directed antibodies, as determined by BLI (top) and percent neutralization of authentic SARS-CoV-2 at a 100 nM concentration (bottom). (C) Antibody inhibition of SARS-CoV-2 S binding to endogenous ACE2 expressed on Vero E6 cells, as determined by flow cytometry. Antibodies were mixed with recombinant SARS-CoV-2 S bearing a Twin-Strep tag at a molar ratio of 10:1 before adding to Vero E6 cells. An anti-ebolavirus antibody (KZ52) was used as an isotype control. The “no antigen” control indicates secondary-only staining. The asterisk indicates that no detectable binding was observed. Bars are colored according to epitope specificity, as determined in the BLI competition assay. Data represent three technical replicates. (D) Percent authentic SARS-CoV-2 neutralization in the presence of 100 nM IgG. Antibodies are grouped according to epitope specificity. RBD-directed antibodies that compete or do not compete with ACE2 are designated as ACE2 and non-ACE2, respectively. (E) Antibody neutralization of SARS-CoV and SARS-CoV-2 MLV pseudovirus (strain n-CoV/USA_WA1/2020) using HeLa-ACE2 _target cells, and neutralization of authentic SARS-CoV, SARS-CoV-2, and WIV1-CoV using Vero E6 _target cells. Data represent two technical replicates. (F) Binding EC50s for cell-surface SARS-CoV-2 S are plotted against the percent neutralization of authentic SARS-CoV-2 at 100 nM. Background binding was assessed using mock-transfected HEK-293 cells. Data points are colored according to epitope specificity. RBD-directed antibodies are further categorized based on their competition group: hACE2 indicates hACE2-only competitors; CR3022 indicates CR3022-only competitors; hACE2/CR3022 indicates antibodies that compete with hACE2 and CR3022; other indicates hACE2 and CR3022 noncompetitors. Antibodies with cell-binding EC50s >100 nM are designated as weak binders (w.b.) on the plot. (G) Antibody binding activity to cell-surface SARS-CoV-2 S over time, as determined by flow cytometry. IgGs were incubated with cells expressing WT SARS-CoV-2 over the indicated time intervals. Binding MFI was assessed at 240 min for all samples. CR3022 is included for comparison. Curves are colored by epitope specificity, as in (F). Data represent two technical replicates.
Fig. 4
Fig. 4. Structures of cross-neutralizing antibodies bound to SARS-CoV-2 S.
(A) Negative-stain EM 2D class averages of SARS-CoV-2 S bound by Fabs of indicated antibodies. The Fabs have been pseudocolored for ease of visualization. (B and C) 3D reconstructions of Fab:SARS-CoV-2 S complexes are shown in transparent surface representation (light gray) with the structure of the SARS-CoV-2 S trimer (white surface) and Fabs (ribbon) docked into the density. S-bound Fabs of ADI-55689 (B) and ADI-56046 (C) are colored in orange and purple, respectively. The hACE2 and CR3022 binding sites on S are shaded in red and light blue, respectively.
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