SARS-CoV-2 can recruit a heme metabolite to evade antibody immunity

Abstract

The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of heme metabolism, with nanomolar affinity. Using cryo-electron microscopy and x-ray crystallography, we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that SARS-CoV-2 spike NTD harbors a dominant epitope, access to which can be controlled by an allosteric mechanism that is regulated through recruitment of a metabolite.

Fig. 1. Cryo-EM structures of the SARS-CoV-2 spike-biliverdin complex.

Three-dimensional reconstructions of trimeric SARS-CoV-2 spike ectodomain in 3RBD-down (left) and 1RBD-up (right) conformations determined under saturation with biliverdin. Spike protomers are color-coded. Biliverdin and glycans are shown in green and gray, respectively.

Fig. 2. Crystal structure of isolated SARS-CoV-2 spike NTD bound to biliverdin.

Details of the biliverdin binding pocket in the crystal structure refined at 1.8-Å resolution. SARS-CoV-2 NTD is shown as cartoons with selected amino acid residues and biliverdin in sticks. Carbon atoms of the protein chain, sugars (N-acetylglucosamine, NAG), and biliverdin are in purple, gray, and green, respectively; the remaining atoms are colored as follows: oxygen, red; nitrogen, blue; and sulfur, yellow. Dark gray dashes are hydrogen bonds.

Fig. 3. Biliverdin strongly down-modulates the reactivity of SARS-CoV-2 spike with antibodies present in immune sera.

(Left) Mean fluorescence intensity (MFI) of IgG staining of human embryonic kidney (HEK) 293T cells expressing full-length WT or N121Q SARS-CoV-2 spike by individual patient sera in the absence or the presence of 10 μM biliverdin. Each symbol represents an individual patient (n = 17), and colored dotted lines represent the linear regression for each spike variant. The inset shows posterior probability density plots of values for pairwise contrasts (±biliverdin) for the WT and N121Q spikes. Black dots indicate the median of the distribution, and thick and thin line ranges correspond to the 85 and 95% highest density interval, respectively; the dotted vertical line indicates a zero difference. (Right) Changes in MFI caused by the addition of 10 μM biliverdin, as percent of staining without biliverdin, for serum for IgG antibodies. Each pair of connected symbols represents an individual patient. The P value reported above the plot was calculated using a two-tailed paired Student’s t test comparing the effect of biliverdin (percent change in binding) on the WT spike versus the effect of biliverdin on the N121Q spike for each serum sample.

Fig. 4. Biliverdin decreases binding to SARS-CoV-2 spike by a group of human monoclonal IgGs.

(A) Antibodies were titrated sixfold and assayed by direct ELISA for binding to recombinant S1 biliverdin-depleted by purification under acidic conditions (−biliverdin), same protein but supplemented with biliverdin (+biliverdin) or N121Q S1. Area under the curve (AUC) is shown for IgG that were sensitive to biliverdin and two unaffected control IgGs. AUC values are color-coded as per the key; fold change compared to WT protein are reported. (B) Biliverdin-sensitive IgGs were titrated 10-fold and incubated with 293T cells expressing full-length WT or N121Q SARS-CoV-2 spike with or without 10 μM biliverdin. Binding was detected using an anti-IgG antibody and reduction in binding in the presence of biliverdin is shown as % MFI reduction and color-coded as a heatmap of the quartile values. (C) Enzyme-linked immunosorbent assay (ELISA) titration curves for four neutralizing IgG including the biliverdin-insensitive control COVA1-18. (D) Relative MFI dose-dependent curves for four neutralizing IgG including the biliverdin-insensitive control COVA1-18. Relative MFI calculated by normalizing to the MFI of the biliverdin-insensitive COVA1-18 at the highest concentration against spike. (E) IgG indicated above each graph were titrated fivefold against SARS-CoV-2 spike pseudotype, in the presence and absence of 10 μM biliverdin, and a version of spike encoding the mutation N121Q. COVA1-18 was used as a biliverdin-insensitive control IgG. (F) Neutralization of SARS-CoV-2 (England 02/2020/407073) by IgGs was measured in the absence and presence of 10 μM biliverdin in Vero-E6 cells. P003_027 was used as a biliverdin-insensitive control IgG.

Fig. 5. Cryo-EM structure of the spike-Fab complex.

(A) Reconstruction obtained with multibody refinement in Relion (left) and a zoom on the spike-Fab interface in the structure obtained by consensus refinement (fig. S12D). (B) Refined model of the spike-Fab complex shown as cartoon, with selected amino acid side chains in sticks and indicated. Carbon atoms of the gate and lip NTD elements that relocate to allow Fab binding (arrows) are shown in black. Fab heavy (HV) and light (LV) chains are shown in blue and beige, respectively.