SARS-CoV-2 recruits a haem 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 haem 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 the virus co-opts the haem metabolite for the evasion of humoral immunity via allosteric shielding of a sensitive epitope and demonstrate the remarkable structural plasticity of the NTD.

Figure 1.. Structures of SARS-CoV-2 spike-biliverdin (A,B) and spike-P008_056 Fab (C) complexes.

(A) Cryo-EM 3D 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 grey, respectively. (B) Details of the biliverdin binding pocket in the crystal structure. SARS-CoV-2 NTD is shown as cartoons with selected amino acid residues and biliverdin in sticks. Carbon atoms of the protein chain, sugars (NAG), and biliverdin are in purple, grey and green, respectively; the remaining atoms are coloured as follows: oxygen, red; nitrogen, blue; and sulphur, yellow. Dark grey dashes are hydrogen bonds.

Figure 2.. Biliverdin strongly downmodulates the reactivity of SARS-CoV-2 spike with antibodies present in immune sera.

Left: Mean fluorescence intensity (MFI) of IgG staining of HEK293T 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 coloured 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, 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 IgM and IgA antibodies. Each pair of connected symbols represents an individual patient.

Figure 3.. Biliverdin decreases binding to SARS-CoV-2 spike by a group of human monoclonal IgGs.

(A) Antibodies were titrated 6-fold 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 colour-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 colour-coded as a heatmap of the quartile values. (C) 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 normalising to the MFI of the biliverdin-insensitive COVA1–18 at the highest concentration against spike. (E) IgG indicated above each graph were titrated 5-fold 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.

Figure 4.. 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. S11d). (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.