Circulating SARS-CoV-2 spike N439K variants maintain fitness while evading antibody-mediated immunity

Abstract

SARS-CoV-2 can mutate and evade immunity, with consequences for efficacy of emerging vaccines and antibody therapeutics. Here, we demonstrate that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is a highly variable region of S and provide epidemiological, clinical, and molecular characterization of a prevalent, sentinel RBM mutation, N439K. We demonstrate N439K S protein has enhanced binding affinity to the hACE2 receptor, and N439K viruses have similar in vitro replication fitness and cause infections with similar clinical outcomes as compared to wild type. We show the N439K mutation confers resistance against several neutralizing monoclonal antibodies, including one authorized for emergency use by the US Food and Drug Administration (FDA), and reduces the activity of some polyclonal sera from persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics.

Keywords: COVID-19; N439K; SARS-CoV-2; Spike; monoclonal antibody escape; mutation; protein structure; receptor binding motif; variant.

Graphical abstract

Figure 1

The RBM exhibits significant natural diversity in circulating SARS-CoV-2 viruses SARS-CoV-2 variants (retrieved from CoV-GLUE) are based on 209,239 high-quality sequences downloaded from GISAID on November 30, 2020. (A) Structure of the SARS-CoV-2 RBD-hACE2 complex (PDB: 6M0J) highlighting the RBM (blue) and residue N439 (yellow). (B) Thirty-four residues (the size of the RBM) were randomly sampled without replacement 50,000 times from the mature S protein (excluding the RBM). Median entropies were computed for each draw. The resulting 50,000 median entropies were used to build the entropy distribution of residues other than the RBM. The top 10% medians are highlighted in red. The median entropy of RBM residues was compared with the non-RBM entropy distribution to determine the variability of the RBM relative to non-RBM residues. To allow for a fair comparison, sampling was performed without enforcing residue contiguity, as the RBM is not contiguous in sequence space. Therefore, in any given sample, residues are unlikely to share any functional relationship. (C) Per-residue entropies of the mature S protein were smoothed by plotting medians of a 25-aa center-aligned sliding window. Smoothing allows visualizing local peaks of variability. The RBM residues and the NTD, RBD, and S2 domains are highlighted. Due to the non-contiguous nature of the RBM in sequence space, the sliding window median at RBM residues is diluted by neighboring non-RBM residues. (D) Boxplot of per-residue entropies in four S domains (or full mature S protein). The lower and upper hinges correspond to the first and third quartiles. The lower/upper whiskers extend from the hinge to the smallest/largest value no further than 1.5 times the inter-quartile range. Outliers beyond the end of the whiskers are not plotted but are retained for statistical testing. Pairwise comparisons by Mann-Whitney U tests. p value thresholds are 0.05 (^∗), 0.01 (^∗∗) and 0.001 (^∗∗∗); ns, not significant. See also Figures S1 and S2.

Figure S1

High RBM variability in deposited SARS-CoV-2 sequences is consistent with a dynamic RBD:hACE2 binding interface, related to Figures 1 and 2 (A) Number of observed variants in four S domains (or full mature S protein) normalized by the total number of residues in each domain, where the number of observed isolates required to call a variant is varied along the x axis. (B) Distributions of distances observed for RBD (gray):hACE2 (gold) residue pairs: K417-D30, E484-K31, Q493-K31, Q493-E35, G496bb-K353, G502bb-K353bb, Y449-Q42, Y449-D38, K31-E35 (bb = backbone interaction). RBD:ACE2 residue pairs were chosen based on RBM residues with high binding energies as determined by the binding energy % column (green) in Figure 2. Distances were computed every 2.5 ns from 118.7 μs of molecular dynamics simulation data. Dashed lines indicate a distance of 3.5 Å and the percentage of distances below and above 3.5 Å are annotated to the left and right of the lines, respectively.

Figure 2

RBM functional constraints compared to RBM natural diversity Each residue in the RBM is annotated by several metrics, depicted as a heatmap. DMS scores: outlined in black boxes (center) are summaries of hACE2 binding and RBD expression deep mutational scanning (DMS) experimental results (Starr et al., 2020b). DMS score is the binding or expression fold change of a variant over WT on a log10 scale (red indicating improvement and blue indicating loss as compared to WT). In the “mutagenesis” columns, DMS results are given for each residue as either the minimum (most disruptive variant) or the average score across all possible variants of a residue, except for the reference residue and the stop codon. In the “observed variants” columns, minimum and average scores are computed only across variants that have been observed in GISAID (same set of sequences as used for Figure 1). When no natural variants have been observed, cells are gray. Data were sorted on the leftmost DMS column. Frequency: each RBM position is annotated with the frequency of non-reference amino acids in deposited sequences (darker red indicating higher frequency; at least 1 supporting sequence per 25,000 deposited sequences is required to call a variant). The number of countries in which variants have been observed is also annotated (darker purple indicating more countries). Binding energy: a re-refined SARS-CoV-2 RBD:hACE2 complex X-ray structure (PDB: 6M0J) was used to determine the approximate, decomposed binding free energy associated with each RBM residue. Results for each RBM residue are expressed as a percentage of the total binding interface interaction energy (darker green indicating stronger contribution to the binding energy). See also Figures S1 and S2.

Figure S2

RBDs from bat and pangolin Sarbecovirus isolates bind to hACE2 despite RBM divergence, related to Figures 1 and 2 (A) Top – Percent identity to SARS-CoV-2 using a sliding window size of 30 amino acids for seven related Sarbecoviruses (see figure key, ^∗: viruses which bind to hACE2) across the RBD region of the Spike protein. Bottom – Site-specific entropy plot across the RBD protein alignment of SARS-CoV-2 and 68 related viruses (Table S1). Sites constituting the RBM are annotated in blue; the x axis refers to absolute positions in the SARS-CoV-2 Spike protein sequence. Right – boxplot of site-specific entropy values for the RBM sites (blue) and the full RBD (gray). (B) Sequence alignment (left) and identity for RBM and RBD (right) to SARS-CoV-2 of the RBD sequences showing binding to hACE2. RBM residues indicated by blue boxes. (C) Binding of hACE2 to human, pangolin, and bat Sarbecovirus RBDs by BLI. Bat CoV RaTG13, Bat CoVs ZC45, BtKY72 and BGR2008 have also been tested and did not bind hACE2.

Figure 3

The N439K RBM mutation has arisen independently multiple times, twice forming significant lineages (A) Phylogenetic tree (de-duplicated and down-sampled) showing the relationship among representative global SARS-CoV-2 variants, with N439K variants highlighted in color. Two significant N439K lineages, one in Scotland (>500 sequences, blue circles) and one in 32 countries (>6,000 sequences, yellow circles) were detected as of January 6, 2021. The N439K mutation has also emerged independently on at least seven occasions (red circles show four of these) bringing the total country count to 34. Vertical bars indicate global lineage, the presence of N439K (same colors as tree), D614G (orange) or D614N (dark gray). The scale bar corresponds to a single nucleotide polymorphism (SNP). (B) Frequency of N439K variants relative to sampling time and their geographical area of occurrence (see key): Africa (Morocco, Nigeria), Americas (Brazil, USA), Asia (Japan, Singapore, South Korea), the European countries Denmark, England, Republic of Ireland and Scotland and other European countries (Belgium, Bosnia-Herzegovina, Croatia, Czech Republic, Faroe Islands, Finland, France, Germany, Hungary, Italy, Luxembourg, Netherlands, Northern Ireland, Norway, Poland, Romania, Slovakia, Sweden, Switzerland, Wales), and Oceania (Australia, New Zealand). The prominent light gray bars correspond to other European countries. See Table S2 for total numbers for each country. (C) Frequency of the two N439K lineages (same colors as A) over time relative to all sequences for that country (gray) and their normalized contributions (lower panels) in Scotland, England, Republic of Ireland, and Denmark. See also Figure S3.

Figure S3

Virological and clinical results stratified by positions 439 and 614, related to Figures 3 and 5 (A) Phylodynamic analysis showing lineage growth rates relative to sampling times for UK lineages in Scotland. Data used for analysis were sampled between Feb 28, 2020 and Aug 18, 2020 (see STAR methods and http://sars2.cvr.gla.ac.uk/RiseFallScotCOVID/). The Scottish N439K lineage i (which co-occurs with D614G) is indicated in black along with whether wild-type N439 lineages are D614 (red) or D614G (blue). The inset shows a boxplot for the distributions of these genotypes. Note, only the growth rates between −50 and 50 are plotted. (B) Comparison of clinical severity between D614/N439, D614G/N439 and D614G/N439K genotypes by patient age group for 1591 patients whose diagnostic samples were sequenced. Ordinal scale scored by oxygen requirement: 1. No respiratory support, 2: Supplemental oxygen, 3: Invasive or non-invasive ventilation or oxygen delivery by high flow nasal cannulae, 4: Death.

Figure 4

N439K creates a new RBD:hACE2 salt bridge and enhances RBD:hACE2 affinity (A–C) X-ray structures of the SARS-CoV (A), SARS-CoV-2 WT (B), and SARS-CoV-2 N439K (C) RBD in complex with hACE2 (based on 2AJF, 6M0J, and current work, respectively). Select interface residues are shown as sticks. hACE2 is shown in orange and RBD in gray. The inset in (C) shows the 2Fo-Fc electron density contoured at 1σ for the K439-E329 salt bridge. (D) Binding affinity of RBD and Spike variants for hACE2 measured by surface plasmon resonance. Monomeric hACE2 is injected successively at 11, 33, 100, and 300 nM onto surface-captured spike extracellular domain (ECD) or RBD; alternately, RBD is injected successively at 3.1, 12.5, and 50 nM onto surface-captured hACE2. All spike ECD contain the D614G mutation. Bar graph: affinity measurements (averages of 3–4 replicates) expressed as a fold change relative to WT binding within each experiment format, where >1 indicates improved binding (smaller K_D) relative to WT. WT K_D values measured as: 95 ± 1.6 nM (Spike surface), 63 ± 1.0 nM (RBD surface), 19 ± 3.3 nM (hACE2 surface); errors are SEM. See also Table S3.

Figure 5

Clinical outcomes and virological evaluation of N439K lineage i indicate maintenance of fitness relative to WT virus (A) Epidemiological growth of the N439/D614, N439/D614G, or N439K/D614G virus in the National Health Service (NHS) Greater Glasgow and Clyde (GGC) Health Board area relative to sampling time in epidemiological (epi) weeks (top) and their relative contributions (bottom) for 1,918 patients whose diagnostic samples were sequenced. (B) Top: real-time PCR data for N439/D614, N439/D614G, and N439K/D614G groups, same patient population as in (A). The N439K genotype was associated with marginally lower Ct values than the N439 genotype (posterior mean Ct value difference between N439K/D614G and N439/D614G: −0.65, 95% CI: −1.22, −0.07). Bottom: correlation between Ct and quantitative viral load. (C) Severity of disease within NHS GGC for a subset of 1,591 patients. Ordinal scale scored by requirement for supplementary oxygen: (1) no respiratory support, (2) supplemental oxygen, (3) invasive or non-invasive ventilation or oxygen delivered by high-flow nasal cannula, and (4) death. Ordinal regression analysis indicated that the N439K viral genotype was associated with similar clinical outcomes compared to the N439 genotype (posterior mean of N439K/D614G genotype effect: 0.06, 95% CI: −1.21, 1.33). (D) Growth curves for GLA1 (N439/D614G) or GLA2 (N439K/D614G) virus isolates in Vero E6 cells with ACE2 and TMPRSS2 overexpression (+TMPRSS2 +ACE2), ACE2 overexpression (+ACE2), or no overexpression. Error bars are SD from three replicates. (E) Competition of GLA1 and GLA2 virus isolates for growth in Vero E6 cells with ACE2 and TMPRSS2 overexpression (+TMPRSS2 +ACE2), ACE2 overexpression (+ACE2), or no overexpression, after inoculation at a matched MOI. Quantification of each virus was performed by tracking the frequency of N439K within the spike gene using metagenomic NGS. Error bars are SD from three replicates. See also Figure S3 and Tables S4–S6.

Figure 6

RBM variants exhibit escape from monoclonal antibodies and sera binding (A and B) Binding of serum and plasma samples from 442 SARS-CoV-2 infected individuals against WT and N439K RBD plotted as (A) ELISA ED₅₀ for each RBD (cut-off for positive binding to WT set at 30) and (B) fold change relative to WT. Data shown are the average of two independent replicates (source data given in Data S1). Blue dots indicate sera with at least 2-fold loss of binding to the N439K RBD variant as compared to WT in both replicates. Purple dots indicate sera from individuals infected with SARS-CoV-2 N439K variant. (C and D) Binding of 140 mAbs from SARS-CoV-2 infected individuals and four clinical-stage or EUA-approved mAbs against WT, N439K, K417V, and N439K/K417V RBD, plotted as (C) ELISA AUC for each RBD and (D) fold change relative to WT. Data shown are the average of two independent replicates (source data given in Data S1). For all, the colored dots indicate mAbs demonstrating at least 2-fold loss of binding to the variant RBD as compared to WT (counted if the average of both replicates is at least 2-fold and each individual replicate is at least 1.7-fold). (E) Kinetics of binding to RBD variants by Octet of six representative mAbs (representative of n = 2 independent experiments). (F) Distribution of the 144 mAbs based on binding to RBD variants (expressed as fold-change over WT) and hACE2 competition (expressed as the mAb concentration blocking 80% of hACE2 binding, BC80, also indicated as a blue gradient; source data in Data S1). Higher BC80 values (lighter blue) correspond to less hACE2 competition, with mAbs indicated at the top of the panels (white) showing no competition at all. See also Figures S4, S5, and S6.

Figure S4

Sera ELISA results, related to Figure 6 ELISA binding of the 33 human sera with a > 2-fold reduction of binding to RBD N439K (A) and of the 6 sera of individuals infected with SARS-CoV-2 N439K viruses (B) to RBD WT (gray), N439K (blue), K417V (yellow) and N439K/K417V (red). Representative of n = 2 independent experiments.

Figure S5

mAb ELISA results, related to Figure 6 ELISA binding of 80 out of the 144 mAbs to RBD WT (gray), N439K (blue), K417V (yellow) and N439K/K417V (red). AUC used for quantification is highlighted between dotted lines. Representative of n = 2 independent experiments. See Data S1 for results of all 144 mAbs.

Figure S6

mAb BLI results, related to Figure 6 Binding of 13 selected mAbs to RBD WT (gray), N439K (blue), K417V (yellow) and N439K/K417V (red) as measured by BLI. Representative of n = 2 independent experiments.

Figure 7

Neutralization of four RBM variants by a panel of antibodies and a nanobody (A) Neutralization of four VSV-pseudovirus variants by six of the mAbs tested. Data shown are representative of n = 3 biological replicates, bars = SD of technical duplicate (Data S1). (B) Correlation of ELISA-binding fold change and neutralization fold change for each variant relative to WT. (C) Top: neutralization IC₅₀ of the D614G virus determined as the geometric mean of three biological replicates. Bottom: neutralization results for all mAbs tested, expressed as a fold-change relative to D614G (all variants are in the background of D614G) (Data S1). The individual values of the three replicates are shown as open circles, their geometric mean as colored bars and the geometric SD as error bars. Each antibody is annotated according to its hACE2 competition (as shown in Figure 6F) as well as its epitope (site I, II, or IV) (Data S1). Gray boxes with a slash indicate not tested for hACE2 competition or epitope analysis. See also Figure S7.

Figure S7

VSV pseudovirus neutralization curves of all mAbs tested, related to Figure 7 Representative of n = 3 biological replicates, bars = SD of technical duplicate.