The alpha/B.1.1.7 SARS-CoV-2 variant exhibits significantly higher affinity for ACE-2 and requires lower inoculation doses to cause disease in K18-hACE2 mice

Abstract

The alpha/B.1.1.7 SARS-CoV-2 lineage emerged in autumn 2020 in the United Kingdom and transmitted rapidly until winter 2021 when it was responsible for most new COVID-19 cases in many European countries. The incidence domination was likely due to a fitness advantage that could be driven by the receptor-binding domain (RBD) residue change (N501Y), which also emerged independently in other variants of concern such as the beta/B.1.351 and gamma/P.1 strains. Here, we present a functional characterization of the alpha/B.1.1.7 variant and show an eightfold affinity increase towards human angiotensin-converting enzyme-2 (ACE-2). In accordance with this, transgenic hACE2 mice showed a faster disease progression and severity after infection with a low dose of B.1.1.7, compared to an early 2020 SARS-CoV-2 isolate. When challenged with sera from convalescent individuals or anti-RBD monoclonal antibodies, the N501Y variant showed a minor, but significant elevated evasion potential of ACE-2/RBD antibody neutralization. The data suggest that the single asparagine to tyrosine substitution remarkable rise in affinity may be responsible for the higher transmission rate and severity of the B.1.1.7 variant.

Keywords: SARS-CoV-2; epidemiology; human; immunology; inflammation; mouse; mouse model.

Figure 1.. Biophysical characterization of recombinant receptor-binding domain (RBD) variants.

(A) Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) total protein stain of RBD variants and angiotensin-converting enzyme-2 (ACE-2). (B) Size-exclusion chromatography (SEC) profiles of the purified proteins run in a BioSep-SEC-S3000 column. Purity was determined by peak integration with the Empower software. (C) Thermal denaturation curves of the RBD wild-type (wt), N439K, and N501Y variants. Data are represented as the first derivative of the intrinsic fluorescence ratio 350:330 nm of the mean of three replicates. Vertical dashed lines represent the inflection temperatures (Ti). Biolayer interferometry (BLI) sensorgrams of RBD wt (D), N439K (E), and N501Y (F) binding to ACE-2-Fc immobilized in anti-human Fc capture (AHC) sensors. ACE-2-immobilized sensors were dipped into 7- to 11-point dilution series of RBD for 500 s, followed by dissociation for another 500 s.

Figure 2.. Development of COVID-19-like disease in mice.

(A) Weight evolution of K18-hACE2 mice challenged intranasally with 2.5 × 10³ (low, n = 9 per group) or 2.5 × 10⁴ p.f.u. (high, n = 5 per group) SARS-CoV-2 B.1 (Freiburg isolate, FR-4286) or B.1.1.7. Global differences between the groups were analysed with the Kruskal–Wallis test. (B) Viral load measured in the lungs on day 2 post-infection. The mice were infected with the same doses as described in (A). The expression of SARS-CoV-2 RNA was analysed by real-time quantitative polymerase chain reaction (qPCR). Values normalized to 18S-rRNA are presented as mean ± standard error of the mean (SEM). * = 0.019.

Figure 3.. Antibody-mediated inhibition potency of recovered COVID-19 patient sera.

(A) Inhibition of wild-type (wt), N439K, and N501Y receptor-binding domain (RBD) towards angiotensin-converting enzyme-2 (ACE-2) in serum from convalescent COVID-19 individuals (n = 140). Friedman test with Dunn’s multiple comparisons. Orange lines represent medians. ****p < 0.0001. Linear regression and Spearman correlation analyses for N439K vs wt (B) and N501Y vs wt (C). Trend line represents linear regression. (D) Neutralization of serum from convalescent COVID-19 individuals (n = 10) against B.1 and B.1.1.7 calculated by the plaque reduction neutralization test (PRNT) and analysed using Wilcoxon matched-pairs signed ranked tests with multiple comparisons corrections. NS, no serum.

Figure 4.. Antibody-mediated inhibition potency of polyclonal sera and monoclonal antibodies (mAbs) isolated from mice immunized against wild-type (wt) receptor-binding domain (RBD) or spike protein.

IC₅₀ comparison of the inhibition of polyclonal mice sera from an animal vaccine model based on RBD (A) or spike (B) challenged with RBD wt, N439K, and N501Y. Connecting lines represent non-linear fits using the equation [inhibitor] vs normalized response with variable slope. Vertical dashed lines delimit the IC₅₀ values, where they intersect the horizontal 50% inhibition dashed line. Data are presented as mean ± standard error of the mean (SEM). Comparison of the inhibition potency (log[IC₅₀]) of mouse mAbs (n = 8) calculated from three independent experiments against RBD wt and RBD N439K (C) or RBD N501Y (D), analysed by linear regression and Spearman correlation. The trend represents a linear regression. Dashed line signals equidistance between axis (i.e. slope 1). Neutralization fold changes over 1.5 are highlighted. Only mAbs with IC₅₀ values within the range of concentrations tested were included in the statistical analyses (i.e. 8 out of 18 tested mAbs).

Author response image 1.. Inhibitory potency of COVID-19 convalescent patient sera against RBD variants.

Antibody-mediated inhibition of serum from recovered COVID-19 patients (n = 150) against RBD wt, N501Y and P.1 (harboring the Y417N, E484K, and N501Y mutations). Statistical comparisons between groups were performed using the Friedman test with Dunn’s multiple comparisons. Orange lines represent medians.

Author response image 2.