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

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.

transmitted rapidly until winter 2021 when it was responsible for most new COVID-19 23 cases in many European countries. The incidence domination was likely due to a fitness 24 advantage that could be driven by the RBD residue change (N501Y), which also emerged show an eight-fold affinity increase towards human ACE-2. In accordance with this, 28 transgenic hACE-2 mice showed a faster disease progression and severity after infection 29 with a low dose of B.1.1.7, compared to an early 2020 SARS-CoV-2 isolate. When 30 challenged with sera from convalescent individuals or anti-RBD monoclonal antibodies, the 31 N501Y variant showed a minor, but significant elevated evasion potential of ACE-2/RBD 32 In order to examine whether the increased affinity of the N501Y variant for ACE-2 was 119 associated with a more efficient establishment of infection and development of disease, 120 we challenged transgenic ACE-2 humanized K18-hACE2 mice with the early 2020 SARS-121 CoV2 B.1 (Freiburg isolate, FR-4248 31 ) and the B1.1.7 (alpha) strains. The model has been 122 reported to reflect many aspects of COVID-19, including viral replication and 123 histopathological changes in the lungs 32 . Upon infection with two different doses of the B.1 124 strain, we observed no weight loss as an indication of disease development ( Fig. 2A). 125 However, infection with B.1.1.7 at the same doses led to severe disease development in 126 the mice. Mann-Whitney pair-wise comparisons (B.1 vs B.1.1.7) showed a significant 127 difference in weight at low viral doses at days 6 and 7 (multiple comparisons corrected q 128 values 0.009743 and 0.000291, respectively, n = 9 per group). The same tendency, albeit 129 not statistically significant, was observed for the high viral doses (n = 5 per group). We also 130 observed that the virus replicates to higher levels in the lungs at day 2 post-infection with 131 B.1.1.7 compared to B.1 when infected with low viral doses (Fig. 2B). Next, we sought to clarify whether the residue changes could affect the folding and binding 146 properties of the RBD and viral fitness by providing immune evasion. To do so, we first 147 determined the antibody-mediated inhibition potency, measured as the inhibition of the 148 ACE-2/RBD interaction, in sera of recovered COVID-19 patients (n = 140) using a validated 149 in vitro antibody inhibition assay 33 (Fig. 3A, B, C). There was a statistically significant 150 reduction in the inhibition of the N439K and N501Y RBD compared to the wt (p < 0.0001 for both) (Fig. 3A). The inhibition potencies towards the wt and the variants had a highly 152 significant correlation (ρ = 0.9774 and ρ = 0.9581 for N439K and N501Y respectively, p < 153 0.0001), with the best-fit X-intercept ranging from 12.38 to 15.89 for the N439K and N501Y 154 respectively (Fig. 3B, C). However, analyses of convalescent sera (n = 10) using a PRNT virus 155 neutralization platform 34 showed no significant difference in the neutralization potency 156 towards the B.1 and B.1.1.7 strains (Fig. 3D). Next, we analysed the variants' evasion capacity using a previously established vaccine 169 mouse model 20 . Briefly, mice were immunized with wt RBD (n = 3) or wt prefusion-170 stabilized spike protein ectodomain (n = 3). Polyclonal sera were collected after 3 rounds 171 of immunizations, and monoclonal antibodies (mAbs) were developed and characterized 172 from cloned hybridomas. As shown previously 33,35 , polyclonal sera from RBD immunized 173 mice was approx. 4-fold more effective than spike-immunized mice sera, with IC 50 values of 174 2.4-2.6x10 4 (RBD) and 6.6-8.2x10 3 (spike), respectively (Fig. 4A, B). Sera from RBD-175 immunized mice showed no difference in the inhibitory potency against the wt and the 176 variants. However, best-fit IC 50 values from sera from mice immunized with spike differed 177 slightly between strains, as 11% and 19% higher serum concentration was required for the 178 N439K and N501Y variants, respectively, to achieve the same inhibition levels as for the wt 179 RBD. We also evaluated the inhibition potency of mouse mAbs raised against wt RBD and 180 wt spike (n = 18) (Fig. 4C, D). The mAbs were screened for high affinity towards the SARS -181 CoV-2 RBD wt, and their epitopes mapped via competition assays 33 . Linear regression and 182 Spearman correlation analyses of the mAbs with best-fit IC50s within the range of 183 concentration tested (n = 8) showed that the N439K and N501Y mutations had very minor effects on the inhibition potency of the mAbs (N439K R 2 = 0.9777, ρ = 1, p < 0.0001; N501Y 185 R 2 = 0.9832, ρ = 0.9762, p < 0.0001). Emerging clusters of genetically drifted SARS-CoV-2 variants have received much attention due to 203 concerns of enhanced adaptive fitness and viral escape of neutralizing antibodies or T-cell 204 mediated responses. A specific focus has been on the spike protein changes that interact with the 205 ACE-2 receptor and mediate host cell entry. This is also the target for the vast majority of the 206 vaccine strategies. A worldwide effort to sequence viral strains has revealed many emerging 207 variants. However, although many spike variants have been reported so far, there seems to be a 208 limitation in terms of the "freedom" possibilities to which and how many non-synonymous 209 mutations are emerging in the S gene and particularly in the RBD coding region 28 . One of these 210 RBD residue changes is N501Y, which has appeared by convergent evolution in three of the so-211 We aimed to characterize the functional properties of the B.1.1.7 (alpha) variant and 222 address any potential evasion capacity of antibody-mediated neutralization. We also did 223 this for the prevalent RBD mutation N439K and we found a two-fold affinity increase to ACE-2 224 and a partial evasion of antibody-mediated neutralization, lending support to a recently published 225 When we did BLI measurements of the 1:1 interaction of N501Y RBD on ACE-2 immobilized 227 sensors, we found a remarkable eight-fold affinity increase of the variant (2.2 nM) compared to

ACE-2/RBD affinity determination by biolayer interferometry
Binding kinetics experiments were performed on an Octet RED383 system (ForteBio, 313 California, USA) as described elsewhere with minor modifications 35 . Briefly, 13 µg/ml ACE-2-314 Fc was loaded on anti-human Fc capture (AHC) sensors (Pall Life Sciences, California, USA) for 500 315 s, followed by baseline for 60 s, association to 12-point 1.5-fold serial dilutions starting at 150 316 nM for RBD wt, N439K, and N501Y for 500 s, and finally dissociation for another 500 s. For each 317 16-channel column sensor, four sensors loaded with ACE-2-Fc were assigned as a reference and 318 dipped into buffer during the association and dissociation phases. Final sensorgrams were 319 reference subtracted column-wise and globally fitted to a 1:1 binding model. 320

ACE-2/RBD antibody inhibition assay 322
The antibody neutralization potency, calculated from the degree of inhibition of the ACE- CoV-2 to a final titer of 100 TCID 50% /well, and incubated at 4 °C overnight. A "no serum" and a "no 397 virus" (uninfected) control samples were included. TCID 50% control plates (in triplicates) of each of 398 the viruses were included to control for actual virus titer of B.1 and B.1.1.7, respectively. The 399 following day, virus:serum mixtures were added in octuplicates to 2 x 10 4 VeroE6-hTMPRSS2 cells 1 400 seeded in flat-bottomed 96-well plates (Thermo Fisher), and incubated 72 h in a humidified CO 2 401 incubator at 37 °C, 5% CO 2 . Cytopathic effect (CPE) was scored after fixing with 5% formalin 402 (Sigma-Aldrich) and crystal violet stain (Sigma-Aldrich), using a light microscope (Leica DMi1). 403

Blood samples 405
The antibody neutralization potency was assessed in 140 randomly-selected convalescent serum 406 samples (the patient cohort has been described elsewhere 42 ) with IC 50 values for the ACE-2/RBD 407 wt interaction ranging from low to high (estimated from 6-point 4-fold dilution series done as part 408 of a previous study 33 ). A serum pool from healthy individuals was used as a negative control. All aspects of this study were approved by the office of the Danish Working Environment 422 Authority, Landskronagade 33, 2100 Copenhagen Ø, before the initiation of this study. Work with 423 SARS-CoV-2 was performed in a biosafety level 2+ laboratory by personnel equipped with 424 powered air-purifying respirators. 425

Statistics 427
Statistical analyses were performed with GraphPad Prism 9 (GraphPad Software, California, 428 USA). Global differences in the weight of K18-hACE2 mice exposed to high and low doses of 429 SARS-CoV-2 were analyzed with Kruskal-Wallis. Pair-wise comparisons of the effect of SARS-CoV-2 430 variants in weight loss were performed with multiple Mann-Whitney tests (ranks computed for 431 each day) and false discovery rate approach using the two-stage step-up method of Benjamini, 432