Evidence for an ACE2-Independent Entry Pathway That Can Protect from Neutralization by an Antibody Used for COVID-19 Therapy

Abstract

SARS-CoV-2 variants of concern (VOC) acquired mutations in the spike (S) protein, including E484K, that confer resistance to neutralizing antibodies. However, it is incompletely understood how these mutations impact viral entry into host cells. Here, we analyzed how mutations at position 484 that have been detected in COVID-19 patients impact cell entry and antibody-mediated neutralization. We report that mutation E484D markedly increased SARS-CoV-2 S-driven entry into the hepatoma cell line Huh-7 and the lung cell NCI-H1299 without augmenting ACE2 binding. Notably, mutation E484D largely rescued Huh-7 but not Vero cell entry from blockade by the neutralizing antibody Imdevimab and rendered Huh-7 cell entry ACE2-independent. These results suggest that the naturally occurring mutation E484D allows SARS-CoV-2 to employ an ACE2-independent mechanism for entry that is largely insensitive against Imdevimab, an antibody employed for COVID-19 therapy. IMPORTANCE The interaction of the SARS-CoV-2 spike protein (S) with the cellular receptor ACE2 is considered essential for infection and constitutes the key target for antibodies induced upon infection and vaccination. Here, using a surrogate system for viral entry, we provide evidence that a naturally occurring mutation can liberate SARS-CoV-2 from ACE2-dependence and that ACE2-independent entry may protect the virus from neutralization by an antibody used for COVID-19 therapy.

Keywords: ACE2; COVID-19; antibody; neutralization; spike.

FIG 1

Spike mutation E484D leads to cell line-dependent enhancement of infection in a potentially ACE2-independent manner and allows escape from neutralization by Imdevimab. (a) Spike (S) protein scheme (abbreviations: RBD = receptor binding domain, TD = transmembrane domain) and location of residue E484 in the context of the three-dimensional S protein structure (color code: Light blue = S1 subunit [non-RBD], dark blue = RBD, gray = S2 subunit, red = residue E484). (b) Frequency of mutations at S protein residue E484 (letters indicate amino acid exchanges, single letter code). The dashed line shows the threshold for selection of mutants for in-depth analysis (minimum frequency = 75 entries in the GISAID database as of 29.09.2021). (c) Mutations at position E484 lead to cell line-dependent augmentation of infection. Particles pseudotyped with the indicated S proteins were inoculated onto H1299 (human, lung) and Huh-7 (human, liver) cells. At 16–18h postinoculation, transduction efficiency was analyzed by measuring virus-encoded luciferase activity in cell lysates. Presented are the average (mean) data from three biological replicates (each conducted with four technical replicates), for which transduction was normalized against wild-type (WT) SARS-CoV-2 S (set as 1). Error bars indicate the standard error of the mean (SEM). (d) Mutation E484D enables evasion from Imdevimab-mediated neutralization in Huh-7 but not Vero cells. Particles pseudotyped with the indicated S proteins were preincubated (30 min, 37°C) with different concentrations of monoclonal antibodies used for COVID-19 therapy (Casirivimab, Imdevimab, Bamlanivimab, Etesevimab) or an unrelated control antibody (hIgG), before being inoculated onto Vero and Huh-7 cells. Transduction efficiency was quantified at 16–18h postinoculation as described for panel c and normalized against samples that did not contain antibody (= 0% inhibition). Presented are the average (mean) data from a single experiment conducted with four technical replicates. Results were confirmed in a separate experiment. Error bars indicate the standard deviation (SD). (e) Evidence that mutation E484D allows for ACE2-independet cell entry. Vero and Huh-7 were preincubated (30 min, 37°C) with different concentrations of anti-ACE2 antibody, before particles pseudotyped with the indicated S proteins were added on top. Transduction efficiency was quantified at 16–18h postinoculation as described for panel c of Fig. 1. Presented are the average (mean) data from three biological replicates (each conducted with four technical replicates), for which transduction was normalized against samples that did not contain antibody (= 100% pseudotype entry). Error bars indicate the SEM. (f) S protein-driven entry into Huh-7 cells depends on heparan sulfate. Particles pseudotyped with the indicated S proteins (or VSV-G) were preincubated (30 min, 37°C) with different concentrations of heparin before being inoculated on to Vero and Huh-7 cells. Transduction efficiency was quantified at 16–18h postinoculation as described for panel c of Fig. 1. Presented are the average (mean) data from three biological replicates (each conducted with four technical replicates), for which transduction was normalized against samples that did not contain heparin (= 100% pseudotype entry). Error bars indicate the SEM. Statistical analysis: For panel c, statistical significance was assessed by two-tailed Student's t test with Welch’s correction, whereas for panels e and f, statistical significance was assessed by two-way analysis of variance (ANOVA) with Sidak’s post hoc test (P > 0.05, not significant [ns; not indicated in panel c]; P ≤ 0.05, *; P ≤ 0.01, **; P ≤ 0.001, ***).