Fig. 1: Host cell entry and antibody evasion by the spike protein of SARS-CoV-2 AY.4.2.

a Monthly and cumulative numbers of globally reported AY.4.2 isolates. b Distribution of reported AY.4.2 isolates at the global (left) and European (right) levels. Numerical values in brackets indicate the number of isolates per country (* = twenty countries with < 100 isolates). c Schematic illustration of the SARS-CoV-2 spike protein in which the locations of functional domains (RBD, receptor binding domain; TD, transmembrane domain) and cleavage sites (S1/S2 and S2’) are highlighted. Mutations found in the spike protein of B.1.617.2 (Delta variant, EPI_ISL_1921353) are highlighted in red, while the additional mutations found in the Delta sublineage AY.4.2 (EPI_ISL_5633764) are highlighted in green. d Location of the amino acid changes in the context of the trimeric spike protein. e Pseudotyped particles bearing the indicated S proteins (equipped with a C-terminal HA epitope tag) were subjected to immunoblot analysis to analyze S protein incorporation and cleavage. S proteins and VSV-M (loading control) were detected using anti-HA and anti-VSV-M antibodies, respectively, in combination with a peroxidase-conjugated anti-mouse secondary antibody. The results from a single experiment are presented, and the results were confirmed in two additional experiments. For quantification of S protein incorporation, in all experiments, the S protein signals were first normalized against the corresponding VSV-M signals, and further incorporation of B.1 S protein was set as 1 (data represent the mean ± standard deviation, SD). For quantification of S protein cleavage, total S protein signals (bands representing unprocessed [S0] and processed [S2] S protein) for each S protein were set as 100%, and the respective proportions of S0 and S2 were calculated. Statistical significance was assessed by two-tailed Student’s t test with Welch’s correction; p > 0.05, not significant [ns]). f Four different human cell lines were inoculated with pseudotyped particles bearing the indicated spike proteins. At 16–18 h postinoculation, particle entry efficiency was analyzed by measuring the activity of virus-encoded luciferase in cell lysates. Presented are the average (mean) data from 4–5 independent experiments (each performed with four technical replicates) for which particle entry driven by the B.1 spike protein was set as 1. Error bars indicate the standard error of the mean. Statistical significance was assessed by two-tailed Student’s t test with Welch’s correction (p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***; please see also Supplemental information, Fig. S1a). g Pseudotyped vectors bearing the indicated spike proteins were incubated (30 min, 37 °C) in the presence of different concentrations of monoclonal antibody or medium alone (control) before being added to Vero cells. Vector entry efficiency was analyzed at 16–18 h postinoculation and normalized against the respective control (set as 0% inhibition). Presented are the average (mean) data for a single experiment (with four technical replicates). The data were confirmed in a separate experiment. Error bars indicate the SD. Curves were calculated using a nonlinear regression model (variable slope). h Pseudotyped vectors bearing the indicated spike proteins were incubated (30 min, 37 °C) in the presence of different dilutions of convalescent plasma or only medium (control) before being added to Vero cells. Vector entry efficiency was analyzed at 16–18 h postinoculation and normalized to the respective control (set as 0% inhibition, please see Supplemental information, Fig. S1b for individual data). Furthermore, the plasma dilution that causes a reduction of 50% in vector entry (neutralizing titer 50, NT50) was calculated. Presented are the combined data for 10 convalescent plasma (each analyzed in four technical replicates). Black lines and numerical values indicate the median NT50. In addition, the data were normalized to reflect the relative change in neutralization sensitivity with the neutralization of B.1 spike serving as reference (set as 1, identical plasma are connected by lines). Statistical significance was assessed by Kruskal–Wallis analysis with Dunn’s multiple comparison test (p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.001, ***). i The experiment was performed as described in (h), but serum from vaccinated individuals (BNT162b2/BNT162b2, n = 10) was analyzed (please see Supplemental information, Fig. S1c for individual data)