Fig. 2: Titration and neutralization of SARS-CoV-2 viral stocks in VeroE6 and HAT-24 cell lines.
Virus stocks were serially diluted in 5-fold steps and added to cells in octuplicate. Cell nuclei were enumerated with high-content microscopy and cell numbers normalized to mock-infected controls where 100% represents cell numbers for mock-infected controls and 0% represents cell numbers for the highest viral concentration. a, Dose-dependent loss of nuclei in VeroE6-TMPRSS2, VeroE6 and HAT-24 cells at 20 h post infection (hpi). b,c, Titrations of a panel of SARS-CoV-2 isolates including the ancestral virus strain, VOC and VUI. Readout occurred at 72 hpi for VeroE6 (b) and 20 hpi for HAT-24 (c) cells. d, Representative fluorescence images of HAT-24 cells stained with Hoechst-33342, showing progressive loss of nuclei with increasing virus concentrations. e, Enumeration of stained nuclei with high-content imaging platform (IN Carta, Cytiva) using image thresholding and segmentation algorithms. Scale bars for d and e, 50 μm. f,g, Visual scoring of CPE at 72 hpi was done to calculate TCID50 ml−1 values in VeroE6 (f) and HAT-24 (g) cells (coloured bars). LD50 values at 20 hpi are also displayed (small bars with pattern). Shown are the mean ± s.d. from at least n = 3 experiments. h–p, Neutralization assays were performed in high-throughput format with both VeroE6 and HAT-24 cells using live virus isolates from the VOC: Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2), as well as the VUI: Epsilon (B.1.429), Zeta (P2), Eta (B.1.525), Kappa (B.1.617.1), Lambda (C.37) and C.36. ‘Wildtype’ virus from the same clade containing the dominant D614G mutation (Clade B - B.1.319) and ancestral Wuhan-like virus with the original D614 background (Clade A - A.2.2) were also included. h,i, Neutralization assay of SARS-CoV-2 isolates with monoclonal AB-3467 in HAT-24 (h) and VeroE6 cells (i). j, Spearman correlation of IC50 values in h and i (r = 0.9371, P < 0.0001, two-tailed). k,l, Neutralization assay of SARS-CoV-2 isolates by WHO control sample G in HAT-24 (k) and VeroE6 cells (l). m, Spearman correlation of IC50 values in k and l (r = 0.8671, P = 0.0005, two-tailed). n,o, Neutralization assay of SARS-CoV-2 isolates by the Plasma Alliance control sample in HAT-24 (n) and VeroE6 (o) cells. p, Spearman correlation of IC50 values in n and o (r = 0.7972, P = 0.0029, two-tailed). Shown are the mean ± s.d. of technical replicates done in quadruplicate. Each panel is representative of a minimum of three independent experiments. In h, i, k, l, n and o, dose-response curves and interpolated IC50 values were determined using the sigmoidal 4PL model of regression analysis in GraphPad Prism software version 9.1.2.
