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Figure 1. Generation of VSV pseudotyped viruses bearing truncated SARS-CoV-2 Spike proteins. (A) Preparation of rVSV-ΔG-G* in BHK21/W1 cell line. (B) Packaging of rVSV-ΔG-Sdel21 in HEK293T cell line. (C) Infection of pseudovirus carrying GFP reporter and sequential passaging of pseudovirus to eliminate parental virus (rVSV-ΔG-G*) using plasmids encoding Sdel21 and T7 polymerase transfected Vero E6. (D)SARS-CoV-2 Spike pseudovirus neutralization assay using human convalescent plasma samples and GFP signal reading.
Figure 2. Viral entry assay in Vero E6 cell line. (A)Vero-E6 cells were inoculated with VSV-ΔG-Sdel21 pseudotyped virus. Cells were treated with hydroxychloroquine at 20 min before inoculation. After 24h, GFP signal detected under fluorescence microscope is shown in representative cellular images. (B) For quantification, GFP signal from 25 images per sample was analyzed by using Image J at 24h of post-inoculation. Values are presented as % of total population. Scale bar: 100µm. Data are represented as means of ±S.D. of at least three independent experiments. *p < 0.05; ****p < 0.0001 - using One-Way ANOVA with Bonferroni’s post-hoc test.
Figure 3. Colocalization of pseudovirus with hACE-2 receptor or SARS-CoV-2 Spike protein in Vero E6 cell line. Cells were incubated with anti-SARS-CoV-2 Spike, human anti-ACE2 and DAPI. (A–D)Mock-treated VeroE6 cells with hACE2 staining. (E-H)VSV-ΔG-Sdel21 infected Vero E6 cells with hACE2 staining. (I-L)VSV-ΔG-Sdel21 infected Vero E6 cells with SARS-CoV-2 Spike staining. Yellow color shows viral S protein (J ), and red colors represents human ACE2 (B, F ). Scale bars, 20 μm. Data inA–L are representative images of two independent experiments.
Figure 4. In vitro pseudovirus neutralization activity of human plasma samples (1-13) against SARS-CoV-2 Spike variants; Wuhan(A ), Alpha (B ), Beta (C ). Neutralization curves of human convalescent plasma samples against pseudotyped SARS-CoV-2 variants; (A) VSV-ΔG-Sdel21 Wuhantype, (B) VSV-ΔG-Sdel21 Alpha variant carrying ΔH69-V70, N501Y, D614G, ΔY144 and (C) VSV-ΔG-Sdel21 Beta variant carrying E484K, N501Y, D614G, K417N mutations were measured as GFP signal in Vero E6 cells, and the percentage of neutralization was calculated. Data are represented as means of ±S.D. of at least two independent experiments.
Figure 5 . Comparison of pseudovirus neutralization assays with the plaque reduction assay. Pseudovirus neutralization assays of Wuhan (A) , Alpha (B) andBeta variants (C) were plotted against the virus neutralization activities determined by PRA with authentic Wuhanvirus. The R2 value indicates the certainty of the values to be at the trend line.
Table 1. Vaccination status of human plasma samples used for neutralization assays
Table 2. PRA Titers and Vaccination Profile of Human Plasma Samples
Figure 1S. Spike expressing construct map
Figure S2 . Positive control images of viral entry assay in Vero E6 cell line. Vero-E6 cells were inoculated with VSV-ΔG-Sdel21 pseudotyped particles. After 24 h, the GFP signal was observed using Axio Observer Fluorescence Microscope. The representative cellular images were given.
Table S1. The sequences of the mutation primers