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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