Figure 1.
SARS-CoV-2 TS mutants show attenuated phenotype and induce protective immunity
(A) Comparison of the growth kinetics of the four TS mutants with those of the B-1 strain at different temperatures. Vero cells were infected with each strain at multiplicity of infection (MOI) = 0.01. The viral titer of the supernatant was evaluated by TCID50 using Vero cells. Symbols represent the average of three independent experiments, and error bars depict the mean SDs. For samples below the limit of detection (LOD), the assay’s LOD was used to calculate the mean. The LOD is 2.05 log10 TCID50/mL, indicated in the x-axis. TCID50: 50% tissue culture infectious dose. To compare titers on the same day between the B-1 and the other strains, two-way ANOVA was performed.
(B) Overview of the evaluation for pathogenicity and immunogenicity of TS mutants (n = 5).
(C) Weight changes in the Syrian hamsters infected with the TS mutant or B-1 strains. The average weight change is plotted, and error bars represent the SDs. To compare weight change on the same day between the mock and the other groups, two-way ANOVA was performed (∗∗p < 0.01 and ∗∗∗∗p < 0.0001).
(D and E) The viral titers of nasal wash specimens (D) and lung homogenates (E) three days post-infection. Infectious viruses were evaluated by plaque formation assays. PFU: plaque-forming units. Bars depict the mean values, and symbols represent individual viral titers. Error bars represent SDs. For statistical analysis, one-way ANOVA was performed (∗∗p < 0.01 and ∗∗∗p < 0.001).
(F) Schematic diagram for the evaluation of mucosal tissue damage (n = 4).
(G) Damage scores of each section. The percentage of the disrupted area in the entire visual field was classified as 0: not remarkable (< 10%), 1: minimal (10%–50%), and 2: mild (50%–70%), respectively.
(H) Representative hematoxylin and eosin (HE)-stained images of sections at level 3. Scale bar: 1000 μm (left panel), and 200 μm (right panel). Right panels are higher magnification images of the marked areas.
(I) Neutralization titers of the sera from hamsters infected with each strain and mock-treated hamsters. Median values are plotted, and error bars represent the 95% CIs. For statistical analysis, the Kruskal-Wallis test was performed (n.s.: not significant). The LOD is indicated in the x-axis.
(J) Weight change in hamsters after B-1 virus reinfection. The average weight change is plotted, and error bars represent mean SDs. To compare weight change on the same day between the naive and the other groups, two-way ANOVA was performed.