Table 1.
Virulence attenuation in BALB/c mice of Y. pestis 201 sORF mutants.
Fig 1.
The survival curves and body weight loss of mice s.c inoculated with the EV76-derived candidate vaccines.
(A) BALB/c mice (n = 10, female) were inoculated s.c. with 1×107 CFU of Y. pestis EV76, EV76Δyp1, EV76Δyp2 or EV76Δyp1&yp2, respectively; (B) Body weight was monitored continuously for 14 days. The weight changes on days 2 and 3 were shown in detail in the right panel. One-way ANOVA with Tukey post hoc test was used to analyze the significance of differences in body weight losses among the infected groups. ns indicates no statistical significance. ****P<0.0001.
Fig 2.
The survival curves of mice inoculated with the EV76-derived candidate vaccines under iron overload conditions.
BALB/c mice (n = 5 per group, females) were inoculated s.c. with 1×107 CFU of EV76, EV76Δyp2, EV76Δyp1 or EV76Δyp1&yp2, respectively, and subsequently intraperitoneally (i.p.) injected daily with 100 μg of FeCl2 (A) or sterile PBS (B). Kaplan–Meier analysis with log-rank (Mantel-Cox) test was used to calculate P values, comparing the results to the EV76-inoculated groups. **P<0.01.
Fig 3.
Bacterial loads in organs of mice inoculated with the EV76-derived candidate vaccines.
Mice (n = 10 per time point in each group) were inoculated s.c. with 1×107 CFU of EV76, EV76Δyp2, EV76Δyp1, or EV76Δyp1&yp2. At different time points, mice were sacrificed, and bilateral inguinal lymph nodes (A), spleens (B) and liver (C) were collected, and the number of living bacteria was determined. Two-way ANOVA with Tukey’s post hoc test was used to calculate significant differences in bacterial loads. ns indicates no statistical significance, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Fig 4.
Histopathological analysis on mouse tissues following inoculation with the EV76-derived candidate vaccines.
Mice (n = 5 per time point in each group) were inoculated s.c. with 1×107 CFU EV76, EV76Δyp2, EV76Δyp1, or EV76Δyp1&yp2. Representative hematoxylin-eosin (HE) staining results for the different tissues at 3 dpi. (A) and 6 dpi. (B) are shown as indicated. HE staining of the lymph nodes tissue sections revealed various degrees of inflammatory cell infiltration (red arrows), necrosis (black arrows), connective tissue hyperplasia (yellow arrows), and congestion (blue arrows). The spleen tissue sections showed different degrees of inflammatory cell infiltration (red arrows), massive extramedullary hematopoiesis (black arrows), and a mild increase of multinucleated giant cells (yellow arrows). The liver tissue sections showed degeneration of hepatocytes (black arrows), few foci of extramedullary hematopoiesis (red arrows), and few venous congestion and vasodilatation (blue arrows), inflammatory cell infiltration (yellow arrows). Pathological examination of lung tissues from mice revealed mild inflammatory cell infiltration (black arrows), with limited thickening of alveolar walls observed. The histopathological scores of various organs at (C) 3 dpi. and (D) 6 dpi. were determined using Kruskal-Wallis with Dunn’s post hoc test to assess statistical significance. *P<0.05.
Fig 5.
Humoral immune responses in mice administrated s.c. with the EV76-derived candidate vaccines in short-term study.
Mice (n = 10 per group) were immunized s.c. with two doses of 5×106 CFU of EV76, EV76Δyp2, EV76Δyp1 or EV76Δyp1&yp2 at a 21-day interval. Sera were collected on day 41 after the initial immunization. Titers of IgG specific to F1 (A), LcrV (B), and 201Δcaf1-WCL (C) were measured using ELISA. (D) For Western blotting analysis, blotted antigens were obtained from the whole cell lysates of 201 or 201Δcaf1, and pooled sera from immunized mice were used as primary antibodies. (E) IgG subclasses to 201-WCL antigen were analyzed by ELISA. (F) The serum IgG2a/IgG1 ratios in the immunized mouse groups were determined. Statistical analysis was conducted using One-way ANOVA with Tukey post hoc to determine the significance of differences. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.
Fig 6.
Evaluation of cellular immune responses in mice inoculated s.c. with EV76-derived candidate vaccines.
Splenocytes were harvested and stimulated with 201-WCL and the supernatants were evaluated for cytokines levels by Luminex assay (n = 5 for each group). One-way ANOVA with Tukey post hoc was used to determine the significance of differences. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Fig 7.
Efficacy of EV76-derived candidate vaccines in protecting immunized mice against exposure to Y. pestis 201 or 201Δcaf1.
Mice were immunized s.c. twice at 21-day interval with 5×106 CFU of EV76, EV76Δyp2, EV76Δyp1, or EV76Δyp1&yp2, respectively. Vaccinated mice were subjected to s.c. or i.n. challenge 42 days after the initial immunization. Groups of mice injected with PBS instead of the bacterial suspension served as a control. Vaccinated mice (n = 9~10 per group) were challenged s.c. with 1000 CFU (322 LD50) (A), 1×107 CFU (3.22×106 LD50) (B) of Y. pestis 201, 1000 CFU (403 LD50) of Y. pestis 201Δcaf1 (C), or i.n. challenged with 5000 CFU (11.4 LD50) (D), 2×104 CFU (45.6 LD50) (E) of Y. pestis 201, or challenged i.n. with 7×104 CFU (207 LD50) of Y. pestis 201Δcaf1 (F). Kaplan–Meier analysis with log-rank (Mantel-Cox) test was used to determine the significance of differences between the survival curves. ns, no significance, *P<0.05, **P<0.01, ***P<0.001.
Fig 8.
Evaluation of rapid protection efficacy in mice immunized with a single-dose of EV76-derived candidate vaccines.
Mice (n = 10 per group) were immunized s.c. with 1×107 CFU of EV76, EV76Δyp2, EV76Δyp1, or EV76Δyp1&yp2. After 21 days, the vaccinated mice were subjected to i.n. challenge with 3×104 CFU (68LD50) of Y. pestis 201. Kaplan–Meier analysis with log-rank (Mantel-Cox) test was used to determine the significance of differences. ns, no significance, *P<0.05.
Fig 9.
Efficacy of EV76-derived candidate vaccines in protecting mice from i.n. challenge with Y. pestis 201-lux.
Mice (n = 10 each group) were immunized s.c. twice at a 21-day interval with 5×106 CFU either of EV76, EV76Δyp2, EV76Δyp1&yp2, or PBS. The IgG titers against F1 antigen in the sera of mice were then determined by ELISA. (A) The IgG titers against F1 after initial and booster immunization. (B) The kinetics of IgG titers against F1 were monitored over 112 days. Two-way ANOVA with Tukey post hoc was used to determine the significance of differences in IgG titers between the different groups (C) On day 120 post initial-immunization, the vaccinated mice were challenged i.n. with 1.7×104 CFU (19.7 LD50) of Y. pestis 201-lux. Mouse survival was monitored daily for 14 days and the survival curves were plotted using GraphPad 8.0.1 Kaplan–Meier analysis with log-rank (Mantel-Cox) test was used to determine the significance of differences. One-year post initial immunization, IgG titers against 201-WCL (D) or F1 antigen (E) were measured in the sera of mice (n = 3 or 4 per group) immunized with the indicated Y. pestis strains. One-way ANOVA with Dunnett was used to determine the significance of differences. (F) In vivo luminescence imaging of mice i.n. challenged with Y. pestis 201-lux. On 3 and 12 dpi, surviving mice were imaged, and those exhibiting luminescence signals were denoted with red asterisks. The luminescent intensity ranged from 1.5×e6 (red) to 1.5×e5 (violet) as indicated. ns, no significance, *P<0.05, **** P<0.0001.