Fig 1.
Expression of recombinant ZIKV envelope proteins and immune strategy.
(A) Schematics of plasmid constructs expressing E80_S and E80_E. (B) Purified recombinant ZIKV E80 proteins were verified by SDS-PAGE (left panel) and Western Blot with anti-His antibody (middle) or anti ZIKV envelope antibody (right). Experimental strategies: (C) immunization and measurement of cellular immune responses; (D) Immunization and measurement of humoral immune responses and protection against ZIKV challenge.
Fig 2.
Measurement of ZIKV specific and DENV cross-reactive antibody responses elicited by ZIKV E80_S and E80_E proteins.
(A) and (B) Serum samples from different groups of mice (n = 5 for each group) were 2-fold serially diluted from 1:320 to 1:40960 and measured for (A) end-point dilution titers and (B) OD450 values by using ZIKV E80 protein coated ELISA. (C) and (D) Serum sample obtained from mice received PBS (n = 5), 50μg E80_E (n = 4) and 50μg E80_S (n = 5) were 2-fold serially diluted from 1:160 to 1:20480 and measured for (C) end-point dilution titers and (D) OD450 values by using DENV-3 E80 protein coated ELISA. The statistical differences between PBS control and immunization groups were determined by Student’s t test, and marked with black stars. A p<0.05 value was designated as *, p<0.01 as **, and p<0.001 as ***. Red stars indicate statistical differences between the 10μg E80_E group and other immunization groups. The data were presented as mean ± SEM. (A) and (B) Each sample was assayed in duplicates and the figures are representative results of 4 independent experiments. (C) and (D) Each sample was assayed in duplicates and the figures are representative results of 2 independent experiments.
Fig 3.
Assessment of serum neutralization activity by plaque reduction neutralization test (PRNT).
Serum samples were 3-fold serially diluted from 1:10 to 1:2430, and mixed with an equal volume of medium containing 100 PFU Zika virus then applied on Vero cells. Neutralizing activity was calculated as described in the Materials and Methods. (A) Summary of PRNT50 titer of each mouse in different experimental groups. Red stars represent significant differences (Student’s t tests) between the PBS control group and the indicated vaccination group against serum dilutions. (C) Two-way ANOVA test, followed by Tukey's correction for multiple comparisons were used to determine statistical differences among curves plotted in (B). The data were presented as mean ± SEM. Each sample was assayed in duplicates and the figures are representative results of 4 independent experiments.
Fig 4.
Measurement of antigen specific T cell responses by ELISPOT and ICS assays.
(A) Representative ELISPOT results. Splenocyte of PBS, 50μg E80_E and 50μg E80_S immunized mice were stimulated with PBS (lane 1), DENV E80 protein (lane 2), HIV synthesized polypeptide (lane 3), ZIKV E80 protein (lane 4) and Con A (lane 5). (B) Summary of the ELISPOT results. Splenocytes were cultured at 37°C for 48 hours with the same dose of (10μg/ml) ZIKV E protein, DENV E protein, HIV protein or PBS. IFNγ ELISPOT assay was performed to quantify antigen specific T cells at 48 hours after stimulation. (C) Summary of the ICS results. Splenocytes from immunized mice were stimulated with either 10μg/ml of ZIKV E protein, or 10μg/ml of DENV E proteins, or 10μg/ml of HIV protein or PBS for 6 hours. Golgi stop was supplemented 2 hours after stimulation. Statistical differences were analyzed by Student’s t tests. Different p values were indicated by * (p<0.05), or ** (p<0.01), or *** (p<0.001). The data were presented as mean ± SEM. The figures are representative results of 2 independent experiments. In ELISPOT assay, each sample was assayed in duplicates; in ICS assay, each sample was measured once.
Fig 5.
Protective efficacy of vaccines against ZIKV viremia.
Viremia levels of immunized mice after ZIKV challenge were presented by percentage of infected Vero cells. 10μl of serum samples obtained at various days post infection were applied to Vero cells in DMEM containing 1% FBS for 4 days. The percentage of infected cells was determined by staining with an anti-E antibody 4G2, followed by an anti-mouse AF488 conjugated antibody, and then analyzed by flow cytometry. (A) 10μg E80_E group (n = 4), (B) 50μg E80_E group (n = 4), (C) PBS group (n = 5), (D) 10μg E80_S group (n = 5), and (E) 50μg E80_S group (n = 5). Each line represented one mouse. (F) Area under the curve (AUC) was calculated by Graphpad Prism 6 software. (G) Maximum percentage of infected cells from 1 to 5 DPI for each experimental group. (H) Duration of detectable viremia. (I) Summary of viremia data for each of the groups at days 1 to 5 post infection. The data were presented as mean ± SEM. Statistical differences were determined by Student’s t test and p values were indicated by * (p<0.05), or ** (p<0.01), or *** (p<0.001). The figures are representative results of 2 independent experiments.
Fig 6.
Weight changes of mice in each experimental group.
(A) Summary of weight changes of immunized mice upon ZIKV infection from day 1 to 14 DPI. (B) Two-way ANOVA test was performed on data in (A), followed by Tukey's correction for multiple comparisons. The data were presented as mean ± SEM. The figures are representative results of 2 independent experiments.
Fig 7.
Assessment of protection by adoptively transfer of ZIKV immune sera.
Each mouse received 300μl of pooled sera from either control (A) or 50μg ZIKV E80 immune (B, C) mice and then challenged. Viremia level of each group of recipient mice (n = 3) was measured and presented as percentage of infected cells. Each line represented one mouse. (D) AUC was calculated by using the Graphpad Prism 6 software. (E) Maximum percentage of infected cells from 1 to 5 DPI was measured for each experimental group. (F) Duration of detectable viremia. The data were presented as mean ± SEM. Statistical differences were determined by Student’s t test. The p values were indicated by * (p<0.05), or ** (p<0.01), or *** (p<0.001).