Table 1.
Characteristics of study subjects.
Fig 1.
Serotype-specific neutralization activities of serum samples from convalescent dengue patients.
(A) Serotype-specific neutralization potency against DENV-1 or DENV-3 was determined by performing focus-forming assays on Vero cells with 2-fold serially diluted DENV-naïve sera (n = 3) and DENV immune sera of DENV-1 early (n = 10), DENV-1 late (n = 10), DENV-3 early (n = 10) and DENV-3 late (n = 10) groups, respectively. (B) The 50% plaque reduction neutralization end-point titers (PRNT50) against either DENV-1 or DENV-3 were calculated by probit analysis using the SPSS software. Small horizontal solid lines indicated the mean values of PRNT50, and the dotted lines represented the initial serum dilution (1/100 for DENV-1 and 1/50 for DENV-3). Statistical significances were identified using unpaired two-tailed Student’s t test. “*” represents p<0.05.
Fig 2.
ZIKV binding and neutralization potency by DENV immune sera.
(A) Binding capacity of DENV immune sera in DENV-1 early group (n = 10), DENV-1 late group (n = 10), DENV-3 early group (n = 10), DENV-3 late group (n = 10) or DENV-naïve donors (n = 3) were detected by ELISA using plates coated with ZIKV culture supernatant. The binding activity was presented as the optical density value at 450nm (OD450). (B) Summary of ELISA results as mean endpoint titers. Statistical significances were identified using the unpaired two-tailed Student’s t test. “**” represents p<0.01, and “***” represents p<0.001. (C) Neutralization potency of DENV-immune sera in each group and three DENV-naïve sera against ZIKV was assessed by performing a plaque-forming assay on Vero cells with 2-fold diluted serum samples.
Table 2.
ZIKV-susceptibility in subsets of human PBMCs.
Fig 3.
Enhancement of ZIKV infection in primary monocytes triggered by DENV immune sera.
Freshly isolated PBMCs from healthy donors were either uninfected or infected with ZIKV at an MOI of 2 in the presence or absence of serially diluted DENV immune serum samples. Cells were harvested and stained with anti-E antibody in combination with antibodies to CD3, CD14, CD19 and then analyzed by flow cytometry. (A) and (C) Representative flow cytometry analyses of ZIKV-infected (Anti-E) monocytes (CD14+CD3-CD19-), T cells (CD14-CD3+CD19-) and B cells (CD14-CD3-CD19+) in the presence of a DENV immune serum sample D1-E01 (A) and a control serum Naïve-01 (C) were presented. (B) and (D) Verification of enhancement in ZIKV infection by the boosted assay on Vero cells. Culture supernatants from ZIKV-infected PBMCs were harvested and used to infect Vero cells for two days followed by intracellular staining with an anti-E antibody and flow cytometry analysis. Representative results of the boosted infection assay on Vero cells for the DENV immune serum D1-01 (B) and the control serum Naïve-01 (D) were shown.
Fig 4.
CD14+ monocytes are the principle target cells mediating ADE of ZIKV infection by DENV immune sera.
(A) PBMCs were sorted by magnetic beads into CD14+ and CD14- fractions through a positive selection process. The percentage of different cell subtypes (viable cells) in each fraction and unfractionated PBMCs were presented. (B and C) PBMCs as well as CD14+ and CD14- fractions were infected with ZIKV at varying MOIs in the absence of serum samples or an MOI of 2 in the presence of DENV immune sera (D1-E01 at a dilution of 1/2,500, or D1-E06 at a dilution of 1/2,500, or D3-L01 at a dilution of 1/100) or a control serum (Naïve-01, 1/100 dilution). Cell culture supernatants from infected PBMCs (B), CD14+ and CD14- fractions (C) were collected and inoculated on Vero cells and analyzed for ZIKV-infected cells by flow cytometry. The mean and SEM of results from three independent experiments were presented. Statistical significances were determined using unpaired two-tailed Student’s t test. “*” represents p<0.05, and “***” represents p<0.001.
Fig 5.
Immature DCs do not mediate ADE of ZIKV infection.
(A) Immature DCs were infected with ZIKV (MOI 2) in the presence or absence of serially diluted DENV immune serum (D1-E01) or DENV-naïve serum (Naïve-01). 48 h post-infection, cells were harvested and intracellularly stained with an anti-E antibody and subjected to flow cytometry analysis. A representative infection panel of two independent experiments was presented. (B) Supernatants from ZIKV-infected immature DCs were collected and inoculated to Vero cells for 2 days. Vero cells were collected and intracelullarly stained with an anti-E antibody for flow cytometry analysis. Data shown were representative results of three independent experiments.
Fig 6.
Mature DCs are not infected by ZIKV in the presence or absence of enhancing serum.
(A) Mature DCs were infected by ZIKV at an MOI of 2 with or without serially diluted DENV immune serum (D1-E01) or DENV-naïve serum (Naïve-01) and cultured for 48 h. Cells were collected and stained for ZIKV-infected cells (anti-E positive) and then analyzed by flow cytometry. (B) Supernatants from ZIKV or virus/sera immune complex treated mature DCs were used to infect Vero cells for 48 h, and cells were stained for flow cytometry to identify ZIKV-infectivity. Data shown were representative results of three independent experiments.
Fig 7.
DENV immune sera from patients under different stages of convalescent phase exhibit divergent enhancing activities to ZIKV infection.
All DENV immune sera and DENV-naïve sera were evaluated for ADE of ZIKV infection in PBMCs first and then by the boosted assay on Vero cells. The fold enhancement of each serum sample was presented under serial dilutions in monocytes (A) and Vero cells (B). (C) Summary of enhancing activities in monocytes and the results of boosted assay on Vero cells by calculating the area under curve (AUC) of the data from (A) and (B). The solid line represented the mean value of AUC in each group. Statistical significances were determined using unpaired two-tailed Student’s t test. “*” represents p<0.05, and “***” represents p<0.001.
Fig 8.
FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) are all involved in ADE of ZIKV infection by DENV immune sera.
PBMCs from healthy donors (n = 3) were treated with monoclonal antibodies (1 μg/ml, 3 μg/ml or 10 μg/ml) against each FcγR types or combined (1 μg/ml, 3 μg/ml or 10 μg/ml in total), or with IgG control antibody prior to performing the boosted ZIKV ADE assay with DENV-1 immune serum (D1-E01, 1/2,500 dilution). The percentage of anti-E positive Vero cells in the control group, where no anti-FcγR antibodies were applied at the ADE condition was defined as 100%. The mean and standard deviation of results from three independent experiments without blocking antibody treatment (A) or with blocking antibody treatment (B) were presented. Statistical significances were determined by comparison of the relative proportion of infected cells with blocking antibody treatment to that of the control IgG antibody treatment under the same antibody concentration using unpaired two-tailed Student’s t test and designated as “*”for p<0.05, “**” for p<0.01, and “***” for p<0.001.