Cells and viruses
African monkey kidney (Vero), K562 and C6/36 cells were kindly provided by Xiao-Hui Zou of the National Institute for Viral Disease Control and Prevention, China CDC, Beijing. Cells were grown at 37 °C in Eagle’s minimal essential medium (MEM) or RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). The viral strains GZ (DENV-1), ZS (DENV-2), H-87 (DENV-3) and H241 (DENV-4) were used for the plaque-reduction neutralization test (PRNT), animal inoculation and animal challenge experiment. Virus propagation was implemented in C6/36 cell and viral titers were determined by plaque assay on Vero cells.
Animals and ethics approval
BALB/c mice (female, 6–8 weeks old) were purchased from Pengyue Laboratory Animal Co., Ltd. (Jinan), and housed in appropriate animal care facilities during the experimental period. The animals were kept at the Laboratory Animal Center of Weifang Medical University. All of the mouse experiments were carried out in strict accordance with the guidelines of the Animal Committee of Weifang Medical University. The study was reviewed and approved by the Ethic Committee of Weifang Medical University. All mice were euthanized by cervical dislocation after the experiment. All sections of this report adhere to the ARRIVE Guidelines for reporting animal research. A completed ARRIVE guidelines checklist is included in Checklist S1.
The design, cloning, expression and purification of the tetravalent dengue protein were described previously . In brief, the DIII regions were fused with linker to generate a rEDIII chimeric protein of each DENV serotype with a 6-His-tag at their C-terminal ends. The recombinant plasmid was transfected sf9 cells and the target protein was secreted into the supernatant culture. The supernatant fluid was clarified and subjected to chromatography on a Ni-NTA column attached to an AKTA purifier system (GE Healthcare Life Sciences, Sweden). At last, protein were collected and quantified. All recombinant protein were lyophilized and stored at -20 °C until use. The tetravalent formulations were prepared with different quantities of the rEDIII protein and incubated with 500 µg aluminum for 36 hours at 4 °C (on a rocking platform), clarifed by centrifugation (5,000 rpm, 5 min) and re-suspended in 100 µl sterile 1 × PBS.
The immunogen was injected by the intraperitoneal route into five groups of 16 female BALB/c mice on days 0, 15 and 45. Groups 1 to 4 received different tetravalent formulations containing rEDIII protein and aluminum adjuvant. Mice that were injected with aluminum alone (without vaccine candidates) served as negative controls. Blood and spleens of ten mice from each group were obtained for further immunological analysis 15 days after the last dose injection. Meanwhile, eight mice from each group were prepared for the animal challenge experiment. All sera were prepared and stored at -20 °C until use.
Measurement of humoral immune response
The presence of rEDIII-specifc IgG in the sera was determined by enzymelinked immunosorbent assay (ELISA). A 2-fold serial dilution (starting at 1:10 for negative control or 1:100 for other groups) of each sample was prepared. Polystyrene 96-well plates (Costar, USA) were each coated with 100 µl of EDIII(D1-D4) protein (3 µg/mL) overnight at 4 °C in coating buffer (0.16% Na2CO3, 0.29% NaHCO3, pH 9.5), respectively. Then they were blocked in coating buffer containing 5% skimmed milk for 1 h at 37 °C. After five washes with PBS containing 0.05% Tween 20 (PBS-T), 100 µl per well of sera from each group were tested by serial dilutions in PBS-T, starting at 1:5000. The sera of healthy mice were used as negative control. Plates were incubated for 1 h at 37 °C and washed as described above. Later, 100 µl per well of 1:30 000 diluted Goat anti mouse IgG peroxidase conjugate (Amersham Pharmacia, Beckinghamshire, UK) were added and the plates were incubated for 1 h at 37 °C. After washing, 100 µl per well of substrate, 3, 3′, 5, 5′-tetramethylbenzidine (TMB), was added for color development. The plates were kept for 15 min at room temperature and the reaction was stopped with 50 µl per well of 2.5 M H2SO4. Absorbance was read at 450 nm in a SensIdent Scan device (Merck, Helsinki, Finland). The positive cutoff value was set as twice the mean absorbance value of negative control sera.
Plaque reduction neutralization test
Neutralizing antibody titers were measured by plaque reduction neutralization test (PRNT) in Vero cells as previously described . In brief, Vero cells (2 × 103 cells/well) were dispensed into 96-well plates and incubated for 16 h at 37 °C in a humidified 5% CO2 environment. Equal amounts of diluted serum sample and diluted DENV were mixed and incubated at 37 °C for 1 h. Thereafter, 100 µl of the neutralized liquid was inoculated into three wells per dilution and allowed to adsorb for 36 h at 37 °C and 5% CO2. Neutral red staining was then performed before final plaque counts were made. The neutralizing antibody titers were identified as the highest serum dilution that reduced the number of virus plaques by 50% (PRNT50) compared with control samples containing the virus alone. The sera were diluted by the continuous double dilution method starting at 1:2.
Measurement of cellular immune response
Mice were sacrificed and splenocytes were prepared 30 days after the third dose injection. The frequency and intensity of IFN-γ-producing cells were determined by mouse IFN-γ ELISPOT kits (Dakewe). All operations were conducted according to the manufacturer’s procedures. In brief, 100 µl of IFN-ɣ-specific mAb was coated onto 96-well plates with PVDF membranes and incubated overnight at 4 °C. The plates were washed three times with PBS and blocked with RPMI medium supplemented with feta bovine serum (10%) at 37 °C for 1 h to prevent nonspecific binding in later steps. The splenocytes were seeded at a concentration of 1 × 106 cells/well with each EDIII protein of DENV 1–4. Quadruplicate wells were set up for each stimulation and PBS or concanavalin A (5 µg/mL) were included as controls in parallel. After stimulation for 2 days at 37 °C in a 5% CO2 humidified incubator, the cells were removed from the plates by washing five times with 0.05% (w/v) Tween 20 in PBS. Secondary biotin-conjugated antibody was added to each well and incubated at 37 °C for 2 h. The wells were washed five times with 0.05% Tween 20 in PBS, and peroxidase-labeled streptavidin was added at a 1:1000 dilution for 1 h. Then the plates were washed five times with 0.05% (w/v) Tween 20 in PBS and three times with PBS alone. Finally, a 100 µl aliquot of 3-amine-9-ethylcarbazole staining solution was added to each well to develop the spots. The reaction was stopped after 30 min by placing the plates under tap water. Plates were dried, and spots were counted under an ELISPOT reader. The results were expressed as the number of spot-forming units (SFU) per 106 splenocytes.
Animal challenge with DENV-2 infected K562 cells
Eight immunized mice from each group received a intraperitoneal injection of 5 × 107 DENV-2 infected K562 cells that were suspended in 0.5 mL of serum-free RPMI medium one month after the last dose injection, of which the mice immunized with aluminum (without vaccine candidates) were taken as negative control. One day after challenge, the mice were euthanized to measure viral loads in plasma by quantification on Vero cells. The blood (0.2 mL) was immediately mixed with 0.02 mL of 3.8% sodium citrate pre-chilled on ice. The plasma was isolated, and the viremia level was evaluated using PRNT with Vero cells.
Prism software version 5.00 (GraphPad Software, San Diego, CA, USA) was used for calculating the means, standard deviations, standard errors, and statistics analyses. Direct or transformed (Log2, Log10) data that passed the normality test (Kolmogorov–Smirnov or D’Agostino and Pearson omnibus normality test) and showed variance homogeneity (Bartlett’s test) were analyzed by ANOVA parametric tests. Data that did not fulfill normality and/or variance homogeneity tests, even after transformations, were analyzed by the nonparametric test (Kruskal-Wallis test with LSD’s multiple comparison tests). Differences with p < 0.05 were considered to be statistically significant.