Cells and viruses
293T cells (ATCC No.CRL-11268) were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C with 5% CO2. C6/36 Aedes albopictus cells (ATCC No.CRL-1660) were grown at 28°C without CO2 in Eagle's Minimum Essential Medium (EMEM; Gibco) supplemented with FBS, penicillin and streptomycin as well.
Each serotype of dengue virus was passaged and propagated in C6/36 cells. The DENV-1 strain GZ01/95 and DENV-2 strain ZS01/01 were supplied by the Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, China. DENV-1 strain Hawaii, DENV-2 strain NGC, DENV-3 strain H87 and DENV-4 strain H241 were preserved by our laboratory. Strain GZ01/95, ZS01/01, H87 and H241 were used for RNA extraction and then VLPs expression plasmids construction while strain Hawaii, NGC, H87 and H241 were used for neutralization analysis. Japanese encephalitis virus (JEV) strain SA14-14-2 was also propagated in C6/36 cells and mainly used for cDNA cloning.
Construction of DENV VLP expression plasmids
The QIAamp Viral RNA Kit (Qiagen, Santa Clarita, CA) was used to extract genomic RNA of DENV1-4 and JEV from 140 μl C6/36 cells culture supernatant infected with each virus. The extracted RNA was subjected to RT using Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Cat. No. 05081955001) to generate cDNA templates for amplification of target genes. The PCR segments were digested with NheI and NotI enzymes and inserted into NheI and NotI sites of pcDNA5/FRT vector.
Three types of expression plasmids were constructed for each serotype of DENV (Figure 1A). The first type of plasmids was named pD1-D4prME, with entire prM and E genes of each DENV serotype cloned into the pcDNA5/FRT vector. The second type of plasmids was pJD1-D4prME, with entire prM and E genes of each DENV serotype and an optimized JEV signal sequence(JESS) [17, 20, 21] gene from SA14-14-2 strain. To further compare the impact of E protein transmembrane and cytoplasmatic domains, the third type of plasmids was designed as chimeric constructs pJD1-D4prMEΔ20%JEV, which contained JESS and full length of prM, but replaced the 3' terminal 20% region of E gene with the corresponding sequence of JEV (strain SA14-14-2).
Transient transfection of 293T cells with DENV VLP expression plasmids
293T cells were prepared in wells of 6-well plates one day earlier and were transfected with pD1-D4prME, pJD1-D4prME or pJD1-D4prMEΔ20%JEV for each well using lipofectamin2000 (Invitrogen, Cat no.11668019) according to instructions supplied by the manufacturer. Briefly, for each plasmid transfection, 8 μl lipofectamin2000 was diluted in 200 μl Opti-MEM (Gibco), and after incubating for 5 min at room temperature the diluted lipofectamin2000 was combined with diluted plasmid DNA (4 μg diluted in 200 μl Opti-MEM). After 20 min's incubation at room temperature, the mixture was added to each well with 80%-90% confluence of 293T cells. 48 hours post-transfection, cells and supernatants were harvested for future use.
Western blot analysis
Concentrated culture supernatants were applied to a NuPAGE 4-12% Bis-Tris gradient gel, and followed by electroblotting onto PVDF membrane. Non-denatured proteins were then probed with E protein specific rabbit polyclonal sera which were produced in our laboratory. A goat anti-rabbit IgG conjugated to HRP was used as the secondary antibody. The reactions were detected by 3,3' diaminobenzidine (DAB) reagent according to the manufacturer's instructions.
Purification of DENV VLPs and virions
For DENV1-4 VLPs purification, 293T cells in T175 flasks were transiently transfected with optimized plasmids of each serotype. The culture supernatants containing extracellular VLPs were harvested routinely every 2 days, continuously repeated 3 times. To purify DENV1-4 virions which were used for electron microscopy and mice immunization, C6/36 cells in T175 flasks were infected with DENV strain Hawaii, NGC, H87 and H241 respectively. At 7 days after infection, the supernatants were harvested and inactivated with 1:2000 β-propionolactone.
Cell supernatants collected from dengue VLPs production cells or dengue virus infected C6/36 cells were clarified by centrifugation at 10 000× g for 30 min followed by concentration using ultrafiltration system Vivaflow 200 (Sartorius stedim biotech). The concentrated culture supernatants were then purified by a two-step ultracentrifugation. The first step was a rate zonal centrifugation. Samples were added on the top of a 15-60% sucrose gradient and ultracentrifuged in a SW41 rotor (Beckman Coulter Inc.) at 38,000 rpm at 4 °C for 4 h. About 1 ml fractions were collected and pelleted by a second ultracentrifugation and then resuspended in PBS. The total protein concentrations of authentic virions and dengue VLPs were determined by Pierce BCA Protein Assay Kit.
Transmission Electron microscopy (TEM)
Purified dengue VLPs and dengue virions from sucrose density-gradient fractions were fixed with 2% glutaraldehyde. Small droplets of fixed samples were placed on copper formvar-coated grids for 1 min, then the grids were stained with sodium phosphotungstate for 1 min (excess samples of each step were removed). At last, the grids were visualized by TEM.
Mice immunization
Four to six-week-old female BALB/c mice were purchased from Chinese Academy of Medical Sciences Breeding Laboratories and were intraperitoneally (i.p.) inoculated with monovalent DENV VLPs (100 μg per dose) or a tetravalent combination (25 μg of each serotype per dose) in Freund's complete adjuvant (Sigma) for priming and in Freund's incomplete adjuvant for two times of boosting at an interval of 2 weeks. Equal amount of DENV virions (100 μg for monovalent vaccine and 25 μg of each serotype for tetravalent vaccine) were used as controls with the same regimen. On days 0, 14 and 28, blood samples were collected through tail vein for measurement of serum IgG. At 2 weeks after the last inoculation, mice were sacrificed to collect serum for the neutralizing antibodies assay and separated splenocytes for testing cytotoxic T cell responses.
ELISA to measure serum IgG
VLPs specific serum IgG antibodies were titred by the binding capacity with rEIII protein, a recombinant protein that chimericly expressed DENV1-4 EIII domains in a certain order and previously produced in our laboratory. IgG titers were measured using enzyme-linked immunosorbent assay (ELISA). Briefly, 200 ng purified rEIII per well was coated on 96-well plates at 4°C overnight. Then, the plates were blocked with 5% skimmed milk in PBS for 1 h, and incubated with 2-fold serial diluted serum samples (starting from 1:50) at 37°C for 1 h. Bound IgG was detected by HRP-conjugated goat anti-mouse IgG (Sigma). After addition of 3,3', 3,5'-tetramethylbenzidine (TMB), absorbance was measured at 450 nm. The value which exceeds the mean+2 S.D. of negative control was considered positive.
Antibody neutralization assay
The neutralization ability of serum antibodies against DENV was determined using CPE-determination assays. Briefly, mice sera from all groups were heat-inactivated at 56°C for 30 min, then the sera were two-fold serial diluted from 1:5 to 1:160 in Eagle's medium supplemented with 1% heat-inactivated FBS, penicillin and streptomycin and mixed with 100TCID50 virus. After 1 h incubation at 37°C, 100 μl of virus-serum mixture was inoculated to the confluent monolayer of BHK-21 cells in 96-well plates. Every dilution of each serum was performed in quadruplicate. The plates were then incubated in a CO2 incubator at 37°C for 7 days. The neutralization titer was expressed as the maximum serum dilution at which the CPE of the virus was not observed in all four wells.
Enzyme Linked Immunospot (ELISPOT) Assay
The ELISPOT 96-well plates (BD) were coated with 100 μl of anti-mouse IFN-γ (5 μg/ml in coating buffer) at 4°C overnight. The following day, plates were washed and blocked with blocking solution for 2 h. Then, 100 μl freshly isolated splenocytes (5 × 105 cells) from the immunized mice were added to each well and stimulated with DENV VLPs at 37°C for 40 h. After cells were washed out, biotinylated anti-mouse IFN-γ was added to each well and incubated for 2 h at room temperature. Thereafter, the plates were washed and incubated for 1 h at room temperature with streptavidin-HRP. Finally, AEC substrate solution (BD) was added and spots were counted by ImmunoSpot® Analyzer (Cellular Technology Ltd.).
Statistical analysis
Statistical comparisons among groups were analyzed by one way ANOVA using SPSS 11.5. A P value less than 0.05 was considered statistically significant.