The highest serum dilution that reduced in at least 50% the number of plaques was considered the final neutralization titer. Lymphoid spleen cells from immunized and control mice were collected, washed twice in RPMI 1640 containing 10% heat-inactivated FBS. After wash, the cells were resuspended at a final concentration of 1 × 106 cells/ml with RPMI 1640 and 100 μl aliquots were plated into 96-well culture plates. Then we added different stimuli to the culture, 1 × 106 PFU of DENV-4 (heat inactivated) as specific stimulus or concanavalin BKM120 supplier A 2 μg/ml (Sigma–Aldrich) as mitogenic stimulus, the plates were covered and incubated at 37 °C in a 5%

CO2 atmosphere. After 48 h of stimulation, aliquots of supernatants were removed and stored at −70 °C for subsequent analysis. Sandwich-type ELISAs (DuoSet™, R&D Systems) were used to estimate the IFN-γ, IL-2 and IL-10 levels in virus-stimulated and control cell supernatants, according to the manufacturer’s instructions. Briefly, serial dilutions of cytokine standards, samples and controls were added to 96-well ELISA microplates coated with specific monoclonal antibody and incubated for 2 h at room temperature. Plates were then washed five times with PBS/T (PBS/0.5% Tween) and 100 μl of horseradish peroxidase-linked polyclonal anti-mouse

antibody was added. After 2 h at room temperature, the plates were washed five times and 100 μl of a substrate solution were added to each well. The plates were incubated for 30 min at room temperature, CX5461 and then read at 450 nm. The levels of cytokines in the supernatants were calculated by comparing their O.D. to a standard calibration curve. The DENV-4 specific lymphoproliferative

responses from vaccine and control immunized mice were determined by standard CFSE staining in two different experiments. Spleens were harvested from the same mice (4 mice per group) inoculated with recombinant DENV-4-DNAv, inactivated DENV-4, and pCI, as previously described in the Imunization of mice heading. Spleen cell suspensions were treated with Tris-buffered ammonium chloride to eliminate the red blood cells, washed, and resuspended in RPMI 1640 supplemented with 5% FBS, HEPES buffer, l-glutamine, penicillin and streptomycin. Cells all were cultured in triplicate in 96-well microtiter plates (1 × 105 cells/well) in the presence of heat inactivated DENV-4 (1 × 105 PFU), control RPMI medium, or ConA 2 μg/ml. Specific T cell proliferation of DENV-4-DNAv-immunized mice and control groups were evaluated by staining the cells with 5-(and-6) carboxy-fluorescein diacetate, succinimidyl ester (CFSE) (Molecular Probes, Oregon, USA). The reading was performed after 3 days of stimulus in a flow cytometry (FACscan) with software Cellquest (both from Becton-Dickinson Immunocytometry Systems Inc., San Jose, CA), and the statistical analysis was accomplished using the program WinMDI version 2.8.