Cell culture and virus production

C6/36 Aedes albopictus cells were grown in L-15 medium (Cultilab, Brazil) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Cultilab, Brazil), 100 μg/mL penicillin and 100 μg/mL streptomycin at 28°C. The DENV-1, 2, 3 and 4 were isolated from dengue infected patients in Brazil and were kindly donated by Dr. Erna G. Kroon (Lab Virus, Federal University of Minas Gerais, Brazil). The propagation of each serotype of DENV was carried out in separate C6/36 cell cultures flasks. The cells were infected with DENV-1, 2, 3 or 4 at a multiplicity of infection (MOI) of 0.1 and incubated at 28°C for a week. After the development of cell syncytia, the supernatants were harvested, and titrated by standard plaque assay in LLC-MK2 cells [7]. Heat-inactivated virus was prepared by incubating virus samples in a 55°C water bath for one hour as described previously [8].

Nanoparticle preparation and characterization

The nanoparticles were obtained by the addition of ethanol dropwise (ethanol:water relation 1,5:1) to an aqueous solution of bovine serum albumin (BSA) (2% w/v). The coacervates were hardened by adding 50 μL of 25% glutaraldehyde while stirring for 2 hours at room temperature. The BSA-nanoparticles were purified by three cycles of centrifugation at 13,000 g for 30 minutes to eliminate free BSA and the excess of the crosslinking agent. The supernatants were removed and the pellets resuspended in sterile PBS (final concentration of 20 mg/mL). For adsorption of inactivated viral particles to the surface of the nanoparticles (NP+DENV), 1 mL suspension of the tetravalent DENV antigenic suspension (equivalent of 1. 2 × 10⁴ plaque forming units (PFU) for each serotype) was incubated with 1 mL of NP at 20 mg/mL. After rapid homogenization (30 seconds), the nanoparticles were purified by three successive centrifugations, each at 13,000 g for 30 minutes, 20°C. The supernatants were collected after each centrifugation and tested by Bradford assay [9]. To determine the amount of protein adsorbed to the nanoparticles, a colorimetric assay was used and compared to the input protein and that recovered in the supernatants after centrifugation.

The presence of viral particles on the surface of NPs was demonstrated by ELISA assays using the NP+DENV as antigen. Briefly, 96-well microtiter plates were coated with NP+DENV (200 μg/well) in carbonate buffer, incubated overnight at 4°C and then washed with PBS containing 0.05% Tween-20 (PBST). Subsequently, the plates were blocked with 100 μl/well of blocking buffer (5% dry milk in PBST) for 2 h at 37°C. After three washes, 100 μl of monoclonal anti-DENV-1 (E95), anti-DENV-2 (E96), anti-DENV-3 (E51) or DENV-4 (E88) was added at 1 μg/mL [10–12] in triplicate and incubated for 1 h at 37°C. After three washes, the plates were incubated for 1 hour at 37°C with 100 μl/well of horseradish-peroxidase (HRP)-labeled anti-mouse IgG (Sigma) diluted 1:5000 in PBST. After three additional washes, the plates were incubated for 15 minutes with 0.1 ml of 0.045% H₂O₂ and 0.4 mg/ml of o-phenylenediaminedihydrochloride (OPD) in phosphate-citrate buffer (0.1 M citric acid, 0.2 M sodium phosphate dibasic, pH 5.0). The reaction was stopped by adding 50 μl of 2 M H₂SO₄. The optical densities were read at 450 nm with a microplate reader. The zeta potential of the NPs were determined with a Zetasizer 3000 HS (Malvern Instruments, UK). The surface morphology and the diameter of the formulated nanoparticles were visualized by scanning electron microscopy (Model JEOL 560).

Immunization protocol and quantification of anti-DENV and anti-BSA IgG

Six-week-old male Swiss Webster mice were used for immunizations. All mice were maintained with free access to sterile food and water, and protocols were approved by the local Animal Experimentation Ethics Committee. Mice were immunized via the subcutaneous route using 100 μg of NP (n = 15) or NP+DENV (n = 20) on days 0, 7 and 14. The mice were sacrificed 7 days after the last immunization (at day 21). Additional mice were immunized with PBS (negative control) (n = 10) or 1.2 × 10⁴ PFU of the tetravalent DENV heat-inactivated antigenic suspension (positive control) (n = 10). Blood was collected by cardiac puncture and sera were separated for ELISA. To perform IgG quantification, 96-well microtiter plates were coated with 500 PFU/well of the inactivated DENV particles (serotypes 1 to 4) in carbonate buffer or BSA (2 μg/well). An ELISA was performed exactly as described above for quantitation of viral particles on NP, except virions were captured directly and sera from mice were used as the primary antibody. An analysis of ELISA results was performed using analysis of variance (ANOVA), and the differences between groups were evaluated with Tukey's test.

Plaque Reduction Neutralization assay (PRNA)

Briefly, heat-inactivated mouse sera were serially diluted two-fold from 1:20 to 1:320 in media and then mixed with a single DENV serotype (20 to 50 PFU/well) and incubated for 1 hour at 37°C. The mixture was inoculated onto LLC-MK2 cells in 24-well plates. Plates were incubated for 1 h before media was aspirated and replaced with 0.5 mL of 0.8% carboximethylcellulose medium (with 2% FBS). The plates are incubated at 37°C (5% CO2) for five days. At that time, cells were stained with a crystal violet solution (0.5% crystal violet, 10% ethanol and 1% paraformaldehyde) for 20 minutes, washed again, and the viral plaques were counted. The positive-control serum sample was a serum sample derived from a human patient and the antibody titers were 1:40 for DENV-1 and 1:20 for DENV-2, 3 and 4. Experiments were performed in duplicate.