Table 1.
Dengue specific monoclonal antibodies.
Table 2.
Particulated rE induced a balanced serotype specific tetravalent neutralizing antibody response in mice.
Fig 1.
Expression and characterization of DENV1-4 rE.
A) Schematic representation of the DENV1-4 rE expression constructs. The ectodomain of DENV1-4 E was expressed together with the homotypic prM protein, upstream an IL2 leader peptide. Recombinant proteins were equipped with a C-terminal His-tag and expressed under control of a CAG promoter on a pαH-expression vector in EXPI293 cells. B) Purified rE was subjected to SDS-PAGE and analyzed with CBB and WB using a 1M7 human derived mAb. C) DENV1-4 rE purified proteins fractions were loaded on Ni2+-coated ELISA plates and analyzed with a panel of cross-reactive or serotype specific mAbs (DENV1-1F4; DENV2-3H5, 2D22; DENV3-8A1, 5J7; DENV4-DV4 141, 5H2).
Fig 2.
A) rE proteins from all serotypes were individually adsorbed to the PLGA nanoparticles in variable srE/NP (w/w%) ratios. Adsorption efficiency was determined by the amount of non-adsorbed rE after the spinning down the particles. B) Mice were immunized (subcutaneous) with 5 μg of rE or 5 μg rE adsorbed to PLGA nanoparticles. Animals were boosted on day 21 and day 63 and serum samples were taken at indicated time points.
Fig 3.
Monovalent NP-rE formulations induce robust neutralizing antibody titers.
A) DENV1-4 specific IgG titers were determined for week 3, 4, 10 and 16. B) The neutralizing activity of the mice sera at week 16 was determined with a neutralization assay where DENV is incubated with serially diluted mice sera and subsequently allowed to infect Vero-cells. Neutralizing activity was expressed as the dilution where 50% of the virus was neutralized (Neut50). * DENV2 data adapted from previously published data [15]. Statistical differences were determined by one-way ANOVA followed by Tukeys test (p<0.05).
Fig 4.
Tetravalent DENV1-4 rE formulations.
The tetravalent formulations were divided into 2 groups. The tetra rE group (TR) combined 5 μg soluble rE of each serotype (n = 5). The tetra NP-rE group (TNR) combines four individually adsorbed NP-rE formulations of each DENV serotype (NP-rEDENV1 + NP-rEDENV2 + NP-rEDENV3 + NP-rEDENV4 (500 μg NPs + 5 μg rE).
Fig 5.
Tetravalent NP-rE immunization induces robust neutralizing IgG responses.
A) DENV1-4 specific IgG responses were determined for week 3, 4, 8, 10 and 16. B) DENV1-4 specific IgG end-point dilution titers (EPD) for total IgG, IgG1 and IgG2a were determined for week 16 sera. C) The neutralizing activity of the mice sera at week 16 was determined with a neutralization assay where DENV is incubated with serially diluted mice sera and subsequently allowed to infect Vero-cells. Neutralizing activity was expressed as the dilution where 50% of the virus was neutralized (Neut50). Statistical differences were determined by one-way ANOVA followed by Tukeys test (p<0.05).
Fig 6.
Tetra NP-rE induces serotype specific IgG responses in a tetravalent background.
A DENV rE depletion assay was used to estimate the proportions of serotype-specific and cross-reactive antibody panels in sera of mice immunized with tetravalent vaccine formulations. Serum was incubated with magnetic beads coated with rE of DENV1, 2, 3 or 4 and serotype specific IgG titers were determined against all 4 rE serotypes for A) TR and B) TNR tetravalent formulations. For example, to establish complete exhaustion (red bars) of all serotype-specific and cross-reactive DENV1 binding antibodies, sera was depleted with DENV1 rE. To determine the DENV1 serotype-specific portion (green bars), the same sera was depleted with DENV2, 3 and 4 rE. A negative control or handling control depletion (yellow bars) was performed using maltose binding protein (MBP). Statistical differences were determined by one-way ANOVA followed by Tukeys test (p<0.05).
Fig 7.
DENV rE particulation enhances serotype specificity in tetravalent formulations.
To determine the portion of serotype-specific neutralizing antibodies, sera from A) TR and B) TNR immunized mice were depleted from serotype-specific and cross-reactive (red lines), cross-reactive (green lines) neutralizing antibodies. Serum neutralization was determined using a flow based approach where serially diluted serum was incubated with virus, which was subsequently allowed to infect vero-cells. Neutralizing capacity of the sera is expressed as Neut50 values, indicating the serum dilution where 50% of the virus is neutralized (Table 1).