Fig 1.
Expression and characterization of sRecE.
A) Schematic representation of the sRecE expression construct. The DENV2 prM sequence leads the DENV2 E ectodomain (aa1-395) that is equipped with a C-terminal His-tag. The prM/sRecE is N-terminally fused to the IL2-leader sequence. B) Purified sRecE was subjected to SDS-PAGE and analyzed with CBB and WB using a 4G2 mouse derived Mab. C) Purifed sRecE was loaded on a Ni2+ coated ELISA plate and analyzed with a panel of mouse and human derived Mab. 8A1 is a DENV3 specific Mab and was used as a negative control.
Table 1.
Dengue specific monoclonal antibodies.
Fig 2.
sRecE adsorption to PRINT PLGA nanoparticles.
A) TEM analysis of 80×320 nm, 80×180 nm, 55×70 nm and 200×200 nm PLGA particles. B) sRecE was adsorbed to the PLGA nanoparticles in variable sRecE/PLGA (w/w%) ratios. Increasing sRecE/PLGA ratios were obtained by fixing sRecE and decreasing the particle mass. Adsorption efficiency was determined by the amount of non-adsorbed sRecE after the spinning down the particles.
Fig 3.
Long lasting IgG antibody titers induced by PLGA-RecE.
A) Mice were immunized (subcutaneous) with 5 μg sRecE or 5 μg RecE adsorbed to PLGA particles. Animals were boosted with similar doses at day 21 and day 63 and mice were blead on indicated time points. B-C) DENV specific IgG end point dilution titers were determined at day 28, 70, 98, 154 and 210. Statistical differences were determined by one-way ANOVA followed by Tukeys test (p<0.05).
Fig 4.
PLGA-RecE induces a long lasting neutralizing antibody response.
The neutralizing activity of the mice sera was determined by a neutralization assay where DENV is incubated with serially diluted sera and subsequently allowed to infect Vero cells. Neutralizing activity was expressed as the dilution where 50% of the virus was neutralized (Neut50). A) At day 70 post immunization, 1 week post 2nd boost and B) at day 210, 20 weeks post 2nd boost. Statistical differences were determined by one-way ANOVA followed by Tukeys test (p<0.05).
Fig 5.
Cross-neutralization of RecE induced IgG antibodies.
The capability of the cross reactive antibodies to neutralize DENV1, DENV2, DENV3 or DENV4 was tested in a neutralization assay using Vero cells. Neutralizing activity was expressed as the dilution where 50% of the virus was neutralized (Neut50).
Fig 6.
Bioavailability of sRecE and PLGA-RecE in PLNs and injection site.
A) sRecE was tagged with Alexa Fluor 647 and adsorbed to PLGA nanoparticles and inoculated into to footpad. At indicated time points, the draining popliteal lymph nodes (PLNs) were isolated and analyzed for total fluorescence. The upper panel comparing sRecE and 80×320 nm PLGA-RecE is representative for the measure of antigen presence in the PLNs. The comparison of all nanoparticle sizes is depicted in the lower panel. B) The foot pads were analyzed at similar time points to analyze the bio-availability at the inoculation site. Statistical analysis was done by two-way ANOVA followed by Bonferroni posttests. * p<0.05, ** p<0.01, *** p<0.001.