Data for: "Rational design of adjuvants for subunit vaccines: the format of cationic adjuvant affects the induction of antigen-specific antibody responses"

A range of cationic delivery systems have been investigated as vaccine adjuvants, though few direct comparisons exist. To investigate the impact of the delivery platform, we prepared four cationic systems (emulsions, liposomes, polymeric nanoparticles and solid lipid nanoparticles) all containing equal concentrations of the cationic lipid dimethyldioctadecylammonium bromide in combination with the Neisseria adhesin A variant 3 subunit antigen. The formulations were physico-chemically characterized and their ability to associate with cells and promote antigen processing was compared in vitro and their vaccine efficacy (antigen-specific antibody responses and IFN-γ production) and biodistribution (antigen and adjuvant) were evaluated in vivo. Due to their cationic nature, all delivery systems gave high antigen loading (> 85%) with liposomes, lipid nanoparticles and emulsions being < 200 nm, whilst polymeric nanoparticles were larger (~350 nm). In vitro, the particulate systems tended to promote cell uptake and antigen processing, whilst emulsions were less effective. Similarly, whilst the particulate delivery systems promoted a depot (of both delivery system and antigen) at the injection site, the cationic emulsions did not. However, the cationic emulsions promoted the highest antibody responses. These results demonstrate that the whilst cationic lipids can have strong adjuvant activity, their formulation platform influences their immunogenicity.

Figure Legends
Figure 1. In vitro association of adjuvants with THP-1 cells and induction of antigen degradation. Association efficiency of emulsions, liposomes, polymeric nanoparticles (PNPs) or solid lipid nanoparticles (SLNs) expressed as A) Percentage of DiD+ cells, B) DiD mean florescence intensity (MFI) or C) Relative number of associated adjuvants (Nr). Antigen degradation efficiency of emulsions, liposomes, PNPs or SLNs is expressed as D) DQ-OVA+ cells and E) DQ-OVA mean fluorescence intensity (MFI). F) Plots the % of cell association versus % antigen processing at all time points measured (data from A and D). Results are represented as mean ± SD of three independent experiments. Refer to Figure S1 in the supplementary for representative histograms of DiD and DQ-OVA positive THP-1 cells incubated with different adjuvants.
Figure 2. Biodistribution of DiD-labelled adjuvants in mice following intramuscular administration. Female BALB/c mice were administered NadA3 adjuvanted with DiD-labelled emulsions, liposomes, polymeric nanoparticles (PNPs) and solid lipid nanoparticles (SLNs) intramuscularly in the right quadricep. The fluorescent signals were evaluated by IVIS over 11 days p.i. A) Pharmacokinetic profiles of DiD-labelled adjuvants at the injection site. B) Results were normalized and replotted as percentage of signal ratio. C) Images acquired at selected time points. Results are represented as the mean ± SD of three animals per group. Refer to Figure S2 in the supplementary for images of mice administered with adjuvanted NadA3 acquired at all time points over 11 days p.i.
Figure 3. Biodistribution of adjuvanted NadA3 antigen. Female BALB/c mice were administered unadjvuanted AF790-labelled NadA3 or adjuvanted with DiD-labelled emulsions, liposomes, polymeric nanoparticles (PNPs) and solid lipid nanoparticles (SLNs) intramuscularly in the right quadricep. The fluorescent signals were evaluated by IVIS over 11 days p.i. A) Pharmacokinetic profiles of either unadjuvanted or adjuvanted NadA3 at the injection site. B) Results were normalized and replotted as percentage of signal ratio. C) Images acquired at selected time points. Results are represented as the mean ± SD of three animals per group. Refer to Figure S3 in the supplementary for images of mice administered with either unadjuvanted or adjuvanted NadA3 acquired at all time points over 11 days p.i.

Figure 4. Comparison of AUC for both antigen and adjuvant after intramuscular injection. The retention of the antigen promoted by the adjuvant was calculated as area under the curve (AUC) for each formulation for both adjuvant and antigen (AUC; flux.day).

Figure 5. Biodistribution of radiolabeled cationic formulations. Female BALB/c mice were administered 3H-cholesterol-labelled emulsions, liposomes, polymeric nanoparticles (PNPs) and solid lipid nanoparticles (SLNs) intramuscularly in the right quadriceps. The radioactivity was evaluated in the injection site, liver, spleen, popliteal lymph node (PLN), inguinal lymph node (ILN) and whole carcass over 48 hours p.i. A) Pharmacokinetic profiles of 3H-cholesterol-labelled adjuvants. Percentage of dose determined at B) popliteal lymph node (PLN) and C) inguinal lymph node (ILN). Results are expressed as the mean ± SD of four animals. Data of biodistribution of liposomes were previously published by (43).

Figure 6. In vivo humoral responses promoted by four different cationic delivery platforms. Female BALB/c mice were immunized twice i.m. with unadjuvanted NadA3 (10 µg/dose) or NadA3 adjuvanted with cationic emulsions, liposomes, polymer nanoparticles (PNPs) and solid lipid nanoparticles (SLNs), with a booster immunization four weeks after the prime immunization. The antigen-specific total IgG, IgG1 and IgG2a levels in the blood were evaluated two weeks after the final immunization by using ELISA. Antigen specific A) total IgG levels, B) IgG1 and C) IgG2a average of mice in repeated studies, n=3-6, mean ± SD and end point titers for D) total IgG, E) IgG1 and F) IgG2a, n=4-6, lines denote mean ± SD. p ≤ 0.05 (*).