mRNA-based vaccines combine the positive attributes of both live-attenuated and subunit vaccines. In order for these to be applied for clinical use, they require to be formulated with engineered delivery systems. However, there are limited in vivo studies which compare different delivery platforms. Therefore, we have compared four different cationic platforms: 1) liposomes, 2) solid lipid nanoparticles, 3) polymeric nanoparticles and 4) emulsions to deliver a self-amplifying mRNA (SAM) vaccine. All formulations contained either the non-ionizable cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or dimethyldioctadecylammonium bromide (DDA) and they were characterized in terms of physico-chemical attributes, in vitro transfection efficiency and in vivo vaccine potency. Our results showed that SAM encapsulating DOTAP NPs, DOTAP liposomes and DDA liposomes induced the highest antigen expression in vitro and, from these, DOTAP NPs were the most potent in triggering humoral and cellular immunity among candidates in vivo. This dataset supports our recent publication in this area and the data sets map to the following figures.
Table 1. Physico-chemical properties of cationic lipid based delivery systems. All formulations were prepared at 1 mg/mL final cationic lipid concentration. Results are represented as mean ± SD of three independent batches of each formulation. NPs (polymeric nanoparticles), SLNs (solid lipid nanoparticles), size (Z-average diameter), ZP (zeta potential), PDI (polydispersity), E.E. (encapsulation efficiency), AE (adsorption efficiency).
Figure 1. Cell viability and cell association of delivery platforms with BHK cells. Cytotoxicity (A and B) and cell association (C and D) in BHK cell of DOTAP based (A and C) and DDA (B and D) based liposomes, SLNs, NPs and emulsions. Lipofectamine 2000 (LF2000) was used as positive control. For cell viability, cationic lipid concentration of 3.7 to 100 µg/mL (N:P ratio of 8:1) were tested. For cell association, the results represent percentage of DilC positive BHK cells after 16 hours incubation with 11 µg/mL of DOTAP (C) based and DDA (D) formulations in either 5% FCS or FCS-free media. Results are represented as mean ± SD of 3 independent experiments.
Figure 2. Cell association of formulations in BHK cell line expressed as mean fluorescence intensity (MFI). Mean fluorescence intensity of BHK cells after 16 hours incubation with DOTAP based (A and C) and DDA based (B and D) liposomes, SLNs, NPs and emulsions in either 5% FCS (A and B) or FCS free media (C and D). Results are represented as mean ± SD of 3 independent experiments.
Figure 3. In vitro potency (IVP) in BHK cell line. IVP in BHK in either 5% FCS (A and B) or FCS free (C and D) medium of DOTAP based (A and C) and DDA based (B and D) formulations prepared with SAM-RVG at different concentrations. Lipofectamine2000 (LF2000) was used as positive control. Results are represented as mean ± SD of 3 independent experiments.
Figure 4. Immunogenicity of SAM-RVG vaccine delivered by different cationic carriers. Groups of ten BALB/c mice were immunized i.m. on days 0 and 28 with either 1.5 or 0.15 μg of self-amplifying RNA encoding the rabies virus G protein formulated in DOTAP polymeric nanoparticles (NPs), DOTAP Liposomes or DDA Liposomes and compared to the commercial vaccine Rabipur (1/20 of human dose). GSK trademark CNE56 was used as positive control. Specific IgG titres were measured by enzyme-linked immunosorbent assay (ELISA). Data are from pools of two mice (depicted as dots), and the geometric mean titres (GMTs) are solid lines. Sera were collected and analysed A) 4 weeks after the first immunization and B) 2 weeks after the second immunization. Titres < 0.125 EU/mL (dotted blue line) were below the limit of detection, while titres >0.5 EU/mL (dotted red line) were an indication of protection. Intergroup comparison was analysed using the one-way ANOVA test (Dunnett's multiple comparison test).
Figure 5. Percentages of antigen-specific CD4+ or CD8+ T cells. Splenic A) CD4+ T cells, and B) CD8+ T cells 2 weeks after two intramuscular immunizations spaced 4 weeks apart in BALB/c mice (N=3). Mice were immunized with either 1.5 or 0.15 μg/dose of self-amplifying RNA expressing rabies G glycoprotein adjuvanted with either polymeric nanoparticles (NPs), DOTAP Liposomes or DDA Liposomes. Candidates were compared with the commercial vaccine Rabipur (1/20 of human dose). GSK trademark CNE56 was used as positive control. Splenocytes were stimulated with rabies G1-G2-G3 peptide, stained for intra-cellular cytokines, and subjected to flow cytometry. Colors code indicates the different combinations of cytokine produced by the respective cells. Unstimulated cells were used as control.
Figure 6. Percentages of cytotoxic CD4+ or CD8+ T cells. The induction of rabies-specific CD4+ or CD8+ T cells by either 1.5 μg or 0.15 μg/dose of SAM encapsulating nanoparticles (NPs) DOTAP liposomes and DDA liposomes was characterized 2 weeks after the second immunization. Candidates were compared with the commercial vaccine Rabipur (1/20 of human dose). GSK trademark CNE56 was used as positive control. Surface expression of CD107a on splenocytes stimulated in vitro with rabies G1-G2-G3 peptide was assessed by flow cytometry. Data show the frequency of cytokine-secreting A) CD4+ or B) CD8+ T cells that express (blue bars) or not (orange bars) CD107a. Unstimulated cells were used as control.