Self-amplifying RNA (SAM) represents a versatile tool that can be used to develop potent vaccines, potentially able to elicit strong antigen-specific humoral and cellular-mediated immune responses to virtually any infectious disease. To protect the SAM from degradation and achieve efficient delivery, lipid nanoparticles (LNPs), particularly those based on ionizable amino-lipids, are commonly adopted. Herein, we compared commonly available cationic lipids, which have been broadly used in clinical investigations, as an alternative to ionizable lipids. To this end, a SAM vaccine encoding the rabies virus glycoprotein (RVG) was used. The cationic lipids investigated including 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), dimethyldioctadecylammonium (DDA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), 1,2-stearoyl-3-trimethylammonium-propane (DSTAP) and N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ). Whilst all cationic LNP (cLNP) formulations promoted high association with cells in vitro, those formulations containing the fusogenic lipid 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE) in combination with DOTAP or DDA were the most efficient at inducing antigen expression. Therefore, DOTAP and DDA formulations were selected for further in vivo studies and were compared to benchmark ionizable LNPs (iLNPs). Biodistribution studies revealed that DDA-cLNPs remained longer at the injection site compared with DOTAP-cLNPs and iLNPs when administered intramuscularly in mice. However, both the cLNP formulations and the iLNPs induced strong humoral and cellular-mediated immune responses in mice that were not significantly different at a 1.5 µg SAM dose. In summary, cLNPs based on DOTAP and DDA are an efficient alternative to iLNPs to deliver SAM vaccines.
Table 1. Physicochemical characterization of SAM-LNPs produced by microfluidics. Formulations were composed of DOPE, a cationic lipid and DMG-PEG2000 at 49:49:2 molar ratio or DSPC, Chol, a cationic lipid/ionizable lipid and DMG-PEG2000 at 10:48:40:2 molar ratio. E.E. (encapsulation efficiency); ZP (zeta-potential). Results are represented as mean ± of three independent experiments.
Figure 1. Cellular uptake RVG-SAM cLNPs and iLNPs in presence (A, B) and absence of 5% FBS (C, D) represented in terms of percentage of Dil-C18+ cells (A, C) and mean fluorescence intensity (B, D). cLNPs were composed of DOPE, a cationic (DOTAP, DDA, DC-Chol, DMTAP or DOBAQ) and DMG-PEG2000 at 49:49:2 mole % or DSPC, Chol, a cationic lipid and DMG-PEG2000 at 10:48:40:2 mole %. Results are represented as mean ± SD of three experiments. Statistical significance: P < 0.05 (*).
Figure 2. In vitro potency of RVG-SAM iLNPs and DOPE-cLNPs in presence (A) and absence of 5% FBS (B). LF2000 (Lipofectamine2000). Results are represented as mean ± SD of three independent experiments.
Figure 4. Total anti-RVG IgG titers in mice upon intramuscular injection of SAM formulated in cLNPs, DOTAP-CNE, iLNPs or the commercial vaccine Rabipur on days 0 and 28. Sera were collected after 27 (A) and 42 (B) days and total IgG titers were quantified using PLATELIA RABIES II KIT (Bio-Rad). Dots depict measurements from pools of 2 sera each. The solid lines represent the geometric mean titer (GMT) of each group (n=5). Dotted lines at 0.5 and 0.125 EU/mL correspond to protection threshold and limit of quantification respectively. HD (human dose). C) Frequencies of RVG-specific cytokine producing CD8+ (C) and CD4+ T cells (D) analyzed with Boolean gates. CD4+ T cells were represented as Th1 and Th0 cells according to secreted cytokines. T cell results are represented as mean ± SD of three replicates. For the statistical analysis of T cell responses, DOTAP-cLNPs, DDA-cLNPs and DOTAP-CNE were compared to iLNPs at the same SAM dose: non-significant (ns); p < 0.05 (*)
Figure 5. Biodistribution of DOTAP-cLNPs, DDA-cLNPs and benchmark iLNPs in mice following intramuscular administration. A) Images acquired at relevant time points. B) Biodistribution pharmacokinetics. C) Calculated areas under the curve for each formulation. Results are represented as total flux in the region of interest, in pink, as mean ± SD of five animals per group. Statistical significance of DDA-cLNPs compared to DOTAP-cLNPs and iLNPs: p < 0.05 (*).