The role of nanoparticle format and route of administration on self-amplifying mRNA vaccine potency

Abstract

The efficacy of RNA-based vaccines has been recently demonstrated, leading to the use of mRNA-based COVID-19 vaccines. The application of self-amplifying mRNA within these formulations may offer further enhancement to these vaccines, as self-amplifying mRNA replicons enable longer expression kinetics and more potent immune responses compared to non-amplifying mRNAs. To investigate the impact of administration route on RNA-vaccine potency, we investigated the immunogenicity of a self-amplifying mRNA encoding the rabies virus glycoprotein encapsulated in different nanoparticle platforms (solid lipid nanoparticles (SLNs), polymeric nanoparticles (PNPs) and lipid nanoparticles (LNPs)). These were administered via three different routes: intramuscular, intradermal and intranasal. Our studies in a mouse model show that the immunogenicity of our 4 different saRNA vaccine formulations after intramuscular or intradermal administration was initially comparable; however, ionizable LNPs gave higher long-term IgG responses. The clearance of all 4 of the nanoparticle formulations from the intramuscular or intradermal administration site was similar. In contrast, immune responses generated after intranasal was low and coupled with rapid clearance for the administration site, irrespective of the formulation. These results demonstrate that both the administration route and delivery system format dictate self-amplifying RNA vaccine efficacy.

Keywords: Immunogenicity; Lipid nanoparticles; Polymeric nanoparticles; RNA vaccines; Route of administration; Self-amplifying RNA; Solid lipid nanoparticles; saRNA.

Graphical abstract

Fig. 1

Physicochemical characterization of saRNA formulations. SLNs, PNPs, cLNPs and iLNPs were prepared as outlined in (A) and characterized in terms of B) particles size (d.nm) and polydispersity index (PDI), C) zeta-potential (mV) and D) encapsulation efficiency (EE%). Results are represented as mean ± SD of two different batches used for first and second vaccination respectively.

Fig. 2

Immunogenicity of RVG-saRNA loaded SLNs, PNPs and LNPs. Humoral immune responses elicited by RVG-saRNA formulated in either DOTAP-based SLNs, PNPs or LNPs following intramuscular (IM, 0.15 μg), intradermal (ID, 0.15 μg) or intranasal (IN, 1.5 μg) administration in mice. Mice were also immunized with benchmark iLNPs [11] or 2% (IM and ID) or 5% (IN) of the human dose of Rabipur. Mice were vaccinated four weeks apart and total anti-RVG IgG titers were quantified four weeks after the first vaccination (A), two weeks after the second vaccination (B) and 10 weeks after the second vaccination (C). Markers depict measurements from pools of 2 mice each. The solid lines represent the geometric mean titer of each group (n = 4–5). Dotted lines at 0.5 and 0.125 EU/mL correspond to the correlate of protection and limit of quantification, respectively.

Fig. 3

Cellular immune response elicited by RVG-saRNA loaded nanoparticles after IM, ID or IN administration. Splenic CD8+ and CD4+ T cell responses elicited by RVG-saRNA formulated in either DOTAP-based SLNs, PNPs or LNPs following intramuscular (IM, 0.15 μg), intradermal (ID, 0.15 μg) or intranasal (IN, 1.5 μg) administration in mice. Mice were also immunized with benchmark iLNPs [11] and either 2% (IM and ID) or 5% (IN) of the human dose of Rabipur. Splenocytes were collected two weeks after the second vaccination and re-stimulated in vitro with an RVG peptide pool. A) Frequencies of cytokine-producing CD8+ T cells. B) Frequencies of CD107+ CD8+ T cells. C) Frequencies of CD4+ T cells expressed as Th1 and Th0 according to the cytokines expressed. Results are represented as mean ± SD of three samples. Refer to Fig. S1 in the supplemental material for the gating strategy.

Fig. 4

Lung CD8+ and CD4+ T cell responses following intranasal vaccination. Lung cells were collected two weeks after the second vaccination and re-stimulated in vitro with an RVG peptide pool. A) Frequencies of cytokine-producing CD8+ T cells. B) Frequencies of CD107+ CD8+ T cells. C) Frequencies of CD4+ T cells expressed as Th1 and Th0 according to the cytokines expressed. Results are represented as mean ± SD of three samples. Refer to Fig. S2 in the supplemental material for the gating strategy.

Fig. 5

Biodistribution of RVG-saRNA loaded SLNs, PNPs and LNPs in a mouse model. Representative IVIS images of groups of 5 BALB/c mice injected with either saRNA-SLNs, saRNA-PNPs or saRNA-LNPs by the intramuscular (IM), intradermal (ID) or intranasal (IN) route at selected time points. Mice received 25 μg of nanoparticles, corresponding to the administration of 1 μg of saRNA. The total flux was calculated in the regions of interest highlighted in blue. Scale of fluorescence is reported. Refer to Fig. S3 in the supplementary for enlarged images of mice at all time points over 10 days p.i. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Pharmacokinetic profile at the site of injection of RVG-saRNA loaded SLNs, PNPs and LNPs. Pharmacokinetic profile at the site of injection of either saRNA-SLNs, saRNA-PNPs or saRNA-LNPs following A) intramuscular, B) intradermal or C) intranasal administration. Mice received 25 μg of nanoparticles, corresponding to the administration of 1 μg of saRNA. A naive mouse was used as a negative control. D) Calculated areas under the curve at the site of injection for saRNA encapsulating LNPs, PNPs and SLNs administered by intramuscular (IM), intradermal (ID) or intranasal (IN) route. The total flux was normalised by dividing each time point by the value at 4 h time point as it was the highest in each group. This was considered as 100%Dotted line represents the background value. Results are represented as mean ± SD of five animals per group.