Polymeric and lipid nanoparticles for delivery of self-amplifying RNA vaccines

Abstract

Self-amplifying RNA (saRNA) is a next-generation vaccine platform, but like all nucleic acids, requires a delivery vehicle to promote cellular uptake and protect the saRNA from degradation. To date, delivery platforms for saRNA have included lipid nanoparticles (LNP), polyplexes and cationic nanoemulsions; of these LNP are the most clinically advanced with the recent FDA approval of COVID-19 based-modified mRNA vaccines. While the effect of RNA on vaccine immunogenicity is well studied, the role of biomaterials in saRNA vaccine effectiveness is under investigated. Here, we tested saRNA formulated with either pABOL, a bioreducible polymer, or LNP, and characterized the protein expression and vaccine immunogenicity of both platforms. We observed that pABOL-formulated saRNA resulted in a higher magnitude of protein expression, but that the LNP formulations were overall more immunogenic. Furthermore, we observed that both the helper phospholipid and route of administration (intramuscular versus intranasal) of LNP impacted the vaccine immunogenicity of two model antigens (influenza hemagglutinin and SARS-CoV-2 spike protein). We observed that LNP administered intramuscularly, but not pABOL or LNP administered intranasally, resulted in increased acute interleukin-6 expression after vaccination. Overall, these results indicate that delivery systems and routes of administration may fulfill different delivery niches within the field of saRNA genetic medicines.

Keywords: Immunogenicity; Lipid nanoparticle; Polyplex; Protein expression; Replicon; SARS-CoV-2; Self-amplifying RNA; Vaccine.

Graphical abstract

Fig. 1

Schematic illustration of VEEV self-amplifying RNA (A), polymeric and lipid nanoparticle formulations (B) and pABOL chemical structure (C).

Fig. 2

Characterization of saRNA polymeric and lipid nanoparticle formulations: particle size (A) and zeta potential (B) as determined by dynamic light scattering (DLS) and encapsulation efficiency of saRNA relative to initial loading (C) as determined by RiboGreen assay. Bars represent mean ± standard deviation for n = 3. For (A), bars and error bars correspond to the left axis (particle diameter) and dots correspond to the right axis (PDI).

Fig. 3

Effect of polymeric and lipid nanoparticle formulations on saRNA protein expression in vivo. Quantification of fLuc expression from pABOL or LNP (LM01PE-LM03PC) 7 days after injection. Mice were injected intramuscularly with 5 μg of saRNA with an N:P ratio of 45:1 for pABOL and 8:1 for the LNP. For imaging, mice were injected IP with D-luciferin substrate, allowed to rest for 10 min, anesthetized using isoflurane and imaged on an In Vivo Imaging System (IVIS) FX Pro as described in the Methods section. Each circle represents one leg of one animal, and line represents mean ± SD, n = 5. *Indicates significance of p < 0.05 compared to pABOL as determined by a Kruskal-Wallis test adjusted for multiple comparisons.

Fig. 4

Immunogenicity of pABOL and LNP formulations against influenza HA (Cal/09). (A) HA antigen-specific IgG antibody titers following IM immunization with a prime and boost of saRNA formulated with pABOL or LNP (LM01PE-LM03PC). Line represents mean ± SD for n = 5. (B) Change in body weight after IN challenge with Cal/09 flu virus for either mice injected IM with pABOL or LNP formulations, or naïve mice. Dots represent mean percentage of body weight normalized to day 0 for each mouse, ± SD for n = 5. *Indicates significance of p < 0.05 compared to pABOL as determined by a Kruskal-Wallis test adjusted for multiple comparisons.

Fig. 5

Dose titration and systemic and mucosal humoral immunogenicity of pABOL and LNP formulations against SARS-CoV-2 after IM or IN inoculation. (A,B) Systemic (A) or mucosal (B) SARS-CoV-2 spike antigen-specific IgG antibody titers following IM or IN immunization with a prime and boost of saRNA formulated with pABOL or LNP (LM03PE). Line represents mean ± SD for n = 5. (C) Neutralization IC₅₀ against pseudotyped SARS-CoV-2 virus following IM or IN immunization with a prime and boost of saRNA formulated with pABOL or LNP (LM03PE). Line represents mean ± SD for n = 5. *Indicates significance of p < 0.05 compared to pABOL as determined by a Kruskal-Wallis test adjusted for multiple comparisons.

Fig. 6

Cellular immunogenicity to SARS-CoV-2 after prime and boost of saRNA formulated with pABOL or LNP. Quantification of IFN-γ secretion by splenocytes upon restimulation with SARS-CoV-2 peptides, expressed as sport forming units (SFU) per 10⁶ cells. Naïve animals were used as a negative control. Line represents mean ± SD for n = 5.

Fig. 7

Cytokine profile in sera of mice 4 h after immunization with saRNA formulated with pABOL or LNP and administered IM or IN. Line represents mean ± SD for n = 5. * indicates significance of p < 0.05 compared to naïve control as determined by a Kruskal-Wallis test.