Innate Inhibiting Proteins Enhance Expression and Immunogenicity of Self-Amplifying RNA

Abstract

Self-amplifying RNA (saRNA) is a cutting-edge platform for both nucleic acid vaccines and therapeutics. saRNA is self-adjuvanting, as it activates types I and III interferon (IFN), which enhances the immunogenicity of RNA vaccines but can also lead to inhibition of translation. In this study, we screened a library of saRNA constructs with cis-encoded innate inhibiting proteins (IIPs) and determined the effect on protein expression and immunogenicity. We observed that the PIV-5 V and Middle East respiratory syndrome coronavirus (MERS-CoV) ORF4a proteins enhance protein expression 100- to 500-fold in vitro in IFN-competent HeLa and MRC5 cells. We found that the MERS-CoV ORF4a protein partially abates dose nonlinearity in vivo, and that ruxolitinib, a potent Janus kinase (JAK)/signal transducer and activator of transcription (STAT) inhibitor, but not the IIPs, enhances protein expression of saRNA in vivo. Both the PIV-5 V and MERS-CoV ORF4a proteins were found to enhance the percentage of resident cells in human skin explants expressing saRNA and completely rescued dose nonlinearity of saRNA. Finally, we observed that the MERS-CoV ORF4a increased the rabies virus (RABV)-specific immunoglobulin G (IgG) titer and neutralization half-maximal inhibitory concentration (IC₅₀) by ∼10-fold in rabbits, but not in mice or rats. These experiments provide a proof of concept that IIPs can be directly encoded into saRNA vectors and effectively abate the nonlinear dose dependency and enhance immunogenicity.

Keywords: RNA; gene delivery; immunomodulation; innate immunity; interferon; nanoparticles; replicon; self-amplifying; vaccines.

Graphical abstract

Figure 1

Schematic and In Vitro Protein Expression from WT and IIP VEEV Replicons (A) Schematic of wild-type and cis-encoding IIP VEEV replicons. (B) Schematic of innate sensing of self-amplifying RNA. (C) In vitro transfection of firefly luciferase saRNA in HEK293T.17, HeLa, and MRC5 cells measured as relative light units (RLU). Bars represent mean fold change ± standard deviation normalized to wild-type VEEV control, for n = 3.

Figure 2

Dose Titration of WT and MERS-CoV ORF4a Replicon in C57BL6/J Mice Protein expression was quantified at days 7 and 10 after intramuscular injection of either 0.2, 2, or 20 μg of RNA. Each dot represents a single mouse and the bar represents the mean ± SEM with n = 10. ∗p < 0.05 as evaluated using a Kruskal-Wallis test with multiple comparisons.

Figure 3

Co-formulation of WT and MERS-CoV ORF4a Replicons with JAK Inhibitor Ruxolitinib in C57BL6/J Mice (A–D) Protein expression was quantified at days (A) 4, (B) 7, (C) 10, and (D) 14 after intramuscular injection of either 5 μg of RNA with 100 μg of ruxolitinib. Each dot represents a single mouse leg and the bar represents the mean ± SEM with n = 10. ∗p < 0.05 compared to WT fLuc using a Kruskal-Wallis test adjusted for multiple comparisons.

Figure 4

Protein Expression of EGFP in Human Skin Explants with or withouot IIPs and Ruxolitinib (A–D) Protein expression of EGFP with and without MERS-CoV ORF4a RNA (0.2, 2, or 20 μg) (A and B) or with and without ruxolitinib (0.1, 1, 10, or 100 μg) (C and D) in human skin explants. Number of EGFP expression cells (%GFP⁺ cells) (A and C) and total protein expression per cell (GFP median fluorescent intensity [MFI]) (B and D) were quantified 72 h after injection. Each dot represents a single explant and the bar represents the mean ± SEM with n = 3. ∗p < 0.05 as evaluated using a Kruskal-Wallis test with multiple comparisons.

Figure 5

Immunogenicity of RABV with and without MERS-CoV ORF4a in Rabbits (A) RABV antigen-specific IgG antibody titers following intramuscular immunization with prime and boost of 20 μg at 0 and 4 weeks, with n = 5. (B) Neutralization IC₅₀ against pseudotyped RABV with n = 5; gray dotted line represents the limit of detection. Each dot represents one rabbit and the bar represents mean ± SEM with n = 5. ∗p < 0.05 as evaluated using a Kruskal-Wallis test with multiple comparisons.

Figure 6

Mechanism of IIP Innate Inhibition of saRNA (A) Schematic of proposed mechanism of PIV-5 V and MERS-CoV ORF4a on saRNA sensing. (B and C) Quantity of NF-κB (B) and IRF3 (C) in MRC5 cell nuclear extracts 4, 24, and 48 h after transfection, with n = 3. Bars represent mean ± standard deviation. ∗p < 0.05 compared to WT fLuc using a two-way ANOVA adjusted for multiple comparisons.