Innate inhibiting proteins enhance expression and immunogenicity of self-amplifying RNA

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. Here, we screen a library of saRNA constructs with cis-encoded innate inhibiting proteins (IIPs) and determine the effect on protein expression and immunogenicity. We observed that the PIV-5 V and MERS-CoV ORF4a proteins enhance protein expression 100-500-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 JAK/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 RABV-specific IgG titer and neutralization IC50 by ~10-fold in rabbits, but not 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.