Self-amplifying mRNA (saRNA) represents a promising platform for nucleic acid delivery of vaccine immunogens. Unlike plasmid DNA, saRNA does not require entry into the nucleus of target cells for expression, having the capacity to drive higher protein expression compared to mRNA as it replicates within the cytoplasm. In this study, we examined the potential of stabilized native-like HIV-1 Envelope glycoprotein (Env) trimers to elicit immune responses when delivered by saRNA polyplexes (PLXs), assembled with linear polyethylenimine. We showed that Venezuelan equine encephalitis virus (VEEV) saRNA induces a stronger humoral immune response to the encoded transgene compared to Semliki Forest virus saRNA. Moreover, we characterized the immunogenicity of the soluble and membrane-bound ConSOSL.UFO Env design in mice and showed a faster humoral kinetic and an immunoglobulin G (IgG)2a skew using a membrane-bound design. The immune response generated by PLX VEEV saRNA encoding the membrane-bound Env was then evaluated in larger animal models including macaques, in which low doses induced high IgG responses. Our data demonstrated that the VEEV saRNA PLX nanoparticle formulation represents a suitable platform for the delivery of stabilized HIV-1 Env and has the potential to be used in a variety of vaccine regimens.

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We introduce aqueous phases comprising single, highly compacted self-amplifying messenger RNA (saRNA) molecules, that are suitable for prophylactic and therapeutic application, specifically for vaccination against infectious diseases. The formulations are formed in the presence of the positively charged polymer polyethylenimine (PEI), which leads to condensation of the single saRNA molecules into a globular organization with high packing density, low mass fraction of polymer, and, consequently, very small size. In this format, they display improved biological activity in comparison to previously described saRNA/PEI nanoparticlulate formulations, both in vitro and in vivo. Application of the ultra-compacted single saRNA formulation for vaccination, via intramuscular route, results in relevant titers for practical use at lower doses compared to the nanoparticle formulations. These novel saRNA vaccine products can be obtained by straight-forward manufacturing routes, and they can be readily frozen or lyophilized. With these characteristics they can be particularly of interest for future vaccine products even for application under challenging conditions, where requirements such as high activity, good thermostability, low cost of goods, and facilitated logistics need to be fulfilled at the same time. One-Sentence Summary: single saRNA molecules, condensed into an unusually compact globular organization with very small size by an oppositely charged polyelectrolyte (polyethylenimine), are applicable as vaccines, which induce very strong immunological responses at very low doses.