Alphaviral backbone of self-amplifying RNA enhances protein expression and immunogenicity against SARS-CoV-2 antigen

Self-amplifying RNA (saRNA) vectors are a next-generation RNA technology that extends the expression of heterologous genes. Clinical trials have shown the dose-sparing capacity of saRNA vectors in a vaccine context compared with conventional messenger RNA. However, saRNA vectors have historically been based on a limited number of alphaviruses, and only the Venezuelan equine encephalitis virus-based saRNA vaccines have been used clinically. Here, we designed genotypically distinct alphaviral saRNA vectors and characterized their performance in mammalian cell lines, human skin explants and mice. Five of the 12 vectors had substantial luciferase expression in mice with variable pharmacokinetics, enabling modulation of both the magnitude and duration of protein expression. Additionally, we demonstrated that the alphaviral genotype of the saRNA significantly impacts the immunogenicity of saRNA vaccines, including the humoral and cellular responses in mice. Given the differences in RNA reactogenicity and expression between mice and humans, we assessed the saRNA vectors in human skin explants obtained from patients and observed high transgene expression. saRNA bioluminescence and immunogenicity in different mice strains were highly correlative, while minimal correlation was observed when compared with human explants and mammalian cell lines. This work demonstrates that efficacious saRNA vaccines and therapies can be produced by adapting genetically diverse alphaviruses into vectors.