Abstract
Self-amplifying RNA (saRNA)-based vaccines have emerged as a potent and durable RNA vaccine platform relative to first generation mRNA vaccines. However, RNA vaccine platforms trigger undesirable side effects at protective doses, underscoring the need for improved tolerability. To address this, we leveraged the Cardiovirus leader protein, which is well-characterized to dampen host innate signaling by modulating nucleocytoplasmic transport (NCT). Co-administration of a leader-protein-encoding mRNA (which we have named "RNAx") delivered alongside vaccine cargo saRNA reduced interferon production while enhancing Influenza hemagglutinin (HA) expression in human primary cells and murine models. RNAx potently decreased serum biomarkers of reactogenicity after immunizations with an HA-expressing saRNA-LNP vaccine while maintaining the magnitude of the antibody and cellular response. RNAx also consistently enhanced binding antibody titers after a single injection and in some conditions enhanced binding antibody and neutralization titers post-boost. These findings support RNAx as a promising platform approach for improving tolerability of saRNA-LNP vaccines while preserving or enhancing immunogenicity.