A localizing replicon RNA vaccine harnesses the intramuscular immune microenvironment to drive adaptive responses

Intramuscular administration of self-amplifying replicon RNA (repRNA) formulated with the cationic nanocarrier LION™ represents a promising strategy for inducing durable protective immunity with minimal doses and limited systemic reactogenicity. However, the cellular and molecular mechanisms by which it elicits adaptive immune responses remain incompletely understood. Here, we demonstrate that repRNA delivery generates a microenvironment within skeletal muscle that promotes adaptive immune responses. In this local environment, repRNA-transfected muscle cells transfer antigen-encoding RNA and proteins to antigen-presenting cells to cross-prime CD8⁺ T cells, while CD4⁺ T cells and CD138⁺ cells expand in parallel with antibody production. CD4⁺ T cells are required for humoral immunity, and intramuscular CD138⁺ cell frequencies correlate with serum antibody levels. These findings reveal a coordinated mechanism by which the muscle microenvironment orchestrates adaptive immunity following localized repRNA delivery and provide mechanistic insight for the rational design of next-generation RNA vaccines and therapeutics.