Skeletal muscle as an active immune niche orchestrating adaptive responses to replicon RNA vaccination

Introduction/Rationale: Self-amplifying replicon RNA (repRNA) vaccines formulated with the cationic nanocarrier LION™ induce strong adaptive immunity with low systemic reactogenicity. However, the mechanisms linking localized intramuscular delivery to systemic T- and B-cell responses remain poorly understood.

Methods: We combined in vivo murine immunization models, engineered repRNA variants, flow cytometry, in situ hybridization, and in vitro muscle–APC–T cell co-culture systems to dissect interactions between transfected muscle cells, antigen-presenting cells (APCs), and lymphocytes.

Results: Intramuscular administration of repRNA/LION generated a muscle-resident immune niche. Transfected myocytes transferred RNA and protein antigens to infiltrating monocyte-derived dendritic cells, which cross-primed CD8⁺ T cells. Antibody induction required CD4⁺ T cells, which expanded locally and exhibited follicular helper–like features. CD138⁺ antibody-secreting cells accumulated in muscle, and their frequencies correlated with serum IgG titers.

Conclusion: Our findings reveal skeletal muscle as an active immunological hub rather than a passive site of antigen expression. Local antigen transfer and immune cell interactions orchestrate systemic T- and B-cell responses to repRNA/LION vaccination, providing mechanistic insights to guide safer and more durable RNA vaccine design.