Incorporation of 5-methylcytidine alleviates RIG-I–mediated innate immune responses to a self-amplifying RNA vaccine

To improve existing synthetic RNA-based vaccines, we previously developed a self-amplifying RNA (saRNA)–based vaccine expressing a membrane-anchored (TM) receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein (S-RBD-TM) and demonstrated that a low dose of this saRNA vaccine elicits robust immune responses. Moreover, a recent clinical trial with an saRNA vaccine incorporating 5-methylcytidine (5mC) (saRNA-5mC) has demonstrated reduced vaccine reactogenicity while maintaining robust humoral responses. In this study, we investigate the mechanisms by which 5mC incorporation attenuates adverse effects while maintaining immunogenicity. We found that incorporation of 5mC into the saRNA platform led to prolonged and robust expression of antigen and attenuated induction of type I interferon, a key driver of reactogenicity, specifically in plasmacytoid dendritic cells (pDCs). As a result, saRNA-5mC alleviated excessive innate immune responses in vivo without impairing B cell and T cell responses against the SARS-CoV-2 RBD. Mechanistically, we demonstrated that the detection of unmodified saRNA in pDCs was mediated by a host cytosolic RNA sensor, RIG-I, and this sensing was abolished with 5mC incorporation. In contrast, saRNA-5mC induced robust innate activation in professional antigen-presenting cells, such as macrophages, in a RIG-I–independent manner, highlighting distinct host sensing mechanisms for synthetic RNAs. Our study provides support for the potential clinical use of saRNA-5mC vaccine platforms.