CircRNA based bi-antigen vaccines against mpox virus induce potent and durable cross-protection in mice

The ongoing epidemic of mpox highlights the urgency of developing novel and effective vaccines against mpox virus (MPXV). Circular RNA (circRNA) has emerged as a promising novel vaccine platform due to its high stability, resistance to exonuclease-mediated degradation, and potential to support prolonged antigen expression. Based on our lipid nanoparticle (LNP)-encapsulated circRNA vaccine platform, we developed two novel bi-antigen circRNA vaccines cirEV and cirMV, which respectively encode the combination of MPXV extracellular enveloped virion (EEV) antigens (A35R and B6R) or intracellular mature virion (IMV) antigens (A29L and M1R). The two MPXV circRNA vaccines alone or mixed as a combination vaccine successfully induce high levels of antigen-specific antibody responses and cellular immune responses against the MPXV antigens and protect mice from lethal vaccinia virus (VACV) challenge in a dose-dependent manner. Furthermore, both circRNA vaccines can provide complete long-term cross-protection against lethal VACV challenge at day 260 after first immunization, indicating the durability of vaccine-induced protective immunity. Notably, vaccine-induced cellular immune responses play a crucial role in immune protection and virus clearance in mice. Our study provides critical insights to understand the protective mechanisms underlying circRNA vaccines in an orthopoxvirus surrogate model, demonstrating that bi-antigen circRNA vaccines represent a promising multivalent vaccine platform.