UNLABELLED: ARNAX is a synthetic nucleotide-based Toll-like receptor 3 (TLR3) ligand that specifically stimulates the TLR3/TIR domain-containing adaptor molecule 1 (TICAM-1) pathway without activating inflammatory responses. ARNAX activates cellular immunity via cross-presentation
hence, its practical application has been demonstrated in cancer immunotherapy. Given the importance of cellular immunity in virus infections, ARNAX is expected to be a more effective vaccine adjuvant for virus infections than alum, an adjuvant approved for human use that mainly enhances humoral immunity. In the present study, the trimeric recombinant spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was prepared as a vaccine antigen and formulated with ARNAX. When T-cell and neutralizing antibody responses were evaluated in immunized mice, antigen formulated with ARNAX generated significantly larger numbers of antigenspecific CD4 IMPORTANCE: Cellular immunity is a critical immunological defense system against virus infections. However, aluminum salts, the most widely used adjuvant for vaccines for human use, do not promote strong cellular immunity. To prepare for the next pandemic of viral origin, the development of Th1-type adjuvants with low adverse reactions that induce cellular immunity is necessary. ARNAX is a TLR3 agonist consisting of DNA-RNA hybrid nucleic acid, which is expected to be an adjuvant that induces cellular immunity. The present study using a coronavirus disease 2019 mouse model demonstrated that ARNAX potently induces cellular immunity in addition to humoral immunity with minimal induction of inflammatory cytokines. Therefore, ARNAX has the potential to be used as a potent and welltolerated adjuvant for vaccines against pandemic viruses emerging in the future.