A novel double-stranded RNA adjuvant markedly amplifies the immunogenicity of nucleoside-modified mRNA-LNP vaccines 2256634
mRNA vaccines have transformed vaccinology in recent years, shortening timelines and enabling precise response to infectious diseases. One key modification that made this platform so successful is the replacement of uridine (U) with N1-methylpseudouridine (m¹ψ), which improved antigen expression and tolerability. The use of adjuvants for mRNA platforms represents a promising approach to increase immunogenicity of these vaccines, lowering the doses and offering more potent CD8+ T cell priming. However, successful adjuvant strategies remain largely undefined. Here, we developed a novel and structurally defined double-stranded RNA (dsRNA) and evaluated its immunogenic potential to induce immune responses against infectious diseases using an mRNA encoding influenza hemagglutinin (HA) as a model. C57BL/6 mice were immunized with 3 doses of HA m¹ψ mRNA-LNP (HA-LNP) alone or in co-administered with dsRNA-LNP using intramuscular-prime/ intravenous-boost administration. Peripheral blood mononuclear cells (PBMCs) were collected after each dose and stimulated ex vivo with HA peptides for intracellular cytokine staining (ICS) or ELISpot. Sera was collected 21 days after immunization for humoral analyses. Co-administration of HA-LNP and dsRNA-LNP induces more robust humoral responses with ∼3-fold higher specific IgG-antibody titers and increased neutralizing ability compared to HA-LNP alone. Evaluation of cellular arm showed that dsRNA-LNP potentializes the cellular immune responses with enhanced frequency of IFNγ production by CD4+ and CD8+ subsets, T cell activation (CD69+), CD8+ T cell polyfunctionality (IFNγ/TNFα), and higher cytotoxic profile (Granzyme B+/ Perforin+). Overall, the dsRNA-LNP acts as a potent adjuvant for nucleoside-modified mRNA vaccination, inducing a strong immune activation, representing a potent platform for eliciting balanced, durable antiviral immunity. This work was supported by the National Institute of Allergy and Infectious Disease (NIAID) and National Cancer Institute (NCI) of the US National Institutes of Health (NIH) under award numbers R01AI153064 (NP) and R01CA283736 (NP and CGR), respectively. Vaccines and Immunotherapy (VAC)