Double-stranded RNA (dsRNA) contamination generated during in vitro transcription (IVT) poses a significant challenge to the efficacy and safety of messenger RNA (mRNA) vaccines. This study systematically examined how varying dsRNA levels influence mRNA translation and immunogenicity using influenza hemagglutinin (HA) and human papillomavirus (HPV) E7 antigens as model systems, representing preventive and therapeutic vaccine targets, respectively. IVT-produced mRNA was synthesized using either uridine or N1-methyl-pseudouridine (m1Ψ) nucleosides, purified by cellulose chromatography, and supplemented with increasing synthetic dsRNA concentrations. In vivo experiments in mice revealed that high dsRNA concentrations markedly decreased antigen expression and increased pro-inflammatory cytokine production, regardless of nucleoside modification. Preventive HA mRNA vaccines containing elevated dsRNA levels showed decreased antibody titers and impaired T-cell responses. Conversely, therapeutic HPV E7 mRNA vaccines showed antigen-dependent variability; moderate dsRNA levels enhanced CD8T-cell responses, whereas higher concentrations were suppressive. Structural analysis of the RNA indicated that the HPV construct, with higher UTP content and reduced thermodynamic stability, generated more dsRNA during IVT, contributing to the observed immunostimulatory effects. Our findings indicate that dsRNA impurities act as a double-edged sword: low levels can function as beneficial immune adjuvants, whereas excessive amounts significantly hinder antigen expression and adaptive immunity. These results underscore the need for optimized dsRNA purification strategies tailored to specific antigens to balance immunogenicity and vaccine safety effectively. This study provides essential insights into refining mRNA vaccine production, highlighting the necessity of stringent control over dsRNA levels to maximize both preventive and therapeutic vaccine efficacy and minimize unintended inflammation. +
mRNA Technology