A systemic evaluation of the effect of dsRNA contamination on mRNA vaccine expression and immunogenicity

Nov 28, 2025Journal of controlled release : official journal of the Controlled Release Society

How double-stranded RNA contamination may affect mRNA vaccine production and immune response

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mRNA Technology on OpenScience ↗PubMed ↗DOI ↗

Abstract

High concentrations of double-stranded RNA (dsRNA) significantly decreased antigen expression and increased pro-inflammatory cytokine production in mice.

Varying levels of dsRNA during in vitro transcription influence mRNA translation and immune responses.
Preventive mRNA vaccines for influenza showed decreased antibody levels and T-cell responses with high dsRNA concentrations.
Therapeutic mRNA vaccines for HPV demonstrated that moderate dsRNA levels could enhance CD8 T-cell responses, while excessive amounts suppressed them.
Structural analysis revealed that the HPV construct generated more dsRNA due to higher UTP content and lower thermodynamic stability.
Low levels of dsRNA may act as beneficial immune adjuvants, but excessive amounts hinder antigen expression and adaptive immunity.
Optimized dsRNA purification strategies are necessary to balance immunogenicity and safety in mRNA vaccine production.

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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. +