Highly pure mRNA vaccine provides robust immunization against P. aeruginosa by minimizing type I interferon responses.

Developing effective vaccines against bacteria is critical given the growing threat of antimicrobial resistance (AMR). In this study, we developed mRNA vaccines targeting Pseudomonas aeruginosa (P. aeruginosa), a key AMR pathogen, using PureCap mRNA encapsulated in lipid nanoparticles (LNPs). The PureCap technology offers a facile method for removing immunostimulatory impurities from in vitro transcribed mRNA, such as uncapped RNA and double-stranded RNA (dsRNA). Following intramuscular vaccination of mice with mRNA encoding a model antigen, PureCap mRNA elicited antibody titers that were 26-fold higher than those induced by conventional ARCA-capped mRNA. Mechanistic analyses revealed that both uncapped RNA and dsRNA impurities in ARCA-capped mRNA were responsible for the reduced humoral immune responses. While PureCap mRNA enhanced protein expression efficiency and reduced pro-inflammatory responses compared to ARCA-capped mRNA, minimizing pro-inflammatory responses was particularly critical. When anti-interferon-α/β receptor antibodies were administered, antibody responses to ARCA-capped mRNA vaccination were restored to levels comparable to those achieved with PureCap mRNA vaccination, highlighting the negative impact of type I interferon responses on antibody responses following vaccination with ARCA-capped mRNA. In a vaccination targeting the PcrV protein of P. aeruginosa, PureCap mRNA, but not ARCA-capped mRNA, significantly prolonged the survival of mice following bacterial challenges, presumably due to enhanced antibody production. Furthermore, PureCap mRNA vaccination significantly reduced bacterial loads in the lungs and mitigated tissue damage, edema, and inflammatory responses. These findings underscore the potential of PureCap mRNA as a promising platform for bacterial vaccination, offering a valuable strategy to combat AMR.