Residual DNAs as adjuvants in existing mRNA vaccines - a multidisciplinary analysis

mRNA vaccines have been celebrated for their safety and efficacy. Their rapid utilization wasenabled by improved purification processes and modifications to the mRNAs which minimizes recognition and excessive responses by innate immune sensors. It is believed that the required innate immune activation is realized by the inherent adjuvanticity of this platform, which has been linked to both components and residual byproducts such as dsRNAs, although prompting many questions even years after their global deployment.This article analyzes the Gutschi hypothesis that residual DNAs from manufacturing processes mayplay a decisive role in enhancing mRNA-LNP vaccine immunity. Interestingly, the co-administrationof DNA adjuvants has previously been suggested by Karik`o et al. to facilitate the use of chemicallymodified mRNA for vaccines. Specific DNAs consisting of unmethylated pairs of cytosine and guanineoligodeoxynucleotides (ODNs) have been extensively studied as vaccine adjuvants in more traditionalbut not mRNA vaccines.Towards gaining a deeper insight into the immunopotentiation activity of residual DNAs, this studyperforms a multi-disciplinary rational analysis, building on experiences and insights about CpG ODNsincluding their immune characteristics. It highlights difficulties in extending these approaches to prodrugs such as mRNA vaccines and the key prerequisite of antigen-adjuvant co-signaling. Related to residual DNAs from manufacturing, special emphasis is placed on their uptake, the possible pathways and cellular localizations, as facilitated by the vaccine LNPs, and how this supports the required cosignaling in antigen-presenting cells. A special focus is placed on regulatory guidelines to determine residual DNA fragments that were developed in unrelated technologies targeting genotoxicity concerns. Here, it is found that synergistic effects via mRNA vaccine components and contaminants can substantially increase immunopotentiation activities and result in extraordinary dose-sparing effects. Established experiments involving notably low CpG ODN dosages, combined with peculiar immune signatures found in some mRNA vaccine studies reminiscent of CpG adjuvant activities, but difficult to explain otherwise, this indicates the real potential of residual DNAs as under-appreciated mRNA vaccine adjuvants via TLR9 and cGAS-STING pathways. The potential adjuvanticity of residual DNAs has far-reaching implications, spanning foundational issues from the modus operandi of these platforms, clinical sequelae, to regulatory guidelines.