Drawing from Karik ò et al.'s 2008 suggestion to explore DNA adjuvants with modified mRNA and the hypothesis that manufacturing residuals function as inherent vaccine components, this Review synthesizes decades of CpG DNA adjuvant research, cancer mRNA vaccine strategies, antigen-presenting cell (APC) biology, and intramuscular (i.m.) administration challenges to examine residual manufacturing CpG DNAs as inherent adjuvants for generating prophylactic CD8+ T cell immunity.  This analysis comprehensively details CpG DNA activation of both endosomal TLR9 and cytosolic cGAS-STING pathways, which drive the essential vacuolar and cytosolic cross-presentation  routes required for effective CD8+ T cell priming. Through lipid nanoparticle (LNP) delivery,  manufacturing byproducts may benefit from the analogous co-localization effects as does the  vaccine mRNA itself, facilitating antigen-adjuvant co-signaling to amplify APC maturation following  i.m. vaccination.  Conversely, the pro-drug nature of i.m. mRNA vaccines presents a fundamental challenge  absent from conventional vaccine platforms: antigens produced in-situ within transfected cells  cannot be physically conjugated to adjuvants. CpG DNAs, effective triggers of IFN-Is, risk excessive signaling without the antigen. Deleterious IFN-I effects – previously attributed to “premature”  IFN-I signaling relative to TCR engagement or suppression of vaccine/antigen uptake in transfected/bystander dendritic cells (DC) – trigger T cell apoptosis, bystander DC suppression, and  reactogenicity. Because of the pro-drug concern, the dual aspects of interferons are intimately linked to magnitude-dependent effects: IFN-I signaling facilitates DC maturation, antigen cross-presentation, and  CD8+ T cell differentiation/expansion; excessive concentrations trigger deleterious sequelae,  thus creating an extraordinarily narrow “Goldilocks” window. This extreme dose sensitivity renders low-level manufacturing CpG DNAs uniquely feasible for prophylactic applications: unlike high-dose adjuvants requiring physical antigen linkage, LNP-delivered  residuals may achieve functional co-localization through natural spatiotemporal proximity. Independent studies on unrelated platforms confirm CpG DNA adjuvanticity at concentrations substantially  lower than conventional assumptions. Conversely, when in higher concentrations, synergizing with  other interferon-provoking agents, or in a pre-existing elevated interferon milieu, residual CpG  DNAs could impair vaccine immunity or engender adverse clinical sequelae. In sum, the inherent adjuvant paradigm can help resolve conflicting IFN-I literature through  context-dependent magnitude effects that can be critically impacted by residual CpG-containing  DNAs. Testable predictions establish a foundational framework for mRNA vaccine immunogenicity  analysis.

A 19-ntd genome portion that encompasses the Furin cleavage site (FCS) of SARS-CoV-2 is 100% identical to a reverse complement proprietary sequence of a human DNA mismatch repair gene. Even though this sequence overlap is usually interpreted as implying a laboratory event, an independent analysis, separate from the origin question, allows a detailed expose of unappreciated mechanisms and relationships. Once these are known, they automatically engender sensitive information that, in an adversarial context, may result in their unknown malicious exploitation. Therefore, this article scrutinizes the feasibility of related scenarios, even if entirely based on a theoretical analysis and completely unrelated to any potential past events. Instead, it describes unappreciated aspects of RNA viruses and their nuclear role, including their hijacking of DNA damage response (DDR) processes, expected characteristics of escape mutants against specific antivirals or other types of lab-imposed pressure, and how these are conducive to CoVs. The article also highlights novel biorisks of viruses employed in cancer research more generally, including regulatory RNAs employed in knockdown experiments, which may foster unaccounted or covert viral evolution. Exposed to certain drugs, such as DDR antagonists, or in the context of cell culture experiments involving large libraries of small interfering RNAs (siRNAs) aimed at targeting critical host genes during viral infection, viral escape mutants can be endowed with specific advantages that may be even superior to those seen in natural CoV evolution. Aspects of the postulated framework are congruent with experiments described for an influenza virus, which implicated analogous MMR genes as necessary for viral clearance. Motivated by the above sequence overlap, the analysis of feasible mechanistic underpinnings exposes novel gaps in biorisk policies, even those unaccounted for by the recently developed potent "Sequences of Concern" paradigm. Key concerning attributes, including genome multi-functionality, such as the NLS/FCS overlap in SARS-CoV-2, antisense sequences, and their combination, are further described in more general terms. The article concludes with recommendations based on feasible mechanistic underpinnings of recombination events inspired by the seeming sequence overlap and those gleaned from other disciplines, calling for a combination of technical advances with traditional or even ancient core values.

The potential of unmethylated CpG DNAs to amplify vaccine immunity has been actively pursued for several platforms. Human applications have been constrained by differences between species and concerns over adverse effects, including the risk of autoimmune induction. Their reported advantages encompass enhancing antibody-mediated and Th1-biased CD4+ T helper cell responses, as well as stimulating cytotoxic CD8+ T cell immunity. In a vero cell-derived EV71 inactivated vaccine, DNA byproducts from manufacturing processes, present in residual amounts in accordance with official guidelines, were sufficient to act as potent CpG adjuvants. The analogous situation for currently licensed mRNA vaccines has not been investigated.

One of the many novel features of the COVID-19 mRNA injections is their inherent self-adjuvanticity. It is believed that the adjuvant activity of the lipid nanoparticles (LNPs) containing ionizable or cationic lipids can explain their superiority in the induction of adaptive immune responses. Several studies have provided evidence of various immunostimulatory effects via the mRNA-LNP complexes or their individual compounds. Specifically, a 2023 study in Nature Communications implicated the mRNA compounds acting as adjuvants for the cellular immunity of mRNA technologies. In addition, this study found these injections trigger some unexpected antigen processing pathways which remained unaccounted for by the authors. A recent rational analysis of these unexpected processes identified a significant potential for adverse implications with clinical and environmental ramifications. These concerns have not been described elsewhere. Very recently, a Nature Communication study revealed some results that are difficult to align with the modus operandi of these platforms and their inherent adjuvanticity. Several logical arguments are provided to identify the nature of these gaps and how they can be explained by the unaccounted immune activation processes identified earlier. In all, this study extends the previous theoretical considerations related to several underappreciated findings about the modus operandi of mRNA technologies and seamlessly aligns existing gaps and apparently paradoxical data from recent studies. Poorly understood aspects of antigen processing and adjuvanticity converge to cause unaccounted immune responses towards targeted and off-target (self)antigens and challenge specificity and type (immunogenic vs. tolerogenic) of mRNA vaccine immunity.