Immune sensing of DNA and strategies for fish DNA vaccine development
Abstract
The ambitious endeavor of developing highly effective and durable DNA vaccines fundamentally hinges upon a profound and comprehensive understanding of the intricate molecular and cellular mechanisms that underpin their immunological actions. This mechanistic insight is unequivocally recognized as the cornerstone for advancing vaccine design from empirical trial-and-error to a more rational, targeted, and predictable developmental paradigm. DNA vaccines, by virtue of delivering genetic material encoding target antigens directly into host cells, offer distinct advantages, including remarkable stability, ease of production, and the capacity to elicit both humoral and cellular immune responses, making them particularly attractive candidates for prophylactic and therapeutic applications across various species, including aquaculture.
Current scientific knowledge has elucidated several critical components involved in the immune response orchestrated by DNA vaccines, particularly following intramuscular administration, a common route for fish DNA vaccines. At the site of injection within the muscle tissue, a dynamic interplay involving antigen presenting cells (APCs) is initiated. These specialized immune cells, such as dendritic cells and macrophages, play a pivotal role in the uptake and processing of the expressed foreign antigen, subsequently presenting it to lymphocytes. This crucial antigen presentation event serves as the primary trigger for the activation and differentiation of both B and T cells, leading to the development of a robust and specific adaptive immune response. Concurrently with the initiation of adaptive immunity, a critical innate immune response is rapidly upregulated, characterized notably by the production of type I interferons (IFN-I). This family of potent cytokines is absolutely essential for orchestrating a rapid and effective antiviral response, acting as a crucial first line of defense against viral threats. Beyond their immediate antiviral effects, IFN-I also plays a profound role in shaping and benefiting the subsequent adaptive immune responses, acting as a crucial molecular bridge that primes and amplifies antigen-specific immunity in vaccinated fish.
The induction of type I interferons in response to DNA vaccination can be triggered through two primary, yet interconnected, pathways. Firstly, IFN-I production may be initiated directly by the *expressed antigen* itself. For instance, in the context of rhabdovirus G protein-encoded DNA vaccines, the intracellular expression and processing of this viral antigen can lead to the generation of pathogen-associated molecular patterns (PAMPs) that are recognized by host innate immune receptors, thereby stimulating IFN-I pathways. Secondly, and perhaps more broadly, IFN-I may be directly triggered by the *plasmid DNA itself*. This recognition occurs when the bacterial plasmid DNA, which differs structurally from host DNA, gains access to the cytosol of host cells. This intracellular localization allows the foreign DNA to be sensed as a danger signal through a sophisticated array of cytosolic DNA sensing mechanisms, thereby activating innate immune pathways that culminate in IFN-I production.
Recent investigative efforts have significantly deepened our understanding of these intricate DNA sensing mechanisms in fish. Specifically, the roles of Toll-like receptor 9 (TLR9) and Toll-like receptor 21 (TLR21) have been extensively characterized. These receptors are known for their ability to recognize unmethylated CpG motifs, which are abundant in bacterial and viral DNA but rare in eukaryotic DNA, serving as critical pathogen recognition receptors. While TLR9 is a well-established CpG-motif sensor in many vertebrate species, including various fish, TLR21 has emerged as a particularly prominent and functional CpG sensor in numerous fish species, highlighting species-specific variations in immune recognition. Beyond the endosomal TLRs, the elucidation of key cytosolic DNA receptors has provided even more profound insights. The DExD/H-box helicase DDX41 has been identified as a crucial initial sensor of cytosolic DNA. Upon binding to foreign DNA, DDX41 undergoes conformational changes and initiates a signaling cascade that ultimately converges on the stimulator of interferon genes (STING). STING, upon activation, acts as a pivotal adaptor protein that recruits and activates TANK-binding kinase 1 (TBK1). The activation of TBK1 subsequently leads to the phosphorylation and activation of interferon regulatory factors, primarily IRF3 and IRF7. These activated transcription factors then translocate to the nucleus, where they bind to specific promoter regions, initiating the transcription of genes encoding type I interferons and other antiviral effectors. This intricate STING-TBK1-IRF signaling axis thus represents a central and highly conserved pathway for robust IFN-I production in response to cytosolic DNA.
This review article synthesizes and describes these recent and significant findings regarding the critical receptors and downstream signaling pathways involved in cytosolic DNA sensing, particularly emphasizing the detailed elucidation of the STING-TBK1-IRF axis. By systematically consolidating this cutting-edge knowledge, the article aims to underscore the profound implications of these discoveries for the future development of DNA vaccines. A comprehensive understanding of these innate immune activation pathways opens up unprecedented opportunities for rational vaccine design. This includes strategies for optimizing plasmid DNA constructs to enhance their recognition by specific cytosolic sensors, engineering more potent DNA vaccine adjuvants that specifically engage these pathways, and ultimately, designing highly effective DNA vaccines tailored to elicit robust and protective immune responses against a wide array of pathogens, thereby translating fundamental immunological insights into practical advancements in vaccine technology.
Keywords: Cytosolic DNA sensing; DDX41; DNA vaccine adjuvant; Fish DNA vaccine; STING signaling; TLR21; TLR9; Type I interferon.
Introduction
The field of fish aquaculture has significantly benefited from advancements in prophylactic strategies, particularly in the development of vaccines against prevalent viral and bacterial pathogens that inflict substantial economic losses. Previous investigations have extensively assessed the efficacy of experimental DNA vaccines in fish against a range of these diseases. While traditional vaccine approaches, such as inactivated viral vaccines, have demonstrated some utility, they often necessitate the administration of relatively high doses to achieve sufficient protective immunity. Such high-dose regimens can quickly become economically prohibitive, rendering them impractical for widespread application in large-scale commercial fish farming operations. In contrast, inactivated bacterial vaccines have shown a degree of success in controlling certain bacterial infections. However, a significant limitation of these inactivated preparations is their inability to effectively trigger antigen-specific cellular immunity. This deficiency is particularly problematic for controlling intracellular bacterial pathogens, as cell-mediated immune responses, involving cytotoxic T lymphocytes, are crucial for identifying and eliminating infected host cells, thereby preventing the persistent survival and proliferation of these intracellular invaders.
Among the various vaccine platforms, live attenuated vaccines are widely recognized as the most effective, as they closely mimic natural infection, thereby eliciting a comprehensive and robust immune response that integrates both humoral (antibody-mediated) and cell-mediated immunity. This broad spectrum of immunity provides superior and often longer-lasting protection compared to inactivated or subunit vaccines. Despite their demonstrated efficacy, the widespread adoption of live attenuated vaccines in the aquatic environment for fish farming has been severely constrained by inherent safety concerns. The risk of reversion to virulence, shedding of attenuated organisms into the aquatic ecosystem, and potential transmission to non-target species presents significant ecological and health hazards. Consequently, only a limited number of attenuated vaccines are currently approved and utilized within the fish farming industry. Notable examples include vaccines targeting *Edwardsiella ictaluri* and *Flavobacterium columnare* in channel catfish (*Ictalurus punctatus*), and a viral vaccine against koi herpesvirus in common carp (*Cyprinus carpio*).
In this challenging landscape, DNA vaccines have emerged as a highly promising and transformative alternative. These innovative vaccines possess the unique capacity to immunize fish by delivering plasmid DNA that encodes the desired antigen directly into host cells. Once transcribed and translated within the host, the expressed antigen then elicits both humoral and cell-mediated immune responses, mirroring the comprehensive protection afforded by live attenuated vaccines but circumventing the inherent safety risks associated with live organisms. The numerous advantages associated with DNA vaccines make them particularly appealing. These include their remarkable versatility, allowing for rapid adaptation to new pathogens; an excellent safety profile due to the non-replicating nature of the plasmid DNA; relative ease of production, as they can be manufactured through well-established molecular biology techniques; and a significantly lower cost compared to many traditional vaccine formulations, making them more economically viable for aquaculture. Furthermore, DNA vaccines provide an invaluable research platform for studying microorganisms that are notoriously difficult to culture *in vitro* or those that are highly pathogenic, by enhancing the accessibility of their genetic information for vaccine development.
The practical success of DNA vaccines in fish aquaculture is exemplified by the first licensed DNA vaccine, which was developed to combat Infectious Hematopoietic Necrosis Virus (IHNV). This highly virulent rhabdovirus caused a severe and devastating epidemic in Atlantic salmon (*Salmo salar*) farming operations in British Columbia between 2001 and 2003, inflicting immense economic losses. However, the widespread and strategic deployment of the IHNV DNA vaccine proved highly effective, leading to the successful elimination of the disease in the affected regions. More recently, another significant milestone in fish DNA vaccine technology was achieved with the approval of Clynav (Elanco), a DNA vaccine designed to protect Atlantic salmon farming. This vaccine garnered considerable attention after its authorization for use in the European Union by the European Medicines Agency. In 2018, Clynav received further approval for use against Salmon Pancreas Disease Virus (SPDV) in Norway, marking its widespread clinical application. Clynav is ingeniously designed as a DNA plasmid, specifically named PUK-SPDV-poly2#1, which ingeniously encodes multiple proteins from salmon alphavirus (SAV3), enabling a broad-spectrum protective response. The company responsible for Clynav’s development has asserted its capacity to significantly increase the survival rates of Atlantic salmon and effectively mitigate the clinical syndromes caused by SAV3 infection in farmed fish.
These successful experimental trials and commercial authorizations unequivocally underscore that fish DNA vaccine technology is currently undergoing a period of vigorous and dynamic development. A primary research objective in this burgeoning field is to engineer DNA vaccines that can elicit truly comprehensive and robust immunity. Achieving this level of protection is critically dependent on a deeper understanding of the sophisticated cellular machinery involved in cytosolic DNA sensing and the intricate pathways that lead to DNA vaccine-triggered immune responses. Advancing our knowledge in this specific area is considered pivotal for substantially enhancing the immunogenicity and overall efficacy of future DNA vaccine formulations. Recent groundbreaking studies have illuminated the profound capacity of DNA vaccines to promote type I interferon (IFN-I) mediated immunity. This is achieved through the rapid initiation of an early innate immune response, which, in turn, plays a critical and multifaceted role in orchestrating immune cell functioning to effectively modulate and amplify the subsequent adaptive immune response in fish. Researchers have further demonstrated that the IFN-I response can be triggered either directly by the expressed antigen proteins encoded by the vaccine plasmid or, importantly, by the direct sensing of the plasmid DNA itself within the cytosol of host cells. These fundamental insights into how innate immune pathways are activated are expected to play an essential role in guiding future vaccine development efforts.
Against this backdrop, the present review article aims to systematically consolidate and present the most recent findings in this rapidly evolving field. We will first provide a concise overview of the current landscape of fish DNA vaccine development, highlighting key achievements and ongoing challenges. Subsequently, a significant portion of this review will focus specifically on studies that have meticulously assessed the mechanisms by which DNA vaccines trigger the robust type I interferon (IFN-I) response. This will include an in-depth discussion of research efforts aimed at identifying the specific host sensing receptors responsible for recognizing vaccine DNA and initiating these critical innate immune cascades. Finally, we will conclude by discussing the profound implications of these current findings, elaborating on how a deeper understanding of cytosolic DNA sensing and IFN-I induction can be strategically leveraged to inform and guide the rational design and development of next-generation DNA vaccines, ultimately leading to improved disease control and enhanced sustainability in aquaculture.
2. Immune Response Initiated by DNA Vaccination
A traditional DNA vaccine is fundamentally comprised of a meticulously engineered plasmid DNA, typically derived from bacterial sources. This plasmid is designed to harbor a gene that encodes a specific antigen protein, the target against which an immune response is desired. The expression of this antigen gene within host cells is precisely controlled by an active promoter sequence, ensuring efficient transcription, and it also includes a terminator sequence that facilitates the proper termination of messenger RNA (mRNA) transcription. Once this recombinant plasmid DNA is delivered into the host organism, it enters target cells, where the genetic machinery of the cell is harnessed to transcribe the plasmid-encoded gene into mRNA, which is subsequently translated into the foreign antigen protein. It is the intracellular expression of this antigen protein that then triggers the desired and specific immune response.
In mammalian systems, the intricate immune response to DNA vaccination is primarily modulated by the crucial action of antigen presenting cells (APCs). These specialized immune cells, upon taking up the antigen-encoding plasmid, process the expressed polypeptides. This processing typically occurs through two main pathways: the proteasomal pathway, which processes endogenous proteins and presents them on major histocompatibility complex class I molecules (MHC-I), and the endosomal pathway, which processes exogenous proteins for presentation on major histocompatibility complex class II molecules (MHC-II). Once processed, these antigen fragments are presented by MHC-I molecules to naive CD8 T cells, leading to the generation of cytotoxic T lymphocytes that can directly kill infected cells. Simultaneously, antigens presented by MHC-II molecules engage naive CD4 T cells, which then differentiate into helper T cells, providing essential support for both cellular and humoral (antibody-mediated) immune responses.
In addition to this direct uptake and presentation by professional APCs, the phenomenon of “cross-presentation” of antigen polypeptides is also a significant contributor to the immune response. This occurs when antigen-encoding plasmids are primarily transfected into stromal cells at the injection site, such as muscle cells, which are not professional APCs. These muscle cells then express the antigen. Alternatively, antigens can be released from apoptotic or necrotic transfected stromal cells, and subsequently taken up by professional APCs in the vicinity. These APCs can then “cross-present” these exogenously acquired antigens on their MHC-I molecules to activate CD8 T cells, further diversifying and amplifying the cellular immune response. Beyond antigen-specific responses, the bacterial plasmid DNA itself inherently triggers “built-in” adjuvant effects. These intrinsic immunostimulatory properties are largely derived from the presence of unmethylated CpG-motifs and double-stranded DNA structures within the bacterial plasmid backbone. These pathogen-associated molecular patterns (PAMPs) are recognized by specific host pattern recognition receptors (PRRs). In mammals, the Toll-like receptor 9 (TLR9), an endosomal receptor, is a primary sensor for unmethylated CpG-motifs. Upon activation, TLR9 can direct a potent T helper 1 (Th1) immune response, characterized by the production of pro-inflammatory cytokines that favor cellular immunity. Furthermore, the presence of double-stranded DNA in the cytosol is detected by various cytosolic DNA receptors that mediate signaling through TANK-binding kinase 1 (TBK1). TBK1 plays a critical role in mediating the production of type I interferons (IFN-I), which significantly enhances the cytotoxicity of antigen-specific CD8 T cells, thereby bolstering the cell-mediated arm of immunity. Recent groundbreaking studies have elucidated that the stimulator of interferon genes (STING; also known by various aliases such as MITA, ERIS, TMEM173, or MPYS) is a crucial resident transmembrane protein located in the endoplasmic reticulum. STING functions as a pivotal DNA receptor adaptor, facilitating the formation of a multiprotein complex known as the STING-TBK1-IRF3 complex. The assembly and activation of this complex are central to the robust induction of type I interferons, serving as a critical upstream event in the antiviral innate immune response.
Regardless of whether the immune response is primarily triggered by direct antigen presentation or through cross-presentation by professional antigen presenting cells or muscle cells at the injection site, the outcome is a dual activation: both robust adaptive immunity specific to the target antigen and a potent induction of type I interferons. The intricate interplay and cooperative action of the DNA vaccine and the host’s cytosolic DNA sensing machinery collectively orchestrate this multifaceted immune response, as broadly conceptualized.
3. DNA Vaccine-Mediated IFN-I Response in Fish
Numerous studies have consistently reported that the inherent immunogenicity of DNA plasmids in fish can be largely attributed to their remarkable capacity to activate the crucial type I interferon (IFN-I) response. This fundamental observation underscores the intrinsic adjuvant properties of the DNA vaccine backbone. A seminal investigation into the DNA vaccine encoding the rhabdovirus glycoprotein G from Infectious Hematopoietic Necrosis Virus (IHNV) provided compelling evidence. This study demonstrated that the G protein-encoding plasmid was a potent inducer of IFN-I, triggering a rapid and non-specific antiviral response shortly after DNA immunization. This early and robust innate immune activation is a critical first step towards protection against viral pathogens. Subsequent research further elucidated the immune response profiles elicited by the injection of a G protein-encoding plasmid from Viral Haemorrhagic Septicaemia Rhabdovirus (VHSV) through comprehensive transcriptome analysis. Their data unequivocally indicated that the G protein actively promoted the widespread upregulation of an enormous number of genes. This included genes involved in the TNFα pathway, the IkappaB kinase/NF-kappaB cascade, and most notably, a significant induction of the IFN-I response, as evidenced by the upregulation of *Ifn1* and *Ifn2* genes, along with a multitude of other immune response activating genes.
Interestingly, even the empty plasmid (pMCV1.4), devoid of a specific antigen gene, was observed to induce a slight yet discernible IFN-I and antiviral response at an early time point following administration. This finding strongly suggested that the mere presence of the backbone plasmid itself possesses inherent immunostimulatory properties, which could contribute to and potentially improve the overall immunogenicity of a DNA vaccine. A comparative study investigating DNA vaccines encoding the G and nucleocapsid (N) proteins from hirame rhabdovirus further illuminated these dynamics. It was shown that the G protein-encoding plasmid effectively induced the IFN-I system, initiating a powerful antiviral response early on, and thereby likely enhancing adaptive immunity against viral infection. In stark contrast, while the N protein plasmid also induced a set of immune-related genes, it notably lacked a significant IFN-I response and provided diminished protective effects compared to the G protein-encoding plasmid. This difference might be attributed to inhibitory mechanisms employed by certain viral proteins, as exemplified in IHNV, where the N protein is known to directly degrade MITA (also known as STING), a crucial component of the IFN-I signaling pathway, via the ubiquitin-proteasome pathway, thus subverting the host’s antiviral response.
Transcriptome analysis has also been instrumental in investigating the adjuvant properties of various backbone plasmids in fish. For instance, a comparison between the plain pcDNA3.3 backbone plasmid and a plasmid encoding the envelope glycoprotein, hemagglutinin esterase (pHE), of Infectious Salmon Anaemia Virus (ISAV) revealed significant differences. Genes corresponding to the IFN-I response, including interferon regulatory factor 3 (IRF3), IRF7, signal transducer and activator of transcription 1 (STAT1), and STAT2, were found to be expressed at considerably higher levels in cells containing the empty pcDNA3.3 plasmid than in those containing the HE-encoded plasmid. Subsequently, the inhibitory activity of the HE protein on interferon signaling was directly confirmed, which provided a plausible explanation for why the pHE DNA vaccine, when administered alone, is often not effective unless it is co-injected with an IFN-I-encoding plasmid.
In a different context, DNA vaccination utilizing a plasmid encoding the structural polyprotein C-E3-E2-6K-E1 (pCSP) of the Salmonid Alphavirus subtype 3 (SAV3) produced a robust antibody-mediated response against the viral E2 protein and significantly elevated levels of SAV3 neutralization activity in serum, ultimately providing enhanced protection against SAV3 infection. Transcriptome analysis of this vaccine indicated that the observed protective effects could be attributed to a synergistic immune response induced by both the pCSP-containing plasmid and the intrinsic adjuvant properties of the backbone plasmid (pcDNA3.3). Specifically, pCSP strongly induced the expression of the chemokine CXCL10 and the crucial pro-inflammatory cytokines IFN-γ, IL-1β, and TNFα. Further analysis of cell markers for B cells, T cells, and antigen presenting cells (APCs) at the muscle injection site strongly suggested that a potent chemoattraction of immune cells occurred as a direct result of the DNA vaccination. Moreover, these results particularly underscored that a significant portion of the IFN-I response originated from the backbone plasmid itself, highlighting its intrinsic potential to improve the overall immunogenicity of the pCSP DNA vaccine.
Taken collectively, these diverse research findings unequivocally illustrate that DNA vaccination in fish effectively triggers a multifaceted host immune response, fundamentally driven by the robust induction of type I interferons. Furthermore, they highlight an intriguing, albeit initially undefined, mechanism by which the backbone plasmid itself is sensed by the host immune system. Building upon these crucial observations, researchers subsequently embarked on studies to directly evaluate the functional significance of IFN-I by administering IFN-I encoding plasmids directly into fish. This direct delivery strategy successfully promoted a strong antiviral response and conferred significant protection against several formidable viral diseases. Transcriptome analysis of these direct IFN-I administrations further demonstrated that the IFN-I-encoding plasmid not only induced a potent antiviral response but also notably promoted the accumulation of various chemokines at the muscle injection site, including those related to mammalian chemokines such as CCL5, CCL8, CCL19, and CXCL10. This attraction of critical immune cells, whose functions closely parallel those observed in mammalian IFN-I responses, solidified the role of IFN-I as a key immunomodulator. Moreover, the profound effects of IFN-I as a DNA vaccine adjuvant have been definitively demonstrated in Atlantic salmon. The adjuvant effect of IFN-I was conclusively confirmed by its co-administration with the ISAV-derived hemagglutinin-esterase (HE) gene in salmon pre-smolts. This co-administration resulted in a remarkable enhancement of the antigen-specific antibody response observed 10 weeks post-immunization, coupled with a significantly stronger protective effect against ISAV infection. Immunization strategies augmented with the addition of the IFN-I plasmid also led to an enhanced transcription of crucial B cell markers, including IgM and IgT, as well as cytotoxic T cell-related genes, such as CD8α, perforin, and granzyme A. These compelling findings strongly suggested that the elevated levels of IFN-I generated at the muscle injection site actively attracted and subsequently activated both B and T cells, thereby orchestrating a more comprehensive and protective adaptive immune response.
While the critical role of the externally induced IFN-I response in DNA vaccination has been extensively explored, a more complete and nuanced understanding of the underlying mechanisms through which the host’s DNA sensing machinery (representing the internal IFN-I response) functions is still being actively pursued and remains crucial for the further improvement of DNA vaccines. Recent pivotal work has therefore meticulously examined the intricate processes involved in the recognition of cellular DNA, encompassing the specific recognition of unmethylated CpG-motifs, the direct sensing of cytosolic double-stranded DNA (dsDNA), and the downstream signaling cascades that culminate in the robust IFN-I response. Continued advancements in elucidating these precise molecular pathways are expected to be transformative for the future of DNA vaccine development.
Overview of Toll-like Receptors (TLRs)
Before delving into a detailed description of the profound effects of DNA sensing in various fish tissues and their specialized immune cells, it is imperative to establish a foundational understanding of the fish immune system. It is noteworthy that fish possess immune organs and a diverse repertoire of leukocytes that share remarkable similarities, both structurally and functionally, with their mammalian counterparts. These include key antigen presenting cells (APCs) such as dendritic cells, macrophages, and neutrophils, as well as crucial lymphocyte populations like monocytes, thrombocytes, T cells, and B cells, all of which are fundamental components of a vertebrate immune defense.
In fish, the head kidney serves as the primary hematopoietic organ, analogous to mammalian bone marrow, responsible for the genesis and maturation of various immune cells. The thymus, on the other hand, is the central organ dedicated to the development and education of T lymphocytes, ensuring their proper functionality and self-tolerance. Interestingly, dendritic cell-like features, including the expression of MHC-II molecules and various Toll-like receptors (TLRs), have been identified in hematopoietic tissues of species such as rainbow trout. Furthermore, in Atlantic salmon-derived dendritic cell-like cells, it has been unequivocally demonstrated that the upregulation of type I interferons (IFN-I) can be robustly induced upon stimulation with specific TLR ligands, underscoring their role in initiating innate antiviral responses. Research has also clearly established that B-cell production, maturation, and the subsequent synthesis of immunoglobulins are predominantly localized within the head kidney. The spleen functions as a critical secondary lymphoid organ, serving as a hub for the development and residence of lymphocytes, macrophages, and plasma cells, playing a vital role in filtering blood-borne pathogens and mounting systemic immune responses.
A unique and intriguing aspect of the fish immune system is the presence of a distinct T-cell-rich organ located at the base of the gills, which is postulated to contain mucosal-associated immune tissues. This anatomical specialization highlights the importance of mucosal immunity in aquatic environments. Furthermore, similar to mammals, a distinct lineage of B cells has been identified in fish that are responsible for the production of the mucosal-specific antibody IgT/Z, emphasizing a specialized mucosal defense. T cells are also widely distributed in various mucosal-associated tissues, including the skin, gills, and intestines of fish, forming critical immune surveillance outposts at the primary interfaces with the aquatic environment. The comprehensive understanding of the presence, distribution, and functional characteristics of these immune cells and, crucially, the various cytosolic DNA receptors across diverse fish tissues, has provided invaluable information. This knowledge is indispensable for researchers to effectively evaluate whether vaccine efficacies can be substantially improved by strategically incorporating agonists that directly target these receptors or by overexpressing key signaling molecules as potent adjuvants within DNA vaccine formulations. Such targeted approaches aim to harness and amplify the intrinsic immunostimulatory properties of DNA vaccines.
TLRs are a crucial family of pattern recognition receptors (PRRs) that play a sentinel role in innate immunity. These proteins are characterized by their location, either embedded within the surface membranes of cells or localized within endosomal compartments. Structurally, TLRs are modular proteins, typically comprising an ectodomain, which is exposed to the extracellular environment or within the endosomal lumen. This ectodomain contains a distinctive leucine-rich repeat (LRR) region, a motif rich in cysteine residues, followed by a transmembrane region that anchors the receptor to the membrane. Crucially, on the cytoplasmic side, TLRs possess a highly conserved Toll/interleukin-1 receptor (TIR) domain. The LRR domain is responsible for the specific interaction with various pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs), acting as the primary recognition interface. In contrast, the TIR domain is the intracellular signaling hub, mediating the recruitment of adaptor molecules and initiating downstream signaling cascades. The vast majority of TLRs transduce their signals via myeloid differentiation primary response protein 88 (MyD88)-dependent signaling mechanisms, leading to the activation of transcription factors like NF-κB and the production of pro-inflammatory cytokines. An important exception is TLR3, which is uniquely embedded in cell-surface membranes or within endosomes and signals primarily via the TIR-domain-containing adapter-inducing interferon-β (TRIF) protein. This TRIF-dependent pathway specifically activates TANK-binding kinase 1 (TBK1) and interferon regulatory factors (IRFs), ultimately leading to the robust production of type I interferons and NF-κB.
In mammalian systems, TLR9 is the canonical TLR sensor for DNA, specifically unmethylated CpG motifs, and its expression is predominantly, if not exclusively, observed in plasmacytoid dendritic cells (pDCs), specialized APCs known for their prodigious IFN-I production capacity. TLR9 activation is a complex process that necessitates the proteolytic cleavage of its ectodomain, which then enables the receptor to undergo dimerization, a conformational change absolutely required for the effective recognition and binding of CpG-DNA. Studies have compellingly demonstrated that this direct interaction between TLR9 and CpG-containing DNA necessitates the proteolytic cleavage of an undefined region within the ectodomain to induce crucial conformational changes in TLR9, facilitating its transformation into an activated dimer. Upon the activation of TLR9 within plasmacytoid dendritic cells, the ensuing signaling cascade is predominantly driven by MyD88-dependent pathways, involving the crucial transcription factor IRF7 and culminating in the robust expression of type I interferons, particularly IFN-α.
Recent genomic sequencing and annotation efforts in fish have revealed a remarkable expansion of the TLR family, with at least 22 distinct TLRs identified to date. While TLR1-3, TLR5, and TLR7-9 in fish exhibit functional similarities to their mammalian counterparts, a unique characteristic of aquatic animals is the presence of TLR13, TLR14, and TLR18–TLR28, which are conspicuously absent in mammals, reflecting species-specific evolutionary adaptations to diverse pathogen challenges. In fish, the functions of TLR7/TLR8 have been investigated, demonstrating their involvement in the activation of the downstream MyD88 signaling pathway after interacting with IRF3 and IRF7, as observed in Atlantic salmon and Japanese flounder (*Paralichthys olivaceus*). Regarding the TRIF-dependent pathway, TLR3 in fish has been shown to be activated via stimulation with double-stranded RNA (dsRNA) or its synthetic analog, polyI:C, followed by the recruitment of the TRIF protein, which subsequently activates NF-κB and leads to IFN expression in zebrafish (*Danio rerio*). Interestingly, TLR4 in fish exhibits a distinct compartmentalization compared to mammalian TLR4 protein, and has been implicated in negatively regulating NF-κB activity, highlighting a divergence in its immune function. Among the non-mammalian specific TLRs, the functions of TLR19 and TLR22 have been explored in species such as grass carp (*Ctenopharyngodon idella*) and fugu (*Takifugu rubripes*). In both these fish species, these TLR proteins were found to be capable of recognizing long dsRNA molecules, effectively inducing IFN production to prevent reoviral and birnaviral infections, respectively, underscoring their critical roles in antiviral immunity.
5. The Fish Cytosolic CpG-Motif Sensors TLR9 and TLR21
Over five decades ago, pioneering research unequivocally established that exogenously introduced cytosolic DNA possessed the remarkable capacity to stimulate potent immune activities in mammalian cells. This profound stimulatory effect was initially and most definitively demonstrated through the use of unmethylated CpG-motifs derived from *Bacillus Calmette–Guérin*, which were found to activate the host immune response primarily through endosomal Toll-like receptor signaling. These CpG-oligodeoxynucleotides (CpG-ODNs) are now well-recognized for their potent immune stimulating properties. They are naturally abundant in bacterial genomic DNA, which, unlike mammalian DNA, contains numerous unmethylated CpG dinucleotides. These specific motifs are critically associated with the immunity-mediated activation of cellular sensing receptors: TLR9 in mammals and its functional homolog, TLR21, in avian species such as chickens. A striking evolutionary divergence is observed here: mammals, while possessing TLR9, lack TLR21, whereas chickens, which have TLR21, do not possess TLR9. Intriguingly, both TLR9 and TLR21 can be found co-existing within various fish species, suggesting a broader and potentially redundant or complementary role in immune surveillance.
The fish TLR9 gene often exhibits structural variations, notably containing zero to four introns. Furthermore, the phenomenon of intron retention, through the alternative splicing of two specific introns within the TLR9 gene, can lead to the formation of a shorter isoform, TLR9B, in addition to the full-length TLR9A. This TLR9B isoform contains a truncated TIR domain, which is hypothesized to interfere with the crucial TIR-TIR dimerization necessary for downstream signaling, potentially resulting in an antagonistic effect on the immune response. This negative regulatory aspect may need careful consideration when contemplating the incorporation of CpG-motifs as vaccine adjuvants.
Alignment analysis of the ectodomain in fish TLR9 proteins has revealed that the “undefined regions” (also referred to as “Z-loops” in mammalian TLRs), which are situated between LRR14 and LRR15, are highly variable in fish, and significantly longer than those found in mammalian TLR9s. However, these regions bear structural similarities to TLR9 genes of phylogenetically related species. A similar phenomenon concerning Z-loop variation has been observed in mammalian TLR8 genes, though not in mammalian TLR9s, and these undefined regions have been similarly reported to be important for TIR dimerization and subsequent ligand activation. Therefore, the significant diversity observed in the undefined region of fish TLR9 suggests that its specific activities might exhibit considerable intrinsic variation among different fish species, impacting their immune recognition profiles. In contrast, the undefined regions have not been identified in fish TLR21 proteins, akin to their absence in the homologous chicken TLR21. Furthermore, the ectodomain regions, LRR14 and LRR15, of fish TLR21 are highly conserved, which strongly suggests that the fundamental biological function of TLR21s is largely conserved across different fish species, implying a more consistent immune recognition role.
Toll-like receptors play an unequivocally vital role for sentinel cells, functioning as a critical first line of defense in various tissues. They are responsible for the immediate triggering of a cascade of diverse signaling pathways within the innate immune response, primarily through the precise recognition of structurally-conserved, microbe-derived molecular patterns. In human tissues, TLR9 exhibits its highest expression levels in lymphoid organs such as the lymph node, spleen, tonsil, and appendix. Furthermore, TLR9 expression in response to microbes or CpG-ODNs primarily occurs in specialized immune cells, including B cells, T cells, NK cells, and professional APCs, all of which mediate robust cytokine production following immune stimulation, thereby orchestrating broader immune responses. The recognition profiles of fish TLR9 and TLR21, particularly regarding their CpG-ODNs-mediated functioning, have been extensively investigated across numerous fish species.
Studies in zebrafish have shown that both TLR9 (DrTLR9) and TLR21 (DrTLR21) are expressed during the early embryonic developmental stage (as early as 3 hours post-fertilization), indicating their fundamental role in innate immunity from very early life. In adult zebrafish, the expression levels of both DrTLRs were found to be higher in immune-rich organs such as the spleen, kidney, and intestine compared to other tissues. The deepening understanding of how CpG-motifs or pathogen-associated molecular patterns engage their corresponding receptors has directly led researchers to develop sophisticated vaccine strategies specifically tailored to elicit a particular type of immune response. Various approaches have been established to rigorously assess these immune responses, including cell-based NF-κB activation luciferase reporter assays. Using such assays, researchers were able to precisely determine that DrTLR9 broadly recognizes various CpG-ODN motifs, exhibiting a slightly enhanced capacity to recognize specific sequences such as GACGT (CpG-2000) and AACGTT (CpG-HC4040). Conversely, DrTLR21 showed a preferential recognition for GTCGTT (CpG-2006, CpG-2007) motifs, highlighting distinct ligand specificities between the two TLRs. Moreover, *in vivo* injection of CpG-ODNs in zebrafish induced a significant upregulation of a panel of cytokines, including IFN-1β, TNF-α, and IFN-γ, particularly in the kidney and intestine. This cytokine induction indicated a robust activation of the cellular arm of the innate immune system, which subsequently led to protective immunity against *Edwardsiella tarda* in CpG-ODN-immunized fish, demonstrating the therapeutic potential of targeting these pathways.
In salmonids, the TLR9 genes typically consist of three introns, as observed in Atlantic salmon (SsTLR9) and rainbow trout (*Oncorhynchus mykiss*). SsTLR9 transcripts are most highly detected in the spleen and ovary and can be induced via stimulation with IFN-γ and phosphorothioate (PS)-modified CpG-2006 ODNs in head kidney leukocytes. Interestingly, SsTLR9 has been shown to bind to PS-modified ODNs even in the absence of CpG-motifs, a finding similar to observations in mammals, where CpG-motifs are known to promote the conformational changes crucial for downstream signaling processes. More recently, SsTLR9 was revealed to be constitutively expressed at higher levels than SsTLR21 in salmon IgM+ B cells. Crucially, these IgM+ B cells demonstrated responsiveness to PS-modified CpG-2006 ODNs, with subsequent upregulation of both IgM and IFN-I. These results collectively suggest that PS-modified CpG-2006 ODNs could be strategically incorporated as vaccine adjuvants to significantly enhance B cell stimulation and antibody production in salmonids.
In Japanese flounder, a similar investigative approach was employed to study TLR9 and TLR21 (PoTLR9, PoTLR21). It was demonstrated that both PoTLR9 and PoTLR21 could be upregulated by GTCGTT (CpG-1668 ODNs) sequences, leading to the activation of MyD88 signaling and subsequent TNF-α expression. Intriguingly, upon *Edwardsiella tarda* infection, a rapid infiltration and assemblage of PoTLR9-expressing cells were observed at the surface of accreted secondary gill lamellae. This spatial and temporal pattern strongly indicated that PoTLR9 is intricately involved in the first line of defense within the mucosal-associated tissues of the gills, a critical site for pathogen entry. This suggests that understanding PoTLR9′s role in mucosal immunity could be pivotal for developing DNA vaccines that target mucosal surfaces.
Two distinct isoforms of TLR9 were identified in the orange-spotted grouper (*Epinephelus coioides*), designated EcTLR9A and EcTLR9B. This isoform diversity arises from the alternative RNA splicing of two introns, resulting in the production of EcTLR9A (the full-length functional form) and EcTLR9B (a shorter isoform with a truncated C′-terminal signal transducing domain). Both EcTLR9 isoforms were found to be responsive to inactivated *Vibrio vulnificus* and CpG-ODNs stimulation in grouper kidney cells. Furthermore, their subcellular localization for both EcTLR9A and EcTLR9B included the perinuclear endoplasmic reticulum (ER) region extending to the endosome. However, a critical observation was that EcTLR9B was unable to recruit downstream effector proteins such as IRAK-4 and TRAF6 after interacting with MyD88. This suggested that the truncated EcTLR9B might function as a negative regulator, potentially by competitively interfering with the signaling of the full-length EcTLR9A in the endosome. This potential negative regulatory effect is an important consideration when incorporating CpG-motifs as vaccine adjuvants, as it could influence the desired immune outcome.
A study focusing on juvenile Pacific red snapper (*Lutjanus peru*) TLR9 (LpTLR9) revealed that LpTLR9 was predominantly expressed in immune tissues, with the highest levels found in the intestine, followed by blood leukocytes, liver, skin, head kidney, and, least abundantly, in the gills. *Aeromonas veronii* infection of Pacific red snapper resulted in a significant increase in LpTLR9 expression in all analyzed organs except the liver. The most substantial increase in LpTLR9 was observed in the intestine 48 hours post-*A. veronii* infection, which may be a consequence of the secretion of bacterial lectin proteins known to bind with high affinity to M-like cells present in the intestinal epithelium, gills, and intestine of rainbow trout. These findings collectively indicated that fish TLR9 is actively involved in the modulation of mucosal immunity, particularly within the intestinal tract.
Similar results regarding TLR9 expression and its involvement in immune responses have also been observed in cobia (*Rachycentron canadum*) TLR9 (RcTLR9). The upregulation of RcTLR9 was demonstrated in the spleen, kidney, liver, and intestine following *Photobacterium damselae* subsp. *piscicida* infection. The immunostimulatory effect of CpG-ODNs in cobia was further evidenced by the injection of specific CpG-ODN sequences (CpG-ODN 1668, 2006, and 2395), which led to increased expression of RcTLR9, IL-1β, and chemokine CC in the spleen and liver. Notably, CpG-ODN 2006 and CpG-ODN 2395 specifically induced IgM and Mx expression, respectively, indicating distinct and robust immune activation profiles. In common carp TLR9 (CcTLR9), transfection of a CcTLR9-encoding plasmid successfully activated the IRF3, IRF7 signaling pathways, and subsequently induced the expression of antiviral proteins such as ISG15 and Mx1 in epithelioma papulose cyprini cells. This confirmed that the TLR9 signaling pathway in fish operates in a manner consistent with that observed in mammals. Similar observations concerning the functional roles of TLR9 and TLR21 have been consistently reported across a number of studies, collectively assessing the profound effects of CpG-motifs and various pathogenic stimulations on fish immune responses.
6. Update on Cytosolic DNA Sensor and the Fish DDX41
In addition to recognition by Toll-like receptors, DNA derived from intracellular bacterial and viral infections can escape the normal metabolic degradation pathways—such as degradation by DNase II in the lysosome or exonuclease TREX1 in the cytoplasm—and gain access to the cytosol. Once in the cytosol, this foreign DNA may then be sensed by a sophisticated network of cytosolic DNA receptors, triggering potent innate immune responses. The standard B-form double-stranded DNA is a classical immune stimulator, and its presence in the cytosol is associated with the activation of a distinct set of receptors. These include Z-DNA-binding protein 1, also known as DNA-dependent activator of IFN-regulatory factors (DAI), various DExD/H box helicases, and cyclic GMP-AMP synthase (cGAS). Furthermore, other cytosolic DNA receptors, including cGAS, Interferon Gamma Inducible Protein 16 (IFI16), DExH-Box Helicase 9 (DHX9), and DEAD-Box Helicase 41 (DDX41), have been shown to be abundantly expressed in the cytosol following recognition of various DNA forms, such as Z-form DNA, poly(dA:dT), poly(dC:dG), and viral DNA. Most of these cytosolic DNA receptors function upstream of the critical STING-dependent signaling pathway, ultimately leading to the production of type I interferons (IFN-I). These pathways are absolutely essential for the robust activation of dendritic cells and macrophages, key players in initiating adaptive immune responses. A recent significant finding in fish immunology is the identification of a fish-specific DNA sensor protein kinase containing Z-DNA binding domains (PKZ). This protein may play roles similar to DAI in mammals, particularly in its ability to induce apoptosis through phosphorylating eukaryotic initiation factor 2α kinase. PKZ has been shown to specifically recognize poly(dA:dT) and poly(dG:dC) but notably not poly I:C (a dsRNA analog), and interacts with and activates mediators within the IRF3-dependent or ISGF3-like dependent pathway, thereby initiating IFN-I expression.
The DExD/H-box helicase superfamily encompasses a vast array of proteins classified as DEAD, DEAH, DExH, and DExD based on their conserved amino acid motifs. These proteins include both DNA and RNA helicases and are characterized by a DExD/H-box domain that has been implicated in a wide range of crucial cellular processes, including the regulation of gene induction, the modulation of signal transduction, promoter regulation, mRNA splicing, and translational regulation. Within this superfamily, several DDX proteins, such as the RIG-I-like helicases (including RIG-I, MDA5, and LGP2), are well-known RNA sensors. Another DDX signaling molecule, DDX3, while primarily involved in RNA metabolism, has been shown to contribute to the DNA sensor DAI-dependent IFN response, highlighting its multifaceted roles. More recently, studies have focused on the cytosolic sensor, DDX41, particularly in myeloid dendritic cells. DDX41 has been demonstrated to interact directly with synthetic double-stranded DNA (dsDNA) and is an essential component for the activation of the DNA-dependent IFN-I response. Notably, DDX41 can also directly bind to bacterial cyclic dinucleotides (CDNs) such as cyclic-di-GMP, and the CDN-triggered IFN response has been determined to be DDX41-dependent in mammals. While DDX41 may not always be absolutely necessary to initiate the initial DNA or CDNs sensing event, it clearly plays a vital role as a signaling molecule in the STING-dependent DNA or CDNs responses, acting as a critical bridge.
In fish, while only a limited number of studies have specifically investigated the role of cellular DEAD-box helicases in DNA sensing, it is undeniable that fish possess similar features for robust cytosolic DNA recognition. In Japanese flounder DDX41 (PoDDX41), the protein was found to be ubiquitously expressed in all tested tissues, including the brain, eye, gills, heart, intestine, head kidney, trunk kidney, liver, muscle, skin, spleen, and stomach, indicating its widespread physiological importance. Furthermore, the expression of PoDDX41 was significantly induced by lymphocystis disease virus infection, highlighting its role in antiviral immunity. Phylogenetic analysis of the PoDDX41 gene revealed that its functional domains, including the DEAD box and helicase motifs, are highly similar to those found in human DDX41, suggesting functional conservation across species. The stimulatory effect of Ranavirus (a DNA virus) on flounder monocyte-like cells resulted in the significant upregulation of PoDDX41, Mx, IFN-I, IL-6, and IL-1β. Moreover, overexpression of PoDDX41 in a flounder embryo cell line led to a sensitization to cyclic-di-GMP stimulation, culminating in the upregulation of IFN-I, Mx, IL-6, and IL-1β. These results collectively demonstrated that PoDDX41 is actively involved in CDNs sensing and contributes to the IFN-I-mediated antiviral and inflammatory responses in Japanese flounder.
In zebrafish DDX41 (DrDDX41), an ortholog containing conserved functional motifs was identified, sharing a high sequence identity of 82–85% with its mammalian counterpart and 88–92% identity with PoDDX41 and other fish species. The DrDDX41 protein possesses crucial domains, including CC, DEADc, helicase superfamily c-terminal, and ZnF_C2HC domains, making it remarkably similar to human DDX41. A comparison of the crystal structure of the DEADc domain of these proteins further revealed that their C-terminal regions exhibited identical conformations (amino acid residues 224–390), underscoring structural conservation. Furthermore, several specific amino acid residues critical for dsDNA and CDNs recognition were precisely identified within the DEADc domain of DrDDX41. Transcripts of DrDDX41 were found to be widely expressed across all tested tissues in zebrafish, with the highest levels of expression observed in the brain and immune tissues, including the liver, gills, spleen, and skin. A functional study of DDX41, involving the stimulation of human HEK293T cells and zebrafish embryos with poly(dA:dT), yielded surprising and significant results. Reporter assays revealed that DrDDX41 is capable of initiating the STING-dependent activation of NF-κB and IFN signaling in both human and zebrafish cells, providing strong evidence for the functional conservation of DDX41 between fish and mammals. The study also tested the effect of knocking down the expression of DrDDX41 and the STING/STAT6 in zebrafish embryos, which led to diminished antibacterial activities and a reduction in embryo survival following *Aeromonas hydrophilia* or *Edwardsiella tarda* infection. These findings from both Japanese flounder and zebrafish unequivocally suggest that fish DDX41 actively participates in a wide array of physiological and immunological activities, mirroring the critical roles played by mammalian DDX41. A comprehensive summary of DNA receptors and their known ligands in both fish and mammals, along with their main upregulations in cytokine signaling, is provided in Table 1 for ease of reference and comparison.
7. The Finding of STING-Mediated Signaling in Fish
The stimulator of interferon genes (STING) protein functions as a crucial adaptor molecule for many, if not all, likely cytosolic DNA sensors. While STING itself does not directly interact with DNA, it plays an absolutely central and indispensable role in intracellular signaling cascades, ultimately leading to the initiation of robust innate immune responses. The pivotal role of STING has been extensively demonstrated in the mammalian immune response to various pathogens and, notably, in self-DNA-triggered autoimmunity, highlighting its involvement in both protective immunity and pathological inflammatory conditions. Structurally, the STING protein consists of a transmembrane domain, which is strategically positioned within its N-terminal region and is intimately associated with the endoplasmic reticulum (ER) membrane. In contrast, the carboxy-terminal region of its C-terminal tail extends into the cytosol, providing a critical scaffold for the recruitment of key signaling molecules such as IRF3 and for the activation of TBK1. This intricate assembly and subsequent phosphorylation of IRF3 culminates in its translocation to the nucleus, where it drives the transcription of essential type I interferon genes. While one line of research has revealed a direct interaction of STING with cyclic dinucleotides (CDNs), the precise mechanism for CDNs binding and subsequent TBK1 activation still remains an active area of exclusive investigation and detailed characterization.
The immunostimulatory effect of cyclic-di-GMP in human dendritic cells has been meticulously studied, revealing its capacity to significantly increase the expression of MHC-II molecules, enhance the production of T helper 1 (Th1) cytokines, and upregulate several chemokine receptors, including C–C chemokine receptor type 1, C–C chemokine receptor type 7, and C-X-C chemokine receptor type 4. Furthermore, *in vivo* studies involving the co-injection of cyclic-di-GMP and *Staphylococcus aureus* in a mouse model resulted in a robust upregulation of the antibody response and a significant reduction of *S. aureus* bacterial burden within infected tissues. The compelling findings from several studies strongly suggest that DDX41 functions as a primary receptor for cyclic-di-GMP. The subsequent formation of a complex involving DDX41 and CDNs is thought to be essential for the activation of the STING-dependent IFN signaling pathway, which then robustly interacts with the STING-TBK1-IRF3 axis, leading to the vigorous production of IFN-I and the subsequent initiation of a comprehensive innate immune response. In zebrafish, the STING protein has been definitively shown to be required for the herpes simplex virus 1-triggered type I interferon response. The identification of STING orthologs in zebrafish provides compelling evidence that fish and mammals share highly conserved signaling mechanisms involving the RIG-I-like receptor pathway culminating in robust IFN-I activation.
The orange-spotted grouper STING (EcSTING) has been characterized, demonstrating a significant sequence homology of 46%–80% with STING from large yellow croaker (*Larimichthys crocea*) and 46%–76% homology to other known STING amino acid sequences of various fish species, underscoring its evolutionary conservation. EcSTING possesses four transmembrane domains at its N-terminal region, consistent with its association with the ER, and notably contains a cyclic-di-GMP binding domain and a C-terminal tail domain within its C-terminal region, architectural features strikingly similar to mammalian STINGs. More importantly, two critical serine residues in human STING, which are essential for TBK1 and IRF3 activation, were also found to be perfectly conserved in EcSTING and other fish species, highlighting the functional importance of these specific sites. EcSTING transcripts were found in all analyzed organs and were particularly abundant in the gills, spleen, brain, skin, and liver, indicating its widespread role in fish immunity. Splenic EcSTING was significantly upregulated after challenge with Singapore grouper iridovirus (a large DNA virus), lipopolysaccharide, and poly I:C, demonstrating its responsiveness to diverse pathogen-associated molecular patterns. Furthermore, transfection of an EcSTING-encoded plasmid into splenic grouper cells induced the expression of key antiviral proteins such as ISG15, Mx1, and viperin, and crucially, protected these cells from iridovirus infection. This result strongly suggested that EcSTING functions via the IRF3/IRF7-dependent IFN-I response, consistent with the mammalian paradigm.
In another study focusing on Japanese flounder, the structure of STING (PoSTING) was shown to possess similar features to previously characterized STING proteins. PoSTING contained three transmembrane domains in its N-terminal region and a STING superfamily domain, including a cyclic-di-GMP binding domain, and a C-terminal tail, all consistent with its functional role. The amino acid sequence of PoSTING shared a high identity of 82.4% with STING from large yellow croaker and over 41% identity with other known fish STING orthologs, further emphasizing conservation. PoSTING was found in all analyzed organs, but was most abundantly accumulated in the spleen, a major lymphoid organ. Moreover, using infection models with *Edwardsiella tarda* and VHSV in Japanese flounder, it was revealed that VHSV specifically stimulated PoSTING and IFN-I expression. This compelling finding suggests that PoSTING signaling is similar to mammalian STING, which is predominantly involved in mounting a robust immune response to viral infection. In the Cyprinidae family, STING has been well-described in both grass carp and crucian carp (*Carassius auratus* L.). The amino acid sequences of STING proteins from these two species shared 100% identity, and consisted of an N-terminal transmembrane domain and a highly conserved STING superfamily domain within the C-terminal portion of the proteins. The crucian carp STING (CaSTING) was found to be capable of activating the IRF3/7-dependent IFN response and restricting GCRV and SVCV replication in epithelioma papulosum cyprini cells. CaSTING-mediated activation of the IFN response was further confirmed in an IFN promoter assay, where co-transfection of CaSTING, TBK1, and IRF3-encoding plasmids resulted in enhanced promoter activity, unequivocally demonstrating that the STING-TBK1-IRF3 axis is functionally conserved from fish to mammals. In grass carp STING (CiSTING), the distribution of CiSTING was widespread across all analyzed tissues and could be robustly upregulated by GCRV and poly I:C stimulation, but notably not by lipopolysaccharide, suggesting a specificity towards nucleic acid PAMPs. Interestingly, CiSTING could also be upregulated by peptidoglycan, which suggested that CiSTING-mediated TBK1–IRF3/IRF7 signaling may modulate both antiviral and antibacterial immune responses in grass carp, highlighting its broader role in innate immunity.
8. Adjuvants Targeting the DNA Sensing Pathways
Adjuvants are critical components in modern vaccine formulations, specifically designed to provide an additional immune stimulating effect. They play a pivotal role in enhancing the immunogenicity of vaccine preparations, thereby leading to a stronger, more comprehensive, and often more durable protective immune response. Functionally, adjuvants stimulate the maturation of antigen presenting cells (APCs) and facilitate the robust activation of both B- and T-cells, which are essential for developing adaptive immunity. Pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) have been broadly utilized in vaccine adjuvants for subunit and inactivated vaccines. These adjuvants interact with host pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and various cytosolic receptors, thereby facilitating the rapid activation of innate immune responses, which in turn prime and amplify the subsequent adaptive immune response.
In the context of DNA vaccines, the overall function of adjuvants typically involves either the direct incorporation of immunostimulatory molecules encoded within the plasmid DNA itself to intrinsically alter vector-directed innate immune activation, or the co-injection of external immune stimulators. Both strategies aim to significantly enhance signal transduction pathways, thereby improving the overall immune responses elicited by the DNA vaccine. Several studies have compellingly demonstrated the efficacy of adjuvants specifically designed to target DNA sensing pathways. For instance, a study assessing the use of a CpG-enriched plasmid for antigen expression in a cancer vaccine context resulted in the robust induction of protective and antigen-specific T cell responses specifically directed against melanoma cells, highlighting the utility of CpG motifs as intrinsic adjuvants. The direct incorporation of TLR adaptor molecules, such as TRIF or MyD88, alongside the influenza HA antigen, has also shown significant adjuvant effects in a mouse model. Their results indicated that MyD88 was the most potent inducer of NF-κB activation, while TRIF induced the strongest activation of the IFN-β promoter in a luciferase reporter assay conducted in HEK293 cells. Furthermore, antigen-specific cytokines, specifically IFN-γ and IL-4, were found to be increased after *in vitro* re-stimulation of splenocytes obtained from mice post-DNA vaccination, unequivocally indicating that MyD88 and TRIF can function as effective genetic adjuvants to DNA vaccines. Moreover, the co-expression of TRIF and the influenza HA antigen within a DNA vaccine significantly increased hemagglutinin inhibition titers, which ultimately conferred superior protection against influenza challenge. A similar strategic approach was taken to assess the use of the cytoplasmic DNA sensor DAI as a DNA vaccine adjuvant in conjunction with the model antigen ovalbumin. This study demonstrated that the co-expression of DAI led to a potent induction of the IFN-I response and a significant proliferation of antigen-specific CD8+ T cells in the mouse model used, further validating the strategy of targeting cytosolic DNA sensors.
In fish, similar successful strategies have been implemented. For example, the co-injection of a CpG-enriched plasmid (pCN5; containing CpG-ODN 205) alongside the *Vibrio harveyi* subunit vaccine has demonstrably exhibited powerful adjuvant effects. These studies reported the upregulation of MHC-IIα, IgM, Mx, and IL-8 receptors, coupled with significantly enhanced protection against bacterial challenge in turbot (*Scophthalmus maximus*). Interestingly, injecting fish solely with CpG-ODN 205 (the synthetic oligonucleotide) produced a minor immune response compared to fish injected with the pCN5 plasmid. This difference might be attributed to the fact that the backbone plasmid, by virtue of its larger and more stable structure, prolongs the half-life of the CpG-motifs by providing resistance to nuclease digestion, thereby allowing for sustained immune stimulation. Furthermore, the double-stranded DNA backbone itself may interact with other cytosolic DNA receptors and trigger STING-mediated signaling pathways, contributing to the enhanced adjuvant effect. Similar results were found when the plasmid DNA pcDNA3.1, specifically engineered to contain five repetitions of CpG-ODN and 1670A motifs, was co-administered with the inactivated GCRV vaccine in grass carp fingerlings. This CpG-enriched plasmid provided substantial adjuvant effects, leading to increased levels of IgM in serum and the spleen and head kidney, as well as the upregulation of TLR9 and Mx2 expression, which collectively contributed to inhibiting GCRV proliferation. In Japanese flounder, the co-administration of a DNA plasmid encoding DDX41 with the VHSV-G protein-encoding plasmid resulted in increased levels of IFN-I and IFN-γ, along with heightened expression of the antiviral genes ISG15 and Mx in the kidney and spleen, and upregulated pro-inflammatory cytokines IL-1β and IL-6 in the kidney. However, the flounder exhibited only slightly higher survival rates following VHSV infection compared to fish that received only the G protein plasmid. This somewhat muted additional benefit might be due to the fact that rhabdovirus G proteins are themselves known to be strong IFN-I stimulators, making it challenging to precisely distinguish the additive effects from those associated with the backbone plasmid or other inherent signaling processes. A similar approach involving the incorporation of the MyD88 genetic adjuvant within a DNA vaccine has been pursued in orange-spotted grouper, specifically targeting the accessory colonization factor (AcfA) against *Vibrio alginolyticus* infection. The co-injection of plasmids encoding MyD88 and AcfA led to a significant increase in antigen-specific antibody responses and the expression of MHC-Iα, MHC-IIα, CD4, CD8α, IL-1β, and TNF-α, which collectively enhanced the fish survival rate in challenge experiments.
Beyond targeting cytosolic DNA sensing pathways directly through CpG-motifs and genetic adjuvants, a novel and promising strategy for enhancing the immunogenicity of DNA vaccines involves directly triggering dendritic cell activation using biodegradable microspheres. In mammalian systems, it has been demonstrated that cationic polysaccharide chitosan (forming cationic polymer microspheres) is capable of stimulating the intracellular release of DNA, which then initiates cGAS-STING signaling and robustly promotes dendritic cell maturation via the IFN-I-dependent response. Another study highlighted the synergistic effects of a combination of chitosan and CpG molecules, which successfully promoted NLRP3-dependent antigen-specific Th1 and Th17 responses, indicative of a potent and diversified cellular immune activation. To date, MRT67307 fish vaccination research has increasingly focused on utilizing microspheres for immersion and oral immunization, encapsulating DNA or subunit vaccines, as these methods are considered the most convenient and scalable delivery approaches for aquaculture. Recent findings indicating the presence of antigen-sampling cells in the gill and intestinal epithelium of rainbow trout and zebrafish, which exhibit similar characteristics to Ulex europaeus agglutinin-1 binding M cells and express IL-1β, IL-12, CD83, and MHC-II, are reminiscent of professional APCs. Although the precise mechanism by which cationic polymer microspheres (e.g., chitosan, poly lactic-co-glycolic acid, alginate) trigger specific signaling processes has not yet been fully investigated in fish, a study of an oral chitosan-encapsulated DNA vaccine against nodavirus in European sea bass (*Dicentrarchus labrax*) was able to upregulate the expression of IFN-I, IFN-γ, IgM, MHC-I, and genes related to cell-mediated cytotoxicity (TCRβ and CD8α). Furthermore, a similar enhancement was observed in the control group receiving chitosan-encapsulated backbone plasmid, indicating that the chitosan microspheres might be taken up by APCs, subsequently initiating non-specific IFN-I mechanisms, thereby functioning as an adjuvant.
Overall, a deeper and more comprehensive understanding of adjuvants and their corresponding signaling pathways is critically important for the rational design and development of truly improved DNA vaccines. The identification of key cytosolic DNA sensors such as TLR9, TLR21, and the newly characterized DDX41, along with the detailed elucidation of STING signaling in fish, as comprehensively described in this review, provides researchers with powerful tools and a mechanistic basis to significantly enhance the efficacy of DNA vaccines. Moreover, continued studies into the intricate machinery involved in modulating APCs in the context of DNA vaccines through the use of suitable receptor agonists, such as CpG-motifs, various DNA molecules, and innovative potential biomaterials for IFN-I activation, are indispensable for guiding and accelerating future vaccine development efforts in aquaculture.