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Background: The FAST (food allergy-specific immunotherapy) project aims at developing safe and effective subcutaneous immunotherapy for fish allergy, using recombinant hypoallergenic carp parvalbumin, Cyp c 1. Objectives: Preclinical characterization and good manufacturing practice (GMP) production of mutant Cyp (mCyp) c 1. Methods:Escherichia coli-produced mCyp c 1 was purified using standard chromatographic techniques. Physicochemical properties were investigated by gel electrophoresis, size exclusion chromatography, circular dichroism spectroscopy, reverse-phase high-performance liquid chromatography and mass spectrometry. Allergenicity was assessed by ImmunoCAP inhibition and basophil histamine release assay, immunogenicity by immunization of laboratory animals and stimulation of patients' peripheral blood mononuclear cells (PBMCs). Reference molecules were purified wild-type Cyp c 1 (natural and/or recombinant). GMP-compliant alum-adsorbed mCyp c 1 was tested for acute toxicity in mice and rabbits and for repeated-dose toxicity in mice. Accelerated and real-time protocols were used to evaluate stability of mCyp c 1 as drug substance and drug product. Results: Purified mCyp c 1 behaves as a folded and stable molecule. Using sera of 26 double-blind placebo-controlled food-challenge-proven fish-allergic patients, reduction in allergenic activity ranged from 10- to 5,000-fold (1,000-fold on average), but with retained immunogenicity (immunization in mice/rabbits) and potency to stimulate human PBMCs. Toxicity studies revealed no toxic effects and real-time stability studies on the Al(OH) 3 -adsorbed drug product demonstrated at least 20 months of stability. Conclusion: The GMP drug product developed for treatment of fish allergy has the characteristics targeted for in FAST: i.e. hypoallergenicity with retained immunogenicity. These results have warranted first-in-man immunotherapy studies to evaluate the safety of this innovative vaccine.

The complete germline repertoires of the channel catfish, Ictalurus punctatus , T cell receptor (TR) loci, TRAD, TRB, and TRG were obtained by analyzing genomic data from PacBio sequencing. The catfish TRB locus spans 214 kb, and contains 112 TRBV genes, a single TRBD gene, 31 TRBJ genes and two TRBC genes. In contrast, the TRAD locus is very large, at 1,285 kb. It consists of four TRDD genes, one TRDJ gene followed by the exons for TRDC, 125 TRAJ genes and the exons encoding the TRAC. Downstream of the TRAC, are 140 TRADV genes, and all of them are in the opposite transcriptional orientation. The catfish TRGC locus spans 151 kb and consists of four diverse V-J-C cassettes. Altogether, this locus contains 15 TRGV genes and 10 TRGJ genes. To place our data into context, we also analyzed the zebrafish TR germline gene repertoires. Overall, our findings demonstrated that catfish possesses a more restricted repertoire compared to the zebrafish. For example, the 140 TRADV genes in catfish form eight subgroups based on members sharing 75% nucleotide identity. However, the 149 TRAD genes in zebrafish form 53 subgroups. This difference in subgroup numbers between catfish and zebrafish is best explained by expansions of catfish TRADV subgroups, which likely occurred through multiple, relatively recent gene duplications. Similarly, 112 catfish TRBV genes form 30 subgroups, while the 51 zebrafish TRBV genes are placed into 36 subgroups. Notably, several catfish and zebrafish TRB subgroups share ancestor nodes. In addition, the complete catfish TR gene annotation was used to compile a TR gene segment database, which was applied in clonotype analysis of an available gynogenetic channel catfish transcriptome. Combined, the TR annotation and clonotype analysis suggested that the expressed TRA, TRB, and TRD repertoires were generated by different mechanisms. The diversity of the TRB repertoire depends on the number of TRBV subgroups and TRBJ genes, while TRA diversity relies on the many different TRAJ genes, which appear to be only minimally trimmed. In contrast, TRD diversity relies on nucleotide additions and the utilization of up to four TRDD segments.

While robust interferon (IFN) responses in fish are critical for viral clearance, dysregulated signalling can trigger detrimental hyperinflammation, necessitating precise immunoregulatory mechanisms. This study identified Toll-interacting protein (TOLLIP) as a pivotal negative regulator of IFN production in grass carp (Ctenopharyngodon idella) . Upon grass carp reovirus (GCRV) infection, TOLLIP expression increases significantly in tissues and cells. Furthermore, TOLLIP overexpression reduced GCRV- and polyinosinic-polycytidylic acid (poly I:C)-induced IFN expression, whereas tollip knockdown increased the cellular IFN production capacity. TOLLIP subsequently binds and degrades STING. Further mechanistic studies revealed that TOLLIP degrades STING in a dose-dependent manner via an autophagy-lysosome-dependent pathway. Interestingly, autophagy-related protein 5 (ATG5) was found to interact with TOLLIP and reduce TOLLIP-mediated STING degradation after atg5 knockdown. In addition, TOLLIP attenuated STING-driven IFN activation and compromised antiviral efficacy. These findings demonstrate that fish TOLLIP plays a specialized regulatory role in antiviral innate immunity, balancing immune defence with homeostasis maintenance.

Polyinosinic:polycytidylic acid (poly(I:C)) is a ligand of toll-like receptor (TLR) 3 that has been used as an immunostimulant in humans and mice against viral diseases based on its ability to enhance innate and adapt immunity. Antiviral effect of poly(I:C) has also been observed in teleost, however, the underling mechanism is not clear. In this study, we investigated the potential and signaling mechanism of poly(I:C) as an antiviral agent in a model of Japanese flounder (Paralichthys olivaceus) infected with megalocytivirus. We found that poly(I:C) exhibited strong antiviral activity and enhanced activation of head kidney macrophages and peripheral blood leukocytes. In vivo studies showed that (i) TLR3 as well as MDA5 knockdown reduced poly(I:C)-mediated immune response and antiviral activity to significant extents; (ii) when Myd88 was overexpressed in flounder, poly(I:C)-mediated antiviral activity was significantly decreased; (iii) when Myd88 was inactivated, the antiviral effect of poly(I:C) was significantly increased. Cellular study showed that (i) the NF-κB activity induced by poly(I:C) was upregulated in Myd88-overexpressing cells and unaffected in Myd88-inactivated cells; (ii) Myd88 overexpression inhibited and upregulated the expression of poly(I:C)-induced antiviral genes and inflammatory genes respectively; (iii) Myd88 inactivation enhanced the expression of the antiviral genes induced by poly(I:C). Taken together, these results indicate that poly(I:C) is an immunostimulant with antiviral potential, and that the immune response of poly(I:C) requires TLR3 and MDA5 and is negatively regulated by Myd88 in a manner not involving NK-κB. These results provide insights to the working mechanism of poly(I:C), TLR3, and Myd88 in fish.

Teleost fish possess a highly diverse innate immune system, which is well-adapted to the pathogen-rich aquatic environment in which they reside. NOD-like receptors (NLRs), a conserved family of cytosolic pattern recognition receptors, are at the center of this defense mechanism, activating immune responses, recognizing pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Here, we present an integrative overview of the current state of fish NLRs in terms of their evolutionary diversification, structural framework, signaling pathways, and functional roles in the context of bacterial, viral, and parasitic pathogens. We discuss six principal NLRs: nucleotide-binding oligomerization domain-containing protein 1 (NOD1), NOD2, NLRC3, NLRC5, NLR family member X1 (NLRX1), and NLR family pyrin domain-containing 1 (NLRP1), highlighting their domain structures, 3D conformations, and downstream signal chains. We focused on the immune regulatory roles of NLR family acidic transactivation domain-containing (NLRA) and NLR family CARD domain-containing (NLRC) subfamily components, the formation of the NLRP1 inflammasome, and the new roles of mitochondrial-specific NLRs in antiviral immunity. We discuss future directions for NLRs as immunological targets in aquaculture, referencing known NLR-activating adjuvants, exploring their ligand specificity, and highlighting challenges like functional redundancy. Much of the insight into the fish NLRs in this review comes from their well-researched mammalian counterparts. NLR-based immune modulation represents the ability of these receptors to detect microbial or danger signals and regulate key signaling pathways, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein 1 (AP-1), interferon regulatory factors (IRFs), and inflammasome activation. These pathways help shape the immune response by negatively or positively altering cytokine production and improving antigen presentation. By bringing together what we know about NLR evolution, structure, and function, this review aims to support new ideas and research into how fish defend themselves from disease and how we might strengthen that defense through improved vaccine and adjuvant design.

Trim47, a TRIM C-VII subgroup protein characterized by a conserved SPRY domain, has been primarily studied for its ubiquitin-dependent roles in mammals. This study reports a paradigm-shifting finding in teleost immunology: grass carp Trim47 (gcTrim47) employs its SPRY domain to execute a novel, ubiquitin-independent antiviral pathway, selectively degrading GCRV-I nonstructural proteins NS38/NS80 via autophagy-mediated clearance. Unlike mammalian TRIMs, gcTrim47 antiviral activity is strictly dependent on its SPRY domain—devoid of RING/B-box domains critical for E3 ligase function—revealing an evolutionarily divergent mechanism where substrate-targeting specificity, not ubiquitination, drives viral replication factory (viral inclusion body, VIB) dismantling. Functional assays demonstrated that gcTrim47 overexpression in CIK cells reduced viral titers and suppressed VIB formation, with SPRY domain deletion ablating these effects. In vivo , a yeast surface-display platform presenting gcTrim47-PYD1 conferred 32.94% relative percent survival (RPS) against GCRV-II infection, the first reported use of a TRIM family protein as an antiviral immunogen in grass carp. This strategy mitigated splenic/kidney viral loads and alleviated histopathological damage, including tubular necrosis and inflammatory infiltration. The successful application of this mechanism into a yeast-based immunization strategy highlights its potential for developing novel antiviral biotherapeutics in aquaculture.

Background The function of Toll-like receptor 2 (TLR2) in host defense against pathogens, especially Mycobacterium tuberculosis (Mtb) is poorly understood. To investigate the role of TLR2 during mycobacterial infection, we analyzed the response of tlr2 zebrafish mutant larvae to infection with Mycobacterium marinum (Mm), a close relative to Mtb, as a model for tuberculosis. We measured infection phenotypes and transcriptome responses using RNA deep sequencing in mutant and control larvae. Results tlr2 mutant embryos at 2 dpf do not show differences in numbers of macrophages and neutrophils compared to control embryos. However, we found substantial changes in gene expression in these mutants, particularly in metabolic pathways, when compared with the heterozygote tlr2 +/− control. At 4 days after Mm infection, the total bacterial burden and the presence of extracellular bacteria were higher in tlr2 −/− larvae than in tlr2 +/− , or tlr2 +/+ larvae, whereas granuloma numbers were reduced, showing a function of Tlr2 in zebrafish host defense. RNAseq analysis of infected tlr2 −/− versus tlr2 +/− shows that the number of up-regulated and down-regulated genes in response to infection was greatly diminished in tlr2 mutants by at least 2 fold and 10 fold, respectively. Analysis of the transcriptome data and qPCR validation shows that Mm infection of tlr2 mutants leads to decreased mRNA levels of genes involved in inflammation and immune responses, including il1b , tnfb , cxcl11aa/ac , fosl1a , and cebpb . Furthermore, RNAseq analyses revealed that the expression of genes for Maf family transcription factors, vitamin D receptors, and Dicps proteins is altered in tlr2 mutants with or without infection. In addition, the data indicate a function of Tlr2 in the control of induction of cytokines and chemokines, such as the CXCR3-CXCL11 signaling axis. Conclusion The transcriptome and infection burden analyses show a function of Tlr2 as a protective factor against mycobacteria. Transcriptome analysis revealed tlr2 -specific pathways involved in Mm infection, which are related to responses to Mtb infection in human macrophages. Considering its dominant function in control of transcriptional processes that govern defense responses and metabolism, the TLR2 protein can be expected to be also of importance for other infectious diseases and interactions with the microbiome.

Stimulator of interferon genes (STING) is a key connector protein in interferon (IFN) signaling, crucial for IFN induction during the activation of antiviral innate immunity. In mammals, ring finger protein 5 (RNF5) functions as an E3 ubiquitin ligase, mediating STING regulation through K150 ubiquitylation to prevent excessive IFN production. However, the mechanisms underlying RNF5’s regulation of STING in teleost fish remain unknown. This study investigated the regulatory role of the mandarin fish ( Siniperca chuatsi ) RNF5 ( sc RNF5) in the STING-mediated antiviral immune response and identified the specific regulatory sites on sc STING. Furthermore, an examination of sc RNF5 expression patterns in virus-infected cells revealed its responsiveness to mandarin fish ranavirus (MRV) infection. The ectopic expression of sc RNF5 suppressed sc STING-mediated IFN signaling and facilitated MRV replication. Co-immunoprecipitation experiments indicated an interaction between sc RNF5 and sc STING. The further experiments demonstrated that sc RNF5 exerted its inhibitory effect by promoting the degradation of sc STING, which was observed to be blocked by MG132 treatment. Ubiquitination assays with various sc STING mutants showed that sc RNF5 catalyzed the ubiquitination of sc STING at K135 and K155 residues. Furthermore, we provided evidence that sc RNF5 significantly attenuated sc STING-dependent antiviral immunity by targeting negative regulators within the sc STING signaling cascade. This study underscored that RNF5 negatively regulated the STING-mediated IFN signaling pathway in mandarin fish, attenuated STING’s antiviral activity, and facilitated STING degradation via the ubiquitin-proteasome pathway at two novel lysine sites (K135 and K155). Our work offered valuable insights into the regulatory mechanisms of STING-mediated signaling in teleost fish, paving the way for further research.

remains a pervasive aquatic pathogen precipitating catastrophic economic depletion and threats to global food security. Conventional therapeutic modalities are constrained by inadequate stability, prohibitive costs, and biosafety risks. To address such challenges, an engineered strain was developed to constitutively express the outer membrane protein OmpAI through a tandem promoter system to ensure optimized antigen delivery. Anal intubation with engineered in stimulated compartmentalized mucosal immunity, evidenced by a six-fold elevation in hindgut IgT transcripts, significant infiltration of IgT B cells, and pIgR mediated transcytosis, synchronized with systemic IgM activation. The probiotic treament accelerated pathogen clearance, improved survival rates to 42.9% compared to 12.5% in control group, and reorganized the commensal microbiome through a specific enrichment of beneficial . This study elucidated a novel engineered-probiotic mucosal vaccination strategy for teleosts, providing a noninvasive, mucosal targeted immunoprophylactic strategy to decrease antibiotic dependency in aquaculture.

Zinc finger NFX1-type containing 1 (ZNFX1) has been established as a critical mediator of the antiviral response in mammals, functioning through dsRNA recognition and priority activation of the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling pathway. However, the role of its fish ortholog, particularly in relation to aquatic virus interactions, remains elusive. The absence of the RIG-I homolog, a key pattern recognition receptor, in multiple Actinopterygii may compromise their innate antiviral immune responses. Here, ZNFX1 in Epinephelus coioides (EcZNFX1) is identified as an evolutionarily primitive, interferon (IFN)-stimulated dsRNA sensor that compensates for the absence of RIG-I in Actinopterygii. EcZNFX1 is rapidly upregulated by orange-spotted grouper nervous necrosis virus (OGNNV) infection and restricts viral replication in grouper brain-derived (GB) and spleen-derived (GS) cells after binding to viral dsRNA intermediates via its conserved P-loop NTPase domain. Notably, EcZNFX1 exerts a dual immunoregulatory role in modulating virus-induced inflammatory responses in diverse cellular contexts. In GB cells that are highly susceptible to OGNNV and have attenuated regenerative capacity, EcZNFX1 suppresses IFN-I/ISGs production and pyroptosis mediated by viral infection, thereby limiting neurotoxicity by precise tuning of the antiviral response. Conversely, in GS cells with stronger resistance to OGNNV, EcZNFX1 directly interacts with TBK1 to promote its phosphorylation and subsequent nuclear translocation of IRF3, activating a robust IFN-I signaling. Overall, this study elucidates that ZNFX1 is a compensatory receptor for dsRNA sensing in RIG-I-deficient teleost, which exerts context-dependent antiviral effects in cell-type-specific microenvironments, providing mechanistic insights for aquatic virus countermeasures.