Young Rim Kim

LGP2 Expression is Enhanced by Interferon Regulatory Factor 3 in Olive Flounder, Paralichthys olivaceus

In innate immunity, LGP2 (laboratory of genetics and physiology 2) plays a very important role in the production of type I interferon (IFN) through recognition of cytosolic viral RNA. Although viral infection or stimulation with double-strand RNA dramatically induces expression of the LGP2 gene, the underlying transcriptional mechanism has never been studied. Here, we cloned and characterized the 5′-upstream region (−1,337 bp) of the LGP2 gene in olive flounder (Paralichthys olivaceus). Numerous canonical motifs for IFN-regulatory factors (IRFs) were found in this region, and reporter assays identified a poly I:C-responsive promoter region (−506 to −398) that regulated LGP2 transcription. Transcriptional activity of the LGP2 promoter was strongly enhanced by IRF3, which bound to IRF3 motif #3 (−480). The LGP2 promoter was also responsive to viral infection in vitro. These results suggest that LGP2 transcriptional control is crucially involved to regulated by IRF3 function after viral infection or stimulation with poly I:C.

Identification and determination of antigenic proteins of Korean ranavirus-1 (KRV-1) using MALDI-TOF/TOF MS analysis

Ranaviruses are serious pathogens of fish, amphibians, and reptiles, and pose a major threat to global biodiversity. A ranavirus isolated from tissues of diseased tadpoles and frogs in Gangwon province, Korea, in 2006 and 2007, was designated Korean ranavirus-1 (KRV-1) and was infectious in a variety of fish cell lines with highest titers (10(10)TCID(50)/ml) in Epithelioma papulosum cyprini cells (EPCs) and baby hamster kidney-21 (BHK-21) cells. Bullfrog (Rana catesbeiana) tadpoles challenged by immersion in 10(5)TCID(50)/ml of KRV-1 showed 60% mortality within 10 days. SDS-PAGE of frog virus 3 (FV3) and KRV-1 proteins yielded several bands 35-49kDa in size, which were identified as major capsid proteins (MCPs) by MALDI-TOF MS. Immunoblotting of FV3 proteins showed antigenic bands 34kDa and 93kDa in size which were identified by MALDI-TOF/TOF MS as MCP and neurofilament triplet H1-like protein (NF-H1), respectively. In KRV-1, antigenic bands at 32kDa, 69kDa, and 72kDa were identified as MCP, Hypothetical protein, and NF-H1, respectively. The genes encoding these KRV-1 proteins were sequenced. KRV-1 appeared to be closely related to the soft-shelled turtle iridovirus (STIV), based on alignments of amino acid sequences from various ranaviruses. Variability in ranavirus antigenic proteins was apparent in an earlier study. It is expected that use of the methods employed here, together with the results of the present work, will contribute to an understanding of the pathogenesis of ranaviruses, and will further the development of DNA- or protein-based bait vaccines for conservation of natural habitats.

Enhancement of glycoprotein-based DNA vaccine for viral hemorrhagic septicemia virus (VHSV) via addition of the molecular adjuvant, DDX41

ABSTRACT The use of molecular adjuvants to improve the immunogenicity of DNA vaccines has been thoroughly studied in recent years. Glycoprotein (G)‐based DNA vaccines had been proven to be effective in combating infection against Rhabdovirus (especially infectious hematopoietic necrosis virus, IHNV) in salmonids. DDX41 is a helicase known to induce antiviral and inflammatory responses by inducing a type I IFN innate immune response. To gain more information regarding G‐based DNA vaccines in olive flounder (Paralicthys olivaceus), we tried to develop a more efficient G‐based DNA vaccine by adding a molecular adjuvant, DDX41. We designed a DNA vaccine in which the VHSV glycoprotein (G‐protein) and DDX41 were driven by the EF‐1&agr; and CMV promoters, respectively. Olive flounders were intramuscularly immunized with 1 &mgr;g of plasmids encoding the G‐based DNA vaccine alone (pEF‐G), the molecular adjuvant alone (pEF‐D), or the vaccine‐adjuvant construct (pEF‐GD). At two different time points, 15 and 30 days later, the fish were intraperitoneally infected with VHSV (100 &mgr;L; 1 × 106 TCID50/mL). Our assays revealed that the plasmid constructs showed up‐regulated expression of IFN‐1 and its associated genes at day 3 post‐vaccination in both kidney and spleen samples. Specifically, pEF‐GD showed statistically higher expression of immune response genes than pEF‐G and pEF‐D treated group (p < 0.05/p < 0.001). After VHSV challenge, the fish group treated with pEF‐GD showed higher survival rate than the pEF‐G treated group, though difference was not statistically significant in the 15 dpv challenged group however in the 30 dpv challenged group, the difference was statistically significant (p < 0.05). Together, these results clearly demonstrate that DDX41 is an effective adjuvant for the G‐based DNA vaccine in olive flounder. Our novel findings could facilitate the development of more effective DNA vaccines for the aquaculture industry. HighlightsThe adjuvant effect of DDX41 was assessed in this study.Simultaneous expression of VHSV glycoprotein and DDX41 showed enhanced IFN‐mediated immune response.The vaccine‐adjuvant construct showed enhanced regulation of type I interferon and IFN‐related genes.

Investigation of variable lymphocyte receptors in the alternative adaptive immune response of hagfish

Jawless vertebrates have an alternative adaptive immune system mediated by variable lymphocyte receptors (VLRs), VLRA, VLRC and VLRB. In investigation on the adaptive immunity of hagfish, avian influenza virus hemagglutinin (H9N2-HA1) was used as a model antigen, with mRNA expression levels of VLRA, VLRC and Ikaros were up-regulated in the first week post-immunization. CD45 was up-regulated after the first week; and expression of VLRB progressively increased over the course of the trial. The transcriptional/translational activation of VLRB in blood was verified. The VLRBs cloned from these transcripts showed diversity in their leucine-rich repeats (LRRs). The production of specific VLRB increased in a time- and dose-dependent manner, detected by an anti-VLRB antibody (11G5). The plasma VLRB could distinguish H9N2-HA1 from unrelated proteins, but not from other HA1 subtypes. Together, our findings show that VLRs play a major role in the alternative adaptive immune system of hagfish by responding to specific foreign substances, such as H9N2-HA1.

Development of a monoclonal antibody against the CD3ε of olive flounder (Paralichthys olivaceus) and its application in evaluating immune response related to CD3ε

&NA; The T cell receptor (TCR) is the binding site of antigen and is responsible for specifically activating the adaptive immune response. CD3, an essential component of the CD3‐TCR complex, is known to be composed of &ggr;&dgr; and &egr; chains in teleost. However, there are few monoclonal antibodies (mAb) available to identify these molecules on T cells, so we aimed to produce a mAb against CD3&egr; to improve our understanding of T cell immune response in olive flounder (Paralichthys olivaceus). CD3&egr; recombinant protein was expressed in yeast, the expression of which was confirmed by SDS‐PAGE, MALDI‐TOF/TOF MS and Western blot analysis. A CD3&egr;‐specific mAb 4B2 was selected, the specificity of which was examined by confocal microscopy, flow cytometry and RT‐PCR, and the mAb was subsequently used to examine the CD3&egr; lymphocyte population in several different immune organs, with relatively high percentages of these cells seen in trunk‐kidney and spleen, while lower percentages were seen in the liver and peripheral blood of olive flounder. During a viral hemorrhagic septicemia virus (VHSV) infection in olive flounder, the number of CD3&egr; lymphocytes was seen to gradually increase in the liver, spleen and trunk‐kidney of infected fish until 7 days post infection (dpi). In peripheral blood, on the other hand, the increase in CD3&egr; lymphocyte numbers peaked by 3 dpi. These results suggest that CD3&egr; lymphocytes might be involved in the immune response against VHSV. HighlightsRecombinant CD3&egr; was produced using yeast expression system.Monoclonal antibody 4B2 specifically detects the CD3&egr; lymphocyte in olive flounder.CD3&egr; lymphocytes might be involved in immune response related to VHSV.

Development of an immunochromatography assay kit for rapid detection of ranavirus

Ranaviruses are large, double-stranded DNA viruses of the family Iridoviridae and are known to be primary pathogens in frogs, fish and other amphibians. These viruses have been shown to be highly adaptable and have the ability to cross species barriers, making them a potent threat to global biodiversity. There is therefore, a need for rapid and efficient diagnostic methods to control the spread of these viruses. To address this, monoclonal antibodies (MAbs) were developed against ranavirus strain FV-3 (standard frog virus 3) to detect the major capsid protein and FV-3gorf19R related hypothetical protein in both the FV-3 and KRV-1 (Korean ranavirus) strains. The antibodies were then applied on a colloidal gold-immunochromatographic assay (GICA) as a kit for the detection of ranaviruses. The kit was able to detect low concentrations of the virus (10(1)TCID50/ml) and showed analytical specificity when tested against other viral pathogens, including those belonging to the same family. It was possible to detect ranavirus in experimentally infected frogs within 30 min using the kit. The kit described here is expected to be a valuable and informative tool for on-site detection of ranavirus in frog.

Generation and characterization of hagfish variable lymphocyte receptor B against glycoprotein of viral hemorrhagic septicemia virus (VHSV)

HIGHLIGHTSSuperhydrophobic C‐termini of hagfish VLRB leads to extremely low expression level.C4bp oligomerization domain mediates heptameric VLRB with high binding ability and producttivity.In vitro affinity maturation was efficiently carried out by LRRCT mutagenesis.Fine epitope mapping revealed 37DWDTPL42 is the recognition epitope of selected arVLRBs.The resulting arVLRBs can be used as diagnostic tools or therapeutic agents of VHSV. ABSTRACT Variable lymphocyte receptors B (VLRBs) are non‐immunoglobulin components of the humoral immune system in jawless vertebrates including hagfish (Eptatretus burgeri) and lamprey (Petromyzon marinus). Hagfish VLRBs consist of leucine rich repeat (LRR) modules with a superhydrophobic C‐terminal tail, the latter of which leads to extremely low expression levels in recombinant protein technology. Here, we present an artificially oligomerized VLRB (arVLRB) that conjugates via the C4bp oligomerization domain derived from human C4b‐binding protein (hC4bp) rather than the superhydrophobic tail. The resulting arVLRB had a tightly multimerized form with seven monomeric VLRB arms and showed high expression and secretion levels in a mammalian expression system. To isolate antigen‐specific arVLRB, we constructed large VLRB libraries from hagfish immunized with the fish pathogen, viral hemorrhagic septicemia virus (VHSV). The selected arVLRBs were found to recognize various types of antigens, including the recombinant target protein, purified viruses, and progeny viruses, with high antigen binding abilities and specificities. We also performed in vitro affinity maturation of the arVLRBs through LRRCT mutagenesis, and found that this enhanced their antigen‐binding properties by at least 125‐fold. Our epitope mapping analysis revealed that 37DWDTPL42, which is located in a region conserved among the glycoproteins of all VHSV isolates, is the recognition epitope of the arVLRBs. Thus, our newly developed arVLRB could prove useful in the development of universal diagnostic tools and/or therapeutic agents for the virus. Together, our novel findings provide valuable insights into hagfish VLRB and its potential use as a novel alternative to conventional antibodies for biotechnological applications.

Globular-shaped variable lymphocyte receptors B antibody multimerized by a hydrophobic clustering in hagfish

In hagfish and lampreys, two representative jawless vertebrates, the humoral immunity is directly mediated by variable lymphocyte receptors B (VLRBs). Both monomeric VLRBs are structurally and functionally similar, but their C-terminal tails differ: lamprey VLRB has a Cys-rich tail that forms disulfide-linked pentamers of dimers, contributing to its multivalency, whereas hagfish VLRB has a superhydrophobic tail of unknown structure. Here, we reveal that VLRBs obtained from hagfish plasma have a globular-shaped multimerized form (approximately 0.6 to 1.7 MDa) that is generated by hydrophobic clustering instead of covalent linkage. Electron microscopy (EM) and single-particle analysis showed that the multimerized VLRBs form globular-shaped clusters with an average diameter of 28.7 ± 2.2 nm. The presence of VLRBs in the complex was confirmed by immune-EM analysis using an anti-VLRB antibody. Furthermore, the hydrophobic hagfish C-terminus (HC) was capable of triggering multimerization and directing the cellular surface localization via a glycophosphatidylinositol linkage. Our results strongly suggest that the hagfish VLRB forms a previously unknown globular-shaped antibody. This novel identification of a structurally unusual VLRB complex may suggest that the adaptive immune system of hagfish differs from that of lamprey.

Potential Use of Genetically Engineered Variable Lymphocyte Receptor B Specific to Avian Influenza Virus H9N2

The variable lymphocyte receptor (VLR) B of jawless vertebrates functions as a secreted Ab of jawed vertebrates and has emerged as an alternative Ab with a single polypeptide chain. After observing an upregulated VLRB response in hagfish immunized with avian influenza virus (AIV) subtype H9N2, we screened AIV H9N2–specific VLRB using a mammalian expression system. To improve the binding avidity of the Ag-specific VLRB to the Ag, we enabled multimerization of the VLRB by conjugating it with C-terminal domain of human C4b-binding protein. To dramatically enhance the expression and secretion of the Ag-specific VLRB, we introduced a glycine–serine linker and the murine Ig κ leader sequence. The practical use of the Ag-specific VLRB was also demonstrated through various immunoassays, detected by anti-VLRB Ab (11G5). Finally, we found that the Ag-specific VLRB decreased the infectivity of AIV H9N2. Together, our findings suggest that the generated Ag-specific VLRB could be used for various immunoapplications.

Immunostimulatory effect of DDX41 of olive flounder (Paralichthys olivaceus)

ABSTRACT The cellular DEAD-box helicase DDX41, which functions as an initial sensor for cytoplasmic DNA, is involved in the activation of type I interferon (IFN-1) immune response in olive flounder. A plasmid encoding DDX41 (pEF-D) was introduced into flounder cells in vitro and in vivo. Immune responses induced by DDX41 were evaluated by relative quantification value (ΔΔCt) method of RT-qPCR using specific IFN-related gene primers. Results in in vitro, transcript levels of IFN-1, IRF-3, ISG-15 and IL-1β were significantly higher in pEF-D- than pEF-A-transfected cells, with 15-, 4-, 10- and 32-fold changes, respectively. In vivo, elevated expression of these genes was observed in the kidney of pEF-D group on days 1 and 3 post-treatment. The viral challenge test revealed higher survival rate in fish treated with pEF-D (67.5%) than controls, PBS- and pEF-A-treated fish (45%). Conclusively DDX41 elicits a robust IFN-mediated immune response, validating its adjuvant property.