Jae Wook Jung

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.

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.

Rapid MALDI biotyper-based identification and cluster analysis of Streptococcus iniae

Streptococcus iniae causes severe mortalities among cultured marine species, especially in the olive flounder (Paralichthys olivaceus), which is economically important in Korea and Japan. Recently, there has been growing concern regarding the emergence of S. iniae as a zoonotic pathogen. Here, 89 S. iniae isolates obtained from diseased olive flounders collected from 2003 to 2008 in Jeju Island, South Korea, were characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results were aligned both with the available Bruker Daltonics data-base and with a new set of S. iniae data entries developed in our laboratory, and the results were compared. When we used the Bruker Daltonics database, the 89 isolates yielded either “no reliable identification” or were incorrectly identified as Streptococcus pyogenes at the genus level. When we used the new data entries from our laboratory, in contrast, all of the isolates were correctly identified as S. iniae at the genus (100%) and species (96.6%) levels. We performed proteomic analysis, divided the 89 isolates into cluster I (51.7%), cluster II (20.2%), and cluster III (28.1%), and then used the MALDI Biotyper software to identify specific mass peaks that enabled discrimination between clusters and between Streptococcus species. Our results suggest that the use of MALDI TOF MS could outperform the conventional methods, proving easier, faster, cheaper and more efficient in properly identifying S. iniae. This strategy could facilitate the epidemiological and taxonomical study of this important fish pathogen.

Outer membrane vesicles from β-lactam-resistant Escherichia coli enable the survival of β-lactam-susceptible E . coli in the presence of β-lactam antibiotics

Outer membrane vesicles (OMVs) containing various bacterial compounds are released from mainly gram-negative bacteria. Secreted OMVs play important roles in the ability of a bacterium to defend itself, and thus contribute to the survival of bacteria in a community. In this study, we collected OMVs from β-lactam antibiotic-resistant Escherichia coli established by conjugation assay and the parental β-lactam antibiotic-susceptible strain, and performed comparative proteomic analysis to examine whether these OMVs carried β-lactam-resistant compounds. We also investigated whether both types of OMVs could protect susceptible cells from β-lactam-induced death and/or directly degrade β-lactam antibiotics. Several proteins that can be involved in degrading β-lactam antibiotics were more abundant in OMVs from β-lactam-resistant E. coli, and thus OMVs from β-lactam resistant E. coli could directly and dose-dependently degrade β-lactam antibiotics and fully rescue β-lactam-susceptible E. coli and other bacterial species from β-lactam antibiotic-induced growth inhibition. Taken together, present study demonstrate that OMVs from β-lactam-resistant E. coli play important roles in survival of antibiotic susceptible bacteria against β-lactam antibiotics. This finding may pave the way for new efforts to combat the current global spread of antibiotic resistances, which is considered to be a significant public health threat.

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.

Characterization of a specific monoclonal antibody against immunoglobulin light kappa/L1 chain in olive flounder (Paralichthys olivaceus)

ABSTRACT Immunoglobulins (Ig) are heterodimeric proteins that play critical roles in the adaptive immune system of vertebrates. Because of their plasticity, teleostean Igs are more diverse, and thus do not conform to mammalian classifications. Because of this, mammalian‐based Ig cell markers cannot be used successfully to study immune responses in fish. There is therefore a need to produce Ig‐specific cell markers for fish. Here, we attempted to identify the specific isotype detected by an Ig light chain‐specific monoclonal antibody (anti‐olive flounder IgL‐mAb: M7C3–4) that we had previously produced [11]. Three newly identified sequences of the Ig light chain from olive flounder were classified according to their isotypes. Subsequent analyses revealed that M7C3–4 was able to specifically detect lymphocytes expressing one of the &kgr; chains (Ig&kgr;‐a) in olive flounder. Interestingly, Ig&kgr;‐a+ B cells were more abundant in spleen and trunk‐kidney than in peripheral blood, indicating a distribution different from that of IgM+ B cells. Our work reveals interesting aspects of B cell distribution and differentiation, and may aid in the production of suitable and effective cell markers for olive flounder. HIGHLIGHTSOlive flounder has three isotypes of IgL, two of &kgr;, and one of &sgr;.M7C3–4 produced against serum immunoglobulins of olive flounder specifically recognizes Ig&kgr;‐a isotype.Ig&kgr;‐a+ B cells are more abundant in organs than in peripheral blood.

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.

Expression and characterization of monomeric variable lymphocyte receptor B specific to the glycoprotein of viral hemorrhagic septicemia virus (VHSV)

Monomeric variable lymphocyte receptor B (VLRB) is one of the smallest binding scaffold (20-25 kDa) from jawless vertebrates, hagfish and lamprey. This relatively new class of binding scaffold has various advantages: i) it has a single peptide composition, amenable to molecular engineering for enhancing its stability and affinity; ii) it has a small size, contributing better tissue penetration and easier production using microorganism expression system. Monomeric arVLRB142, which can specifically bind to the glycoprotein of viral hemorrhagic septicemia virus (VHSV), was expressed in Pichia pastoris. High quantity recombinant monomeric arVLRB142 (rVLR142mono) was purified from 100 ml of culture with a resulting yield of 2.6 ±1.3 mg of target protein. Functional studies revealed that the purified rVLR142mono can specifically recognize low levels of the target antigen (recombinant glycoprotein) (i.e. as low as 0.1 nM), but also the native glycoprotein of VHSV. The expressed rVLR142mono exhibited high levels of stability and it retained it binding capacity over broad temperature (4 °C ~ 60 °C) and pH ranges (pH 1.5-12.5). We developed an effective expression system for mass production of monomeric VLRB based on P. pastoris. The recombinant protein that was obtained offers promising binding avidity and biophysical stability and its potential use in various biotechnological applications.

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.