Immunostimulatory effect of DDX41 of olive flounder (Paralichthys olivaceus)

Lazarte, Jassy Mary S.,Kim, Young Rim,Lee, Jung Seok,Im, Se Pyeong,Kim, Si Won,Jung, Jae Wook,Kim, Jaesung,Lee, Jeong-Ho,Jung, Tae Sung Taylor Francis 2017 FOOD AND AGRICULTURAL IMMUNOLOGY Vol.28 No.5

Efficacy enhancement of glycoprotein-based DNA vaccinevia addition of molecular adjuvant DDX41 - invitro challenge test

Jassy Mary S.Lazarte,Young Rim Kim,Jung Seok Lee,Se Pyeong Im,Si Won Kim,Jae Wook Jung,Tae Hun Kim,Tae Sung Jung 대한수의학회 2015 대한수의학회 학술대회발표집 Vol.2015 No.-

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

Lazarte, Jassy Mary S.,Kim, Young Rim,Lee, Jung Seok,Im, Se Pyeong,Kim, Si Won,Jung, Jae Wook,Kim, Jaesung,Lee, Woo Jai,Jung, Tae Sung Elsevier 2017 Fish & shellfish immunology Vol.62 No.-

<P><B>Abstract</B></P> <P>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 <I>(Paralicthys olivaceus)</I>, 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-1α and CMV promoters, respectively. Olive flounders were intramuscularly immunized with 1 μg 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 μL; 1 × 10<SUP>6</SUP> TCID<SUB>50</SUB>/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.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The adjuvant effect of DDX41 was assessed in this study. </LI> <LI> Simultaneous expression of VHSV glycoprotein and DDX41 showed enhanced IFN-mediated immune response. </LI> <LI> The vaccine-adjuvant construct showed enhanced regulation of type I interferon and IFN-related genes. </LI> </UL> </P>

Matrix-assisted laser desorption ionization-time of flight mass spectrometry based identification of Edwardsiella ictaluri isolated from Vietnamese striped catfish (Pangasius hypothalamus)

TRUONGQUYNH NHU,Seong Bin Park,김시원,이정석,Se Pyeong Im,Jassy Mary S. Lazarte,Jong Pyo Seo,Woo-Jai Lee,김재성,Tae Sung Jung 대한수의학회 2016 Journal of Veterinary Science Vol.17 No.3

Edwardsiella (E.) ictaluri is a major bacterial pathogen that affects commercially farmed striped catfish (Pangasius hypothalamus) in Vietnam. In a previous study, 19 strains of E. ictaluri collected from striped catfish were biochemically identified with an API-20E system. Here, the same 19 strains were used to assess the ability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS; applied using a MALDI Biotyper) to conduct rapid, easy and accurate identification of E. ictaluri. MALDI-TOF MS could directly detect the specific peptide patterns of cultured E. ictaluri colonies with high (> 2.0, indicating species-level identification) scores. MALDI Biotyper 3.0 software revealed that all of the strains examined in this study possessed highly similar peptide peak patterns. In addition, electrophoresis (SDS-PAGE) and subsequent immuno-blotting using a specific chicken antibody (IgY) against E. ictaluri revealed that the isolates had highly similar protein profiles and antigenic banding profiles. The results of this study suggest that E. ictaluri isolated from striped catfish in Vietnam have homologous protein compositions. This is important, because it indicates that MALDI-TOF MS analysis could potentially outperform the conventional methods of identifying E. ictaluri.

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

Lee, Jung Seok,Kim, Jaesung,Im, Se Pyeong,Kim, Si Won,Lazarte, Jassy Mary S.,Jung, Jae Wook,Gong, Tae Won,Kim, Young Rim,Lee, Jeong Ho,Kim, Hyoung Jun,Jung, Tae Sung Elsevier 2018 Molecular immunology Vol.99 No.-

<P><B>Abstract</B></P> <P>Variable lymphocyte receptors B (VLRBs) are non-immunoglobulin components of the humoral immune system in jawless vertebrates including hagfish (<I>Eptatretus burgeri</I>) and lamprey (<I>Petromyzon marinus</I>). 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 <I>via</I> 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 <I>in vitro</I> 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 <SUP>37</SUP>DWDTPL<SUP>42</SUP>, 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.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Superhydrophobic C-termini of hagfish VLRB leads to extremely low expression level. </LI> <LI> C4bp oligomerization domain mediates heptameric VLRB with high binding ability and producttivity. </LI> <LI> <I>In vitro</I> affinity maturation was efficiently carried out by LRRCT mutagenesis. </LI> <LI> Fine epitope mapping revealed <SUP>37</SUP>DWDTPL<SUP>42</SUP> is the recognition epitope of selected arVLRBs. </LI> <LI> The resulting arVLRBs can be used as diagnostic tools or therapeutic agents of VHSV. </LI> </UL> </P>

Dual functionality of lamprey VLRB C-terminus (LC) for multimerization and cell surface display

Lee, Jung Seok,Kim, Jaesung,Im, Se Pyeong,Kim, Si Won,Jung, Jae Wook,Lazarte, Jassy Mary S.,Lee, Jeong Ho,Thompson, Kim D.,Jung, Tae Sung Elsevier 2018 Molecular immunology Vol.104 No.-

<P><B>Abstract</B></P> <P>Lamprey, one of the living representatives of jawless vertebrates, uses variable lymphocyte receptors B (VLRB) for antigen recognition, rather than immunoglobulin (Ig) based receptors as used by higher vertebrates. The C-terminus of lamprey VLRB (LC) possess a glycosylphosphatidylinositol (GPI) signal sequence and seven cysteine residues providing dual functionality of the VLRB antibody in the form of a humoral agglutinin and cell membrane receptors. Here, we show that the LC can be either secreted or be membrane anchored as a heterologous fused protein in a multimeric form comprising of eight or ten monomeric units. Using serially truncated LC variants, we showed that the LC, in which the last three amino acid “RKR” were deleted, referred to as LC7, was the most suitable domain for multimeric construction, whereas, the intact LC is more tailored for applications involving membrane anchorage. We show that an antibody specific for viral hemorrhagic septicemia virus (VHSV) (VLR43), displayed on HEK-293F cells using a PiggyBac (PB) transposase system, exhibited a dose-dependent reaction with its antigen, verifying that the LC can be applied in antibody display technology. Therefore, the present report provides valuable insight into the structure of the lamprey VLRB and highlights its potential use as a novel fusion partner for multimerization and membrane anchorage of chimeric proteins.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Lamprey VLRB has dual functionality like as humoral agglutinin and membrane receptors. </LI> <LI> The LC can perform multimerization of fused protein via disulfide-linkages. </LI> <LI> The LC can mediate cell surface localization of fused proteins via GPI anchoring. </LI> <LI> The LC can be used at an antibody display system as membrane tethering domain. </LI> </UL> </P>

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

Lee, Jung Seok,Kim, Jaesung,Im, Se Pyeong,Kim, Si Won,Jung, Jae Wook,Lazarte, Jassy Mary S.,Lee, Jeong-Ho,Thompson, Kim D.,Jung, Tae Sung Elsevier 2018 Journal of immunological methods Vol.462 No.-

<P><B>Abstract</B></P> <P>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 <I>Pichia pastoris</I>. High quantity recombinant monomeric arVLRB142 (rVLR142<SUP>mono</SUP>) 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 rVLR142<SUP>mono</SUP> 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 rVLR142<SUP>mono</SUP> 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 <I>P. pastoris</I>. The recombinant protein that was obtained offers promising binding avidity and biophysical stability and its potential use in various biotechnological applications.</P>

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

Im, Se Pyeong,Kim, Jaesung,Lee, Jung Seok,Kim, Si Won,Jung, Jae Wook,Lazarte, Jassy Mary S.,Kim, Jong Yong,Kim, Young Rim,Lee, Jeong Ho,Chong, Roger S. M.,Jung, Tae Sung American Association of Immunologists 2018 Journal of Immunology Vol. No.

<P>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.</P>

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

Jung, Jae Wook,Lee, Jung Seok,Kim, Young Rim,Im, Se Pyeong,Kim, Si Won,Lazarte, Jassy Mary S.,Kim, Jaesung,Thompson, Kim D.,Suh, Jong Pyo,Jung, Tae Sung Elsevier 2017 Fish & shellfish immunology Vol.65 No.-

<P><B>Abstract</B></P> <P>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 γδ and ε 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 CD3ε to improve our understanding of T cell immune response in olive flounder (<I>Paralichthys olivaceus)</I>. CD3ε recombinant protein was expressed in yeast, the expression of which was confirmed by SDS-PAGE, MALDI-TOF/TOF MS and Western blot analysis. A CD3ε-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 CD3ε 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 CD3ε 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 CD3ε lymphocyte numbers peaked by 3 dpi. These results suggest that CD3ε lymphocytes might be involved in the immune response against VHSV.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Recombinant CD3ε was produced using yeast expression system. </LI> <LI> Monoclonal antibody 4B2 specifically detects the CD3ε lymphocyte in olive flounder. </LI> <LI> CD3ε lymphocytes might be involved in immune response related to VHSV. </LI> </UL> </P>

Characterization of Hagfish (<i>Eptatretus burgeri</i>) Variable Lymphocyte Receptor–Based Antibody and Its Potential Role in the Neutralization of Nervous Necrosis Virus

Jung, Jae Wook,Lee, Jung Seok,Kim, Jaesung,Im, Se Pyeong,Kim, Si Won,Lazarte, Jassy Mary S.,Kim, Young Rim,Chun, Jin Hong,Ha, Min Woo,Kim, Hyeong Su,Thompson, Kim D.,Jung, Tae Sung American Association of Immunologists 2020 Journal of Immunology Vol.204 No.3

<P><B>Key Points</B></P><P><OL><LI>NNV-specific VLRBs were developed using a library screening system.</LI><LI>The binding ability of this Ab was enhanced through modular engineering.</LI><LI>VLRB could be an alternative neutralizing Ab against Betanodavirus infection.</LI></OL></P><P>The variable lymphocyte receptor (VLR) mediates the humoral immune response in jawless vertebrates, including lamprey (<I>Petromyzon marinus</I>) and hagfish (<I>Eptatretus burgeri</I>). Hagfish VLRBs are composed of leucine-rich repeat (LRR) modules, conjugated with a superhydrophobic C-terminal tail, which contributes to low levels of expression in recombinant protein technology. In this study, we screened Ag-specific VLRBs from hagfish immunized with nervous necrosis virus (NNV). The artificially multimerized form of VLRB was constructed using a mammalian expression system. To enhance the level of expression of the Ag-specific VLRB, mutagenesis of the VLRB was achieved in vitro through domain swapping of the LRR C-terminal cap and variable LRR module. The mutant VLRB obtained, with high expression and secretion levels, was able to specifically recognize purified and progeny NNV, and the Ag binding ability of this mutant was increased by at least 250-fold to that of the nonmutant VLRB. Furthermore, preincubation of the Ag-specific VLRB with NNV reduced the infectivity of NNV in E11 cells in vitro, and in vivo experiment. Our results suggest that the newly developed Ag-specific VLRB has the potential to be used as diagnostic and therapeutic reagents for NNV infections in fish.</P>