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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>
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>
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.