Viral hemorrhagic septicemia (VHS), caused by viral hemorrhagic septicemia virus (VHSV), is a viral disease affecting teleosts, and is the major cause of virus-related deaths in olive flounder (
). ...Research has focused on ways to control VHS, and recently, the use of polyinosinic-polycytidylic acid poly (I:C)-potentiated vaccination has been investigated, whereby fish are injected with poly (I:C) and then with live pathogenic virus, resulting in a significant decrease in VHSV-related mortality. T cell responses were investigated in the present study after vaccinating olive flounder with poly (I:C)-potentiated vaccination to understand the ability of poly (I:C) to induce T cell immunity. Stimulation of T cell responses with the poly (I:C)-potentiated vaccination was confirmed by examining levels of CD3
T cells, CD4-1
T cells and CD4-2
T cells. Higher levels of CD4-2
T cells were found in vaccinated fish than CD4-1
T cells, believed to result from a synergistic effect between poly (I:C) administration and pathogenic VHSV immunization. More importantly, the role of CD4-2
T cells in the antiviral response was clearly evident. The results of this study suggest that the outstanding protection obtained with the poly (I:C)-potentiated vaccination is due to the robust immune response initiated by the CD4-2
T cells.
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.
The variable lymphocyte receptor (VLR) mediates the humoral immune response in jawless vertebrates, including lamprey (
) and hagfish (
). 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.
•Superhydrophobic 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.
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.
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-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 × 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.
•The 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.
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 (Paralichthys olivaceus). 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.
•Recombinant CD3ε was produced using yeast expression system.•Monoclonal antibody 4B2 specifically detects the CD3ε lymphocyte in olive flounder.•CD3ε lymphocytes might be involved in immune response related to 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.
The variable lymphocyte receptor B (VLRB) of jawless vertebrates has a similar function to the antibodies produced by jawed vertebrates, and has been considered as an alternative source to mammalian ...antibodies for use in biological research. We developed a modified yeast display vector system (pYD8) to display recombinant hagfish VLRB proteins on the extracellular surface of yeast for the isolation of antigen-specific VLRBs. After observing an up-regulation in the VLRB response in hagfish immunized with hemagglutinin 1 of avian influenza virus H9N2 subtype (H9N2-HA1), the antigen-specific VLRBs decorated on the yeast's surface were selected by quantitative library screening through magnetic-activated cell sorting (MACS) and fluorescent-activated cell sorting (FACS). We also demonstrated a strong specificity of the antigen-specific VLRBs, when expressed as a secreted protein using a mammalian expression system. Together, our findings suggest that the pYD8 vector system could be useful for screening antigen-specific hagfish VLRBs, and the specificity of secreted VLRB may have potential for a variety of biological applications.
•The transcription levels of VLRB were up-regulated in immunized hagfish.•The newly developed pYD8 system displayed recombinant hagfish VLRB on the yeast surface.•The antigen-specific hagfish VLRBs were screened by the pYD8 system.•The antigen-specific VLRB secreted from mammalian system showed strong specificity to the antigen.
Avian pathogenic Escherichia coli (APEC) is a major pathogen that causes avian colibacillosis and is associated with severe economic losses in the chicken-farming industry. Here, bacteriophage ...KBNP1315, infecting APEC strain KBP1315, was genomically and functionally characterized. The evolutionary relationships of KBNP1315 were analyzed at the genomic level using gene (protein)-sharing networks, the Markov clustering (MCL) algorithm, and comparative genomics. Our network analysis showed that KBNP1315 was connected to 30 members of the Autographivirinae subfamily, which comprises the SP6-, T7-, P60-, phiKMV-, GAP227- and KP34-related groups. Network decomposition suggested that KBNP1315 belongs to the SP6-like phages, but our comparison of putative encoded proteins revealed that key proteins of KBNP1315, including the tail spike protein and endolysin, had relative low levels of amino acid sequence similarity with other members of the SP6-like phages. Thus KBNP1315 may only be distantly related to the SP6-like phages, and (based on the difference in endolysin) its lysis mechanism may differ from theirs. To characterize the lytic functions of the holin and endolysin proteins from KBNP1315, we expressed these proteins individually or simultaneously in E. coli BL21 (DE3) competent cell. Interestingly, the expressed endolysin was secreted into the periplasm and caused a high degree of host cell lysis that was dose-dependently delayed/blocked by NaN3-mediated inhibition of the SecA pathway. The expressed holin triggered only a moderate inhibition of cell growth, whereas coexpression of holin and endolysin enhanced the lytic effect of endolysin. Together, these results revealed that KBNP1315 appears to use a pin-holin/signal-arrest-release (SAR) endolysin pathway to trigger host cell lysis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
•Lamprey VLRB has dual functionality like as humoral agglutinin and membrane receptors.•The LC can perform multimerization of fused protein via disulfide-linkages.•The LC can mediate cell surface ...localization of fused proteins via GPI anchoring.•The LC can be used at an antibody display system as membrane tethering domain.
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.
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