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Immune response of heterologous recombinant antigenic protein of viral hemorrhagic septicemia virus (VHSV) in mice Immune response of heterologous recombinant antigenic protein of viral hemorrhagic septicemia...
Shin, Chunha; Kang, Yangjoo; Kim, Heui-Soo; Shin, Yong Kyoo; Ko, Kisung
2019-03-04 00:00:00
NEUROBIOLOGY & PHYSIOLOGY ANIMAL CELLS AND SYSTEMS 2019, VOL. 23, NO. 2, 97–105 https://doi.org/10.1080/19768354.2019.1575904 Immune response of heterologous recombinant antigenic protein of viral hemorrhagic septicemia virus (VHSV) in mice a a b c a Chunha Shin , Yangjoo Kang , Heui-Soo Kim , Yong Kyoo Shin and Kisung Ko a b Department of Medicine, College of Medicine, Chung-Ang University, Seoul, South Korea; Department of Biological Sciences, College of Natural Sciences, Pusan National University, Busan, South Korea; Department of Pharmacology, College of Medicine, Chung-Ang University, Seoul, South Korea ABSTRACT ARTICLE HISTORY Received 16 July 2018 Viral hemorrhagic septicemia (VHS) is an important infectious disease in fish worldwide caused by Revised 4 September 2018 viral hemorrhagic septicemia virus (VHSV). VHSV is the causative agent of serious systemic diseases Accepted 5 September 2018 in fish, affecting a number of teleost fish species. In this study, VHSV glycoprotein (G), including its epitope, as a subunit vaccine candidate, was expressed in tobacco plant (Nicotiana tabacum). The KEYWORDS recombinant gene, VHSVG, was fused to the immunoglobulin Fc fragment and extended with the Plant expression system; endoplasmic reticulum (ER) retention signal (KDEL) to generate VHSVG-FcK. The recombinant vaccine; VHSV; VHSVG-FcK expression vector for VHSVG-FcK was transferred into Agrobacterium tumefaciens (LBA4404), and plant transformation was conducted N. tabacum. Polymerase chain reaction (PCR) was performed to confirm gene insertion and VHSVG-FcK protein expression was confirmed by immunoblot analysis. VHSVG-FcK protein was successfully purified from tobacco plant leaves. Furthermore, ELISA analysis showed that mice serum immunized with the plant-derived VHSVG-FcK (VHSVGP- FcK) had a high absorbance against VHSVG-FcK, indicating that the plant-derived recombinant subunit vaccine protein VHSVG-FcK can induce immune response. Taken together, this recombinant vaccine protein can be expressed in plant expression systems and can be appropriately assembled to be functional in immunogenicity. Introduction (Boxall 2004; Cabello 2006). Application of antibiotics to Viral hemorrhagic septicemia (VHS) is serious infectious fish culture systems is not an environmentally friendly disease in fish worldwide caused by viral hemorrhagic approach and the antibiotics can be transferred to septicemia virus (VHSV) (Walker and Winton 2010). humans via fish (Kemper 2008). Therefore, it is necessary VHSV is the causative agent of serious systemic diseases to develop vaccines that can effectively prevent viral dis- in fish, affecting a number of teleost fish species, includ- eases in fish. Recently, several studies are being con- ing the olive flounder (Paralichthys olivaceus) (Skall et al. ducted on the development of fish vaccines (Gudding 2005). VHSV belongs to the Novirhabdovirus genus of the and Van Muiswinkel 2013). Several vaccines are available, Rhabdoviridae family and has a negative-sense, single- such as an attenuated viral vaccine, bacterial vaccine, stranded RNA genome (Caipang et al. 2003). The virus DNA vaccine, recombinant subunit vaccine, and virus- genome is composed of six parts: nucleoprotein (N), like particle vaccine (Lorenzen et al. 1993; Biering et al. phosphoprotein (P), matrix protein (M), glycoprotein 2005; Håstein et al. 2005; Lorenzen and LaPatra 2005; (G), RNA polymerase (L), and non-structural viral Crisci et al. 2012). Among these, the recombinant protein (NV) (Schu et al. 1999). Among the components, subunit vaccine is produced by using the virus glyco- the glycoprotein (G) is located on the surface of the virus protein-that contains the causative immunogenic part and can induce immune response because of the pres- of the disease in a heterologous expression system. In ence of epitopes (Coll 1995; Lorenzen et al. 1999; Byon addition, this vaccine can efficiently induce immune et al. 2006). responses (Lecocq-Xhonneux et al. 1994; Brun et al. 2011). To date, fish are treated with excessive antibiotics in In particular, to produce the recombinant protein, the aquaculture to manage the fish disease problem; this Escherichia coli system or mammalian-derived cell can lead to various side effects and increase the cost system has been used. However, existing approaches CONTACT Yong Kyoo Shin syk@cau.ac.kr; Kisung Ko ksko@cau.ac.kr Supplemental data for this article can be accessed at https://doi.org/10.1080/19768354.2019.1575904. © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 98 C. SHIN ET AL. have some limitations such as high cost, function, and immunofunction of VHSVG-Fc protein were investigated safety issues (Zhu 2012; Rosano and Ceccarelli 2014). In in tobacco plant. the E. coli bacterial expression system, the prokaryotic cell cannot perform the post-translational modifications, Materials and methods such a glycosylation, and thus recombinant glyco- proteins produced from E. coli cannot function effectively Plasmid construction (Yin et al. 2007). The mammalian cell expression system is The gene encoding VHSV glycoprotein (G), including epi- expensive and can potentially be contaminated by topes, was fused to the human IgG Fc fragment human pathogens (Twyman et al. 2003). However, extended with KDEL, the endoplasmic reticulum (ER) plants can be easily cultivated for scale-up production, retention signal, to generate VHSVG-FcK (Figure 1). resulting in lower production costs without any human VHSVG-FcK was cloned under the control of the pathogenic contaminant concerns (Sharma and Sharma enhanced cauliflower mosaic virus (CaMV) constitutive 2009; Obembe et al. 2011). Plants have post-translational 35S promoter with the untranslated leader sequence of modification and glycosylation processes similar to alfalfa mosaic virus RNA 4 (AMV) (Figure 1). The humans (Gomord and Faye 2004; Walsh and Jefferis expression cassette was cloned in the plant binary 2006; Arcalis et al. 2013). Because of the many advan- vector pBI121 by HindIII and EcoRI to produce pBI tages, we used the tobacco plant expression system for VHSVG-FcK and transferred into DH5α E. coli cells. the expression and purification of the recombinant vaccine candidate protein of VHSV. In this study, the plant expression system was estab- Agrobacterium-mediated plant transformation lished for the production of the VHSV vaccine candidate to determine in in vivo animal. The VHSV glycoprotein (G) The recombinant expression vector pBI VHSVG-FcK was fused to the immunoglobulin Fc fragment (VHSVG-Fc) transferred into Agrobacterium tumefaciens (LBA4404) fusion protein was expressed in the tobacco plant. Its using electroporation method. Plant transformation gene insertion, expression, purification, and was conducted via the Agrobacterium-mediated leaf Figure 1. Schematic diagram of plant expression vector for VHSVG-FcK protein. VHSVG-FcK gene expression cassette was introduced to plant expression vector pBI121. E/35S-P, the Cauliflower mosaic virus 35S promoter with duplicated enhancer region; A, an alfalfa mosaic virus untranslated leader sequence (AMV) of RNA4; K, endoplasmic reticulum retention signal (KDEL); NOS-Ter, the nopaline synthase gene terminator. Expected protein structure of the recombinant fusion protein VHSVG-FcK: overlapped X shape bar, VHSVG; white oval region, Fc; and spring-shaped region, KDEL. Expected glycan structure: the symbols of the glycan structures are as follows: N-acetylglucosamine, black square; mannose, white circle. ANIMAL CELLS AND SYSTEMS 99 Figure 2. Schematic diagram of production process of VHSVG-FcK transgenic plants. After the Agrobacterium-mediated plant trans- formation, plants were regenerated under kanamycin and cefotaxime pressure and shoots were induced from callus. Shoots were trans- ferred to magenta vessel GA-7 and stem and root were induced in kanamycin cefotaxime containing media. Transgenic plants expressing VHSVG-FcK were selected to grow for mass production. disc method using tobacco plant (Nicotiana tabacum) (Lu used: forward primer, 5 -GTC GAC ATG GAA TGG AAT ′ ′ et al. 2012). The inoculated explants were placed on Mur- ACT TTT TC-3 , and reverse primer, 5 -GCC AAA TGT ashige and Skoog (MS) medium [30 g/L of sucrose, 6 g/L TTG AAC GAT CGG-3 . PCR was performed under the fol- of phyto agar, and 4.8 g/L of MS B5 vitamin (Duchefa Bio- lowing conditions: initial denaturation at 94°C for 2 min, chemie, Haarlem, Netherlands)] containing 100 mg/L of denaturation at 94°C for 20 s, annealing at 59°C for 30 s, kanamycin and 250 mg/L of cefotaxime. All plants were extension at 72°C for 2 min, and final extension at 72°C grown in a chamber under constant temperature (23°C) for 5 min. The pBI VHSVG-FcK vector was used as a and maintained in a 16-h light and 8-h dark cycle. All positive control and the genomic DNA fragment from leaf explants were subcultured every week and devel- non-transgenic tobacco plant was used as a negative oped to callus over time (Figure 2). The regenerated control. The expected size of VHSVG-FcK gene was shoots were removed from the callus and placed for 2286 bp. stem and root induction in magenta vessel GA-7 (Sigma, St. Louis, MO) containing MS medium with Immunoblot analysis 100 mg/L of kanamycin and 250 mg/L of cefotaxime (Figure 2). Leaves (100 mg) of transgenic plant expressing VHSVG- FcK or non-transgenic plant were collected and hom- ogenized with 300 µL of 1X PBS (137 mM NaCl, 10 mM Genomic DNA extraction and PCR Na HPO , 2.7 mM KCl, and 2 mM KH PO ) to extract the 2 4 2 4 Genomic DNA was extracted from 100 mg of transgenic total soluble proteins. A volume of 16 µL of grinded and non-transgenic plant leaves. The isolated genomic samples were mixed with the sample buffer (1 M DNA was analyzed by polymerase chain reaction (PCR) Tris-HCl, 50% glycerol, 10% SDS, 5% 2-mercaptoethanol, using a DNA extraction kit (iNtRON Biotechnology, and 0.1% bromophenol blue) and loaded on 10% sodium Seoul, Korea) according to the manufacturer’s rec- dodecyl sulfate (SDS) polyacrylamide gel. Proteins were ommendations. The extracted genomic DNA was separated by SDS-polyacrylamide gel electrophoresis amplified by PCR to confirm the recombinant VHSVG- and transferred to a nitrocellulose membrane (Millipore FcK gene insertion and the following primer pairs were Corp, Billerica, MA). The membrane was blocked with 100 C. SHIN ET AL. 5% skim milk (Sigma, St. Louis, MO) in TBS-T buffer [1X blue R-250) for 30 min at room temperature. The gel TBS plus 0.5% (v/v) Tween 20] for 1 h 30 min at room was destained with destaining solution (10% acetic temperature. The blot was incubated for 2 h at room acid and 30% methanol) through a 3 times buffer temperature with horseradish peroxidase (HRP)-conju- exchange for 1 h 30 min. gated goat anti-human IgG, Fcγ fragment antibody (Jackson ImmunoResearch, West Grove, PA) diluted in Immunization of plant-derived VHSVG-FcK blocking buffer at 1:5000 and then incubated for 2 h at protein in mice room temperature. The blot was then incubated for 2 h at room temperature with rabbit anti-VHSV antibody All mice experiments were approved by the Institutional (Amsbio, Abingdon, UK) diluted in blocking buffer at Animal Care and Use Committee (IACUC) at Chung-Ang 1:1000. Lastly, the blot was treated with HRP-conjugated University, Seoul, Korea (Approval number: 2017- anti-rabbit IgG (H + L) antibody (Bethyl, Montgomery, TX) 00050). Plant-derived VHSVG-FcK (VHSVG -FcK) protein diluted in blocking buffer at 1:5000 for 1 h 30 min. The and 1X PBS used as a negative control were administered protein bands were detected using the chemilumines- with an adjuvant (aluminum hydroxide) to mice through cence substrate (Bio-Rad, Hercules, CA) and the signal intraperitoneal injection. The samples were injected into was visualized by exposing the membrane to an X-ray 8-week-old female BALB/c mice three times with 10 μgof film (Fuji, Tokyo, Japan). The non-transgenic plant VHSVG -FcK and 1X PBS with a total volume of 300 μlat sample was used as a negative control. The expected intervals of two weeks. Blood sample collection was per- VHSVG-FcK protein size was 80 kDa. formed 10 days after the second immunization and 10 days after the third immunization by the retro-orbital sinus bleeding method. Purification of recombinant VHSVG-FcK protein from plant leaf ELISA for confirmation of immune response of Transgenic tobacco plants expressing the VHSVG-FcK VHSVG -FcK in mice protein were transferred to pots and grown in a green- house (Figure 2). The leaves were harvested after 1 P Purified VHSVG -FcK sample was diluted in 0.2 M sodium month. The 250 g of harvested leaves were ground in carbonate/sodium bicarbonate buffer and applied to an HR2094 aluminum blender (Philips, Seoul, Korea) MaxiSorp 96-well plates (Nunc, Rochester, NY) at a con- with extraction buffer (37.5 mM Tris-HCl pH 7.5, 50 mM centration of 100 ng. The ELISA plates were incubated NaCl, 15 mM EDTA, 75 mM sodium citrate, and 0.2% overnight at 4°C and washed with 1X PBS-T [1X PBS sodium thiosulfate) and centrifuged at 8800g for plus 0.5% (v/v) Tween 20]. The plates were blocked 30 min at 4°C. The supernatant was filtered with a Mira- using 5% skim milk in 1X PBS-T (Sigma, St. Louis, MO) cloth (Biosciences, La Jolla, CA). The filtered supernatant for 2 h at room temperature. Half serial diluted mouse was adjusted to pH 5.1 with acetic acid (pH 2.4) and cen- serum made from 1 μl was applied to each well as a trifuged at 10,200g for 30 min at 4°C. After centrifu- primary antibody for 2 h at room temperature. HRP-con- gation, the supernatant solution was readjusted to pH jugated anti-mouse IgG Fc antibody (Jackson, West 7.0 using 3 M Tris-HCl and ammonium sulfate was Grove, PA) as a secondary antibody was added to the added to 8% saturation. After the 2 h incubation at 4°C, plates. After 2 h incubation, TMB substrate solution (Ser- the solution was centrifuged at 8800g at 4°C and acare, Milford, MA) was added to the sample for 5 min, ammonium sulfate was added to the supernatant to and the reaction was stopped by TMB stop solution (Ser- 22.6% saturation. After overnight incubation at 4°C, the acare, Milford, MA). The 96-well plate was read by the solution was centrifuged at 8800g for 30 min at 4°C ELISA reader Epoch (Biotek, Winooski, VT) at an absor- and the precipitate was resuspended in extraction bance of 450 nm. buffer to one-tenth of the original volume. The final sol- ution was centrifuged at 10,200g for 30 min at 4°C and the supernatant was filtered through a 0.45-μm filter Results (Millipore, Bedford, MA). The obtained sample was PCR confirmation of VHSVG-FcK gene insertion in applied to a protein A Sepharose 4 Fast Flow (GE Health- plant care, Sweden, NJ) and performed according to the man- ufacturer’s recommendations. SDS-PAGE was performed Agrobacterium-mediated transformation was conducted for the visualization of purified samples. The gel was three times to obtain transgenic lines expressing stained with Coomassie brilliant blue staining solution VHSVG-FcK. Each transformation event produced three (10% acetic acid, 30% methanol, and 0.05% Coomassie cocultivation plates containing 20 leaf cut pieces for ANIMAL CELLS AND SYSTEMS 101 Figure 3. Confirmation of VHSVG-FcK gene insertion and protein expression. (A) Polymerase chain reaction (PCR) analysis was per- formed to confirm the VHSVG-FcK gene existence. The genomic DNA, isolated from fresh leaf, was amplified and target gene was confirmed by 1% agarose gel electrophoresis. VHSVG-FcK (2286 bp): lane 1, positive control (+), pBI VHSVG-FcK recombinant vector in DH5α competent cell; lane 2, negative control (−), genomic DNA fragment from non-transgenic tobacco plants; lane 3–10, genomic DNA fragments of transgenic plants. The 3 μl of sample was loaded on each well. (B) Immunoblot analysis was performed to confirm VHSVG-FcK protein expression in transgenic tobacco plants. Leaf samples from transgenic plants were ground with 1X PBS and 20 μl of samples were loaded on each well. VHSVG-FcK (>70 kDa) was detected with HRP-conjugated goat anti-human IgG Fc antibodies. Lane 1, negative control (−), non-transgenic tobacco plants; Lane 2–9, transgenic tobacco plants. Expected protein struc- tures were illustrated with arrow (Right). each plate. From each transformation event, 10, 9, and 7 peroxidase as a secondary antibody (Figure 3(B)). The regenerated shoots were obtained on regeneration expected protein band size was 80 kDa. The putative media. The transformants were transplanted to root- VHSVG-FcK protein band was detected at approximately inducing media containing kanamycin antibiotic. In >70 kDa in all tested transgenic plant samples. However, around two weeks after transplanting, new leaf and the band density of each transgenic plant varied. Among root growth were observed from transformants. PCR the tested transgenic lines, T810 showed the strongest amplification of genomic DNA extracted from leaves of density, whereas T815 and T816 had the weakest the putative transgenic tobacco plant was performed density. Thus, T810 was selected for mass production. to confirm the VHSVG-FcK gene insertion (Figure 3(A)). No protein band was detected in the non-transgenic The PCR products were separated on a 1% agarose gel plant as a negative control. Non-specific protein bands (Figure 3(A)). The expected size of the amplified PCR (35 kDa) were detected in all tested transgenic plants. band was 2286 bp. The PCR amplified band for VHSVG- These had similar or slightly more prominent protein FcK was detected in all transformants with healthy leaf band densities compared to the >70 to 80 kDa protein and root growth (Figure 2). T811, T812, and T813 were (Figure 3(B)). transferred to root-inducing media but did not survive without any root induction. No PCR band was detected Purification of VHSVG-FcK protein from in the genomic DNA sample of the non-transgenic transgenic tobacco plant plant (NT) (Figure 3(A)). VHSVG-FcK protein was purified from leaves produced from mass production of tobacco plant by using Immunoblot analysis for the confirmation of protein A affinity chromatography (Figure 2). SDS-PAGE VHSVG-FcK protein expression was performed with eluted fraction samples to observe In order to confirm VHSVG-FcK protein expression in the purified protein band (Figure 4(A)). The expected transgenic plants, immunoblot analysis was conducted protein band of VHSVG-FcK was detected at >70 kDa in with anti-human IgG Fc antibody conjugated horseradish the fraction samples #1 and 2. Non-specific total 102 C. SHIN ET AL. Figure 4. Purification of VHSVG-FcK protein from plant biomass production. (A) SDS-PAGE for detection of VHSVG-FcK protein from purified samples. M, protein marker; BSA, 2 μg; 1–5, eluted fraction sample number; CT, Column through sample. Each sample was loaded with 16 μl, respectively. (B) Immunoblot analysis was performed to confirm the presence of VHSVG-FcK proteins from the eluted samples. VHSVG-FcK (>70 kDa) was detected with rabbit anti-VHSV antibodies. Lane 1–5, fraction number 1–5 of purified samples; CT, Column through sample. The sample was loaded 16 μl, respectively. (C) Immunoblot analysis was conducted with HRP-conjugated goat anti-human IgG Fc antibodies. Lane 1–5, fraction number 1–5 of purified samples; CT, Column through sample. The sample was loaded 8 μl, respectively. soluble protein bands were detected in column. samplings, respectively) were applied to a 96-well plate However, unexpected protein bands were also detected coated with 100 ng of VHSVG -FcK (Figure 5(A,B)). The at around 35 kDa (Figure 4(A)). Immunoblot analysis with secondary antibody used was HRP-conjugated anti- both anti-VHSVG antibody and anti-IgG Fc antibody was mouse IgG Fc to detect the VHSVG -FcK specific anti- performed to confirm the specific VHSVG-FcK protein bodies in the mice serum. The absorbance of the mice band in each eluted fraction (Figure 4(B,C), respectively). injected with the VHSVG -FcK group was significantly The VHSVG-FcK protein band was detected at approxi- higher than the mice injected with 1X PBS. The absor- mately >70 kDa in the fraction 1–5 samples by both anti- bance in the 2nd bleeding sample was slightly higher bodies. The 35 kDa protein band also was detected in the than that in the 1st. In both 1st and 2nd bleeding fraction 1–5 samples. >70 and 35 kDa bands were not samples, the absorbances were sequentially decreased detected in the column through (CT). along with the decreasing serum amount added to the plates. Immune response of mice against plant-derived subunit vaccine candidate VHSVG-FcK Discussion ELISA was performed to confirm the specific antibodies In this study, we successfully established a plant production against the VHSVG-FcK in mice after the expression system for the production of the recombinant plant-derived VHSVG-FcK injection (Figure 5). The sera VHSVG-FcK fish vaccine candidate. Although many collected from mice at 10 days after the 2nd injection immunotherapeutic proteins have been produced in and 10 days after the 3rd injection (1st and 2nd bleed plant expression systems that are safe, cost efficient, ANIMAL CELLS AND SYSTEMS 103 Figure 5. Schematic diagram of the immunization of VHSVG-FcK in mice and ELISA to confirm immune response of VHSVG-FcK in mice. (A) VHSVG -FcK was injected to eight-week-old female BALB/c mice three times via intraperitoneal injection. 1X PBS was administered as a negative control using the same injection method. The 10 μg of VHSVG -FcK and 1X PBS in a total volume of 300 μl were injected with an adjuvant (aluminum hydroxide). The samples were injected at intervals of 2 weeks and blood samples were collected 10 days after the second immunization and 10 days after the third immunization by the retro-orbital sinus bleeding method. (B) ELISA result of 1X PBS and VHSVG-FcK immunization. Ninety-six-well plate wells were coated with VHSVG-FcK (100 ng/well), applied with mouse serum as a capture antibody, and treated with HRP-conjugated anti-mouse IgG Fc. and easy to scale up, expression of fish vaccines has not 2015). In addition, the Fc region can be applied in been studied extensively in plants. The present study protein-G/A affinity chromatography to purify the Fc demonstrated that the recombinant subunit vaccine fused recombinant protein from plant leaf biomass (Lu protein VHSVG-FcK as a fish vaccine candidate was et al. 2012). Our previous studies showed that the expressed in the tobacco plant, and this plant-derived various antigenic proteins fused to the Fc fragment VHSVG-FcK purified from tobacco plant leaves induced expressed in plant (Lee and Ko 2017) and insect cell an immune response in mice. (Moussavou et al. 2018) were efficiently purified by The subunit vaccine candidate, VHSVG, was fused to protein A or G affinity chromatography column (Flana- the immunoglobulin Fc fragment and cloned under the gan et al. 2007; Czajkowsky et al. 2012). The function of control of the enhanced CaMV 35S promoter with KDEL-tagging is to retain the tagged protein to ER from AMV. Fusion of Fc to the recombinant subunit vaccine cis-Golgi, eventually enhancing protein accumulation endows the molecules to interact with Fc-receptors levels (Tekoah et al. 2004). Thus, the KDEL (Lys-Asp-Glu- (FcRs) on immune cells (Nimmerjahn and Ravetch Leu) ER retention motif was fused to enhance protein 2007). Thus, it is expected that the Fc-region bound to expression level and retain the recombinant vaccine, Fc-receptors should efficiently prepare antigen delivery inducing oligo-mannose type of glycan structures to vehicles to antigen presenting cells (DiLillo and Ravetch relieve immune response in other species (Figure 1). 104 C. SHIN ET AL. The ER-retained proteins have typically Man GlcNAc second bleeding sample, after the third injection, 8 2 (Man8) N-glycans (Gomord et al. 2010). However, once showed the stronger absorbance signal suggesting exposed to cis-Golgi processing enzymes such as α that the third injection boosted the immune responses. (1,2)-mannosidase, the KDEL-tagged proteins carry These results were consistent with previous studies Man7 and Man6 N-glycans, which are partially trimmed where the third injection with anti-cancer antigen as N-glycans (De Meyer and Depicker 2014). a vaccine candidate induced immune responses com- To confirm the VHSVG-FcK gene insertion, PCR analy- pared to the first and second injections in mice (Lee sis was conducted. Some regenerants did not undergo et al. 2016). The VHSVG -FcK injection group showed transgene insertion. These results are consistent with notable absorbance compared to the 1X PBS injection previous studies where some regenerants had nptII group as a negative control. The immune responses genes but not the targeted transgene of interest (Ko seemed to produce anti-human Fc IgGs. According to et al. 2003). The immunoblot analysis result confirmed the western blot with serum from the VHSVG-FcK VHSVG-FcK protein expression in all tested plants with injected mice (Supplemental Figure 1), the human IgG transgene insertion. The intensity of each band was heavy chains (HC) were detected by the serum. different, indicating that expression levels by each trans- However, the density of VHSVG-FcK protein bands genic plant varied. The variation of transgene expression detected by the serum was stronger than the HC has been reported in many studies. The variation of band of human mAb, indicating that the VHSVG-FcK transgene expression is due to the position effect and induces more anti-VHSVG IgG immune responses com- copy number (Matzke and Matzke 1998; Kooter et al. pared to the Fc fragments in mice. Taken together, 1999). For further study, T810 with the highest these results suggest that the recombinant VHSVG- expression was chosen for leaf biomass production. A FcK vaccine candidate can have an immunogenicity. non-specific protein band (35 kDa) was also detected in In conclusion, our results showed that the recombi- all samples. According to the detected protein band nant VHSVG-FcK subunit vaccine protein can be size (35 kDa) by both anti-Fc and anti-VHSVG antibodies, expressed in transgenic plants, purified using protein A it is speculated the cleaved fragment (35 kDa) consists of affinity chromatography, and evoke the immune partial fragments of the VHSVG and Fc region response in mice. Fusion of VHSV domain to the Both SDS-PAGE and immunoblot analyses were human IgG Fc fragment is an ideal strategy to purify performed to investigate whether the VHSVG-FcK was the recombinant vaccine candidate protein expressed successfully purified using protein A affinity chromato- in transgenic plants. However, the stability of VHSVG-Fc graphy. The expected VHSVG-FcK band (80 kDa) was fusion protein without degradation and cleavage still detected in the fraction samples #1 and 2. However, remains to be determined. Nevertheless, a plant the unexpected protein band (35 kDa) was also detected. expression system can be applied to develop recombi- The band pattern was similar to that produced by anti- nant fish vaccine. human IgG Fc antibody conjugated to HRP in immuno- blot analysis. Since protein A can bind to the Fc region of immunoglobulin, it is speculated that the detected Disclosure statement 35 kDa protein bands are only FcK fragments. However, No potential conflict of interest was reported by the authors. both anti-VHSG and anti-human IgG Fc antibodies detected the 35 kDa proteins, suggesting that the 35 kDa protein is composed of the C-terminus part of Funding the VHSV and FcK domains. This research was supported by the Chung-Ang University The immunological properties of VHSVG -FcK after Research Scholarship Grants in 2014; National Research Foun- the second or third injections in mice were investi- dation of Korea (NRF) grant funded by the Korea government gated. The VHSVG domain has not been studied as a (MSIT) [grant number NRF-2017R1A2A2A0569788]. target for anti-virus recombinant vaccines. Thus, it is crucial to investigate whether the VHSVG domain can induce immune responses. Indeed, in the current References study, the VHSVG domain fused to the Fc region Arcalis E, Stadlmann J, Rademacher T, Marcel S, Sack M, induced immune responses. Immunization with viral Altmann F, Stoger E. 2013. Plant species and organ antigen is a potential strategy for prevention against influence the structure and subcellular localization of recom- fish virus infection (Plant and LaPatra 2011). ELISA binant glycoproteins. 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