Biotechnol Lett (2015) 37:539–544 DOI 10.1007/s10529-014-1720-1
ORIGINAL RESEARCH PAPER
Oral vaccination of mice with Tremella fuciformis yeast-like conidium cells expressing HBsAg Dong-Il Shin • Kyu-Seon Song • Hee-Sung Park
Received: 28 August 2014 / Accepted: 28 October 2014 / Published online: 6 November 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Tremella fuciformis yeast-like conidium (YLC) cells were transformed by co-cultivation with Agrobacterium cells harboring the hepatitis B surface antigen (HBsAg) gene construct under the control of the CaMV35S promoter. Integration of HBsAg DNA into the YLC genome was confirmed by PCR and dotblot hybridization. Immunoblotting verified expression of the recombinant protein. Oral administration of YLC cells expressing HBsAg in mice significantly increased anti-HBsAg antibody titer levels using a double prime-boost strategy that combined parenteral and oral HBsAg boosters. Keywords Hepatitis B surface antigen Oral administration Transformation Tremella fuciformis
Introduction Hepatitis B virus is widespread and highly detrimental to human health. Although a safe and effective hepatitis B vaccine is readily available in many
D.-I. Shin K.-S. Song H.-S. Park (&) Department of Biotechnology, Catholic University of Daegu, Gyeongsan 712-702, Korea e-mail: [email protected]
countries, the associated cost makes it less accessible in poorer countries. As an alternative, a cost-effective plant-based oral vaccine with a simple administration process and enhanced compliance has been developed (Thanavala et al. 2005; Mishra et al. 2008). However, public concerns regarding contamination of the food supply by transgenic plants have resulted in strict regulations restricting the use of transgenic plant products. Plant cell and hairy root expression systems may serve as alternatives, as they are generally cultivated in controlled facilities (Hellwig et al. 2004; Sunil Kumar et al. 2006; Tele and Timko 2010). Tremella fuciformis, a traditional edible mushroom found in China, grows very rapidly by vegetative propagation. Submerged fermentation systems of T. fuciformis are optimized to achieve high yields of bioactive exopolysaccharides, which are associated with immune strengthening and antitumor activity (Cho et al. 2006). Yeast-like conidium (YLC) cells of T. fuciformis can potentially be used as microbial cell factory systems, considering their monokaryotic nature, their yeast-like growth in submerged fermentation devices, and their human-like post-translational modification system (Berends et al. 2009). We previously reported a highly efficient Agrobacteriummediated method coupled with mechanical wounding to efficiently transform YLC cells (Shin and Park 2013). Here, we developed a YLC cell-based factory system by delivering the HBsAg gene into YLC cells and assessed the oral immunogenicity of HBsAgexpressing YLC transformants in mice.
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Materials and methods Fungal growth Tremella fuciformis strain KACC 42232 was obtained from the RDA-GeneBank Information Center (Suwon, Korea). T. fuciformis YLC cells were maintained in either potato/dextrose/agar (PDA) or broth (PDB) at 25 °C. DNA construction and transformation HBsAg coding regions were PCR-amplified from pAM6 (ATCC 40101) using HBsAg-specific primers (forward, 50 -ACGGATCCCGGGTCAACGAACATG GAGAACATCACATCA-30 ; reverse, 50 -CCGGAG CTCTAGGGTTTAAATGTATACCCAAAGAC-30 ). The resulting PCR products were cloned as BamHI/ Fig. 1 HBsAg expression in Tremella fuciformis yeast-like conidium (YLC) cells. a Schematic diagram of the pBHBsAg and pCHBsAg construct. b Introduction of pBHBsAg into YLC cells (left panel) was confirmed by PCR amplification (right upper panel) of 700 bp DNA fragments (arrow) from transformed (1–10) and nontransformed (c) YLC cells. M = 250 bp DNA ladder. The right middle panel shows the results of dot blot hybridization to verify DNA integration in transformed (1–10) and nontransformed (c) YLC cells. pBHBsAg plasmid DNA is indicated as ?c. The right lower panel shows detection of HBsAg expression by western blotting. Recombinant HBsAg (Ag) and nontransformed (c) and transformed (1–8) samples are indicated. The arrow indicates bands migrating at an apparent molecular weight identical to that of yeast-derived rHBsAg
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SacI fragments into pBI121DGUS, thereby generating the expression vector pBHBsAg. The HindIII/EcoRI fragment containing the CaMV35S promoter: HBsAg:nopaline synthase terminator region was removed from pBHBsAg and subcloned into the corresponding restriction sites of pCambia1300, thereby constructing pCHBsAg (Fig. 1a). To transform YLC cells, pCHBsAg, which harbors the hygromycin phosphotransferase (hph) gene, was introduced into the Agrobacterium tumefaciens strain LBA4404 by the freeze–thaw method. Following mechanical wounding-assisted Agrobacterium-mediated transformation (Shin and Park 2013), transformants were selected with the appropriate antibiotics (30 lg hygromycin ml-1 and 250 lg cefotaxime ml-1). Similarly, YLC cells were transformed with pBHBsAg, which harbors the neomycin phosphotransferase (nptII) gene. Transformants were selected with the
Biotechnol Lett (2015) 37:539–544
appropriate antibiotics (200 lg kanamycin ml-1 and 250 lg cefotaxime ml-1). PCR and dot blot hybridization Genomic DNA was isolated from YLC cells according to the glass bead yeast DNA preparation method (Hoffman and Winston 1987). DNA integration was confirmed by PCR using HBsAg-specific primers under the following thermocycling conditions: 94 °C, 5 min; 30 9 (94 °C, 30 s; 52 °C, 30 s; 72 °C, 1 min); and 72 °C, 3 min. For dot-blot hybridization, 10–15 lg boiled genomic DNA was loaded onto a nitrocellulose membrane and hybridized in hybridization buffer (5 9 SSC, 0.1 % SDS, and 5 % dextran sulfate) for 24 h at 60 °C. Hybridization signals were detected using the BrightStar detection system (Ambion Inc., USA). The 700 bp DNA that was PCRamplified from the pBHBsAg plasmid DNA was labeled using the BrightStar Psoralen-Biotin Non radioisotopic Labeling Kit (Ambion Inc., USA). Western blotting To detect HBsAg protein, YLC cells grown in PDB for 48 h with shaking (180 rpm, 25 °C) were harvested by centrifugation (15,0009g, 10 min). Pelleted cells were lysed and solubilized in hot alkali buffer condition (Kushnirov 2000). Protein extracts were resolved by SDS-PAGE on 12 % gels and electoblotted onto PVDF membranes for immunoreaction with anti-HBsAg IgG. Immunoreactive bands were detected using the ECL western blotting detection system (GE Healthcare, UK). The protein samples were dialyzed against PBS, and the concentration of HBsAg was measured using an AxSYM HBsAg (V2) kit and an IMx detector (Abbott, USA). Oral vaccination BALB/c mice were fasted overnight before being fed YLC cells. Non-transformed YLC cells were used as the control. For each experiment, ten mice were fed transformed YLC cells containing the equivalent of approx. 0.14 lg HBsAg twice per week according to the immunization protocol. We selected this immune dose of HBsAg based on preliminary oral vaccination studies. Mice were injected with a subimmunogenic dose (0.5 lg of yeast-derived rHBsAg) of commercial
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vaccine (Green Cross, Korea) as a primer or booster. Anti-HBsAg antibodies from mouse serum were titrated using the AUSAB EIA in an IMx detector (Abbott, USA). The AUSAB standard panel was included with each assay performed, and this permitted the conversion of OD values to mIU ml-1. All of our mice maintenance and experimental procedures were approved by the IACUC of Catholic University of Daegu (Approval No. CUD2011-003).
Results Cell transformation and selection The hph gene, which has been used extensively to transform many different fungi, allows clear discrimination between transformed and nontransformed cells (Hynes 1996). Initially, we delivered the pCHBsAg plasmid into YLC cells, which were then subjected to hygromycin selection. Unfortunately, many selected transformants that were verified for HBsAg DNA integration by PCR grew slower than the wild type cells (data not shown). It is therefore possible that hygromycin displayed some level of toxicity even to transformants containing the hph gene. Use of hygromycin selection was thus determined to be impractical for YLC transformation. We therefore delivered pBHBsAg to the YLC cells and selected with kanamycin. The growth of wild-type YLC cells was not influenced by 100 lg kanamycin ml-1, but was severely suppressed by 200 lg kanamycin ml-1. We expected that the NOS promoter would direct the expression of the nptII gene to avoid kanamycin toxicity at higher concentrations. The results indicated that large, medium and small transformed colonies were present (Fig. 1b). Large colonies were selected because no significant difference was observed in the growth rates between wildtype and large colonies. DNA integration and protein expression The presence of the transferred HBsAg gene was confirmed by PCR. Successful amplification of a 700 bp DNA product is shown in Fig. 1b. No amplification products were obtained for wild-type cells. The integrated HBsAg gene was also successfully detected in all transformants by dot blot hybridization
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Biotechnol Lett (2015) 37:539–544 b Fig. 2 Measurement of plasma anti-HBsAg titer in mice.
Single prime-boost vaccination (a): 1 week following commercial vaccine priming, mice were given HBsAg YLC or the vehicle for YLC dissolution (distilled water) twice a week for 4 weeks. After a 2-week resting period, they were given YLC twice a week for 4 weeks. Blood was obtained through retroorbital bleeding 3 days after the last administration of the YLCs. Values represent mean ± SEM for ten mice per group. The HBsAg antibody titers of the mice administered HBsAg YLC were significantly higher (p \ 0.05) than those of the control mice (prime only). Double prime-boost vaccination (b): 1 week following commercial vaccine priming, mice were given HBsAg YLC twice a week for 4 weeks. A second commercial vaccine priming was performed 4 days after the last administration of HBsAg YLC. The next-day administration of HBsAg YLC was continued twice a week for 4 weeks. Cardiac puncture was performed to collect blood 3 days after the last HBsAg YLC administration. Values represent mean ± SEM for ten mice per group. The HBsAg antibody titers measured after eight and 16 repetitions of double prime-boost vaccination were significantly higher (p \ 0.05) than those measured after single prime-boost vaccination
Immunogenicity of YLC cells
(Fig. 1b). The stability of the transferred HBsAg gene was confirmed by PCR after several passages over 3 months (data not shown). Western blotting was used to investigate the production of HBsAg protein in transgenic YLC cells. Immunoreactive bands from the transformants were compared to yeast rHBsAg as a positive control and a non-transformant as a negative control (Fig. 1b). All tested transformants yielded bands migrating at an apparent molecular weight identical to that of yeast-derived rHBsAg. The concentration of HBsAg ranged from 0.4 to 1.4 lg g-1 dried YLC cells for different transformants, which was consistent with the expression levels reported in transgenic plant studies (Guana et al. 2010).
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The immunogenicity of YLC cells was tested by orally administering YLC cells expressing the highest HBsAg protein levels to mice. Retro-orbital bleeding was used to assess the presence of specific antibodies. Detectable primary antibodies were not measured after oral administration of transgenic YLC cells alone within 4 weeks. In the current study, the mice were injected with a subimmunogenic dose (0.5 lg HBsAg) of commercial vaccine and then boosted with transgenic YLC cells for various time periods. After a single injection for priming, their antibody titers increased to 68 ± 27 mIU ml-1 and then declined. Within several weeks, the mice boosted with transgenic YLC cells developed serum antibody concentrations of 301 ± 125 mIU ml-1 and 332 ± 167 mIU ml-1 after being boosted 8 and 16 times, respectively (Fig. 2a). The levels of specific antibodies exceeded the protective level of 100 mIU ml-1 when we followed the vaccination scheme consisting of three doses in humans (van Hatrtum 1995). Oral booster immunizations augmented antibody titer levels. It was evident that parenteral priming led to a recall response induced by YLC-derived HBsAg. There was no booster response in mice fed with wild-type YLC cells. We administered an additional booster with commercial vaccine and performed subsequent YLC immunization for 4 weeks after the first primer and booster. Antibody titer levels in serum were
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6,369 ± 1,962 mIU ml-1, while they were only 665 ± 203 mIU ml-1 after the first primer and booster (Fig. 2b). This almost 10-fold increase indicated that transgenic YLC was an excellent immuno-booster effector in combination with a primer and booster injection of the commercial vaccine.
Discussion The use of plants as bioreactors to produce edible vaccines and biopharmaceuticals is well established. The products can be given directly by oral delivery (Guana et al. 2010). Plants are increasingly used to express antigens for vaccines, as demonstrated by the rising number of reports of transgenic plant expression of various antigens. The potato plant has been extensively studied for the production of edible plant vaccines since the first report on oral immunization with plant-based vaccines. Potato-based HBsAg was determined to be orally immunogenic in mice (Kong et al. 2001) and humans (Thanavala et al. 2005). Tremella fuciformis is an edible mushroom that grows rapidly in liquid cultivation. Submerged fermentation systems of T. fuciformis have been well optimized. YLC cells can be transformed into a potential biofactory system using the protoplast method (Guo et al. 2009), electroporation (Guo et al. 2008) or Agrobacterium (Shin and Park 2013). In this study to develop a YLC-based vaccine, we delivered the HBsAg gene under the control of the CaMV35S promoter into T. fuciformis YLC cells using the Agrobacterium method. It should be noted that the hph gene has been employed extensively for fungal transformation. In contrast, the nptII gene, which is widely used in the direct transformation of various plant species for selection after Agrobacterium-mediated transformation, has never been employed for fungal species because of their resistance to extremely high concentrations of kanamycin. However, selection of YLC transformants with kanamycin at higher concentrations yielded the best results in terms of cell growth and genetic stability. We confirmed HBsAg DNA integration and protein expression using standard molecular techniques in transformed YLC cells. The glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter is one of the most widely used promoters in basidiomycete vectors (Frandsen 2011). Gpd promoters from various basidiomycete species
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have been used for laccase (Alves et al. 2004; Kilaru et al. 2006) and peroxidase production (Sollewijn Gelpke et al. 1999) or for expression of the green fluorescent protein gene (gfp) (Cormack 1998; Ma et al. 2001) or the hph gene (Hirano et al. 2000; Kuo et al. 2004). Current data do not reveal the requirements for highly efficient expression of heterologous genes in basidiomycetes (Kilaru and Ku¨es 2005). In plants, the CaMV35S promoter is preferred because it is a strong, constitutive promoter. Some basidiomycete vectors also have employed the CaMV35S promoter to express hph (Sharma and Kuhad 2010) or b-glucuronidase (GUS) reporter gene (Sun et al. 2002). We also used it for driving HBsAg expression in T. fuciformis in this study. Based on the small number of available studies, it is not evident that the CaMV35S promotercontaining vector efficiently improves heterologous protein expression. The gpd promoter from T. fuciformis has been isolated and used for expression of EGFP protein in T. fuciformis (Sun et al. 2009). Like the CaMV35S promoter, this promoter can be tested for heterologous gene expression in T. fuciformis. Further investigation will be conducted.
Conclusions Oral administration of HBsAg-expressing YLC cells to mice significantly elevated their serum anti-HBsAg antibody titer levels. The appropriate combinations of parenteral and oral delivery could provide a successful immune response. The lack of toxicity of T. fuciformis YLC cells makes it an attractive vaccine vehicle. Its safety in humans has been widely established from its widespread consumption. It can also be easily engineered to express antigen in large quantities, can be grown rapidly, and is very stable. In this context, a YLC-based system might be a potential vaccine vector for direct and rapid application. Acknowledgments This work was supported by the Research Funds for 2011 from the Catholic University of Daegu.
References Alves AM, Record E, Lomascolo A, Scholtmeijer K, Asther M, Wessels JG, Wo¨sten HA (2004) Highly efficient production of laccase by the basidiomycete Pycnoporus cinnabarinus. Appl Environ Microbiol 70:6379–6384
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544 Berends E, Scholtmeijer K, Wo¨sten HAB, Bosch D, Lugones LG (2009) The use of mushroom-forming fungi for the production of N-glycosylated therapeutic proteins. Trends Microbiol 17:439–443 Cho EJ, Oh JY, Chang HY, Yun JW (2006) Production of exopolysaccharides by submerged mycelial culture of a mushroom Tremella fuciformis. J Biotechnol 127:129–140 Cormack B (1998) Green fluorescent protein as a reporter of transcription and protein localization in fungi. Curr Opin Microbiol 1:406–410 Frandsen RJN (2011) A guide to binary vectors and strategies for targeted genome modification in fungi using Agrobacterium tumefaciens-mediated transformation. J Microbiol Methods 87:247–262 Guana ZJ, Guoa B, Huoc YL, Guand ZP, Weia YH (2010) Overview of expression of hepatitis B surface antigen in transgenic plants. Vaccine 28:7351–7362 Guo L, Liu Y, Zhao S, Liu E, Lin J (2008) Highly efficient transformation of intact yeast-like conidium cells of Tremella fuciformis by electroporation. Sci China Ser C Life Sci 51:932–940 Guo L, Liu E, Wang J, Lin J (2009) Development of highly efficient transformation system of yeast-like conidia of Tremella fuciformis. Agric Sci China 8:268–275 Hellwig S, Drossard J, Twyman RM, Fischer R (2004) Plant cell culture for the production of recombinant proteins. Nat Biotechnol 22:1415–1422 Hirano T, Sato T, Yaegashi K, Enei H (2000) Efficient transformation of the edible basidiomycete Lentinus edodes with a vector using a glyceraldehyde-3-phosphate dehydrogenase promoter to hygromycin B resistance. Mol Gen Genet 263:1047–1052 Hoffman CS, Winston F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272 Hynes MJ (1996) Genetic transformation of filamentous fungi. J Genet 75:297–311 Kilaru S, Ku¨es U (2005) Comparison of gpd genes and their protein products in basidiomycetes. Fungal Genet Newsl 52:18–23 Kilaru S, Hoegger PJ, Majcherczyk A, Burns C, Shishido K, Bailey A, Foster GD, Ku¨es U (2006) Expression of laccase gene lcc1 in Coprinopsis cinerea under control of various basidiomycetous promoters. Appl Microbiol Biotechnol 71:200–210 Kong Q, Richter L, Yang YF, Arntzen CJ, Mason HS, Thanavala Y (2001) Oral immunization with hepatitis B surface
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ORIGINAL RESEARCH PAPER
Oral vaccination of mice with Tremella fuciformis yeast-like conidium cells expressing HBsAg Dong-Il Shin • Kyu-Seon Song • Hee-Sung Park
Received: 28 August 2014 / Accepted: 28 October 2014 / Published online: 6 November 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Tremella fuciformis yeast-like conidium (YLC) cells were transformed by co-cultivation with Agrobacterium cells harboring the hepatitis B surface antigen (HBsAg) gene construct under the control of the CaMV35S promoter. Integration of HBsAg DNA into the YLC genome was confirmed by PCR and dotblot hybridization. Immunoblotting verified expression of the recombinant protein. Oral administration of YLC cells expressing HBsAg in mice significantly increased anti-HBsAg antibody titer levels using a double prime-boost strategy that combined parenteral and oral HBsAg boosters. Keywords Hepatitis B surface antigen Oral administration Transformation Tremella fuciformis
Introduction Hepatitis B virus is widespread and highly detrimental to human health. Although a safe and effective hepatitis B vaccine is readily available in many
D.-I. Shin K.-S. Song H.-S. Park (&) Department of Biotechnology, Catholic University of Daegu, Gyeongsan 712-702, Korea e-mail: [email protected]
countries, the associated cost makes it less accessible in poorer countries. As an alternative, a cost-effective plant-based oral vaccine with a simple administration process and enhanced compliance has been developed (Thanavala et al. 2005; Mishra et al. 2008). However, public concerns regarding contamination of the food supply by transgenic plants have resulted in strict regulations restricting the use of transgenic plant products. Plant cell and hairy root expression systems may serve as alternatives, as they are generally cultivated in controlled facilities (Hellwig et al. 2004; Sunil Kumar et al. 2006; Tele and Timko 2010). Tremella fuciformis, a traditional edible mushroom found in China, grows very rapidly by vegetative propagation. Submerged fermentation systems of T. fuciformis are optimized to achieve high yields of bioactive exopolysaccharides, which are associated with immune strengthening and antitumor activity (Cho et al. 2006). Yeast-like conidium (YLC) cells of T. fuciformis can potentially be used as microbial cell factory systems, considering their monokaryotic nature, their yeast-like growth in submerged fermentation devices, and their human-like post-translational modification system (Berends et al. 2009). We previously reported a highly efficient Agrobacteriummediated method coupled with mechanical wounding to efficiently transform YLC cells (Shin and Park 2013). Here, we developed a YLC cell-based factory system by delivering the HBsAg gene into YLC cells and assessed the oral immunogenicity of HBsAgexpressing YLC transformants in mice.
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Materials and methods Fungal growth Tremella fuciformis strain KACC 42232 was obtained from the RDA-GeneBank Information Center (Suwon, Korea). T. fuciformis YLC cells were maintained in either potato/dextrose/agar (PDA) or broth (PDB) at 25 °C. DNA construction and transformation HBsAg coding regions were PCR-amplified from pAM6 (ATCC 40101) using HBsAg-specific primers (forward, 50 -ACGGATCCCGGGTCAACGAACATG GAGAACATCACATCA-30 ; reverse, 50 -CCGGAG CTCTAGGGTTTAAATGTATACCCAAAGAC-30 ). The resulting PCR products were cloned as BamHI/ Fig. 1 HBsAg expression in Tremella fuciformis yeast-like conidium (YLC) cells. a Schematic diagram of the pBHBsAg and pCHBsAg construct. b Introduction of pBHBsAg into YLC cells (left panel) was confirmed by PCR amplification (right upper panel) of 700 bp DNA fragments (arrow) from transformed (1–10) and nontransformed (c) YLC cells. M = 250 bp DNA ladder. The right middle panel shows the results of dot blot hybridization to verify DNA integration in transformed (1–10) and nontransformed (c) YLC cells. pBHBsAg plasmid DNA is indicated as ?c. The right lower panel shows detection of HBsAg expression by western blotting. Recombinant HBsAg (Ag) and nontransformed (c) and transformed (1–8) samples are indicated. The arrow indicates bands migrating at an apparent molecular weight identical to that of yeast-derived rHBsAg
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SacI fragments into pBI121DGUS, thereby generating the expression vector pBHBsAg. The HindIII/EcoRI fragment containing the CaMV35S promoter: HBsAg:nopaline synthase terminator region was removed from pBHBsAg and subcloned into the corresponding restriction sites of pCambia1300, thereby constructing pCHBsAg (Fig. 1a). To transform YLC cells, pCHBsAg, which harbors the hygromycin phosphotransferase (hph) gene, was introduced into the Agrobacterium tumefaciens strain LBA4404 by the freeze–thaw method. Following mechanical wounding-assisted Agrobacterium-mediated transformation (Shin and Park 2013), transformants were selected with the appropriate antibiotics (30 lg hygromycin ml-1 and 250 lg cefotaxime ml-1). Similarly, YLC cells were transformed with pBHBsAg, which harbors the neomycin phosphotransferase (nptII) gene. Transformants were selected with the
Biotechnol Lett (2015) 37:539–544
appropriate antibiotics (200 lg kanamycin ml-1 and 250 lg cefotaxime ml-1). PCR and dot blot hybridization Genomic DNA was isolated from YLC cells according to the glass bead yeast DNA preparation method (Hoffman and Winston 1987). DNA integration was confirmed by PCR using HBsAg-specific primers under the following thermocycling conditions: 94 °C, 5 min; 30 9 (94 °C, 30 s; 52 °C, 30 s; 72 °C, 1 min); and 72 °C, 3 min. For dot-blot hybridization, 10–15 lg boiled genomic DNA was loaded onto a nitrocellulose membrane and hybridized in hybridization buffer (5 9 SSC, 0.1 % SDS, and 5 % dextran sulfate) for 24 h at 60 °C. Hybridization signals were detected using the BrightStar detection system (Ambion Inc., USA). The 700 bp DNA that was PCRamplified from the pBHBsAg plasmid DNA was labeled using the BrightStar Psoralen-Biotin Non radioisotopic Labeling Kit (Ambion Inc., USA). Western blotting To detect HBsAg protein, YLC cells grown in PDB for 48 h with shaking (180 rpm, 25 °C) were harvested by centrifugation (15,0009g, 10 min). Pelleted cells were lysed and solubilized in hot alkali buffer condition (Kushnirov 2000). Protein extracts were resolved by SDS-PAGE on 12 % gels and electoblotted onto PVDF membranes for immunoreaction with anti-HBsAg IgG. Immunoreactive bands were detected using the ECL western blotting detection system (GE Healthcare, UK). The protein samples were dialyzed against PBS, and the concentration of HBsAg was measured using an AxSYM HBsAg (V2) kit and an IMx detector (Abbott, USA). Oral vaccination BALB/c mice were fasted overnight before being fed YLC cells. Non-transformed YLC cells were used as the control. For each experiment, ten mice were fed transformed YLC cells containing the equivalent of approx. 0.14 lg HBsAg twice per week according to the immunization protocol. We selected this immune dose of HBsAg based on preliminary oral vaccination studies. Mice were injected with a subimmunogenic dose (0.5 lg of yeast-derived rHBsAg) of commercial
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vaccine (Green Cross, Korea) as a primer or booster. Anti-HBsAg antibodies from mouse serum were titrated using the AUSAB EIA in an IMx detector (Abbott, USA). The AUSAB standard panel was included with each assay performed, and this permitted the conversion of OD values to mIU ml-1. All of our mice maintenance and experimental procedures were approved by the IACUC of Catholic University of Daegu (Approval No. CUD2011-003).
Results Cell transformation and selection The hph gene, which has been used extensively to transform many different fungi, allows clear discrimination between transformed and nontransformed cells (Hynes 1996). Initially, we delivered the pCHBsAg plasmid into YLC cells, which were then subjected to hygromycin selection. Unfortunately, many selected transformants that were verified for HBsAg DNA integration by PCR grew slower than the wild type cells (data not shown). It is therefore possible that hygromycin displayed some level of toxicity even to transformants containing the hph gene. Use of hygromycin selection was thus determined to be impractical for YLC transformation. We therefore delivered pBHBsAg to the YLC cells and selected with kanamycin. The growth of wild-type YLC cells was not influenced by 100 lg kanamycin ml-1, but was severely suppressed by 200 lg kanamycin ml-1. We expected that the NOS promoter would direct the expression of the nptII gene to avoid kanamycin toxicity at higher concentrations. The results indicated that large, medium and small transformed colonies were present (Fig. 1b). Large colonies were selected because no significant difference was observed in the growth rates between wildtype and large colonies. DNA integration and protein expression The presence of the transferred HBsAg gene was confirmed by PCR. Successful amplification of a 700 bp DNA product is shown in Fig. 1b. No amplification products were obtained for wild-type cells. The integrated HBsAg gene was also successfully detected in all transformants by dot blot hybridization
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Biotechnol Lett (2015) 37:539–544 b Fig. 2 Measurement of plasma anti-HBsAg titer in mice.
Single prime-boost vaccination (a): 1 week following commercial vaccine priming, mice were given HBsAg YLC or the vehicle for YLC dissolution (distilled water) twice a week for 4 weeks. After a 2-week resting period, they were given YLC twice a week for 4 weeks. Blood was obtained through retroorbital bleeding 3 days after the last administration of the YLCs. Values represent mean ± SEM for ten mice per group. The HBsAg antibody titers of the mice administered HBsAg YLC were significantly higher (p \ 0.05) than those of the control mice (prime only). Double prime-boost vaccination (b): 1 week following commercial vaccine priming, mice were given HBsAg YLC twice a week for 4 weeks. A second commercial vaccine priming was performed 4 days after the last administration of HBsAg YLC. The next-day administration of HBsAg YLC was continued twice a week for 4 weeks. Cardiac puncture was performed to collect blood 3 days after the last HBsAg YLC administration. Values represent mean ± SEM for ten mice per group. The HBsAg antibody titers measured after eight and 16 repetitions of double prime-boost vaccination were significantly higher (p \ 0.05) than those measured after single prime-boost vaccination
Immunogenicity of YLC cells
(Fig. 1b). The stability of the transferred HBsAg gene was confirmed by PCR after several passages over 3 months (data not shown). Western blotting was used to investigate the production of HBsAg protein in transgenic YLC cells. Immunoreactive bands from the transformants were compared to yeast rHBsAg as a positive control and a non-transformant as a negative control (Fig. 1b). All tested transformants yielded bands migrating at an apparent molecular weight identical to that of yeast-derived rHBsAg. The concentration of HBsAg ranged from 0.4 to 1.4 lg g-1 dried YLC cells for different transformants, which was consistent with the expression levels reported in transgenic plant studies (Guana et al. 2010).
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The immunogenicity of YLC cells was tested by orally administering YLC cells expressing the highest HBsAg protein levels to mice. Retro-orbital bleeding was used to assess the presence of specific antibodies. Detectable primary antibodies were not measured after oral administration of transgenic YLC cells alone within 4 weeks. In the current study, the mice were injected with a subimmunogenic dose (0.5 lg HBsAg) of commercial vaccine and then boosted with transgenic YLC cells for various time periods. After a single injection for priming, their antibody titers increased to 68 ± 27 mIU ml-1 and then declined. Within several weeks, the mice boosted with transgenic YLC cells developed serum antibody concentrations of 301 ± 125 mIU ml-1 and 332 ± 167 mIU ml-1 after being boosted 8 and 16 times, respectively (Fig. 2a). The levels of specific antibodies exceeded the protective level of 100 mIU ml-1 when we followed the vaccination scheme consisting of three doses in humans (van Hatrtum 1995). Oral booster immunizations augmented antibody titer levels. It was evident that parenteral priming led to a recall response induced by YLC-derived HBsAg. There was no booster response in mice fed with wild-type YLC cells. We administered an additional booster with commercial vaccine and performed subsequent YLC immunization for 4 weeks after the first primer and booster. Antibody titer levels in serum were
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6,369 ± 1,962 mIU ml-1, while they were only 665 ± 203 mIU ml-1 after the first primer and booster (Fig. 2b). This almost 10-fold increase indicated that transgenic YLC was an excellent immuno-booster effector in combination with a primer and booster injection of the commercial vaccine.
Discussion The use of plants as bioreactors to produce edible vaccines and biopharmaceuticals is well established. The products can be given directly by oral delivery (Guana et al. 2010). Plants are increasingly used to express antigens for vaccines, as demonstrated by the rising number of reports of transgenic plant expression of various antigens. The potato plant has been extensively studied for the production of edible plant vaccines since the first report on oral immunization with plant-based vaccines. Potato-based HBsAg was determined to be orally immunogenic in mice (Kong et al. 2001) and humans (Thanavala et al. 2005). Tremella fuciformis is an edible mushroom that grows rapidly in liquid cultivation. Submerged fermentation systems of T. fuciformis have been well optimized. YLC cells can be transformed into a potential biofactory system using the protoplast method (Guo et al. 2009), electroporation (Guo et al. 2008) or Agrobacterium (Shin and Park 2013). In this study to develop a YLC-based vaccine, we delivered the HBsAg gene under the control of the CaMV35S promoter into T. fuciformis YLC cells using the Agrobacterium method. It should be noted that the hph gene has been employed extensively for fungal transformation. In contrast, the nptII gene, which is widely used in the direct transformation of various plant species for selection after Agrobacterium-mediated transformation, has never been employed for fungal species because of their resistance to extremely high concentrations of kanamycin. However, selection of YLC transformants with kanamycin at higher concentrations yielded the best results in terms of cell growth and genetic stability. We confirmed HBsAg DNA integration and protein expression using standard molecular techniques in transformed YLC cells. The glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter is one of the most widely used promoters in basidiomycete vectors (Frandsen 2011). Gpd promoters from various basidiomycete species
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have been used for laccase (Alves et al. 2004; Kilaru et al. 2006) and peroxidase production (Sollewijn Gelpke et al. 1999) or for expression of the green fluorescent protein gene (gfp) (Cormack 1998; Ma et al. 2001) or the hph gene (Hirano et al. 2000; Kuo et al. 2004). Current data do not reveal the requirements for highly efficient expression of heterologous genes in basidiomycetes (Kilaru and Ku¨es 2005). In plants, the CaMV35S promoter is preferred because it is a strong, constitutive promoter. Some basidiomycete vectors also have employed the CaMV35S promoter to express hph (Sharma and Kuhad 2010) or b-glucuronidase (GUS) reporter gene (Sun et al. 2002). We also used it for driving HBsAg expression in T. fuciformis in this study. Based on the small number of available studies, it is not evident that the CaMV35S promotercontaining vector efficiently improves heterologous protein expression. The gpd promoter from T. fuciformis has been isolated and used for expression of EGFP protein in T. fuciformis (Sun et al. 2009). Like the CaMV35S promoter, this promoter can be tested for heterologous gene expression in T. fuciformis. Further investigation will be conducted.
Conclusions Oral administration of HBsAg-expressing YLC cells to mice significantly elevated their serum anti-HBsAg antibody titer levels. The appropriate combinations of parenteral and oral delivery could provide a successful immune response. The lack of toxicity of T. fuciformis YLC cells makes it an attractive vaccine vehicle. Its safety in humans has been widely established from its widespread consumption. It can also be easily engineered to express antigen in large quantities, can be grown rapidly, and is very stable. In this context, a YLC-based system might be a potential vaccine vector for direct and rapid application. Acknowledgments This work was supported by the Research Funds for 2011 from the Catholic University of Daegu.
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