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Oral immunisation of mice with transgenic rice calli expressing cholera toxin B subunit fused to consensus dengue cEDIII antigen induces antibodies to all four dengue serotypes

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Abstract

Dengue virus (DENV) infection is an emerging global health threat. DENV consists of four distinct serotypes, necessitating a tetravalent vaccine. In this study, expression of consensus envelope protein domain III (cEDIII) fused to cholera toxin B subunit (CTB) in transgenic rice calli was improved using the luminal binding protein BiP at the N-terminus and the SEKDEL signal sequences at the C-terminus, targeting the recombinant protein to endoplasmic reticulum (ER). We found that the fusion protein showed higher levels of expression when compared to the fusion proteins using rice amylase 3D (RAmy3D) or CTB native signal sequence only. The CTB-cEDIII fusion protein was evaluated as an oral dengue vaccine candidate in mice. Serotype specific systemic IgG antibodies and specific IgA response in feces were detected and furthermore, T cell proliferation and high frequency antibody-secreting B cells were detected in the spleen. These results suggest the possible use of plant-based dengue tetravalent vaccine targeted to the mucosal immune system for induction of systemic and mucosal immune responses to DENV infection.

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References

  • Blanchard TG, Lycke N, Czinn SJ, Nedrud JG (1998) Recombinant cholera toxin B subunit is not an effective mucosal adjuvant for oral immunization of mice against Helicobacter felis. Immunology 94:22–27

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bourguin I, Chardes T, Bout D (1993) Oral immunization with toxoplasma-gondii antigens in association with cholera-toxin induces enhanced protective and cell-mediated-immunity in C57bl/6 mice. Infect Immun 61:2082–2088

    CAS  PubMed  PubMed Central  Google Scholar 

  • Boyhan D, Daniell H (2011) Low-cost production of proinsulin in tobacco and lettuce chloroplasts for injectable or oral delivery of functional insulin and C-peptide. Plant Biotechnol J 9:585–598

    Article  CAS  PubMed  Google Scholar 

  • Capeding MR, Tran NH, Hadinegoro SR, Ismail HI, Chotpitayasunondh T, Chua MN, Luong CQ, Rusmil K, Wirawan DN, Nallusamy R, Pitisuttithum P, Thisyakorn U, Yoon IK, van der Vliet D, Langevin E, Laot T, Hutagalung Y, Frago C, Boaz M, Wartel TA, Tornieporth NG, Saville M, Bouckenooghe A (2014) Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial. Lancet 384:1358–1365

    Article  CAS  PubMed  Google Scholar 

  • Elson CO, Ealding W (1984) Generalized systemic and mucosal immunity in mice after mucosal stimulation with cholera-toxin. J Immunol 132:2736–2741

    CAS  PubMed  Google Scholar 

  • Guo L, Liu KM, Xu GX, Li XK, Tu JJ, Tang F, Xing YY, Xi T (2012) Prophylactic and therapeutic efficacy of the epitope vaccine CTB-UA against Helicobacter pylori infection in a BALB/c mice model. Appl Microbiol Biotechnol 95:1437–1444

    Article  CAS  PubMed  Google Scholar 

  • Hadinegoro SR, Arredondo-Garcia JL, Capeding MR, Deseda C, Chotpitayasunondh T, Dietze R, Muhammad Ismail HI, Reynales H, Limkittikul K, Rivera-Medina DM, Tran HN, Bouckenooghe A, Chansinghakul D, Cortes M, Fanouillere K, Forrat R, Frago C, Gailhardou S, Jackson N, Noriega F, Plennevaux E, Wartel TA, Zambrano B, Saville M (2015) Efficacy and long-term safety of a dengue vaccine in regions of endemic disease. N Engl J Med 373:1195–1206

    Article  CAS  PubMed  Google Scholar 

  • Haq TA, Mason HS, Clements JD, Arntzen CJ (1995) Oral immunization with a recombinant bacterial antigen produced in transgenic plants. Science 268:714–716

    Article  CAS  PubMed  Google Scholar 

  • Holmgren J, Czerkinsky C, Lycke N, Svennerholm AM (1994) Strategies for the induction of immune-responses at mucosal surfaces making use of cholera-toxin-B subunit as immunogen, carrier, and adjuvant. Am J Trop Med Hyg 50:42–54

    CAS  PubMed  Google Scholar 

  • Huang KJ, Yang YC, Lin YS, Huang JH, Liu HS, Yeh, Chen SH, Liu CC, Lei HY (2006) The dual-specific binding of dengue virus and target cells for the antibody-dependent enhancement of dengue virus infection. J Immunol 176:2825–2832

    Article  CAS  PubMed  Google Scholar 

  • Huang LF, Tan CC, Yeh JF, Liu HY, Liu YK, Ho SL, Lu CA (2015) Efficient secretion of recombinant proteins from rice suspension-cultured cells modulated by the choice of signal peptide. PLos One 10:e0140812

    Article  PubMed  PubMed Central  Google Scholar 

  • Jackson RJ, Fujihashi K, Xuamano JC, Kiyono H, Elson CO, Mcghee JR (1993) Optimizing oral vaccines-induction of systemic and mucosal B Cell and antibody-responses to tetanus toxoid by use of cholera-toxin as an adjuvant. Infect Immun 61:4272–4279

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kapusta J, Modelska A, Pniewski T, Figlerowicz M, Jankowski K, Lisowa O, Plucienniczak A, Koprowski H, Legocki AB (2001) Oral immunization of human with transgenic lettuce expressing hepatitis B surface antigen. Prog Basic Clin Immunol 495:299–303

    Article  CAS  Google Scholar 

  • Kashima K, Yuki Y, Mejima M, Kurokawa S, Suzuki Y, Minakawa S, Takeyama N, Fukuyama Y, Azegami T, Tanimoto T, Kuroda M, Tamura M, Gomi Y, Kiyono H (2016) Good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants. Plant Cell Rep 35:667–679

    Article  CAS  PubMed  Google Scholar 

  • Kim TG, Huy NX, Kim MY, Jeong DK, Jang YS, Yang MS, Langridge WH, Lee JY (2009) Immunogenicity of a cholera toxin B subunit Porphyromonas gingivalis fimbrial antigen fusion protein expressed in E. coli. Mol Biotechnol 41:157–164

    Article  CAS  PubMed  Google Scholar 

  • Kim SH, Seo KW, Kim J, Lee KY, Jang YS (2010) The M cell-targeting ligand promotes antigen delivery and induces antigen-specific immune responses in mucosal vaccination. J Immunol 185:5787–5795

    Article  CAS  PubMed  Google Scholar 

  • Kim TG, Kim MY, Yang MS (2010) Cholera toxin B subunit-domain III of dengue virus envelope glycoprotein E fusion protein production in transgenic plants. Protein Expr Purif 74:236–241

    Article  CAS  PubMed  Google Scholar 

  • Kim MY, Yang MS, Kim TG (2012) Expression of a consensus dengue virus envelope protein domain III in transgenic callus of rice. Plant Cell Tiss Organ Cult 109:509–515

    Article  CAS  Google Scholar 

  • Kim MY, Chung ND, Yang MS, Kim TG (2013) Expression of a cholera toxin B subunit and consensus dengue virus envelope protein domain III fusion gene in transgenic rice callus. Plant Cell Tiss Organ Cult 112:311–320

    Article  CAS  Google Scholar 

  • Lam SK (2013) Challenges in reducing dengue burden; diagnostics, control measures and vaccines. Expert Rev Vaccine 12:995–1010

    Article  CAS  Google Scholar 

  • Leng CH, Liu SJ, Tsai JP, Li YS, Chen MY, Liu HH, Lien SP, Yueh A, Hsiao KN, Lai LW, Liu FC, Chong P, Chen HW (2009) A novel dengue vaccine candidate that induces cross-neutralizing antibodies and memory immunity. Microbes Infect 11:288–295

    Article  CAS  PubMed  Google Scholar 

  • Mason HS, Ball JM, Shi JJ, Jiang X, Estes MK, Arntzen CJ (1996) Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proc Natl Acad Sci USA 93:5335–5340

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • McGarvey PB, Hammond J, Dienelt MM, Hooper DC, Fu ZF, Dietzschold B, Koprowski H, Michaels FH (1995) Expression of the rabies virus glycoprotein in transgenic tomatoes. Biotechnology (NY) 13:1484–1487

    Article  CAS  PubMed  Google Scholar 

  • Merlin M, Gecchele E, Capaldi S, Pezzotti M, Avesani L (2014) Comparative evaluation of recombinant protein production in different biofactories: the green perspective. Biomed Res Int. doi:10.1155/2014/136419

    PubMed  PubMed Central  Google Scholar 

  • Messina JP, Brady OJ, Scott TW, Zou C, Pigott DM, Duda KA, Bhatt S, Katzelnick L, Howes RE, Battle KE, Simmons CP, Hay SI (2014) Global spread of dengue virus types: mapping the 70 year history. Trends Microbiol 22:138–146

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Min MK, Kim SJ, Miao Y, Shin J, Jiang LW, Hwang I (2007) Overexpression of Arabidopsis AGD7 causes relocation of golgi-localized proteins to the endoplasmic reticulum and inhibits protein trafficking in plant cells. Plant Physiol 143:1601–1614

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Moeller L, Gan QL, Wang K (2009) A bacterial signal peptide is functional in plants and directs proteins to the secretory pathway. J Exp Bot 60:3337–3352

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mutreja A, Kim DW, Thomson NR, Connor TR, Lee JH, Kariuki S, Croucher NJ, Choi SY, Harris SR, Lebens M, Niyogi SK, Kim EJ, Ramamurthy T, Chun J, Wood JL, Clemens JD, Czerkinsky C, Nair GB, Holmgren J, Parkhill J, Dougan G (2011) Evidence for several waves of global transmission in the seventh cholera pandemic. Nature 477:462–465

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Peltola H, Siitonen A, Kyronseppa H, Simula I, Mattila L, Oksanen P, Kataja MJ, Cadoz M (1991) Prevention of travellers’ diarrhoea by oral B-subunit/whole-cell cholera vaccine. Lancet 338:1285–1289

    Article  CAS  PubMed  Google Scholar 

  • Rask C, Holmgren J, Fredriksson M, Lindblad M, Nordstrom I, Sun JB, Czerkinsky C (2000) Prolonged oral treatment with low doses of allergen conjugated to cholera toxin B subunit suppresses immunoglobulin E antibody responses in sensitized mice. Clin Exp Allergy 30:1024–1032

    Article  CAS  PubMed  Google Scholar 

  • Sanchez J, Holmgren J (2008) Cholera toxin structure, gene regulation and pathophysiological and immunological aspects. Cell Mol Life Sci 65:1347–1360

    Article  CAS  PubMed  Google Scholar 

  • Shin YJ, Hong SY, Kwon TH, Jang YS, Yang MS (2003) High level of expression of recombinant human granulocyte–macrophage colony stimulating factor in transgenic rice cell suspension culture. Biotechnol Bioeng 82:778–783

    Article  CAS  PubMed  Google Scholar 

  • Soejima Y, Lee JM, Nagata Y, Mon H, Iiyama K, Kitano H, Matsuyama M, Kusakabe T (2013) Comparison of signal peptides for efficient protein secretion in the baculovirus-silkworm system. Cent Eur J Biol 8:1–7

    CAS  Google Scholar 

  • Sun JB, Holmgren J (1994) Czerkinsky C: cholera-toxin B-subunit-an efficient transmucosal carrier-delivery system for induction of peripheral immunological-tolerance. Proc Natl Acad Sci USA 91: 10795–10799

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tacket CO, Mason HS, Losonsky G, Estes MK, Levine MM, Arntzen CJ (2000) Human immune responses to a novel Norwalk virus vaccine delivered in transgenic potatoes. J Infect Dis 182:302–305

    Article  CAS  PubMed  Google Scholar 

  • Tacket CO, Pasetti MF, Edelman R, Howard JA, Streatfield S (2004) Immunogenicity of recombinant LT-B delivered orally to humans in transgenic corn. Vaccine 22:4385–4389

    Article  CAS  PubMed  Google Scholar 

  • Villar L, Dayan GH, Arredondo-Garcia JL, Rivera DM, Cunha R, Deseda C, Reynales H, Costa MS, Morales-Ramirez JO, Carrasquilla G, Rey LC, Dietze R, Luz K, Rivas E, Miranda Montoya MC, Cortes Supelano M, Zambrano B, Langevin E, Boaz M, Tornieporth N, Saville M, Noriega F (2015) Efficacy of a tetravalent dengue vaccine in children in Latin America. N Engl J Med 372:113–123

    Article  PubMed  Google Scholar 

  • Weiner HL, da Cunha AP, Quintana F, Wu H (2011) Oral tolerance. Immunol Rev 241:241–259

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang P, Yang QB, Balkovetz DF, Lewis JP, Clements JD, Michalek SM, Katz J (2005) Effectiveness of the B subunit of cholera toxin in potentiating immune responses to the recombinant hemagglutinin/adhesin domain of the gingipain Kgp from Porphyromonas gingivalis. Vaccine 23:4734–4744

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This study was supported by NRF-2014K1B1A1073861 through the National Research Foundation (NRF) funded by Korean Ministry of Science, ICT & Future Planning. The study was also supported by the BK21 Plus program in the Departments of Bioactive Material Sciences award to Mr. B-Y Kim. We thank Dr Hwang (POSTECH, Pohwang, Korea) for providing the genomic DNA of BiP signal sequence, Dr. Kim (Chonbuk National University) for scientific advice and Mr. Park (Chonbuk National University) for helping with the analysis of cellular responses.

Author contributions

M-Y. Kim, M-S. Yang and Y-S. Jang conceived and designed the overall study. M-Y. Kim and S-M Oh generated the constructs and performed expression experiments. B-Y. Kim performed the animal experiments and analysed the data. M-S. Yang, Y-S. Jang and R.R. advised on dengue vaccine strategy. M-Y. Kim, B-Y. Kim and R. R. wrote the manuscript.

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Correspondence to Moon-Sik Yang.

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The authors declare that they have no conflict of interest.

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Mi-Young Kim and Beyng-Young Kim have contributed equally to this study.

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Kim, MY., Kim, BY., Oh, SM. et al. Oral immunisation of mice with transgenic rice calli expressing cholera toxin B subunit fused to consensus dengue cEDIII antigen induces antibodies to all four dengue serotypes. Plant Mol Biol 92, 347–356 (2016). https://doi.org/10.1007/s11103-016-0517-0

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  • DOI: https://doi.org/10.1007/s11103-016-0517-0

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