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Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 127, Issue 2, pp 369–380 | Cite as

Immunogenicity of an S1D epitope from porcine epidemic diarrhea virus and cholera toxin B subunit fusion protein transiently expressed in infiltrated Nicotiana benthamiana leaves

  • Nguyen-Xuan Huy
  • Nguyen-Quang-Duc Tien
  • Mi-Young Kim
  • Tae-Geum Kim
  • Yong-Suk Jang
  • Moon-Sik Yang
Original Article

Abstract

Porcine epidemic diarrhea virus (PEDV) belongs to the Coronaviridae family and causes acute enteritis in pigs. A fragment of the large spike glycoprotein, termed the S1D epitope (aa 636–789), alone and fused with cholera toxin B subunit, were independently cloned into plant expression vectors, yielding plasmids pMYV717 and pMYV719, respectively. Plant expression vectors were transformed into Agrobacterium tumefaciens and subsequently infiltrated into Nicotiana benthamiana leaves. The highest expression level of S1D was found at 2 days post infiltration (dpi), reached 0.04 % of total soluble protein, and rapidly decreased thereafter. The expression and assembly of CTB–S1D fusion protein were confirmed by Western blot and GM1-ELISA. The highest expression level of CTB–S1D fusion protein was 0.07 % of TSP at 4 dpi, with a rapid decrease thereafter. In the presence of p19 protein from tomato bushy stunt virus, the S1D and CTB–S1D protein levels peaked at 6 dpi and were fourfold to sevenfold higher than in the absence of p19, respectively. After oral administration of transiently expressed CTB–S1D fusion protein, or with bacterial cholera toxin or rice callus expressing mutant cholera toxin 61F, mice exhibited significantly greater serum IgG and sIgA levels against bacterial CTB and S1D antigen, peaking at week 6. Transiently expressed CTB–S1D fusion protein will be administered orally to pigs to assess the immune response against PEDV.

Keywords

Edible vaccine Agroinfiltration Cholera toxin PEDV S1D 

Notes

Acknowledgments

This research was supported by the Agriculture, Food and Rural Affairs Research Center Support Program, Ministry of Agriculture, Food and Rural Affairs, and Nguyen-Quang-Duc Tien was supported by the BK21 plus program, Republic of Korea.

Author contributions

Nguyen-Xuan Huy, Nguyen-Quang-Duc Tien and Moon-Sik Yang conceived, designed and performed the overall study. Mi-Young Kim generated the transformed rice callus expressing mutant cholera toxin 61F. Tae-Geum Kim and Yong-Suk Jang designed the mouse experiment. Nguyen-Xuan Huy wrote the manuscript.

References

  1. Arakawa T, Chong DK, Merritt JL, Langridge WH (1997) Expression of cholera toxin B subunit oligomers in transgenic potato plants. Transgenic Res 6(6):403–413PubMedCrossRefGoogle Scholar
  2. Beam A, Goede D, Fox A, McCool MJ, Wall G, Haley C, Morrison R (2015) A porcine epidemic diarrhea virus outbreak in one geographic region of the united states: descriptive epidemiology and investigation of the possibility of airborne virus spread. Plos One 10(12):e0144818. doi: 10.1371/journal.pone.0144818 PubMedPubMedCentralCrossRefGoogle Scholar
  3. Chang SH, Bae JL, Kang TJ, Kim J, Chung GH, Lim CW, Laude H, Yang MS, Jang YS (2002) Identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus. Mol Cells 14(2):295–299PubMedGoogle Scholar
  4. Chasey D, Cartwright SF (1978) Virus-like particles associated with porcine epidemic diarrhoea. Res Vet Sci 25(2):255–256PubMedGoogle Scholar
  5. Chen Q, Lai H, Hurtado J, Stahnke J, Leuzinger K, Dent M (2013) Agroinfiltration as an effective and scalable strategy of gene delivery for production of pharmaceutical proteins. Adv Tech Biol Med 1(1):103 doi: 10.4172/atbm.1000103 PubMedPubMedCentralCrossRefGoogle Scholar
  6. Debouck P, Pensaert M (1980) Experimental infection of pigs with a new porcine enteric coronavirus, CV 777. Am J Vet Res 41(2):219–223PubMedGoogle Scholar
  7. Duarte M, Laude H (1994) Sequence of the spike protein of the porcine epidemic diarrhoea virus. J Gen Virol 75(5):1195–1200 doi: 10.1099/0022-1317-75-5-1195 PubMedCrossRefGoogle Scholar
  8. Ducatelle R, Coussement W, Charlier G, Debouck P, Hoorens J (1981) Three-dimensional sequential study of the intestinal surface in experimental porcine CV 777 coronavirus enteritis. Zentralbl Vet B 28(6):483–493CrossRefGoogle Scholar
  9. Elson CO, Ealding W (1984) Cholera toxin feeding did not induce oral tolerance in mice and abrogated oral tolerance to an unrelated protein antigen. J Immunol 133(6):2892–2897PubMedGoogle Scholar
  10. Follis KE, York J, Nunberg JH (2006) Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell–cell fusion but does not affect virion entry. Virology 350(2):358–369 doi: 10.1016/j.virol.2006.02.003 PubMedGoogle Scholar
  11. Haan Ad, Renegar KB, Small PA Jr, Wilschut J (1995) Induction of a secretory IgA response in the murine female urogenital tract by immunization of the lungs with liposome-supplemented viral subunit antigen. Vaccine 13(7):613–616. doi: 10.1016/0264-410X(94)00062-R PubMedCrossRefGoogle Scholar
  12. Hernández M, Rosas G, Cervantes J, Fragoso G, Rosales-Mendoza S, Sciutto E (2014) Transgenic plants: a 5-year update on oral antipathogen vaccine development. Expert Rev Vaccines 13(12):1523–1536. doi: 10.1586/14760584.2014.953064 PubMedCrossRefGoogle Scholar
  13. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227(4691):1229–1231. doi: 10.1126/science.227.4691.1229 CrossRefGoogle Scholar
  14. Huy NX, Kim YS, Jun SC, Jin Z, Park SM, Yang MS, Kim TG (2009) Production of a heat-labile enterotoxin B subunit-porcine epidemic diarrhea virus-neutralizing epitope fusion protein in transgenic lettuce (Lactuca sativa). Biotechnol Bioprocess Eng 14(6):731–737CrossRefGoogle Scholar
  15. Huy NX, Yang MS, Kim TG (2011) Expression of a cholera toxin B subunit-neutralizing epitope of the porcine epidemic diarrhea virus fusion gene in transgenic lettuce (Lactuca sativa L.) Mol Biotechnol 48(3):201–209. doi: 10.1007/s12033-010-9359-1 PubMedCrossRefGoogle Scholar
  16. Huy NX, Kim SH, Yang MS, Kim TG (2012) Immunogenicity of a neutralizing epitope from porcine epidemic diarrhea virus: M cell targeting ligand fusion protein expressed in transgenic rice calli. Plant Cell Rep 31(10):1933–1942. doi: 10.1007/s00299-012-1306-0 PubMedCrossRefGoogle Scholar
  17. Isaka M, Komiya T, Takahashi M, Yasuda Y, Taniguchi T, Zhao Y, Matano K, Matsui H, Maeyama JI, Morokuma K, Ohkuma K, Goto N, Tochikubo K (2004) Recombinant cholera toxin B subunit (rCTB) as a mucosal adjuvant enhances induction of diphtheria and tetanus antitoxin antibodies in mice by intranasal administration with diphtheria–pertussis–tetanus (DPT) combination vaccine. Vaccine 22(23–24):3061–3068. doi: 10.1016/j.vaccine.2004.02.019 PubMedCrossRefGoogle Scholar
  18. Jespersgaard C, Hajishengallis G, Greenway TE, Smith DJ, Russell MW, Michalek SM (1999) Functional and immunogenic characterization of two cloned regions of Streptococcus mutans glucosyltransferase I. Infect Immun 67(2):810–816PubMedPubMedCentralGoogle Scholar
  19. Kang T-J, Loc N-H, Jang M-O, Yang M-S (2004) Modification of the cholera toxin B subunit coding sequence to enhance expression in plants. Mol Breed 13(2):143–153. doi: 10.1023/B:MOLB.0000018762.27841.7a CrossRefGoogle Scholar
  20. Kang T-J, Kim Y-S, Jang Y-S, Yang M-S (2005) Expression of the synthetic neutralizing epitope gene of porcine epidemic diarrhea virus in tobacco plants without nicotine. Vaccine 23(17–18):2294–2297. doi: 10.1016/j.vaccine.2005.01.027 PubMedCrossRefGoogle Scholar
  21. 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(2):157–164 doi: 10.1007/s12033-008-9102-3 PubMedCrossRefGoogle Scholar
  22. Kim TG, Kim BG, Kim MY, Choi JK, Jung ES, Yang MS (2010) Expression and immunogenicity of enterotoxigenic Escherichia coli heat-labile toxin B subunit in transgenic rice callus. Mol Biotechnol 44(1):14–21 doi: 10.1007/s12033-009-9200-x PubMedCrossRefGoogle Scholar
  23. Kocherhans R, Bridgen A, Ackermann M, Tobler K (2001) Completion of the porcine epidemic diarrhoea coronavirus (PEDV) genome sequence. Virus Genes 23(2):137–144PubMedCrossRefGoogle Scholar
  24. Lal P, Ramachandran VG, Goyal R, Sharma R (2007) Edible vaccines: current status and future. Indian J Med Microbiol 25(2):93–102PubMedCrossRefGoogle Scholar
  25. Lee S, Lee C (2014) Outbreak-related porcine epidemic diarrhea virus strains similar to US strains, South Korea, 2013. Emerg Infect Dis 20(7):1223–1226. doi: 10.3201/eid2007.140294 PubMedPubMedCentralCrossRefGoogle Scholar
  26. Lee S, Kim Y, Lee C (2015) Isolation and characterization of a Korean porcine epidemic diarrhea virus strain KNU-141112. Virus Res 208:215–224. doi: 10.1016/j.virusres.2015.07.010 PubMedCrossRefGoogle Scholar
  27. Leenaars M, Hendriksen CFM (2005) Critical steps in the production of polyclonal and monoclonal antibodies: evaluation and recommendations. ILAR J 46(3):269–279. doi: 10.1093/ilar.46.3.269 PubMedCrossRefGoogle Scholar
  28. Leuzinger K, Dent M, Hurtado J, Stahnke J, Lai H, Zhou X, Chen Q (2013) Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins. J Vis Exp doi: 10.3791/50521 PubMedPubMedCentralGoogle Scholar
  29. Li W, Li H, Liu Y, Pan Y, Deng F, Song Y, Tang X, He Q (2012) New variants of porcine epidemic diarrhea virus, China, 2011. Emerg Infect Dis 18(8):1350–1353. doi: 10.3201/eid1808.120002 PubMedPubMedCentralCrossRefGoogle Scholar
  30. Maclean J, Koekemoer M, Olivier AJ, Stewart D, Hitzeroth II, Rademacher T, Fischer R, Williamson AL, Rybicki EP (2007) Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: comparison of the suitability of different HPV-16 L1 gene variants and different cell-compartment localization. J Gen Virol 88(5):1460–1469. doi: 10.1099/vir.0.82718-0 PubMedCrossRefGoogle Scholar
  31. Marillonnet S, Giritch A, Gils M, Kandzia R, Klimyuk V, Gleba Y (2004) In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium. Proc Natl Acad Sci U S A 101(18):6852–6857. doi: 10.1073/pnas.0400149101 PubMedPubMedCentralCrossRefGoogle Scholar
  32. Mohammadzadeh S, Roohvand F, Memarnejadian A, Jafari A, Ajdary S, Salmanian A-H, Ehsani P (2015) Co-expression of hepatitis C virus polytope–HBsAg and p19-silencing suppressor protein in tobacco leaves. Pharm Biol:1–9 doi: 10.3109/13880209.2015.1048371
  33. Munro S, Pelham HR (1987) A C-terminal signal prevents secretion of luminal ER proteins. Cell 48(5):899–907PubMedCrossRefGoogle Scholar
  34. Pensaert MB, de Bouck P (1978) A new coronavirus-like particle associated with diarrhea in swine. Arch Virol 58(3):243–247PubMedCrossRefGoogle Scholar
  35. Poland GA, Murray D, Bonilla-Guerrero R (2002) New vaccine development. BMJ. Br Med J 324(7349):1315–1319CrossRefGoogle Scholar
  36. Puranaveja S, Poolperm P, Lertwatcharasarakul P, Kesdaengsakonwut S, Boonsoongnern A, Urairong K, Kitikoon P, Choojai P, Kedkovid R, Teankum K, Thanawongnuwech R (2009) Chinese-like strain of porcine epidemic diarrhea virus, Thailand. Emerg Infect Dis 15(7):1112–1115. doi: 10.3201/eid1507.081256 PubMedPubMedCentralCrossRefGoogle Scholar
  37. Sala F, Manuela Rigano M, Barbante A, Basso B, Walmsley AM, Castiglione S (2003) Vaccine antigen production in transgenic plants: strategies, gene constructs and perspectives. Vaccine 21(7–8):803–808. doi: 10.1016/S0264-410X(02)00603-5 PubMedCrossRefGoogle Scholar
  38. Steinrigl A, Revilla Fernández S, Stoiber F, Pikalo J, Sattler T, Schmoll F (2015) First detection, clinical presentation and phylogenetic characterization of porcine epidemic diarrhea virus in Austria. BMC Vet Res 11:310. doi: 10.1186/s12917-015-0624-1 PubMedPubMedCentralCrossRefGoogle Scholar
  39. Stevenson GW, Hoang H, Schwartz KJ, Burrough ER, Sun D, Madson D, Cooper VL, Pillatzki A, Gauger P, Schmitt BJ, Koster LG, Killian ML, Yoon KJ (2013) Emergence of porcine epidemic diarrhea virus in the United States: clinical signs, lesions, and viral genomic sequences. J Vet Diagn Investig 25(5):649–654 doi: 10.1177/1040638713501675 CrossRefGoogle Scholar
  40. Strugnell R, Zepp F, Cunningham A, Tantawichien T (2011) Vaccine antigens. Perspect Vaccinol 1(1):61–88 doi: 10.1016/j.pervac.2011.05.003 CrossRefGoogle Scholar
  41. Sun D, Feng L, Shi H, Chen J, Cui X, Chen H, Liu S, Tong Y, Wang Y, Tong G (2008) Identification of two novel B cell epitopes on porcine epidemic diarrhea virus spike protein. Vet Microbiol 131(1–2):73–81. doi: 10.1016/j.vetmic.2008.02.022 PubMedCrossRefGoogle Scholar
  42. Takahashi K, Okada K, Ohshima K (1983) An outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in Japan. Nihon Juigaku Zasshi 45(6):829–832PubMedCrossRefGoogle Scholar
  43. Turgeon DC, Morin M, Jolette J, Higgins R, Marsolais G, DiFranco E (1980) Coronavirus-like particles associated with diarrhea in baby pigs in Quebec. Can Vet J 21(3):100PubMedPubMedCentralGoogle Scholar
  44. Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33(5):949–956PubMedCrossRefGoogle Scholar
  45. Woof JM, Mestecky J (2005) Mucosal immunoglobulins. Immunol Rev 206:64–82. doi: 10.1111/j.0105-2896.2005.00290.x PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Nguyen-Xuan Huy
    • 1
    • 5
  • Nguyen-Quang-Duc Tien
    • 2
  • Mi-Young Kim
    • 1
  • Tae-Geum Kim
    • 3
    • 4
  • Yong-Suk Jang
    • 1
    • 2
    • 3
  • Moon-Sik Yang
    • 1
    • 2
    • 3
  1. 1.Department of Molecular BiologyChonbuk National UniversityJeonjuRepublic of Korea
  2. 2.Department of Bioactive Material ScienceChonbuk National UniversityJeonjuRepublic of Korea
  3. 3.Research Center of Bioactive MaterialsChonbuk National UniversityJeonjuRepublic of Korea
  4. 4.Center for Jeongup Industry-Academy-Institute CooperationChonbuk National UniversityJeonjuRepublic of Korea
  5. 5.Biology DepartmentHue University of EducationHueVietnam

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