Recombinant adenovirus carrying a core neutralizing epitope of porcine epidemic diarrhea virus and heat-labile enterotoxin B of Escherichia coli as a mucosal vaccine

  • Van Tan Do
  • Jisung Jang
  • Jeongho Park
  • Hoai Thu Dao
  • Kiju Kim
  • Tae-Wook HahnEmail author
Original Article


Porcine epidemic diarrhea virus (PEDV) targets the intestinal mucosa in pigs. To protect against PEDV invasion, a mucosal vaccine is utilized effectively. In this study, we generated a recombinant adenovirus vaccine encoding the heat-labile enterotoxin B (LTB) and the core neutralizing epitope (COE) of PEDV (rAd-LTB-COE). The fusion protein LTB-COE was successfully expressed by the recombinant adenovirus in HEK293 cells, and the immunogenicity of the vaccine candidate was assessed in BALB/c mice and piglets. Three intramuscular or oral vaccinations with rAd-LTB-COE at two-week intervals induced robust humoral and mucosal immune responses. Moreover, a cell-mediated immune response was promoted in immunized mice, and the neutralizing antibody inhibited both the vaccine strain and the emerging PEDV isolate. Immunization experiments in piglets revealed that rAd-LTB-COE was immunogenic and induced good immune responses in piglets. Further studies are required to evaluate the efficacy of rAd-LTB-COE against a highly virulent PEDV challenge.



We would like to acknowledge the assistance of Mr. Bogyo Jung (College of Veterinary Medicine, Kangwon National University, Korea) in preparing the documents for the registration of the animal experiments.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.


  1. 1.
    Pensaert M, de Bouck P (1978) A new coronavirus-like particle associated with diarrhea in swine. Arch Virol 58:243–247CrossRefGoogle Scholar
  2. 2.
    Lee C (2015) Porcine epidemic diarrhea virus: An emerging and re-emerging epizootic swine virus. Virol J 1:13–19Google Scholar
  3. 3.
    Cho YY, Lim SI, Kim YK, Song JY, Lee JB, An DJ (2014) Complete genome sequence of K14JB01, a novel variant strain of Porcine Epidemic Diarrhea Virus in South Korea. Genome Announce. CrossRefGoogle Scholar
  4. 4.
    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 Invest 25:649–654CrossRefGoogle Scholar
  5. 5.
    Vlasova AN, Marthaler D, Wang Q, Culhane MR, Rossow KD, Rovira A, Collins J, Saif LJ (2014) Distinct characteristics and complex evolution of PEDV strains, North America, May 2013–February 2014. Emerg Infect Dis 20:1620–1628CrossRefGoogle Scholar
  6. 6.
    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 14:295–299PubMedGoogle Scholar
  7. 7.
    Cruz DJM, Kim C, Shin H (2008) The GPRLQPY motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus. Virus Res 132:192–196CrossRefGoogle Scholar
  8. 8.
    Song D, Moon H, Kang B (2015) Porcine epidemic diarrhea: a review of current epidemiology and available vaccines. Clin Exp Vaccine Res 4:166–176CrossRefGoogle Scholar
  9. 9.
    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 131:73–81CrossRefGoogle Scholar
  10. 10.
    Sun DB, Feng L, Shi HY, Chen JF, Liu SW, Chen HY, Wang YF (2007) Spike protein region (aa 636–789) of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies. Acta Virol 51:149–156PubMedGoogle Scholar
  11. 11.
    Lavelle E, Sharif S, Thomas N, Holland J, Davis S (1995) The importance of gastrointestinal uptake of particles in the design of oral delivery systems. Adv Drug Deliv Rev 18:5–22CrossRefGoogle Scholar
  12. 12.
    Mestecky J, Michalek SZ, Moldoveanu Z, Russell MW (1997) Routes of immunization and antigen delivery systems for optimal mucosal immune responses in humans. Behring Inst Mitt 98:33–43Google Scholar
  13. 13.
    Kang T, Han S, Yang M, Jang Y (2006) Expression of synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of Escherichia coli heat-labile enterotoxin in tobacco plants. Protein Expr Purif 46:16–22CrossRefGoogle Scholar
  14. 14.
    Spangler BD (1992) Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin. Microbiol Rev 56:622–647PubMedPubMedCentralGoogle Scholar
  15. 15.
    Bagdasarian MM, Nagai M, Frey J, Bagdasarian M (1999) Immunogenicity of Actinobacillus ApxIA toxin epitopes fused to the E. coli heat-labile enterotoxin B subunit. Vaccine 17:441–447CrossRefGoogle Scholar
  16. 16.
    Yu D, Jin C, Ramachandran M, Xu J, Nilsson B et al (2013) Adenovirus serotype 5 vectors with Tat-PTD modified Hexon and serotype 35 Fiber show greatly enhanced transduction capacity of primary cell cultures. PLoS One 1:4. CrossRefGoogle Scholar
  17. 17.
    Alba R, Bosch A, Chillon M (2005) Gutless adenovirus: last-generation adenovirus for gene therapy. Gene Ther 12:18–27CrossRefGoogle Scholar
  18. 18.
    Douglas JT (2007) Adenoviral vectors for gene therapy. Mol Biotechnol 36:71–80CrossRefGoogle Scholar
  19. 19.
    Wesley RD, Tang M, Lager KM (2004) Protection of weaned pigs by vaccination with human adenovirus 5 recombinant viruses expressing the hemagglutinin and the nucleoprotein of H3N2 swine influenza virus. Vaccine 22:3427–3434CrossRefGoogle Scholar
  20. 20.
    Berk AJ (1986) Adenovirus promoters and E1A transactivation. Annu Rev Genet 20:45–77CrossRefGoogle Scholar
  21. 21.
    Ferguson A, Humphreys K, Croft N (1995) Technical report: results of immunological tests on faecal extracts are likely to be extremely misleading. Clin Exp Immunol 99:70–75CrossRefGoogle Scholar
  22. 22.
    Lee S, Kim Y, Lee C (2015) Isolation and characterization of a Korean porcine epidemic diarrhea virus strain KNU-141112. Virus Res 208:215–224CrossRefGoogle Scholar
  23. 23.
    Huy N, Yang M, Kim T (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 48:201–209CrossRefGoogle Scholar
  24. 24.
    Kang T, Kang K, Kim J, Kwon T, Jang Y, Yang M (2004) High-level expression of the neutralizing epitope of porcine epidemic diarrhea virus by a tobacco mosaic virus-based vector. Protein Expr Purif 38:129–135CrossRefGoogle Scholar
  25. 25.
    Lv C, Xiao Y, Li X, Tian K (2016) Porcine epidemic diarrhea virus: current insights. Virus Adapt Treat 8:1–12Google Scholar
  26. 26.
    Chen Q, Li G, Stasko J, Thomas JT, Stensland WR, Pillatzki AE, Gauger PC, Schwartz KJ, Madson D, Yoon KJ, Stevenson GW, Burrough ER, Harmon KM, Main RG, Zhang J (2014) Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the United States. J Clin Microbiol 52:234–243CrossRefGoogle Scholar
  27. 27.
    Pan Y, Tian X, Li W, Zhou Q, Wang D, Bi Y, Chen F, Song Y (2012) Isolation and characterization of a variant porcine epidemic diarrhea virus in China. Virol J. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Sun R, Cai R, Chen Y, Liang P, Chen D, Song C (2012) Outbreak of porcine epidemic diarrhea in suckling piglets, China. Emerg Infect Dis 18:161–163CrossRefGoogle Scholar
  29. 29.
    Liu R, Wu L, Huang B, Huang J, Zhang Y, Ke M, Wang J, Tan W, Zhang R, Chen H (2005) Adenoviral expression of a truncated S1 subunit of SARS-CoV spike protein results in specific humoral immune responses against SARS-CoV in rats. Virus Res 112:24–31CrossRefGoogle Scholar
  30. 30.
    Callebaut P, Enjuanes L, Pensaert M (1996) An adenovirus recombinant expressing the spike glycoprotein of porcine respiratory coronavirus is immunogenic in swine. J Gen Virol 77:309–313CrossRefGoogle Scholar
  31. 31.
    Hashigucci K, Ogawa H, Ishidate T, Yamashita R, Kamiya H, Watanabe K, Hattori N, Sato T, Suzuki Y, Nagamine T (1996) Antibody responses in volunteers induced by nasal influenza vaccine combined with Escherichia coli heat-labile enterotoxin B subunit containing a trace amount of the holotoxin. Vaccine 14:113–119CrossRefGoogle Scholar
  32. 32.
    Jawale CV, Lee JH (2014) Salmonella enterica serovar enteritidis ghosts carrying the Escherichia coli heat-labile enterotoxin B subunit are capable of inducing enhanced protective immune responses. Clin Vaccine Immunol 21:799–807CrossRefGoogle Scholar
  33. 33.
    Jeon BW, Jawale CV, Kim SH, Lee JH (2012) Attenuated Salmonella Gallinarum secreting an Escherichia coli heat-labile enterotoxin B subunit protein as an adjuvant for oral vaccination against fowl typhoid. Vet Immunol Immunopathol 150:149–160CrossRefGoogle Scholar
  34. 34.
    Tamura S, Asanuma H, Tomita T, Komase K, Kawahara K, Danbara H, Hattori N, Watanabe K, Suzuki Y, Nagamine T (1994) Escherichia coli heat-labile enterotoxin B subunits supplemented with a trace amount of the holotoxin as an adjuvant for nasal influenza vaccine. Vaccine 12:1083–1089CrossRefGoogle Scholar
  35. 35.
    Ge J, Liu D, Li Y (2012) Construction of recombinant lactobacilli expressing the core neutralizing epitope (COE) of porcine epidemic diarrhea virus and a fusion protein consisting of COE and Escherichia coli heat-labile enterotoxin B, and comparison of the immune responses by orogastric immunization. Can J Microbiol 58:1258–1267CrossRefGoogle Scholar
  36. 36.
    Wang X, Wang L, Huang X, Ma S, Yu M, Shi W, Qiao X, Tang L, Xu Y, Li Y (2017) Oral delivery of probiotics expressing dendritic cell-targeting peptide fused with porcine epidemic diarrhea virus COE antigen: a promising vaccine strategy against PEDV. Viruses 9:312. CrossRefPubMedCentralGoogle Scholar
  37. 37.
    Song D, Oh JS, Kang BK, Yang JS, Moon HJ, Yoo HS, Jang YS, Park BK (2007) Oral efficacy of Vero cell attenuated porcine epidemic diarrhea virus DR13 strain. Res Vet Sci 82:134–140CrossRefGoogle Scholar
  38. 38.
    Lin H, Chen L, Gao L, Yuan X, Ma ZHE, Fan H (2016) Epidemic strain YC2014 of porcine epidemic diarrhea virus could provide piglets against homologous challenge. Virol J 13:68. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Veterinary Medicine, College of Veterinary Medicine and Institute of Veterinary ScienceKangwon National UniversityChuncheon-siSouth Korea
  2. 2.Gangwon Veterinary Service LaboratoryChuncheonSouth Korea
  3. 3.Department of Pathobiology, School of Veterinary MedicineUniversity of PennsylvaniaPhiladelphiaUSA

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