Applied Microbiology and Biotechnology

, Volume 100, Issue 17, pp 7457–7469 | Cite as

A phase trial of the oral Lactobacillus casei vaccine polarizes Th2 cell immunity against transmissible gastroenteritis coronavirus infection

  • Xinpeng Jiang
  • Xingyu Hou
  • Lijie Tang
  • Yanping Jiang
  • Guangpeng MaEmail author
  • Yijing LiEmail author
Biotechnological products and process engineering


Transmissible gastroenteritis coronavirus (TGEV) is a member of the genus Coronavirus, family Coronaviridae, order Nidovirales. TGEV is an enteropathogenic coronavirus that causes highly fatal acute diarrhoea in newborn pigs. An oral Lactobacillus casei (L. casei) vaccine against anti-transmissible gastroenteritis virus developed in our laboratory was used to study mucosal immune responses. In this L. casei vaccine, repetitive peptides expressed by L. casei (specifically the MDP and tuftsin fusion protein (MT)) were repeated 20 times and the D antigenic site of the TGEV spike (S) protein was repeated 6 times. Immunization with recombinant Lactobacillus is crucial for investigations of the effect of immunization, such as the first immunization time and dose. The first immunization is more important than the last immunization in the series. The recombinant Lactobacillus elicited specific systemic and mucosal immune responses. Recombinant L. casei had a strong potentiating effect on the cellular immunity induced by the oral L. casei vaccine. However, during TGEV infection, the systemic and local immune responses switched from Th1 to Th2-based immune responses. The systemic humoral immune response was stronger than the cellular immune response after TGEV infection. We found that the recombinant Lactobacillus stimulated IL-17 expression in both the systemic and mucosal immune responses against TGEV infection. Furthermore, the Lactobacillus vaccine stimulated an anti-TGEV infection Th17 pathway. The histopathological examination showed tremendous potential for recombinant Lactobacillus to enable rapid and effective treatment for TGEV with an intestinal tropism in piglets. The TGEV immune protection was primarily dependent on mucosal immunity.


Transmissible gastroenteritis coronavirus Phase trial Lactobacillus casei vaccine T helper cell 



This work was supported by the National Natural Science Foundation of China (31472226).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical statement

The piglets were handled and maintained under strict ethical conditions according to international recommendations for animal welfare. This article does not contain any studies with human participants performed by any of the authors.


  1. Anton IM, Sune C, Meloen RH, Borras-Cuesta F, Enjuanes L (1995) A transmissible gastroenteritis coronavirus nucleoprotein epitope elicits T helper cells that collaborate in the in vitro antibody synthesis to the three major structural viral proteins. Virology 212(2):746–751. doi: 10.1006/viro.1995.1535 CrossRefPubMedGoogle Scholar
  2. Anton IM, Gonzalez S, Bullido MJ, Corsin M, Risco C, Langeveld JP, Enjuanes L (1996) Cooperation between transmissible gastroenteritis coronavirus (TGEV) structural proteins in the in vitro induction of virus-specific antibodies. Virus Res 46(1–2):111–124CrossRefPubMedGoogle Scholar
  3. Bengmark S, Gil A (2006) Bioecological and nutritional control of disease: prebiotics, probiotics and synbiotics. Nutr Hosp 21(Suppl 2):72–84, 73–86PubMedGoogle Scholar
  4. Berthon P, Bernard S, Salmon H, Binns RM (1990) Kinetics of the in vitro antibody response to transmissible gastroenteritis (TGE) virus from pig mesenteric lymph node cells, using the ELISASPOT and ELISA tests. J Immunol Methods 131(2):173–182CrossRefPubMedGoogle Scholar
  5. Blum S, Schiffrin EJ (2003) Intestinal microflora and homeostasis of the mucosal immune response: implications for probiotic bacteria? Curr Issues Intest Microbiol 4(2):53–60PubMedGoogle Scholar
  6. Braat H, Rottiers P, Hommes DW, Huyghebaert N, Remaut E, Remon JP, van Deventer SJ, Neirynck S, Peppelenbosch MP, Steidler L (2006) A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn's disease. Clin Gastroenterol Hepatol 4(6):754–759. doi: 10.1016/j.cgh.2006.03.028 CrossRefPubMedGoogle Scholar
  7. Cavanagh D (1997) Nidovirales: a new order comprising Coronaviridae and Arteriviridae. Arch Virol 142(3):629–633PubMedGoogle Scholar
  8. Charley B, Laude H (1988) Induction of alpha interferon by transmissible gastroenteritis coronavirus: role of transmembrane glycoprotein E1. J Virol 62(1):8–11PubMedPubMedCentralGoogle Scholar
  9. Chen CC, Baylor M, Bass DM (1993) Murine intestinal mucins inhibit rotavirus infection. Gastroenterology 105(1):84–92CrossRefPubMedGoogle Scholar
  10. Corthesy B, Gaskins HR, Mercenier A (2007) Cross-talk between probiotic bacteria and the host immune system. J Nutr 137(3 Suppl 2):781S–790SPubMedGoogle Scholar
  11. Crowe CR, Chen K, Pociask DA, Alcorn JF, Krivich C, Enelow RI, Ross TM, Witztum JL, Kolls JK (2009) Critical role of IL-17RA in immunopathology of influenza infection. J Immunol 183(8):5301–5310. doi: 10.4049/jimmunol.0900995 CrossRefPubMedPubMedCentralGoogle Scholar
  12. de Vrese M, Marteau PR (2007) Probiotics and prebiotics: effects on diarrhea. J Nutr 137(3 Suppl 2):803S–811SPubMedGoogle Scholar
  13. Di-Qiu L, Xin-Yuan Q, Jun-Wei G, Li-Jie T, Yan-Ping J, Yi-Jing L (2011) Construction and characterization of Lactobacillus pentosus expressing the D antigenic site of the spike protein of Transmissible gastroenteritis virus. Can J Microbiol 57(5):392–397. doi: 10.1139/W11-027 CrossRefPubMedGoogle Scholar
  14. Dirisala VR, Jeevan A, Ramasamy SK, McMurray DN (2013) Molecular cloning, expression, and in silico structural analysis of guinea pig IL-17. Mol Biotechnol 55(3):277–287. doi: 10.1007/s12033-013-9679-z CrossRefPubMedGoogle Scholar
  15. Dock DB, Aguilar-Nascimento JE, Latorraca MQ (2004) Probiotics enhance the recovery of gut atrophy in experimental malnutrition. Biocell 28(2):143–150PubMedGoogle Scholar
  16. Friedrich MJ (2013) Genomes of microbes inhabiting the body offer clues to human health and disease. JAMA 309(14):1447–1449. doi: 10.1001/jama.2013.2824 CrossRefPubMedGoogle Scholar
  17. Grodeland G, Fossum E, Bogen B (2015) Polarizing T and B cell responses by APC-targeted subunit vaccines. Front Immunol 6:367. doi: 10.3389/fimmu.2015.00367 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Gunzer F, Hennig-Pauka I, Waldmann KH, Sandhoff R, Grone HJ, Kreipe HH, Matussek A, Mengel M (2002) Gnotobiotic piglets develop thrombotic microangiopathy after oral infection with enterohemorrhagic Escherichia coli. Am J Clin Pathol 118(3):364–375. doi: 10.1309/UMW9-D06Q-M94Q-JGH2 CrossRefPubMedGoogle Scholar
  19. Hori T, Kiyoshima J, Shida K, Yasui H (2001) Effect of intranasal administration of Lactobacillus casei Shirota on influenza virus infection of upper respiratory tract in mice. Clin Diagn Lab Immunol 8(3):593–597. doi: 10.1128/CDLI.8.3.593-597.2001 PubMedPubMedCentralGoogle Scholar
  20. Isolauri E, Kirjavainen PV, Salminen S (2002) Probiotics: a role in the treatment of intestinal infection and inflammation? Gut 50(Suppl 3):III54–III59PubMedPubMedCentralGoogle Scholar
  21. Jiang TJ, Zhang JY, Li WG, Xie YX, Zhang XW, Wang Y, Jin L, Wang FS, Zhao M (2010) Preferential loss of Th17 cells is associated with CD4 T cell activation in patients with 2009 pandemic H1N1 swine-origin influenza A infection. Clin Immunol 137(3):303–310. doi: 10.1016/j.clim.2010.07.010 CrossRefPubMedGoogle Scholar
  22. Jiang X, Yu M, Qiao X, Liu M, Tang L, Jiang Y, Cui W, Li Y (2014) Up-regulation of MDP and tuftsin gene expression in Th1 and Th17 cells as an adjuvant for an oral Lactobacillus casei vaccine against anti-transmissible gastroenteritis virus. Appl Microbiol Biotechnol 98(19):8301–8312. doi: 10.1007/s00253-014-5893-2 CrossRefPubMedGoogle Scholar
  23. Kaila M, Isolauri E, Soppi E, Virtanen E, Laine S, Arvilommi H (1992) Enhancement of the circulating antibody secreting cell response in human diarrhea by a human Lactobacillus strain. Pediatr Res 32(2):141–144. doi: 10.1203/00006450-199208000-00002 CrossRefPubMedGoogle Scholar
  24. Kararli TT (1995) Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals. Biopharm Drug Dispos 16(5):351–380CrossRefPubMedGoogle Scholar
  25. Kiros TG, van Kessel J, Babiuk LA, Gerdts V (2011) Induction, regulation and physiological role of IL-17 secreting helper T-cells isolated from PBMC, thymus, and lung lymphocytes of young pigs. Vet Immunol Immunopathol 144(3–4):448–454. doi: 10.1016/j.vetimm.2011.08.021 CrossRefPubMedGoogle Scholar
  26. Lafdil F, Wang H, Park O, Zhang W, Moritoki Y, Yin S, Fu XY, Gershwin ME, Lian ZX, Gao B (2009) Myeloid STAT3 inhibits T cell-mediated hepatitis by regulating T helper 1 cytokine and interleukin-17 production. Gastroenterology 137(6):2125–2135. doi: 10.1053/j.gastro.2009.08.004, e1-2CrossRefPubMedPubMedCentralGoogle Scholar
  27. Lavelle EC, O'Hagan DT (2006) Delivery systems and adjuvants for oral vaccines. Expert Opin Drug Deliv 3(6):747–762. doi: 10.1517/17425247.3.6.747 CrossRefPubMedGoogle Scholar
  28. Lee BM, Han YW, Kim SB, Rahman MM, Uyangaa E, Kim JH, Roh YS, Kim B, Han SB, Hong JT, Kim K, Eo SK (2011) Enhanced protection against infection with transmissible gastroenteritis virus in piglets by oral co-administration of live attenuated Salmonella enterica serovar Typhimurium expressing swine interferon-alpha and interleukin-18. Comp Immunol Microbiol Infect Dis 34(4):369–380. doi: 10.1016/j.cimid.2011.05.001 CrossRefPubMedGoogle Scholar
  29. Liu D, Wang X, Ge J, Liu S, Li Y (2011) Comparison of the immune responses induced by oral immunization of mice with Lactobacillus casei-expressing porcine parvovirus VP2 and VP2 fused to Escherichia coli heat-labile enterotoxin B subunit protein. Comp Immunol Microbiol Infect Dis 34(1):73–81. doi: 10.1016/j.cimid.2010.02.004 CrossRefPubMedGoogle Scholar
  30. Malik DK, Baboota S, Ahuja A, Hasan S, Ali J (2007) Recent advances in protein and peptide drug delivery systems. Curr Drug Deliv 4(2):141–151CrossRefPubMedGoogle Scholar
  31. McKinstry KK, Strutt TM, Buck A, Curtis JD, Dibble JP, Huston G, Tighe M, Hamada H, Sell S, Dutton RW, Swain SL (2009) IL-10 deficiency unleashes an influenza-specific Th17 response and enhances survival against high-dose challenge. J Immunol 182(12):7353–7363. doi: 10.4049/jimmunol.0900657 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Moore-Connors JM, Fraser R, Halperin SA, Wang J (2013) CD4(+)CD25(+)Foxp3(+) regulatory T cells promote Th17 responses and genital tract inflammation upon intracellular Chlamydia muridarum infection. J Immunol 191(6):3430–3439. doi: 10.4049/jimmunol.1301136 CrossRefPubMedGoogle Scholar
  33. Mouricout M, Petit JM, Carias JR, Julien R (1990) Glycoprotein glycans that inhibit adhesion of Escherichia coli mediated by K99 fimbriae: treatment of experimental colibacillosis. Infect Immun 58(1):98–106PubMedPubMedCentralGoogle Scholar
  34. Nagata T, McKinley L, Peschon JJ, Alcorn JF, Aujla SJ, Kolls JK (2008) Requirement of IL-17RA in Con A induced hepatitis and negative regulation of IL-17 production in mouse T cells. J Immunol 181(11):7473–7479CrossRefPubMedGoogle Scholar
  35. Olivares M, Diaz-Ropero MP, Sierra S, Lara-Villoslada F, Fonolla J, Navas M, Rodriguez JM, Xaus J (2007) Oral intake of Lactobacillus fermentum CECT5716 enhances the effects of influenza vaccination. Nutrition 23(3):254–260. doi: 10.1016/j.nut.2007.01.004 CrossRefPubMedGoogle Scholar
  36. Oswald IP, Desautels C, Laffitte J, Fournout S, Peres SY, Odin M, Le Bars P, Le Bars J, Fairbrother JM (2003) Mycotoxin fumonisin B1 increases intestinal colonization by pathogenic Escherichia coli in pigs. Appl Environ Microbiol 69(10):5870–5874CrossRefPubMedPubMedCentralGoogle Scholar
  37. Ouwehand AC (2007) Antiallergic effects of probiotics. J Nutr 137(3 Suppl 2):794S–797SPubMedGoogle Scholar
  38. Qiao X, Li G, Wang X, Li X, Liu M, Li Y (2009) Recombinant porcine rotavirus VP4 and VP4-LTB expressed in Lactobacillus casei induced mucosal and systemic antibody responses in mice. BMC Microbiol 9:249. doi: 10.1186/1471-2180-9-249 CrossRefPubMedPubMedCentralGoogle Scholar
  39. Ruan X, Zhang W (2013) Oral immunization of a live attenuated Escherichia coli strain expressing a holotoxin-structured adhesin-toxoid fusion (1FaeG-FedF-LTA(2):5LTB) protected young pigs against enterotoxigenic E. coli (ETEC) infection. Vaccine 31(11):1458–1463. doi: 10.1016/j.vaccine.2013.01.030 CrossRefPubMedGoogle Scholar
  40. Saarela M, Mogensen G, Fonden R, Matto J, Mattila-Sandholm T (2000) Probiotic bacteria: safety, functional and technological properties. J Biotechnol 84(3):197–215CrossRefPubMedGoogle Scholar
  41. Saif LJ (1996) Mucosal immunity: an overview and studies of enteric and respiratory coronavirus infections in a swine model of enteric disease. Vet Immunol Immunopathol 54(1–4):163–169CrossRefPubMedGoogle Scholar
  42. Sartor RB (2005) Probiotic therapy of intestinal inflammation and infections. Curr Opin Gastroenterol 21(1):44–50PubMedGoogle Scholar
  43. Schwegmann-Wessels C, Herrler G (2006) Transmissible gastroenteritis virus infection: a vanishing specter. Dtsch Tierarztl Wochenschr 113(4):157–159PubMedGoogle Scholar
  44. Sheil B, Shanahan F, O'Mahony L (2007) Probiotic effects on inflammatory bowel disease. J Nutr 137(3 Suppl 2):819S–824SPubMedGoogle Scholar
  45. Siddell SG, Anderson R, Cavanagh D, Fujiwara K, Klenk HD, Macnaughton MR, Pensaert M, Stohlman SA, Sturman L, van der Zeijst BA (1983) Coronaviridae. Intervirology 20(4):181–189CrossRefPubMedGoogle Scholar
  46. Superti F, Marziano ML, Tinari A, Donelli G (1993) Effect of polyions on the infectivity of SA-11 rotavirus in LCC-MK2 cells. Comp Immunol Microbiol Infect Dis 16(1):55–62CrossRefPubMedGoogle Scholar
  47. Tadros T, Traber DL, Heggers JP, Herndon DN (2003) Effects of interleukin-1alpha administration on intestinal ischemia and reperfusion injury, mucosal permeability, and bacterial translocation in burn and sepsis. Ann Surg 237(1):101–109. doi: 10.1097/01.SLA.0000041039.16815.69 CrossRefPubMedPubMedCentralGoogle Scholar
  48. Tsunetsugu-Yokota Y, Ato M, Takahashi Y, Hashimoto S, Kaji T, Kuraoka M, Yamamoto K, Mitsuki YY, Yamamoto T, Oshima M, Ohnishi K, Takemori T (2007) Formalin-treated UV-inactivated SARS coronavirus vaccine retains its immunogenicity and promotes Th2-type immune responses. Jpn J Infect Dis 60(2–3):106–112PubMedGoogle Scholar
  49. Tukler Henriksson J, Coursey TG, Corry DB, De Paiva CS, Pflugfelder SC (2015) IL-13 stimulates proliferation and expression of mucin and immunomodulatory genes in cultured conjunctival goblet cells. Invest Ophthalmol Vis Sci 56(8):4186–4197. doi: 10.1167/iovs.14-15496 CrossRefPubMedPubMedCentralGoogle Scholar
  50. Villena J, Racedo S, Aguero G, Alvarez S (2006) Yoghurt accelerates the recovery of defence mechanisms against Streptococcus pneumoniae in protein-malnourished mice. Br J Nutr 95(3):591–602CrossRefPubMedGoogle Scholar
  51. Wells JM, Mercenier A (2008) Mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria. Nat Rev Microbiol 6(5):349–362. doi: 10.1038/nrmicro1840 CrossRefPubMedGoogle Scholar
  52. Wen K, Azevedo MS, Gonzalez A, Zhang W, Saif LJ, Li G, Yousef A, Yuan L (2009) Toll-like receptor and innate cytokine responses induced by lactobacilli colonization and human rotavirus infection in gnotobiotic pigs. Vet Immunol Immunopathol 127(3–4):304–315. doi: 10.1016/j.vetimm.2008.10.322 CrossRefPubMedGoogle Scholar
  53. Yang W, Ding X, Deng J, Lu Y, Matsuda Z, Thiel A, Chen J, Deng H, Qin Z (2011) Interferon-gamma negatively regulates Th17-mediated immunopathology during mouse hepatitis virus infection. J Mol Med (Berl) 89(4):399–409. doi: 10.1007/s00109-010-0711-5 CrossRefGoogle Scholar
  54. Yigang XU, Yijing LI (2008) Construction of recombinant Lactobacillus casei efficiently surface displayed and secreted porcine parvovirus VP2 protein and comparison of the immune responses induced by oral immunization. Immunology 124(1):68–75. doi: 10.1111/j.1365-2567.2007.02738.x CrossRefPubMedPubMedCentralGoogle Scholar
  55. Yuan TL, Zhu YH, Shi M, Li TT, Li N, Wu GY, Bazer FW, Zang JJ, Wang FL, Wang JJ (2015) Within-litter variation in birth weight: impact of nutritional status in the sow. J Zhejiang Univ Sci B 16(6):417–435. doi: 10.1631/jzus.B1500010 PubMedPubMedCentralGoogle Scholar
  56. Zhang W, Azevedo MS, Wen K, Gonzalez A, Saif LJ, Li G, Yousef AE, Yuan L (2008) Probiotic Lactobacillus acidophilus enhances the immunogenicity of an oral rotavirus vaccine in gnotobiotic pigs. Vaccine 26(29–30):3655–3661. doi: 10.1016/j.vaccine.2008.04.070 CrossRefPubMedPubMedCentralGoogle Scholar
  57. Zhang Y, Wang X, Wang H, Jiao J, Li Y, Fan E, Zhang L, Bachert C (2015) TMEM16A-mediated mucin secretion in il-13-induced nasal epithelial cells from chronic rhinosinusitis patients. Allergy Asthma Immunol Res 7(4):367–375. doi: 10.4168/aair.2015.7.4.367 CrossRefPubMedPubMedCentralGoogle Scholar
  58. Zheng BJ, Du LY, Zhao GY, Lin YP, Sui HY, Chan C, Ma S, Guan Y, Yuen KY (2008) Studies of SARS virus vaccines. Hong Kong Med J 14(Suppl 4):39–43PubMedGoogle Scholar
  59. Zhou X, Chen Q, Moore J, Kolls JK, Halperin S, Wang J (2009) Critical role of the interleukin-17/interleukin-17 receptor axis in regulating host susceptibility to respiratory infection with Chlamydia species. Infect Immun 77(11):5059–5070. doi: 10.1128/IAI.00403-09 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Department of Preventive Veterinary Medicine, College of Veterinary MedicineNortheast Agricultural UniversityHarbinPeople’s Republic of China
  2. 2.Agricultural High Technology DepartmentChina Rural Technology Development CenterBeijingPeople’s Republic of China

Personalised recommendations