Applied Microbiology and Biotechnology

, Volume 98, Issue 19, pp 8301–8312 | Cite as

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

  • Xinpeng Jiang
  • Meiling Yu
  • Xinyuan Qiao
  • Min Liu
  • Lijie Tang
  • Yanping Jiang
  • Wen Cui
  • Yijing Li
Applied microbial and cell physiology


The role of muramyl dipeptide (MDP) and tuftsin in oral immune adjustment remains unclear, particularly in a Lactobacillus casei (L. casei) vaccine. To address this, we investigated the effects of different repetitive peptides expressed by L. casei, specifically the MDP and tuftsin fusion protein (MT) repeated 20 and 40 times (20MT and 40MT), in mice also expressing the D antigenic site of the spike (S) protein of transmissible gastroenteritis virus (TGEV) on intestinal and systemic immune responses and confirmed the immunoregulation of these peptides. Treatment of mice with a different vaccine consisting of L. casei expressing MDP and tuftsin stimulated humoral and cellular immune responses. Both 20MT and 40MT induced an increase in IgG and IgA levels against TGEV, as determined using enzyme-linked immunosorbent assay. Increased IgG and IgA resulted in the activation of TGEV-neutralising antibody activity in vitro. In addition, 20MT and 40MT stimulated the differentiation of innate immune cells, including T helper cell subclasses and regulatory T (Treg) cells, which induced robust T helper type 1 and T helper type 17 (Th17) responses and reduced Treg T cell immune responses in the 20MT and 40MT groups, respectively. Notably, treatment of mice with L. casei expressing 20MT and 40MT enhanced the anti-TGEV antibody immune responses of both the humoral and mucosal immune systems. These findings suggest that L. casei expressing MDP and tuftsin possesses substantial immunopotentiating properties, as it can induce humoral and T cell-mediated immune responses upon oral administration, and it may be useful in oral vaccines against TGEV challenge.


Muramyl dipeptide Tuftsin Lactobacillus casei Transmissible gastroenteritis virus T helper cell 



This work was supported by the National Natural Science Foundation of China (31272594) and the Science and Technology Innovation Team of Heilongjiang Province. The authors wish to thank NIZO Food Research for providing the pPG612 plasmids and Food Science College of Northeast Agricultural University (NEAU) for providing the L. casei bacterial strain.

Conflict of interest

The authors declare no financial or commercial conflicts of interest.


  1. Bernard S, Laude H (1995) Site-specific alteration of transmissible gastroenteritis virus spike protein results in markedly reduced pathogenicity. J Gen Virol 76(Pt 9):2235–41PubMedCrossRefGoogle Scholar
  2. Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK (2006) Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441(7090):235–8PubMedCrossRefGoogle Scholar
  3. Castilla J, Sola I, Enjuanes L (1997) Interference of coronavirus infection by expression of immunoglobulin G (IgG) or IgA virus-neutralizing antibodies. J Virol 71(7):5251–8PubMedPubMedCentralGoogle Scholar
  4. 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):392–7PubMedCrossRefGoogle Scholar
  5. Dzierzbicka K, Wardowska A, Rogalska M, Trzonkowski P (2012) New conjugates of muramyl dipeptide and nor-muramyl dipeptide linked to tuftsin and retro-tuftsin derivatives significantly influence their biological activity. Pharmacol Rep : PR 64(1):217–23PubMedCrossRefGoogle Scholar
  6. Esplugues E, Huber S, Gagliani N, Hauser AE, Town T, Wan YY, O’Connor W Jr, Rongvaux A, Van Rooijen N, Haberman AM, Iwakura Y, Kuchroo VK, Kolls JK, Bluestone JA, Herold KC, Flavell RA (2011) Control of TH17 cells occurs in the small intestine. Nature 475(7357):514–8PubMedCrossRefPubMedCentralGoogle Scholar
  7. Iwakura Y, Nakae S, Saijo S, Ishigame H (2008) The roles of IL-17A in inflammatory immune responses and host defense against pathogens. Immunol Rev 226:57–79PubMedCrossRefGoogle Scholar
  8. Korn T, Bettelli E, Oukka M, Kuchroo VK (2009) IL-17 and Th17 Cells. Annu Rev Immunol 27:485–517PubMedCrossRefGoogle Scholar
  9. Kotani Y, Shinkai S, Okamatsu H, Toba M, Ogawa K, Yoshida H, Fukaya T, Fujiwara Y, Chaves PH, Kakumoto K, Kohda N (2010) Oral intake of Lactobacillus pentosus strain b240 accelerates salivary immunoglobulin A secretion in the elderly: a randomized, placebo-controlled, double-blind trial. Immun Ageing: I & A 7:11CrossRefGoogle Scholar
  10. Kukowska-Kaszuba M, Dzierzbicka K, Serocki M, Skladanowski A (2011) Solid phase synthesis and biological activity of tuftsin conjugates. J Med Chem 54(7):2447–54PubMedCrossRefGoogle Scholar
  11. Kumar M, Kumar A, Nagpal R, Mohania D, Behare P, Verma V, Kumar P, Poddar D, Aggarwal PK, Henry CJ, Jain S, Yadav H (2010) Cancer-preventing attributes of probiotics: an update. Int J Food Sci Nutr 61(5):473–96PubMedCrossRefGoogle Scholar
  12. Ley RE, Peterson DA, Gordon JI (2006) Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 124(4):837–48PubMedCrossRefGoogle Scholar
  13. Lise LD, Audibert F (1989) Immunoadjuvants and analogs of immunomodulatory bacterial structures. Curr Opin Immunol 2(2):269–74PubMedCrossRefGoogle Scholar
  14. 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):73–81CrossRefGoogle Scholar
  15. Maragkoudakis PA, Chingwaru W, Gradisnik L, Tsakalidou E, Cencic A (2010) Lactic acid bacteria efficiently protect human and animal intestinal epithelial and immune cells from enteric virus infection. Int J Food Microbiol 141(Suppl 1):S91–7PubMedCrossRefGoogle Scholar
  16. Mayer L (2000a) Mucosal immunity and gastrointestinal antigen processing. J Pediatr Gastroenterol Nutr 30(Suppl):S4–12PubMedCrossRefGoogle Scholar
  17. Mayer L (2000b) Oral tolerance: new approaches, new problems. Clin Immunol 94(1):1–8PubMedCrossRefGoogle Scholar
  18. Mazmanian SK, Round JL, Kasper DL (2008) A microbial symbiosis factor prevents intestinal inflammatory disease. Nature 453(7195):620–5PubMedCrossRefGoogle Scholar
  19. Menendez-Arias L, Mas A, Domingo E (1998) Cytotoxic T-lymphocyte responses to HIV-1 reverse transcriptase (review). Viral Immunol 11(4):167–81PubMedCrossRefGoogle Scholar
  20. Mills KH (2008) Induction, function and regulation of IL-17-producing T cells. Eur J Immunol 38(10):2636–49PubMedCrossRefGoogle Scholar
  21. Mora JR, Iwata M, Eksteen B, Song SY, Junt T, Senman B, Otipoby KL, Yokota A, Takeuchi H, Ricciardi-Castagnoli P, Rajewsky K, Adams DH, von Andrian UH (2006) Generation of gut-homing IgA-secreting B cells by intestinal dendritic cells. Science 314(5802):1157–60PubMedCrossRefGoogle Scholar
  22. Mucida D, Pino-Lagos K, Kim G, Nowak E, Benson MJ, Kronenberg M, Noelle RJ, Cheroutre H (2009) Retinoic acid can directly promote TGF-beta-mediated Foxp3(+) Treg cell conversion of naive T cells. Immunity 30(4):471–2, author reply 472–3PubMedCrossRefPubMedCentralGoogle Scholar
  23. Neurath MF, Finotto S, Glimcher LH (2002) The role of Th1/Th2 polarization in mucosal immunity. Nat Med 8(6):567–73PubMedCrossRefGoogle Scholar
  24. Nissen JC, Selwood DL, Tsirka SE (2013) Tuftsin signals through its receptor neuropilin-1 via the transforming growth factor beta pathway. J Neurochem 127(3):394–402PubMedCrossRefGoogle Scholar
  25. Owen RL, Bhalla DK (1983) Cytochemical analysis of alkaline phosphatase and esterase activities and of lectin-binding and anionic sites in rat and mouse Peyer’s patch M cells. Am J Anat 168(2):199–212PubMedCrossRefGoogle Scholar
  26. Peine M, Rausch S, Helmstetter C, Frohlich A, Hegazy AN, Kuhl AA, Grevelding CG, Hofer T, Hartmann S, Lohning M (2013) Stable T-bet(+)GATA-3(+) Th1/Th2 hybrid cells arise in vivo, can develop directly from naive precursors, and limit immunopathologic inflammation. PLoS Biol 11(8):e1001633PubMedCrossRefPubMedCentralGoogle Scholar
  27. Posthumus WP, Lenstra JA, Schaaper WM, van Nieuwstadt AP, Enjuanes L, Meloen RH (1990) Analysis and simulation of a neutralizing epitope of transmissible gastroenteritis virus. J Virol 64(7):3304–9PubMedPubMedCentralGoogle Scholar
  28. Pouwels PH, Leunissen JA (1994) Divergence in codon usage of Lactobacillus species. Nucleic Acids Res 22(6):929–36PubMedCrossRefPubMedCentralGoogle Scholar
  29. 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 9:249PubMedCrossRefPubMedCentralGoogle Scholar
  30. Qu N, Xu M, Mizoguchi I, Furusawa J, Kaneko K, Watanabe K, Mizuguchi J, Itoh M, Kawakami Y, Yoshimoto T (2013) Pivotal roles of T-helper 17-related cytokines, IL-17, IL-22, and IL-23, in inflammatory diseases. Clin Dev Immunol 2013:968549PubMedCrossRefPubMedCentralGoogle Scholar
  31. Rahimi RA, Leof EB (2007) TGF-beta signaling: a tale of two responses. J Cell Biochem 102(3):593–608PubMedCrossRefGoogle Scholar
  32. Sakaguchi S, Baba K, Ishikawa M, Yoshikawa R, Shojima T, Miyazawa T (2008a) Focus assay on RD114 virus in QN10S cells. J Vet Med Sci / J Soc Vet Sci 70(12):1383–6CrossRefGoogle Scholar
  33. Sakaguchi S, Yamaguchi T, Nomura T, Ono M (2008b) Regulatory T cells and immune tolerance. Cell 133(5):775–87PubMedCrossRefGoogle Scholar
  34. Sarra M, Pallone F, Macdonald TT, Monteleone G (2010) IL-23/IL-17 axis in IBD. Inflamm Bowel Dis 16(10):1808–13PubMedCrossRefGoogle Scholar
  35. Siemion IZ, Kluczyk A (1999) Tuftsin: on the 30-year anniversary of Victor Najjar’s discovery. Peptides 20(5):645–74PubMedCrossRefGoogle Scholar
  36. Su B, Wang J, Wang X, Jin H, Zhao G, Ding Z, Kang Y, Wang B (2008) The effects of IL-6 and TNF-alpha as molecular adjuvants on immune responses to FMDV and maturation of dendritic cells by DNA vaccination. Vaccine 26(40):5111–22PubMedCrossRefGoogle Scholar
  37. Van Eden W, Van Der Zee R, Van Kooten P, Berlo SE, Cobelens PM, Kavelaars A, Heijnen CJ, Prakken B, Roord S, Albani S (2002) Balancing the immune system: Th1 and Th2. Ann Rheum Dis 61(Suppl 2):i25–8Google Scholar
  38. Wardowska A, Dzierzbicka K, Trzonkowski P, Mysliwski A (2006) Immunomodulatory properties of new conjugates of muramyl dipeptide and nor-muramyl dipeptide with retro-tuftsin (Arg-Pro-Lys-Thr-OMe). Int Immunopharmacol 6(10):1560–8PubMedCrossRefGoogle Scholar
  39. Wardowska A, Dzierzbicka K, Menderska A, Trzonkowski P (2013) New conjugates of tuftsin and muramyl dipeptide as stimulators of human monocyte-derived dendritic cells. Protein Pept Lett 20(2):200–4PubMedCrossRefGoogle Scholar
  40. Wilson-Welder JH, Torres MP, Kipper MJ, Mallapragada SK, Wannemuehler MJ, Narasimhan B (2009) Vaccine adjuvants: current challenges and future approaches. J Pharm Sci 98(4):1278–316PubMedCrossRefGoogle Scholar
  41. 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):68–75PubMedCrossRefPubMedCentralGoogle Scholar
  42. Yin JC, Ren XF, Li YJ (2005) Molecular cloning and phylogenetic analysis of ORF7 region of chinese isolate TH-98 from transmissible gastroenteritis virus. Virus Genes 30(3):395–401PubMedCrossRefGoogle Scholar
  43. Yorulmaz E, Adali G, Yorulmaz H, Ulasoglu C, Tasan G, Tuncer I (2011) Metabolic syndrome frequency in inflammatory bowel diseases. Saudi J Gastroenterol: Off J Saudi Gastroenterol Assoc 17(6):376–82CrossRefGoogle Scholar
  44. Zhang N, Hou X, Yu L, Wang G, Zhao Z, Gao Y (2010) Colonization and distribution of recombinant Lactobacillus casei with green fluorescent protein in mice intestine. Wei sheng wu xue bao = Acta Microbiologica Sinica 50(9):1232–8PubMedGoogle Scholar
  45. Zhu J, Paul WE (2010) Peripheral CD4+ T-cell differentiation regulated by networks of cytokines and transcription factors. Immunol Rev 238(1):247–62PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Xinpeng Jiang
    • 1
  • Meiling Yu
    • 1
  • Xinyuan Qiao
    • 1
  • Min Liu
    • 1
  • Lijie Tang
    • 1
  • Yanping Jiang
    • 1
  • Wen Cui
    • 1
  • Yijing Li
    • 1
  1. 1.Department of Preventive Veterinary Medicine, College of Veterinary MedicineNortheast Agricultural UniversityHarbinPeople’s Republic of China

Personalised recommendations