Nanogram Doses of Alum-Adjuvanted HBs Antigen Induce Humoral Immune Response in Mice When Orally Administered

  • Józef Kapusta
  • Tomasz Pniewski
  • Jacek Wojciechowicz
  • Piotr Bociąg
  • Andrzej Płucienniczak
Original Article


Mucosal immunity elicited by plant-based and other orally administered vaccines can serve as the first line of defense against most pathogens infecting through mucosal surfaces, but it is also considered for systemic immunity against blood–borne diseases such as hepatitis B (HB). Previous oral immunization trials based on multiple administration of high doses of HBs antigen elicited an immune response; however, a reproducible and long-lasting immunization protocol was difficult to design. The objective of this study was to evaluate the effect of dose and timing of orally delivered alum-adsorbed antigen on the magnitude of the anti-HBs humoral response. Mice were immunized orally by gavage intubation or parenterally by intramuscular injection three times, once every 2 weeks, with doses of 5, 50, or 500 ng alum-adjuvanted HBsAg. A low dose (10 ng) of HBsAg was orally administered three times in different time intervals: 2, 4, 6, and 8 weeks. The three consecutive 5-ng oral doses of the antigen induced immune response at the protective level (≥10 mIU/ml), significantly higher than the reaction elicited by three 50 or 500 ng doses. In contrast, intramuscular delivery of these doses did not differ significantly; however, they induced a five to six times higher immune response than oral immunization. The 8-week period between each of the three oral immunizations appeared to be favorable to the anti-HBs humoral responses compared with the shorter schedules. The results presented here clearly identify the importance of low doses of antigen administered orally in extended intervals for a significantly higher anti-HBs response. This finding provides some indications concerning the strategy of orally administered vaccines, including plant-based ones.


HBs antigen HBsAg Oral immunization Anti-HBV oral vaccine 


HBs, HBsAg

Hepatitis B surface antigen


Hepatitis B


Hepatitis B virus


Milli-international unit/ml–unit of anti-HBs antibody titer


Secretory IgA


Gut-associated lymphoid tissue


Virus-like particles


Antigen-presenting cells


Cholera toxin subunit B


Heat-labile enterotoxin B


Phosphate-buffered saline



This study was supported by grant Nos. 6 P04B 012 16 and 2 P04B 001 27 of the Polish Ministry of Science and Higher Education.


  1. Agnello D, Hervé CA, Lavaux A et al (2006) Intrarectal immunization with rotavirus 2/6 virus-like particles induces an antirotavirus immune response localized in the intestinal mucosa and protects against rotavirus infection in mice. J Virol 80:3823–3832CrossRefPubMedGoogle Scholar
  2. Arakawa T, Chong DK, Langridge WH (1998) Efficacy of a food plant-based oral cholera toxin B subunit vaccine. Nat Biotechnol 16:292–297CrossRefPubMedGoogle Scholar
  3. Azis MA, Midha S, Waheed SM et al (2007) Oral vaccines: new needs, new possibilities. Bioessays 29:591–604CrossRefGoogle Scholar
  4. Bachmann MF, Zinkernagel RM (1996) The influence of virus structure on antibody responses and virus serotype formation. Immunol Today 17:553–558CrossRefPubMedGoogle Scholar
  5. Ball JM, Graham DY, Opekun AR et al (1999) Recombinant Norwalk virus-like particles given orally to volunteers: phase I study. Gastroenterology 117:40–48CrossRefPubMedGoogle Scholar
  6. Beyer AJ, Wang K, Umble AN et al (2007) Low-dose exposure and immunogenicity of transgenic maize expressing the Escherichia coli heat-labile toxin B subunit. Environ Health Perspect 115:354–360PubMedCrossRefGoogle Scholar
  7. Boisgerault F, Moron G, Leclerc C (2002) Virus-like particles: a new family of delivery systems. Expert Rev Vaccines 1:101–109CrossRefPubMedGoogle Scholar
  8. Borges O, Tavares J, de Sousa A et al (2007) Evaluation of the immune response following a short oral vaccination schedule with hepatitis B antigen encapsulated into alginate-coated chitosan nanoparticles. Eur J Pharm Sci 32:278–290CrossRefPubMedGoogle Scholar
  9. Brandtzaeg P (2003) Role of secretory antibodies in the defence against infections. Int J Med Microbiol 293:3–15CrossRefPubMedGoogle Scholar
  10. Brandtzaeg P (2007) Induction of secretory immunity and memory at mucosal surfaces. Vaccine 25:5467–5484CrossRefPubMedGoogle Scholar
  11. Brocke P, Schaefer S, Melber K (2005) Recombinant hepatitis B vaccines: disease characterization and vaccine production. In: Gellissen G et al (eds) Production of recombinant proteins. Novel microbial and eukaryotic systems. Wiley-CH Verlag GmbH & Co KGaA, Weinheim, pp 319–359Google Scholar
  12. Chen Y, Inobe J, Marks R et al (1995) Peripheral deletion of antigen-reactive T cells in oral tolerance. Nature 376:177–180CrossRefPubMedGoogle Scholar
  13. Choi AH, McNeal MM, Basu M et al (2002) Intranasal or oral immunization of inbred and outbred mice with murine or human rotavirus VP6 proteins protects against viral shedding after challenge with murine rotaviruses. Vaccine 20:3310–3321CrossRefPubMedGoogle Scholar
  14. Franco L, Benedetti R, Ferek GA et al (1998) Priming or tolerization of B- and Th2-dependent immune response by the oral administration of OVA-DNP is determined by the antigen dosage. Cell Immunol 190:1–11CrossRefPubMedGoogle Scholar
  15. Friedman A, Weiner HL (1994) Induction of anergy or active suppression following oral tolerance is determined by antigen dosage. Proc Natl Acad Sci USA 91:6688–6692CrossRefPubMedGoogle Scholar
  16. Garside P, Mowat AM (2001) Oral tolerance. Semin Immunol 13:177–185CrossRefPubMedGoogle Scholar
  17. Goldstein ST, Fiore AE (2001) Toward the global elimination of hepatitis B virus transmission. J Pediatr 139:343–345CrossRefPubMedGoogle Scholar
  18. Gotsman I, Beinart R, Alper R et al (2000) Induction of oral tolerance towards hepatitis B envelope antigens in a murine model. Antiviral Res 48:17–26CrossRefPubMedGoogle Scholar
  19. Gotsman I, Alper R, Klein A et al (2002) Inducing oral immune regulation of hepatitis B virus envelope proteins suppresses the growth of hepatocellular carcinoma in mice. Cancer 94:406–414CrossRefPubMedGoogle Scholar
  20. Heath WR, Carbone FR (2001) Cross-presentation, dendritic cells, tolerance and immunity. Annu Rev Immunol 19:47–64CrossRefPubMedGoogle Scholar
  21. Hollinger FB (1996) Hepatitis B virus. In: Fields BN, Knipe DM, Howley PM et al (eds) Fields virology. Lippincott, Philadelphia, pp 2739–2806Google Scholar
  22. Holmgren J, Czerkinsky C, Lycke N et al (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 50:42–54PubMedGoogle Scholar
  23. Huang Z, Elkin G, Maloney BJ et al (2005) Virus-like particle expression and assembly in plants: hepatitis B and Norwalk viruses. Vaccine 23:1851–1858CrossRefPubMedGoogle Scholar
  24. Huibregtse IL, Snoeck V, de Creus A et al (2007) Induction of ovoalbumin-specific tolerance by oral administration of Lactococcus lactis secreting ovoalbumin. Gastroenterology 133:517–528CrossRefPubMedGoogle Scholar
  25. International Group (1988) Immunisation against hepatitis B. Lancet 1:875–876Google Scholar
  26. Jiang XL, He ZM, Peng ZQ et al (2007) Cholera toxin B in transgenic tomato fruit induces systemic immune response in mice. Transgenic Res 16:169–175CrossRefPubMedGoogle Scholar
  27. Joung YH, Youm JW, Jeon JH et al (2004) Expression of the hepatitis B surface S and preS2 antigens in tubers of Solanum tuberosum. Plant Cell Rep 22:925–930CrossRefPubMedGoogle Scholar
  28. Kao JH, Chen DS (2002) Global control of hepatitis B virus infection. Lancet Infect Dis 2:395–403CrossRefPubMedGoogle Scholar
  29. Kapusta J, Modelska A, Figlerowicz M et al (1999) A plant-derived edible vaccine against hepatitis B virus. FASEB J 13:1796–1799PubMedGoogle Scholar
  30. Kapusta J, Modelska A, Pniewski T et al (2001) Oral immunization of human with transgenic lettuce expressing hepatitis B surface antigen. Adv Exp Med Biol 495:299–303PubMedGoogle Scholar
  31. Kew OM, Sutter RW, De Gourville EM et al (2005) Vaccine-derived polioviruses and the endgame strategy for global polio eradication. Annu Rev Microbiol 59:587–635CrossRefPubMedGoogle Scholar
  32. Kirk DD, McIntosh K, Walmsley AM et al (2005) Risk analysis for plant-made vaccines. Transgenic Res 14:449–462CrossRefPubMedGoogle Scholar
  33. Kong Q, Richter L, Yang YF et al (2001) Oral immunization with hepatitis B surface antigen expressed in transgenic plants. Proc Natl Acad Sci USA 98:11539–11544CrossRefPubMedGoogle Scholar
  34. Koprowski H (2002) Old and new prescriptions for infectious diseases and the newest recipes for biomedical products in plants. Arch Immunol Ther Exp 50:365–369Google Scholar
  35. Koprowski H, Jervis GA, Norton TW (1952) Immune responses in human volunteers upon oral administration of a rodent-adapted strain of poliomyelitis virus. Am J Hyg 55:108–126PubMedGoogle Scholar
  36. Lamphear BJ, Streatfield SJ, Jilka JM et al (2002) Delivery of subunit vaccines in maize seed. J Control Release 85:169–180CrossRefPubMedGoogle Scholar
  37. Li T, Takeda N, Miyamura T (2001) Oral administration of hepatitis E virus-like particles induces a systemic and mucosal immune response in mice. Vaccine 19:3476–3484CrossRefPubMedGoogle Scholar
  38. Lubeck MD, Davis AR, Chengalvala M et al (1989) Immunogenicity and efficacy testing in chimpanzees of an oral hepatitis B vaccine based on live recombinant adenovirus. Proc Natl Acad Sci USA 86:6763–6767CrossRefPubMedGoogle Scholar
  39. Mason HS, Haq TA, Clements JD et al (1998) Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene. Vaccine 16:1336–1343CrossRefPubMedGoogle Scholar
  40. Mason HS, Thanavala Y, Arntzen CJ et al (2003) Expression of immunogenic hepatitis B surface antigen in transgenic plants. US patent 6 551 820 B1Google Scholar
  41. Matzinger P (1994) Tolerance, danger and extended family. Annu Rev Immunol 12:991–1045PubMedGoogle Scholar
  42. McAleer WJ, Buynak EB, Maigetter RZ et al (1984) Human hepatitis B vaccine from recombinant yeast. Nature 307:178–180CrossRefPubMedGoogle Scholar
  43. McCluskie MJ, Brazolot Millan CL, Gramzinski RA et al (1999) Route and method of delivery of DNA vaccine influence immune responses in mice and non-human primates. Mol Med 5:287–300CrossRefPubMedGoogle Scholar
  44. Mestecky J, Russell MW, Elson CO (2007) Perspectives on mucosal vaccines: is mucosal tolerance a barrier? J Immunol 179:5633–5638PubMedGoogle Scholar
  45. Mestecky J, Nguyen H, Czerkinsky C et al (2008) Oral immunization: an update. Curr Opin Gastroenterol 24:713–719CrossRefPubMedGoogle Scholar
  46. Moss B, Smith GL, Gerin JL et al (1984) Live recombinant vaccinia virus protects chimpanzees against hepatitis B. Nature 311:67–69CrossRefPubMedGoogle Scholar
  47. Mowat AM (2003) Anatomical basis of tolerance and immunity to intestinal antigens. Nat Rev Immunol 3:331–341CrossRefPubMedGoogle Scholar
  48. Nardelli-Haefliger D, Benyacoub J, Lemoine R et al (2001) Nasal vaccination with attenuated Salmonella typhimurium strains expressing the hepatitis B nucleocapsid: dose response analysis. Vaccine 19:2854–2861CrossRefPubMedGoogle Scholar
  49. Obst R, van Santen HM, Mathis D et al (2005) Antigen persistence is required throughout the expansion phase of a CD4(+) T cell response. J Exp Med 201:1555–1565CrossRefPubMedGoogle Scholar
  50. Pamer EG (2007) Immune responses to commensal and environmental microbes. Nat Immunol 8:1173–1178CrossRefPubMedGoogle Scholar
  51. Peng HJ, Turner MW, Strobel S (1989) The kinetics of oral hyposensitization to a protein antigen are determined by immune status and the timing, dose and frequency of antigen administration. Immunology 67:425–430PubMedGoogle Scholar
  52. Perrie Y, Obrenovic M, McCarthy D et al (2002) Liposome (Lipodine™)-mediated DNA vaccination by the oral route. J Liposome Res 12:185–197CrossRefPubMedGoogle Scholar
  53. Poonam P (2007) The biology of oral tolerance and issues related to oral vaccine design. Curr Pharm Des 13:2001–2007CrossRefPubMedGoogle Scholar
  54. Rask C, Fredriksson M, Lindblad M et al (2000) Mucosal and systemic antibody responses after peroral or intranasal immunization: Effects of conjugation to enterotoxin B subunits and/or of co-administration with free toxin as adjuvant. APMIS 108:178–186CrossRefPubMedGoogle Scholar
  55. Richman LK, Chiller JM, Brown WR et al (1978) Enterically induced immunologic tolerance. I. Induction of suppressor T lymphocytes by intragastric administration of soluble proteins. J Immunol 121:2429–2434PubMedGoogle Scholar
  56. Richter LJ, Thanavala Y, Arntzen CJ et al (2000) Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nat Biotechnol 18:1167–1171CrossRefPubMedGoogle Scholar
  57. Shukla A, Khatri K, Gupta PN et al (2008) Oral immunization against hepatitis B using bile salt stabilized vesicles (bilosomes). J Pharm Pharm Sci 11:59–66PubMedGoogle Scholar
  58. Strobel S (2001) Immunity induced after a feed of antigen during early life: oral tolerance v. sensitization. Proc Nutr Soc 60:437–442CrossRefPubMedGoogle Scholar
  59. Swarbrick ET, Stokes CR, Soothill JF (1979) Absorption of antigens after oral immunisation and the simultaneous induction of specific systemic tolerance. Gut 20:121–125CrossRefPubMedGoogle Scholar
  60. Taams LS, van Rensen AJ, Poelen MC et al (1998) Anergic T cells actively suppress T cell responses via the antigen-presenting cell. Eur J Immunol 28:2902–2912CrossRefPubMedGoogle Scholar
  61. Tacket CO, Pasetti MF, Edelman R et al (2004) Immunogenicity of recombinant LT-B delivered orally to humans in transgenic corn. Vaccine 22:4385–4389CrossRefPubMedGoogle Scholar
  62. Thanavala Y, Mahoney M, Pal S et al (2005) Immunogenicity in humans of an edible vaccine for hepatitis B. Proc Natl Acad Sci USA 102:3378–3382CrossRefPubMedGoogle Scholar
  63. Wang L, Coppel RL (2008) Oral vaccine delivery: can it protect against non-mucosal pathogens? Expert Rev Vaccines 7:729–738CrossRefPubMedGoogle Scholar
  64. Wang Y, Wang W, Li N et al (2002) Activation of antigen-presenting cells by immunostimulatory plant DNA: a natural resource for potential adjuvant. Vaccine 20:2764–2771CrossRefPubMedGoogle Scholar
  65. Wee JL, Scheerlinck JP, Snibson KJ et al (2008) Pulmonary delivery of ISCOMATRIX influenza vaccine induces both systemic and mucosal immunity with antigen dose sparing. Mucosal Immunol 1:489–496CrossRefPubMedGoogle Scholar
  66. Wu X, Roelofs-Haarhuis K, Zhang J et al (2007) Dose dependence of oral tolerance to nickel. Int Immunol 19:965–975CrossRefPubMedGoogle Scholar
  67. Yao Q, Zhang R, Guo L et al (2004) Th cell-independent immune responses to chimeric hemagglutinin/simian human immunodeficiency virus-like particles vaccine. J Immunol 173:1951–1958PubMedGoogle Scholar
  68. Yoneda A, Tuchiya K, Takashima Y et al (2008) Protection of mice from rabies by intranasal immunization with inactivated rabies virus. Exp Anim 57:1–9CrossRefPubMedGoogle Scholar
  69. Yusibov V, Hooper DC, Spitsin SV et al (2002) Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine. Vaccine 20:3155–3164CrossRefPubMedGoogle Scholar
  70. Zinkernagel RM (2000) Localization dose and timing of antigens determine immune reactivity. Semin Immunol 12:163–171CrossRefPubMedGoogle Scholar

Copyright information

© L. Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland 2010

Authors and Affiliations

  • Józef Kapusta
    • 1
    • 2
  • Tomasz Pniewski
    • 2
  • Jacek Wojciechowicz
    • 3
  • Piotr Bociąg
    • 2
  • Andrzej Płucienniczak
    • 1
  1. 1.Institute of Biotechnology and AntibioticsWarsawPoland
  2. 2.Institute of Plant Genetics, Polish Academy of SciencesPoznanPoland
  3. 3.DNA Research CentrePoznanPoland

Personalised recommendations