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Graduate Symposium Presentation


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In between the rotavirus and multiple sclerosis projects.

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Graduate Symposium Presentation

  1. 1. Immunity vs. Tolerance: Production of a vaccine antigen and autoantigen in tobacco and lettuce chloroplasts. Wesley Schonborn PI: Dr. Henry Daniell
  2. 2. Chloroplasts <ul><li>Up to 100 copies of the chloroplast genome in each chloroplast and up to 100 chloroplasts per plant cell allows accumulation of large amounts of foreign protein (10,000 copies!) </li></ul><ul><li>Transgenes can be placed in tandem </li></ul><ul><li>Maternal inheritance prevents unwanted gene transmission </li></ul>
  3. 3. Bombardment
  4. 4. Gene Integration <ul><li>Homologous recombination allows the gene of interest to be precisely placed </li></ul><ul><li>Placed between trnI and trnA in the inverted repeat region </li></ul>
  5. 5. Selection <ul><li>Spectinomycin used for selection </li></ul><ul><li>Concentration depends on the plant species being transformed </li></ul>
  6. 6. Oral Delivery <ul><li>Orally deliverable plant material reduces processing and storage costs </li></ul><ul><li>Easy administration </li></ul><ul><li>Immunological advantages </li></ul>
  7. 7. CTB <ul><li>Cholera toxin B subunit (CTB) has been used as a mucosal adjuvant to induce both immunity and tolerance . </li></ul><ul><li>CTB attaches to GM1 receptors on intestinal walls and enhances the immune response (Holmgren et al 1993, Sun et al 1994) </li></ul>
  8. 8. Mucosal Immunology
  9. 9. Oral Tolerance <ul><li>It has been demonstrated in EAE mice that tolerance can be induced upon feeding of autoantigens and specific cytokines (Inobe et al 1998, Slavin et al 2000) </li></ul><ul><li>Regulatory T cells are induced in the gut and keep the immune system from reacting to self antigens </li></ul><ul><li>But, not all antigens introduced into the gut are tolerated </li></ul>
  10. 10. Multiple Sclerosis <ul><li>Chronic autoimmune demyelinating disease affecting 2.5 million people worldwide </li></ul><ul><li>Often, attacks occur infrequently, usually months to years apart </li></ul><ul><li>The most common animal model used to study MS is experimental autoimmune/allergic encephalomyelitis (EAE). </li></ul><ul><li>Myelin basic protein (MBP) is one of the major autoantigens </li></ul>Myelin sheath
  11. 11. IL-4 and IL-10 <ul><li>The cytokines IL-4 and IL-10 both reduce of inflammation and stimulate a Th2 response </li></ul><ul><li>Current delivery methods for cytokines involve intravenous injection </li></ul><ul><li>Oral delivery of IL-4 and IL-10 would simplify delivery </li></ul>
  12. 12. MS Project Vector Construction IL-4/10, MBP PCR Add SmaI and XbaI IL-4/10, MBP SmaI XbaI IL-4/10, MBP pCR Blunt II Topo pBR322 Cut with XbaI and SmaI Ligate into vector with CTB IL-4/10, MBP SmaI XbaI NdeI NotI SmaI XbaI CTB psbA(T)
  13. 13. MS Project Vector Construction IL-4/10, MBP SmaI XbaI NdeI NotI Cut with NdeI and NotI IL-4/10, MBP XbaI NotI SmaI NdeI SalI Ligate into LsLf vector CTB psbA(T) CTB psbA(P) psbA(T)
  14. 14. Rotavirus (CTB-NSP4) <ul><li>Major cause of diarrheal deaths of about 440,000 children annually with a disproportionate number (82%) in poor countries </li></ul><ul><li>The first rotavirus vaccine, RotaShield, was removed from the market </li></ul><ul><li>Two new vaccines, Rotarix and RotaTeq, have been approved </li></ul><ul><li>Using CTB as an adjuvant also has the beneficial side-effect of immunizing for cholera </li></ul>
  15. 15. NSP4 <ul><li>The first discovered viral enterotoxin </li></ul><ul><li>Cause of diarrhea </li></ul><ul><li>Helps folding of viral proteins </li></ul><ul><li>Released from infected cell </li></ul>NSP4 ( N on- S tructural P rotein 4 )
  16. 16. Vector Map NSP4
  17. 17. 4.3 kb 2 kb 5 kb
  18. 18. Western/Quantification There is some degradation over time (in older leaves). Young and bottom full-sized leaves were separated since they were several weeks apart in age. 50kDa 25kDa 15kDa Young Mature Old Also want to compare tobacco species.
  19. 19. Age of Leaf Quantification Young Mature Old
  20. 20. Age of Leaf Quantification <ul><li>CTB-NSP4 is under control of a light and developmentally controlled promoter, psbA </li></ul><ul><li>Summer plants have higher expression than Fall/Winter plants </li></ul><ul><li>Sampling at each stage shows which is the best for CTB-NSP4 </li></ul>
  21. 21. Time of Day Quantification <ul><li>CTB-NSP4 is under control of the light-regulated promoter, psbA </li></ul><ul><li>Protein generally increase throughout the day </li></ul>
  22. 22. Future Studies <ul><li>Animal study on CTB-NSP4 to evaluate passive immunity in pups </li></ul><ul><ul><li>Low-cost dual oral vaccine for cholera and rotavirus </li></ul></ul><ul><ul><li>Correlate protection and immunoglobulins </li></ul></ul><ul><li>Complete and bombard the MS vectors </li></ul><ul><li>Animal study to evaluate the effectiveness of orally delivered CTB-MBP and cytokines </li></ul><ul><ul><li>Restore oral tolerance to treat multiple sclerosis </li></ul></ul><ul><ul><li>Determine whether cytokines given orally are effective </li></ul></ul>
  23. 23. ACKNOWLEDGEMENTS <ul><li>Dr. Daniell and the Daniell lab members. </li></ul>
  24. 24. References Holmgren J & C Czerkinsky. Mucosal Immunity and Vaccines. Nature Medicine 11:S45-S53 (2005). Holmgren J, N Lycke & C Czerkinsky. Cholera toxin and cholera B subunit as oral-mucosal adjuvant and antigen vector systems. Vaccine 11:1179-1206 (1993). Inobe J-i, AJ Slavin, Y Komagata, Y Chen, et al. IL-4 is a differentiation factor for transforming growth factor- secreting Th3 cells and oral administration of IL-4 enhances oral tolerance in experimental allergic encephalomyelitis. European Journal of Immunology 28:2780-2790 (1998). Slavin AJ, R Maron & HL Weiner. Mucosal administration of IL-10 enhances oral tolerance in autoimmune encephalomyelitis and diabetes. International Immunology 13:825-833 (2001). Sun J-B, J Holmgren & C Czerkinsky. Cholera toxin B subunit: an efficient transmucosal carrier-delivery system for induction of peripheral immunological tolerance. PNAS 91:10795-10799 (1994).