Kathleen L. Hefferon Cornell University

1. Plant- derived Pharmaceuticals for Developing Countries Kathleen L. Hefferon Cornell University

2. Vaccines? In Plants? Concept: vaccine proteins which are expressed in plants can safely mount an immune response in humans

3. Why Plant-derived Vaccines ? -the World Health Organization estimates that approximately 4 million people die every year from preventable diseases. -traditional vaccines are expensive and In short supply -circumvents the need for injection, refrigeration, needles, trained medical workers: all stumbling blocks for providing vaccines to poor, developing countries

4. Heat-stable plant-made therapeutic proteins are inexpensive to produce, can be administered orally and could be utilized to enhance the availability of inexpensive pharmaceuticals, particularly to those in developing countries.

5. Advantages of Plant-derived Vaccines • Relatively low cost of production in plants • -Large quantities of vaccine proteins can be produced • -Safer, less chance of contamination • No refrigeration required, no medical infrastructure required • Plants have potential of producing multiple vaccine epitopes; • can therefore confer protective immunity against several diseases at once • Plant-derived vaccines possess post-translational modifications similar to • animal-derived vaccines

6. A selective advantage of plant-made pharmaceuticals such as vaccine proteins is that the plant tissue they reside in can deliver the antigen to the mucosal immune system and at the same time prevent the antigen from degradation as it passes through the gut.

7. Clinical Trials with Plant-derived Hepatitis B Surface Antigen Construct used to create transgenic line BL BR Antibiotic resistance gene Potato HBsAg Termination promoter signal Richter et al., 2000 Nature Biotechnology Human Clinical Trials Thanavala et al., 2005 PNAS

8. Plant Virus Expression Vectors Advantages: -Higher levels of protein expressed over conventional transgenic plants -Peak protein expression levels can be achieved within a short period of time -Ability to produce proteins which are potentially toxic to plants at high levels -Many biosafety concerns associated with transgenic plants can be avoided

9. Vaccine epitopes or full-length proteins can be expressed from plant virus expression vectors Cowpea Mosaic Virus Tobacco Mosaic Virus George Lomonossoff’s lab Gleba et al., Current Opinion in Biotechnology, Volume 18, Issue 2, April 2007, Pages 134–141

10. Geminivirus Expression System Bean Yellow Dwarf Geminivirus (BeYDV) -recently isolated from South Africa -member of the Mastreviridae Structural Studies of Plant Geminiviruses: Carolina G. Casado, Javier Ortiz, Mavis Agbandje-McKenna

11. Genomic Organization of Bean Yellow Dwarf Virus (BeYDV) -single-stranded circular DNA molecule -2.6 kbp in length - 4 genes LIR, SIR: long and short intergenic regions C-sense strand: Rep (replication initiator protein): required for replication RepA: role in cell cycle control V-sense strand: MP: movement protein CP: coat protein Rep, LIR and SIR are required for replication MP LIR V1 Rep RepA C1 V2 intron C2 CP SIR

12. BeYDV replicon system for foreign protein expression Absence of Rep Presence of Rep integrated BeYDV expresssion vector gene of interest chromosome Plant nucleus

13. Construction of BeYDV-based expression vector LIR 35S GOI SIR (Rep) LIR T GFP expression in NT-1 cells post-cobombardment with GFP pSKBYD1.4 p35SRep p35SRep p35SΔRep

14. Staphylococcus Enterotoxin B (SEB) ‘Toxic Shock Syndrome’ Expression of SEB in plant cell lines using BeYDV expression system pBYSEB+p35SRep pBYSEB nonbombarded

15. Applications for BeYDV Replicon Expression System ● HBV surface protein in plant bioreactor (O’Neill et al., 2008) ● Expression of HBV core antigen and NV CP as VLPs (Huang et al., 2009) ● HPV-16 major CP L1, HIV p24 antigen (Regnard et al., 2010) ● Heavy and light chain IgG Mab against Ebola virus GP1 (Huang et al., 2011) Expression levels: 1 mg/ g leaf mass 4 days post-agroinoculation

16. Ebola antibody treatment, produced in plants, protects monkeys from lethal disease

17. Downstream Processing of Plant-Made Pharmaceuticals Harvest Crop Storage/Processing Protein extraction, purification

18. Provides a unique opportunity to make an impact on global health through the responsible development of plant biotechnology

19. Applications for rapid, inexpensive, easily stored Plant-Made Vaccines Developing countries: cholera, rotavirus, Norwalk virus vaccines (major killers of children) also rabies, dengue fever virus (poorly funded), HPV, HIV various monoclonal antibodies Pandemics: H1N1 influenza virus Defense: Anthrax, plague, smallpox virus Personalized medicine: Non-Hodgkins Lymphoma The first licensed plant-made vaccine is for Newcastle disease virus, a veterinary vaccine generated to protect poultry.

20. Companies involved in commercialization of biopharmaceuticals from plants: -orally delivered glucocerebrosidase (GCD) enzyme for the potential treatment of Gaucher disease, generated in carrot suspension cells.

21. deconstructed TMV virus vector system to produce Non Hodgkin’s Lymphoma vaccine

22. -PVX used as carrier and adjuvant for Human epidermal growth factor receptor 2 (HER2) epitope presentation Example from BAYER INNOVATION GmbH

23. Plant Virus Nanoparticles can block tumor progression

25. Plant virus nanoparticles can be engineered for pharmaceutical production or for tumor homing CPMV TMV PVX CPMV A. B. TMV tumor PVX Fusion proteins Mab

26. Conclusions and Impact on the Developing World Plant made vaccines could slow the spread of major diseases of the third world, such as HIV, HBV, malaria and in the future, even some cancers. Plant made pharmaceuticals are inexpensive to produce, do not require sophisticated equipment to be grown or harvested and perhaps could even be produced locally, to address the specific needs of a geographical region.

29. Acknowledgements