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Combating Infectious Diseases in Africa: The Contribution of PLANT BIOTECHNOLOGY Koreen Ramessar, Teresa Capell & Paul Christou Departament de Producció.

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Presentation on theme: "Combating Infectious Diseases in Africa: The Contribution of PLANT BIOTECHNOLOGY Koreen Ramessar, Teresa Capell & Paul Christou Departament de Producció."— Presentation transcript:

1 Combating Infectious Diseases in Africa: The Contribution of PLANT BIOTECHNOLOGY Koreen Ramessar, Teresa Capell & Paul Christou Departament de Producció Vegetal I Ciència Forestal University of Lleida, Spain

2 DiseaseWORLD LOW INCOME COUNTRIES Coronary heart disease Stroke & other cerebrovascular diseases Lower respiratory infections Chronic obstructive pulmonary disease Diarrhoeal diseases HIV/AIDS Tuberculosis Malaria Prematurity and low birth weight Top 10 causes of Death # Deaths (in millions) World Health Organization Fact sheet No 310 / November 2008

3 PREVALENCE OF HIV INFECTION AMONG ADULTS (1990–2007) 33.2 million people living with HIV in 2007 World Health Statistics 2008 (WHO)

4 Treatments: Antiretroviral treatment (HAART) – drugs to slow down viral replication Treatments for opportunistic infections Vaccines (antigen & antibody administration) trials Microbicides - Gels, creams, films, suppositories, or vaginal rings; Contraceptive or non-contraceptive Shattock & Moore, Nature Reviews Microbiology (1) 2003 Potential viral targets for microbicide compounds

5 Production capacity shortage Mammalian cell culture protein capacity in Kg Optimistic mAb demand (Dain Rauscher 2000) Realistic mAb demand (CSFB 2001) Kg of mAb

6 Production costs for antibodies Production costscost in $ /gram hybridomas1000 transgenic animals100 transgenic plants50 Daniell et al. (2001) TIPS 6, E. coli & yeast transgenic animals & cells transgenic plants

7 Features / limitations of alternative expression systems 1 Bacteria: no glycosylation of heterologous proteins Yeast: Pichia pastoris: only high mannose type Saccharomyces cerevisiae: hyperglycosylation, no sialyltransferase Insect cells: extensive glycosylation, no sialyltransferase: insect specific glycans, e.g. bee venom-related to anaphylactic shock Mammalian cell cultures: CHO=G0 glycoforms >MBL-RA, NSO = gal  1,3gal epitopes. Glycosylation patterns are dependent on cell line and culturing conditions, glycoengineering done on these systems  closer (naturally) to human glycans. However extensive engineering might be limited due to severe side-effects of altering glycosylation patterns of endogenous proteins

8 Molecular Pharming = Production of pharmaceutical molecules in plants Scale-up technology available for harvesting and processing plants Plant cells resemble mammalian cells in possessing an endomembrane system, allowing the folding, assembly and post-translational modification of complex proteins Simplification of the purification requirement Plants are not infected by potential human pathogens, such as prions or viruses, which reduces production costs, and minimizes health risks Amenable to technology transfer to developing countries Ab production in plants (crude extract for topical application) ~ €0.13 per gram (Epicyte 2001) Easy and cheap to grow Why use plants?

9 Pharmaceutical antibodies currently produced in plants (in R & D) Streptococcus surface antigen tobacco SigA/G (CaroRx) Therapeutic (topical) Herpes simplex virus soybean, rice IgG Therapeutic (topical) Respiratory Syncytial virus maize IgG Therapeutic (inhaled) Sperm maize IgG Contraceptive (topical) Non-Hodgkins lymphoma tobacco scFv Personalised vaccines Herpes simplex virus maize sIgA Therapeutic Human IgG alfalfa IgG Diagnostic Rhesus D Arabidopsis IgG Diagnostic Rabies virus tobacco IgG Therapeutic Carcinoembryonic antigen tobacco, rice, scFV, diabody Therapeutic/Diagnostic wheat, tomato Colon cancer antibody tobacco IgG Therapeutic/Diagnostic CD40 tobacco cell culture scFV-immunotoxin Therapeutic Herpes simplex virus Chlamydomonas scFv Therapeutic Glycophorin barley, potato, scFv-fusion Diagnostic (HIV) tobacco Human chorionic gonadotropin tobacco scFV, diabody, IgG1 Diagnostic/Contraceptive Antigen Plant Antibody form Application Stoger et al. (2002) Current Opinion in Biotechnology 13(2)

10 Costs for recombinant antibody production in maize EPICYTE, 2001 Purification levelPurification process% purity$ cost/gram Maize mealMilled endosperm EnrichedExtraction, ultrafiltration Moderately pureTangential flow filtration High purityIon exchange Rx grade QA/QCAffinity purification>

11 To express functional 2G12 neutralising HIV monoclonal antibody in maize seed; To identify highly expressing plants for purification of the 2G12 antibody for use as topical application (microbicide/vaginal cream) monoclonal HIV neutralizing monoclonal antibodies: b12, 2F5, 4E10 and 2G12

12 2G12 produced in maize seeds High & stable expression in maize seeds (~ 100 µg/g dry seed weight) Correctly processed N-terminus Functionally equivalent to its CHO-derived counterpart Can be efficiently purified (90% purity)  Ramessar et al. (2008) PNAS 105(10): monoclonal2F5, 4E102G12 HIV neutralizing monoclonal antibodies (MAbs): b12, 2F5, 4E10 & 2G12

13  caused by the autoimmune destruction of pancreatic beta cells glutamic acid decarboxylase  smaller isoform of glutamic acid decarboxylase of 65 KDa (GAD65): major autoantigen  mice studies: parenteral administration of GAD65 can prevent (or delay) the onset of diabetes  Poor GAD protein solubility (bacteria) + inadequate production (eukaryotic cells)  Molecular pharming: transgenic plants to be screened (seeds) Insulin-dependent diabetes mellitus Type 1 Diabetes (T1DM) (Bruna Miralpeix) Collaboration: Department of Science and Technology, University of Verona

14  Regulatory approval: Safety and Risk assessment studies  Risk assessment (EC, 2002; Codex Alimentarius, 2001) hazard identification, hazard characterization, exposure assessment and risk characterization  Environmental and food/feed safety assessments  Different between countries  USA and Canada – substantial equivalence  Europe - process (precaution) Biosafety

15 Comparative approach (Substantial equivalence) Comparative approach (Substantial equivalence):  compares GE-derived products with their non-GE counterparts  if substantially equivalent (composition & nutritional characteristics)  regarded as safe as the conventional food (FDA, 1992; OECD, 1993)  does not require extensive safety testing  No absolute safety or zero risk  proposes that safety evaluated as equivalent to common foods is an acceptable risk Precautionary Principle : Wingspread Statement : “When an activity raises threats of harm to human health or the environment, precautionary measures should be taken even if some cause and effect relationships are not fully established scientifically.”  assumes GE product inherently hazardous from beginning  incorporated into Cartagena Protocol

16 Summarized Summarized: Precautionary Principle assumes that a GMO is best treated as unsafe, unless proved otherwise; Comparative approach assumes that a GMO is the same as its non-modified counterpart, unless proved otherwise  Debate continues – What level of precaution is required? What level of scientific evidence for absence of risk is required? Relationship between risk assessment and cost-benefit analysis?  Adventitious presence thresholds: EU mandatory 0.9% labeling USA voluntary 5% labeling

17 Gene transfer to the environment Human & animal health safety issues Inadvertant entry into the food chain Biosafety issues of Molecular Pharming Proper risk management & stewardship Good Manufacturing Practice (GMP) Adherence to USDA & FDA guidelines  prevent entry into foodchain Success in seperation and production of hybrid maize seed US Federal Seed Act (USDA)  95% pure to be labeled as a single hybrid Successful segregation of non-transgenic oilseed rape varieties:  variety for oil (used as lubricant & plasticizer) contains high levels of erucic acid (harmful upon ingestion)  zero erucic acid, zero glucosinolate oilseed rape (canola) – edible oil

18 Acknowledgements European Union (6th Framework) Acciones Complementarias (MEC) Centre CONSOLIDER on Agrigenomics (funded by Spanish Ministry of Education & Science) Generalitat de Catalunya Gates Foundation



21  Chinese hamster ovary (CHO) in vitro cell culture €770  1mg of purified 2G12 = €770 (Polymun Scientific) 2G12 action 4E10 action 90 HIV isolates tested  4E10 (100% inhibition); 2G12 (50% inhibition)

22 Plant Transformation Type-I Callus initiationShoot development Rooting & Regeneration Hardened off Pollination (PPT selection) * Protein analysis Screening (T 1 seeds) Primary transformants (independent events)

23 N-glycosylation in mammals/humans versus plants Mammals Plants   1,6 core fucosylation  proximal 1,4 galactosidation  terminal sialylation  complex glycans dominate   1,3 core fucosylation &  1,2 core xylose  proximal galactosidation not common, only 1,3 type proximal  1,3 fucosylation  no terminal sialylation

24 NoMAbs

25 Advantages of cereals Grown world-wide Well established agricultural and processing infrastructure Easy scale-up High stability of recombinant proteins in dry seeds Easy storage and distribution No toxic compounds GRAS status

26 Larger grain size Higher proportion of endosperm, up to 82% of the seed (Watson et al., 2000) Selective breeding  optimized for increased seed yield Higher biomass yield per hectare & lower production costs (Giddings et al., 2000) C4 photosynthetic pathway  more efficient at biomass production Seeds protected husk: prevents seed shattering reduces likelihood of seed loss during harvesting, helps prevent microbial infections (Sparrow et al., 2007) PMPs stable in maize seeds  cracked, flaked seeds:- 10°C (3 months) no significant loss of activity no loss with high-temperature grinding Stable for at least 6 years  easy transport & storage Why maize seeds ? Reviewed in Ramessar et al., Plant Science 2008

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