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Combating Infectious Diseases in Africa:

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Presentation on theme: "Combating Infectious Diseases in Africa:"— 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 Top 10 causes of Death Disease WORLD LOW INCOME COUNTRIES
# Deaths (in millions) Disease WORLD LOW INCOME COUNTRIES Coronary heart disease 7.20 2.47 Stroke & other cerebrovascular diseases 5.71 1.48 Lower respiratory infections 4.18 2.94 Chronic obstructive pulmonary disease 3.02 0.94 Diarrhoeal diseases 2.16 1.81 HIV/AIDS 2.04 1.51 Tuberculosis 1.46 0.91 Malaria 0.88 0.86 Prematurity and low birth weight 1.18 0.84 World Health Organization Fact sheet No 310 / November 2008

33.2 million people living with HIV in 2007 World Health Statistics 2008 (WHO)

4 Treatments for opportunistic infections
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 Potential viral targets for microbicide compounds Shattock & Moore, Nature Reviews Microbiology (1) 2003

5 Production capacity shortage
10 000 20 000 30 000 40 000 50 000 60 000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 Kg of mAb Mammalian cell culture protein capacity in Kg Optimistic mAb demand (Dain Rauscher 2000) Realistic mAb demand (CSFB 2001)

6 Production costs for antibodies
cost in $ / gram hybridomas 1000 transgenic animals 100 transgenic plants 50 Daniell et al. (2001) TIPS 6, E. coli & yeast transgenic animals & cells transgenic plants

7 Features / limitations of alternative expression systems
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 1

8 Molecular Pharming Why use plants?
= Production of pharmaceutical molecules in plants Why use 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

9 Pharmaceutical antibodies currently produced in plants (in R & D)
Antigen Plant Antibody form Application 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 CD 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 Stoger et al. (2002) Current Opinion in Biotechnology 13(2)

10 Costs for recombinant antibody production in maize
Purification level Purification process % purity $ cost/gram Maize meal Milled endosperm 0.1 0.20 Enriched Extraction, ultrafiltration 25 0.60 Moderately pure Tangential flow filtration 70 2.10 High purity Ion exchange 95 3.70 Rx grade QA/QC Affinity purification >99 20-200 EPICYTE, 2001

11 HIV neutralizing monoclonal antibodies: b12, 2F5, 4E10 and 2G12
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)

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): HIV neutralizing monoclonal antibodies (MAbs): b12, 2F5, 4E10 & 2G12

13 Insulin-dependent diabetes mellitus
Type 1 Diabetes (T1DM) (Bruna Miralpeix) caused by the autoimmune destruction of pancreatic beta cells 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) Collaboration: Department of Science and Technology, University of Verona

14 Biosafety 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)

15 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: 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 Biosafety issues of Molecular Pharming
Gene transfer to the environment Human & animal health safety issues Inadvertant entry into the food chain Proper risk management & stewardship Good Manufacturing Practice (GMP) Adherence to USDA & FDA guidelines  prevent entry into foodchain 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 Success in seperation and production of hybrid maize seed US Federal Seed Act (USDA)  95% pure to be labeled as a single hybrid

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

22 Plant Transformation Type-I Callus initiation Shoot development
(PPT selection) (PPT selection) Rooting & Regeneration (PPT selection) Hardened off * Protein analysis Screening (T1 seeds) Pollination 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 No MAbs

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 Why maize seeds ? 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 Reviewed in Ramessar et al., Plant Science 2008

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