combating infectious diseases in africa: the contribution of plant biotechnology koreen ramessar,...
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Combating Infectious Diseases in Africa:
The Contribution of The Contribution of
PLANT BIOTECHNOLOGYPLANT BIOTECHNOLOGY
Koreen RamessarKoreen Ramessar, Teresa Capell & Paul Christou, Teresa Capell & Paul Christou
Departament de Producció Vegetal I Ciència Forestal University of Lleida, Spain
Disease WORLDLOW 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
Top 10 causes of DeathTop 10 causes of Death
# Deaths (in millions)
World Health Organization Fact sheet No 310 / November 2008
PREVALENCE OF HIV INFECTION PREVALENCE OF HIV INFECTION
AMONG ADULTS (1990–2007)AMONG ADULTS (1990–2007)
33.2 million people living with HIV in 2007World Health Statistics 2008 (WHO)
TreatmentsTreatments::
• 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
Production capacity shortage
Mammalian cell culture protein capacity in KgMammalian cell culture protein capacity in Kg
Optimistic mAb demand (Dain Rauscher 2000)Optimistic mAb demand (Dain Rauscher 2000)
Realistic mAb demand (CSFB 2001)Realistic mAb demand (CSFB 2001)
0
10 000
20 000
30 000
40 000
50 000
60 000
2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Kg
of m
Ab
Production costs for antibodiesProduction costs for antibodies
Production costs cost in $ / gram
hybridomas 1000
transgenic animals 100
transgenic plants 50
Daniell et al. (2001) TIPS 6, 219-226
E. coli & yeast transgenic animals & cells transgenic plants
Features / limitations of Features / limitations of alternative expression systemsalternative 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 = gal1,3gal 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
Molecular PharmingMolecular 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?Why use plants?
Pharmaceutical antibodies currently produced Pharmaceutical antibodies currently produced in plants (in R & D)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)
Costs for recombinant antibody Costs for recombinant antibody production in maizeproduction in maize
EPICYTE, 2001
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
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)
HIV neutralizing monoclonalmonoclonal antibodies: b12, 2F5, 4E10 and 2G12
2G12 produced in maize seeds 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):3727 - 3732
HIV neutralizing monoclonalmonoclonal antibodies (MAbs): b12, 2F5, 4E102F5, 4E10 & 2G122G12
caused by the autoimmune destruction of pancreatic beta cells
smaller isoform of glutamic acid decarboxylaseglutamic 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 Insulin-dependent diabetes mellitus
Type 1 Diabetes (T1DM)Type 1 Diabetes (T1DM)(Bruna Miralpeix)(Bruna Miralpeix)
Collaboration: Department of Science and Technology, University of Verona
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)
BiosafetyBiosafety
Comparative approachComparative approach (Substantial equivalence)(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 PrinciplePrecautionary 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
SummarizedSummarized:
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
• Gene transfer to the environment
• Human & animal health safety issues
• Inadvertant entry into the food chain
Biosafety issues of Molecular PharmingBiosafety 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
Acknowledgements
European Union (6th Framework)
Acciones Complementarias (MEC)
Centre CONSOLIDER on Agrigenomics (funded by Spanish Ministry of Education & Science)
Generalitat de Catalunya
Gates Foundation
[email protected]@pvcf.udl.es
Chinese hamster ovary (CHO) in vitro cell culture
1mg of purified 2G12 = €770€770 (Polymun Scientific)
2G12 2G12
actionaction
4E10 4E10
actionaction
90 HIV isolates tested 4E10 (100% inhibition); 2G12 (50% inhibition)
Plant Transformation Plant Transformation
Type-I Callus initiation Shoot development
Rooting & Regeneration
Hardened off
Pollination
(PPT selection) (PPT selection)
(PPT selection)
* Protein analysis* Protein analysis
Screening (T1 seeds) Primary transformants
(independent events)
N-glycosylation in mammals/humansN-glycosylation in mammals/humansversus versus plants 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
Advantages of cereals 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
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 ?Why maize seeds ?
Reviewed in Ramessar et al., Plant Science 2008