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

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 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