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

Koreen.Ramessar@pvcf.udl.esKoreen.Ramessar@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

No No

MAbsMAbs

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