PhD Andrés WigdorovitzWorkshopArgentina -Japan3 rd August 2009

Institute of Virology and Institute of Genetic CICVyA, INTA, Castelar, Argentina

Institute of Genetic and Molecular Biology INGEBI, Buenos Aires, Argentina

Workshop Argentina-Japan“Bioscience and Biotechnology for the Promotion

for Agriculture and Food Production”August 3rd to 7th, 2009

Veterinary vaccines produced in plants

Summary

Production of nanobodies (Llama –derived Single –Chain Antibody Fragments), in tobacco transplantomic plants.

Production of experimental vaccines in alfalfa transgenic plants

Foot and mouse disease virus

Development of transgenic alfalfa plants containing the foot and mouth disease virus structural polyprotein P1 and its utilization as an experimental immunogen.

Bovine diarrhea virus First report of an experimental vaccine against BVDV produced in alfalfa transgenic plants that induce neutralizing antibodies and protection in cattle.

Bovine RotavirusPassive protection against bovine rotavirus (BRV) induced by a peptide edible vaccine produced in transgenic alfalfa.

Agriculture and Food Production in Argentina

WorkshopArgentina -Japan3 rd August 2009

ARGENTINAARGENTINA

•• 3,760,000 km3,760,000 km22

•• Population~ 39 M Population~ 39 M

•• GovernmentGovernment::

RepresentativeRepresentative

RepublicanRepublican

FederalFederal

•• LanguageLanguage

SpanishSpanish

NOA• Sugar

• Tobacco

• Citrus

• Oilseeds: Soybean, Olive

• Wine

• Bovine Meat

• Vegetables

• Native Forest

• Honey

• Camelids

• Cotton

NEA• Bovine Meat

• Cotton

• Oilseed: Soybean

• Implanted Forest

• Native Forest

• Yerba Mate (Ilexparaguaiensis)

• Tea

• Rice

• Citrus

• Vegetables

• TobaccoCUYO• Pip Fruit

• Wine

• Oilseed: Olive

• Vegetables

• Bovine Meat

PATAGONIA• Wool

• Ovine Meat

• Apple and Pear

• Berries

• Native Forest

• Bovine Meat

• Wine

• Honey

PAMPEANA• Wheat and Maize

• Oilseeds: Soybean-Sunflower

• Bovine Meat

• Bovine Milk

• Vegetables

• Porcine Meat

• Fruits

• Rice

• Poultry and Eggs

• Implanted Forest

ARGENTINE PRODUCTIONARGENTINE PRODUCTION

AGRICULTURAL PRODUCTIONAGRICULTURAL PRODUCTION

Grains:Grains: 41,912 million tons41,912 million tons

Oilseeds:Oilseeds: 51,502 million tons51,502 million tons

Pip fruit: Pip fruit: 1,936 million tons1,936 million tons

Citrus: Citrus: 3,091 million tons3,091 million tons

Honey: Honey: 115,000 tons115,000 tons

Wines: Wines: 15,400 million hectoliters15,400 million hectoliters

Beef: Beef: 3,117 million tons3,117 million tons

Pork: Pork: 198,050 tons198,050 tons

Poultry: Poultry: 1,160,000 tons1,160,000 tons

Milk: Milk: 9.600 million liters9.600 million liters

Wool:Wool: 76.500 tons76.500 tons

AGRICULTURAL EXPORTSAGRICULTURAL EXPORTS

Agricultural and livestock exports: 23.870 million USDAgricultural and livestock exports: 23.870 million USD

•• First world exporter of flour and oil soybean, First world exporter of flour and oil soybean, sunflower oil, honey, pears and lemon.sunflower oil, honey, pears and lemon.

•• Second world exporter of maize and sorghum.Second world exporter of maize and sorghum.

•• Third world exporter of soybean.Third world exporter of soybean.

•• Fifth world exporter of wheat and beef.Fifth world exporter of wheat and beef.

Gross Domestic Product: 213.172 million USDGross Domestic Product: 213.172 million USD

Agricultural, Livestock and AgroAgricultural, Livestock and Agro--industry GDP: industry GDP: 30%30%

INTA INTA HeadquartersHeadquarters15 Regional 15 Regional CentersCenters4747 Experimental StationsExperimental Stations4 4 ResearchResearch CentersCenters15 15 Research Institutes Research Institutes > 300> 300 Extension Agencies Extension Agencies

2 2 PrivatePrivate OrganizationsOrganizations

Alfalfa (Medicago sativa)

It is the most important forage grass in Argentine and it presents high levels of total soluble protein.

General plant advantages :

Economic.

Easy to obtain in big amounts.

It can be used as an oral vaccine.

General plants disadvantages:Low levels of heterologue protein

expression.

Production of experimental vaccines in alfalfa transgenic plants

Introduction FOOT AND MOUTH DISEASE VIRUS

FMDV is a picornavirus with a single-stranded positive sense RNA genome.

Icosaedrical capsid, 30nm size

4 structural proteins compose its capsid:VP1, VP2, VP3 and VP4.

It is responsable for the most important economiclosses of cattle production in the world

ObjectivesExpression of P1 polyprotein of Foot and Mouth Diseasevirus in transgenic alfalfa plants for the production ofexperimental vaccines.

Evaluation in a murine model.

Construction # transformed plants

pROK-P1.2A.2B.3C 20

Electronicmicroscopy

Plant A

FMDV empty capsid

Non Related gene

30 nm

Plant A

30 nm

Detection of neutralizing antibodies induced in I.P immunized mice with plant A extractsNeutralizing

assayMice IP immunized with:

Mouse Nº* SNI

Alfalfa Plant A 1 2.2

2 2.3

3 2.1

4 2.1

Alfalfa expressing a non related gene

5 0.6

6 0.6

Antibody

responseVP1 epitope

0

0,5

1

1,5

2

2,5

3

1 2 3 4 5 6 7 8 9 10 11 12 13Mouse Nº

Tit

er

(lo

g1

0)

ELISA

0,05- 0,2 mg/g TSP

Immune response in mice I.P. immunized with P1

polyprotein transgenic alfalfa plants

Complete FMDV particle

Murine model Protection against FMDV challenge in miceimmunized with P1 polyprotein transgenic plants

Mice IP immunized with:

Protection (%)

Alfalfa Plant A 13/13 (100%) 9/9 (100%)

Alfalfa expressing a non related gene

0/10 (0%) 0/6 (0%)

Buffer 0/6 (0%) 0/6 (0%)

pending patent P040102842

Introduction BOVINE ROTAVIRUS

BRV is a rotavirus with a RNA positive genome.

2 structural proteins compose its outer capsid: VP4, and VP7.

VP4 and VP7 carry critical epitopes responsible for the induction of neutralizing antibodies.

VP 6

VP 7

VP 4

Rotavirus infections are the primary cause of severe diarrhea in young animals and children in the world.

Objective To develop an experimental edible vaccine based on the use of recombinant BRV peptide fused to the enzyme β-GUS expressed in transgenic alfalfa plants.

To assess the ability of the chimerical protein to induce passive protective immunity against virulent bovine rotavirus in suckling mice.

BRV C486 oral infection

weeks42 6 750

copulatenew born

85

Presence ofdiarrhea

Experimental Vaccine

Murine model

Antibody

response

Immune response in mice intraperitoneally immunized with

eBRVP4-BGUS leaves extracts

0,000

0,200

0,400

0,600

0,800

1,000

1,200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20mice

Abs

490n

m

Immune response in mice fed with eBRVP4-BGUS fresh

leaves

vaccinated dams

respective offspring

Negative control dams

0,000

0,200

0,400

0,600

0,800

1,000

1,200

1,400

1,600

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

mice

Abs

490

nm

ELISA

Mice IP immunizedwith:

Route ofimmunization

Protection (%) Fisher´sTest

Alfalfa expressingeBRV4-βGUS

Oral 22/29 (76%) p=0.003

IP 10/14 (71%) P= 0.0063

Alfalfa expressinga non related gene

oral 4/19 (21%) p=0.003

IP 2/13 (21%) P= 0.0063

Murine model Evaluation of passive protection against BRV C486

Introduction I

BVDV is a pestivirus with a RNA positive genome.

Spherical shape 40 -60 nanometers (nm) of diameter, icosaedral capsid and external envelope.

E2 glycoprotein carries critical epitopes responsible for the induction of neutralizing antibodies.

BOVINE DIARREA VIRUS

Infects bovines of all ages, causing reproduction problems and altering biological products of high commercial value thereby, resulting in considerableeconomical losses.

Plantsexpression I

Quantification and stability of the selected Transgenic plants obtained using Agrobacterium tumefaciens -

mediated transformation of alfalfa

00,20,40,60,8

11,21,41,61,8

2

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23 P24 P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23 P24 C+ C+ C- C-

Abs

orva

ncia

405

nm

E2T: 1.7 ugr /gr leafE2: 1,5 ugr/ gr leaf

0

0,5

1

1,5

2

2,5

alfalfa E2T alfalfa E2 alfalfa C23selected plants

Abs

490

nm

3.0 0.4 0.05ugr E2T /ml

Plantsexpression I

Aqueous twoAqueous two--phase systems for purification of phase systems for purification of recombinant E2T and APCH1recombinant E2T and APCH1--E2T proteins expressed in E2T proteins expressed in

alfalfa plantsalfalfa plants

0,428

2

0,00189 0,00121

0,314

3,25

0

0,5

1

1,5

2

2,5

3

3,5

ug/m

l

Ext Planta

ATPS

CHO-K1 SD

ProtPurif 2 ug/ml

Lac Z (C-)

C23 (C-)

y = 0,558x - 1,0059R2 = 0,9919

-1

-0,8

-0,6

-0,4

-0,2

0

0,2

0,4

0 0,5 1 1,5 2 2,5

0,428

2

0,00189 0,00121

0,314

3,25

0

0,5

1

1,5

2

2,5

3

3,5

ug/m

l

Planta ext

ATPS

CHO-K1

Purified prot 2 ug/ml

Lac Z (C-)

C23 (C-)

Curve patron for E2T quantification

y = 0,558x - 1,0059R2 = 0,9919

-1

-0,8

-0,6

-0,4

-0,2

0

0,2

0,4

0 0,5 1 1,5 2 2,5

Evaluation of the immunogenicity of the E2T protein expressed in alfalfa plants in cattle

Antibody

Response I

42 60

weeks

Experimental Vaccine

8

Challenge

3 ug3Negativecontrol (C23-VP8)4

-3BVDV INTA 107/ml3

1,5 ugr3E2T cc alfalfa2

3 ug3E2T cc alfalfa 1

CATTLE DOSISnANTIGENGROUP

3 ug3Negativecontrol (C23-VP8)4

-3BVDV INTA 107/ml3

1,5 ugr3E2T cc alfalfa2

3 ug3E2T cc alfalfa 1

CATTLE DOSISnANTIGENGROUP

Evaluation of the immunogenicity of the E2T protein expressed in alfalfa plants in cattle

Antibody

Response I

Viral neutralizationtest

0

0,5

1

1,5

2

2,5

3

3,5

3 ugr 1.5 ugr control (-) Com. 1 Fortdoge INTA

Títu

lo A

N

T0

30dpv

60dpv

Challenge I Evaluation of the protection induced by the experimental vaccines in cattle

We have the approbation from CONABIA and SENASA for the immunization and challenge experience.

Six-month–old Hereford calves were challenge two weeks after the last

immunization with 25 ml 1 X 10 -6 TCID 50/ml BVDV 98/124 type 1 by aerosolization

* Virus isolation from either plasma or buffy coat. DPI: Days Post-Infection.

0/31/32/33/30/3Ctrl

1/30/30/30/30/3BVDV Inact. (106.5)

0/31/31/30/30/3Alfalfa E2t 1.5 ug

0/30/30/30/30/3Alfalfa E2t 3 ug

0/31/31/32/3*0/3mammalianE2t 1,5 ug

DPI 11DPI 8DPI 6DPI 4DPI 0

Antigen

Viirus shedding after BVDV challenge

Challenge IEvaluation of the protection induced by the experimental vaccines in cattle

pending patent P 090100556

To express a fusion protein between a Single Chain Antibody Fragment (APCH1) against MHC class II using Cassava promoter, in order to optimize the accessibility of the recombinant protein to the immune system

Objective II

Agreement ALGENEX – INTA.

PCT/ES2008/070053. Pending patent P080101073

Vaccine antigen

Plantsexpression II

Transgenic plants obtained using Agrobacteriumtumefaciens - mediated transformation of alfalfa

Construction # transformed plants

33

pCar (cassava) –E2t

pCar (cassava) APCH1–E2t

pCar (cassava) –E2

9

12

APCH1 E2T and E2T: 1.7 ugr /gr leafE2: 1,5 ugr/ gr leaf

3,0 0,4 0,05

ugr/ ml E2T

0

0,5

1

1,5

2

2,5

3

alfalfa ScFvE2T

alfalfa E2T alfalfa E2 alfalfa C23

selected plants

Abs

490

nm

Alfalfa APCH1

0

10

20

30

40

50

60

70

80

90

100

Bovine pig horse Sheep Guinea pig

Pos

itive

cel

ls(%

)

Control (-) E2T APCH1-E2T

Binding of APCH1-E2T to MHCII surface of peripheral blood mononuclear cells (PBMCs) from different species

by flow cytometry

Flow cytometry

Booster Week 4 (dilution 1/40)

ELISA

Antibody

Response II

Comparison of the immunogenicity of the E2T and APCH1-E2T protein expressed in alfalfa plants in cattle

Dosis 1 ug

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

0 1 2 3 4 5 6 7

E2T ScFv-E2T

Booster week 4 (dilución 1/40)

Dosis 0.2 ug

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

0 1 2 3 4 5 6 7Semana

OD

(4

05 n

m)

E2T APCH1-E2T

34

33

32

ug31

CATTLE DOSISnANTIGENGROUP

0,2 ug34

0,2 ug33

1 ug3APCH1- E2T 2

1 ug3E2T 1

CATTLE DOSISnANTIGENGROUP

E2T

APCH1- E2T

Negative control 5 1 ug

APCH1-E2T

3

E2T APCH1-E2T

Cattle number

0

0,5

1

1,5

2

2,5

3

3,5

E2T (527) E2T (632) E2T (644) APCH1-E2T

(563)

APCH1-E2T

(567)

APCH1-E2T

(587)

Tit

er

Elisa titer

NA titer

Comparison of the immunogenicity of 0,2 ug of E2T and APCH1 E2T protein expressed in alfalfa plants in cattle

Antibody

Response II

It was possible to express E2T and APCH1-E2T protein in alfalfa transgenic plants

Conclusions

APCH1-E2T recognize the MHCII molecule on the surface ofperipheral blood mononuclear cells (PBMCs) from differentspecies

Without any concentration steps we obtain 1,5 mgr/ kgr of E2t in alfalfa plants

E2t experimental vaccine induce neutralizing antibodies and protection in cattle

APCH1 E2T optimize the immune response in cattle

Results obtained allow us to establish the conditions for recuperation of both protein E2T and APCH1-E2T with high yield, low cost and the possibility to carry out the scale-up easily.

• Small sice molecule (15KDa)

• simple structure

• High thermic and chemic resistance

• Recombinant monoclonal antibodies

VHH: VHH: Fragment corresponding to a variable portion of light chain Fragment corresponding to a variable portion of light chain from from camelidaecamelidae

CH2

CH3

VHHVHHTECNOLOGYTECNOLOGY

Production of nanobodies, in tobacco transplantomic plants.

VHHVHH TECNOLOGYTECNOLOGY

MONOMERIC VHH ANTIBODIES AGAINST ROTAVIRUS VP6 FORVIRUS DETECTION AND PASSIVE PROTECTION

Chloroplast transformation

Production of antigens and antibodies for veterinary applications in tobacco transplastomic plants.

Construction pBSW5’UTR / βGUS-E-VHH

Production of antigens and antibodies for veterinary applications in tobacco transplastomic plants.

PhD Andres Wigdorovitz

Maria José Dus Santos PhD

Pecora Andrea

Cristina Gómez

Pérez Aguirreburualde Maria Sol

Fernando Ardila, PhD IGEAF INTA

José Angel Escribano, PhD, ALGENEXINIA

Fernando Bravo Almonacid, PhDCONICET, INGEBI

Mozgovoj Marina PhD

Our Group Our Collaborators

Viviana Viviana ParreParreññoo

Fernando FernandezFernando Fernandez