Veterinary vaccines produced in plants Institute of Virology and - - PowerPoint PPT Presentation

PhD Andrés Wigdorovitz

Workshop Argentina -Japan

3 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

Sum m ary

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 Rotavirus

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

Agriculture and Food Production in Argentina

Workshop Argentina -Japan

3 rd August 2009

ARGENTI NA ARGENTI NA

• 3,760,000 km

3,760,000 km2

2

• Population~ 39 M

Population~ 39 M

• Government

Government: : Representative Representative Republican Republican Federal Federal

• Language

Language Spanish Spanish

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 (Ilex

paraguaiensis)

• Tea
• Rice
• Citrus
• Vegetables
• Tobacco

CUYO

• Pip Fruit
• Wine
• Oilseed: Olive
• Vegetables
• Bovine Meat

PATAGONI A

• 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

ARGENTI NE PRODUCTI ON ARGENTI NE PRODUCTI ON

AGRI CULTURAL PRODUCTI ON AGRI CULTURAL PRODUCTI ON

Grains: Grains: 41,912 million tons 41,912 million tons Oilseeds: Oilseeds: 51,502 million tons 51,502 million tons Pip fruit: Pip fruit: 1,936 million tons 1,936 million tons Citrus: Citrus: 3,091 million tons 3,091 million tons Honey: Honey: 115,000 tons 115,000 tons Wines: Wines: 15,400 million hectoliters 15,400 million hectoliters Beef: Beef: 3,117 million tons 3,117 million tons Pork: Pork: 198,050 tons 198,050 tons Poultry: Poultry: 1,160,000 tons 1,160,000 tons Milk: Milk: 9.600 million liters 9.600 million liters Wool: Wool: 76.500 tons 76.500 tons

AGRI CULTURAL EXPORTS AGRI CULTURAL EXPORTS

Agricultural and livestock exports: 23.870 million USD Agricultural 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 USD Gross Domestic Product: 213.172 million USD Agricultural, Livestock and Agro Agricultural, Livestock and Agro-

• industry GDP:

industry GDP: 30%

30%

INTA

INTA Headquarters Headquarters

• 15 Regional

15 Regional Centers Centers

• 47

47 Experimental Stations Experimental Stations

• 4

4 Research Research Centers Centers

• 15

15 Research Institutes Research Institutes

• > 300

> 300 Extension Agencies Extension Agencies

• 2

2 Private Private Organizations Organizations

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

I ntroduction FOOT AND MOUTH DI SEASE VI RUS

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. I t is responsable for the m ost im portant econom ic losses of cattle production in the w orld

Objectives Expression of P1 polyprotein of Foot and Mouth Disease virus in transgenic alfalfa plants for the production of experimental vaccines. Evaluation in a murine model.

Construction # transformed plants pROK-P1.2A.2B.3C 20

Electronic m icroscopy Plant A FMDV em pty capsid

Non Related gene

30 nm

Plant A

30 nm

Detection of neutralizing antibodies induced in I .P im m unized m ice w ith plant A extracts

Neutralizing assay

Mice I P im m unized w ith: 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 response

VP1 epitope

0,5 1 1,5 2 2,5 3 1 2 3 4 5 6 7 8 9 10 11 12 13

Mouse Nº Titer ( log10)

ELI SA

0 ,0 5 - 0 ,2 m g/ g TSP

I m m une response in m ice I .P. im m unized w ith P1 polyprotein transgenic alfalfa plants

Complete FMDV particle

Murine m odel

Protection against FMDV challenge in m ice im m unized w ith P1 polyprotein transgenic plants

Mice I P im m unized w ith: 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 P0 4 0 1 0 2 8 4 2

I ntroduction BOVI NE ROTAVI RUS

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

• f

neutralizing antibodies. VP 6 VP 7 VP 4 Rotavirus infections are the prim ary cause of severe diarrhea in young anim als and children in the w orld.

Objective To develop an experimental edible vaccine based

• n 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 weeks 4 2 6 7 5 copulate new born 8 5 Presence of diarrhea

Experimental Vaccine

Murine m odel

Antibody response

I m m une response in m ice intraperitoneally im m unized w ith 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 20

mice Abs 490nm

I m m une response in m ice fed w ith 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 490nm

ELISA

Mice I P im m unized w ith: Route of im m unization Protection ( % ) Fisher´s Test

Alfalfa expressing eBRV4-βGUS Oral 22/29 (76%) p=0.003 IP 10/14 (71%) P= 0.0063 Alfalfa expressing a non related gene

• ral

4/19 (21%) p=0.003 IP 2/13 (21%) P= 0.0063

Murine m odel

Evaluation of passive protection against BRV C4 8 6

I ntroduction 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

• f

neutralizing antibodies.

BOVI NE DI ARREA VI RUS

I nfects bovines of all ages, causing reproduction problem s and altering biological products of high com m ercial value thereby, resulting in considerable econom ical losses.

Plants expression I Quantification and stability of the selected Transgenic plants obtained using Agrobacterium tum efaciens - m ediated transform ation of alfalfa

0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2

P1 P2 P3 P4 P5 P6 P7 P8 P9 P1 P1 1 P1 2 P1 3 P1 4 P1 5 P1 6 P1 7 P1 8 P1 9 P20 P21 P22 P23 P24 P1 P2 P3 P4 P5 P6 P7 P8 P9 P1 P1 1 P1 2 P1 3 P1 4 P1 5 P1 6 P1 7 P1 8 P1 9 P20 P21 P22 P23 P24 C+ C+ C- C-

Absorvancia 405 nm

E2T: 1.7 ugr /gr leaf E2: 1,5 ugr/ gr leaf

0,5 1 1,5 2 2,5 alfalfa E2T alfalfa E2 alfalfa C23 selected plants Abs 490 nm 3.0 0.4 0.05 ugr E2T /ml

Plants expression I Aqueous tw o Aqueous tw o-

• phase system s for purification of

phase system s for purification of recom binant E2 T and APCH1 recom binant E2 T and APCH1 -

• E2 T proteins expressed in

E2 T proteins expressed in alfalfa plants alfalfa plants

0,428 2 0,00189 0,00121 0,314 3,25 0,5 1 1,5 2 2,5 3 3,5 ug/ml

Ext Planta ATPS CHO-K1 SD ProtPurif 2 ug/ml Lac Z (C-) C23 (C-)

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

• 1
• 0,8
• 0,6
• 0,4
• 0,2

0,2 0,4 0,5 1 1,5 2 2,5

0,428 2 0,00189 0,00121 0,314 3,25 0,5 1 1,5 2 2,5 3 3,5 ug/ml

Planta ext ATPS CHO-K1 Purified prot 2 ug/ml Lac Z (C-) C23 (C-)

Curve patron for E2T quantification

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

• 1
• 0,8
• 0,6
• 0,4
• 0,2

0,2 0,4 0,5 1 1,5 2 2,5

Evaluation of the im m unogenicity of the E2 T protein expressed in alfalfa plants in cattle Antibody Response I

4 2 6 weeks

Experimental Vaccine

8

Challenge

3 ug 3 Negativecontrol (C23

• VP8)

4

• 3

BVDV INTA 10

7/ml

3 1,5 ugr 3 E2T cc alfalfa 2 3 ug 3 E2T cc alfalfa 1 CATTLE DOSIS n ANTIGEN GROUP 3 ug 3 Negativecontrol (C23

• VP8)

4

• 3

BVDV INTA 10

7/ml

3 1,5 ugr 3 E2T cc alfalfa 2 3 ug 3 E2T cc alfalfa 1 CATTLE DOSIS n ANTIGEN GROUP

Evaluation of the im m unogenicity of the E2 T protein expressed in alfalfa plants in cattle Antibody Response I

Viral neutralization test

0,5 1 1,5 2 2,5 3 3,5 3 ugr 1.5 ugr control (-)

• Com. 1

Fortdoge INTA Título AN

T0 30dpv 60dpv

Challenge I Evaluation of the protection induced by the experim ental 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.

0/3 1/3 2/3 3/3 0/3 Ctrl 1/3 0/3 0/3 0/3 0/3 BVDV Inact. (106.5) 0/3 1/3 1/3 0/3 0/3 Alfalfa E2t 1.5 ug 0/3 0/3 0/3 0/3 0/3 Alfalfa E2t 3 ug 0/3 1/3 1/3 2/3* 0/3 mammalian E2t 1,5 ug DPI 11 DPI 8 DPI 6 DPI 4 DPI 0 Antigen Viirus shedding after BVDV challenge

Challenge I Evaluation of the protection induced by the experim ental vaccines in cattle

pending patent P 090100556

To express a fusion protein betw een a Single Chain Antibody Fragm ent ( APCH1 ) against MHC class I I using Cassava prom oter, in order to optim ize the accessibility of the recom binant protein to the im m une system Objective I I

Agreement ALGENEX – INTA.

PCT/ES2008/070053. Pending patent P080101073

Vaccine antigen

Plants expression I I

Transgenic plants obtained using Agrobacterium tum efaciens - m ediated transform ation 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 leaf E2: 1,5 ugr/ gr leaf

3,0 0,4 0,05 ugr/ ml E2T 0,5 1 1,5 2 2,5 3 alfalfa ScFv E2T alfalfa E2T alfalfa E2 alfalfa C23 selected plants Abs 490 nm Alfalfa APCH1

10 20 30 40 50 60 70 80 90 100

Bovine pig horse Sheep Guinea pig

Positive cells(%)

Control (-) E2T APCH1-E2T

Binding of APCH1 -E2 T to MHCI I surface of peripheral blood m ononuclear cells ( PBMCs) from different species by flow cytom etry Flow cytom etry

Booster Week 4 (dilution 1/40)

ELI SA

Antibody Response I I Com parison of the im m unogenicity of the E2 T and APCH1 -E2 T protein expressed in alfalfa plants in cattle

Dosis 1 ug

0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 2 3 4 5 6 7 E2T ScFv-E2T

Booster week 4 (dilución 1/40) Dosis 0.2 ug

0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1 2 3 4 5 6 7 Semana OD (405 nm) E2T APCH1-E2T

3 4 3 3 3 2 ug 3 1 CATTLE DOSIS n ANTIGEN GROUP 0,2 ug 3 4 0,2 ug 3 3 1 ug 3 APCH1- E2T 2 1 ug 3 E2T 1 CATTLE DOSIS n ANTIGEN GROUP E2T APCH1- E2T Negative control 5 1 ug

APCH1-E2T

3

E2T APCH1-E2T

Cattle num ber

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)

Titer

Elisa titer NA titer

Com parison of the im m unogenicity of 0 ,2 ug of E2 T and APCH1 E2 T protein expressed in alfalfa plants in cattle Antibody Response I I

It was possible to express E2T and APCH1-E2T protein in alfalfa transgenic plants Conclusions APCH1-E2T recognize the MHCII molecule on the surface of peripheral blood mononuclear cells (PBMCs) from different species 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: Fragm ent corresponding to a variable portion of light chain Fragm ent corresponding to a variable portion of light chain from from cam elidae cam elidae

CH2 CH3

VHH VHHTECNOLOGY

TECNOLOGY

Production of nanobodies, in tobacco transplantomic plants.

VHH VHH TECNOLOGY

TECNOLOGY MONOMERIC VHH ANTIBODIES AGAINST ROTAVIRUS VP6 FOR VIRUS 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 W igdorovitz Maria José Dus Santos PhD Pecora Andrea Cristina Góm ez Pérez Aguirreburualde Maria Sol Fernando Ardila, PhD I GEAF I NTA José Angel Escribano, PhD, ALGENEX I NI A Fernando Bravo Alm onacid, PhD CONI CET, I NGEBI Mozgovoj Marina PhD

Our Group Our Collaborators

Viviana Viviana Parre Parreñ ño

• Fernando Fernandez

Fernando Fernandez