Veterinary vaccines produced in plants 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 3 rd to 7 th , 2009 Workshop PhD Andrés Wigdorovitz Argentina -Japan 3 rd August 2009
Sum m ary Agriculture and Food Production in Argentina 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 Rotavirus Passive protection against bovine rotavirus (BRV) induced by a peptide edible vaccine produced in transgenic alfalfa . Bovine diarrhea virus First report of an experimental vaccine against BVDV produced in alfalfa transgenic plants that induce neutralizing antibodies and protection in cattle. Production of nanobodies (Llama –derived Single –Chain Workshop Antibody Fragments), in tobacco transplantomic plants. Argentina -Japan 3 rd August 2009
ARGENTI NA ARGENTI NA 2 3,760,000 km 2 • 3,760,000 km • • Population~ 39 M Population~ 39 M • • Government Government: : • Representative Representative Republican Republican Federal Federal • Language Language • Spanish Spanish
NOA NEA • Sugar ARGENTI NE PRODUCTI ON ARGENTI NE PRODUCTI ON • Tobacco • Bovine Meat • Citrus • Cotton • Oilseeds: Soybean, Olive • Oilseed: Soybean • Wine • Implanted Forest • Bovine Meat • Native Forest • Vegetables • Yerba Mate (Ilex paraguaiensis) • Native Forest • Tea • Honey • Rice • Camelids • Citrus • Cotton • Vegetables CUYO • Tobacco • Pip Fruit • Wine • Oilseed: Olive PAMPEANA • Vegetables • Wheat and Maize • Bovine Meat • Oilseeds: Soybean- PATAGONI A Sunflower • Bovine Meat • Wool • Bovine Milk • Ovine Meat • Vegetables • Apple and Pear • Porcine Meat • Berries • Fruits • Native Forest • Rice • Bovine Meat • Poultry and Eggs • Wine • Implanted Forest • Honey
AGRI CULTURAL PRODUCTI ON AGRI CULTURAL PRODUCTI ON Grains: 41,912 million tons Grains: 41,912 million tons Oilseeds: 51,502 million tons Oilseeds: 51,502 million tons Pip fruit: 1,936 million tons Pip fruit: 1,936 million tons Citrus: 3,091 million tons Citrus: 3,091 million tons Honey: Honey: 115,000 tons 115,000 tons Wines: 15,400 million hectoliters Wines: 15,400 million hectoliters Beef: 3,117 million tons Beef: 3,117 million tons Pork: 198,050 tons Pork: 198,050 tons Poultry: 1,160,000 tons Poultry: 1,160,000 tons Milk: 9.600 million liters Milk: 9.600 million liters Wool: 76.500 tons Wool: 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 30% Agricultural, Livestock and Agro- -industry GDP: industry GDP: 30% Agricultural, Livestock and Agro
� 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 Private Private Organizations Organizations � 2 �
Production of experimental vaccines in alfalfa transgenic plants 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.
I ntroduction FOOT AND MOUTH DI SEASE VI RUS I t is responsable for the m ost im portant econom ic losses of cattle production in the w orld 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 .
Expression of P1 polyprotein of Foot and Mouth Disease Objectives 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 Plant A Non Related gene m icroscopy Plant A FMDV em pty capsid 30 nm 30 nm
I m m une response in m ice I .P. im m unized w ith P1 Antibody polyprotein transgenic alfalfa plants 3 response VP1 epitope 2,5 Complete FMDV 2 particle Titer ( log10) ELI SA 1,5 1 0,5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 0 ,0 5 - 0 ,2 m g/ g TSP Mouse Nº 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 Mouse Nº * SNI w ith: Alfalfa Plant A 1 2.2 2 2.3 3 2.1 4 2.1 Alfalfa expressing 5 0.6 a non related gene 6 0.6
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 Protection ( % ) w ith: Alfalfa Plant A 13/13 (100%) 9/9 (100%) Alfalfa expressing 0/10 (0%) 0/6 (0%) a non related gene Buffer 0/6 (0%) 0/6 (0%) pending patent P0 4 0 1 0 2 8 4 2
I ntroduction BOVI NE ROTAVI RUS Rotavirus infections are the prim ary cause of severe diarrhea in young anim als and children in the w orld. VP 6 BRV is a rotavirus with a RNA positive genome. VP 7 2 structural proteins compose its outer capsid: VP4, and VP7. VP4 and VP7 carry critical VP 4 epitopes responsible for the induction of neutralizing antibodies.
To develop an experimental edible vaccine based Objective 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. Experimental Murine m odel Vaccine weeks 0 2 4 5 6 7 new born copulate Presence of diarrhea 5 8 BRV C486 oral infection
Antibody I m m une response in m ice 1,200 intraperitoneally im m unized w ith response eBRVP4 -BGUS leaves extracts 1,000 0,800 Abs 490nm 0,600 0,400 vaccinated dams 0,200 respective offspring 0,000 Negative control dams 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 ELISA mice 1,600 I m m une response in m ice fed 1,400 w ith eBRVP4 -BGUS fresh 1,200 leaves 1,000 Abs 490nm 0,800 0,600 0,400 0,200 0,000 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 mice
Evaluation of passive protection against BRV C4 8 6 Murine m odel Fisher ´ s Mice I P Route of Protection ( % ) im m unized im m unization Test w ith: Alfalfa expressing Oral 22/29 (76%) p=0.003 eBRV4 - β GUS IP 10/14 (71%) P= 0.0063 Alfalfa expressing oral 4/19 (21%) p=0.003 a non related gene IP 2/13 (21%) P= 0.0063
I ntroduction I 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. 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.
Quantification and stability of the selected Transgenic Plants expression I plants obtained using Agrobacterium tum efaciens - m ediated transform ation of alfalfa 2,5 2 Abs 490 nm 1,5 1 0,5 0 alfalfa E2T alfalfa E2 alfalfa C23 3.0 0.4 0.05 selected plants ugr E2T /ml E2T: 1.7 ugr /gr leaf E2: 1,5 ugr/ gr leaf 2 1,8 1,6 1,4 Absorvancia 405 nm 1,2 1 0,8 0,6 0,4 0,2 0 P1 P2 P3 P4 P5 P6 P7 P8 P9 P1 0 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 0 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-
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