Production of antigens and antibodies in plants: alternative technology?

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1 Production of antigens and antibodies in plants: alternative technology? George Lomonossoff John Innes Centre Norwich, UK ECOPA, Alicante 29 th Sept. 2006

2 Why use Plants as Biofactories? Produce large biomass Require minimal inputs Keep themselves sterile Any product will not be contaminated with an animal pathogen

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5 Classical Methods for Expression of Foreign Proteins in Plants Creation of lines of plants transgenic for gene of interest Use of modified viral genomes for introduction of gene of interest

6 Advantages of Transgenic Approach for Expression Introduced sequence is heritable and truebreeding lines of plants can be created Transgene present in every cell of plant. Site of expression can be controlled by use of tissue-specific promoters No limit on size or complexity of proteins which can be expressed

7 Disadvantages of Transgenic Approach for Expression Transformation/regeneration can be difficult and time-consuming especially for crop species Large variability in properties of resultant transgenic plants making evaluation of different constructs difficult Often get only low level of expression of inserted gene

8 Advantages of Plant Virus Vectors for Expression Viruses multiply in their hosts and therefore high level of expression is expected. Genetic modification of viral genomes is straightforward and quick. Infections can be easily passaged to fresh plants for bulking up material. Particles are stable and have defined structure.

9 Disadvantages of Plant Virus Vectors for Expression Limits on size of sequence that can be inserted into a viral genome. Multiple rounds of replication may lead to the accumulation of mutations within inserted sequence. If a vector based on a wild-type virus is used, there may be problems of containment.

10 Alternative methods Chloroplast transformation + Increased copy number of inserted genes; no pollen transfer - can be difficult to obtain homoplasty; no glycosylation Transient Agrobacterium-based expression + Very quick; can test many different constructs high throughput - Problems of scale up; need efficient method of infiltration Combined transgene/virus systems + Potentially high level expression; can use disabled virus for biocontainment - Requires the production of transgenic lines

11 Choice of host Whole food plant (e.g. potato, soya, maize) Whole non-food plant (e.g. tobacco, Arabidopsis) Unusual plants (Duckweed, moss, algae)

12 Containment? Physical e.g. in containment greenhouses, tissue culture vessels. Problems of expense. Biological e.g. chloroplast transformation o prevent pollen transfer, use of disabled viral vectors

13 Examples Norwalk virus in transgenic potatoes

14 Production of a vaccine against Small RNA virus which causes intestinal infection Results in diarrhoea and vomiting (Aurora) Cannot grow virus in culture No vaccine or treatment available norwalk virus

15 Use of plants to produce oral vaccines

16 Results Expression detected in plants Expressed protein forms NV particles Plant-derived particles stimulate mucosal immune response when orally administered to mice When peeled raw potatoes were fed to human volunteers increased levels of NV antibodies were found.

17 Examples Norwalk virus in transgenic potatoes Production of anti-tgev SIP in cowpea using a viral vector

18 TGEV is a coronavirus which infects via the enteric tract. Newborn animals are protected by passive immunisation with antibodies in milk Oral administration of antibodies may be an effective therapy IF antibodies can be produced at sufficiently low cost. What is TGEV?

19 Structures of scfv and SIPs scfv s have advantage that they are small molecules (approx. 250 amino acids). However, they are monovalent and therefore have low avidity. SIPs are larger molecules (approx. 380 amino acids) but have the advantage that they can dimerise to increase avidity. SIPs containing CH4 dimerise rapidly due to C-terminal Cys. CH4

20 -SIP expression in Cowpea ApaI StuI EcoRI 35S Pr 58K/48K L S 2A SIP -SIP Dimer -SIP Monomer

21 In vivo protection in vivo protection assay: 100g plant tissue 100 ml protein extract lyophilised mixed with milk fed challenged newborn piglets determine virus titres in gut and lung

22 In vivo protection in vivo protection assay: g plant tissue PFU/gr tissue ml protein extract PFU/gr tissue lyophilised 100 mixed with milk 10 PFU/gr tissue PFU/gr tissue GUT LUNG 1 human asip 6AC 3 swine asip 6AC 3 1 swine asip 1C10 fed challenged newborn piglets human mab 6AC asip3 6AC 3 swine P3 asip 6AC 3 1 swine Serum asip #560 1C10 1 Serum human mab C- PVX-rIgA swine P3 Cowpea Serum human swine Serum Cowpea swine mabc- PVX-rIgA swine P3 Cow Serum mab C- pea Cow Serum PVX- P3 pea C- PVX-rIgA Serum Serum Cowpea C- C- PVX-rIgA Cowpea 6AC asip 3 asip asip He-SIP #560 asip 1C10 6AC3 asip3 asip 1C10 He-SIP 6AC #560 3 HaSIP #560 He-SIP HaSIP 6AC 3 6AC 3 6AC3 6AC 3 C H determine virus titres in gut and lung

23 Examples Norwalk virus in transgenic potatoes Production of anti-tgev SIP in cowpea using a viral vector Production of cholera toxin in chloroplasts

24 Chloroplast transformation Usually achieved by biolistic bombardment Integration into chloroplast genome is by homologous recombination Plants selected by rounds of antibiotic selection

25 Expression of cholera toxin B B subunit targets toxin to ganglioside GM1 in gut Non-toxic without A subunit Pentamer of 11.6kDa protein subunits Used to orally immunise against cholera subunit

26 Expression of CT-B from chloroplasts Tobacco chloroplasts transformed with CT- B sequence Expressed protein was shown to oligomerise to give pentamers Levels reached at least 4.1% soluble leaf protein Expressed protein shown to bind to GM1 in vitro

27 Examples Norwalk virus in transgenic potatoes Production of anti-tgev SIP in cowpea using a viral vector Production of cholera toxin in chloroplasts Production of Newcastle Disease vaccine in cell culture (Dow AgroScience)

28 Design of a Plant cell produced antigen Fusion Matrix DNA Plasmid Genome Plasmid containing HN gene for the protective antigen prepared for insertion into plant cells Hemagglutinin/ Neuramindase (HN) Newcastle disease virus (NDV) Membrane Phospholipid bilayer Russell Nightly Media

29 Concert tm Plant-Cell-Produced Production System Isolate gene / Transform plant cells Disease-causing agent (containing protective antigen protein) Transfer antigen gene into Agrobacterium plasmid LB Agrobacterium inserts antigen gene into plant cells RB Select transformed cells Characterize & Grow plant cells Scale-up plant cells Characterize / prepare transformed cells to build master cell stock Extract antigen Formulate product Harvest Break open cells to extract antigen Remove non-essential material UNRESTRICTED - May be shared with anyone -- Formulate / package / test Example product

30 Prospects The science behind plant-expressed pharmaceuticals has developed greatly. The granting of a license to DowAgrosciences for NDV vaccine is a breakthrough There is renewed interest in Plant-based vaccines e.g. FP6 PharmaPlanta