Ebola virus currently has no licenced vaccine or cure, however several potential therapies are in development. Why does this merit mention on a blog about botany? For one good reason; scientists used plants in a bio-pharming approach to produce the potential Ebola treatment which was recently given to 3 patients infected with the virus. Bio-pharming uses genetic modification to introduce genes coding for pharmaceutical proteins (e.g. antibodies, or when produced in plants; ‘plantibodies’) into plants, the plant will then produce these proteins as if they were its own – essentially acting as a protein factory. The plants are harvested, the pharma-protein extracted and purified to a level comparable to any other medicinal protein.
Bio-pharming is currently in the news as ZMapp (Mapp Pharmaceuticals), an experimental antibody cocktail targeted against Ebola virus and produced in tobacco plants, was used in the treatment of two US aid workers and a priest infected with the virus. ZMapp is a combination of three antibodies which recognise different parts of Ebola glycoprotein; a protein present on the outside of the virus which allows the virus to attach to and enter into cells. The antibodies attach strongly to the Ebola glycoprotein thereby inactivating the virus, preventing it from entering cells and acting as a beacon to the patient’s immune system that the virus needs to be destroyed.
The starting point for this particular biopharming process was to isolate Ebola specific antibodies from mice. The genetic code of these antibodies was determined and the bits that make an antibody recognisably ‘mouse’ were swapped for the equivalent human sections whilst retaining the Ebola binding portions. Once the human versions of the Ebola antibody sequences were established they were cloned into a virus-based plant transformation system (magnICON from ICON Genetics) producing a ‘rapid antibody manufacturing system’ (RAMP). The magnICON system uses two modified plant viruses (tobacco mosaic virus and potato virus X; Giritch et al 2006) that don’t compete with each other when in the same plant, allowing high level expression of different parts of an antibody in the same cell. The RAMP system is potentially scalable, so could allow production of large amounts of target protein as some of the process can be automated. ZMapp is being produced by Kentucky Bioprocessing.
There are concerns associated with producing pharmaceutical proteins in plants, particularly about potential alteration of the protein’s overall activity. In almost every cell proteins have sugar structures added to them by enzymes called glycosyl transferases; a process called glycosylation. Glycosylation influences a protein’s final function, stability and activity – this process is happening within you right now thanks to a wonderful organelle, the Golgi apparatus. Plants add slightly different sugars to their proteins compared to mammals and other organisms – and this is a point of concern as to how pharmaceutical proteins might be affected by this different glycosylation (making them weaker or stronger, changing how long they remain active or how long they persist in the body). It has also been suggested that the plant glycosylation itself could be allergenic, though studies have not shown evidence of this in animal models (Chargelegue et al. 2000). These concerns have been tackled by ‘humanising’ plant glycosylation through modifying the sugar processing enzyme activity present in the Golgi of tobacco plants (Strasser et al 2008). Components of the ZMapp plantibody cocktail used in successful animal trials were produced in tobacco with humanised glycosylation (Olinger et al. 2012).
A panel of medical ethics experts gathered by the World Health Organisation have supported the use of such untested drugs given the scale and spread of the current Ebola outbreak. There is currently a very limited supply of ZMapp (production time for further antibodies is likely to be several months), only when this is more widely available will it be possible to determine whether the treatment will be a help in the fight against Ebola.
Whilst ZMapp is at an early stage in its development pathway, several pharmaceutical proteins for treatment of other diseases expressed in plants or plant cells are further along the route to commercialisation. Elelyso from Protalyix Biotherapeutics and rights bought by Pfizer is a treatment for Type 1 Gaucher’s disease produced in carrot cells, which gained FDA approval in 2012. A Lemna (duckweed) production system was used to make interferon alpha2b (commercial name: Locteron developed by Biolex Therapeutics – since declared bankrupt, but taken on by OctoPlus N.V.), which has finished phase IIb clinical trials and showed better clinical data than current treatments. Canadian based firm Medicago Ltd have a number of virus-like particles in development in a tobacco based system, including potential seasonal and pandemic flu vaccines, with the pandemic vaccine authorised form emergency usage. Planet Biotechnology has CaroRX™ in phase II trials in the U.S., it is a secretory antibody produced in plants which binds to Streptococus mutans (the bacteria that cause tooth decay). Treatment with CaroRX™ has been shown the effectively eliminate S. mutans for 2 years – this could be a revolution in dental treatment.
Chargelegue D., Vine N.D., van Dolleweerd C.J., Drake P.M. & Ma J.K. A murine monoclonal antibody produced in transgenic plants with plant-specific glycans is not immunogenic in mice., Transgenic research, PMID: http://www.ncbi.nlm.nih.gov/pubmed/11032367
Giritch A., C. Engler, G. van Eldik, J. Botterman, V. Klimyuk & Y. Gleba (2006). Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors, Proceedings of the National Academy of Sciences, 103 (40) 14701-14706. DOI: http://dx.doi.org/10.1073/pnas.0606631103
Olinger G.G., D. Kim, C. Working, O. Bohorov, B. Bratcher, E. Hiatt, S. D. Hume, A. K. Johnson, J. Morton & M. Pauly & (2012). Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques, Proceedings of the National Academy of Sciences, 109 (44) 18030-18035. DOI: http://dx.doi.org/10.1073/pnas.1213709109
Strasser R., J. Stadlmann, R. Kunert, H. Quendler, P. Gattinger, J. Jez, T. Rademacher, F. Altmann, L. Mach & H. Steinkellner & (2009). Improved Virus Neutralization by Plant-produced Anti-HIV Antibodies with a Homogeneous 1,4-Galactosylated N-Glycan Profile, Journal of Biological Chemistry, 284 (31) 20479-20485. DOI: http://dx.doi.org/10.1074/jbc.m109.014126