The second conference of the
International Society for
Plant Molecular Farming
2016Ghent
th
th
25 -27 May
Courtesy of C. Asensio
Welcome to the second conference
of the ISPMF!
Society organising committee:
President: Julian Ma
Secretary: Joachim Schiemann
Treasurer: Penelope Hundleby
Communications officers: Heribert Warzecha and Inga Hitzeroth
Biennial meeting organiser: Ann Depicker
Conference organising committee:
Ann Depicker
Vikram Virdi
Paloma Juarez
Veronique Boudolf
Sylvie De Buck
Annick Bleys
Sponsors:
QC code for the URL of the abstract book:
Programme
Wednesday May 25th, 2016
09.00 – 09.50 Registration / Coffee
09.50 – 10.00 Welcome by Ann Depicker
Session 1: Plant, animal and Pichia production platforms
Chairs: Pascal Drake & Vikram Virdi
Invited speakers
10.00 – 10.30 Andy Hiatt, MaPP Biopharmaceuticals Inc., USA
“Ebola antibodies and clinical trials”
10.30 – 11.00 Luc Kupers, Genzyme, Belgium
“Animal cell technology for the manufacturing of biopharmaceuticals”
11.00 – 11.30 Willem Van de Velde, Ablynx, Belgium
“Double digit-titers and highly cost/quality-effective production of nanobodies® via
Pichia pastoris”
11.30 – 12.00 Coffee break
12.00 – 12.20 Markus Sack, Fraunhofer Institute, Aachen, Germany
“Of products and processes”
12.20 – 12.40 Andreas Schaaf, Greenovation Biotech GmbH, Germany
“Bryotechnology: technology update and clinical pipeline
manufacturing”
of
moss-based
12.40 – 13.00 Estelle Nisse, Université de Lorraine – INRA, France
“Carnivorous plants as alternative models for the production of plant recombinant
proteins”
13.00 – 13.50 Lunch
Session 2: Greenhouse to market: translational aspects
Chair: Sylvie De Buck
Invited speakers
13.50 – 14.00 Marc Van Montagu, International Plant Biotechnology Outreach, Belgium
“Discover, innovate, communicate”
14.00 – 14.20 Lars von Borcke, Plant Bioscience Limited, UK
“Progress and opportunities in molecular pharming”
14.20 – 14.40 Els Beirnaert, VIB, Belgium
“VIB new ventures: translating scientific excellence into economic value”
14.40 – 15.00 Joachim Schiemann, Julius Kühn-Institut, Germany
“Plant genome editing: application, risk assessment and regulation”
Session 3: Plant-made vaccines
Chairs: George Lomonossoff & Eva Stöger
Invited speakers
15.00 – 15.30 Xavier Saelens, Medical Biotechnology, VIB, UGent, Belgium
“Prevention and treatment of respiratory viral infections: plants come to the rescue”
15.30 – 16.00 Group Picture - Coffee break
16.00 – 16.20 Inga Hitzeroth, University of Cape Town, South Africa
“Development of a plant-made recombinant virus-like particle (VLP) vaccine against
African horse sickness”
16.20 – 16.40 Rima Menassa, Agriculture and Agri-Food Canada, Canada
“Veterinary vaccines for enterohemorrhagic E. coli - Improving accumulation of type
3 secretion proteins in plants”
Selected short talks
16.40 – 16.50 Renate Lamprecht (I. Hitzeroth), University of Cape Town, South Africa
“Production of Human papillomavirus pseudovirions in plants and their use in
pseudovirion-based neutralisation assays in mammalian cells”
16.50 – 17.00 Hoang Trong Phan (U. Conrad), IPK Gatersleben, Germany
“Multimerization strategy to create avian flu vaccines in plants”
17.10 – 17.20 Johanna Marsian (G. Lomonossoff), John Innes Centre, Norwich, UK
“Developing a synthetic polio vaccine using two plant-based expression systems”
17.20 – 17.30 Zayn Khamis* (R. Menassa), Agriculture and Agri-Food Canada, Canada
“Porcine epidemic diarrhea virus: Producing the membrane protein as a subunit
vaccine, and producing virus-like particles”
17.30 – 17.40 Eva Thuenemann (G. Lomonossoff), John Innes Centre, Norwich, UK
“Developing plant-expressed bluetongue virus particles as a delivery system for
foreign proteins”
17.40 – 17.50 Keith Saunders (G. Lomonossoff), John Innes Centre, Norwich, UK
“The generation of modified plant virus-like particles by transient expression for
potential bionanotechnological applications”
17.50 – 18.00 Matilde Merlin (L. Avesani), University of Verona, Italy
“GAD65-based edible vaccine: a new autoimmune diabetes perspective
* received an ISPMF bursary
2-min poster presentations for ISPMF bursaries: Adam Chin-Fatt and Cornelius J. Gunter
18.00 – 21.30 Poster walk with Mediterranean buffet
Busses have been arranged to bring everybody back to the Gent Sint-Pieters railway station.
Thursday May 26th, 2016
Free morning or Knowledge for Growth or Visit to Food Pilot at ILVO
12.30 – 13.30 ISPMF annual general meeting
Session 4: Innovative expression strategies and applications
Chairs: Alexandra Castilho & Inga Hitzeroth
Invited speakers
14.00 – 14.30 Yuri Gleba, Nomad Bioscience GmbH, Germany
“Antimicrobials made in plants”
14.30 – 14.50 Udo Conrad, IPK Gatersleben, Germany
“Nanobodies in and from plants: production of nanobody multimers and selected
protein degradation in planta by nanobody-F-Box fusions”
14.50 – 15.10 Diego Orzaez, IBMCP (CSIC-UPV), Spain
“Consistent production of plant-made recombinant polyclonal antibodies against
snake venoms”
15.10 – 15.30 Ann Meyers, University of Cape Town, South Africa
“Functional HRP-fused anti-rabbit IgG scFvs produced in plants”
Selected short talks
15.30 – 15.40 Nicola Weichert (U. Conrad), IPK Gatersleben, Germany
“Seeds are capable to produce non-cytotoxic synthetic spider silk biopolymers”
15.40 – 15.50 Roberta Zampieri (L. Avesani), University of Verona, Italy
“Modified plant-viruses nanoparticles for the Sjogren’s syndrome diagnosis”
15.50 – 16.00 Axel Masloboy* (I. Broer), Department of Agrobiotechnology, Rostock, Germany
“Spaying with the aid of plant-made vaccines”
16.00 – 16.10 Henrik Nausch* (I. Broer), Department of Agrobiotechnology, Rostock, Germany
“Regulated degradation of plant-derived Cyanophycin to release dipeptides as feed
additives”
16.10 – 16.20 Shruti Bakshi (A. Depicker), Ghent University/VIB, Belgium
“Engineering chimeric antibodies aimed for passive mucosal immunization against
HRSV”
16.20 – 16.30 Jorge Palaci (A. Depicker), Ghent University/VIB, Belgium
“Stability evaluation of orally given plant-made antibodies by faecal analysis”
* received an ISPMF bursary
2-min poster presentations for ISPMF bursaries: Reza Saberianfar, J. Hendrik Els and Ahmad
Ibrahim
16.30 – 17.00 Coffee break
Session 5: Boosting expression levels and optimizing processing
Chairs: Inge Broer & Markus Sack
Invited speakers
17.00 – 17.20 Renier van der Hoorn, University of Oxford, UK
“The extracellular protease complement of agroinfiltrated Nicotiana benthamiana”
17.20 – 17.40 Eva Stöger, University of Natural Resources and Life Sciences, Austria
“Utilizing endogenous and ectopic protein storage organelles to boost recombinant
protein performance”
17.40 – 18.00 Jussi Joensuu, VTT , Finland
“Plant-based production of bi-functional fusion proteins with hydrophobin fusion
library”
18.00 – 18.20 Johannes Felix Buyel, Fraunhofer Institute for Molecular Biology and Applied
Ecology, Germany
“Heat precipitation of tobacco host cell proteins facilitates target protein recovery and
purification, which can be implemented into an automated large-scale manufacturing
process”
Selected short talks
18.20 – 18.30 Philippe Jutras (D. Michaud), Université Laval, Québec, Canada
“The multiple uses of tomato cystatin SlCYS8 as a co-expression partner in plant
protein biofactories”
18.30 – 18.40 Kouki Matsuo (T. Matsunura), National Institute of Advanced Industrial Science
and Technology (AIST) Japan
“Dicer-Like 2/4 suppressed tobacco plants for high expression of recombinant
proteins”
18.40 – 18.50 Lotte Westerhof (A. Schots), Plant Science Group, Wageningen University, The
Netherlands
“Codon use and mRNA structure analyses across kingdoms indicates selection on
both mRNA stability and translatability”
18.50 – 19.00 Noriho Fukuzawa (T. Matsumura), National Institute of Advanced Industrial
Science and Technology, Sapporo, Japan
“CMV-Agroinfection triggers plant defense mechanism suppressing
expression”
gene
19.00 – 19.10 Hamideh Ofoghi* (P. Ehsani), Biotechnology Dept., IROST. Tehran, Iran
“Production of Human Nerve Growth Factor in Nicotiana benthamiana”
19.10 – 19.20 Luis Matías Hernández (R. Aiese Cigliano), Parc Científic Barcelona (PCB), Spain
“TrichoPharming and OrtoPharming by Sequentia: trichomes as natural factories
and bioinformatics tools to ease plant molecular pharming research”
* received an ISPMF bursary
2-min poster presentations for ISPMF bursaries: Mahsid Amiri
19.20 – 21.30 Poster walk with Belgian buffet with beer
Busses have been arranged to bring everybody back to the Gent Sint-Pieters railway station.
Friday May 27th, 2016
Session 6: Plant glycan-engineering
Chairs: Veronique Boudolf & Eva Decker
Invited speakers
09.00 – 09.30 Francis Santens, Medical Biotechnology, VIB, UGent, Belgium
“Using GlycoDelete for pharming of proteins without plant-specific N-glycan
modification”
09.30 – 10.00 Richard Strasser, BOKU, Austria
“O-glycan engineering in Nicotiana benthamiana”
10.00 – 10.20 Svend Dam, Carbohydrate Recognition & Signaling, University of Aarhus,
Denmark
“Engineering the N- and O-glycosylation pathways in plants for mammalian-like
glycoprotein farming”
10.20 – 10.40 Alexandra Castilho, BOKU, Austria
“Complex sialylation of plant-derived recombinant proteins”
10.40 – 11.00 Coffee break
Selected short talks
11.00 – 11.10 Szymon Stelter (J. Ma), St George's, University of London
“Plant antibodies in mediating FcγR functions and engaging neonatal Fc receptor”
11.10 – 11.20 Sunghwa Choe, Seoul National University, Korea
“Optimization of protein glycosylation pattern in host plants through CRISPR/Cas9mediated genome editing for production of edible influenza vaccines”
11.20 – 11.30 Chiara Lonoce (M. Donini), Laboratory of Biotechnology ENEA Research Center,
Casaccia 00123 Rome, Italy
“Production of tumour targeting antibodies”
11.30 – 11.40 Catherine Navarre (M. Boutry), Institute of Life Sciences, Université de Louvain,
Belgium
“Towards humanization of glycoproteins secreted in Nicotiana tabacum BY-2 cells”
11.40 – 11.50 Ruud Wilbers (A. Schots), Wageningen University and Research Centre, The
Netherlands
“Engineering of plants for the expression of helminth glycoproteins with their native
N-glycan structures”
11.50 – 12.00 Kim van Noort (A. Schots), Wageningen University and Research Centre, The
Netherlands
“Characterization of Schistosoma mansoni fucosyltransferases for glyco-engineering
of ‘native’ helminth N-glycan structures in planta”
12.00 – 12.10 Sébastien Mercx (M. Boutry), Institute of Life Sciences, Université de Louvain,
Belgium
“Gene inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 suspension cells”
Session 7: Secondary metabolite farming
Chairs: Diego Orzaez & Renier van der Hoorn
Selected short talks
12.10 – 12.20 Moran Oliva (G. Galili), Plant Sciences, Weizmann Institute of Science, Rehovot,
Israel
“Enhancing levels of aromatic amino acids in whole plants and cell cultures. A case
study in flowering plants and grape derived cell culture”
12.20 – 12.30 Suvi T. Häkkinen (H. Rischer), VTT Technical Research Centre of Finland
“Plant-based production of natural flavours – case raspberry ketone”
12.30 - 13.30 Lunch
Invited speakers
13.30 – 14.00 Gilbert Gorr, Phyton Biotech GmbH, Germany
“Plant cell culture as a highly controllable tool for the sustainable production of
anticancer products”
14.00 – 14.30 David Craik, Institute for Molecular Bioscience, University of Queensland,
Australia
“Design and plant-based production of therapeutic cyclic peptides”
14.30 – 15.00 Alain Goossens, Plant Systems Biology, VIB, UGent, Belgium
“How jasmonates provide the key to harness plant chemistry”
15.00 – 15.20 Heribert Warzecha, Technische Universität Darmstadt, Germany
“Production of halogenated indigoid precursors in metabolically engineered tobacco
plants”
15.20 – 15.40 Closing remarks by Julian Ma
15.40 – 16.30 Farewell drink
Session 1
Plant, animal and Pichia
production platforms
Session 1
Invited speaker
Ebola antibodies and clinical trials
Hiatt A.
Mapp Biopharmaceutical, San Diego, USA
This review describes the history of Ebola monoclonal antibody (mAb) development leading up to the
recent severe Ebola outbreak in West Africa. The Ebola virus has presented numerous perplexing
challenges in the long effort to develop therapeutic antibody strategies. Since the first report of a
neutralizing human anti-Ebola mAb in 1999, the straightforward progression from in vitro
neutralization resulting in in vivo protection and therapy has not occurred. A number of mAbs,
including the first reported, failed to protect non-human primates (NHPs) in spite of protection in
rodents. An appreciation of the role of effector functions to antibody efficacy has contributed
significantly to understanding mechanisms of in vivo protection. However a crucial contribution, as
measured by post-exposure therapy of NHPs, involved the comprehensive testing of mAb cocktails.
This effort was aided by the use of plant production technology where various combinations of mAbs
could be rapidly produced and tested. Introduction of appropriate modifications, such as specific
glycan profiles, also improved therapeutic efficacy. The resulting cocktail, ZMapp™, consists of three
mAbs that were identified from numerous mAb candidates. ZMApp™ is now being evaluated in human
clinical trials but has already played a role in bringing awareness to the potential of antibody therapy
for Ebola.
Session 1
Animal cell technology for the manufacturing of biopharmaceuticals
Kupers L.
Genzyme, Geel, Belgium
Invited speaker
Session 1
Invited speaker
Double digit-titers and highly cost-effective production of nanobodies® via Pichia
Pastoris
Van de Velde W.
Albynx NV, Gent, Belgium
Ablynx is a biopharmaceutical company engaged in the discovery and development of Nanobodies®, a
novel class of therapeutic proteins based on single-domain antibody fragments, for a range of serious
human diseases, including inflammation, haematology, oncology and pulmonary disease. The company
has >40 pharmaceutical programs in the pipeline and several of our Nanobodies are already in Phase
I, Phase II and Phase III clinical trials. Because of their small size, Nanobodies have several advantages
in comparison to conventional antibodies, such as ease of manufacturing, flexible formatting, excellent
stability and possibility to use alternative routes of delivery.
Pichia pastoris is currently Ablynx’ preferred production host for Nanobodies, mainly because of its
high expression yields and low amount of secreted host cell proteins, resulting in short process
development timelines. Production processes are fully developed in-house, starting from host creation
to fermentation optimization and development of the downstream process. Non-cGMP productions
are performed at Ablynx up to 100 L-scale, while larger cGMP productions for early clinical studies are
being performed externally at >1000 L-scale.
This presentation will address the different aspects of Pichia process development for Nanobody
production, from host creation to process development and analytics, with the main focus on the
optimization of product yield and quality.
Session 1
Invited speaker
Of products and processes
Sack M.
RWTH Aachen University, Aachen, Germany
Plant molecular farming is maturing, as demonstrated by recent trends in the building of manufacturing
capacity and investments from large pharmaceutical companies. As the field advances, the demand for
a supply industry comprising hardware, RTD services and auxiliary technologies will increase in parallel,
possibly comparable to the growth of the CHO/mammalian cell culture industry. However, plant
molecular farming products and applications are much more diverse, which can be seen both as an
advantage and a drawback. In any case, products and manufacturing processes must advance to a stage
where they become economically viable. Different strategies and tools to achieve this will be
presented, including the use of protein engineering to increase yields, and the application of a recently
developed platform based on plant suspension cell packs, known as the “cookie method”.
Session 1
BryoTechnology:
manufacturing
Schaaf A.
Invited speaker
technology
update
and
clinical
pipeline
of
moss-based
Greenovation Biotech GmbH, Freiburg, Germany
BryoTechnology, i.e. moss-based production of biopharmaceuticals has evolved into a GMP
manufacturing technology for complex biopharmaceuticals. Whilst leveraging the unchallenged
advantages of phototrophic moss based production, comparability to mammalian cell based
technologies was a major focus in process development. As a result today’s moss process is initiated
from cryo-conserved master cell banks and realized in unmodified single-use equipment from wellknown bioprocess suppliers. These facts among others allow for straightforward establishment of the
technology at any production site and facilitate its adoption by biopharmaceutical producers.
Greenovation, developer of BryoTechnology, generated a clinical pipeline of currently four moss-made
products. All of them are in rare indications and potentially possess a therapeutic advantage from being
produced in moss. Moss-aGal, a moss-produced version of human alpha-galactosidase A is the furthest
advanced in development and has been approved by German regulatory authority (BfArM) for a firstin-men clinical phase I study.
The presentation will give an update on the current technological status of BryoTechnology and
summarize the preclinical and clinical status of Greenovation`s products.
Session 1
Invited speaker
Carnivorous plants as alternative models for the production of plant recombinant
proteins
Nisse E.1,&, Miguel S.1,&, Mignard B.1, Hehn A.2,3 and Bourgaud F.1,2,3
1Plant
Advanced Technologies SA, Vandœuvre-lès-Nancy, France; 2INRA UMR 1121, Laboratoire
Agronomie et Environnement, Vandœuvre-lès-Nancy, France; 3Université de Lorraine UMR 1121,
Laboratoire Agronomie et Environnement, Vandœuvre-lès-Nancy, France.
&Equal contributions to this work.
Carnivorous plants are able to attract, trap, retain, kill, and digest prey (Juniper et al., 1989). They are
found all around the world growing on nutrient-poor soils. They have established an original way to
circumvent the shortage of mineral nitrogen resources: their leaves have evolved to form traps for
catching prey. These preys (insects or small animals) are subsequently digested allowing acquiring
substantial amounts of nitrogen. Drosera and Nepenthes are two genera of carnivorous plants able to
produce and excrete out of their tissues a digestive fluid containing proteins which are mostly
hydrolytic enzymes. Drosera leaves are covered on their upper face by stalked glands secreting sticky
and viscous digestive mucilage. Nepenthes leaves are differentiated in pitchers, the lower internal part
being covered by glands secreting a digestive liquid (Juniper et al., 1989).
One of the major issues related to the production of recombinant proteins from plants is the cost
associated to the extraction, separation, and purification of the recombinant proteins. Downstream
processes can represent up to 80% of the total cost of production of a recombinant protein (Hellwig et
al., 2004). To overcome this bottleneck, we aim to exploit the natural ability of carnivorous plants to
secrete proteins in order to develop Drosera and Nepenthes plants as new plant recombinant protein
platforms. These systems address two main drawbacks: 1) extraction is made easier since the digestive
fluid is readily accessible and 2) purification is simpler because the digestive fluid contains only proteins
(Hatano and Hamada, 2008). However, these proteins are mainly constituted of hydrolytic enzymes
which represent a potential threat in a protein recovery process.
An early proof of concept was acquired with Drosera rotundifolia, for which a protocol for genetic
transformation was already available (Hirsikorpi et al., 2002). Our experimental studies clearly
highlighted the presence of Green Fluorescent Protein (GFP) and β-glucuronidase (GUS) in the digestive
secretions of transgenic plants (Biteau, 2009). These preliminary results were patented as the PAT
Friday® technology in 2008 (Biteau et al., 2008). The technology was further extended to two additional
plants: 1) Drosera capensis, which has longer leaves and hence higher quantities of mucilage available
than Drosera rotundifolia, and 2) Nepenthes mirabilis, the digestive liquid of this latter species being
harvestable just by pouring the pitchers. For both species, an original protocol for the genetic
transformation has been established (Miguel, 2013; Nisse, 2014) and GFP could be detected in their
digestive fluids.
A major drawback of this system relies on proteases naturally present in the secretions which could
damage the recombinant proteins. Although our ultimate results show that these hydrolytic enzymes
can be inactivated by modulating the pH of the plant secretions, it is important to characterize them
at the molecular level. Therefore, we have investigated the exact composition of the digestive liquids
from Drosera capensis and Nepenthes mirabilis through transcriptomic and proteomic approaches
(Rottloff et al., 2016).
Our ultimate goal is to offer the PAT Friday® technology for the production of relevant recombinant
therapeutic proteins. The production of a monoclonal antibody, of a cytokine, and a gastric protein
among others are currently under way.
Biteau, F. (2009). Production de protéines recombinantes par des plantes carnivores génétiquement transformées:
Application à Drosera rotundifoila et transfert de la technologie à Nepenthes alata. Université de Nancy.
Biteau, F., Bourgaud, F., Gontier, E., and Fevre, J.P. (2008). Process for the Production of Recombinant Proteins Using
Carnivorous Plants. WO/2008/040599A1.
Hatano, N., and Hamada, T. (2008). Proteome analysis of pitcher fluid of the carnivorous plant Nepenthes alata. J. Proteome
Res. 809–816.
Hellwig, S., Drossard, J., Fischer, R., and Twyman, M. (2004). Plant cell cultures for the production of recombinant proteins.
Nat. Biotechnol. 22 (11).
Hirsikorpi, M., Kämäräinen, T., Teeri, T., and Hohtola, A. (2002). Agrobacterium-mediated transformation of round leaved
sundew (Drosera rotundifolia L.). Plant Sci. 162, 537–542.
Juniper, B.E., Robins, R.J., and Joel, D.M. (1989). The Carnivorous Plants (San Diego). Academic press Editions.
Miguel, S. (2013). Développement d’une nouvelle plateforme végétale de production de protéines recombinantes par
l’utilisation des plantes carnivores du genre Nepenthes. Université de Lorraine.
Nisse, E. (2014). Développement d’un nouveau système de production de protéines recombinantes par la plante carnivore
Drosera capensis. Université de Lorraine.
Rottloff S. et al. (2016). Proteome analysis of digestive fluids in Nepenthes pitchers. Ann. Bot. 117, 479-495.
Session 2
Greenhouse to market:
translational aspects
Session 2
Invited speaker
Discover, innovate, communicate
Van Montagu M.
International Plant Biotechnology Outreach (IPBO), VIB - Ghent University, Gent, Belgium
The recent opening up of the “RNA –World” brings tremendous tools for epigenetic gene expression,
targeted mutagenesis and pathway constructions in all living organisms.
This breakthrough in fundamental research will however only be appreciated by society if we can
convince the private sector to get involved in the production and commercialisation of the novel
compounds now accessible. Finding financial support for such endeavours will depend on the socioeconomic acceptability of these new technologies by a substantial part of our communities. To obtain
this, rational arguments alone are rarely convincing enough. Our R&D scientists need direct interaction
with social scientist and economists, staged in the units where the innovative products are developed.
A similar effort in pointing out the dangers of technophobia and aversion for new knowledge, to the
students in human sciences, will be needed.
Session 2
Invited speaker
Progress and opportunities in molecular pharming
von Borcke L.
Plant Bioscience Limited, Norwich, United Kingdom
The production of heterologous proteins for pharmaceutical and other uses in plants has been a
scientific and commercial target for over 25 years. Over this time the industry has encountered a range
of issues. A defining moment was the discovery that some of the planted transgenic corn producing
trypsin was still present in the field after harvesting. This event in 2002 resulted in great concerns about
producing heterologous proteins in transgenic crop plants and was a major setback for the Plant
Molecular Pharming community.
The concerns over regulatory and safety issues dramatically reduced the active companies in the field
of molecular pharming about 12 years ago as investors were reluctant to continue to support these
companies. While the industry is now much smaller than in the early 2000s when around 200 Biotech
companies were active in the Plant Molecular Pharming field it has certainly recovered from the early
setbacks and is now focussed on a range of promising products.
This presentation will give an overview of the industry, its current state and opportunities for products
and applications, as well as different approaches to produce heterologous proteins in plants.
Session 2
VIB new ventures: Translating scientific excellence into economic value
Beirnaert E.
VIB, Gent, Belgium



VIB approach towards start-ups
Opportunities for innovative start-ups
Challenges in financing and fundraising
Invited speaker
Session 2
Invited speaker
Plant genome editing: application, risk assessment and regulation
Schiemann J.
Julius Kühn-Institut (JKI), Institute for Biosafety in Plant Biotechnology, Quedlinburg, Germany
The genome editing and modification techniques are tools for sequence-specific changes in the plant
genome. These techniques enable breeders to introduce a single point mutation or a new DNA
sequence at a specific location in the plant genome, thereby circumventing the negative side effects of
conventional mutagenesis. The potential risks of exploring these new genome editing techniques are
comparable to conventional mutagenesis or genetic engineering. Considering these techniques and
emerging new breeding techniques, the GMO-legislation framework in the EU, which is mainly
interpreted and executed as being based on the technique which is used to produce a new plant, is not
reflecting the progress made in recent development of genome editing.
In its recent Statement on New Breeding Techniques EASAC (European Academies Science Advisory
Council) requests that the EU policy development for agricultural innovation should be transparent,
proportionate and fully informed by the advancing scientific evidence and experience worldwide.
EASAC demands to resolve current legislative uncertainties and asks EU regulators to confirm that the
products of genome editing, when they do not contain foreign DNA, do not fall within the scope of
GMO-legislation. In contrast, in an Open Letter to the Commission on new genetic engineering
methods the anti-GMO Non-Governmental Organizations call on the Commission to reject any attempt
to exclude these new techniques from EU regulation. In particular, they urge the Commission to ensure
that organisms produced by these new techniques will be regulated as GMOs under existing EU
regulations and that current GMO health and environmental safety testing requirements are
strengthened in light of the enhanced ability of these new techniques to alter the genetic code.
The European Plant Science Organisation (EPSO) as well as National academies of science e.g. in
Germany such as the Leopoldina, acatech and the Union of the German Academies of Sciences and
Humanities support the application of genome editing for future crop improvement and share the legal
interpretation that the Directive 2001/18/EC should be interpreted as process- as well as productbased.
To date (March 2016) a clarifying legal opinion of the European Commission is still pending. Until the
legal opinion is released the legal status of living organisms and products deriving from genome editing
approaches is unclear.
Session 3
Plant-made vaccines
Session 3
Invited speaker
Prevention and treatment of respiratory viral infections: plants come to the rescue
Saelens X.
Medical Biotechnology Center, VIB, Gent, Belgium; Department of Biomedical Molecular Biology,
Ghent University, Gent, Belgium
Human influenza viruses and human respiratory syncytial virus (HRSV) circulate globally and cause
serious economical and clinical problems. Prophylactic vaccines are available against influenza. Most
of these are based on the growth of live influenza viruses in embryonated chicken eggs. The only
currently available medical intervention that is licensed to reduce the risk of hospitalization due to
HRSV infection is a prophylactic therapy with a monoclonal antibody. Recently recombinant influenza
virus-like particles produced in plants have been evaluated in early stage clinical trials with promising
outcome. There are also several small molecule antivirals available to treat human influenza. We have
explored the use of single domain antibodies directed against the influenza glycoproteins and against
the HRSV fusion protein for prophylactic and therapeutic use against these viruses. Some of these
single domain antibodies were genetically fused to a conventional IgG Fc domain and produced in
transgenic Arabidopsis plants. These single domain antibodies inhibited virus replication in vitro and in
a mouse model of influenza and HRSV infection.
Session 3
Invited speaker
Development of a plant-made recombinant virus-like particle (VLP) vaccine against
African horse sickness
Hitzeroth I.I., Dennis S.J., Meyers A.E. and Rybicki E.P.
Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town,
Rondebosch, South Africa
African horse sickness is a devastating, infectious, but non-contagious disease that causes great
suffering and many deaths among horses in sub-Saharan Africa. The aetiological agent is a dsRNA virus
of the same name, African horse sickness virus (AHSV), an orbivirus of the family Reoviridae. The
disease has significant economic consequences for the equine industry both in southern Africa, and
increasingly further afield as its midge vector spreads with global warming. Live attenuated vaccines
have been in use with relative success for more than 5 decades, but there is a risk of reversion to
virulence as well as reassortment of the segmented genome between outbreak and vaccine strains.
Furthermore, the vaccines lack DIVA capacity, the ability to distinguish between vaccine-induced
immunity and that induced by natural infection. Several studies have demonstrated the potential for
the use of plant expression systems for the prod uction of VLPs which are excellent vaccine candidates,
as they don’t contain the virus genetic material and there is no risk of reversion to virulence or
reassortment with wild virus strains. Even though recombinant VLPs have the potential to be excellent
vaccine candidates, they usually only provide protection against one serotype.
In this study, we investigated the formation of AHSV VLPs in N. benthamiana by expressing the four
structural proteins VP2, VP3, VP5 and VP7 of two serotypes. We successfully cloned Nicotiana spp.
codon-optimised AHSV capsid protein genes into a replicating geminivirus-derived plant expression
vector pRIC3.0 and into pEAQ-HT. Recombinant Agrobacterium tumefaciens harbouring the VP genes
were co-infiltrated into N. benthamiana and shown to successfully express in plants by western blot
analysis. The formation of AHSV VLPs was observed by transmission electron microscopy,
demonstrating the potential of this system for the production of a novel AHSV VLP vaccine.
Session 3
Invited speaker
Veterinary vaccines for enterohemorrhagic E.coli – Improving accumulation of type 3
secretion proteins in plants
Menassa R., Miletic S., MacDonald J., Kaldis A., Leuthreau A., Gaildry T., Huenerberg M.
and McAllister T.
Agriculture and Agri-Food Canada, London, Canada
Enterohemorrhagic E. coli (EHEC) are human enteropathogens that colonize the digestive tract of
animals and are shed in their manure, frequently contaminating meat, vegetables, and drinking water.
EHEC does not affect animal health, but causes disease in humans which can vary from mild diarrhea
to kidney failure and death. Immunizing cattle against EHEC is a strategy to reduce colonization and
therefore decrease food contamination. Such veterinary vaccines can dramatically reduce human
cases, yet adoption of these vaccines has been low due to cost of the vaccine and necessity of animal
handling for injection. The goal of this project is to produce a plant-made vaccine for oral
administration to ruminants. Several recombinant proteins from the type 3 secretion system (T3SS) of
EHEC were designed as vaccine candidates and expressed transiently in Nicotiana benthamiana and
transplastomically in N. tabacum. Only one of these protei ns accumu lated at levels that could be used
for oral immunization of animals. This vaccine candidate, EspA, accumulates in transplastomic lines to
220.7 mg/kg of fresh leaf weight and is stable in lyophilized leaf tissue. Oral immunization of sheep
with EspA shows potential for reducing EHEC shedding.
To improve accumulation of T3SS proteins, we co-expressed T3SS chaperones with EspD, Tir, and EspA.
We found that co-expression with the chaperone CesT significantly increased accumulation of
recombinant Tir when both proteins were transiently expressed in the nucleus and targeted to the
chloroplast of N. benthamiana. CesT also helped maintain higher levels of a Tir:GFP fusion protein over
time both in vivo and ex vivo, indicating that the favourable effect of CesT on accumulation of Tir is not
specific to a single time point or to fresh material. To validate the effect of CesT on Tir, we transformed
the nuclear genome of Tir transplastomic plants with CesT targeted to the chloroplasts, and found that
while Tir was undetectable in the original transplastomic lines, it became clearly detectable in the CesT
lines. These results can contribute to the production of recombinant T3SS-associated proteins in plants,
and may have implications for understanding interactions between T3 SS chaper ones and their targets.
In particular, our findings highlight the potential of chaperones to increase accumulation of
recombinant proteins in heterologous systems.
Session 3
Short Talk - Poster 3.1
Production of Human papillomavirus pseudovirions in plants and their use in
pseudovirion-based neutralisation assays in mammalian cells
Lamprecht R.L.1, Kennedy P.1, Huddy S.M.1, Hendrikse M.1, Bethke S.2, Hitzeroth I.I.1 and
Rybicki E.P.1,3
Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town,
Rondebosch, South Africa; 2Pharmaceutical Product Development, Fraunhofer IME, Aachen, Germany;
3Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch, South
Africa
Human papillomaviruses (HPV) are the causative agents of cervical cancer and have recently been
implicated in several other cancers, including mouth, laryngeal and anogenital cancers. Presently there
are two prophylactic vaccines that show good efficacy, but further work is needed to develop second
generation HPV vaccines that are more affordable, stable and display cross-neutralizing activity across
a broader range of HPV types. Candidate vaccines need to be tested for their ability to neutralize HPV
pseudovirions (PsVs) in a pseudovirion-based neutralisation assay (PBNA). The HPV PsVs consist of HPV
L1 and L2 capsid proteins that encapsidate ~8 kbp plasmids encoding reporter genes. Successful
neutralisation of the PsVs is ultimately observed as a loss of reporter gene function. Currently, the
accepted PsV production method utilizes mammalian cell culture to produce HPV PsVs, which is
expensive and time-consuming. Transient re combinant protein expression of the L1 and L2 capsid
proteins and of the reporter genes in plants as a self-replicating plasmid may offer a more rapid and
cost-effective alternative to the currently accepted method.
In this study, we aimed to manufacture plant-produced HPV-16 PsVs for use in the PBNA, with a view
to developing a cheaper alternative to the current assay approach. A self-replicating geminivirusderived viral vector was modified to include the secreted embryonic alkaline phosphatase (SEAP)
reporter gene required for the PBNA. Autonomous replication of the SEAP construct resulted in
replicons of a size that were able to be packaged by assembling HPV virions. This replicating vector was
co-infiltrated with the non-replicating vectors expressing L1 and L2 in plants and resulting PsVs were
purified and tested for DNA encapsidation to confirm the assembly of PsVs in plants. The purified PsVs
were able to infect mammalian cells and were also successfully neutralised by different HPV antibodies.
To our knowledge, this is the first demonstration of the production of HPV PsVs in plants and their
successful use in a PBNA and the first demonstration of the potential of plants to make DNA vaccines.
Session 3
Short talk - Poster 3.2
Multimerization strategy to create avian flu vaccines in plants
Phan H.T.1,2, Schinkoethe A.1, Gresch U.1 and Conrad U.1
1Department
of Molecular Genetics, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK),
Gatersleben, Germany; 2Department of Plant Cell Biotechnology, Institute of Biotechnology (IBT),
Vietnam Academy of Science and Technology (VAST), Hanoi, Vietnam
The H5N1 avian influenza A virus is a highly contagious, deadly pathogen in poultry, transmitted from
to humans from poultry causing high mortality (ca 60%) and pose a pandemic threat. Vaccination is the
most effective approach to reduce illness and death from pandemic influenza. Influenza vaccines in
general use today are derived from viruses grown in hens’ eggs. However this approach exposes several
obstacles such as complex processes, long preparation time and limited capacity. To address these
points, the subunit vaccines are an alternative approach to meet the global demand for the influenza
vaccines. However, the subunit vaccines generally induce low immunogenicity against influenza. To
overcome this effect, we are presenting here strategies to generate oligomers of main surface antigen,
hemagglutinin, in plants. New oligomers show high-molecular-weight and have super functionality
rather than a counter trimer. This includes hem agglutination activity and induction of mouse sera
providing hemagglutination inhibition activity.
Session 3
Short talk - Poster 3.3
Developing a synthetic polio vaccine using two plant-based expression systems
Marsian J.D. and Lomonossoff G.P.
John Innes Centre, Norwich, United Kingdom
Poliomyelitis is a serious infectious disease caused by the poliovirus. It attacks the central nervous
system of mostly infants and children and in the worst case it can lead to paralysis of the extremities
or death. Poliovirus is a non-enveloped virus that is 27-30 nm in diameter. The initiative by the World
Health Organisation (WHO) to eradicate poliomyelitis has been very successful, decreasing cases by
over 99% since 1988. However, that story of success has been disturbed with numerous outbreaks in
the last 3 years in East and West Africa and in the Middle East. WHO is therefore seeking novel polio
vaccines because the existing Sabin oral polio vaccine (OPV) and the parentally administered Salk
inactivated polio vaccine (IPV) have major disadvantages and their continued deployment is
incompatible with eradication. Failure to implement strategic approaches has led to ongoing
transmission of the virus. Endemic transmission is continui ng in Pakistan and Afghanistan.
The overall aim of this project is to produce a novel vaccine against poliovirus using a plant expression
system. In particular, it is aimed at the generation of non-infectious poliovirus-like particles (PV VLPs)
that have the capability of eliciting a protective immune response against polio. To execute this plan,
stabilized mutant versions of the poliovirus gene P1, encoding the precursor of the structural genes
and the necessary proteinase. 3CD, were cloned into separate pEAQ-HT expression vectors and
transformed into A.tumefaciens. Recombinant strains were co-infiltrated into N.benthamiana and the
leaves harvested 5 days after infiltration. VLPs were purified by Nycodenz gradient centrifugation giving
a purified PV VLP yield of 70 mg/kg fresh weight tissue. Mice carrying the gene for the human PV
receptor were protected of wild-type PV when immunized with the plant made PV VLPs. This
represents the first report of the production of immunologically effective non-infectiou s poliovi ruslike particles in plants.
To make the product more attractive to the vaccine industry, tobacco BY-2 cells have been successfully
tested for the transient expression of the above-mentioned PV mutant VLPs using the cell-pack
method. Work is currently ongoing on examining the properties of these PV VLPs. BY-2 cells grow in
suspension culture and have no limit for mass production which is a potential advantage over whole
plants.
Session 3
Short talk - Poster 3.4
Porcine epidemic diarrhea virus: Producing the membrane protein as a subunit
vaccine, and producing virus-like particles
Khamis Z. and Menassa R.
London Research & Development Centre, Agriculture and Agri-Food Canada, London, Canada;
Department of Biology, University of Western Ontario, London, Canada
Porcine epidemic diarrhea has caused widespread economic losses due to death of neonatal piglets
and weight loss of fattening pigs. A subunit vaccine against its causative agent, porcine epidemic
diarrhea virus (PEDv), a coronavirus, could help protect herds through vaccinating sows, providing
lactogenic immunity to suckling newborns. Research to date has focused on producing the spike (S)
protein. However, while S is prone to mutations, the membrane (M) protein is highly conserved, and
also has a virus-neutralizing epitope. As well, the membrane protein of coronaviruses has been shown
to play a critical role in viral assembly and budding, and thus is necessary for production of virus-like
particles (VLPs). VLPs resemble the virion, and due to this stimulate strong immune responses, making
them the ideal vaccine. Expression of M alone or in combination with the envelope protein (E), the
nucleocapsid protein (N), or the spike pr otein (S) of other coronaviruses has resulted in VLP assembly.
Using plants as a platform to produce the protein allows for oral administration, a method that was
shown to elicit a strong mucosal immune response. Therefore, I have undertaken the production of
the M protein in Nicotiana benthamiana, and found that M accumulates to high levels in the insoluble
membrane-rich fraction of leaf extracts. I then undertook the production of M co-expressed with the
E, N and/or S proteins. Similar to what was shown previously with SARS virus, another coronavirus,
thermal aggregates form with the membrane protein when boiled. In addition, the choice of detergent
for membrane protein extraction has a significant impact, increasing extraction efficiency and allowing
a more precise determination of accumulation levels of over 0.7 mg/g fresh weight. Co-expressing M
and E has shown promise, and transmission electron microscopy will be used to determine if VLPs are
produced.
Session 3
Short talk - Poster 3.5
Developing plant-expressed bleutongue virus particles as a delivery system for foreign
proteins
Thuenemann E.C. and Lomonossoff G.P.
Department of Biological Chemistry, John Innes Centre, Norwich, United Kingdom
Biocompatible nanoshells based on a variety of virus-like particles (VLPs) have been developed as drugdelivery and bioimaging agents over the past few years. In most cases the cargo that can be loaded
into the nanoshells is restricted to small molecules due to size constraints. The use of VLP-derived
nanoshells to encapsulate larger molecules, such a proteins, requires that the particles have a
comparatively large internal volume. The bluetongue virus (BTV) subcore structure, with its large
(approx. 40 nm diameter) internal cavity encased by only 120 subunits of the VP3 structural protein,
makes it particularly suited for this application. Furthermore, we have previously shown that BTV
particles can be efficiently assembled after the plant-based transient expression of VP3 with or without
the other three main structural proteins (Thuenemann et al., 2013). Thus we have investigated
whether proteins fused to VP3 can be efficiently encap sulated.
We have followed an approach of N-terminal fusion, similar to that previously used in insect cells (Kar
et al., 2013). Green fluorescent protein fused to VP3 has allowed us to efficiently produce fluorescent
subcore-, core- and virus-like particles that harbour GFP on the inside without altering the size or
surface structure of the particles. Such constructs can be used to study particle assembly in plants, or
to visualise attachment and entry into host cells.
Beyond fluorescent proteins, BTV particles can be made to encapsulate even larger proteins and,
depending on the cargo, such particles could have a range of applications from vaccine carriers to nanoscale reaction vessels.
Thuenemann, E. C., A. E. Meyers, J. Verwey, E. P. Rybicki and G. P. Lomonossoff (2013). "A method for rapid production of
heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles." Plant Biotechnol
J 11(7): 839-846.
Kar, A. K., N. Iwatani and P. Roy (2005). "Assembly and intracellular localization of the bluetongue virus core protein VP3."
Journal of Virology 79(17): 11487-11495.
Session 3
Short talk - Poster 3.6
The generation of modified plant virus-like particles by transient expression for
potential bionanotechnological applications.
Saunders K., Castells-Graells R. and Lomonossoff G.P.
Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, United Kingdom
A key property of single-stranded RNA plant viruses is to multiply and produce high yields of virus
particles that encapsidate the viral genome. The transient expression of the viral coat proteins induces
the synthesis of virus-like particles (VLPs) that lack the infectious genome. This technology, which is
now well established, can be used to produce high yields of VLPs that have a morphology essential
identical to that of the virus from which they have been derived.
We have utilised genome components of two plant viruses of differing morphology, turnip crinkle virus
(TCV, a spherical virus) and tobacco mosaic virus (TMV, a ridge rod-shaped virus) to generate VLPs in
infiltrated plant leaves. Both viruses consist of a capsid shell composed of multiple copies of a single
coat protein entity. By transiently expressing modified forms of the coat protein of these plant viruses
and altering the length of the RNAs designed to be encapsidated, it is possible to demonstrate the
formation of T=1 or T=3 spherical shells and nano-rods of a differing geometry, compared to the
morphology that these viruses naturally exhibit. Nano-rod length is regulated by the length of its
encapsidated RNA. Additional changes to coat protein sequence allows for the expression of defined
amino acid sequences on the capsid surface. In the case of TCV, it is also possible to extend the range
of modification which can be incorporated into the coat protein through the cr eation of mosaic
spherical VLPs consisting of both the wild-type and modified coat proteins.
Session 3
Short talk - Poster 3.7
GAD65-based edible vaccine: a new autoimmune diabetes perspective
Bertini E., Merlin M., Gecchele E., Klimyuk V., Pezzotti M. and Avesani L.
University of Verona, Verona, Italy
Plants have emerged as competitive production platforms for pharmaceutical proteins that are
required in large quantities. One example is the 65-kDa isoform of human glutamic acid decarboxylase
(GAD65), a major autoimmune diabetes autoantigen that has been developed as a vaccine candidate
for the primary prevention of diabetes. The production of GAD65 in plants has been optimized by the
expression of a mutated catalytically inactive form of the protein (GAD65mut), but large-scale
purification is hampered by its tendency to associate with thylacoid membranes.
More recently, an N-terminal truncated form of GAD65mut (Δ87GAD65mut) has been expressed in
Nicotiana spp. aiming at obtaining a more soluble form of the protein. In this framework, the truncated
form of the protein revealed to be also more stable and to accumulate at higher levels than the fulllength one when expressed in N. benthamiana with the MagnICON system.
Here, we investigated the potential of combining two different approaches: the high yields obtained
with the MagnICON system, particularly with GAD65mut and its truncated form Δ87GAD65mut, and
the use of edible tissues as expression platforms to set-up an edible vaccine, thus bypassing purification
of recombinant protein from plant material.
The plant systems tested in the project as hosts for target molecule expression were spinach and red
beet. The growing conditions for each plant system were firstly selected to improve biomass
production, then optimal infiltration conditions were identified (e.g. Agrobacterium dilution, day of
sampling, performance comparison of manual/vacuum infiltration systems).
The best expressing system comprising the combination of target molecule, plant species and bestperforming protocol for transient expression in terms of recombinant protein accumulation, was
selected for the first steps of set-up of a putative edible vaccine for type 1 autoimmune diabetes.
Session 3
Poster 3.8
Leaf Systems®: industrialisation of the Hypertrans® transient gene expression system
Jamieson G.
Leaf Systems, Norwich, United Kingdom
The Hypertrans® (HT-CPMV) gene expression system was developed by Professor George Lomonossoff
and Dr Frank Sainsbury at The John Innes Centre, Norwich for the transient expression of proteins in
plants.
The technology is already being used commercially in Canada and the US, where licensee Medicago
Inc. is using plant-based expression for the rapid development and production of pandemic and
seasonal influenza vaccines, the most advanced of which are in phase II clinical trials.
Now the John Innes Centre has partnered with its main strategic funder the Biotechnology and
Biological Sciences Research Council (BBSRC) and Plant Bioscience Ltd to bring the use of the
technology closer to home.
The three partners have established a new company – Leaf Systems® which will comprise state of the
art plant-growing areas and development laboratories. This is a translational facility enabling the
transition of the Hypertrans system from research use to production of materials for onward evaluation
and potential commercialisation.
Target products include therapeutic proteins, vaccines and also plant natural products. Work at the
John Innes Centre by Professor Anne Osbourn’s group has combined the Hypertrans technology with
insights into the clustering of plant natural product genes to enable the facile manipulation of
secondary metabolite pathways to access the enormous diversity in plant bioactive molecules.
Session 3
Poster 3.9
The transient expression of Bluetongue virus proteins in Nicotiana benthamiana for
plant-based vaccine development
Maranyane H., Meyer A., Regnard G., Hitzeroth I. and Rybicki E.
The Biopharming Research Unit (BRU), Department of Molecular and Cell Biology, Faculty of Sciences,
University of Cape Town, Rondebosch, South Africa
The Bluetongue virus is a prototype virus of the genus Orbivirus. It is transmitted to vertebrate hosts
(sheep, cattle, horse and deer) via arthropod vector bites (Culicoides species) in a blood meal. Host
infections cause fatal illness characterized by damage to small blood vessels, oral ulceration, facial and
pulmonary oedema, vascular thrombosis and necrosis of infected tissues. There is currently no
treatment for the disease other than supportive care. Bluetongue is mainly controlled by vaccination
with live-attenuated or inactivated virus vaccines however these come with disadvantages. Live
attenuated vaccines are known to revert to virulence or recombine with circulating strains of the virus
thereby leading to the development of progeny strains and inactivated virus vaccines are difficult to
make. There are currently 26 known serotypes of the virus, with serotypes 2, 4 and 8 being of global
importance. We aimed to express BTV vira l proteins (VP’s) in Nicotiana benthamiana to synthesize
virus-like particles (VLPs) in order to create DIVA complaint subunit vaccines against these three
serotypes. We successfully cloned Nicotiana spp. codon-optimised BTV8 capsid protein genes VP2, VP5,
VP3 and VP7 and BTV2-VP2 into a replicating Geminivirus plant-expression vector pRIC3.0.
Recombinant Agrobacterium tumefaciens GV3101::pMP90RK strains were generated. Combinations of
these recombinant strains were co-infiltrated into N. benthamiana and shown to successfully express
in plants by western blot analysis. Co-infiltration experiments were then optimized for VLP production
and purification.
Session 3
Poster 3.10
Development of a plant-produced vaccine candidate for beak and feather disease virus
Regnard G.L.1, Hitzeroth I.I.1 and Rybicki E.P.1,2
1Biopharming
Research Unit, Department of Molecular & Cell Biology, University of Cape Town,
Rondebosch, South Africa; 2Institute of Infectious Disease and Molecular Medicine, Faculty of Health
Sciences, University of Cape Town, Observatory, Rondebosch, South Africa
Psittacine beak and feather disease (PBFD), the most prevalent viral disease affecting psittacines, is
caused by beak and feather disease virus (BFDV), a small spherical virus with a circular ssDNA genome.
The main clinical features are beak and feather abnormalities accompanied by immunosuppression. At
present no treatment or vaccine is commercially available, largely because the virus is not culturable,
and it is difficult to produce the coat protein (CP) in bacterial or animal cell expression systems.
Accordingly, in this study we investigated the feasibility of making a plant-produced BFDV subunit
vaccine candidate.
The capsid gene (cp) was optimised based on protein localisation, infiltration density and time of
harvest for transient Agrobacterium-mediated expression in whole-plant Nicotiana benthamiana (N.
benthamiana). Virus-like particles (VLPs) were purified using sucrose cushion centrifugation and CsCl
density gradient centrifugation, and analysed using transmission electron microscopy. As a control,
virions from a naturally-infected Palm cockatoo (Probosciger aterrimus) were also purified. The CP was
successfully expressed in N. benthamiana, and expression was increased through optimisation of
Agrobacterium infiltration density and the co-infiltration of the tomato spotted wilt virus NSs silencing
suppressor. Expression and accumulation of CP was highest on day 3, when Agrobacterium containing
cp was co-infiltrated at an absorbance value of 0.5 together with NSs at an absorbance value of 0.25.
These plant-produced BFDV VLPs were shown to be morphologically similar to those produced in insect
cells and infectious virions. It is possible that the VLPs are spontaneously incorporating amplicon DNA
produced from the replicating geminivirus-based plant expression vector. The resulting pseudovirions
could feasibly be used to further the efficacy of vaccines against BFDV. The CP was successfully
produced in planta and presence of VLPs suggests the possibility of developing pseudovirions. This is
the first report of BFDV VLP production in plants.
Session 3
Poster 3.11
Expression in Nicotiana benthamiana and oral immunogenicity of a porcine epidemic
diarrhea virus epitope (S1D) fused with Cholera toxin B subunit
Tien N.-Q.-D., Kim M.-Y. and Yang M.S.
Department of Molecular Biology, Chonbuk National University, Jeonju-si, Republic of Korea
Porcine epidemic diarrhea virus (PEDV) is a coronavirus, that causes very severe diarrhea, vomiting,
dehydration, and high mortality rates in sucking pigs. It had resulted in significant economic losses in
many swines-raising countries in the world. Currently, there are no licensed vaccines and substantial
PEDV vector control effort has not stopped its rapid emergence and global spreading. Plant cells are
ideal bioreactors for the production and oral delivery of vaccines, eliminating the need for expensive
fermentation, purification, cold storage, transportation and sterile delivery. Co-administration of a
plant-made vaccine antigen with a carrier protein such as cholera toxin, it can increase the
immunogenicity and compensate in part for low antigen recognition. In this study, a new epitope of
PEDV (S1D) alone or fused with Cholera toxin B subunit were rapidly transient co-expressed with p19
protein of tomato bushy stunt virus in Nicot iana benthamiana as an oral vaccine candidate against
PEDV. The expression and GM1-ganglioside receptor binding activity of CTB-S1D fusion protein were
confirmed by Western blot analysis and GM1-ELISA. Importantly, following oral immunization of mice,
the CTB-S1D fusion protein or with bacterial cholera toxin or mutant cholera toxin 61F from rice callus,
the immunized mice were induced significantly serum IgG and sIgA levels against bacterial CTB and S1D
antigen, peaking at sixth week. Our results suggest that new epitope S1D of PEDV fused with cholera
toxin B subunit have potential to be developed as plant-based vaccines candidate against porcine
epidemic diarrhea virus.
Session 3
Poster 3.12
Porcine circovirus capsid protein production in plants
Gunter C.J., Regnard G.L., Hitzeroth I.I. and Rybicki E.P.
Biopharming Research Unit, Department of Molecular and Cell Biology, Faculty of Science, University
of Cape Town, Rondebosch, South Africa.
Porcine circovirus type 2 (PCV-2) is the main causative agent associated with diseases collectively
known as porcine circovirus associated disease. There is significant economic strain on the global swine
industry due to high production costs of currently available PCV-2 vaccines. Plant expression systems
are a viable technology for the production of recombinant proteins as pharmaceutical agents and
vaccine development. These systems are free of mammalian pathogens as well as cost-effective with
rapid and scalable production potential. Plant expressed, self-assembled virus like particles (VLP) may
elicit strong antibody and cellular immune responses. Our objective in this study was to express the
PCV-2 capsid protein (CP) in Nicotiana benthamiana plants so that expression would produce VLP.
Recombinant Agrobacterium tumefaciens delivered the modified pEAQ-HT vector which ensured high
levels of transiently expressed foreign proteins in N. benthamiana. Immunoblotting confirmed the
expression of PCV-2 CP which was optimised for days post infiltration, and the OD600 of infiltrated
recombinant A. tumefaciens. The self-assembled virus-like particles (VLP) would then be purified by
CsCl density purification and visualised by transmission electron microscopy.
Optimum protein expression of the 27-kDa PCV-2 CP has been confirmed at A. tumefaciens OD600
infiltration of 0.5 three days post infiltration.
The impact of successfully producing PCV-2 CP self-assembling into VLP’s will not only further our
understanding of foreign protein and DNA production in plants, but may well serve as viable alternative
for commercial vaccine development.
Session 3
Poster 3.13
Screening for a plant-based subunit vaccine biobetter with improved stability against
STEC O157:H7
Chin-Fatt A. and Menassa R.
Department of Biology, University of Western Ontario, London, Canada
Shiga toxin producing Escherichia coli (STEC) O157:H7 is a pathogen that harbors asymptomatically in
the recto-anal junction of cattle and is commonly associated with widespread meat recalls and food
poisoning affecting approximately 2.8 million people globally each year. EspB is a bacterial secretory
protein involved in the type III secretion system and has previously been validated to be effective at
immunizing cattle against STEC O157:H7. Its production in a plant-based system would be
advantageous because of the potential for oral delivery to cattle, the lack of animal pathogens and
endotoxin contaminants as well as the potential for scalability. A major hurdle of recombinant protein
production is low accumulation levels, due at least in part to structural instability of the protein upon
folding, which may result in degradation and aggregation. Given the cost of producing these molecules,
even small increases in stability, and resul tant yield and product homogeneity, can translate to
significant savings. The transient transformation of chloroplast targeted native EspB into Nicotiana
benthamiana leaf tissue results in accumulation of approximately 100mg/kg FW. To further improve
accumulation, stabilizing point mutations were predicted in silico by first modelling the structure with
the I-TASSER software and then predicting the free energy changes upon all possible amino acid
substitutions. The top 22 candidates predicted to have the most improved stability were chosen for
site-directed mutagenesis of EspB. The mutants were transiently transformed into N. benthamiana leaf
tissue, extracted and then assessed by immunoblot. Two asparagine to aspartate mutations were
found to improve accumulation by ~20% (~120mg/kg FW) compared to native EspB. Removal of the
transmembrane and coiled coil domains was also investigated as part of the screen since these domains
can resist solubilisation upon extraction leavi ng the pr otein trapped in the pellet but no change in
accumulation was observed. The screening for a plant-based subunit vaccine biobetter provides an
avenue for improved yield that could potentially translate to better cost appeal for cattle farmers and
processors. Development of a robust and affordable STEC O157:H7 subunit vaccine that can be
administered orally to cattle has the potential to improve food safety and reduce outbreak recurrence
of O157:H7.
Session 3
Poster 3.14
Towards Pepino mosaic virus virion-like particles production
Méndez López E., Sanchez-Pina M.A., Valle M. and Aranda M.A.
Plant Pathology Unit, Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), Murcia, Spain;
and Structural Biology Unit, Center for Cooperative Research in Biosciences, CIC bioGUNE, Derio, Spain
We have described Pepino mosaic virus (PepMV; genus Potexvirus, family Alphaflexiviridae) particles
at 3.9 Å resolution using electron cryomicroscopy (Agirrezabala et al., 2015). The cryoEM map revealed
a left-handed helix with a diameter of 130 Å and an inner narrow channel of 13 Å, with a pitch of 34.6
Å and 8.7 coat protein (CP) copies. The single stranded RNA runs in a helix of 70 Å and resides in a
continuous groove with a high electropositive potential. The helical assembly of PepMV is also
mediated by protein-protein interactions involving the CP N-terminal arm and C-terminal extension.
The N-terminal arm establishes the main side-by-side contact in the helical arrangement, whereas the
C-terminal extension builds the inner wall of the virus and creates a network of small and local
interactions. No specificity in the ssRNA sequence is required to stabilise viral particles; still, expression
in plants of the CP on its own did not result in the formation of virion-like particles (VLPs), whereas coexpression of CP with viral genomic RNA does, suggesting the existence of an origin of assembly (OAS).
A 5´-deletion analysis of viral genomic RNA identified a stem loop between nucleotides 53 and 87 as
potential OAS. Indeed, expression of the CP from an RNA containing this stem loop resulted in the
formation of VLPs. Tagged versions of PepMV expressing N-terminal GFP-CP fusions are viable
(Sempere et al., 2011), thus the VLPs described here have significant potential for epitope display in
plants.
Agirrezabala et al., 2015, eLIFE 4:e11795
Sempere et al., 2011, Plant Methods 7: 6
Session 3
Poster 3.15
Polymeric IgG-Ag85B fusion proteins as TB vaccine candidates
Webster G., Teh A., Reljic R., Craig van Dolleweerd C. and Ma J.
Molecular Immunology Unit, Institute for Infection and Immunity, St. George’s University of London,
London, United Kingdom
Tuberculosis (TB) remains a major global health issue, despite the widespread use of BCG vaccine and
effective drug therapies. The development of a new vaccine would be an important component of TB
control in the future. Ag85B, the most abundantly expressed protein in mycobacterial culture fluids, is
a leading vaccine candidate currently used in various TB subunit vaccines in clinical trials. In this project,
Ag85B was used to form a novel polymeric immune complex-like structure with immunoglobulin
(polymeric immunoglobulin or PIgs). IgG γ-chain was fused to IgM μ-tail piece to facilitate polymeric
structure formation. Polymeric antibodies such as IgM fix complement much more effectively than
monomeric IgG. Conversely, IgG activates other immune responses through binding to Fc receptors,
such as antibody dependent cell mediated cytotoxicity (ADCC). PIgs were cloned and expressed in
Nicotiana benthamiana and complexes of expected size were observed and could be purified using
Protein G chromatography. They were shown to be biologically active as they bind to C1q component
of the complement cascade as well as FcγRs. Furthermore, PIgs were shown to be taken up by THP1
cells, a macrophage cell line. Immunisation and challenge studies in BALB/c and CD64 transgenic mice
are on-going. Future work involves in vitro cell binding and activation assays as well as analysing results
from on-going immunisation and challenge studies in animal models.
Session 3
Poster 3.16
Molecular design of a novel, self-adjuvanting vaccine against dengue infection
Mi-Young K., Craig V.D., Alastair C., Gina W., Jorge R., Moon-Sik Y., Yong-Suk J., Rajko R.
and Julian K-C.M.
Molecular Biology Department of Basic Science, Chonbuk National University, South Korea
Dengue infection is a global health problem with vaccine development and urgent priority. Here,
molecular engineering approach was employed to construct a polymeric molecular scaffold that
incorporates multiple copies of the consensus domain III sequence of DENV glycoprotein E. This new
vaccine construct, termed cEDIII-PIGS (polymeric IgG scaffold) was made, either using murine
immunoglobulin sequence - for preliminary evaluation, or human immunoglobulin sequence - for
subsequent product development. Both versions were successfully expressed in plants and Chinese
Hamster Ovary cells. These molecules were shown to assemble into polymeric structures that retain
important Fc receptor functions associated with immunoglobulins, including binding to C1q
component of the complement cascade and the Fc receptors. Murine and human cEDIII-PIGS were
shown to be highly immunogenic in wild type and CD64 transgenic mice (expressing human F cRI re
ceptor) respectively, with or without an adjuvant. The murine cEDIII-PIGS induced a high level IgG
antibody response in their sera, which showed neutralising activity against DENV serotype 2.
Additionally, these molecules also induced a significant cellular response, as measured by T cell
proliferation, secretion of IFN-and presence of polyfunctional T-cells. A balanced helper/cytotoxic Tcell profiles were were induced by both murine and human cEDIII-PIGS, thus underscoring the potential
of this immunisation approach to induce and modulate a broad-ranging anti-dengue immune response.
Session 3
Poster 3.17
Exploiting Bean pod mottle virus as a transient expression system to produce plantmade vaccines against porcine reproductive and respiratory syndrome virus (PRRSV)
in soybean leaves.
Tran H.-H., Chen H., Menassa R., Huner N. and Wang A.
Department of Biology, Western University, London, Canada; Agriculture and Agri-Food Canada,
London, Canada
PRRSV is a severe pathogen that infects pigs and causes huge economic losses in the swine industry
worldwide. Since no therapy for the disease exists, prevention of outbreaks relies mostly on vaccines.
Plant-made vaccines have garnered increasing interest within the veterinary field, for which regulatory
approval is less cumbersome than human applications. Here, we introduce a transient viral expression
system based on bean pod mottle virus (BPMV) to produce plant-made vaccines against PRRSV. BPMV
belongs to the family of Comoviridae, which possesses a genome of two genomic RNAs, RNA1 and
RNA2. BPMV RNA1 (approximately 6 kb) encodes five mature proteins, protease cofactor (Co-pro),
putative helicase (Hel), viral genome-linked protein (VPg), protease, and putative RNA-dependent RNA
polymerase (RdRp), all of which are necessary for viral replication. BPMV RNA2 (approximately 3.6 kb)
encodes four proteins including a replication c ofactor ( CR), a putative movement protein (MP), a large
coat protein (L-CP) and a small coat protein (S-CP). The full length cDNAs of RNA1 and RNA2 were
cloned into binary vectors under the control of a 35S promoter. The vector containing RNA2 was
modified to carry genes of interest, flanked by two self-cleavage sites in the region between MP and LCP. A combination of modified RNA2 and RNA1 clones was transfected into soybeans via biolistic
bombardment. Using this transient expression system, green fluorescence protein (GFP) was produced
at levels up to 37 mg/kg fresh leaf biomass at 30-45 days post inoculation. Furthermore, the
glycoprotein 5 (GP5) gene of PRRSV was halved into N-terminal GP5 (GP5N) and C-terminal GP5 (GP5C)
fragments and fused with GFP to express GP5N-GFP and GP5C-GFP, respectively. The presence of
GP5C-GFP in soybean was confirmed by western blot, whereas the plants bombarded with the GP5NGFP construct showed no infectivity. The resulting GP5C-GFP will be evalu ated for potential utilization
as an oral vaccine against PRRSV.
Session 3
Poster 3.18
Plant-derived antigen-antibody complex induced anti-cancer protective immune
response through large quaternary structural formation
Kim D.-S.1, Lee K.J.1, Qiao L.1, Ko K.2, Do Y.3, Myeung S.C.4 and Ko K.1
1Department
of Medicine, Therapeutic Protein Engineering Lab, College of Medicine, Chung-Ang
University, Seoul, Korea; 2Department of Stem Cell Biology, School of Medicine, Konkuk University,
Seoul, Korea; 3School of Nano-Bioscience and Chemical Engineering, Ulsan National Institute of Science
and Technology, Ulsan, South Korea; 4Department of Urology, Chung-Ang University, College of
Medicine, Korea
The effectiveness of antigen-antibody complex (AAC)-mediated immunomodulation as a vaccine
spurred the development of diverse quaternary protein structures for vaccination. Herein, we describe
the formation of a large quaternary protein structure, involving the clustering of the colorectal cancer
associated antigen GA733-Fc-KDEL ER retention motif fusion protein (GAP) and the anti-GA733
antibody mAb CO17-1A with KDEL (COP) co-expressed in plants, and its immune functional
characterization. Transgenic plants expressing the GAP and the anti-colorectal cancer COP, respectively,
were crossed to produce F1 plants expressing both proteins (COPGAP). ELISA and surface plasmon
resonance (SPR) revealed that both GAP and COP proteins were assembled into the large quaternary
structure of AAC. Electron micrographs (EM) showed that the AAC formed a ~30 nm circular protein
structure. In addition, atomic force microscope (AFM) confirmed that the sizes of GAP×COP and GAP
were ~15 and ~30 nm, respectively. Furthermore, dynamic light scattering (DLS) also identified larger
molecular size of GAP×COP than individual GAP and mixture GAP+COP. In mice administered with
COPGAP, anti-GA733 IgGs were generated at significantly higher titers than in mice induced by the
independent antigens [mammalian-derived GA (GAM) and GAP] or the in vitro mixtures of GAM and
mammalian-derived mAb CO17-1A (COM) (GAM+COM), and GAP+COP counterparts, respectively. Sera
from the mice immunized with the GAPCOP efficiently inhibited the growth of SW620 human
colorectal cancer cells xenografted in nude mice similar to the anti-GA733 mAbM. These data indicate
that antigen and antibody can be assembled to form large quaternary protein complex structures in
plants, which induce anti-tumor immune responses to inhibit tumor growth.
Session 4
Innovative expression
strategies and applications
Session 4
Invited speaker
Antimicrobials made in plants
Gleba Y.
Nomad Bioscience GmbH, Halle, Germany
Enterohemorrhagic or Shiga toxin–producing Escherichia coli contaminating food products are a
leading cause of bacterial enteric infections in USA and worldwide. There are no effective methods to
control pathogenic bacteria in food chain. Nomad scientists investigated colicins, non-antibiotic
antimicrobial proteins produced by certain E. coli strains and narrowly active against some other strains
of the species, as potential pathogen control agents. Most colicins are expressed at very high yields in
plants, are fully functional and identical to bacterially produced molecules, and simple cocktails of two
or more colicins applied at low concentrations are highly and broadly active against all major
pathogenic E. coli strains causing outbreaks (‚Big Seven‘). Plant-produced colicins are being proposed
as inexpensive food additives for a broad control of pathogenic E. coli bacteria in food products that
can be promptly approved in USA under existing regulatory approval process. In 2015, Nomad has
received its first GRAS (Generally Regarded As Safe) regulatory approval for plant-made colicins. We
have also expressed, in addition to colicins, other antimicrobial proteins, including phage endolysins,
pyocins, pesticins and other, and are in the process of defining antimicrobial cocktails for a range of
Gram-negative and Gram-positive pathogens including Salmonella, Pseudomonas, Listeria and
Clostridium. The products under development may have a broad application in food safety and human
and animal therapy.
Session 4
Invited speaker
Nanobodies in and from plants: production of nanobody multimers and selected
protein degradation in planta by nanobody-F-Box fusions
Conrad U., Baudisch B., Phan H.T., Weselek A. and Hauptmann, V.
Department of Molecular Genetics, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK),
Gatersleben, Germany
Camelidae heavy-chain antibodies have been suggested as an alternative drug format for the
treatment of several diseases. These antibodies recognize the antigen through a single variable heavychain domain (VHH), a molecule which is about ten times smaller than the complete conventional IgG.
In contrast to IgG antibodies, single-domain antibodies lack a light chain instead comprising a single
polypeptide. VHH domains are highly temperature tolerant, and their thermal unfolding is reversible.
The antigen specificities and affinities of VHH and IgG antibodies are very similar, with dissociation
constants lying in the nM range. The multimerization of nanobodies may help to increase the serum
half-life and the stability in organs of application as the intestine. Avidity effects could also be induced.
We produced nanobody multimers by different strategies in planta. Such strategies are
multimerization by trimerization domains combined with disulphide bridges caused by specific cysteins
and the use of trans-splicing strategies by inteins. The specific degradation of proteins in cells is a
valuable tool of cell biology. Specific nanobodies fused to F-Box proteins can be used to target proteins
to the proteasome causing directed degradation. We firstly applied this technology in plants and
demonstrate specific degradation of green fluorescent protein in tobacco cells. This paves the way for
the development of phenotypic mutants for several applications in physiology as well as in pathogen
resistance.
Session 4
Invited speaker
Consistent production of plant-made recombinant polyclonal antibodies against snake
venoms
Julve JM., Huet E., Fernandez del Carmen A., Wei-song P., Venturi M., Jimenez A.,
Gutierrez C., Calvete JJ., Segura A., Gutierrez JM, Granell A. and Orzaez D.
IBMCP-CSIC, Valencia, Spain
Snakebites cause 125000 deaths and 300000 amputations and permanent disabilities per year
worldwide. Unfortunately, shortage of antivenin supplies is a constant threat due to the high
manufacturing costs in confluence with the low income of most target populations. Moreover,
antiserum-based treatments are not free of serious secondary effects as consequence of the exposure
to heterologous antibody constant regions. Alternative antivenins made of recombinant human or
humanized antibody cocktails would be highly desirable, as they would reduce secondary effects while
facilitating product standardization and reproducibility. Unfortunately, manufacturing costs for
complex antibody cocktails are very high because it requires maintenance of parallel production lines
for each component in the cocktail to ensure consistency. As a way to overcome these limitations, we
have developed a new strategy for affordable antivenin production based on recombinant polyclonal
antibodies made in plants. This strategy takes advantage of viral interference, an intrinsic property of
many plant viruses, to induce the formation of somatic expression mosaics in the plant leaf, which in
turn ensures that high levels of recombinant polyclonal antibodies are produced with outstanding
batch-to-batch reproducibility regardless of the complexity of the polyclonal composition. We show
here that this new strategy enables the expression of a selected portion of a mammalian immunized
antibody repertoire (named as “Plantiserum”) on a multi-transgenic plant in a highly reproducibly
manner. Our results indicate that the exploitation of somatic expression mosaics in plants results in
affordable and scalable production of recombinant polyclonal antibodies that distinctively combine
batch-to-batch reproducibility with virtually unlimited complexity.
Session 4
Invited speaker
Functional HRP-fused anti-rabbit IgG scFvs produced in plants
Meyers A.
Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town,
Rondebosch, South Africa
Labelled anti-rabbit IgG secondary antibodies are used extensively in immunochemical applications
such as western blotting, ELISAs and immunofluorescence. These are normally raised in animal hosts,
the species of which have been used to generate the primary antibodies utilised in the particular
immunochemical application. However, animal-sourced reagents are becoming increasingly more
difficult to produce. This is due mostly to increasing ethical limitations, making their production more
time-consuming and expensive. Moreover, there is now a worldwide trend towards producing
laboratory reagents in recombinant systems which do not involve any animals or animal tissue.
We tested the production and functionality of a recombinant plant-made secondary antibody as a
proof of concept for use in western blots and ELISAs. A gene encoding a short chain variable fragment
(scFv) isolated from a chicken Ig phage display library and shown to bind to rabbit IgG protein, was
fused to the 3’ terminus of a horseradish peroxidase (HRP) gene linked to a 6 × histidine tag. This HRP
gene was previously cloned into an expression vector in our laboratory and shown to transiently
express HRP protein at high levels in N. benthamiana. The HRP-IgG-scFv fused construct was cloned
into the pTRAkc-ERH and pTRAc plant expression vectors which were subsequently infiltrated into
Agrobacterium GV3101:pMP90RK. The recombinant constructs were introduced into N. benthamiana
leaves by Agrobacterium–mediated infiltration. Harvesting of infiltrated leaves at three days post
infiltration and preliminary screening of crude leaf extracts by western blots probed with anti- chicken,
anti-HRP and anti-6 × histidine antibodies confirmed expression of the recombinant HRP-IgG-scFv
fusion protein, and activity of the HRP component was also successfully demonstrated. Production of
the protein was scaled up and the protein purified using Ni-NTA affinity chromatography.
The functionality of the purified recombinant fusion protein was verified by western blotting; it was
shown to be able to allow the detection of several different proteins bound by specific primary rabbit
antibodies, using a HRP substrate. The functionality of the fusion protein is currently being tested by
ELISA to detect binding to a selection of antibodies produced in rabbits.
Session 4
Short talk - Poster 4.1
Seeds are capable to produce non-cytotoxic synthetic spider silk biopolymers
Weichert N.1, Hauptmann V.1, Menzel M.2, Reimers K.3, Kürbitz T.2 and Conrad U.1
1Abteilung
für Molekulare Genetik, Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung
(IPK), Gatersleben, Germany; 2Fraunhofer-Institut für Mikrostruktur von Werkstoffen und Systemen
(IMWS), Halle/S., Germany; 3Klinik für Plastische, Ästhetische, Hand- und Wiederherstellungschirurgie,
Medizinische Hochschule Hannover (MHH), Hannover, Germany
Recent biomedical developments in the field of tissue engineering require protein-based biomaterials
as scaffolds that have been optimized for substantial extensibility, biocompatibility as well as long-term
stability. Spider silk proteins represent a potentially ideal biomaterial for medical applications. Our goal
is to produce plant-derived high molecular weight spider silk proteins (spidroins) which have superior
mechanical properties combined with a non-cytotoxic behavior. Miniaturized spidroin genes were
synthesized, combined to form multimers of distinct length and expressed as elastin-like-peptide
fusion proteins in tobacco. The elastic penetration moduli E of biolayers from these different materials
were estimated. The increase of the spidroin content in these fusion proteins lead to an ascending E
value, and, therefore, represents a higher stiffness of these materials. We also show that spidroinbased biopolymers can i nduce hum oral immune responses mainly directed against the fusion partner
ELP. Cytocompatibility assays gave no indication of spidroin-derived cytotoxicity, suggesting that
recombinantly produced biopolymers composed of spider silk-like repetitive elements are suitable for
biomedical applications. This needs also suitable scaluable plant-based production systems. Here, we
explored the benefits of seed-based expression systems to produce high molecular weight spider silk
protein FLAG using intein-based trans-splicing. Multimers larger than 460 kDa in size are routinely
produced. The storage of seeds until 1 year at an ambient temperature of 15°C does neither influence
the accumulation level nor the typical pattern of multimerized bands. Seeds are the method of choice
for stable accumulation of products of complex transgenes and have the capability for long-term
storage at moderate conditions, an important feature for the development of suitable downstream
processes.
Session 4
Short talk - Poster 4.2
Modified plant-viruses nanoparticles for the Sjögren’s syndrome diagnosis
Zampieri R., Tinazzi E., Merlin M., Bason C., Beri R., Lico C., Lunardi C., Pezzotti M. and
Avesani L.
University of Verona, Verona, Italy
Plant viruses can be exploited as self-replicating protinaceous nanomaterials generally stable and easily
produced at high titers in plant hosts (Tinazzi et al., 2015). In this work Potato Virus X (PVX) has been
used as a scaffold to display a linear peptide (lipo) derived from human Lipocalin. Lipo peptide is
immunodominant in Sjögren’s Syndrome (SjS) being recognized by the antibodies present in SjS patient
serum.
The resulting virus-modified nanoparticles (PVX-lipo) have been used to develop a diagnostic kit for SjS
based on a direct enzyme-linked immunosorbent assay (ELISA). We found that the ELISA assay set up
using PVX-lipo were more sensitive than the chemically synthesized immunodominant peptide lipo and
equally specific when used to distinguish between healthy individuals and SjS patients. Our kit
therefore allows the SjS diagnosis with a low-invasive blood test in comparison with the currents
diagnostic techniques which include labial biopsy.
Moreover, laboratory trials have demonstrated that our kit can be storage at 4°C for up to two month
without any loss of sensitivity or specificity. Our results confirm that nanoparticles based on plan
viruses can be exploit in order to improve SjS diagnosis and could also be developed for the diagnosis
of other diseases.
E. Tinazzi, M. Merlin, C. Bason, R. Beri, R. Zampieri, C. Lico, E. Bartolini, A, Puccetti, C. Lunardi, M. Pezzotti, L. Avesani (2015).
Plant-Derived Chimeric Virus Particles for the Diagnosis of Primary Sjogren Syndrome. Frontiers in Plant Science. doi:
10.3389/fpls.2015.01080.
Session 4
Short talk - Poster 4.3
Spaying with the aid of plant-made vaccines
Masloboy A., Unkel K., Broer I. and Huckauf J.
University of Rostock, Faculty of Agricultural and Environment, Department of Agrobiotechnology,
Rostock, Germany
Spaying animals is usually realized by surgical sterilization or hormone therapy, though both are
accompanied by negative side effects1. Vaccine-based contraception using zona pellucida proteins is a
promising alternative2. This method of spaying successfully regulates wild animal population e.g. feral
horse population in USA, but the antigens are purified from pig oocytes3. In order to produce high
amounts of the vaccine we fused the coding regions of two porcine zona pellucida proteins to CTB and
Tetanus toxoid. Both fusion proteins were transiently and stably expressed in plants. The vaccine is
stable at room temperature in freeze-dried material or after purification and evoked an immune
response after parenteral injection in mice without an additional adjuvant.
1
ASA CS (2005) TYPES OF CONTRACEPTION. Wildlife Contraception: Issues, Methods, and Applications: 29.
Kirkpatrick JF, Lyda RO, Frank KM (2011) Contraceptive Vaccines for Wildlife: A Review. AMERICAN JOURNAL OF
REPRODUCTIVE IMMUNOLOGY 66: 40–50.
3
Bechert U et al. (2013) Effects of two porcine zona pellucida immunocontraceptive vaccines on ovarian activity in horses.
Jour. Wild. Mgmt. 77: 1386–1400.
2
Session 4
Short talk - Poster 4.4
Regulated degradation of plant-derived cyanophycin to release dipeptides as feed
additives
Nausch H., Ponndorf D. and Broer I.
University of Rostock, Faculty of Agricultural and Environmental Sciences, Department of
Agrobiotechnology and Risk Assessment for Bio- und Gene Technology, Rostock, Germany
The usage of plants as forage for pig and poultry farming is limited, since (semi-essential amino acids,
such as arginine (arg), are underrepresented (Ufaz and Galili, 2008). Conventional breeding failed to
increase the biosynthesis of free arg due to the regulation by feedback-inhibition and/or the immediate
metabolizing of elevated pools. In contrast to that, the amount of bound arg could be substantially
improved by introducing the biosynthesis of the cyanobacterial polymer cyanophycin (CP) into plant
plastids (Huhns et al., 2008; Huhns et al., 2009). CP is composed of arginine-aspartate dipeptides and
cannot be metabolized by eukaryotic proteases. Its degradation is exclusively restricted to bacterial
cyanophycinases (CPases), releasing the dipeptides (Law et al., 2009). These proved to be superior
compared to free amino acids in fostering animal growth and development (Klang et al., 2005; Broer,
2008; Yagasaki and Hashimoto, 2008; Sal lam and Steinbuchel, 2010).
In order to release the dipeptides in the gastrointestinal tract of animals, CPases need to be codelivered with CP-containing plant feed. Hence, we expressed an intra- (CphBTe) (Richter et al., 1999)
and an extracellular (CphEal) (Sallam and Steinbuchel, 2008) CPase variant in the cytosol of plants. Due
to this spatial separation, plastidic CP should be unaffected by the enzymes as long as the cells are
intact, but degraded during animal feeding, when chloroplasts are decomposed.
CphB was instable in the cytosol and needed to be stabilized by fusion partners such as GFP. Moreover,
even though CphB proved to be active in crude plant extracts, it did not degrade the polymer when
transiently expressed in CP-producing plants after disintegrating the plant material. In contrast to that
CphE was not affected by endogenous proteases, accumulated in significantly higher amounts, showed
a higher enzyme activity and degraded CP in homogenized CP containing tissue.
In first feeding experiment with mice, enhanced levels of arginine-aspartate dipeptides were found in
the blood serum when CP and CPase were added to the feed.
Broer, S. (2008). Physiol Rev 88, 249-286.
Huhns, M. et al. (2009). Plant Biotechnol J 7, 883-898.
Huhns, M. et al. (2008). Plant Biotechnol J 6, 321-336.
Klang, J.E. et al. (2005). J Anim Sci 83, 172-181.
Law, A.M. et al. (2009). J Mol Biol 392, 393-404.
Richter, R. et al. (1999). Eur J Biochem 263, 163-169.
Sallam, A. and Steinbuchel, A. (2008). Appl Environ Microbiol 74, 3434-3443.
Sallam, A. and Steinbuchel, A. (2010). Appl Microbiol Biotechnol 87, 815-828.
Ufaz, S. and Galili, G. (2008). Plant Physiol 147, 954-961.
Yagasaki, M. and Hashimoto, S.-I. (2008). Appl Microbiol Biotechnol 81, 13-22.
Session 4
Short talk - Poster 4.5
Engineering chimeric antibodies aimed for passive mucosal immunization against
HRSV
Bakshi S.1,2, Juarez P.1,2, Palaci J.1,2, Virdi V.1,2, Schepens B.3,4, Saelens X.3,4 and Depicker
A.1,2
1Department
of Plant System Biology, VIB, Gent, Belgium; 2Department of Plant Biotechnology and
Bioinformatics, Ghent University, Gent, Belgium; 3Medical Biotechnology Center, VIB, Gent, Belgium;
4Department of Biomedical Molecular Biology, Ghent University, Gent, Belgium
Human Respiratory Syncytial Virus (HRSV) is the leading cause of acute lower respiratory tract infection
in infants and frequently causes severe disease in the elderly. There is no licensed HRSV vaccine. As an
alternative, the prophylactic treatment of infants that are at risk for developing severe disease
following HRSV infection, with an HRSV fusion protein targeting monoclonal antibody (mAb) is often
recommended. However, the high cost that is associated with the current mammalian cell-based mAb
manufacturing systems hampers the broad implementation of this therapy. Also, it is possible that IgA
type antibodies against HRSV may contribute to protection. Therefore, we aimed to compare the
effectiveness of IgG with secretory IgA based passive immunization against HRSV. In this context, we
choose for the transient expression platform in plants that was shown to permit rapid small scale
production of IgG and IgA based antibody versions.
In this study, we have genetically fused three different single domain antibodies that are specific for
the HRSV fusion protein F [1] to the fragment crystallizable part (Fc) of different murine and human
monomeric IgA and IgG antibodies. In order to obtain all different permutations we took advantage of
the GoldenBraid2.0 cloning system [2]. Since secretory IgA are the predominant antibodies in mucosal
surfaces, they might be more effective than monomeric IgA and IgG in virus neutralization; therefore,
IgA based antibodies will also be tested in their dimeric and secretory forms. A total of 15 different
versions of these chimeric antibodies against HRSV have been engineered and produced in Nicotiana
benthamiana via Agrobacterium tumefaciens – mediated transient expression. Results on the quality,
quantity and HRSV neutralization effectiveness of the produced antibodies in clarified extracts after
transient expression in N. benthamiana leaves will be presented.
1
Schepens, B., et al. (2011). "Nanobodies(R) specific for respiratory syncytial virus fusion protein protect against infection
by inhibition of fusion." J Infect Dis 204(11): 1692-1701.
2
Sarrion-Perdigones, A., et al. (2011). "GoldenBraid: an iterative cloning system for standardized assembly of reusable
genetic modules." PLoS ONE 6(7): e21622.
Session 4
Poster 4.6
Stability evaluation of orally given plant-made antibodies by faecal analysis
Palaci J.1,2, Virdi V.1,2 and Depicker A.1,2
1Department
of Plant Systems Biology, VIB, Gent, Belgium; 2Department of Plant Biotechnology and
Bioinformatics, Ghent University, Gent, Belgium
Because of its numerous advantages, the oral delivery of therapeutic proteins is being explored for
several applications. In this context, production of therapeutic proteins in edible plant tissues is a first
step. While progress has been made in ways to improve production of the recombinant proteins in
plant tissues, there is a lack of information about the effects of gut transit on the target molecule. In
our lab we focused on the production of antibodies in Arabidopsis seeds and investigated the stability
and interactions of plant-made recombinant antibodies with their target during gut transit. More
specifically, the antibodies that were applied to confer protection against post-weaning diarrhea (PWD)
disease in an in vivo challenge experiment in piglets1, were quantified by analyzing the faeces of treated
animals. In the experiment, daily collected faecal samples were processed and the presence of
functional recombinant secretory IgA (SIgA) was evaluated by ELISA. Although the analysis of faecal
samples can be technically challenging, for instance, due to the interferences in the results generated
by competing molecules or due to the presence of proteases, it is a convenient non-invasive method
to prove the integrity and functionality of orally fed recombinant secretory IgA after gut transit.
Moreover, in our experiment, a daily follow-up of the antibody shedding could be performed not only
showing that the antibodies were functional after gut transit, but also suggesting that the antibody
dose delivered to the piglets may be higher than the dose required for their protection against PWD.
1
Virdi, V., et al., Orally fed seeds producing designer IgAs protect weaned piglets against enterotoxigenic Escherichia coli
infection. Proceedings of the National Academy of Sciences of the United States of America, 2013. 110(29), 11809-11814.
Session 4
Poster 4.7
Transient expression of camelid VHH secretory IgA against Clostridium difficile toxin A
Saberianfar R.1, Henry K.2, Hussack G.2 and Menassa R.1
1Agriculture
and Agri-Food Canada, London, Canada; 2National Research Council Canada, Ottawa,
Canada
Clostridium difficile is a gastrointestinal (GI) pathogen that causes C. difficile-associated disease (CDAD)
in humans and animals. Symptoms of CDAD range from mild diarrhea to life-threatening
pseudomembranous colitis, bowel perforation, sepsis and even death, with an estimated $3.2 billion
annual associated health care cost in the US (2012). C. difficile’s virulence factors include toxins A (TcdA)
and B (TcdB), surface layer proteins (SLPs), cell wall proteins and flagellar components. Toxins A and B
are the major therapeutic targets. Currently, antibiotics are used to treat CDAD, which alter the
microflora found in the gut, and select for antibiotic resistant bacteria. An alternative therapeutic
strategy is to neutralize the toxins within the GI tract. Previous studies have shown potential advantages
of oral administration of immunoglobulins such as bovine and human IgA, and chicken IgY against
human pathogens that secrete toxins into the GI lumen. The major limitations of this method are the
susceptibility of antibodies to proteolytic degradation, extreme pH conditions, and high levels of
proteases available in the GI tract.
Secretory IgA antibodies containing camelid heavy chain-only antibodies (VHHs) possess several
characteristics that make them suitable for oral immunotherapy, including stability, high solubility and
resistance to harsh gastrointestinal conditions. We used the previously described llama VHH 5.1 and
VHH 26.8 raised against C. difficile toxin A (TcdA antigen) and their corresponding engineered versions
VHH 5.1m and VHH 26.8m (with increased protease resistance and thermal stability) for production in
Nicotiana benthamiana leaves. To produce the full sIgA molecule, we co-expressed the VHH-IgA Fc with
the joining chain and the secretory component and are currently investigating the assembly of the full
sIgA complex in N. benthamiana leaves.
Session 4
Poster 4.8
Production and characterization of humanized RABV neutralizing monoclonal
antibodies
Ibrahim A., van Dolleweerd C., Banyard A.C., Seldon D., Drake P. and Ma J.
Institute for Infection and Immunity, St George's University of London, London, United Kingdom
Rabies is a central nervous system disease that is caused by a negative stranded virus of the genus
Lyssavirus of the Rhabdoviridae family. From the site of bite, the virus infects neurons and spreads in
trans-synaptic retrograde fashion finally causing lethal encephalitis. The WHO estimates that rabies
causes 55,000 human deaths per year. Universal pre-exposure vaccination is not deemed to be cost
effective and post-exposure prophylaxis (PEP) will continue to be the primary strategy for treatment.
PEP is comprised of both rabies vaccine (ß-propiolactone inactivated virus) to induce virus-neutralising
antibodies and rabies immunoglobulin (RIG) either of human or equine origin. However, due to
socioeconomic reasons less then 1% of all applications of PEP include administration of RIG. As an
alternative, the WHO identified a panel of murine monoclonal antibodies against the viral glycoprotein.
Previously, we generated and tested a plant-derived mouse-human chimaeric version of one of those
monoclonal antibodies, 62-71-3, and demonstrated that it bound to antigenic site I of the RABV
glycoprotein. In the present study, we produced six different humanised versions of this antibody in
Nicotiana benthamiana, and assessed them in virus neutralisation assays.
Session 4
Poster 4.9
Production of an Ebola therapeutic protein using a cell-pack system
Els J.H., Hitzeroth I. and Rybicki E.
Molecular Cell Biology, University of Cape Town, Rondebosch, South Africa
The Ebola virus is part of the Filoviridae family in the order Mononegavirales. The Ebola haemorrhagic
fever virus (EHFV) is a zoonotic virus that has a high fatality rate among those who contract it. There
are five different Ebola species which range widely in lethality, the highest case-fatality rate between
60-90% is the Zaire Ebola virus followed by the Sudan Ebola virus with fatalities from 40-60%; thus,
these are the strains focused on in this study.
The current Ebola epidemic underway in West Africa has revealed the difficulty in treating and
controlling this disease. Despite the fact that the Ebola virus has been studied extensively there are still
no preventative approaches or vaccines available and treatment of the disease revolves around
addressing the current symptoms of the patient. The only therapeutic available during the outbreak
was well publicised, an antibody treatment known as ZMapp, but very few doses were available.
In an attempt to resolve this problem the Biopharming Research Unit (BRU) has endeavoured to
produce a viral inhibitor (NB). It has been shown that antibody-dependant enhancement is required
for Ebola infection. The complement component 1 (C1q) binds the Fc region of the antibody while
interacting with cell surface molecules to mediate the antibody-dependant enhancement. The viral
inhibitor NB406 is highly effective in preventing the immune complex IgG binding to FcRs or C1q by
disrupting of the Fc-Fc interactions which is a factor in Ebola pathogenesis.
NB406 has been shown to inhibit infection of many viruses such as Ebola (Zaire and Sudan), Pandemic
influenza, Dengue and Hepatitis C virus (HCV) core antigen. Problems in producing this protein in E.coli
has prompted investigations into alternative expression systems such as plants.
The inhibitor will be linked to a maize storage protein γ-zein (Zera) to form the fusion protein Zera-NB.
Zera is known to form large protein bodies which allow for simple purification and production of high
levels of protein while it enhances immune mediated response. This project will investigate the ability
of plants and plant cell packs to produce a polypeptide viral inhibitor as a potential therapeutic agent
against the Ebola virus. Pilot production of the candidate therapeutic protein will be followed by
binding studies. Success in these will prompt the development of larger scale production, purification,
and testing in animal models.
Session 4
Poster 4.10
Production and stabilization of Cyanophycinase in Nicotiana benthamiana
Ponndorf D.1, Nausch H.1, Görs S.2, Mettges C.2 and Broer I.1
1University of Rostock, Faculty of Agriculture and Environmental Sciences-Agrobiotechnology, Rostock,
Germany; 2Leibnitz Institut for Farm Animal Biology, Nutritional Physiology, Dummerstorf, Germany
Arginine supplementation of feed, which has proven beneficial effects during the last decade (Wu et
al. 2014), can be optimized by the use of Arg-Asp dipeptides (Sallam and Steinbuchel 2010). These
dipeptides can be produced by the degradation of the bacterial storage peptide Cyanophycin (CP) by
the enzyme Cyanophycinase (Sallam et al. 2009). The production of Cyanopyhicanse in plants would
be an alternative to bacterial expression systems. Different variants of a shortened cphB coding region
(cB) were expressed transiently in Nicotiana benthamina plants. Optimization of translation by
adapting the codon usage of the bacterial coding region (cB-b) to plants (cB-s) and the insertion of the
amino acids Alanin-Serin-Serin (cB-sA) downstream of the start codon, led to an increase of protein
accumulation. Anyhow, Cyanophycinase without N-terminal modifications was highly instable in crude
plant extract. The fusion of the protein to GFP (pGFP -cB-s) and to the transit peptide of the small
subunit of Rubisco (S), resulting in pScB-s for protein production in the chloroplast, led to a high
expression of the protein but no transport to the chloroplast could be demonstrated for pScB-s. In
contrast to that, the combination of cBs-A and S (pScB-sA) led to a decrease of the protein level.
Interestingly, the amount of the native protein, carrying a 55 AA extension at the N-terminus (pcB-s-c),
was lower in Nicotiana benthamiana as well as its activity compared to the shortened version fused to
GFP and S respectively. RNA analysis indicates that differences in protein accumulation are caused by
differences in protein stability. First animal trials indicate that feeding of CP and Cyanophycinase leads
to an increase in β-Asp-Arg dipeptides, though the final evaluation of this experiment is ongoing.
Sallam, A., A. Kast, S. Przybilla, T. Meiswinkel and A. Steinbuchel (2009). "Biotechnological Process for Production of betaDipeptides from Cyanophycin on a Technical Scale and Its Optimization." Appl Environ Microbiol 75(1): 29-38.
Sallam, A. and A. Steinbuchel (2010). "Dipeptides in nutrition and therapy: cyanophycin-derived dipeptides as natural
alternatives and their biotechnological production." Applied Microbiology and Biotechnology 87(3): 815-828.
Wu, G. Y., F. W. Bazer, Z. L. Dai, D. F. Li, J. J. Wang and Z. L. Wu (2014). "Amino Acid Nutrition in Animals: Protein Synthesis
and Beyond." Annual Review of Animal Biosciences, Vol 2 2: 387-417.
Session 4
Poster 4.11
Production of recombinant human acid alpha glucosidase in transgenic rice cell
suspension culture
Jung J.-W. and Yang M.-S.
Bioactive Material Science, Department of Basic Science, Chonbuk National University, Jeonju-si,
Republic of Korea
Recombinant human acid alpha glucosidase (rhGAA) that is used for enzyme replacement therapy for
Pompe disease was produced from transgenic rice cell suspension culture. Pompe disease is fatal
genetic disorder because of deficiency of this enzyme. rhGAA is known as the one of the most
expensive therapeutic enzyme and patient are must treated in every two weeks to decrease lysosomal
in fibroblast. Recently, rice cell suspension culture is thought as an attractive expression system for
production of therapeutic recombinant proteins owing to several advantages such as safety of product,
cost effectiveness, scalability and desirable bio-activity. The target cDNA genes were amplified from
cDNA of human-placental total RNA and cloned into plant expression vector, then introduced into
embryogenic rice callus. Putative transgenic calli was selected on the medium containing hygromycin
-B as selection maker. The rice alpha-amylase 3D pro moter (Ra my3D), signal sequence and terminator
were used for the expression and secretion of rhGAA to high levels into culture medium under sucrosefree conditions. Genomic DNA PCR and Northern blot analysis were used to determine the integration
of target gene and expression of mRNA in the putative transgenic rice callus. Western blot analysis and
SDS-PAGE results indicated that putative 110kDa of GAA precursor form were produced and secreted
into culture medium. The amount of expressed rhGAA was maximized on 11days after sugar starvation
and measured as 37mg/L by ELISA. Alpha-glucosidase activity of rhGAA in cultured medium and
purified rhGAA was approximately 3.48U/mg and 3.313U/mg individually. The rhGAA was purified from
cultured medium by using affinity chromatography with 6histidine on their C-terminal.
In this study, we set up the expression of rhGAA in rice cell suspension culture and characterized it.
However, for the therapeutical purpose, the uptake of rhGAA into target cell requires specific Nglycosylation residue. So strategies for modification of rhGAA is we expressed is on further studying.
Session 4
Poster 4.12
Recombinant production of human blood clotting factor IX in the moss bioreactor
Top O.1,2, Schaaf A.3, Reski R.1,2,4 and Decker E.L.1
1Plant
Biotechnology, Faculty of Biology, University of Freiburg, Freiburg, Germany; 2Spemann
Graduate School of Biology and Medicine (SGBM), University of Freiburg, Freiburg, Germany;
3Greenovation Biotech GmbH, Freiburg, Germany; 4BIOSS Centre for Biological Signalling Studies,
Freiburg, Germany
Hemophilia B is a congenital bleeding disorder caused by a malfunction or deficiency of coagulation
factor IX (FIX), a vitamin K-dependent (VKD) serine protease. Current treatment is restricted to a
protein-replacement therapy. Approximately 1 billion units of FIX were consumed in 2012 (World
Federation of Hemophilia). However, still about 70-80% of the patients, mainly in developing countries,
received inadequate or no treatment because of unavailable and/or unaffordable FIX concentrates.
Our aim is to produce high levels of recombinant FIX in the moss bioreactor. Moss, Physcomitrella
patens, is an important model organism for evolutionary and functional genomics approaches and also
a well established molecular farming platform (Reski et al., 2015). The ease of gene targeting was
employed for precise genome engineering resulting in the elimination of plant-specific protein Nglycan residues. It has been used as a bioreactor for the production of complex recombinant proteins.
For the production of bioactive FIX in a plant-based system, some features have to be taken into
account. In addition to glyco-engineering, the enzyme gamma-glutamyl carboxylase (GGC) is crucial for
post-translational gamma-carboxylation of the first 12 glutamic acid residues within the gammacarboxyglutamic acid (Gla) rich domain of FIX. Moreover, paired basic amino acid cleaving enzyme
(PACE) is a serine protease which is responsible for the processing of the FIX pro-peptide. As both
enzymatic functions were not characterized in plants before, GGC and PACE coding sequences were
introduced into the moss genome. Recombinant FIX-producing moss lines will be generated and
characterized in terms of Gla content and glycosylation by mass spectrometric analyses. The activity of
the moss-derived FIX will be evaluated by activated partial thromboplastin time assay.
Reski, R., Parsons, J. and Decker, E. L. (2015), Moss-made pharmaceuticals: from bench to bedside. Plant Biotechnol J, 13:
1191–1198.
Session 4
Poster 4.13
Plant production of potential Salmonella Typhimurium diagnostic antibodies
Kopertekh L.1, T. Meyer T.3, Freyer C.1, Hust M.2 and Schiemann J.1
1Julius
Kuehn Institute, Federal Research Centre for Cultivated Plants (JKI), Institute for Biosafety in
Plant Biotechnology, Quedlinburg, Germany; 2Technische Universität Braunschweig, Institut für
Biochemie, Biotechnologie und Bioinformatik, Braunschweig, Germany; 3BioNTech AG, Mainz,
Germany
Salmonella infection is one of the most worldwide important food-borne diseases with ten millions
cases every year. The emergence of multi-drug resistant Salmonella Typhimurium strains requires an
enhanced surveillance of bacterial food contamination. Application of new specific antibodies can
improve currently available ELISA tests for S. Typhimurium diagnostic. The aim of this work was plant
production of antibody fragments that can be potentially applied in ELISA diagnostic assay.
The candidate scFv-TM43-E10, scFab-TM43-E10 and scFv-Fc-TM43-E10 antibody derivatives were
transiently expressed in N. benthamiana using PVX-based expression vector. The co-expression of γb
gene silencing suppressor from Poa Semilatent Virus significantly increased the accumulation level of
recombinant proteins. Beside the expression system plant, the antibody derivatives were also
produced in E. coli (scFv-TM43-E10, scFab-TM43-E10) and mammalian HEK293–6E (scFv-Fc-TM43-E10)
cells. The correct expression of the recombinant proteins was confirmed by Western blot. To assess
their functionality the purified proteins were subjected to ELISA test. The antigen binding was
confirmed for all recombinant antibodies. There were no significant difference between binding
capacity of antibody fragments produced in plants and microbial/mammalian cell-based systems.
The results of our experiments demonstrate that the plant-produced scFv-TM43-E10, scFab-TM43-E10
and scFv-Fc-TM43-E10 antibody fragments are functional and could be considered as possible
candidates for development of diagnostic ELISA kits for S. Typhimurium detection.
Session 4
Poster 4.14
In vivo study of two differently shaped plant virus nanoparticles reveals peculiar traits
in biodistribution and safety profiling
Lico C., Giardullo P., Mancuso M., Benvenuto E., Santi L. and Baschieri S.
Laboratory of Biotechnology, Agenzia Nazionale per le Nuove Tecnologie, l’Energia e lo Sviluppo
Economico Sostenibile (ENEA), Rome, Italy
Self-assembling plant virus nanoparticles (pVNPs) have started to be explored as nanometer-sized
“objects” of interest for application in biomedicine, especially for vaccine or drug delivery and imaging.
Plant VNPs are attractive because of the wide diversity of symmetries and dimensions, easy
chemical/biological engineering, easy and rapid production in plants, and may be ideal in terms of
biocompatibility and biodegradability.
During the last years, Tomato bushy stunt virus (TBSV) and Potato virus X (PVX) have been extensively
characterized as ideal, highly ordered, multivalent scaffolds to be used for delivery purpose of peptide
or molecules of biopharmaceutical interest. TBSV is icosahedral with single-stranded positive-sense
(ss(+)) RNA genome embedded in a capsid of about 30 nm in diameter made of 180 identical coat
protein (CP) units, while PVX has a filamentous flexible structure of about 500 nm in length and 13 nm
in diameter, made of a ss(+) RNA wrapped in approximately 1300 units of a single CP. We have
established that TBSV can be useful for the encapsulation of small molecules and the display of
polypeptides, and that genetically-engineered PVX chimeric particles are able to induce the activation
of both antibody and cell-mediated immune responses without the need of adjuvant co-delivery.
Recently, we started to define the safety profile of both viruses, demonstrating, by in vitro and in vivo
assays, they are neither toxic nor teratogenic.
To get a more complete understanding of the behaviour of these nanoparticles in vivo, we report the
results of a study aimed to define the biodistribution of unlabeled, unpegylated, underivatized TBSV
and PVX using immunohistochemistry and Enzyme Linked ImmunoSorbent Assay for detection. The
results revealed that the two pVNPs do not induce major alterations in the cellular and tissue
architectures and have different behaviours in terms of persistence in the blood stream and tissue
localization. Moreover, only PVX associates to erythrocytes. The reported data add new important
information about the in vivo behaviour of these nano-objects and demonstrate that these pVNPs are
endowed with different and peculiar properties. Moreover, this study sets a solid ground reference for
future testing of functionalized pVNPs displaying peptides or active moieties on their external surface,
but also on the inner, designed for biomedical applications.
Session 4
Poster 4.15
A Plant-Produced Bacteriophage Tailspike Protein for the Control of Salmonella
Miletic S.1,2, Simpson D.J.3, Szymanski C.M.3, Deyholos M.K.4 and Menassa R.1,2,*
1Southern
Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London,
Canada;
of Biology, University of Western Ontario, London, Canada; 3Alberta Glycomics
Centre and Department of Biological Sciences, University of Alberta, Edmonton, Canada; 4Department
of Biology, University of British Columbia, Kelowna, Canada
2Department
The receptor binding domain of the tailspike protein Gp9 from the P22 bacteriophage was recently
shown to reduce Salmonella colonization in the chicken gut. In this study, we transiently expressed the
receptor binding domain of the Gp9 tailspike protein in Nicotiana benthamiana, and targeted it to the
endoplasmic reticulum (ER) or to the chloroplasts. Gp9 was also fused to either an elastin-like
polypeptide (ELP) or I tag, which were previously described to improve accumulation levels of
recombinant proteins. The highest levels of recombinant protein accumulation occurred when unfused
Gp9 was targeted to the ER. Lower levels of chloroplast-targeted Gp9 were also detected. ELP-fused
Gp9 was purified and demonstrated to bind to Salmonella enterica serovar Typhimurium in vitro. Upon
oral administration of lyophilized leaves expressing Gp9-ELP to newly hatched chickens, we found that
this tailspike protein has the potential to be used as a therapeutic to control Salmonella contamination
in chickens.
Miletic S, Simpson DJ, Szymanski CM, Deyholos MK and Menassa R (2016) A Plant-Produced Bacteriophage Tailspike Protein
for the Control of Salmonella. Front. Plant Sci. 6:1221. doi: 10.3389/fpls.2015.01221
Session 5
Boosting expression levels
and optimizing processing
Session 5
Invited speaker
The extracellular protease complement of agroinfiltrated Nicotiana benthamiana
Grosse-Holz F., Madeira L., Sueldo D., Chandrasekar B., Hong T.N. and van der Hoorn
R.A.L.
The Plant Chemetics laboratory, Department of Plant Sciences, University of Oxford, Oxford, United
Kingdom
Agroinfiltration of Nicotiana benthamiana is frequently used to transiently express and study proteins
in plants. Besides being a model in plant science, it is also increasingly used as a protein production
platform in Molecular Pharming, because if its flexible, quick expression of (glyco)proteins. Some
proteins are efficiently expressed, accumulating up to 50% of the leaf proteome, but the majority of
the recombinant proteins accumulate to low levels, show truncated products, or declining
accumulation levels, indicating that degradation by endogenous proteases is a major obstacle in the
study of plant proteins and production of recombinant proteins. Here, we aim to identify the proteases
and inhibitors that accumulate in the apoplast of agroinfiltrated leaves. We have analysed leaves that
were infiltrated by disarmed Agrobacterium tumefaciens strains with and without carrying a T-DNA
producing silencing inhibitor P19, and buffer controls at various time points. Transcript, protein, and
activity levels were determined by RNAseq, apoplast proteomics and protease activity profiling,
respectively. We detect 192 annotated proteases and inhibitors in the apoplast, mostly accumulating
at late stages upon agroinfiltration. We detected 11 active papain-like Cys proteases, 7 subtilases, 10
Ser carboxypeptidases, but also vacuolar processing enzymes and various subunits of the proteasome.
Protomap analysis also indicates that numerous processing events in the apoplast. This study reveals
a complex proteolytic network in the apoplast of agroinfiltrated leaves.
Session 5
Invited speaker
Utilizing endogenous and ectopic protein storage organelles to boost recombinant
protein performance
Hofbauer A.1, Tschofen M.1, Arcalis E.1, Melnik S.1, Phan H.T.2, Conrad U.2 and Stöger E.1
1Department
of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences,
Vienna, Austria; 2Department of Molecular Genetics, Leibniz Institute of Plant Genetics and Crop Plant
Research (IPK), Gatersleben, Germany
Naturally occurring storage proteins such as zeins are versatile fusion partners for recombinant
proteins because they induce the formation of ectopic storage organelles known as protein bodies
(PBs) where the proteins are bioencapsulated and stabilized by intermolecular interactions and the
formation of disulfide bonds. Endogenous PBs are derived from the endoplasmic reticulum (ER) of seed
endosperm cells. However, the biogenesis and budding of ectopic PBs does not require seed- or ERspecific factors.
Being water-insoluble polymers, isolated cereal prolamins such as zeins have been widely used to
formulate carrier particles for the delivery of therapeutic molecules. We used an alternative strategy
combining the production and encapsulation of a recombinant model vaccine antigen in planta. A
fusion construct comprising the ectodomain of hemagglutinin subtype 5 and the N-terminal part of γzein was generated to incorporate the recombinant antigen into newly formed zein bodies. The
chimeric protein was transiently produced in tobacco leaves, and H5-containing protein bodies (PBs)
were used to immunize mice. An immune response was achieved in all mice treated with H5-zein, even
at low doses. The fusion to zein markedly enhanced the IgG response compared the soluble H5 control,
and the effect was similar to a commercial adjuvant. The co-administration of adjuvants with the H5zein bodies did not enhance the immune response any further, suggesting that the zein portion itself
mediates an adjuvant effect. While the zein portion used to induce protein body formation was only
weakly immunogenic, our results indicate that zein-induced protein bodies are promising production
and delivery vehicles for subunit vaccines.
Session 5
Invited speaker
Plant-based production of bi-functional fusion proteins with hydrophobin fusion
library
Reuter L., Ritala A. and Joensuu J.J.
VTT Technical Research Centre of Finland LTD, Espoo, Finland
Hydrophobins (HFB) are small globular proteins from filamentous fungi. In fungi HFBs have several
biological functions. While the secreted HFBs decrease the surface tension, the HFB-coating is
functioning as a fungal rain jacket on spores and hyphae to decrease wettability and to support
penetration at air-water interfaces. HFB-coating also serves as camouflage and adhesion agent for the
fungus. These interesting biological functions of HFBs have inspired multitude potential uses in
biotechnology from structure enhancing food additives to coating of sensors and nanoparticles.
Combining the properties of HFBs with activities of other fusion protein partners opens avenue for new
applications. However, the peculiar fold of hydrophobins (surface-exposed hydrophobic patch and four
intramolecular disulphide bridges) have made their heterologous expression challenging.
We have previously shown that plants can provide an excellent platform for manufacturing of HFB
fusion proteins. HFB-fusion strategy has enhanced the expression level of many fusion partners.
Furthermore plants contain only few native hydrophobic proteins enabling very efficient purification
through surfactant based aqueous two phase separation (ATPS) system.
Until now, the HFB fusion technology has relied solely on Trichoderma reesei HFBI leaving the large
diversity of other HFBs unexplored. Here we report plant-based expression of several novel HFB tags.
The amphipathic properties, contributed to the fusion protein by the HFB tag, were evaluated by
testing the performance of the fusions in ATPS. The established library of HFB tags was further used to
engineer bi-functional fusion proteins with human transferrin, human interleukin-22 and
Staphylococcus aureus Protein A. The target proteins were expressed in transient Nicotiana
benthamiana system as well as in stably transformed tobacco BY-2 cells.
Session 5
Invited speaker
Heat precipitation of tobacco host cell proteins facilitates target protein recovery and
purification and can be implemented into an automated large-scale manufacturing
process
Menzel S., Fischer R. and Buyel J. F.
Fraunhofer Institute for Molecular Biology and Applied Ecology IME
Chromatographic techniques are most frequently used for the purification of biopharmaceutical
proteins. However, identifying an effective capture step can be a challenging task for non-antibody
target proteins, especially if these are expressed at low levels or contaminated by a large number of
host cell proteins (HCPs) as it is often the case for plant-derived products. In such cases, several HCPs
can bind to the capture resin and reduce the effective binding capacity resulting in the necessity for
large column dimensions and increasing downstream processing (DSP) costs. A selective removal of
HCPs prior to the initial capture step may help to circumvent this problem and reduce costs in DSP.
Here we present how a heat precipitation step can remove more than 90% of tobacco HCPs and
increase the product stability, i.e. prevent proteolytic degradation. We have optimized the procedure
for three multi-domain malaria vaccine candidate proteins and a fluorescent model protein using a
design-of-experiments (DoE) strategy. In this context, we compare four different variants of heat
precipitation (blanching, hot extraction, heat treatment of extract in a vessel or heat exchanger) along
with several process parameters (incubation time and temperature, various buffer conditions) in terms
of product purity, recovery and compatibility with large scale manufacturing. We also highlight the
impact of heat precipitation on subsequent process steps including the capacity and costs of
subsequent filtration and chromatographic purification. Finally, we present how the best performing
heat precipitation step is automated and currently being implemented into a GMP-compliant
production process for malaria vaccine candidates transiently expressed in Nicotiana benthamiana
with a weekly biomass output of 100 kg.
Session 5
Short talk - Poster 5.1
The multiple uses of tomato cystatin SlCYS8 as a co-expression partner in plant protein
biofactories
Jutras P.V.1, Robert S.1, Goulet M.-C.1, Sainsbury F.1,2 and Michaud D.1
1Département
de Phytologie – Centre de Recherche et d’Innovation sur les Végétaux, Université Laval,
Québec, QC, Canada; 2Centre for Biomolecular Engineering, Australian Institute for Bioengineering and
Nanotechnology, University of Queensland, Brisbane, QLD, Australia
We here discuss the potential of tomato cystatin SlCYS8 as a useful accessory protein for the
stabilization, high yield production and downstream purification of clinically useful recombinant
proteins expressed in plant protein biofactories. The strong inhibitory effects of SlCYS8 against
endogenous Cys proteases in plant tissues make this protein attractive as a co-expression partner for
the in situ stabilization of protease-susceptible proteins, especially during their migration along the cell
secretory pathway. The high translation rates of SlCYS8 transcripts in plant cells make this protein also
useful as an N-terminal translational fusion partner to boost the production of recombinant proteins
recovered at lower levels when expressed alone. Finally, the well defined tertiary structure and intrinsic
stability of SlCYS8 make this protein a convenient and robust fusion template to poly-His tags for the
efficient, single-step IMAC puri fication of recombinant proteins following extraction. These different
applications of SlCYS8 in plant systems are here illustrated with mammalian and human proteins
transiently expressed in leaves of the widely used expression host Nicotiana benthamiana.
Session 5
Short talk - Poster 5.2
Dicer-Like 2/4 suppressed tobacco plants for high expression of recombinant proteins
Matsuo K. and Matsunura T.
National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Japan
Many attempts have been made to improve the expression level of recombinant proteins in plants.
One of the effective approaches, repression of RNA silencing seemed directly linked to the
enhancement of the recombinant protein production.
Many components are involved in post transcriptional gene silencing (PTGS). Dicer-like proteins (DCLs)
are one of the important factors because the DCLs produced small interference RNAs (siRNAs) to start
and maintain RNA silencing. To overcome RNA silencing systems, we have developed DCL2 and DCL4
repressed transgenic Nicotiana benthamiana plants (ΔD2 and ΔD4 plants) using RNA interference.
Furthermore, DCL2 and DCL4 genes double-repressed N. benthamiana plant was also produced
(ΔD2ΔD4 plant). In each transgenic plant, the amount of mRNAs derived from NbDCL2 and/or NbDCL4
genes was greatly decreased. However, significant morphological differences were not observed.
Green fluorescent protein (GFP) was transiently expressed in those transgenic plants. At 3 days after
infiltration (dpi), the high accumulation of GFP observed in the ΔD2ΔD4 plants was higher than those
of ΔD2, ΔD4 and wild type plants. Then acidic fibroblast growth factor (aFGF) was also transiently
expressed in the transgenic plants. The expression levels of aFGF mRNA and protein in ΔD2 and ΔD4
plants were nearly identical to those of WT plants. In contrast, the expression level of aFGF mRNA and
protein in ΔD2ΔD4 plants was significantly higher than those of ΔD2, ΔD4 and WT plants.
The ΔD2ΔD4 plants can express large amounts of recombinant proteins. Thus, ΔD2ΔD4 plants would
be useful for recombinant protein production.
This study was supported by a Grant-in-Aid from Ministry of Economy, Trade and Industry (METI) of Japan.
Session 5
Short talk - Poster 5.3
Codon use and mRNA structure analyses across kingdoms indicates selection on both
mRNA stability and translatability
Westerhof L.B., Sterken M.G., Wilbers R.H.P., Luijben L., van Raaij D.R., L. Snoek L.B.,
Bakker J. and Schots A.
Laboratory of Nematology, Plant Science Group, Wageningen University, Wageningen, The
Netherlands
To boost heterologous protein production the codon use of a gene of interest is often adapted to
reflect the expression host’s codon use in highly expressed genes (optimal codons). However, the
results obtained with this strategy are variable. A comparison between the overall codon use and the
codon use in highly expressed genes of several plant species revealed that optimal codons are not
always the codons of which the use is most increased with expression. Although the codon composition
of highly expressed genes differs between monocots and dicots, often the same codons are linked
strongest to expression (here after named expression codons). We used these conserved expression
codons to optimise the codon composition of three genes, which enhanced protein yield significantly
upon stable and transient expression in plants. Upon stable transformation both transcript levels and
protein yield per transcript increased. Next, we analysed whether this expression-linked codon bias
found in plants also extends to other kingdoms of life. Thereto, expression-linked codon use was
investigated in Escherichia coli (Bacteria), Saccharomyces cerevisiae (Fungi), Caenorhabditis elegans
(Animalia) and Mus musculus (Animalia) using more than 250 microarrays per species. We found that
a common codon use bias exits over all species. In addition, computational analyses of various mRNA
characteristics revealed a similar selection pressure across kingdoms that increases both stability and
translatability. Combining gene expression data with available protein abundance data showed that an
increased number of stem-loop transitions together with a reduction of stem size increases translation
efficiency. An algorithm was developed that combines the use of optimal and expression codons to
create an ideal mRNA structure for any given gene. This algorithm was again tested in plants and lead
to a significant increase in protein production. The spin off company TripleT Biosciences combines this
algorithm with all available knowledge on codon use and offers a codon optimization tool.
Session 5
Short talk - Poster 5.4
CMV-Agroinfection triggers plant defense mechanism suppressing gene expression
Fukuzawa N.1, Masuta C.2, Murota K.1, Itchoda N.1 and Matsumura T.1
1National
Institute of Advanced Industrial Science and Technology, Sapporo, Japan; 2Graduate School
of Agriculture, Hokkaido University, Sapporo, Japan
We previously developed the Cucumber mosaic virus (CMV) vector and have produced a wide variety
of protein using the CMV vector. To enable further improvement in expression levels of gene of interest
(GOI), we have developed the CMV-Agroinfection methods. Here, these methods were used to express
the Green Fluorescence Protein (GFP) in Nicotiana benthamiana plants, results show that the GFP in
plants inoculated with the CMV vector were systemically expressed, whereas the expression of GFP in
plants used for CMV-Agroinfection were observed in vein localization.
We predicted that the occurrence of vein localization of GFP expression was induced by post
transcriptional gene silencing (PTGS) because of transient accumulation of GFP mRNA by CMVAgroinfection. We attempted to overcome the PTGS using RNA silencing suppressers (RSS). Then both
HC-Pro transgenic (Tg) plants and 2b Tg plants were used for CMV-Agroinfection. The 2b Tg plants show
that GFP was detected in all parts of the leaves. However, the HC-Pro Tg plants show that the GFP was
again localized only in the veins. These results suggested that the PTGS was not involved in the GFP
vein localization.
On the other hand, we also observed that the accumulation levels of salicylic acid (SA) had markedly
increased in the plants which were inoculated with Agrobacterium using vacuum infiltration.
Consequently, the large accumulation of SA was presumed to be involved in the GFP vein localization.
Therefore, we produced the Tg plants expressing SA glucosyltransferase (SGT) which catalyzed the
conversion of SA to salicylic acid glucoside (SAG). When the SGT Tg plants were used for CMVAgroinfection, we confirmed that the GFP vein localization was breaking up systemically.
These results indicated that the localized expression of GFP with CMV-Agroinfection is caused by the
large accumulation of SA which was triggered by vacuum infiltration of Agrobacterium, supported by
the spreading of GFP which was induced in both SGT Tg plants and 2b Tg plants. We speculate that 2b
may function as not only a silencing suppressor but also revealing correlation with the response of SA.
This work was supported in part by grants from the Ministry of Economy Trade and Industry in Japan.
Session 5
Short talk - Poster 5.5
Production of Human Nerve Growth Factor in Nicotiana benthamiana
Ofoghi H.1, Zangi M.1, Amini-Bayat Z.1 and Ehsani P.2
1Biotechnology
Department, IROST. Tehran, Iran; 2Molecular Biology Unit, Pasteur Institute of Iran,
Tehran, Iran
Plant-based expression systems proved to be an efficient approach for the production of complex
heterologous proteins). Compared to traditional expression systems based on bacterial and
mammalian cells, they generally offer major advantages such as low costs, especially on large-scale
production and absence of contaminants usually derived from animal cell media. β -NGF (beta-Nerve
growth factor) is a neurotrophic factor important for the development and maintenance of the sensory
neurons. Β -NGF is a target-derived secreted homo-dimeric protein which contains 120-amino acid
polypeptides.
In this study, the gene encoding human NGF protein containing 811 base pair and consists of Pre, Pro
and Mature sequences which was codon optimized and cloned in expression binary vector pGR107
under control of the cauliflower mosaic virus 35S promoter. Human β-NGF was co-expressed by P19
silencing suppressor. The P19 protein was cloned into vector pCAMBIA1304, which contained the
Cauliflower mosaic virus (CaMV) 35S promoter and 5′UTR together with the terminator region of the
nopaline synthase gene of Agrobacterium.
Expression vectors containing the desired genes were transformed to Agrobacterium tumefaciens
strain GV3101 by electroporation. Accuracy of expression cassette was confirmed by restriction
enzyme analysis, PCR and colony PCR. By infiltration of recombinant Agrobacterium cells carrying
human β-NGF gene into Nicotiana benthamiana leaves, transient expression assays were performed
within 3, 4 and 5 DPI (days after infiltration). Our results showed that transient expression of the
recombinant NGF protein yielded about 1.5% of TSP. Co-expression with the p19 suppressor also
significantly increased the β-NGF expression level approximately 5-fold (to about 6-7% of TSP) after
just 4 DPI. A 27 kDa band was detected on SDS-PAGE and confirmed using Western blotting presenting
the precursor form of proNGF and 15 kDa band of Mature NGF was determined in apoplast of Nicotiana
benthamiana leaves. We conclude that the genetically modified plants are capable of producing human
rh NGF with a molecular weight and structure similar to human NGF.
Session 5
Short talk - Poster 5.6
TrichoPharming and OrtoPharming by Sequentia: trichomes as natural factories and
bioinformatics tools to ease plant molecular pharming research
Hernández L.M., Sanseverino W. and Aiese Cigliano R.
Sequentia Biotech, Barcelona, Spain
TrichoPharming, our biofarming research line, use trichomes as natural bio-factories for producing
some interesting compounds to treat malaria and cancer. Trichomes are specialized epidermal
protrusions on the surfaces of leaves and other aerial organs of most of the plants. Trichomes defend
plants against insect herbivores, virus, UV light and excessive water loss. But interestingly, glandular
trichomes are also able to synthesize, store and sometimes secrete large amounts of specialized
metabolites, which could have significant commercial value as pharmaceuticals. Furthermore, the
isolated compartmentalization in trichomes allows the production of potentially detrimental
compounds without affecting plant development. Therefore, TrichoPharming may represent an
unprecedented opportunity to manufacture affordable modern medicines and make these available at
a global scale, particularly in underdeveloped countries where access to medicine s has historically
been limited. We have focused in the use of different medicinal plants, starting with Artemisia annua,
as a cost-effective and valuable system to produce huge amounts of bioactive compounds with anticancer and anti-malarial properties. Artemisinin, a sesquiterpene lactone, is a natural substance only
naturally produced in Artemisia annua plant that is very powerful and efficacious molecule to treat
malaria as well as diverse types of cancer. Indeed, the discovery of this substance has been recently
awarded with the Nobel Prize in Medicine 2015.
Additionally, Sequentia have also started to develop OrtoPharming. OrtoPharming is a bioinformatics
tool which goal is to ease wet-lab work for scientists involved in biofarming research areas.
OrtoPharming will analyze and compare different key enzymes and precursors of interesting plant
molecular pharming pathways in order to look for their possible orthologous in other plant species.
This potent tool will facilitate the improvement of the efficiency for producing valuable products in
plants.
Session 5
Poster 5.7
Adverse impact of silencing suppressor protein 19 on production of Tissue
Plasminogen Activafor in Nicotiana species
Amiri M., Jalali-Javaran M., Ehsani P. and Haddad R
Department of Plant Breeding & Biotechnology, Faculty of Agriculture, Tarbiat Modares University,
Tehran, Iran
P19 protein of Tomato Bushy Stunt Virus (TBSV) suppresses post-transcriptional gene silencing (PTGS)
process and allows rapid and high expression of the gene of interest utilizing transient expression.
However, the influence of the P19 on expression should be assayed in different plant species. Tissue
plasminogen activator (tPA) gene was transferred to pTRAc-ERH binary vector. The expression level
was determined in both co-agroinfiltrated pTRAc-tPA-ERH with and without agrobacteria carrying
pCambia-P19. ELISA results showed that concentration of tPA protein in the absence of P19 was 0.65%
and 0.74% of total soluble protein, versus, 0.141% and 1.36% in presence of P19 in Nicotiana
benthamiana and N. tabacum, respectively. In N. tabacum, co-agroinjection of P19 has increased the
production of tPA two-fold higher and confirmed the synergistic effect of P19 on tPA expression.
However, in N. benthamiana, the presence of P19 has decreased tPA production five-fold lower. N.
bentamiana is well known for less production of proteolytic enzymes especially in endoplasmic
reticulum than other member of genus. In spite that, Western blot analysis has shown that retention
of tPA in endoplasmic reticulum using KDEL peptide could not prohibit the degradation of tPA in
N.benthamiana. Therefore, other transgene specific mechanisms must further be investigated to
improve the production of heterologous protein using gene silencing suppressors. In conclusion, our
results showed that the expression level of tPA ameliorated using virus coded gene silencing
suppressor in N. tabacum.
Session 5
Poster 5.8
Reducing proteolytic activity in Medicago cell cultures for optimized production of
recombinant proteins
Santos R.B.1, Chandrasekar B.2, van der Hoorn R.2, Schiermeyer A.3 and Abranches R.1
1Instituto
de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras,
Portugal;
of Plant Sciences, University of Oxford, Oxford, United Kingdom; 3Fraunhofer
Institute for Molecular Biology and Applied Ecology (IME), Aachen, Germany
2Department
Medicago truncatula is an established model for legume biology studies. It has a small, diploid genome
that is fully sequenced and it is easy to manipulate and transform. Our lab has been working with
Medicago exploring this platform as a plant production platform for biopharmaceuticals. We have
successfully produced fungal and human recombinant proteins in both plant and cell suspensions.
However, some challenges remain so that plant systems become truly competitive, including the
degradation of protein products during production and downstream processing stages, preventing low
yields that are generally obtained.
In Medicago truncatula genome, there are more than 400 known and putative protease genes,
however, the proteolytic content of Medicago cell suspensions is not known. We started by evaluating
the proteolytic activities that hamper the successful production and purification of selected target
proteins. The use of selective protease inhibitors that blocked proteolysis enabled us to classify and
identify the proteases classes responsible for the degradation of the target proteins. Commercial and
in-house made inhibitors were used and inhibition of degradation was achieved for cysteine, aspartyl
and metalloproteases. By mass spectrometry analysis, we concluded that Medicago suspension cells
contain cysteine, serine, aspartyl and metalloproteases. Activity based protein profiling (ABPP) probes
were used to classify specific proteases responsible for degradation and proteolytic activities were
determined in cell extracts and spent culture medium. N-terminal sequencing of target protein
cleavage products was performed in order to understand which protease is responsible for each
particular cleavage.
Knowledge gained about the proteolytic activities will be applied to engineer cell lines with reduced
endogenous protease activities by co-expression of selective protease inhibitors together with the
target protein.
Session 5
Poster 5.9
Quantitative evaluation of constitutive and seed specific promoter activity in
immature soybean cotyledons
Ploužková T., van Haesendonck G., Navrátil O., Plchová H., Jindřichová B., Čeřovská N.
and Moravec T.
Laboratory of Virology, Institute of Experimental Botany, Prague; Department of Biochemistry, Charles
University, Prague
Seed of leguminous crops and particularly that soybean is a very important source of dietary proteins.
Its natural ability to express and store large amounts of proteins makes it especially suitable for
expression of high value proteins such as vaccines or antibodies. However stable transformation of
legumes is technically challenging and takes 9 to 12 months. With the onset of synthetic biology
approaches and combinational cloning there is a strong need to quantitatively evaluate and optimize
large number of genetic constructs in transient expression assay. Here we report a quantitative
comparison of several constitutive and tissue specific promoters in transient assay in developing
soybean seeds performed both in vitro and in vivo.
Session 5
Poster 5.10
The development Pepper mild mottle virus expression vector
Bonnet R.M.V., Duarte M.A., Junqueira, B.R.T. and Nagata T.
Department of Cellular Biology, University of Brasilia, Brazil
The plant virus vector is one of strategic tool for the heterologous protein expression in plant. Some
virus groups were used for this purpose as tobamovirus, potexvirus, comovirus, cucumovirus and so
on. For the natural capacity of high amount coat protein accumulation, tobamovirus and potexvirus
were always leading this technology. In tobamovirus, Tobacco mosaic virus or Tomato mosaic virus are
often used, but other tobamovirus vectors are not well-developed. In this study, Pepper mild mottle
virus (PMMoV) is used for the development of virus vector for protein expression. At first, the
infectious clone of PMMoV was constructed in the small binary vector pJL89 flanking to 35S promoter
of Cauliflower mosaic virus and Hepatitis Delta virus ribozyme using Gibson Assembly technique. After
confirming the infectivity by agro-infiltration, a reporter gene of Green fluorescent protein (GFP) was
inserted to the infectious clone. The resulting construction showed infectivity and GFP signals when
agroinfiltrated. Here we developed the PMMoV-based vector for protein expression.
Session 5
Poster 5.11
Hairy roots as bioreactors for the production of antimicrobial immunotherapeutics for
astronauts
Cerasi M., Lico C., Catellani M., Massa S., Benvenuto E. and Capodicasa C.
ENEA, SSPT-BIOAG, Laboratory of Biotechnology. Research Center Casaccia, Rome, Italy
Diseases caused by opportunistic fungi, expecially in immunocompromised hosts, are in general
increasing and becoming a health emergency. To fight infectious disease, immunotherapy could be a
feasible alternative to replace or integrate the treatment of pathogen with classical chemical drugs.
We recently demonstrated that chimeric murine-human antibodies (a full IgG and a scFvFc format)
conferred significant antifungal protection in vivo against systemic and mucosal Candida albicans
infections (Capodicasa et al. 2011). These chimeric antibodies (Abs), recognising a polysaccharide
present in the cell wall of the majority of fungi, represent promising therapeutic candidates against a
broad spectrum of pathogens. In addition, these Abs were produced in plants that are now wellconsolidated platforms to produce biopharmaceuticals.
In the BIOxTREME project co-funded by ENEA and ASI (Italian Space Agency), one of the major goals is
the production of antimicrobial biomolecules, using roots as bioreactor, for therapeutical application
against infectious diseases contracted by astronauts. During space missions, in fact, the immune
system of the crew tends to be compromised by the stressful conditions of life to which it is subjected,
exposing astronauts to risks for their health such as that of contracting microbial infections. To this
aim, we expressed the antifungal scFvFc format (2G8 Ab) in “Hairy Roots” (HR) derived from Nicotiana
benthamiana and Solanum lycopersicum plants. After transformation of plants, HR clones expressing
higher level of 2G8 Ab were selected. The quantity and quality of 2G8 Ab in the best-expressor HR
clones identified have been evaluated by Western blot and by immunoenzimatic assays (ELISA).
In addition, having fused 2G8 Ab to a signal peptide that direct proteins outside the plant cell, we
verified the rhizosecretion of antifungal Ab in the root culture medium. However, the Ab is found in
the medium only partially and further improvements of secretion are needed. In a future perspective,
growth inhibition assays of C. albicans using both extract and secretion of HR will be developed to
verify their efficacy as ready-to-use antimicrobial immunotherapeutics.
Capodicasa C, Chiani P, Bromuro C, De Bernardis F, Catellani M, Palma AS, Liu Y, Feizi T, Cassone A, Benvenuto E,
Torosantucci A. Plant production of anti-β-glucan antibodies for immunotherapy of fungal infections in humans. Plant
Biotechnol J. 2011 Sep;9(7):776-87).
Session 5
Poster 5.12
Expression of BMP2-HYDROPHOBIN Fusion Protein in Tobacco Plant
Rahimifard P.1, Ofoghi H.2, Solouki M.1 and Ehsani P.3
1Plant
Biotechnology Department, University of Zabol, Zabol, Iran; 2Iranian Research Organization for
Science and Technology Biotechnology Department, Tehran, Iran; 3Molecular Biology Department,
Pasteur Institute of Iran, Tehran, Iran
There is a demand for pharmaceutical proteins produced in different prokaryotes and eukaryotic
systems. Bone Morphogenic Protein2 (BMP2) is an important factor for its major role in restoration
and construction of bone tissues. Studies have shown that the recombinant BMP2 produced in
eukaryotic host has a potential capability for pharmaceutical applications. However, production of
BMP2 in transgenic plants was not satisfactory because of low level of expression. Hydrophobin protein
originated from trichoderma reesei has shown to be effect on yield and stability of its partner fusion
protein when transiently expressed in Nicotiana Benthamiana. We have used Ptrakc-ERH plasmid for
expression of hydrophobin–BMP2 fusion protein with kozak and enterokinase (ent) sequences for
efficient BMP2 purification. In silico studies has shown that the hydrophobin-ent-BMP2 secondary
structure has best results for its primary and secondary structure of mRNA in addition to better
solubility and activity comparing to reversed order of two protein fusion. Immunochemical
experiments have shown that using P19 silencing suppressor has enhancing effect on expression of the
recombinant protein. In addition there were two types (mono and dimer) of BMP2 fusion protein were
expressed as 13 kDa and 26 kDa bands.
Friedrich A, Ripp R, Garnier N, Bettler E, Deléage G, Poch O, Moulinier L. Blast sampling for structural and functional
analyses. BMC Bioinformatics (2007); 8: 8-62.
Khalesi, M., Deckers, S.M., Gebruers, K., Vissers, L., Verachtert, H. and Derdelinckx, G. 2012. Hydrophobins: Exceptional
proteins for many applications in brewery environment and other bio-industries. Cerevisia, 37: 3–9.
Martin GJ, Boden SD, Marone MA, et al. Posterolateral intertransverse process spinal arthrodesis with rhBMP-2 in a
nonhuman primate: important lessons learned regarding dose, carrier, and safety. J Spinal Disord (1999); 12:179–86.
Zhang H, Migneco F, Lin C, Hollister S. J. Chemically-Conjugated Bone Morphogenetic Protein-2 on Three-Dimensional
Polycaprolactone Scaffolds Stimulates Osteogenic Activity in Bone Marrow Stromal Cells. Tissue Eng Part A. 2010
November; 16(11): 3441–3448.
Session 5
Poster 5.13
Versatile tools for in vivo detection of protease activity: Fluorophore Quencher probes
Fernández-Fernández A.1,2, Stael S.1,2,3,4, Willems P.1,2,3,4 and Van Breusegem F.1,2
1Department
of Plant Systems Biology, VIB, Gent, Belgium; 2Department of Plant Biotechnology and
Bioinformatics, Ghent University, Gent, Belgium; 3Department of Medical Protein Research, VIB, Gent,
Belgium; 4Department of Biochemistry, Ghent University, Gent, Belgium
Proteolysis is a universal process found in every organism and it can be executed by single proteases
or the proteasome complex. Despite the old notion that hydrolysis of peptide bonds is performed only
during catabolic degradation of proteins, specific proteolysis is crucial for certain signaling pathways.
Proteolysis is tightly spatiotemporally regulated, which hinders the understanding of protease
activation and activity in vivo, remaining as one of the biggest hurdles to unveil the mechanisms
governing proteolytic processes. We have developed ratiometric GFP-Q (rGFP-Q), a tool that allows
the detection of proteolytic activity in plants. rGFP-Q is based on the co-translational expression of a
ratiometric signal with a protease reporter using a 2A-like peptide. While the ratiometric tag allows to
determine the expression levels of our probe in different tissues, the proteolysis reporter reveals
protease activity. Our probe showed potential metacaspase activation in the root tip and growing root
hairs of Arabidopsis thaliana. The results obtained indicate that ratiometric GFP-Q is a promising tool
that can be easily tailored by researchers fitting their experimental conditions for other proteases.
Callahan, B.P. et al. (2010). Protease activation of split green fluorescent protein. Chembiochem 11: 2259–63.
Minskaia, E., et al. (2013). Optimisation of the foot-and-mouth disease virus 2A co-expression system for biomedical
applications. BMC Biotechnol. 13: 67.
Tsiatsiani, L., et al.(2012). Natural substrates of plant proteases: how can protease degradomics extend our knowledge?
Physiol. Plant. 145: 28–40.
Tsiatsiani, L., et al. (2013).The Arabidopsis metacaspase9 degradome. Plant Cell 25: 2831–47.
Session 5
Poster 5.14
An amiRNA-mediated silencing approach to reduce rhizosecreted recombinant
protein proteolysis in Arabidopsis thaliana.
Lallemand J., Périlleux C. and Tocquin P.
Laboratory of Plant Physiology, PhytoSYSTEMS, University of Liège, Liège, Belgium
Plant-based biopharmaceuticals have gained a lot of interest in the past decade due to their reduced
cost and relative safety compared to mammalian cell cultures. The first plant-made recombinant
proteins have been on the market for the past few years; however, the plant-based production systems
still need improvements to maximize their competitiveness against traditional production platforms.
Optimizing production hosts requires the identification and subsequent inhibition of the most active
endogenous peptidases, proteolysis being one of the main factors limiting yields.
The aim of our study was to identify root-secreted proteases of Arabidopsis thaliana involved in target
protein degradation (BSA) and inhibit them in vivo. Biochemical analyses identified serine proteases as
the main class responsible for BSA degradation. An RT-qPCR experiment led to the choice of the serine
protease gene SBT4.12 and its homologs as targets for an amiRNA-mediated silencing approach.
Arabidopsis amiRNA-expressing lines showed lower levels of expression for SBT4.12 and reduced
proteolytic activity in their rhizosecreted extracts. Crossing these lines with recombinant protein
producing lines could lead to an improved production platform for proteins of interest.
Session 5
Poster 5.15
Tomato hairy root cultures as a platform for the bioproduction of valuable molecules
Massa S.1, Bennici E.1, Villani M.1, Desiderio A.1, Koes R.2, Quattrocchio F.2 and
Benvenuto E.1
1ENEA
Italian National Agency for New Technologies, Energy and Sustainable Economic Development,
Rome, Italy; 2University of Amsterdam, Faculty of Science, Amsterdam, The Netherlands
SUMMARY:
The work takes in consideration the use of plant-based systems for the production of valuable
molecules able to tackle the health issues.
MAIN HYPOTHESIS, METHODOLOGY, MAIN RESULTS AND ADDED VALUE:
In the context of the BIOxTREME project, launched by ENEA and co-funded by ASI (Italian Space
Agency), in order to devise and formulate bioactive products counteracting detrimental effects of the
permanence in the confined environments of the long-term space missions, the optimization of the
production of natural anti-oxidants present in plants was pointed out using plant-based systems as
natural ‘bioreactors’ for the production of bioactive molecules like anti-oxidants.
We exploited an organ culture system, the “hairy root” axenic cultures (HRCs). HRCs represent a robust
platform that combines the benefits of cultivating plant systems with those typical of cell cultures
(sterility, ease of handling) for the bioproduction of valuable molecules.
In cooperation with the University of Amsterdam, a plant-expression vector harbouring a Myb-like
transcription factor from petunia (Petunia hybrida) fused to the reporter gene Green Fluorescent
Protein was used to constitutively express this transcription factor in plant samples. In petunia, the
cooperation between different transcription factors activates genes involved in the biosynthesis of
anthocyanins, valuable anti-oxidants responsible for the purple pigmentation of petals. We have
activated the same genetic switch in tomato by gene-transfer mediated by Agrobacterium rhizogenes
(strain A4RSII). Clonal HRCs were obtained from the cultivar Micro-Tom. HRCs lines having a strong
purple pigmentation were obtained and gene-transfer confirmed by PCR. High levels of anthocyanins
and flavonoids were determined by HPLC and mass spectrometry.
Session 6
Plant glycan-engineering
Session 6
Invited speaker
Using GlycoDelete for pharming of proteins without plant-specific N-glycan
modification
Santens F.
Medical Biotechnology Center, VIB, Gent, Belgium; Department of Biochemistry and Microbiology,
Ghent University, Gent, Belgium
N-glycosylation is one of the most widespread posttranslational protein modifications. This is also
reflected in the field of pharmaceutical protein production, where currently over 65% of the
biopharmaceuticals on the market carry N-glycans. The N-glycans are essential for the correct folding
of the protein, and often have a significant impact on the function of the protein, so in most cases it is
not possible to remove them. However, glycans tend to be very heterogeneous (e.g. in CHO cells,
approximately 3600 different N-glycoforms are possible) and additionally, cells from different species
modify N-glycans differently. Therefore we implemented our GlycoDelete system, which was
previously developed in mammalian cells, in plants. This enables the production of very homogeneous
glycoproteins in seeds without the plant specific N-glycan modifications.
Session 6
Invited speaker
O-glycan engineering in Nicotiana benthamiana
Strasser R.
Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences,
Vienna, Austria
Current mammalian cell-based expression systems for recombinant glycoproteins typically produce a
mixture of heterogeneous glycosylation variants that are neither identical to human glycans nor
suitable for the analysis of their biological function. In terms of glycosylation, plants offer certain
advantages compared to other organisms as the N-glycosylation pathway of plants can easily be
modified towards the generation of homogenous human-type N-glycans and a typical mammalian
mucin-type O-glycosylation pathway does not exists at all. In this project, we focus on the de novo
generation of structurally defined mucin-type O-glycan structures on recombinant glycoproteins
produced in plants. For the generation of desired O-glycans the missing mammalian biosynthetic
pathway is transiently expressed in Nicotiana benthamiana leaves. Co-expression of different human
glycoproteins like human IgA1, erythropoietin or mucin-1 with the mammalian O- glycosylation
machinery resulted in the production of different human-like mucin-type O-glycans. Our study
demonstrates that N. benthamiana are amenable to extensive O-glycosylation engineering and a
valuable platform for the production of recombinant human glycoproteins with defined N- and Oglycans for therapeutic use and structure-function studies.
Session 6
Invited speaker
Engineering the N- and O-glycosylation pathways in plants for mammalian-like
glycoprotein farming
Pedersen C.T.1, Thaysen-Andersen M.2, Lorentzen A.3, Packer N.H.2, Petersen B.L.4,
Roepstorff P.3, Clausen H.5, Stougaard J.1 and Dam S.1
1Centre
for Carbohydrate Recognition and Signalling, Department of Molecular Biology and Genetics,
Aarhus University, Aarhus C, Denmark; 2Department of Chemistry and Biomolecular Sciences,
Macquarie University, Sydney, Australia; 3Department of Biochemistry and Molecular Biology,
University of Southern Denmark, Odense, Denmark; 4Department of Plant and Environmental Sciences,
University of Copenhagen, København, Denmark; 5Centre for Glycomics, Department of Cellular and
Molecular Medicine, University of Copenhagen, København, Denmark
N- and O-glycosylation of proteins can have an impact on protein/enzyme functionality, activity, and
stability. The two corresponding pathways differ between plants and mammalians, and plant specific
N- and O-glycans may trigger an immunogenic response in humans. We have identified 19 different Nglycan structures from seeds of the model legume Lotus japonicus and O-linked arabinosylation of
hydroxyproline-rich glycoproteins using different MS approaches (1 and unpublished data). From the
Lotus LORE1 mutant population2 we obtained knockouts for all genes encoding the 12 enzymes in the
N-glycan maturation pathway and most of the genes in the O-glycosylation pathway in Lotus. For the
majority of the glycosylation genes, we have several independent knockout plant lines.
In general, the genes coding for enzymes in the N-glycan maturation pathway are less redundant than
genes in the O-glycosylation pathway. Clear plant developmental phenotypes were observed for three
of the N-glycan maturation enzyme mutants (data submitted). Currently, we have single and double
mutants for the genes in the N-glycan maturation pathway. Furthermore, single and some double
mutants are generated for the O-glycosylation pathway. One of our goals is to introduce the
mammalian N- and O-glycosylation in Lotus for production of mammalian-like glycoproteins in Lotus
seeds. I will present the current status of our project.
This work was supported by the Danish Council for Independent Research | Technology and Production
Sciences (FTP) and the Danish National Research Foundation grant no. DNRF79.
(1) Dam, S. et al., J. Proteome Res. 12, 3383-3392 (2013)
(2) Urbanski, D et al., Plant J. 69, 731-741 (2012)
Session 6
Invited speaker
Complex sialylation of plant-derived recombinant proteins
Castilho A., Kallolimath S., Strasser R., Grünwald-Gruber C., Altmann F., Strubl S.,
Elisabeth Galuska C.E., Zlatina K., Galuska S.P., Werner S., Gerardy-Schahn R. and
Steinkellner H.
Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences
(BOKU), Vienna, Austria; Department of Chemistry, BOKU, Vienna, Austria; Institute of Biochemistry,
Faculty of Medicine, Justus-Liebig-University, Giessen, Germany; Nambawan Biotech GmbH, Halle
(Saale), Germany; Institute for Cellular Chemistry, Hannover Medical School, Hannover, Germany
Sialic acids (Sia) are essential sugar residues in mammalian cells and confer diverse functions 1. Usually
serum proteins circulate as bi-sialylated molecules with Sia in α2,6- or α2,3-linkage. A particularly
complex form is polysialic acid (polySia), a non-immunogenic and biodegradable homopolymer of sialic
acid in α2,8-linkage2. Such structures enhance the pharmacokinetic values of proteins and confer
protein independent activities, e.g. neural tissue repair. The production of recombinant proteins with
controlled sialylation offers a variety of opportunities in drug development. Naturally, plants to not
synthesize sialylated glycans.
Here we describe a plant-based expression platform that enables the targeted synthesis of sialylated
structures with different inter-linkages and degree of polymerization (DP). The approach relies on a
combination of stably transformed plants with transient expression modules. By the introduction of
multigene vectors carrying the human sialylation pathway3 transgenic plants that sialylate
recombinantly expressed human glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by
the co-expression of human polysialyltransferase genes in planta polysialylation of recombinant
proteins was achieved. Notably, a DP of >40 was detected by high performance liquid chromatography
(HPLC) and fluorescence labeling of Sia polymers. Plant-derived polySia chains are functionally active,
as determined by cellular activity assays (inhibition of histone mediated cytotoxic activation). This
suggests therapeutic applications. Collectively, our approach enables investigations on biologic al
functions of defined sialylation and facilitates a rational design of glycoprotein drugs with optimized
efficacies.
1
Schnaar, R.L., Gerardy-Schahn, R. & Hildebrandt, H. Sialic acids in the brain: gangliosides and polysialic acid in nervous
system development, stability, disease, and regeneration. Physiol Rev 94, 461-518 (2014).
2
Colley, K.J., Kitajima, K. & Sato, C. Polysialic acid: biosynthesis, novel functions and applications. Crit Rev Biochem Mol Biol
49, 498-532 (2014).
3
Castilho, A. et al. Generation of biologically active multi-sialylated recombinant human EPOFc in plants. PLoS One 8, e54836
(2013).
Session 6
Short Talk - Poster 6.1
Plant antibodies in mediating FcγR functions and engaging neonatal Fc receptor
Stelter S.1, Paul M.J.1, Steinkellner H.2, Altmann F.3, Bardor M.4, Allen R.1 and Ma J.K.-C.1
1Institute
for Infection and Immunity, St George’s University of London, London, United Kingdom;
of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences,
Vienna, Austria; 3Division of Biochemistry, Universität für Bodenkultur, Vienna, Austria; 4Laboratoire
de Glycobiologie et Matrice Extracellulaire Végétale, Université de Rouen, Mont-Saint-Aignan, France
2Department
We have previously demonstrated that antibodies with typical plant glycosylation have a reduced
affinity to recombinant human Fc gamma receptors (FcγRs), which can be restored or even improved
by glyco-engineering. The impact of plant glycosylation and glyco-engineering on antibody Fc-mediated
functionality has been further investigated in immunological cell-based assays. Briefly, fluorescent
latex beads were coated with HIV-1 gp140 antigen and different plant-made glycovariants of VRC01,
an anti-HIV antibody, were compared for efficiency in mediating phagocytosis of the beads by THP-1
human monocytes using flow cytometry. The results demonstrated that typical plant glycosylation had
a negative effect on binding of antibody-coated antigen-beads to cell surface receptors and
consequently on their uptake by the cells. However, glyco-engineered antibodies are equally effective
in enhancing cell-mediated phagocytosis as a human cell-made variant.
The next objective was to develop a novel SPR-based assay to investigate the influence of plant
glycosylation on antibody affinity to the neonatal Fc receptor (FcRn), which is involved in determining
the half-life of IgG in the circulation. The results suggest that FcRn binding is independent of plant
glycosylation, however all plant-made glycovariants had a slightly lower affinity to the receptor than a
human cell-made antibody, which correlated with the extent of oxidation of methionine residues
involved in binding. This suggests that plant-based production and purification procedures might
promote antibody oxidation leading to lower affinity to FcRn and points towards a need for process
optimisation to control oxidation levels to improve the quality of plant-produced antibodies.
Session 6
Short talk - Poster 6.2
Optimization of protein glycosylation pattern in host plants through CRISPR/Cas9mediated genome editing for production of edible influenza vaccines
Maharjan P.M., Jeong Y.J., Park A., Sim E. and Choe S.
School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, Republic of
Korea
Production of vaccines using plant systems promises multiple benefits including cost, speed, scalability,
and safety. To better exploit the plant system as a vaccine factory, we sought for humanized plants in
their post translational modification steps. Firstly, we genetically engineer diverse plant species in their
glycosyltransferase systems. The transferase gens of plants exist as multiple copies and expressed
under the different spatiotemporal regulatory programs, thus it is challenging to control this system
simultaneously. In addition, conventional modification of these genes are required to go through
regulations imposed on living modified organisms (LMO) before being utilized as commercial purposes.
To alleviate these concerns, we developed methods entitled DNA-free genome editing in plants. We
first in vitro assembled the two subunits of Cas9 including small guide RNA and Cas9 protein, and this
complex was introduced into cell wall-less plant cells. The RNA-protein complex successfully edited the
target genes without leaving footprints at off-target loci. CRISPR/Cas9-mediated small deletion or
insertion at the target gene was virtually indistinguishable from naturally spontaneous mutations.
Taking advantages of this technology, we are editing plant specific glycosyl transferases in Arabidopsis,
lettuce, and Camelina. Under the various plants with different genetic backgrounds, we express the HA
gene of human influenza virus to produce a library of structurally varying proteins. Combined with the
technology of plant production in controlled environmental systems, the proteins to be produced
should meet impending needs of proteins having diverse structural variations.
Session 6
Short talk - Poster 6.3
Production of tumour-targeting antibodies with a human-type glycosylation profile in
N. benthamiana hairy root cultures
Lonoce C.1 , Marusic C.1, Jutras P.V.2, Morrocchi E.1, Steinkellner H.3, Benvenuto E.1 and
Donini M.1
1Laboratory
of Biotechnology, ENEA Research Center Casaccia, Rome, Italy; 2Département de
Phytologie, Centre de recherche et innovation des végétaux, Université Laval, Québec, Canada;
3Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences,
Vienna, Austria
Among the different approaches used to express heterologous proteins in plant tissues, hairy roots
(HRs) represent an interesting production platform due to their rapid growth, genetic stability and
possibility of working with self-contained systems allowing the accumulation of recombinant proteins
in the culture medium. With the aim of obtaining tumour-targeting monoclonal antibodies (mAbs) with
a human-type glycosylation profile we expressed two different antibodies in HRs generated from glycoengineered ΔXFXT N. benthamiana plants which lack plant typical xylose and fucose residues (Strasser
et al. 2008). Two antibody formats were chosen, full length IgG mAb H10 targeted to several tumors
(Villani et al. 2009) and the single chain version scFv-Fc (2B8-Fc) directed against Non Hodgkin
lymphoma (Marusic et al. 2016). HRs were generated using a multipl e-gene co-transformation strategy
in which leaf disks were co-infected with recombinant Agrobacterium rhizogenes strains carrying the
antibody coding genes (heavy and light chain for mAb H10 or 2B8scFvFc for the anti-CD20 antibody).
In the case of mAb H10, we established in 2 months HRs clones expressing fully assembled antibodies.
The best expressing clone was grown in liquid cultures and an optimised protocol was used for
secretion of mAb H10 in the culture medium with antibody accumulation levels of 2 µg/mL. Nglycosylation profile of mAb H10 purified from liquid media was determined by mass spectrometry
analysis and exhibited human type GnGn structures lacking xylose and fucose residues. HR clones
transformed with 2B8-Fc constructs have been obtained and are currently under evaluation. The data
demonstrate that the infection of ΔXTFT leaves with recombinant A. rhizogenes bearing antibody gene
constructs is a rapid procedure for the generation of HR cultures expressing mAbs with a targeted
glycosylation profile.
Strasser et al. (2008) Plant Biotecnol J. 6: 392-402.
Marusic et al. (2016) Plant Biotechnol J. 14: 240-251.
Villani et al.(2009). Plant Biotechnol. J. 7: 59–72.
Session 6
Short talk - Poster 6.4
Towards the humanization of glycoproteins secreted in Nicotiana tabacum BY-2 cells
Navarre C.1, Smargiasso N.2, Duvivier L.1, Far J.2, De Pauw E.2 and Boutry M.1
1Institut des Sciences de la Vie, Université de Louvain,
Louvain, Belgium; 2Laboratoire de Spectrométrie
de Masse, GIGA-R, Université de Liège, Liège, Belgium
Plant suspension cells have been used to produce various recombinant glycoproteins such as
monoclonal antibodies. One limitation of the system is the addition of the potential immunogenic or
allergenic glycoepitopes alpha-1,3-fucose and beta-1,2-xylose along the secretory pathway. Another
drawback is the absence of human-type N-glycan residues like beta-1,4-galactose or sialic acid.
Although there are several reports on N-glycoengineering in Nicotiana tabacum BY-2 cells, none
analyzed the effect of N-glycoengineering on IgG secreted in the culture medium. The aim of our study
consisted of generating plant cell lines that synthesize and secrete IgG and other glycoproteins with
human type N-glycans into the extracellular medium.
We first analyzed the N-glycans decorating a human IgG2 produced in BY-2 cells under different culture
conditions. IgG2 was purified by protein A affinity from the spent media and digested with trypsin. The
tryptic peptides were separated by nanoLC and analyzed by MS and MS/MS. Glycopeptides were then
manually detected using specific oxonium ions. The complex structure GnGnXF3 represented about
2/3 of all glycan structures secreted in the culture medium, whatever the medium composition.
We then co-expressed the human beta-1,4-galactosyltranferase together with the IgG2 and evaluated
its activity on IgG glycosylation. The beta-1,4-galactosyltransferase was targeted to either the cis- or
the trans-Golgi by fusing the appropriate targeting sequences. We found that beta-1,4galactosyltransferase expression strongly modified the N-glycan profile of IgG secreted in the culture
medium of BY-2 cells, resulting in IgG with galactosylated N-glycans.
Session 6
Short talk - Poster 6.5
Engineering of plants for the expression of helminth glycoproteins with their native Nglycan structures
Wilbers R.H.P., Westerhof L.B., van Noort K., Nguyen D-L., Smant G., Bakker J., Hokke
C.H. and Schots A.
Laboratory of Nematology, Wageningen University and Research Centre, Wageningen, The
Netherlands
Schistosoma mansoni is a parasitic trematode that, like other helminths, secretes immunomodulatory
proteins. These secreted proteins are main topics of research as they are possible vaccine candidates
or may have therapeutic potential to treat inflammatory disorders. Many helminth secretory proteins
carry complex N-glycans, but the exact role of these N-glycans on immunomodulatory properties
remains to be elucidated. As the purification of a single glycoprotein from S. mansoni is inefficient and
unsustainable, a platform is required that enables production of such glycoproteins. Here we show that
S. mansoni-derived glycoproteins can be efficiently produced in plants. Furthermore, we have
engineered the plant glycosylation machinery to synthesise N-glycans carrying structures like Lewis X
or LDNF. Altogether, our results demonstrate that plants are an excellent platform for the expression
of helminth glycoproteins with their native N-gly cans. This opens up a new field of research and might
lead to the identification of novel therapeutic targets.
Session 6
Short talk - Poster 6.6
Characterization of Schistosoma mansoni fucosyltransferases for glyco-engineering of
‘native’ helminth N-glycan structures in planta
van Noort K., Wilbers R.H.P., Westerhof L.B., Smant G., Bakker J. and Schots A.
Laboratory of Nematology, Plant Sciences Group, Wageningen University and Research Centre,
Wageningen, The Netherlands
Secretory glycoproteins of parasitic helminths are in the spotlight as biopharmaceuticals because of
their strong, glycan-dependent immunomodulatory properties. Helminths and their secretions have
been shown to dampen allergic reactions and autoimmune disorders, such as inflammatory bowel
diseases, multiple sclerosis and rheumatoid arthritis. Clinical trials with live parasites and mouse model
studies with excretory/secretory proteins are promising and reveal an urgent need for the large-scale
production of defined secretory glycoproteins from helminths. Helminth N-glycans contribute to
immunomodulation, but have unique structures that cannot be synthesized in current
biopharmaceutical production systems. The trematode Schistosoma mansoni produces complex highly
fucosylated N-glycan structures on its glycoproteins. Therefore, modifications of the N-glycosylation
machinery of the expression host are required for the production of S. mansoni-derived
immunomodulatory glycoproteins with their native N-glycans. This can be achieved by knocking-in or
knocking-out specific glycosyltransferases, allowing synthesis of specific helminth N-glycan structures.
Plants are remarkable versatile as glyco-engineering platform for the synthesis of glycoproteins with
tailored N-glycans. However, for the synthesis of highly fucosylated N-glycans from S. mansoni
knowledge is lacking on how these specific structures are synthesized. For this purpose, we examined
the function of ten selected fucosyltransferases from Schistosoma mansoni using transient coexpression with immunomodulatory omega-1 as a model protein. Two fucosyltransferases were
identified that specifically couple fucoses to the core glucosamine with an α1,3 or α1,6 bond. These
two fucosyltransferases can be used to obtain α1,3- and α1,6-core fucosylated N-glycan structures
found on native helminth secreted immunomodulatory proteins. Our results show the versatility of
plants both as a production system and as a system for characterizing glycosyltransferase functionality.
Further characterization of S. mansoni fucosyltransferases and other glycosyltransferases will expand
our glyco-engineering toolbox and offers perspectives for the synthesis of novel complex helminth Nglycan structures in plants.
Session 6
Short talk - Poster 6.7
Gene inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 suspension cells
Mercx S., Tollet J., Magy B., Navarre C. and Boutry M.
Institute of Life Sciences de la Vie, Université de Louvain, Louvain, Belgium
Plant suspension cells are interesting hosts for the heterologous production of pharmacological
proteins such as antibodies. They have the advantage to facilitate the containment and the application
of good manufacturing practices (GMPs). Furthermore, proteins of interest can be secreted to the
extracellular medium, which makes the purification steps much simpler. However, improvements are
still to be made regarding the quality and the production yield. For instance, the inactivation of
proteases and the humanization of glycosylation are both important targets which requires gene
inactivation. To this purpose, CRISPR-Cas9 is a very promising technique which has been used recently
in a series of plant species, but not yet in plant suspension cells. Here, we sought to use the CRISPRCas9 system for gene inactivation in Nicotiana tabacum BY-2 suspension cells. We transformed a
transgenic line expressing a red fluorescent protein (mCherry) with a binary vector containing genes
coding for Cas9 and three guide RNAs targeting mCherry restriction sites, as well as a bialaphosresistant (bar) gene for selection. To demonstrate gene inactivation in the transgenic lines, the mCherry
gene was PCR-amplified and analyzed by electrophoresis. Seven out of 20 transformants displayed a
shortened fragment, indicating that a deletion occurred between two target sites. We also analyzed
the transformants by restriction fragment length polymorphism and observed that the three targeted
restriction sites were hit. DNA sequencing of the PCR fragments confirmed either deletion between
two target sites or single nucleotide deletion. We therefore conclude that CRISPR-Cas9 can be used in
N. tabacum BY2 cells. This opens an avenue towards the engineering of recipient cell lines more
adapted to the heterologous expression of pharmacological proteins.
Session 6
Poster 6.8
Use of the CRISPR-Cas9 nuclease for targeted transgene integration in tobacco
Bortesi L., Zischewski J. and Fischer R.
RWTH Aachen University, Aachen, Germany
The untargeted nature of current transgene insertion techniques in the nuclear genome of higher
plants represents a challenge that remains to be addressed in plant molecular farming. Not only
different transgenic events often display variable expression levels due to position effects, also
uncontrolled integration of transgenes can interfere with the plant’s metabolism and lead to potential
unintended effects.
Ideally, the establishment of a generic recipient line, characterized in terms of genetic background, in
which the transgene of interest could be integrated in a predetermined site that grants high yield of
recombinant protein, would accelerate both the development and the approval of new plant
production lines.
One method to achieve targeted integration is the use of nucleases to induce a double strand break at
a specific genomic sequence, and then exploit the DNA repair mechanisms of the host to introduce the
desired transgene at the selected position via homologous recombination (HR) or non-homologous end
joining (NHEJ). The development of strategies aimed at promoting either HR or NHEJ, easily
discriminating between HR and NHEJ events, and avoiding random integration of incoming DNA is
necessary.
Here we present our approaches for achieving nuclease-mediated targeted transgene integration in
tobacco exploiting the versatile CRISPR-Cas9 programmable nuclease.
Session 6
Poster 6.9
Identification of genomic loci of suspension plant cells favorable for expressing
heterologous proteins
Tollet J., Navarre C. and Boutry M.
ISV, Université Catholique de Louvain, Louvain, Belgium
Plant suspension cells have been widely used for the expression of pharmacological proteins such as
antibodies. Among all the advantages of this expression system, we can list the low production cost
and the facility to apply the good manufacturing practices because of the cell containment inside the
reactor. However, the low accumulation rates obtained until now limit the possibility of using plant
suspension cells as a commercial system to produce biopharmaceuticals. Improving the accumulation
rate is therefore important.
One way to increase the expression might be to take advantage of the position effect. Indeed it is
known that the heterologous expression is affected by the insertion site of the foreign DNA. Therefore
our objective consists of finding a favorable genomic locus with a high expression level and afterwards
targeting a gene of interest within this specific locus.
We transformed Arabidopsis thaliana and Nicotiana tabacum cell lines with an Agrobacterium
tumefaciens strain carrying within its T-DNA a gene coding for mCherry in the cytosol. We obtained
1600 and 400 transformed cell lines for N. tabacum and A. thaliana, respectively. These transformed
cell lines were screened so as to select the most fluorescent ones. The chosen cell lines were
characterized with a thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) to
determine the number of insertions as well as the insertion sites.
We are currently attempting to target those favorable loci with the CRISPR Cas9 editing technique in
order to insert a reporter gene by homologous recombination.
Session 6
Poster 6.10
Towards higher yields of recombinant products in plant systems: epigenetic
modulation of Medicago truncatula cell suspension cultures
Santos R.B., Nogueira A.C., Ascenso O., Ventura M.R. and Abranches R.
Instituto de Tecnologia Química e Biológica António Xavier (ITQB), Universidade Nova de Lisboa, Oeiras,
Portugal
Plant cell cultures are a promising platform for the production of recombinant proteins; however the
low yields typically obtained remain a major challenge of these systems. Our team has been working
with the model legume Medicago truncatula, and also BY2 tobacco cells, for the production of
recombinant proteins with applications in human health. We are now addressing the issue of low
product accumulation by altering the epigenetic status of the plant cells with the use of small molecule
enhancers (SME). In animal cells, it has been reported that the addition of some chemical compounds
to the culture media, such as sodium butyrate, TSA and SAHA, leads to improved product accumulation,
but they have not yet been tested in plant systems. These molecules are mainly histone deacetylase
inhibitors that promote higher transcription levels, by opening the chromatin conformation. We have
tested both commercial and in-house produced com pounds, based on the structure of SAHA and
containing modifications on the aromatic ring, the carbon chain and substitution of the hydroxamic
acid by a carboxylic acid. Our results on the addition of SMEs to the culture medium of plant cell lines
producing human prostaglandin D synthase, indicate that some of them cause an increase on the levels
of the recombinant product, mainly the compounds with a fluorine at the ortho position of the
aromatic ring, and that this mechanism is associated with histone hyperacetylation. These results
contribute both to the understanding of chromatin-mediated regulation of gene expression as well as
to the field of molecular farming where high yield production is a major challenge.
Session 6
Poster 6.11
A chimeric plant-human enzyme for glyco-engineering in the moss bioreactor
Rodriguez Jahnke F.1,2, Lorenz T.1,3, Parsons J.1, Altmann F.4, Reski R.1,2,3 and Decker E.
L.1
1Plant
Biotechnology, Faculty of Biology, University of Freiburg, Freiburg, Germany; 2Spemann
Graduate School of Biology and Medicine (SGBM), University of Freiburg, Freiburg, Germany; 3BIOSS
Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany, 4Institute of
Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, Austria
Posttranslational modifications of biopharmaceuticals have a great impact on their biological activity,
stability and immunogenicity. This is especially true for protein N-glycosylation, which is known to
differ, depending on the production system, from the patterns known from humans. Due to this reason,
production hosts have to be engineered to match human protein glycosylation.
The moss Physcomitrella patens is especially amenable not only for the production of
biopharmaceuticals in photobioreactors, but also for glyco-engineering due to its high rate of
homologous recombination in vegetative cells, which allows precise gene targeting.
Maturation of N-glycosylation is carried out in a sequential manner in the Golgi apparatus, where it is
crucial that the different glycosyltransferases are located in the appropriate sub-compartments for
them to act in the proper order. Incorrect localisation of these enzymes can lead to aberrations or even
absence of further glycan processing.
As we and others have experienced before, enzymes of mammalian origin are working in plant systems,
however, in their native form they seem to be localised incorrectly within the Golgi sub-compartments.
One of these enzymes is the human β1,4-galactosyltransferase, which has often been observed to act
"too early" in plants, resulting in hybrid- instead of complex-type N-glycosylation.
In order to achieve correct positioning, we have designed a chimeric enzyme by targeted domain
replacement, combining the N-terminus of the last enzyme known to be involved in the N-glycosylation
pathway in plants, the α1,4-fucosyl transferase, with the catalytic domain of the human β1,4galactosyltransferase. N-glycan analysis via mass spectrometry of rhEPO produced by moss lines stably
expressing this chimeric enzyme revealed glycopeptides exhibiting terminally galactosylated complextype N-glycans.
With this approach we show that combining plant-endogenous targeting sequences with catalytic
domains of human origin is an effective way to direct glycosyltransferases to the desired Golgi
compartments.
Session 6
Poster 6.12
Customizing plant genomes for molecular farming: CRISPR/Cas9-mediated knock-out
of plant-specific glycosyltransferases in Nicotiana benthamiana
Zischewski J., Bortesi L. and Fischer R.
Institute for Molecular Biotechnology, RWTH Aachen University, Aachen, Germany
The plant Nicotiana benthamiana can be used for the transient and stable expression of pharmaceutical
proteins like antibodies, presenting an attractive alternative to mammalian expression systems. While
plants are capable of making highly complex post-translational modifications, their N-glycosylation
patterns differ from the ones found on mammalian proteins. Two of these plant-specific glycosylations
are of particular relevance due to their potential immunogenicity. Accordingly, the presence of both
core α-1,3-fucose and β-1,2-xylose is undesirable for many pharmaceutical proteins. In order to
eliminate these unwanted sugar residues from plant glycans, we are using the emerging CRISPR/Cas9
technology to knock-out the genes coding for the responsible enzymes. The RNA-guided nuclease Cas9
is a precise tool that can be easily programmed to introduce a double strand break (DSB) in a sequence
of choice. In plants, the default pathway for repairing DSBs is non-homologous end joining, often
inserting or deleting a few nucleotides during repair. If this happens in a coding region, these indels
can lead to a frameshift mutation and consequently premature stopcodons, thus disrupting the gene.
We are exploiting this mechanism to knock-out all gene copies of the α-1,3-fucosyltransferase and β1,2-xylosyltransferase in the allotetraploid plant Nicotiana benthamiana to facilitate the production of
more pharmaceutically interesting glycoproteins without potentially allergenic N-glycans.
Session 6
Poster 6.13
Genome editing of host plants using programmable CRISPR/Cas9 RNA-guided
endonucleases
Sim E., Cheon J., Slki Park S., Maharjan P.M., Park A.Y., Jeong Y.J. and Choe S.
School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, Korea
Flurry of different species and varying practices in plant molecular farming represent capability to
customize and produce therapeutic proteins; however, at the same time they can also be considered
drawbacks. Lack of a unified platform applicable to different production sites discourage the regulatory
agents to approve the products and methods of production in timely manner. We aim to establish host
plants that can confer the reasonably acceptable homogenousity of proteins by engineering the genes
affecting the post-translational modification of proteins. First, we phylogenetically analyzed amino acid
sequences of the glycosyl transferases in model plants including Arabidopsis thaliana, Camelina sativa,
Lactuca sativa, and Lemna minor. We are deleting the plant-specific glycosyl transferase genes using
CRIPSPR/Cas9 RNA-guided endonucleases. Second, we are screening for the deleterious proteases
activated when disrupting the cells in protein extraction process so that we remove the genetic
information using the molecular scissors. Third, we will be able to edit the genes with which interfere
during the steps of protein purification such as plant secondary metabolites. Once established the
platform, the host plants should help keep the level of complexity relatively low and secure the quality
of plant-produced proteins independent of batches.
Session 7
Secondary metabolite
farming
Session 7
Invited speaker
Plant cell culture as a highly controllable tool for the sustainable production of
anticancer products
Ullisch D.A., Sankar-Thomas Y.D., Wilke S., Leibold T., Pump M., Heckenmüller H.,
Schütte K. and Gorr G.
Phyton Biotech GmbH, Ahrensburg, Germany
Secondary metabolites from plants or derivatives thereof are utilized in numerous medical
applications. In many cases harvest from nature or cultivation is limited by different factors like for
endangered species, low product content, climate impacts or cost intensive extraction. However, in
the pharmaceutical industry the ability to provide sufficient amounts of product and high quality are
inevitable requirements for the supply chain. Whereas the chemical complexity of secondary
metabolites precludes chemical synthesis on a reasonable commercial basis, plant cells provide a
natural chemical factory for a given compound, just by its biosynthetic pathways. Thus plant cells might
allow to overcome the above mentioned supply issues. Moreover, plant cell cultures are an excellent
tool to enrich specific structures out of a number of closely related compounds thus reducing byproducts in a significant manner.
Here, the most prominent example of production by plant cell fermentation (PCF®) – the process for
the anticancer compound paclitaxel – will be presented as one case study. Since 2000 this secondary
metabolite is produced at Phyton Biotech according to cGMP standard by robust and powerful
undifferentiated Taxus chinensis cells at 75,000 l fermentor scale.1
As a second case study the successful development of Thapsigargin expressing plant suspension
cultures will be described. Thapsigargin is a powerful cytotoxin – a SERCA inhibitor – and the precursor
for the derivative ADT, the key ingredient of Mipsagargin (G-202) which is in several clinical trials at
GenSpera, Inc. Currently, it is isolated from seeds of Thapsia garganica. So far the production of
Thapsigargin via plant suspensions cells was not succesfull.2,3
In this study expression of Thapsigargin in cell suspension culture was of particular interest. However,
stability of cell cultures and proportion to Thapsigargicin contents have been characterized as well.
Acknowledgements: The authors gratefully acknowledge GenSpera for funding
1
Imseng N, Schillberg S, Schürch C, Schmid D, Schütte K, Gorr G, Eibl D, Eibl R. Suspension culture of plant cells under
heterotrophic conditions. In: Meyer HP, Schmidhalter DR, editors.
Industrial Scale Suspension Culture of Living Cells. Wiley-VCH, Weinheim, 2014, 224-258
2
Jäger AK, Schottländer B, Smitt UW, Nyman U. Somatic embryogenesis in cell cultures of Thapsia garganica. Plant Cell Rep
1993; 12: 517-520.
3
Andersen TB, López CQ, Manczak T, Martinez K, Simonsen HT. Thapsigargin—From Thapsia L. to Mipsagargin. Molecules
2015, 20: 6113-6127
Session 7
Invited speaker
Design and plant-based production of therapeutic cyclic peptides
Craik D.J., Jia H., Oguis G.K., Smithies B., Qu H., Gilding E.K. and Jackson M.A.
Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia
Until recently it was believed that most naturally occurring cyclic peptides are biosynthesized nonribosomally, as is the case, for example, for the immunosuppressant drug cyclosporin. However, more
than 300 examples of ribosomally synthesized cyclic peptides have been discovered in bacteria, plants
and animals over the last decade1. The cyclotides2 are the largest family of these circular proteins and
have applications in drug design3 and agriculture4. They occur in plants from the Violaceae (violet),
Rubiaceae (coffee), Fabaceae (legume), Solanaceae (nightshade) and Cucurbitaceae (cucurbit) families
and have a diverse range of biological activities, including uterotonic, anti-HIV, and insecticidal
activities, the latter suggesting that their natural function is in plant defense. Cyclotides comprise ~30
amino acids, and incorporate three disulfide bonds arranged in a cystine knot topology. The
combination of this knotted structure with a circular backbone makes cyclotides exceptionally stable
and they have been used as scaffolds to display bioactive (pharmaceutical) epitopes 5. This presentation
will describe the expression of pharmaceutical cyclotides and other cyclic peptides in plant
‘biofactories’, particularly in Arabidopsis, tobacco and petunia.
1
Craik D J: Seamless proteins tie up their loose ends. Science (2006) 311, 1563-1564.
Craik D J (Editor): Advances in Botanical Research, Volume 76, Plant Cyclotides (2015) (Series Editors J P Jacquot and P
Gadal) Elsevier, London UK (ISBN: 978-0-12-800030-4).
3
D’Souza C, Henriques S T, Wang C K, Craik D J: Structural parameters modulating the cellular uptake of disulphide rich cyclic
cell-penetrating peptides: MCoTI-II and SFTI-I. Eur J Med Chem (2014) 88, 10-18.
4
Pinto M F S, Fensterseifer I C M, Migliolo L, Souza D A, de Capdville G, Arboleda-Valencia J W, Colgrave M L, Craik D J,
Magalhaes B S, Dias S C, Franco O L: Identification and structural characterization of a novel cyclotide with activity against
an insect pest of sugar cane. J Biol Chem (2012) 287, 134-147.
5
Northfield S E, Wang C K, Schroeder C I, Durek T, Kan M-W, Swedberg J E, Craik D J: Disulfide-rich macrocyclic peptides as
templates in drug design. European Journal of Medicinal Chemistry (2014) 77, 248-257.
2
Session 7
Invited speaker
How jasmonates provide the key to harness plant chemistry
Goossens A.
Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Bioinformatics,
Ghent University, Gent, Belgium
Across the plant kingdom, the jasmonate hormone steers the delicate balance between growth and
the activation of defence programs, such as the production of bioactive specialized metabolites. These
small organic molecules allow plants to cope with various types of (a)biotic stresses but often also have
biological activities of high interest to human. Plant cells are capable of producing an overwhelming
variety of specialized metabolites, both in terms of complexity and quantity, but this impressive
metabolic machinery is still hardly exploited, mainly because of the limited molecular insight into plant
metabolism.
In our research we try to identify the essential components acting in the jasmonate signalling network
and characterize the molecular mechanisms driving biosynthesis of specialized metabolites in
medicinal, crop and model plants. Extensive collections of genes have been generated that allowed
increasing our fundamental understanding of the central mechanisms that control plant metabolism.
In parallel, these genes serve as a novel resource for metabolic engineering tools that will facilitate the
sustainable production of existing or novel plant-derived molecules with superior bioactivities.
These tools are implemented in a synthetic biology platform that presently focuses on the synthesis of
bioactive triterpenoids and triterpene saponins, and their building blocks in plants and yeasts, in
particular hairy root cultures and Saccharomyces cerevisiae cells, respectively. Through combinatorial
biochemistry and regulatory network engineering, we could, for instance, redirect the yeast
triterpenoid biosynthetic pathway towards the synthesis of new-to-nature molecules or bioactive
intermediates that otherwise do not accumulate in plant cells.
Session 7
Invited speaker
Production of halogenated indigoid precursors in metabolically engineered tobacco
plants
Fräbel S. and Warzecha H.
Plant Biotechnology and Metabolic Engineering, Technische Universität, Darmstadt, Germany
Indigogenic glycosides are useful chromogenic substrates for histochemistry and molecular biology.
Most prominent is X-Gal (5-Bromo-4-chloroindoxyl-ß-D-galactoside) used for blue white selection
during molecular cloning procedures in bacteria. Although the precursor indican is a natural product
widespread in a large number of indigo plants, the halogenated derivatives mainly utilized as enzyme
substrates needs to be chemically synthesized. Synthesis of halogenated aromatic components require
numerous toxic precursors and intermediates as well as organic solvents, rendering the whole process
potentially harmful to the environment. We established a system for the in-planta biosynthesis of a
large array of indigogenic glucosides. Engineering of a three different bacterial halogenases along with
tryptophanase and a monooxygenase lead to the formation of specifically chlorinated indican
derivatives. Moreover, combination of halogen ases enab led chlorination at multiple sites and coadministration of bromine enabled the formation of brominated indican derivatives. Also different
subcellular compartments for optimal precursor supply as well as product formation were evaluated.
Taken together, plants provide a suitable platform for the production of halogenated fine chemicals.
Session 7
Short talk - Poster 7.1
Enhancing levels of aromatic amino acids in whole plants and cell cultures A case study
in flowering plants and grape derived cell culture
Oliva M.*1,2, Fang F.1, Ovadia R.1, Bar E.4, Sikrons-Persi N.5, Perl A.3, Fait A.5, Lewinsohn
E.4, Oren-Shamir M.1 and Galili G.2
1Ornamental
Horticulture, ARO, Volcani center, Bet-Dagan, Israel; 2Plant Sciences, Weizmann Institute
of Science, Rehovot, Israel; 3Fruit Tree Sciences, ARO, Volcani center, Bet-Dagan, Israel; 4Department
of Vegetable Crops, Newe Ya’ar Research Center, ARO, Ramat Yishay, Israel; 5Jacob Blaustein Institutes
for Desert Research, Ben-Gurion University, Sde-Boqer Campus, Israel.
* corresponding author: [email protected]
Specialized metabolites derived from the Aromatic Amino Acid (AAAs) are important compounds
synthesized widely by many agricultural crops. AAAs are involved in the adaptations of plants to biotic
and abiotic stresses and contribute to the plant performance. AAA derived compounds are
commercially important as pigments, aroma and phyto-antioxidants. Despite their significance,
attempts to enhance their levels by classical breeding, without affecting plant’s vigour, gained little
success.
Here, we enhanced levels of AAA derived secondary metabolites by boosting their primary metabolite
precursor in two different plant systems. Flowering plants as a whole plant system and grape cell
culture as cell culture system were studied. Both systems were transformed with a feedbackinsensitive bacterial form of 3-deoxy-diarabino-heptulosonate 7-phosphate synthase (DAHPS) enzyme
(AroG*) of the shikimate pathway, to stimulate plant secondary metabolism via the aromatic amino
acids. In both systems, AroG* enzyme resulted in a significant increase in AAA levels, and enhanced
levels of their downstream specialized metabolites, without affecting plant morphology. Analysis of
petunia and Lisianthus AroG* flowers showed a significant accumulation of fragrant benzenoidphenylpropanoid volatiles. Petunia AroG* characterized more and showed no effect on flavonoids and
anthocyanin levels. In the grape cell culture, AroG* led to accumulation of resveratrol and quercetin,
known promote health properties and did not increase anthocyanin pigmentation. Comparison of the
different plant systems will be presented.
Oliva M., et al., 2014 (Plant Biotechnology Journal): Enhanced formation of aromatic amino acids increases fragrance
without affecting flower longevity or pigmentation in Petunia × hybrid
Manela*, N., Oliva, M*., et al., 2015. *equal contribution. (Frontiers in Plant Sciences): Phenylalanine and tyrosine levels
are rate limiting factors in production of health promoting metabolites in grape cell suspension.
Peled-Zehavi H., Oliva M., et al., 2015. (Bioengineering): Metabolic Engineering of the Phenylpropanoid and Its Primary,
Precursor Pathway to Enhance the Flavor of Fruits and the Aroma of Flowers.
Session 7
Short talk - Poster 7.2
Plant-based production of natural flavours – case raspberry ketone
Häkkinen S.T., Seppänen-Laakso T., Oksman-Caldentey K.-M. and Rischer H.
VTT Technical Research Centre of Finland LTD, Espoo, Finland
Bioconversion, i.e. the use of biological systems to perform chemical changes in synthetic or natural
compounds in mild conditions, is an attractive tool for the production of novel active or high-value
compounds. Raspberry ketone (4-(4-hydroxyphenyl)butan-2-one) – a characteristic aroma component
in raspberry (Rubus idaeus) fruits - is among the most interesting natural flavour compounds, due to
its high demand and significant market value. The biosynthesis of this industrially relevant flavour
compound is relatively well characterised, involving the condensation of 4-coumaryl-CoA and malonylCoA by Type III polyketide synthase to form a diketide, and the subsequent reduction catalysed by an
NADPH-dependent reductase. In order to establishing more efficient and economical production
systems for natural raspberry ketone, bioconversion has shown to be an attractive approach. In this
work, we studied the effect of overexpressed raspberr y ketone reductase (RiZS1) in tobacco on
precursor bioconversion to raspberry ketone. In addition, plant cell cultures representing different
species were studied for their capacity to carry out the bioconversion to raspberry ketone using either
4-hydroxybenzalacetone or betuligenol as a substrate. Apparently plant cells possess rather widely
distributed reductase activity capable of performing the bioconversion to raspberry ketone using
inexpensive and readily available precursors.
Short CV of the invited
speakers
Andy Hiatt
2003-present
1996 - 2003
1994 - 1996
1987 - 1994
1986 - 1987
1984 - 1986
1978 - 1983
1980 - 1982
VP Antibody Research, Mapp Biopharmaceutical, Inc. San Diego, California
Chief Scientific Officer, Epicyte Pharmaceutical, Inc., San Diego, California
Research Director, Rose-Hiatt Biotechnology L.L.C., San Diego, California
Assistant Professor, The Scripps Research Institute, La Jolla, California
Staff Investigator, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Damon Runyon/ Walter Winchell Cancer Fund Fellowship, Cold Spring Harbor
NIH Endocrinology Traineeship, Columbia University, New York, NY
Biochemistry Teaching Assistantship, Columbia University, New York, NY
Willem Van de Velde
Willem Van de Velde obtained his PhD in 2002 at the Department of Plant Systems Biology (VIB-UGent). His
research focused on the “Chitinases and chitinase-like proteins in Sesbania rostrata nodule development”. After
an EMBO short Term Fellow in CNRS (Gif sur Yvette, France), he had three post doc positions at PSB/VIB-UGent
(2003-2005), CNRS (2006-2009) and the Institute of Tropical Medicine Antwerp (2009). In 2010, he became Team
Leader “Downstream Process Development” at Ablynx NV.
Markus Sack
I have studied biology at the RWTH Aachen University and joined the group of Rainer Fischer in 1996, then at
the department of botany headed by Prof. Kreuzaler where I earned my diploma in biology in 1998 and
continued to work at the newly formed department of molecular biology headed by Prof. Fischer. Ever since and
for almost two decades I have been involved in many aspects related to Plant-Molecular Farming. Some
highlights include the pioneering of rapid and scalable Agrobacterium-mediated transient gene expression,
application of surface plasmon resonance spectroscopy for the analysis of recombinant proteins produced in
plants, and process-development for bringing tobacco-derived HIV antibody 2G12 into the clinic. I am currently
engaged in projects to discover, develop and produce antibodies in plants for HIV and Rabies, unlock plantderived products through minimal processing strategies, use of a novel plant cell pack platform (also known as
“the cookie method”) in conjunction with continuous fermentation of plant suspension cells and SPR assay
development for product and process development and MP quality control. Despite many hurdles in the past
and a non-favorable socio-political climate in Europe I have witnessed the enormous progress in the field and
am full of optimism for the future. Plant-molecular farming is too good to fail.
Marc Van Montagu
Em. Prof. Marc Van Montagu is a pioneer in plant molecular biology. He is well known (with J. Schell) as the
discoverer of the Ti-plasmid and the inventor of Agrobacterium tumefaciens transformation technology, now
used worldwide to produce genetically engineered plants. He was Founding Member and Member of the Board
of Directors of two Belgian biotech companies, spin-offs from his laboratory, Plant Genetic System (PGS) and
CropDesign. At PGS he drove front-line innovations for biotech agriculture, such as plants resistant to insects or
tolerant to more environmentally friendly herbicides.
He has won numerous prizes amongst which the Japan prize. He is a member of several academies including the
National Academy of Sciences of the United States, Italy, Brazil and the Third World Academy of Sciences, the
Academies of Agriculture of France and Russia, and the Academy of Engineering of Sweden. In October 2013 he
was awarded the World Food Prize. In November 2014, he became UNIDO Goodwill Ambassador for
Agribusiness. He is ranked as the most cited scientist in the field of Plant & Animal Science until 2004. He has
produced over 1000 publications and the totality of scientific publications has received more than 50.000
citations. Due to his accomplishments he received in 1990 the title of Baron from the King of Belgians. Currently
he is President of the European Federation of Biotechnology and of the Public Research and Regulation Initiative,
and Chairman of the Institute of Plant Biotechnology Outreach. Marc Van Montagu holds a PhD in Organic
Chemistry/ Biochemistry and a BA in Chemistry from Ghent University.
Lars von Borcke
Business Development Manager, PBL (since 2004): Managing a portfolio of over 30 technologies, consisting of
acquiring technologies from academic Institutes, establishing and managing relationships with scientists and
University technology transfer professionals; Coordinating the patent application and patent prosecution
activities for the portfolio of technologies; Developing the commercialisation of technologies through
marketing to the AgBiotech & Life Science industry; Negotiating, drafting and monitoring of license
agreements; Working on projects to commercialise products with partner industry, currently working on the
development of in-field nitrate sensor
Licensing Manager, Out-Licensing, Syngenta, Switzerland (2001 to 2004): Out-licensing of Syngenta’s
biotechnology enabling technologies, including development of licensing strategy and terms, negotiating and
drafting of license agreements
Licensing Manager, Zeneca Agrochemicals, UK (2000 to 2001): Managing all in-licensing activity for North
America, including research agreements and IP licenses
Change Management, Zeneca Agrochemicals, UK (1999 to 2001)
European Quality Co-ordinator, Zeneca Agrochemicals, UK (1997 to 1999)
Graduate Management Trainee, Hazlewood Foods, UK (1995 to 1997)
Els Beirnaert
Els Beirnaert is Senior Manager, New Ventures at VIB, responsible for the establishment of start-up companies
in life sciences. In this role she is engaged in business analysis and market research, identifying product
opportunities, attracting venture capital funding, corporate structuring and negotiations. Previously she was one
of the start-up pioneers of Ablynx, a company established in 2001 focused on the development of biological
therapeutics, where she was leading multidisciplinary project teams and translating several drug development
projects from discovery over pre-clinical development to clinical development. Dr. Beirnaert obtained a Master
in Biotechnology at the University of Ghent and a PhD in Biochemistry at the Institute of Tropical
Medicine/University of Antwerp.
Joachim Schiemann
Prof. Dr. Joachim Schiemann is director of the Institute for Biosafety in Plant Biotechnology at Julius Kuehn
Institute (JKI), Federal Research Centre for Cultivated Plants. Since 2006 he is Honorary Professor at University
of Lüneburg. He has been coordinating several national and EU-funded cluster projects on biosafety research.
From 2000 to 2003 he was member of the Scientific Committee on Plants of the European Commission, Health
& Consumer Protection Directorate-General, and from 2003 to 2009 member of the Panel on Genetically
Modified Organisms of the European Food Safety Authority (EFSA). From 2002 to 2012 he was member of the
Executive Committee of the International Society for Biosafety Research (ISBR), from 2004 to 2008 President of
ISBR. Since 2004 he has been member of the Steering Council of the European Technology Platform “Plants for
the Future”.
Xavier Saelens
Xavier Saelens obtained his PhD in the laboratory of Walter Fiers. His research at VIB and Ghent University
(Belgium) focuses on the development of novel vaccines and antibody-based antivirals against influenza A and
B viruses, human Respiratory Syncytial virus and Junin virus. A strong research focus of his group is to unravel
the mechanisms of actions of these vaccines and antivirals and to understand how influenza viruses evolve in
the immune host. With regard to universal influenza vaccine development his group is well known for proposing
influenza A virus M2e as a broadly protective flu vaccine candidate. Xavier Saelens is a group leader at VIB and
full professor at Ghent University. In 2015 he was awarded the Biennial Prize of the Centre d'études Princesse
Joséphine Charlotte for his research in Virology.
Inga Hitzeroth
Dr. Inga Hitzeroth obtained her PhD in 1988 in Population Genetics in Microbiology at the University of Cape
Town, South Africa. Since 2000, she is senior research officer and deputy director of Prof. E. Rybicki’s
Biopharming Research Unit, Department of Molecular and Cell Biology at the University of Cape Town. The main
focus of her research is to express viral proteins in plants with the aim at producing affordable vaccines for South
Africa. She has worked on HPV expression for the last 15 years and has designed and expressed various HPV
L1/L2 chimaeric proteins with the aim of developing second generation HPV vaccines that will protect against
more than one type. Dr. Hitzeroth also works on the development of therapeutic vaccines for HPV and
expression of various other viral proteins in plants.
Rima Menassa
Dr. Rima Menassa is a senior research scientist at Agriculture and Agri-Food Canada, and holds an adjunct
professor appointment at Western University in London, Ontario, Canada. She obtained her Ph.D. in plant
molecular genetics from McGill University, and then joined Agriculture and Agri-Food Canada as a postdoctoral
fellow with Jim Brandle. She now leads the molecular farming effort for the production of recombinant proteins
in tobacco at AAFC. Her research program is focused on producing veterinary subunit vaccines and antibodies
in plants. Dr. Menassa is funded by AAFC, has co-authored over 45 scientific publications, was involved in three
patent applications, and has co-edited a book. Dr. Menassa serves on the executive board of the Canadian
Association of Plant Biotechnology since 2014 and is the North American representative of ISPMF since 2015.
Yuri Gleba
Dr. Gleba has over 30 years of research and management experience in plant genetics and biotechnology. (M.
Sc., Kiev University, 1971; Ph.D., Institute of Botany, Academy of Sciences of Ukraine, 1974; Prof., D.Sc.,
Leningrad University, 1980).
Dr. Gleba’s pioneering research in plant genetics, physiology and biotechnology was published in more than 200
research papers, books and over 30 patent families, and has earned the respect of the international scientific
community as is evidenced by his election to the World Academy of Arts and Science (Rome), the European
Academy (Academia Europaea, London), the National German Academy Leopoldina (Halle), the National
Ukrainian Academy of Sciences (Kiev), the Lithuanian Academy of Science (Vilnius) and the Bavarian Academy
of Sciences (Munich). He also received numerous international and national awards and prizes, including
Koerber Prize (Hamburg), A. von Humboldt Prize (Bonn), USSR State Prize (Moscow), State Prize of Ukraine
(Kiev), etc.
He founded the Institute of Cell Biology and Genetic Engineering, Ukrainian Academy of Sciences, Kiev, Ukraine,
in 1989, and was serving as its Director until 2010; currently, he is Honorary Director of the Institute.
Dr. Gleba left former USSR in 1991 and joined American Cyanamid Company, Princeton, NJ, where he developed
research efforts in plant biotechnology and genomics, first as a group leader/manager, and, since 1997, as a
Director of Crop Engineering Department. Dr. Gleba left American Cyanamid/American Home Products in 1999
and founded Icon Genetics, Princeton/Munich/Halle, a plant biotechnology company group; he has been serving
since its inception as its CEO. Under his leadership, Icon has developed multiple plant expression technologies,
including the magnICON® transient technology that has been brought to a commercial level and cGMP
compliance and has been used to support clinical trials of the product candidates developed by Icon/Bayer. Icon
has created one of the best IP portfolios in plant biotechnology that currently includes over 450 issued patents
representing 42 patent families of patents/applications. He also founded/founded four other companies,
including Nomad Bioscience GmbH, Germany. In January 2012, Dr. Gleba engineered the acquisition of Icon
Genetics from Bayer by Nomad Bioscience, and in 2015 sale of Icon to Denka, Japan.
During his entire carrier, Dr. Gleba was involved in university education. He was a supervisor of 35 Ph D students
and was lecturing or holding adjunct positions for many years at the universities of USSR, USA, Belgium, Germany
and Ukraine.
Udo Conrad
Udo Conrad has been born in Saxony, East Germany, studied Biology in Greifswald and graduated in yeast
genetics in 1979. From 1979 to1985 he did a PhD in Gatersleben about biochemical genetics of mice, worked
about developmental biology of mammals and molecular immunology and switched to plant sciences in 1990.
Since 1992 he is the leader of the Phytoantibody group at the IPK, habilitated in Botany at the University
Göttingen in 1998 and was a lecturer at the University Halle since 2001. Since 2015 he is teaching as an
Extraordinary Professor at the University Halle.
Diego Orzaez
Diego Orzaez is Tenured Scientist (Científico Titular) at CSIC and co-leads the Plant Genomics and Biotechnology
Group (PGB) at IBMCP. He did his PhD on Plant Programmed Cell Death (PCD) at Granell´s lab. As a Marie-Curie
post-doc he joined E. Woltering lab in Wageningen (NL) for 2 years to study PCD in plant cell cultures. Later he
moved to Wageningen University and joined the A. Schot LMA lab during 4 years, where he started the design
of plants as antibody biofactories. In 2004 he returned to Valencia with a Ramón y Cajal contract and became
Tenured Scientist in 2009. Diego is inventor of 4 patents and has published more than 40 papers in international
journals. He has worked in several research projects at national and international levels as participant or as PI,
and he is currently in charge of the Genetic Engineering and Synthetic Biology projects within the PGB group.
Ann Meyers
Ann Meyers obained a PhD in Microbiology at the University of Cape Town in 1999 After a one year post-doc
position in the department of Biochemistry (UWC), she started her career in the lab of Prof. E. Rubicki where
she became scientific officer in 2004 and research officer in 2010. Her research interests lie mainly in the
expression of recombinant proteins and subunit vaccines in plants using plant expression vectors (biopharming).
Of particular interest are veterinary VLP vaccines against Foot-and-mouth disease virus, Bluetongue virus and
African horse sickness virus. Currently, research is also performed on the expression of various different reagents
in plants which can be used in diagnostic assays. These include Foot-and mouth disease antigens and single chain
variable fragment antibodies (scFvs) used for diagnostic ELISAs, as well as the manipulation and expression of
horseradish peroxidase fusions to different scFvs for immunodetection purposes.
Renier van der Hoorn
Renier Adrianus Leonardus van der Hoorn was born in Leiden in 1971 and was fascinated by plant biology from
early childhood. He studied chemistry at Leiden University and focused soon on plant molecular biology and
biochemistry. After his graduation in 1996, he started his PhD in Molecular Phytopathology (Wageningen
University, Prof. Dr. Pierre de Wit), where he worked on the tomato Cf resistance proteins. He continued working
on Cf proteins in Wageningen as a postdoc, and started his own research program by introducing and applying
activity-based protein profiling in plants. To further develop the technology he joined the phosphoproteomics
group of Dr. Scott Peck for one year (Sainsbury lab, John Innes Centre, Norwich, UK). He initiated the Plant
Chemetics lab in October 2005 at the Max Planck Institutes of Cologne and Dortmund as part of the Chemical
Genomics Centre of the Max Planck Society. His research group operated independently from the departments
at the Max Planck Institutes while he trained twelve MSc students, nine PhD students, eleven postdocs and over
30 visiting scientists. Since October 2013 he is Associate Professor at the Department of Plant Sciences of the
University of Oxford, and Tutor in Plant Sciences at Somerville College. His research focusses on the use of
chemical proteomics to uncover novel host manipulation mechanisms employed by microbes when colonizing
the apoplast.
Eva Stöger
Eva Stöger is currently Professor of Molecular Plant Physiology and head of the Department of Applied Genetics
and Cell Biology at the University of Natural Resources and Life Sciences, Vienna, Austria. After completing her
PhD at the University of Vienna she worked at the University of Florida, Gainesville, US, at the John Innes Centre,
Norwich, UK, and at the Aachen Technical University (RWTH). She received several awards including the Golden
Grain award from the Cerealiers de France and AGPM (France), and the Sofia-Kovalevskaja Prize awarded by the
Alexander-von-Humboldt Foundation (Germany). Her main research interests are in the area of cereal
biotechnology, intracellular protein trafficking and deposition, endomembrane dynamics and the production of
high-value recombinant proteins in seed crops.
Jussi Joensuu
Dr. Joensuu obtained his M.Sc. from Faculty of Agriculture and Forestry from the major of Crop Husbandry at
the University of Helsinki in 2000. His Ph.D. (2006) is from Faculty of Biosciences from the major of Genetics also
at the University of Helsinki. Between years 2006-8 he worked as a Research Fellow at Department of Molecular
and Cellular Interactions, University of Brussels, Belgium and at Agriculture and Agri-Food Canada, London,
Ontario, Canada. Since 2009 he has worked at VTT Technical Research Centre of Finland Ltd and holds an adjunct
position at the University of Helsinki.
His research interests include production of therapeutic and industrial proteins in plants and micro-organisms,
immunobiology, mucosal pathogens, vaccination and host-pathogen interactions. In the recent years Dr.
Joensuu has focused in fusion protein technologies to improve production and purification yields of recombinant
proteins. Dr. Joensuu has co-authored over 30 peer-reviewed publications and patents.
Francis Santens
Francis Santens did his PhD in the lab of Prof. Nico Callewaert, where he co-developed the GlycoDelete platform
both in mammalian cells and plants. Currently he is involved in a collaboration to further mature the GlycoDelete
platform towards a more commercial platform.
Richard Strasser
Prof. Richard Strasser obtained his PhD in 2000 at the University of Natural Resources and Life Sciences (BOKU)
in Vienna at the Department of Applied Genetics and Cell Biology. In that same department, he became
independent group leader in 2006, Assistant Professor in 2009 and Associate Professor (tenure-track position)
in 2012. Since 2014, Prof. Strasser became deputy head of this department.
His research interests include characterization of plant glycosidases and glycosyltransferases, function of Nglycans in plants, N-glycan biosynthesis in plants, N- and O-glycan engineering in plants, Golgi-organization,
glycan dependent ER quality control and degradation pathways. Prof. Strasser has co-authored over 60 peerreviewed papers, book chapters and patents.
Alexandra Castilho
Alexandra Castilho graduated in 1990 from Lisbon University (Portugal) in Applied Plant Genetics. As a young
scientist researcher at the Institut National de la Recherche Agronomique (INRA, Versailles, France) and at the
Agricultural University of Lisbon (ISA, Portugal), she worked in various aspects of cereals Cytogenetics. In 1995
Dr. Castilho earned her PhD from the John Innes Institute in Norwich (UK), working on Molecular Cytogenetics.
She continued to work as a postdoctoral scientist in the group of Prof. Heslop-Harrison until 1999 when she
moved to the Instituto de Tecnologia Química e Biológica ITQB in Oeiras (Portugal). In 2004 she joined the group
of Prof. Herta Steinkellner at the BOKU University in Vienna (Austria) as a senior postdoc. Dr. Castilho is currently
manager of the Laura Bassi Center of Expertise focusing her research on Plant-Produced Glycan-Optimized
Biopharmaceuticals. The research focuses on the development of plant based expression systems that allow
efficient generation of valuable proteins with a “customised” glycosylation profile. This work includes the
molecular characterization and modulation of the N-glycosylation pathway in plants in combination with the
expression of therapeutically relevant target proteins.
Gilbert Gorr
As Chief Scientific Officer of Phyton Biotech GmbH, a subsidiary of the DFB Pharmaceuticals division Phyton
Biotech Inc., Gilbert Gorr is taking care of development projects regarding new plant-based cell cultures –
including acquisition and customer relationship. In addition he provides technical support to the patent
prosecution procedures which so far resulted in far more than 100 granted patents and a number of filings of
new patent applications since 2009, when he joined Phyton.
Prior to starting his career at Phyton Gilbert served as scientific head in small- to medium-sized companies. He
was shareholder and founder of two biotech companies where he realized successful exits.
As CSO and together with his team at that time, Gilbert laid the cornerstone for the success of greenovation.
Gilbert is inventor/co-inventor of 11 patent families comprising secondary metabolites produced by plant cell
cultures, expression technologies, molecular tools and glyco-engineering – including antibody optimization. So
far his work resulted in more than 200 granted patents and numerous scientific publications.
After receiving for his work on the moss bioreactor his doctorate degree in natural sciences at Hamburg
University he was Post doc at the School of Veterinary Medicine Hannover where he worked in the area of
pharmaceutical development.
David Craik
David Craik is a group leader and Professor of Chemistry at the Institute for Molecular Bioscience at The
University of Queensland, Brisbane, Australia. He obtained his PhD in organic chemistry from La Trobe University
in Melbourne, Australia and undertook postdoctoral studies at Florida State and Syracuse Universities before
taking up a lectureship at the Victorian College of Pharmacy in 1983. He was appointed Professor of Medicinal
Chemistry and Head of School in 1988. He moved to University of Queensland in 1995 to set up a new
biomolecular NMR laboratory and is currently an Australian Research Council Laureate Fellow. His research
focuses on applications of circular proteins, toxins and NMR in drug design. He is a Fellow of the Australian
Academy of Science and has received numerous awards for his research, including the Ralph F. Hirschmann
Award from the American Chemical Society. He is author of 580 scientific papers (h-index 74, citations 20,000)
and has trained 60 PhD students.
Alain Goossens
Alain Goossens (born 1971) obtained his Master in Biology-Plant Biotechnology at Ghent University (1992) and
his PhD in Marc Van Montagu’s lab at the Laboratory of Genetics, Ghent University (1998), studying plant seed
storage protein synthesis. Subsequently, he performed postdoctoral studies at the IBMCP-UPV in Valencia
(Spain) in the lab of Ramón Serrano, working on yeast salt tolerance. He returned to VIB-Ghent University and
started his own research group within the VIB Department of Plant Systems Biology at Ghent University in 2003,
focusing on jasmonate signaling, gene discovery in plant specialized metabolism and metabolic engineering. He
is experienced in yeast and plant functional genomics, molecular biology and applied biotechnology. His current
research aspires to understand jasmonate signaling in model, crop and medicinal plants and unravel the
mechanisms that steer plant growth and metabolism in response to developmental and environmental cues to
find novel tools for plant metabolic engineering and synthetic biology. He has been appointed as a part-time Full
Professor at Ghent University in 2015 and is teaching ‘Metabolic Engineering’. He has been mentoring 30 master
students, 24 PhD students and 15 postdoctoral researchers. He has authored 82 peer reviewed ‘A1’ publications,
which have been cited over 3,000 times (H-index 31). He is co-inventor of 10 patent applications. He has been
invited speaker on 26 international conferences and symposia. In 2015 he was included in the prestigious
Thomson Reuters list of Highly Cited Researchers.
Heribert Warzecha
Heribert Warzecha is Professor for Plant Biotechnology and Metabolic Engineering at the Technische Universität
Darmstadt, Germany, Department of Biology. He is a pharmacist and obtained his PhD at the Gutenberg
Universität Mainz in Pharmaceutical Biology, working on alkaloid biosynthesis in medicinal plants and cell
cultures. In 1999 he went for two years to the Boyce Thompson Institute for Plant Research at Cornell University,
Ithaca/NY where he got involved in research in the topic of Molecular Farming and the production of vaccines
in plants. After a stay at Universität Würzburg he moved to TU Darmstadt in 2007. One of his research foci lies
in the metabolic engineering of plant secondary metabolism and the generation of novel medicinal plants. He is
author of numerous scientific publications, chairing the COST Action FA1006 (Plant Engine) between 2011 and
2015, was guest lecturer in Astana/Kasachstan and holds an honorary doctorate from Örebro
University/Sweden.
Participants list
Abranches Rita
ITQB - Universidade Nova de Lisboa, Portugal
[email protected]
Bontinck Michiel
PSB/VIB-UGent, Belgium
[email protected]
Altosaar Illimar
University of Ottawa, Canada
[email protected]
Bortesi Luisa
RWTH-Aachen University, Germany
[email protected]
Amiri Mahshid
Tarbiat Modares University
[email protected]
Boudolf Veronique
PSB/UGent-VIB, Belgium
[email protected]
Aranda Miguel A.
CEBAS-CSIC, Spain
[email protected]
Broer Inge
Universität Rostock, Germany
[email protected]
Ariel Tamar
Protalix, Israël
[email protected]
Buyel Johannes
Fraunhofer IME/RWTH Aachen University, Germany
[email protected]
Avesani Linda
University of Verona, Italy
[email protected]
Callewaert Nico
MBC VIB, VIB-UGent, Belgium
[email protected]
Bakshi Shruti
UGent-VIB, Belgium
[email protected]
Cardon Florian
Root Lines Technology, France
[email protected]
Beirnaert Els
VIB, Belgium
[email protected]
Carreer Roxanne
Bio-Sourcing, Belgium
[email protected]
Bethke Susanne
RWTH Aachen, Germany
[email protected]
Castells Graells Roger
John Innes Centre, UK
[email protected]
Bijora Taise
Universidade Estadual de Maringá, Brazil
[email protected]
Castilho Alexandra
University of Natural Resources and Life Sciences, Austria
[email protected]
Bleys Annick
PSB/VIB-UGent, Belgium
[email protected]
Chin-Fatt Adam
University of Western Ontario, Canada
[email protected]
Bonaventure Gustavo
CropDesign, Belgium
[email protected]
Choe Sunghwa
Seoul National University, Republic of Korea
[email protected]
Ciani Silvano
Dr. Schär R&D Centre, Italy
[email protected]
Donini Marcello
ENEA, Italy
[email protected]
Commandeur Uli
RWTH Aachen University, Germany
[email protected]
Drake Pascal
St George's University of London, UK
[email protected]
Conrad Udo
IPK Gatersleben, Germany
[email protected]
Edgue Gueven
RWTH Aachen, Germany
[email protected]
Contreras Angela
U-Mons / Green2Chem, Belgium
[email protected]
Ehsani Parastoo
Pasteur Institute of Iran, Iran
[email protected]
Cox Eric
UGent, Belgium
[email protected]
El Cheikh Ibrahim Ahmad
St George's University of London, UK
[email protected]
Craik David
University of Queensland, Australia
[email protected]
Els Hendrik
University of Cape Town, South Africa
[email protected]
Dam Svend
Aarhus University, Denmark
[email protected]
Fernández Fernández Álvaro
PSB/VIB-UGent, Belgium
[email protected]
De Buck Sylvie
IPBO/VIB-UGent, Belgium
[email protected]
Fukuzawa Noriho
A.I.S.T., Japan
[email protected]
Decker Eva
University of Freiburg, Germany
[email protected]
Gaildry Typhanie
INRA, France
[email protected]
Depicker Ann
PSB/UGent-VIB, Belgium
[email protected]
Giritch Anatoli
Nomad Bioscience GmbH, Germany
[email protected]
De Wilde Chris
CropDesign, Belgium
[email protected]
Gleba Jurijus
Nomad Bioscience GmbH, Germany
[email protected]
Dickmeis Christina
RWTH Aachen University, Germany
[email protected]
Gohr Florian
RWTH Aachen University, Germany
[email protected]
Gomord Veronique
ANGANY Genetics, France
[email protected]
Hundleby Penny
John Innes Centre, UK
[email protected]
Goossens Alain
PSB/VIB-UGent, Belgium
[email protected]
Jamieson Gordon
Leaf Systems, UK
[email protected]
Gorr Gilbert
Phyton Biotech GmbH, Germany
[email protected]
Jeong Yu Jeong
Seoul National University, Republic of Korea
[email protected]
Gottardo Davide
University of Milan, Italy
[email protected]
Joensuu Jussi
VTT Technical Research Centre of Finland, Finland
[email protected]
Guillet Marina
Root Lines Technology, France
[email protected]
Juarez Paloma
PSB/VIB-UGent, Belgium
[email protected]
Gunter Cornelius
University of Cape Town, South Africa,
[email protected]
Jung Jae-Wan
Chon-Buk National University, Republic of Korea
[email protected]
Häkkinen Suvi
VTT Techical Research Centre, Finland
[email protected]
Jutras Philippe
Laval University, Canada
[email protected]
Helfer Anne
CropDesign, Belgium
[email protected]
Khamis Zayn
The University of Western Ontario, Canada
[email protected]
Hiatt Andrew
Mapp Bio, USA
[email protected]
Kim Miyoung
Chonbuk National University, Republic of Korea
[email protected]
Hitzeroth Inga
University of Cape Town, South Africa
[email protected]
Ko Kisung
Chung-Ang University, Republic of Korea
[email protected]
Hoef Angelique
UGent, Belgium
[email protected]
Kopertekh Lilya
Julius Kühn-Institut, Germany
[email protected]
Holland Tanja
Fraunhofer IME, Germany
[email protected]
Kupers Luc
Genzyme, Belgium
[email protected]
Lallemand Jérôme
University of Liège, Belgium
[email protected]
Lamprecht Renate
University of Cape Town, South Africa
[email protected]
Masloboy Axel
University of Rostock, Germany
[email protected]
Massa Silvia
ENEA, Italy
[email protected]
Lico Chiara
ENEA, Italy
[email protected]
Matías Hernandez Luis
Sequentia Biotech/ Center for Research in AgroGenomics
(CRAG), Spain
[email protected]
Lomonossoff George
John Innes Centre, UK
[email protected]
Matsumura Takeshi
AIST, Japan
[email protected]
Lonoce Chiara
ENEA – Casaccia, Italy
[email protected]
Matsuo Kouki
National Institute of Advanced Industrial Science and
Technology (AIST), Japan
[email protected]
Lorenz Timo
University of Freiburg, Germany
[email protected]
Luijben Lucia
Wageningen UR, Netherlands
[email protected]
Ma Julian
St George's University of London, UK
[email protected]
Maeda Megumi
Okayama University, Japan
[email protected]
Maranyane Hapiloe
University of Cape Town, South Africa
[email protected]
Mariage Pierre-Antoine
Green2Chem, Belgium
[email protected]
Marsian Johanna
John Innes Centre, UK
[email protected]
McLean Michael
PlantForm Corp., Canada
[email protected]
Menassa Rima
Agriculture and Agri-Food Canada, Canada
[email protected]
Méndez López Eduardo
CEBAS-CSIC, Spain
[email protected]
Mercx Sébastien
UCL, Belgium
[email protected]
Merlin Matilde
University of Verona, Italy
[email protected]
Mérot Bertrand
Bio-Sourcing, Belgium
[email protected]
Meyers Ann
University of Cape Town, South Africa
[email protected]
Miletic Sean
Institute of Molecular Biotechnology, Austria
[email protected]
Moravec Tomas
Institute of Experimental Botany, Czech Republick
[email protected]
Nagata Tatsuya
University of Brasilia, Brazil
[email protected]
Nausch Henrik
University of Rostock, Germany
[email protected]
Navarre Catherine
Université catholique de Louvain, Belgium
[email protected]
Nguyen Quang Duc Tien
Chon-Buk National University, Republik of Korea
[email protected]
Nisse Estelle
Plant Advanced Technologies SA, France
[email protected]
Ofoghi Hamideh
Biotechnology Department IROST, Iran
[email protected]
Oksman-Caldentey Kirsi-Marja
VTT Techical Research Centre, Finland
[email protected]
Oliva Moran
Weizmann Institute of Science, Israel
[email protected]
Orzaez Diego
IBMCP-CSIC, Spain
[email protected]
Palaci Jorge
PSB/VIB-UGent, Belgium
[email protected]
Paul Mathew
St George's University of London, UK
[email protected]
Pereira Eridan Orlando
Greenbean Biotechnology, Brazil
[email protected]
Peyret Hadrien
John Innes Centre, UK
[email protected]
Phan Hoang Trong
IPK Gatersleben, Germany
[email protected]
Pietersen Inge
University of Cape Town, South Africa
[email protected]
Ponndorf Daniel
University of Rostock, Germany
[email protected]
Powell Steven
John Innes Centre, UK
[email protected]
Rage Émile
ENSAIA, France
[email protected]
Regnard Guy
University of Cape Town, South Africa
[email protected]
Reski Ralf
University of Freiburg, Germany
[email protected]
Ribeaucourt David
Plant Advanced Technologies, France
[email protected]
Ritala Anneli
VTT Techical Research Centre, Finland
[email protected]
Rodriguez Jahnke Fernando
University of Freiburg, Germany
[email protected]
Rossi Luciana
University of Milan, Italy
[email protected]
Saberianfar Reza
Agriculture and Agri-Food Canada, Canada
[email protected]
Sack Marcus
RWTH Aachen University, Germany
[email protected]
Saelens Xavier
MBC VIB, Belgium
[email protected]
Santens Francis
MBC VIB, Belgium
[email protected]
Santos Rita
ITQB - Universidade Nova de Lisboa, Portugal
[email protected]
Saunders Keith
John Innes Centre, UK
[email protected]
Schaaf Andreas
Greenovation, Germany
[email protected]
Schiemann Joachim
Julius Kuehn Institute, Germany
[email protected]
Schiermeyer Andreas
Fraunhofer IME, Germany
[email protected]
Schots Arjen
Wageningen UR, Netherlands
[email protected]
Sim EunI
Seoul National University, Republic of Korea,
[email protected]
Sion Caroline
ENSAIA, France
[email protected]
Steele John
John Innes Centre, UK
[email protected]
Stelter Szymon
St George's University of London, UK
[email protected]
Stöger Eva
DAGZ/BOKU Vienna, Austria
[email protected]
Strasser Richard
University of Natural Resources and Life Sciences Vienna,
Austria
[email protected]
Teh Audrey
St. George's University of London, UK
[email protected]
Tharad Ashuwini
PSB/VIB-UGent, Belgium
[email protected]
Thuenemann Eva
John Innes Centre, UK
[email protected]
Tollet Jeremie
Université Catholique de Louvain, Belgium
[email protected]
Top Oguz
University of Freiburg, Germany
[email protected]
Walden Miriam
John Innes Centre, UK
[email protected]
Tran Hong-Hanh
Western University, Canada
[email protected]
Warzecha Heribert
Technische Universitaet Darmstadt, Germany
[email protected]
Ullisch David
Phyton Biotech GmbH, Germany
[email protected]
Webster Gina
St George's University of London, UK
[email protected]
van der Hoorn Renier
University of Oxford, UK
[email protected]
Weichert Nicola
IPK Gatersleben, Germany
[email protected]
Van de Velde Willem
Ablynx, Belgium
[email protected]
Westerhof Lotte
Wageningen UR, Netherlands
[email protected]
Van Eerde Andre
Norwegian Institute of Bioeconomy Research, Norway
[email protected]
Wilbers Ruud
Wageningen UR, Netherlands
[email protected]
Van Montagu Marc
IPBO/VIB-UGent, Belgium
[email protected]
Yang Moonsik
Chonbuk National University, Republic of Korea
[email protected]
van Noort Kim
Wageningen UR, Netherlands
[email protected]
Zampieri Roberta
University of Verona, Italy
[email protected]
Virdi Vikram
PSB/VIB-UGent, Belgium
[email protected]
Zischewski Julia
RWTH Aachen University, Germany
[email protected]
von Borcke Lars
Plant Bioscience Limited, UK
[email protected]
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